Title of Invention

ANTIBODIES AND MOLECULES DERIVED THEREFROM THAT BIND TO STEAP-1 PROTEINS

Abstract Antibodies and molecules derived therefrom that bind to novel STEAP-1 protein, and variants thereof, are described wherein STEAP-1 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, STEAP-1 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The STEAP-1 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with STEAP-1 can be used in active or passive immunization
Full Text ANTIBODIES AND MOLECULES DERIVED THEREFROM THAT BIND TO STEAP-1 PROTEINS
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
FIELD OF THE INVENTION ;
The Invention described herein relates to antibodies, as well as bindingfragments hereof and molecules engineered therefrom, that bind proteins, termed STEAP-1. The invention further relates to diagnostic, prognostic, prophylactic and therapeutic methods and compositions useful in the treatment of cancers that express STEAP-1.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. White deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
Worldwide, several cancers stand out as Hie leading killers. In particular, carcinomas of the king, prostate, breast,
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colon, pancreas; ovary, and bladder represent the primary causes of cancer death. These and virtually an other carcinomas share a common lethaj feature. With vejy few exceptions, metastaSc disease from a carcinoma is fatal. Moreover, even for (hose cancer patients who initially survive their primary cancers, common experience has shown (hat their fives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for
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recurrence or treatment failure. Many cancer patients experience physical debflitaSons following treatment Furthermore, many cancer patients experience a recurrence.
Worldwide, prostate cancer is lite fourth most prevalent cancer in men. In North America and Northern Europe, it
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is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease - second only to king cancer. Despite the magnitude of these figures, there is still no effective treatment for metastaCc prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be Ihe main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of (his disease. Although Ihe serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and gerieralirfflity is widely regarded as lacking in several important respects.
Progressln identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease In mice. The LAPC (Los Angeles Fjostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCO) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et at.. 1997, Nat Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et aL, 1996, Proc. NaO. Acad. Sd USA 93:7252), prostate-specific membrane (PSM) antigen (Pinto et aL, Cfin Cancer Res 1996 Sep 2 (9): 1445-

51), STEAP (Hubert, ef at, Proc Na« Acad Sd U S A. 1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter el a/., 1998, Proc. Nal Acad. Sd. USA 95:1735).
White previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and fterapeutfc targets for prostate and related cancers in order to further improve diagnosis and therapy.
Renal ceil carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas.reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The Incidence of renal eel! adenocarcinoma is estimated at more than 29,000 cases in the.United States, and more than 11,600 patients died of this disease in 1998. Transitional cell " ' carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly i.nmunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent In men (fifth most common neoplasm) and 3 percent In women (eighth most common neoplasm). The Incidence is Increasina slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated ,11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and w Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The mulb'focal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.
An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (-2.1% per year). Research suggests ftat these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers* There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11 % of all U.S. cancer deaths.
At present, surgery is the most common form of therapy for colored rancer, and for cancers that have not spread, ft is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel waH or has spread to the lymph nodes. A permanent cctostomy (creation of an abdominal opening for elimination of body wastes) Is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.
There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is dedming signrficantlylnmen.fromahighofSjS.Sper

100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate In women was 42.3 per 100,000.
Lung and bronchial cancer caused an estimated 156,900 deaths in 2000. accounfing for 28% of all cancer deaths.
During 1992-1996, mortality from kmg cancer declined significantly among men (-17% per year) white rates for women were
stfll significantly increasing (0.9% per year). Since1987, more women have died each year of hing cancer than breast
cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing king cancer incidence and
mortality rates most Wcely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking
patterns among women lag behind those of men. Of concern, although toe declines in adult tobacco use have stowed,
tobacco use'inyo^ ' -•--•••••-
Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed In combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small ceD lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There Is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After Increasing about 4% per year tn the 1980s, breast cancer incidence rates In women have leveled off in the 1990s to about 110.6 cases per 100,000.
{|ln the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) hi 2000 due to breast cancer. Breast cancer ranks second among cancer deaths In women. According to.flie most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and Mack. These decreases were probably the result of earlier detection and Improved treatment
Taking into account he medical circumstances and the patient's preferences, treatment of Nr^st cancer may involve lumpectomy {local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical . removal of ihe breast) and removal of the lymph nodes under the arm; radiafion therapy; chemotherapy; or hormone therapy.. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy phis radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances In reconstruction techniques provide several options for breast reconstrucfion after mastectomy. Recently, such reconstruction has been done at the same fine as the mastectomy.
Local excision of ductal carcinoma if? situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of Ihe DCIS. Radiation to the breast and/or tamoxtfen may reduce the chance of DCIS occurring to the remaining breast tissue. This is important because DCIS, If left untreated, may develop into invasive breast cancer. Nevertheless, ttiere are seHousskle effects or sequelae to these treatments. There Is, therefore, a need for efficacious breast cancer treatments.
There were an estimated 23.100 new cases of ovarian cancer h fte United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer Incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually Includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and Ihe uterus (hysterectomy). In some

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very early tumors, wily the involved ovary wffl be removed, espedafly in young women who wish to have children. In ad«/anced disease, an attempt is made to remove all inlra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need tor effective treatment options for ovarian cancer.
There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have deoOned in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over (he past 20 years, there has been a slight but significant decrease in mortality rates among men (about -0.9% per year) whBe rates have increased sfighfly among women.
Surgery, radiation Jherapy,-and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients put are not likely to produce a cure for most There is a significant need for additional therapeutic and diagnostic options for cancers. These include the use of antibodies, vaccines, and small molecules as treatment modalities. Additionally, there is also a need to use these modilities as research tools to diagnose, detect monitor, and further the state of the art In aB areas of cancer treatment and studies.
SUMMARY OF THE INVENTION
The invention provides antibodies as well as binding fragments thereof and molecules engineered therefrom, that bind to STEAP-1 proteins and polypeptide fragments of STEAP-1 proteins. As used herein, the term STEAP-1 is synonamous with 8P1D4. The invention comprises potydonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human anGbodies, and antibodies labeled with a detectable marker or therapeutic agent In certain embodiments, there is a proviso that the entire nucleic acid sequence of Figure 2 is not
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encoded and/or the entire amino add sequence of Figure 2 is not prepared. In certain embodiments, the entire nucleic add sequence of Figure 2 is encoded and/or the entire amino add sequence of Figure 2 is prepared, either of which are in respective human unft dose forms.
The invention further provides methods for detecting the presence and status of STEAP-1 potynudeofides and
proteins in varrousbictogica'aarr^es, as wefl as methods for idenB An embodiment of Ws
Invention provides methods for monitoring STEAP-1 gene products in a fissue or hematology sample hawig or suspected of having some form of growth dysregutafon such as cancer.
The invention further provides various immunogenlc or therapeutic compositions and strategies for treating cancers that express STEAP-1 such as cancers of tissues listed in Table I, inducing therapies aimed at Inhibiting the transcription, translation, processing or function of STEAP-1 as wel as cancer vaccines. In one aspect, the inven$on provides compositions, and methods comprising them, for treating a cancer that expresses STEAP-1 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits die production or function of STEAP-1. Preferably, the carrier Is a uniquely human carrier. In anofter aspect of the invention, the agent is a moiety that is knmunoreactive with STEAP-1 protein. Non-fimiting examples of such moieties Include, but are not United to, antibodies (such as single chain, monoclonal, polydonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations (hereof. The antibodies can be conjugated to a (fiagnostic or therapeutic moiety. In another aspect, tire agent is a smaflmotecute as defined herein.
In another aspect, toe agent comprises one or more than one pepfide which apprises a Us,
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one peptids which comprises a helper T lymphocyte (HTL) epitope which binds an HLA dass II molecule h a human to elicit an HTL response. The pepfides of the invention may be on the same or on one or more separate porypepfide molecules. In a further aspect of the invention, the agent comprises one or more than one nudeic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, fiie one or more than one nudeic add mofecule'may express a moiety that is immunctogically reactive with STEAP-1 as described above. The one or more than one nudeic acid molecule may also be, or encodes, a molecule that inhibits
" * production of STEAP-1. Non-limiting examples of such molecules indude, but are not limited to, those complementary to a nudeotide sequence essential for producflon of STEAP-1 {e.g. antiserise sequences or molecules foat form a triple helix with a nudeofide double helix essential for STEAP-1 production) or a ribozyme effective to lyse-STEAP-1 mRNA.
Another embodiment of Ihe invention is antibody epitopes, which comprise a peptide regions, or an oligonudeoUde encoding Ihe pepfide region, that has one two, three, four, or five of the following characteristics:
I) a peptide region of at least 5 amino acids of a particular peptide of Figure 3, Jn any whole number increment up to the full length of that protein in Figure 3, that includes an amino add position having a value equal to or greater than 0.5, 0.6,0.7,0.8,0.9, or having a value equal to 1.0, in the Hydrophilicity profile of Rgure 5;
II} a pepfide region of at least 5 amino adds of a particular pepSde of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino add position having a value equal lo o- less than 0.5,0.4, 0.3,0.2,0.1, or having a value equal to 0.0, in the Hydropathidty profile of Figure 6;
If) a pepb'de region of at least 5 amino adds of a particular peptide of Figure 3, in any whole number increment up
to the full length of that protein h Figure 3, that indudes an amino add posifion having a value equal to or greater than 0.5,
i 0.6,0.7; 0.8,0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Figure 7;
iv) a peptide region of at teases amino adds of a particular peptide of Figure 3, fa any whofe number increment up to the full length of that protein In Figure 3, that Indudes an amino add position having a value equal to or greater ban 0.5, 0.6,0.7,0.8,0.9, or having a value equal to 1.0, In the Average Flexibility profile of Figure 8; or
v) a pepfide region of at least 5 amino adds of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that Indudes an amino acid position having a value equal to or greater than 0.5, 0.6,0.7,0.8,0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9..
The present invention also relates to a gene, designated STEAP-1, that has been round to be over-expressed in the cancels) fisted in Table I. Northern blot expression analysis of STEAP-1 gene expression In normal tissues shows a restricted expression pattern in aduit tissues. The nudeotide (Figure 2) and amino add (Figure 2, and Figure 3) sequences of STEAP-1 are provided.' The tissue-related profile of STEAP-1 in normal adult tissues, combined with the over-expression observed in the tissues fisted In Table I, shows that STEAP-1 is aberrantly over-expressed in at legist some cancers, and thus serves as a useful diagnosfic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.
The invenfion provides pofynudeofides corresponding or complementary to all or part of (he STEAP-1 genes, mRNAs, and/or coding sequences, preferably ki isolated form, induding poiynudeofides encoding STEAP-1-related proteins and fragments of 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24.95, or more than 25 contiguous amino adds; at least 30,35,40,45,50,55,60,65,70,80,85,90,95,100 or more than 100 contiguous amino adds of a GTEAP-1-related protein, as welt as the pepGdes/proteins themselves; DMA, RNA, DNA/RNA hybrids, and related molecules, Iwlynudeofides or oftgonudeotides complementary or having at least a 90% homotogy to foe STEAP-1 genes or mRNA lequences or parts thereof, ami rxtynucteotk^ OTEAP-1-encoding poiynudeofides. Also provided are means for isolating cONAs and the genes encoring STEAP-1.

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Jfefombinant DMA molecules containing STEAP-1 polynudeotides, cefls transformed or transduced wKh such motecutes, and host^vectar systems for the expression of STEAP-1 gene products ere also provided.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. The STEAP-1 SSH sequence of 436 nudeoFdes.
Figure i The cDNA and amino add sequence of STEAP-1 variant 1 (also called "STEAP-f v.1" or "STEAP-1 ! variant 1") is shown fa Figure 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 66-1065 Including fiie stop codon.
The cDNA and ammo acid sequence of STEAP-1 variant 2 (also called "STEAP-1 V;2*) is shown In Rgure 2B. the codon for the start mefcfonine is underlined. The open reading frame extends from nudeic add 96-872 Induding the stop codon.
The cDNA and amino add sequence of STEAP-1 variant 3 (also caBed "STEAP-1 v.3") is shown in Figure 2C. The codon for the start methionine is underfined. The open reading frame extends from nudeic add 96-944 induding the stop codon.
The cDNA and amino add sequence of STEAP-1 variant 4 (also called "STEAP-1 v.4") is shown in Figure 20. The codon for the start methionine is underlined. The open reading frame extends from nudeic add 96-872 induding the ciop codon.
The cDNA and amino add sequence of STEAP-1 variant 5 (also called 'STEAP-1 v.5") is shown in Figure 2E The codon for the start meihionine is underlined. The open reading frame extends from nucleic add 96-872 induding (he stop codon.
Thai cDNA and amino add sequence of STEAP-1 variant 6 (also called 'STEAP-1 v.6") is shown in Figure 2F. The codon for the start methtanine is underlined. The open reading frame extends from nudeic add 96-872 induding the stop codon.
The cDNA and ammo add sequence of STEAP-1 variant 7 (also called "STEAP-1 v.7") b shown in Figure 2G. The codon for the start methionine is underfined. The open reading frame extends from nudefe add 96-872 induding the stop codon.
The cDNA and amino add sequence of STEAP-1 variant C (also called "STEAP-1 v.81} is shown h Figure 2H. The codon for the start methionine is underlined. The open reading frame extends from nudeic add ae-872 induding the stop codon.
The cDNA and amino add sequence of STEAP-1 variant 9 (also caHed "STEAP-1 v.9") is shown in Figure 21. The codon for the start methionine is underfined The open reading frame extends from nudefc add 96-872 iQduding the stop codon.
The cONA and amino acid sequence of STEAP-1 variant 10 (also called 'STEAP-1 v.10") is shown in Figure 2J. The codon for the start meWonine is underlined. The open reading frame extends from nucleic add 96-872 including the slop codon.
The cONA and amino add sequence of STEAP-1 variant 11 (also called "STEAP-1 v.111 is shown In Figure 2K. The codon for the start methfonine Is underfined. The open reading frame extends from nudeic add 96-872 induding the stop codon.
The cONA and amino add sequence of STEAP-1 variant 12 {also called "STEAP-1 v.12") is shown in Figure 2L
The codon for thfrsiartmethfentoefe underfined. The open readBngframe extends from nucleic add 96-872 induding the
stop codon. -

9-* The cDNAand amino add sequence of STEAP-1 variant 13 (aiso called "STEAM v.13") is shown In Figure 2M.
The codon for the start meihfor.ine Is underfined The open reaolng frame extends from nucleic add 96-872 including the stopcodon.
I The cDNA and amino add sequence of STEAP-1 varianM4 (also cafed "STEAP-1 v.14") Is shown fn Figure 2N.
\ . The codon for the start methfonine is underlined. The open reading frame extends from nudeic add 96-872 including the
I stopcodon.
The cDNA and amino acid sequence of STEAP-I variant 15 (also called "STEAP-1 v.15") is shown in Figure 20.
The codon for the start methtonine is underlined. The open reading frame extends from nuctefc add 96-872 induding the
stop codon. .. -
The cDNA and amino add sequence of STEAP-1 variant 16 (also called "STEAP-1 v.16") is shown in Rgure 2P. The codon for the start methfonine is underlined. The open reading frame extends from nucleic add 96-872 Induding the stopcodon.
The cPNA and amino add sequence of STEAP-1 variant 17 (also called "STEAP-1 v. 171) is shovm in Figure 20. The codon for the start methionine is underlined. The open reading frame extends from nudefc add 96-872 inducing the stop codon. As used herein, a reference to STEAP-1 indudes ail variants thereof, induding those shown in Figures 10,11 and/or 12 unless the context dearly indicates otherwise.
Figure 3. Amino add sequence of STEAP-1 v.1 is shown in Figure 3A; it has 339 amino adds.
The amino add sequence of STEAP-1 v.2 is shown in Figure 3B; it has 2® amino acids.
The amino add sequence of STEAP-1 v.3 is shown h Figure 3C; ft has 282 atn&wadds.
The amino add sequence of STEAP-1 v.4 is shown In Figure 3D; it has 258 amino adds. As used herein, a
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referenced STEAP-1 Indudes all variants thereof, induding those shown in Figures 10,11 and/or 12 unless the context
clearly indicates otherwise.
Figure 4. Figure 4A. The amino add sequence alignment of STEAP-1 v.1 wifli mouse TNFo>induced adipose-
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related, protein (gi/16905133). Figure 4B. The amino add sequence alignment of STEAP-1 v.1 with rat pHyde protein
(gi/21717655/). Figure 4C. Shows Homoiogy of STEAP-1 to mouse six transmembraneepitheliaranfigen of fte prostate (gi|20820492j).
Figure 5. Hydrophifctty amino add profile of STEAP-1 variant 1 (Figure 5(aJ). HydropWScfty amino add profile of STEAP-1 variant 3 (Figure 5(b)), determined by computer aigorilhm sequence analysis using the method of Hopp and Woods (Hopp T.P., Woods K.R., 1981. Proc. Nat). Acad. Sd. U.SA 78:3824-3828) accessed on the ProtScale website located on the work! wide web URL cWcgJ-bWprotscate.pl) through (he ExPasy molecular biology server.
Figures. (Figure 6(a)).Hydropalhid(y amino add profile of STEAP-1 variant 1. (Figure 6(b}}. Hydropathidty amino add profile of STEAP-1 variant 3, determined by computer algorithm sequence analysis using the method of Kyle and Doofittie (Kyte J., DooGttte R.F., 1982. J. Md. Bid. 157:105-132) accessed on the ProtScale website located on the world wktewebURLexpasy.crvcgi-bWprots^
Figure 7. (Figure 7(a)). Percent accessible residues amino add profile of STEAP-1 variant 1. (Figure 7(b)}. Percent accessible residues amino add profile of STEAP-1 variant 3, determined by computer atgorimm sequence analysis using the method of Janin (Janh J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the world wide web URL expasy.cWi^-bin/prolscate.pl) through the ExPasy molecular biology server.
Figures. (Figure 8{aj). Average flexibfflty amino add profile of STEAP-1 variant 1. (Figure 8(b)). Average flexibility amino add proSte of STEAP-1 variant 3, determined by cxxnr^eralgori^sequenoa analysts usirQ«r« method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P.K., 1988. InL J. Pept Protein Has. 32:242-255) accessed

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on the ProtScate website located on the world wide web URL expasy.ch/cgi-bin/protscale.pl) through IheExPasy molecular biology server.
Figure 9. (Figure 9(a)). Beta-turn amino add profile of STEAP-1 variant 1. (Figure 9(b}). Beta-turn amkio acid profile of STEAP-1 variant 3, determined by computer algorithm sequence analysis using the method of Deteage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the world wide web URL expasy.ch/cgi-bin/protscale.pt) through the ExPasy molecular biology server.'
Figure 10. Schematic aGgnmenl of SNP variants of STEAP-1. Variants STEAP-1 v.4 through v.17 are variants with single nudeotide differences as compared to STEAP-1 v.2. Though these SNP variants are shown separately, they jgould also occur in any combinations and in any transcript variants that contains the base pairs, ?.g., STEAP^ v.1 aMv.3.:f Numbers correspond to those of STEAP-1 v.2. Black box shows the same sequence as STEAP-1 v.2. SNPs are indicated above the box.
Figure 11. Exon compositions of transcript variants of STEAP-1. This figure shows the structure of the transcript variants without poly A tail Variants STEAP-1 v.1, v.2 and v.3 are transcript variants that share the same exons 2 and 3. The first exon of STEAP-1 v.1 is 30 bases shorter at 5' end than the first exons of the other two transcript variants. The fourth exon of STEAP-1 v.2 is the same as the combined exon 4, intron 4 and exon 5 of STEAP-1 v.1. Compared with STEAP-1 v.1, variant STEAP-1 v.3 has an additional exon spliced out from intron 4 of STEAP-1 v.1. Lengths of fotrons and exons are not proportional.
Figure 12. Schemafic alignment of protein variants of STEAP-1. Protein variants correspond to nudeofide variants. Nudeotide variants STEAP-1 v.5 through v.17 in Figure 10 code for tie same protein as STEAP-1 v.2. Proteins translated from transcript variants STEAP-1 v.1 and v.3 as shown in Figure 11 may contain amino add F (Phe) or L (Leu) at position 169. Single amino add differences, were indicated above the boxes. Black boxes represent the same sequence as STEAP-1 v.1. Boxes with different patterns of filling show different sequences. Numbers underneath the box correspond to STEAP-1 v.1.
Figure 13. Figures 13{a) - (c). Secondary structure and transmembrane domains prediction for STEAP-1 protein variants. The secondary structure of STEAP-1 protein variants 1 (SEQ ID NO: 46), 2 (SEQ ID NO: 47), and 3 (SEQ ID NO: 48); (Figures 13a-1'3c, respectively) were predicted using Die HNN - Hierarchical Neural Network method (Guermeur, 1997, httpV/pbB.ibcp.fr/cgi-bin/npsa_automaLpl?pacie=npsa_nn.h{ml), accessed from foe ExPasy molecular biology server located on the World Wide Web at (.expasy.cn/tools/). This method predicts the presence and location of alpha hefices, extended strands, and random colls from the primary protein sequence. The percent of the protein in a given secondary structure is also listed. Figures 13{d),13(f), and 13(h): Schematic representations of the probability of existence of transmembrane regions and orientation of STEAP-1 variant 1-3, (Figures 13(d), 13(0 ^ 13{h) respectively, based en the TMored algorithm of Hofrnann and StoffeJ which utilizes TMBASE (K. Hofmann, W. StoffeL TMBASE- A database of membrane spanning protein segments Bid. Chem. Hoppe-Seyter 374:166,1993). Figures 13(e), 13(g), and 13(0: Schematic representations of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of STEAP-1
variants 1-3, Figures 13(e), 1%), and 13(0 respectively, based on the TMHMM algorithm of Sonnhammer, von HeQne, and
-*. Krogh (Erik LL Sonnhammer, Gunnar von He|ne, and Anders Krogh: A hidden Markov model for predicting
transmembrane helices in protein sequences. In Proa of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Utflejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, CA: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy n^ecular biology server lo
Figure 14. Figure 14a. Expression of STEAP-Hn stomach cancer patient specimen. RNA was extracted from normal stomach (N) and from 10 different sfcmach cancer patient specimsns (T). Northern Wot with 10 jig of fctal RNAAane was probed with STEAP-1 sequence. Results show strong expression of an approximately 1.6kb STEAP-1 in the stomach tumor fissues. The lower panel represents ethidium bromide staining of the blot showing quality of the RNA. samples. Figure 14b. STEAP-1 expression In rectum cancer patient tissues. RNA was extracted from normaJnectum (N), rectum cancer patient tumors (T), and rectum cancer metastasis (M). Northern blots with 10 pg of tola! RNA were probed with the STEAP-1 sequence. Results sho'w strong expression of STEAP-1 In the rectum cancer patient tissues. The lower panel represents ethldium bromide staining of the blot showing quality of the RNA samples. Figure 14c. Expression of STEAP-1 in human umbilical vein endothelial cells (HUVEC). First strand cDNA was prepared from HUVEC cells, LAPC-4AD and LAPC-9AD prostate cancer xenografts, as well as from human brain tissues. Normalization was performed by PCR using primers to acfin and GAPDH. Semi-quantitative PCR, using primers to STEAP-1, was performed at 27 and 30 cycles of amplification (A). As a control, PCR using primers to actin is shown in (B). Results show strong expression of STEAP-1 in HUVEC ceils similar to the expression detected in prostate cancer xenograR tissues. Expression of STEAP-1 in HUVEC cells indicates that targeting STEAP-1 may also target endothetial cells of the neovasculature of the tumors. Figure 14(d) and Figure 14{e). STEAP-1 Expression in Normal and Cancer Tissues. First strand cDNA was prepared from normal Gssues (bladder, brain, heart, Kidney, fiver, lung, prostate, spleen, skeletal muscle, tesfis, pancreas, colon and stomach), and from pools of patient cancer specimens (prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pod, ovary cancer pool, breast cancer pod, cancer metastasis pod, pancrecs cancer pod, prostate cancer xenograft pod, and prostate metastasis to lymph node pod. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to STEAP-1, was performed at 26 and 30 cycles of amplification. In (Figure 14d) picture of (he RT-PCR agarqse gel is shown. In (Figure 14e) PCR products were quanfitated using the Alphalmager software. Results show strong of expression of STEAP-1 In normal prostate amongst all (he normal tissues
f
tested. UpregulaSon of STEAP-1 expression was detected in prostate earner pod, bladder cancer pod, kidney cancer pod, colon cancer pod, king cancer pod, ovary cancer pool, breast cancer pod, and pancreatic cancer pod. Strong expression of STEAP-1 was detected in cancer metastasis pool, prostate cancer xenograft pod, and prostate metastasis to lymph node. Figure 14(0: STEAP-1 Expression in rymphoma patient specimens. First strand cDNA was prepared from a panel of "ymphoma patient specimens. Normalization was performed by PCR using primers to actin. Seml-quantitaSve PCR, using primers to STEAP-1, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quanfitated using the Alphalmager software. Expression was recorded as strong or medium, if signal is detected as 26 or 30 cycles of amplification respectively, and absent if no signal is detected even at 30 cycles of amplification. Results show expression of STEAP-1 in 8 of 11 (72.7%) tumor specimens tested. %
Figure 15. Specific cefl Surface staining of STEAP-1 by MAb M2/92.30. Left panels: FACS analysis of recombinant 3T3 and Rat1 celts stably expressing either STEAP1 (dark irtes) or a control neomydn resistance gene (Bght lines) stained with anfi-STEAP MAb M2/92.30 (10 pg/ml) and cell surface bound MAb was detected with a goat anti-mouse IgG-PE conjugalejsecondary reagent The stained cells were then subjected to FACS analysis. As indicated by the fluorescent shift of the Rat1-STEAP1 and 3T3-STEAP1 ceils compared to (he respective control cells, MAb M2/92.30 specifically binds to ceB surface STEAP1. Right panel: Ruoresoent microscopy of 3T3-STEAP1 eels stained with MAb M2/92.30 showing bright ceff surface fluorescence.
Figure 16. STEAP1 M2J92.30 MAb Recognizes Cell-Surface STEAP-1 on Human Prostate Cancer Xenografts. LAPC9 prostate cancer cells were stained with 10 ug/rnl of either MAb M2/92.30 or with a control anfi-KLH MAb. Surface bound MAb was detected with goat-anti-mouse IgG-PE conjugated secondary Ab. Stained cete were hen


subjected to FACS analysis. These results demonstrate that foe antf-STEAPI MAb M2/120.545 specifically btods endogenous cell surface STEAP1 expressed in prostate cancer xenograft cefis.
Figure 17. STEAP1M2J32.30 MAb Recognizes Mouse STEAP-1. 293T cells were transiently transfected with
either pCDNA3.1 encoding the murine STEAP1 cDNA or with an empty vector. 2 days later, (he cells were harvested and
stained with anfi-STEAP1 MAb M2/92.30 (10 ug/ml) and ceB surface bound MAb was detected with a goat anti-mouse IgG-
PE conjugate secondary reagent Cells were then subjected to FACS analysts. As indicated by foe fluorescent shift of the
Z93T cefls transfecfed with murine STEAP1 compared to foe cells transfected with the empty vector, MAb M2/92.30 .
specifically binds to murine STEAP1 protein. ,-_-,-
Figure 18. STEAP1/120.545 MAb recognizes cell surface STEAP-1. Panel A and Panel B. 3T3-neo (A, filled histograms) and 3T3-STEAP1 cells (A, no filf histograms) and Ratf-neo (B, filled histograms} and Rat1-STEAP cells (B, no (HI histograms) were stained with MAb M2/120.545 (10 ug/ml} and surface bound MAb was detected with goat anti-mouse IgG-PE conjugated secondary Ab. Cells were then subjected to FACS analysis. As indicated by the fluorescence shift of the 3T3-STEAP1 and Rat1-STEAP1 cells compared to their respective neo controls, MAb M2/120.545 specifically binds cell surface STEAP1. Panel C. LNCaP cells were stained with either MAb M2/120.545 or a control anb'-KLH MAb and subjected to FACS analysis as above. Panel D. Fluorescence microscopy of (he M2/120.545 stained LNCaP cells showing bright cell surface fluorescence. These results demonstrate that the M2/120.545 MAb specifically binds endogenous cell surface STEAP1 in LNCaP cefls.
Figure 19. Figure 19(a) The cDNA (SEQ ID NO: 49) and amino add sequence (SEQ ID NO: 50) of M2/X9Z30 VHdone#2. Figure 19(b) The cDNA (SEQ ID NO: 51) and amino add sequence {SEQ ID NO: 52)ofM2/X92.30VL done #2* Figure 19(c) The cDNA (SEQ ID NO: 53) and amino add sequence (SEQ ID NO: 54} of M2/X92.30 VL done #6. Figure 19(d) The cDNA (SEQ ID NO: 55) and amino acid sequence (SEQ ID NO: 56)ofM2/X120.545VLdone#8.
Figure 20. Figure 20a. The amino add sequence (SEQ ID NO: 57) of M2/X9Z30 VH done #2. Figure 20b. The amino acid sequence (SEQ ID NO: 58} of M2/X92.30 VI done #2. Figure 20c. The cONA . Figure 21. STEAPI M2/92.30 MAb Recognizes Cell-Surface STEAP-1 on Human Prostate and Bladder Cancer Xenografts. UGB1 bladder cancer cells (left panel) and LAPC9 prostate cancer cells (right panel) were stained with 10 ug/ml of either MAb M2/92.30 or wfth a control anti-KLH MAb. Surface bound MAb was detectedjyiffi goat-anti-mouse IgG-PE conjugated secondary Ab. Stained cells were then subjected to FACS analysis. These results demonstrate that the antf-STEAPI MAb M2/92.30 spedffcaHy binds endogenous ceU surface STEAP1 expressed in bladder and prostate cancer
Figure 21 STEAP-1 Internalization by STEAP1/9130 MAb. 3T3-STEAP1 cells were stained at 4C with M2/92.30 MAb (f 0 ugfcil), washed, then incubated with goat anti-mouse IgG-PE conjugate secondary Ab at 4C. One-half of 8ie cells were moved to 37C for 30 minutes and Ihe other half remained at 4C. Cete from each treatment were Bien subjected to fluorescent microscopy. Cells that remained at 4C showed bright Iring-fflce11 cell surface fluorescence. CeDsthat were moved to 37C showed loss of (he "ring-Cke" celt surface fluorescence and foe appearance of punctate and aggregated fluorescence indicative of capping and Memafeation.
Figure 23. STEAP-1 Internallzation by STEAP1M2/120.545 MAfa. PC3-STEAP1 ce«s were stained at 4C with M2/120.545 MAb (10 ug/ml), washed, then Incubated with goat anti-mouse IgG-PE conjugate secondary Ab. One-half of the


cells were moved to 37C for 30 minutes and the other half remained at 4C. Cells from each treatment were toen subjected to fluorescent microscopy. Cells that remained at 4C showed bright "ring-like" ccS surface fluorescence. Cells that were moved to 37C showed toss of the 'ring-like' cell surface fluorescence and foe appaarance of punctate and aggregated fluorescence Indicative of capping and intemalizafion.
Figure 24. STEAP-1 Internalization. Anti-mouse IgG - saporin conjugates (Advanced Targeting Systems, San Diego, CA) was used to demonstrate that murine Steap-1 M2/120.545 enters target cells via expression of Steap-1 on the surface of LNCaP ceils. The following protocols were used. LNCaP cells were plated at 5000 celis/90 nl /well in 96-weil plate and Incubated ovemighL Secondary tmmunotoxin conjugates (anti-mouse IgG-saporin and anti-goat IgG-saporin) or anfi-mouse IgG were made in cell culture medium to yield a final concentration of 100 ng/mT The primary antibody was added at concentrations ranging from 1-1000 ng/ml. The plates were incubated for 72 hours and the viabaity was determined by MTT assay. The results show that LNCaP cells were killed in the presence of M2/120.545 and anti-mouse IgG-saporin. No effects were detected with either the secondary antibody alone (anti-mouse IgG} or nonspecific secondary antibody conjugates (anti-goat IgG saporin). No toxia'ty was observed with the primary antibody (M2/120.545) alone tested up to 1 pg/ml.
Figure 25. Immunoprecipitatlon of STEAP1 by anti-STEAP-1 MAbs M2/92.30 and M2/120.545. 3T3-STEAP1 and 3T3-neo cells were lysed In RIPA buffer (25mM TnVd pH7.4; 150 mM Nad, 0.5mM EDTA, 1% Triton X-100,0.6% deoxycholic acid, 0.1% SOS, and protease inhibitor cocktaT). The ceB lysates were predeared with protein G sepharose beads and then incubated with 5 ug of either MAb M2/9Z30 or M2/120.545 for 2 hours at room temperature. Protein G beads were added and the mixture was further Incubated for 1 hour. The Immune complexes were washed and solubflized in SDS-PAGE sample buffer The sdubilized samples were (hen subjected b SOS-PAGE and Western blot analysis using a rabbit anti-STEAP pAb. Whole cell lysates of 293T ceDs transfected with STEAP1 was also run as a positive control. An bnmunoreacfive band of -37 KD was seen only n samples derived from 3T3-STEAP1 cells indicative of specific immunopredpitation of STEAP1 by both M2/92.30 and M2/120.545 MAbs.
Figure 26. Effect of STEAP-1 MAbs on the Growth of LAPC9 Human Prostate Cancer Xenografts In Mice. STEAP-1 M2/92.30 and M2/120.545 were tested at two different doses of 100 pg and 500 (jgi PBS and anfi-KLH MAb were used as controls. The study cohort consisted of 6 groups with 10 mice in each group. MAbs were dosed IP twice a week for a total of 12 doses, starBngfte same day as tumor cell injection. Tumorsize was monitored through caTiper measurements twice a week. The kxigest Dimension (L) and the dimension pefpemfioilar to ft (W)we^^ using the formula: W2 x 1/2. Serum PSA concentration at treatment day 40 for each animal was measured using commercial ELISA kit The Kruskal-WalHs test and the Mann-Whitney U test were used to evaluate differences of tumor growth and PSA level among groups. All tests were two-sided with 6=0.05. The data show that STEAP-1 M2/92.30 and M2/120.545 significantly retard the growth of human prostate xenograft in a dose-dependent tumor.
Figure 27. Effect of STEAP-1 MAbs on the Growth of LAPC9 Human Prostate Cancer Xenograft In Mice.
STEAP-1 M2/9Z30 and M2/120.545 were tested at two different doses of 100>jg and 500 jjg. PBS and anO-KLH MAb were
used as controls. JThe study cohort consisted of 6 groups with 10 mice in each group. MAbs were dosed IP twfce a week for
a total of 12 doses, starting foe same day as tumor ceDkijecfion. Tumor size was nxttitc^throuo^cafiperineasuremenls
twice a week. The longest dimension (L) and the dimension perpendfcular to H(W) were taken to calculate tumor volume
using the formula: W2 x 112. Serum PSA concentration at treatment day 40 for each animal was measured using
commercial ELISA kit The Kruskal-Watlis test and the Mann-Whitney U test were used to evaluate differences of tumor
growth and PSA level among groups. AB tests were two-sided with 6=0.05. The results show that STEAP-1 M2/92L30 and
M2/120.545signifk^HyretanJ the grcArth of human pro
-

Figure 28. STEAP-1 induced ERK-1 and ERK-2 phosphorylation. Left panels: PCS ceBs were transfected with neomydn resistance gene alone a with STEAP-1 in pSRa vector. CeHs were grown overnight in 0.5% FBS, then stimulated with 10% FBS for 5 minutes with or without 10 pg/ml MEK inhibitor PD98058. Cell lysates were resolved by 12.5% SOS-PAGE and Western blotted with anfi-phospho-ERK (Cell Signaling) and anfi-ERK(Zymed). Right panels: NIH-3T3 cells were transfected with neomydn resistance gene alone or with STEAP-1 in pSRcr vector. Cells were treated as above but without the MEK inhibitor. In addition, NIH-3T3-Neo cells were treated with 10mg/ml Na salydlate. Expression of STEAP-1 induces the phosphorylation of ERK-1 and ERK-2 in serum and was inhibited by the upstream MEK Wnase inhibitor PD98058.
Figure 29. STEAP-1 Mediates Cell-Cell Communication. PC3 cells were transfeded^'th neomydn resistance gene alone or with STEAP-1 or a control gene in pSRa vector. Redpient cells were labeled with 1 mg/ml dextran-Texas Red and donor cells were labeled with 2.5 yg/ml catcein AM. The donor (green) and recipient (red) cells were co-cultured at 37° C for 1&-24 hours and analyzed by microscopy for the co-localization of fluorescent dyes. Left panel: PC3-Neo cells were used as both donor and recipient. Center panel: PC3-STEAP-1 cells were used as both donor and recipient Right panel: PC3-control cells were used as both donor and recipient STEAP-1 induced the transfer of calcein to ceils containing dextran-Texas Red, indicating that STEAP-1 facilitates cell-cell communication.
Figure 30. Cell Communication Requires STEAP-1 Expression on Donor and Recipient Cells. PC3 cells were transfected with neomydn resistance gene alone or with STEAP-1 in pSRa vector. Recipient cells were labeled with 1 mg/ml dextran-Texas Red arid donor cells were tabled with 2,5 pg/ml calcein AM. The donor (green) and recipient (red) cells were co-cultured at 37° C for 18-24 hours and analyzed by microscopy for the cc4xafizafion of fluorescent dyes. Upper panels: fight microscopy, lower panels: UV fluorescence. Left panels: PC3-Neo cells were both donor and recipient Center panels: p!c341eo were donor cells and PC3-STEAP-1 were recipient Right panels: PC3-STEAP-1 cells were both donor and recipient Only when STEAP-1 was expressed on both donor and recipient was cell-cell communication detected.
Figure 31. STEAP-1/120.545 MAb Effect on Gap Junction. PC3 cells were transfected with neomydn resistance gene alone or with STEAP-1 in pSRa vector. Redpient cells were labeled with 1 mg/ml dextran-Texas Red and donor cells were labeled with 2.5 pg/ml calcein AM. The donor (green) and recipient (red) cells were co-cultured at 37°C for 18-24 hours and analyzed by microscopy for the co-iuualization of fluorescent dyes., (n afl experiments, the same eels were used as donor and acceptor. Cells were incubated with the indicated amounts of STEAP-1/120.545 MAb for 10 minutes prior to plab'ng andMAb was maintained in the culture for 24 hours prior to analysis. STEAP1/120.545 reduces STEAP-1 mediated gap junction in a dose-dependent manner.
Figure 32. Inhibition of ERK-1 and ERK-2 phosphorylation by STEAP-1 MAb and RNAI. PC3 ceils were transfected with neomydn resistance gene alone or with STEAP-1 and MAb in pSRa vector. ForRNAi knockdown, PC3-STEAP-1 cells were stably transfected with a pPUR-Ue-27-STEAP-1 vector containing siRNA to STEAP-1. CeBs were starved in 0.1% FBS for 18 hours at 37°C, placed on tee for 10 minutes without or with 10 pg/mlM2/92.30 MAb, brought to RT for 3 minutes then stimulated with 10% FBS for 5 minutes. Cells were lysed in RIPA buffer, whole ceDlysates resolved by 12.5% SOS-PAGE and proteins detected by Western Wotting. Phospho-ERK was detected with rabbit antiserum (Ceft Signaling) and ERK was delected with rabbit anfi-ERK (Zymed). STEAP-1 was detected with sheep anti-STEAP-1 and acfin was detected with anb'-acfin MAb (Santa Cruz). ERK-1 and ERK-2 phosphorylation were both induced by 10% serum, and were inhibited by M2/9130 MAb and siRNAto STEAP-1.
Figure 33. Effect of STEAP-1 RNAf on Cell-Celt Communication. PC3 cells were transfected with neomydn resistance gene alone or with STEAP-1 ki pSRa vector. For RNAi krwdefown,PCJ3-STEAP-1celswerestabh;ttansfecW with a pPUR-U6-27-STEAP-1 vector containing sJRNA to STEAP-1 or an empty vector. Recipient ceOs were labeled wifo 1


mg/mt dextran-Texas Red and donor cells were labeled with 2.5 Mg/ml calcein AM. The donor (green) and recipient (red) ceHs were co-cultured at 37» C for 18-24 hours and analyzed by microscopy for the co*caRzation ot Muorescent dyes. In all experiments, the same cells were used as donor and acceptor. Specific STEAP-1 RNAi stably expressed in PC3-STEAP-1 cells reduces the STEAP-1 Induced ceH-celt communication.
DETAILED DESCRIPTION OF THE INVENTION Outline of Sections
I.) Definitions
II.) STEAP-1 Polynucleotides
HA) Uses of STEAP-1 Polynucleotides IIA1.) Monitoring of Genetic Abnormalities HA2.) Antisense Embodiments IIA3.) Primers and Primer Pairs
IIA4.) Isolation of STF \P-1-Encoding Nucleic Acid Molecules HAS.) Recombinant Nucleic Add Molecules and Host-Vector Systems HI.) STEAP-1-related Proteins
UIA) Motif-bearing Protein Embodiments
lll.B.) Expression of STEAP-1-related Proteins
lll.C.) Modifications of STEAP-1-related Proteins
III.D.) Uses of STEAP-1-related Proteins 'JrV.) STEAP-1 Antibotfies V.) STEAP-1 Cellular Immune Responses VI.) STEAP-1 Transgenic Animals VII.) Methods for the Detection of STEAP-1
VIII.) Methods for Monitoring the Status of STEAP-1-related Genes and Their Products DC) Identification of Molecules That Interact Wi(h STEAP-1 X) Therapeutic Methods and Compositions
XA) Anti-Cancer Vaccines X.B.) STEAP-1 as a Target for Antibody-Based Therapy XC.) STEAP-1 as a Target for Cellular Immune Responses
X.C.1. Minigene Vaccines %
X.C2. Combinations of CTL Peptides with Helper Peptides
XC.3. Combinations of CTL Peptides with T Cell Priming Agents
XC.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptfdes v XD.) Adoptive Immunotherapy
XE.) Adminletration of Vaccines for Therapeutic or Prophylactic Purposes XI.) Diagnostic and Prognostic Embodiments of STEAP-1. XII.) Inhibition of STEAP-1 Protein Function
XII A) Inhibition of STEAP-1 With Intracellular Antibodies
XILB.) Inhibition of STEAP-1 with Recombinant Proteins
XII.C.) Inhibition of STEAP-1 Transcription or Translation

XII.D.) General Considerations for Therapeutic Strategies XDL) Identification, Characterization and Use of Modulators of STEAP-1 XIV.) RNAi and Therapeutic use of small Interfering RNA (siRNAs) XV.) KITS/Articles of Manufacture
1.) Definitions:
*. *•
Unless otherwise defined, all terms of art notations and oiher scientific terms or terminology used herein are intended to have the meanings commonly understood by those of ski in the art to which this invention pertains. In some -cases, terms with commonly, understood meanings are defined herein for darity and/or for ready reference.'ahdthelhclusiori of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular donfng methodologies described in Sambrook el a/., Molecular Clotting: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring harbor, N.Y. As appropriate, procedures involving the use of commerdally available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or. parameters unless otherwise noted.
The terms "advanced prostate cancer", TocaBy advanced prostate cancer*, "advanced dfeease" and Tocafly advanced disease* mean prostate cancers that have extended through the prostate capsule, and are meant to indude stage C disease under the American Urological Association (AUA) system, stage C1 - C2 disease under the Whltmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system, hi general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is dinicaBy identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally adv?"ced prostate cancer is presently diagnosed pathotwjfcalry following radical prostatectomy If the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades foe seminal vesicles.
"Altering the native glycosylation pattern* is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence STEAP-1 (either by removing the underlying glycosylafion site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence STEAP-1. In addition, the phrase fndudes qualitative changes in ttie glycosylafion of the native proteins, Involving a change in the nature and proportions of the various carbohydrate moieties present
The term 'tonakxj* refers to a molecule which is sfruduraBy similar cr shares sMar a another molecule (e.g. a STEAP-1-related protein). foexan^ananakxjcfaSTEAP-lprotehcanbespecifk^ antibody orTceO that specifically binds to STEAP-1.
The term "anSxxl/is used in Ihetooadest sense unless de^ Therefore, an "antibody" can be
naturaly occurring or man-made such as monoclonal antibodies pnxtoed by corrventirjnalhybnVi^ Anfi-STEAP-1
antibodies cornpriseTbonodonal and pciryctonaianflxxfies as wel as fragments contai^
i "antibody" refers to any form of
antibody or fragment thereof that specifically binds STEAP-1 and/or exhibits the desired Hologtes! Activity and specifically covers monoclonal antibodies (including fufl length monoclonal antibodies), potydonal anfibodies, muffispedfic antibodies (e.g., bispedfic antibodies), and antibody fragments so long as they specifically bind STEAP-1 and/or exhfoit the desired biological activity. Any specific antibody can be used hi the methods and compositions provided herein. Thus, hone

embodiment the term 'anlfood/ encompasses 8 molecule comprising at least one variable region from a light chain bnmunoglobulin molecule and at least one variable region from a heavy chain mdecute that in combination form a specific binding site for the target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody Is a IgGi, IgGi lgG3, or lgG4 antibody. The antibodies useful in the present methods and compositions can be generated In cell culture, In phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. Therefore, hi one embodiment, an anfibody of the present invention is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well Known in the art In one embodiment, the antibody
is produced by reax^naVt^in^rkn^^llhe'arL' For example, a recombinant anfibody can be produced by .transfecb'ng _
a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one VL and one VH region In the host cell. Exemplary descriptions of recombinant means of anfibody generation and production include Delves, ^BODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, ef a/., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Coding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE {Academic Press, 1993); O/RREOT PROTOCOLS wlMMUNOLOGf (John Wiley & Sons, most recent edition). An antibody of the present Invention can be modified by recombinant means to Increase greater efficacy of the antibody kt mediating the aesiiod function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, the substitutions wil be conservative substitutions. For example, at least one amino add In the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551. Application No. WO 9958572; and Angal, ef a/., Mb/. Immunot. 30:105-08 (1993). The modification in amlno adds indudes-deletions, additions, substitutions of amino adds. In some cases, such changes are made to reduce undesJred activities, e.g., comptemenWependent cyfotoxidty. Frequently, the antibodies are labeled by joining, either covatenUy or non-co valentfy, a substance which provides for a detectable signal A wide variety of labels and
ff '
conjugation techniques are known and are repotted extensively in both the sdenBfte and patent literature. These antibodies can be screened for binding to normal or defecfi, a STEAP-1. See e.g., ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies with the desired biologic activities can be identified foe following to vtttv assays induding but not limited to: proliferation, migration, adhesion, soft agar.growth, angtogenesis, cell-cell communication, apoptosis, transport, signal transducfion, and the following in vivo assays such as the inhibition of tumor growth. The antibodies provided herein can also be useful in diagnostic applications. As capture or non-neuuafbdng antibodies, they can be screened for the ablOty to bind to the specific antigen without inhibifing the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful kt competitive binding assays. They can also be used to quantify the STEAP-1 or its receptor.
An 'antibody fragment* Is defined as at least a portion of the variable region of foe bnmunoglobulin molecule that binds to its target, Le., the antigen-binding region. InoieembaJinientftspedfeflytxwefssi^ clones hereof (induoTng agonist, antagonist and neiitraiariganlixx^)andanfi-STEAP-^
pcityepitopta specificity. Theantibodyof fte present methods and oompc^tkmsc^benwxwclonalorpc^yctonal. An antibody
can be in the form of an antigen binding anfibody fragment induding a Fab firagmentF(ab02 fragment, a single chain
variable region, and (he Eke. Fragments of intact molecules can be generated using mefoods wed known in the art and V0
Indude enzymatic digestion and recombinant means.
As used herein, any form of the "antigen" OTte used to generate an ^ Thus,
the efidOrig anfig^ may be a single epllope, mulfiole epitop^ immunogenidty enhancing agents known in the art The elk^tig antigen niay be an isolated M-tengJh protein, a ceB surface
U

protein (e.fl., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extraceSular domain portion of the protein). The anfigen ma/ be produced In a genetically modified cell. The DMA encoding the anfigen may genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "portion" refers to the minimal number of amino acids or nudete acids, as appropriate, to constitute an hununogenfc epitope of .the antigen of interest Any genetic vectors suitable for transformation of Ihe cells of
i
interest may be employed, {rtduduig but not limited to adenoviral vectors, plasmidc, and non-viral vectors, such as cationic
fipids. In one embodiment, Ihe antibody of the methods and compositions herein specifically bind at least a portion of the
extracellular domain of the STEAP-1 of interest ....._„..,,--- -
The antflxxfies or antigen binding fagments thereof provided herein may be conjugated to a "bioactive agent" As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that binds the antigen and/or enhances or mediates a desired biologies! effect to enhance cell-kiPing toxins.
In one embodiment, Ihe binding fragments useful in the present invention are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment that is capable of binding the desired the antigenic epitope and directly or indirectly exerting a biologic effect Direct effects include, but are not limited to the modulation, stimulaSon. and/ or inhibition of a growth signal, the modulation, stimulation, and/ or inhibition of an anfi-apoptotic signal, the modulation, stimulation, and/ or inhibition of an apoptofic or necroBc signal, modulation, stimulation, and/ or inhibition the ADCC cascade, and modulation, stimulation, and.' or inhibifion the CDC cascade.
"Bispecific" antibodies are also useful In the present methods and compositions. As used herein, the term "bispecjfic antibody" refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two differenj antigenic epftopes. In one embodiment, the epltopes are from Ihe same anfigen. In another embodiment, the epitopes are from two different antigens. -Methods for making bispedfic antibodies are known in the art For example, bispectfic antibodies can be produced recombinanfly using the co-expression of two immunogtobufin heavy chain/light chain pairs. See, e.g., Milstein et a/., Nature 205:537-39 (1983). Altemafiveiy, bispedfic antibodies can be prepared using chemical inkage. Se:, e.g., Brennan, et a!., Science £29:61 (1985). Bispecific antibodies include bispectfic antibody fragments. See, e.g., Hoffinger, et a/., Proc. Waff. Acad. Sd U.SA 20:6444-48 (1993), Gruber, ef a/., J. /mmunot 152:5388 (1994). .
The monoclonal antibodies herein specifically include 'chlmeric" antibodies in which a porSon of (he heavy and/or ight chain is identical with or homologous to corresponding sequences In anybodies derived from a parScular species or belongtng to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in anixxfies derived from another species or belonging to another antfoody class or subclass, as weB as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit Ihe desired biological activity (U.S. Pat No. 4,816,567; and Morrison et al., Proc. Nail. Acad. Sd. USA SI: 6851-6855 (1984)).
The term "codon optimized sequences* refers to nudeotkte sequences that haw been optimized for a parficular host species by replacing any codons having a usage frequency of less than about 20%. Nudeofide sequences (hat have been opfimized for expression in a given host species by elimination of spurkws polyadenylation sequences, efimhafion of exon/inlron spfidng signals, e&nlnatfon of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequences."
A 'combinatorial library" is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a inear combinatorial chemical library, such as a potypepGde (e.g., mutein) ixary, is formed by combining a set of chemical txdding blocks called amino adds h every possible way for a given (ttrnpoural length 0-e.,fre number of amftio acids in a

jp* polypepfide confound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Galbp el ai., J. Mad. Chem. 37(9): 1233-1251 (1994)).
Preparation and screening of combinatorial libraries is weB known to those of skill in toe art Such combinatorial chemical libraries include, but are not imited to, peptide libraries (see, e.g., U.S. Patent No, 5,010,175, Furica, PepL ProL Res. 37:487493 (1991), Houghton el al., Nature, 354:84-88 (1991)), peptokfe (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio- digomers (PCT PubiicaSon WO 92/00091), benzodiazepines (U.S. - Pat No. 5,288,514), diversomers such as hydantoins; benzodiazepines and dipepfides (Hobbs et al., Proc. Nat Acad. Sci. USA 90:6909-6913 (1993)), vinytogous pdypeptides (Hagihara etal., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal .. peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et ^^M^.Gim^SQ(^^4^2^^^^2^^i'!^ " analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),
oligocarbamates (Cho, et aL, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell el at, 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 at., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287),carbohydrate libraries (see, e.g., Uang etal.. Science 274:1520-1522 (1996), and U.S. Patent No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); IsoprenokJs, U.S. Patent No. 5,569,588; thiazoGdinones and melathtazanones, 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, U.S. Patent No. 5,288,514; and the Eke). •
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Wotxim, MA; 433A, Applied Biosystems. Foster City, CA; 9050, Plus, Millipbre, Bedford, NIA). A number of .well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems uHizing robotic arms (Zymate H, Zymark Corporation, Hopkinlon, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein wffl be apparent to persons skilled In the relevant art In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc: St Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exfon, PA; MartekBfosdences, Columbia,.MD; etc.).
As used herein, foe term 'conservative substitution" refers to substitutions of amlno adds are known to those of
skOI in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skH in
%
this art recognize that, in general, single amlno add substitutions in non-essential regions of a polypepfide do not substantially alter biological activity (see, e.g., Watson, et a/., MOLECULAR BKXOGY OF THE GENE, The Benjamh/Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary subsfflufions are preferably made hi accordance witti (hose set forth In Tabte(s) lll(a-b). For example, such changes include substituting any of isoleudne (I), vafine (V), and feudne (L) for any other of these hydrophobfc amino adds; aspartic acid (D) for glutarrfeac)d(Q ami vk» versa; ghrtaniine(C9 for asparagine (N) and vice versa; and serine(S) for threorine (T) and vice versa. Oner subsfitufons can also be a>nsWered{xxisefvafive1 depending on the environment of the particular amino acid and its role in the triree-dimenskxia! structure of fteproteh. For example, glydne (G) and alanine (A) can frequently be interchangeable, as can aJanine (A) and vaflne (V). MeMorrine (M), which is relatively hydrophobic, can frequently be interchanged with leudne and isoleudne, and sometimes with vafine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which Ihe significant feature of the amino add

residue is its charge and the differing pK"s of these fora amino acid residues are not significant Still other changes can be consMfirfic! "conservative" in pculicuUir environments (see, e.g. Table lll{a} herein; pages 13-15 "Biochemistry* 2* ED. Lubert Shyer ed (Stanford University); Henikoff ef si, PNAS 1992 Vol 8910915-10919; Lei eta/., J Biol Chem 1995 May 19; 270(20):11882-6). Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.
The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells, lite term is intended to indude radioactive isotopes chemotherapeufc agents, arid toxins such as small molecule toxins or enzymatfcally active toxins of bacterial, fungal, plant or animal o^^jnciujrjfl^^ Jragments.and/or-varianls thereof. Examples of cytotoxic agents Indude, but are not fimited to auristafins, auristafin e, auromydns, maytansinoids, yttrium, bismuth, ridn, ricin A-chain, combrestatin, duocarmydns, doiostafins, doxorubidn, daunorubidn, taxol, dsplatin, cc1065, ethidium bromide, mitomydn, etoposide, tenoposide, vincristine, vinblasfine, colchicine, dihydroxy anthradn dione, actinomydn, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modecdn A chain, alpha-sardn, gefonin, mitogellin, retstrictodn, phenomydn, enomydn, curidn, crofin, calicheamidn, Sapaonaria oflidnalis inhibitor, and glucocorticokJ and e!her chemotherapeutic agents, as well as radioisotopes such as At«i, | w, |« Y», Re1*, Re188, Sm153, BP««r»>, P» and radioacfive isotopes of Lu induding Lu177. Antftxxfies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its acBve form.
As Uoed herein, the term "diabodies" refers to small antibody fragments with two anfigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (Vu) in the same polypeptide chain (Vn-Vt). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully In, e.g^P 404,097; WO 93/11161; and Hoffingeretal, Hoc. Nat!. Acad. Sc/. USA 9Q:644448(1993).
The 'gene product" is used herein to indicate a pepfide/protein or mRNA. For example, a 'gene product of the invention* is sometimes referred to herein as a "cancer amino add sequence*, 'cancer protein*, 'protein of a cancer listed in Table I", a 'cancer mRNA', "mRNA of a cancer fisted in Table I*, etc. In one embodiment the cancer protein is encoded by a nucleic add of Figure 2. The cancer protein can be a fragment, or alternatively, be the'full-length protein encoded by nudeic acids of Figure 2. In one embodiment, a cancer amino add sequence Is used to determine sequence ktenfity or similarity. In another embodiment the sequences are naturally occurring aflelic variants of a protein encoded by a nucleic add of Figure 2. In anofher embodiment, the sequences are sequence variants as further described herein.
"Heteroconjugate" antibodies are useful hi the present methods and compositions. As used herein, fie term "heteroconjugate antibody" refers to two covalentJy joined antibodies. Such antibodies can be preparecfttsing known methods in synthetic protein chemistry, induding using crossfinking agents. Sea. e.g., U.S. Patent No. 4,676,980.
"High throughput screening' assays for the presence, absence, quantification, or other properfies of particular nudeic acids or protein products are weB known to those of skH in theart Similarly, binding assays and reporter gene assays are similarly wed known, thus, e.g., 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 nudeic add binding (le, in arrays); while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for Sgand/anSbody binding.
In addition, high throughput screening systems are commerdafly avaflable
the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of sm*> systems provMe detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting toe modulation of gene transcription, figand binding, and the fike.
The term "homolog" refers to a molecule which exhibits hoenofejgy to arwthern^ecute, by for example, having sequences of chemical residues hat are the same or similar at corresponding positions.
In one embodiment, the antibody provided herein is a "human antibody." As used herein, the term "human
antibody" refers to an antibody in which essentially the entire sequences of the light chain and heavy chain sequences,
"including the complementary determining regions (CDRs}PafetonJiuman genes. In one embodiment, human monoclonal...
antibodies are prepared by the trioma technique, the human B-cell technique (see, e.g., Kozbor, ef aL, Immunol. Today*. 72
(1983), EBV transformation technique (see, e.g., Cole eta!. MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985)),
or using phage display (see, e.g., Marks ef a/., J. Mo/. Biol. £22:581 (1991)). In a specific embodiment, the human antibody
Is generated in a transgenic mouse. Techniques for making such partially io fully human antibodies are known in the art and
• any such techniques can be used According to one particularly preferred embodiment, fully human antibody sequences are
made in a transgenic mouse engineered to express human heavy and light chain antibody genes. An exemplary description
of preparing transgenic mice that produce human antibodies found in Application No. WO 02/43478 and United States
Patent 6,657,103 (Abgenix) and its progeny. B cells from transgenic mice that produce the desired antibody can then be
(used to make hybridoma cell lines for continuous production of the antibody. See, e.g., U.S. Patent Nos. 5,569,825;
5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Jakoboyits, Adv. Drug Del. Rev. 31:33-42 (1998); Green, er a/., J. Exp.
Med.j88;483-95(1998). x , ^
' "Human Leukocyte Antigen" or *HLA" Is a human class I or class II Major HistocompatibOity Comptex^HC) protein (see, e.g., SStes, et a!., IMMUNOLOGY, 8™ ED., Lafige Publishing, Los Altos, CA (1994).
As used herein, the term 'humanized «u lubody* refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobutin. In general, tie humanized antibody wfli comprise substantially all of at least one, and typically two, variable domains, in which aD or substantially all of the hypervariaoie loops correspond to those of a non-human (mmunoglobulin and all or substantially alt of the FR regions are those of a human imrriunogtobulin sequence. The humanized antibody optionally also win comprise at least a portion of an Immunoglobutin constant region (Fc), typically that of a human immunoglobulin. See e.g., Cabilly U.S. Patent No. 4,816,567; Queen et al. (1989) Proc. Natt Acad. Scl. USA 86:1002&-10033; and ANTIBODY ENGINEERING: APRACDCAL APPROACH (Oxford University Press 1996).
The terms "hybridize", "hybridizing", "hybridizes" and the Eke, used in the context of polyjiudeotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% forrnamfdeflBXSSC/0.1% SOS/100 ng/rnl ssDNA. in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1%SDS are above 55 degrees C.
The phrases "isolated* or "biologically pure* refer to material which is substanfially or essenfiafly free from
v -..«-. ..*~*i
components which normally accompany the material as it is found In its native state. Thus, Isolated pepfides h accordance
with the invention preferably do not contain materials normally associated with the peptktes hi their in sfo environment For
example, a pdynudeoGcte is sakl to be Isolated
correspond a are cornpternentary to genes cto thai fceST^^
productor fragments (hereof. Askffled artisan c^readSyemptoynudefca&isc^
pdynudeotide. A protein is saM to be "isolated; for example, wr^r^ysicd

removo the STEAP-1 proteins from ceHular consfituents that are normally associated with the protein. A skilled artisan can readHy employ standard purificat™ methods to obtain an isolated STCAP-1 protein. Alternatively, an isolated protein can be prepared by chemical means.
Suitable labels" include radionudides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemitumlnescent moiefies, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4;275,149; and 4,366,241. In addifion, the antibodies provided herein can be useful as Ihe antigen-binding component of fluorobodies. See e.g.,Zeytunetal., Wat BWechno/. 21:1473-79 (2003).
^-- - The term "mammar refers to any organism classified as a mammal,-induding4nieei«rate,iFabbitsfdogsi cats,-cows/*"-- -
horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of tie invention, the mammal is a human.
The terms "metastatic prostate cancer" and "metastafic disease' mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the ADA system and stage TxNxM+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for predate cancer metastasis is bone. Prostate cancer bone metastases arc often osteobJasBc rather than osteolvtic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis,
rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, Ever and brain. Metastatic
, t ,\ • .
prostate cancer Is typically diagnosed by.open or laparoscopic pelvic rymphadenectomy, whole body radionucfide scans,
skeletal radiography, and/or bone lesion biopsy.
The term "modulator" or 'test compound" or 'drug candidate" or grarunati(^ equhralents as used herein describe any molecule, e.g., protein, ofigopeptide, small organic molecule, polysatx^arkte,polynucleotjde,et(x, to be tested for fte capacity to directly or indirectly alter the cancer phenolype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression,.protein interaction, etc.) In one aspect, a rrodulatorwiH neutralize the effect of a cancer protein of the invention. By "neutralize'is meant that an activity of a protein is Inhibited or blocked, along with me consequent effect on the ceB. In anofter aspect, a rrx>dutatorwl neutralize fiie effect of a gene, and its corresponding protein, of the Invention by normalizing levels of said protein. In preferred embodiments, nxxfulatofc alter expression profiles, w expression pro
pathways, hi one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal fissqe fingerprint In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is ran in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
Modulators, drug candidates or test compounds encompass numerous chemical dasses, though typicaty they are organic molecules, preferably small organic compounds having a molecular weight of nvxe than 100 a«l less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less han 500 0. Candidate agents comprise functional groups necessary for sbiicturalinteracticm with protehs,particulaiV hydros borxiing, and typk^ inducie at teasi an an^, c
chemical groups. The candidate agents often comprise cyclical carbon or heterocyc&c structures amlto aromatic or polyaromafic structures subsfituted with one or more of the above functional groups. Modulators also comprise btomolecules

such as pepfides, saccharides. fatty adds, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One dass of modulators are pepfides, for example of from about five to about 35 amino adds, with from about five to about 20 amino adds being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein Is soluble, indudes a non-transmembrane region, and/or, has an N-termina} Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free add and Ihe N-terminus is a free amine to aid in coupling, ilo., to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an Immunogenic agent as discussed herein. In one embodiment the cancer protein is conjugated to BSA. The peptides of the invention, e.g., of preferred lengths, can be (inked to each other or to other amino adds to create a longer peptide/protein. The modulatory pepfides can be digests of naturally occurring protests as is outlined>above,,raadom pepfides.iorabiased" -random pepb'des. In a preferred embodiment, peptide/prolein-based modulators are antibodies, and fragments thereof, as defined herein.
Modulators of cancer can also be nudeic acids. Nudeic add modulating agents can be naturally occurring nudeic adds, random nudeic acids, or 'biased* random nudeic acids. For example, digests or prokaryotic or eukaryofic genomes can be used in an approach analogous to that outlined above for proteins.
The term "monodona! antibody*, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, le., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single anfigenic epftope. In contrast, conventional (polydonaQ antibody preparations i/pically indude a multitude of antibodies directed against (or spedfic for) different epitopes. -In one embodiment, the polydonal antibody contains a plurality of monodonal antibodies with different epitope spedfidfies, affinities, or avidities within a single antigen that contains multiple anfigenic epftopes. TheNnodifier "monoclonal* indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present Invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant ONA methods (see, e.g., U.S. Pat No. 4,816,567). The 'monodonal antibodies* may also be isolated from phage antibody libraries using the techniques described in Clackson et at., Nature 3§2:624-628 (1991) and Marks et a!., J. Mo/. Bhl. 222:581-597 (1991), tor example. These monodonal antibodies wffi usually bind with at least a Kj of about 1DM,
more usually at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or better, usually determined by ELISA.
A 'motif, as in bfotogfcal motif of a STEAP-1-reJated protein, refers to any pattern of amino adds forming part of the primary sequence of a protein, that Is associated with a particular function (e.g. protein-protein Interaction, protein-ONA interaction, etc) or modificafion (e.g. that is phosphorytated, glycosytated or amidated), or localization (e.g. secretory sequence, nudear localization sequence, etc.) or a sequence that is correlated with being Immunogenic, either humoraliy or cefiulariy. A motif can be either contiguous or capable of being aligned to certain oositions that are genecanycorrelatBd with a certain fancfion oyxoperty. In he context of HLA motifs, "motif refers to the pattern of residues in a peptide of defined tength,usuanyapepfideoffromabout8toabouM3arrito
adds for a dass II HLA motif, which Is recognized by a particular HLA molecule. Peptide moffls for HJ\ binding are typically different for each protein encoded by each human HLA aflete and differ in toe pattern of the primary and secondary anchor residues.
A 'pharmaceutical exdpfenf comprises a material such as an ac^uvanr, a carrier, pH-adjusfing and buffering agents, tonWfy adjusting agents, wetting agents, preservative, and the like.

"Pharmaceutically acceptable" refefs to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
The term 'polynudeoBde' means a polymeric form of nudeofldes of at least 10 bases or base pairs In length, either libonudeotktes or deoxynudeofides or a modified form of either type of nudeoBde, and is meant to hdude single and double stranded forms of DNA and/or RNA. In the art, this term if often used Interchangeably with •ofigonudeoflde". A polynudeofide can comprise a nudeoSde sequence disdosed herein wherein thymkfine (T), as shown for example In Figure 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
The term "porypepdde' means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term fe often used interchangeably with 'pepticle" or "protein".
An HLA "primary anchor residue" is an amino add at a spedfic posifion along a peptide sequence which Is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a "motif for an immunogenic peptide. These residues are understood to fit in dose contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA dass I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal posifion of a 8, 9, 10, 1 1 , or 12 residue peptide epitope in accordance with the invention. Alternatively, hi another embodiment, the primary anchor residues of a peptide binds an HLA dass II molecule are spaced relative to each other, rather than to the termini of a peptide, where the pepfide is generally of at least 9 amino adds in length. The primary anchor positions for each motif and supermofif are set forth In Table IV(a). For example, analog pepb'des can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermofif.
•Radioisotopes" indude, bu» *»re not limited to the following (non-limiting exemplary uses are also set forth in Table
By 'randomized' or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each
nucleic add and peptide consists of essentially random nudeotides and amino adds, respectively. These random pepfides
(or nucleic acids, discussed herein) can Incorporate any nudeotide or amino add at any position. The synthetic process can
be designed to generate randomized proteins or nucleic adds, to allow the formation of alt or most of the possible
combinations over the length of the sequence, thus forming a library of randomized candidate btoacfive proteinaceous
agents. »
in one embodiment, a library is fully randomized,' with no sequence preferences or constants at any position. In another embedment, tte library is a "biased random" Bbrary. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nudeotides or amino add residues are randomized within a defined dass, e.g., of hydrophobic amino adds, hydrophffic residues, stericaBy biased (either smal or large) residues, towards the creation of nudefc add binding domains, the creation of cysteines, for cross-Inking, proBnesfor SH-3 domains, serines, threonines, tyrosines or histidines for phosphoryfation sites, etc., or to purines, etc.
A Iteoxnbinanr Dr^or RNAirnlecule is a Dr^
As used herein, the term "single-chain Fv* or 'scFv" or "single chain" antibody refers to antibody fragments
comprising the VH and VL domains of anGbofy, wher^ Generally,

the Fv potypepfide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to forni the de*i,c Non-Smiting examples of "small molecules' include compounds that bind or Interact with STEAP-1, Bgands Including hormones, neuropeptides, chemoktnes, odorants, phosphdjpids, and functional equivalents thereof that bind and preferably Inhibit STEAP-1 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 1C kDa,more preferably below about 9, about 8, about 7, about 6, about 5 orabout4kDa. Incertain embodiments, small molecules physically associate with, or bind, STEAP-1 protein; are not found in naturally occurring -^metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions.* ^=*^^Si--i^fc * "^ -^—:---:
As used herein, the term "specific" refers to the selective binding of the antibody to fte target antigen epttope. Antibodi3s can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2,5,7, and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific. In one embodiment, a specific antibody is one that only binds the STEAP-1 antigen, but does not bind to the irrelevent antigen. In another embodiment, a specific antibody is one that binds human STEAP-1 antigen but does not bind a non-human STEAP-1 antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid homology with the STEAP-1 antigen. In another embodiment, a specific antibody is one that binds human STEAP-1 antigen and binds murine STEAP-1 antigen, but with a higher degree of binding the human antigen. In another embodiment, a specific antibody is one that binds human STEAP-1 antigen a/id binds primate STEAP-1 antigen, but with a higher degree of binding the human antigen. In another embodiment, the specific antibody binds to human STEAP-1 antigen and any non-human STEAP-1 antigen, but with a higher degree of binding the human antigen or any combination thereof.
"Stringency" of hybridization reactions is readily determinable by one of ordinary ski in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and tail concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present In an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it Mows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel ef a/., Current Protocols In Molecular Biology, Wiley Interscfence Publishers, (1995).
'Stringent conditions' or "high stringency conditions", as defined herein, are identified by» but not imiled to, those that (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodwm chtoride/0.0015 M sodium dtrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% FfcoWJ.1% pdyvinylpynofidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride. 75 mM sodium dlrate at 42^or(^erriptoy50%fonnamide,5x SSC (0.75 M Nad, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DMA (50 ng/ml), 0.1% SOS, and 10% dextran sulfate at 42 °C. with washes at 42°C in 0.2 x SSC (sodium chloride/sodium, citrate) and 50% formamide at 55 °C, followed by a nigh-stringency wash consisting of 0.1 x SSC containing EDTA at 55 °C. •Moderately stringent conditions" are described by, but not hrrited to, those in Sambrook ef at., Molecular Cloning: A Laboratory Manual, New York; Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than

V

those described above. An example of moderately stringent conditions Is overnight incubation at 65°C in a solution comprising: 1% bovine comm albumin, 0.5M «x«um piiosphate pH7.5,1.25mM EDTA, and 7% SDS 5 x SSC (150 mM NaCI, 15 mM trisodium citrate}, followed by washing the fillers In 2 x SSC/1% SDS at 50»C and 0.2 X SSC/0.1% SDS at 50°C. The skilled artisan will recognize how to adjust he temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
An HLA 'supermotif is a pepfide binding specificity shared by HLA molecules encoded by two or more HLA alleles.
Overall phenob/pic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (f). The non-
fimiting constituents of various supertypes are as follows:
„... ... A25;A'02Q1,A*02Q2,A*0203rA*0204,A*0205,A*0206,A*6802,A*6901,A*0207 """ ~"
A3[ A3, A11, A31, A*3301, A*6801, A*0301, AM101, A*3101
il: 87, B*3501-03, B*51,8*5301, B*5401,8*5501, 8*5502, B*5601, B*6701, B*7801, B*0702,8*5101, B*5602
B44: 8*3701,6*4402, B*4403, B*60 (B*4001), B61 (8*4006)
Ml A*0102, A*2604, A*3601, A*4301, A*8001
A24i A*24,A*30,A*2403,A*2404,A*3002,A*3003
B27: 8*1401-02, B*1503, B*1509, B*1510,8*1518,8*3801-02,8*3901,6*3902,8*3903-04,6*4801-02,6*7301. 6*2701-08
B58: 8*1516, BM517, B*5701, B*5702, B58 .
1^1.8*4601,652,8*1501 (B62), 8*1502 (875), 8*1513 (877) Calculated population coverage afforded by different HLA-supertype combinations are set forth In Table IV(g).
As used herein to treat" or therapeutic' and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; as is readily appreciated in the art, full eradication of disease Is a preferred out albeit not a requirement for a treatment act
• A Transgenic animal'(e.g., a nrouse or rat) is an animal having cells that contain a transgene,whic^ was Introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene* is a DMA that fe integrated into the genwne of a cell from which a transgenic animal develops/
As used herein, an HLA or cellular immune response "vaccine* is a composiCon that contains or encodes one or
more pepGdes of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more
individual peplides; one or more pepb'des of the invention comprised by a polyepitopic pepfide; or nucleic adds that encode
such Individual peptRJes or polypepfides, e.g., a minigene that encodes a potyepitopic pepfide. The 'one or more pepfides*
can Include any whole unit Integer from 1-150 or more, e,g., at least 2,3,4,5,6,7,8,9,10,11,12,43,14,15,16,17,18,
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 peptktes of the invention.
TliepeptMesorpdypep6descanoptk>nanybenK>d^,su* HLA
class I peptides of Ihe invention can be admixed with, or inked to, HLA class II peptkies, to faditate activation of botti The term Narianr refers to a mdeoute that exhfcits a variafion from a described type or norm, such as a protein lhat has orre a rrwre differed amir»a 1 protein shown in Figure 2 or Figure 3. An analog is an exampfe of a variant protein. Spfioe isoformsand single nudeofides
polymorphisms (SNPs) are further examples of variants. -

The 'STEAP-1«elated proteins" of the invention include those speofficafly identified herein, as well as altefc variants,
conservafive substitution variants, analogs and homclcgs that con be isolated/generated and characterized without undue
experimentation foliowing he methods outlined herein orreadBy avaSabte in he art. Fusion proteins hat combhe parts of
different STEAP-1 proteins or fragments hereof, as well as fusion proteins of a STEAP-1 rxotehandaheterotogouspolypepfide
are also Induded. Such STEAP-1 proteins are collectively referred to as he STEAP-1-related proteins, he protehs of he
invention, or STEAP-1. The term "STEAP-1-related protein" refers to a polypeptide fragment or a STEAP-1 protein sequence of 4,
5,6,7,8,9,10,11,12,13,14,15,16.17,18.19.20,21,22,23,24,25, or more than 25 amino adds; or, at feast 30,35,40,45,
50,55,60,65,70,80,85,90,95.100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,
190,195,200, 225,250,275,300,325,330,335,339 or more amino adds. -* -,-^^^_., ,, ., ..
II.) STEAP-1 Polynudeotides
One aspect of he invention provides polynudeotides corresponding or complementary to all or part of a STEAP-1
gene, mRNA, and/or coding sequence, preferably in isolated form, inducting polynudeotides encoding a STEAP-1-related protein and fragments hereof, DMA, RNA, DNA/RNA hybrid, and related molecules, polynudeotides or oiigonudeotides complementary to a STEAP-1 gene or mRNA sequence or a part thereof, and polynudeotides or oGgonudeotkJes hat hybridize to a STEAP-1 gene, mRNA, or to a STEAP-1 encoding polynudeotide (collectively, 'STEAP-1 polynudeotides"). hi • all instances when referred to in this section, TcanalsobeU in Rgure 2.
Embodiments of a STEAP-1 polynudeotide indude: a STEAP-1 polynudeotide having he sequence shown in Figure 2, the nudeotide sequence of STEAP-1 as shown In Rgure 2 wherein T is U; at least 10 contiguous nudeofides of a polynudeotide having he sequence as shown in Rgure 2; or; at least 10 contiguous nudeofides of a polynudeotide having he sequence as shown h Figure 2 where T is U. For example, embodiments of STEAP-1 nudeofides comprise, without
Imitation:
(I) a polynudeotide comprising, consisting essentially of, or consisting oi~ it sequence as shown in Rgure 2,
wherein T can also be U;
(II) a polynudeotide comprising, consisting essentially of, or consisting of the sequence as shown hi Rgure
2A, from nucteofide residue number 66 through nudeoOde residue number 1085, including the stop codon, wherein
T ran also be U;
(III) a polynudeotide comprising, consisting essentially of, or consisting of (he sequence as shown in Figure
2B, from nudeotide residue number 96 through nudeotide residue number 872, including foe stop codon, wherein
TcanalsobeU;
»
(IV) a porynudeotjde comprising, consisting essentially of, or consisting of the sequence as shown in Rgure
2C, from nudeofide residue number 96 through nudeotide residue number 944, including fie a stop codon,
wherein T can also be U;
(V) *arx>lynucteotkte 2D, from nudeofide residue number 96 through nudeoflde residue number 872, induoTng he stop codon, wherein V-T can also be U;
(VI) a polynudeotkJe combing, oxisisting essentially of, or assisting of Biesequer^
2E, from nudeotide residue number 96 through nudeofide residue number 872, including he stop codon, wherein TcanalsobeU;

(VII) a potynudeotide comprising, consisting essentiafy of, or consisting of the sequence as shown in Figure
2F, from nudeofide residue number 96 through nudeotide residue number 872, including the slop codon, wherein
T can also be U;
(VIII) a pdynudeofide comprising, consisting essentially of, or consisting of the sequence as shown in Figure
26, from nucfeotide residue number 96 through nudeoGde residue number 872, induding the stop codon, wherein
; T can also be U;
(IX) a polynudeolide comprising, consisting essenGaSy of, or consisting of the sequence as shown in Figure
2H, from nudeotide residue number 96 through nudeotide residue number 872, induding the slop codon, wherein
T can also be U;
(X) a polynudeotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure
21, from nudeofide residue number 96 through nudeotide residue number 872, induding the stop codon, wherein T
can also be U;
(XI) a polynudeotide comprising, consisting essentially of, or consisting of the sequence as shown in Figure
2J, from nudeofide residue number 96 through nudeofide residue number 872, inducting the stop codon, wherein
T can also be U;.
(XII) a polynudeotide comprising, consisting essentially of, or consisting of the sequence as shown in Rgure
2K, from nudeotide residue number 96 through nudeofide residue number 872, induding the stop codon, wherein
]T can also be U; • ,
(XIII) a polynudeofide comprising, consisting essentially of, or consisting of the sequence as shown in Figure
2L, from nudeotide residue number 96 through nudeotide residue number 872, induding the stop codon, wherein
TcanalsobeU;
(XIV) a polynudeotide comprising, consisting essenfiafly of, or consisting of (he sequence as shown in Figure
2M, from nudeotide residue number 96 through nudeofide residue number 872, indudiny ihe stop codon, wherein
*•? T can also be U;
(XV) a polynudeofide comprising, consisting essenfiaBy of, or consisting of the sequence as shown in Rgure
2N, from nudeotide residue number 96 through nudeofide residue number 872, induding the stop codon, wherein
TcanalsobeU;
(XVI) a polynudeoSde comprising, consisting essentiaBy of, or consisting of the sequeTtoe as shown h F^jure
20, from nudeofide residue number 96 through nudeofide residue number 872, Including toe stop codon, wherein
T can also be U;
(XVII) a por/nudeotide comprising, consisting essenfiaBy of, or consisfing of the sequence as shown in Figure
2P,from nudeofide residue number 96 through nudeotide residue number 872, induding the stop codon, wherein
T can also be U;
(XVIII) a polynudeotide comprising, consisting essenfiaBy of, or consisting of the sequence as shown in Figure
20, from nudeotide residue number 96 through nudeofide residue number 872, induding the stop codon, wherein
T can .also be U;

(XIX) a pdynudeotide lhat encodes a STEAP-1-felated protein lhat is at least 90,91,92,93,94,95,96,97,
98,99 or 100% homologous to an entire amino add sequence shown in Figure 2A-Q;
(XX) a polynudeofide that encodes a STEAP-1-related protein that Is at least 90,91,92,93,94,95,96,97,
98,99 or 100% identical to an entire amino add sequence shown in Figure 2A-Q;
. (XXI) a polynucteofide that encodes at least onepepfide set forth in Tables V-XVIII and XXII-U as set forth in United States patent application 10/236,878 filed 06-Sepfember (XXII) a polynudeotide that encodes a pepfide region of at least 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 amino adds of a peptide of Figures 3Ain any
whole number increment up to 339 that includes at least 1,2,3,4,5,6,7.8,9,10,11,12,13,14,15,16,17,18,
19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino acW posifion(s) having a value greater than
0.5 in the HydrophiEctty profile of Figure 5;
(XXIII) a polynudeoBde that encodes a peptide region of atteast 5,6,7,8, Z, 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 amino adds of a peptide of Figure 3A in any whole
number increment up to 339 that Mudes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add positions) having a value less than 0.5 in the
Hydnopattiidty profile of Figure 6;
* 1
(XXIV) a polynudeotide (hat encodes a peptide region of atteast 5,6,7,8,9,10,11,12,13,14,15,16,17,18,
19,20,21,22,23,24,25,2^ 27,23,29,30,31,32,33.34,35 amino adds of a peptide of Figure 3A in any whole
number increment up to 339 thatindudes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add positions) having a value greater than 0.5 in the
Percent Accessible Residues profile of Figure 7;
(XXV) a polymjcfeofide that encodes a peptide region of atteast 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 ammo adds of a peptide of Figure 3A in any whde
number increment up to 399 that indudes 1,2,3,4,5,6,7,3,9,10,11,12,13,14,15,16,17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add positions) having a value greater than 0.5 in the
Average Flexibility profile of Figure 8;
(XXVI) a polynudeotide that encodes a peptide region of at least 5,6,7,8,9,10.11,12,13,14,15,16,17,18,
19,20,21,22,23,24,25,20,27,28,29,30,31,32,33,34,35 amino acids of a peptide of Figure 3A in any whole
number increment up to 339 that indudes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position® having a value greater than 0.5 in the Beta-
turn profile of Figure 9;
(XXVII) a polynucteofide that encodes a peptide region of at least 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 amino adds of a pepfide of Figure 38 and 3D In
any whole number increment up to 258 that indudes 1,2,3,4,5,6,7,8,9,10,11.12,13,14,15,16,17,18,19,
20.21,22,23,24,25,26,27,'28,29,30,31,32,33,34,35amtnoackJposJticHi(s)havingavaluegrealerthan0.5
in the Hydrophadty proffte of Figure 5;

(XXVIII) a polynudeotide that encodes a peptide region of at least 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 amino adds of a peptide of Fgure 3B and 3D in
any whole number increment up to 258 that indudes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,
20,21,22,23.24,25,26,27,28.29,30,31,32,33,34,35 amino add position® having a value tess than 0.5 In
the Hydropathicity profile of Figure 6;
(XXIX) a polynudeotide that encodes a peptide region of at lead 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 amino acids of a peptide of Figure 3B and 3D in
any whole number increment up to 258 that indudes 1,2,3,4,5,6,7,8,9.10,11,12,13,14,15,16,17,18,19,.
20,21,22,23,24,25,26,27,28/29,30,31,32,33,34,35 amino add position(s) having a value greater than 0.5
in the Percent Accessible Residues profile of Rgure 7;
(XXX) a polynudeotide thatencodss apepOde region of at least 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 amino acids of a peptide of Figure 3B and 3D in
any whole number increment up to 258 that indudes 1,2,3,4,5,6,7,8,9,10,11.12,13,14,15,16.17,18,19,
20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position® having a value greater than 0.5
in the Average Flexibility profile of Rgure 8;
(XXXI) a polynudeotide that encodes a peptide region of at least 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 amino adds ofapepGde of Rgure 38 and 3D (n
any whole number increment up to 258 that indudes 1,2,3,4,5.6,7.8,9,10,11,12,13,14,15,16,17,18,19,
20,21,22,23,24,25,26,274 28,29,30,31,32,33,34,35 amino add position® having a value greater than 0.5
' in (he Beta-turn profBe of Figure 9;
(XXXII) a polynudeotide that encodes a peptide region of at least 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 amino adds of a peptide of Figure 3C in any whole
number increment up to 282 that indudes 1,2,3,4,5,6,7,8,9,10,11,12, U14,15,16,17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position® having a value greater than 0.5 In the
Hydrophilidty profile of Figure 5;
(XXXIII) a polynudeotide that encodes a peptide region of at least 5,6.7,8,9,10,11,12,13,14,15,16,17,16,
19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino adds of a peptide of Figure 3C in any whole
number increment up to 282 that indudes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21.22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position® having a value tessthan 0.5 in the
Hydropathicity profile of Figure 6;
(XXXIV) a polynudeotide that encodesapepMe region of atteast 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 amino adds of a peptide of Figure 3C k) any whole
number Increment up to 282 that indudes 1.2,3,4,5,6,7,8,9,10.11,12,13,14,15,16,17,18,19,20,21,22,
23,24,25, ^6,27,28,29.30,31,32,33,34,35 amino add position® having a value greater than 0.5 hi the
Percent Accessible Residues profile of Figure 7;
(XXXV) a polynudeotide that encodes a peptide region of at leasts, 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 amino adds of a peptide of Figure 3C in any whole
number incremenl up to 282 that includes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,

23,24,26,26,27,28,29,30,31,32,33.-S4,35 amino acid postlion(s) having a value greater than 0.5 in the Average Flexibility profile of Figure 8;
(XXXVI) a poiynudeotide that encodes a peptide region of at feast 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 amino adds of a peptide of Figure 3C in any whote number increment up to 282 that includes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, 23,24,25,26,27,28,29,30; 31,32,33,34,35 amino acid posltion(s) having, a value greater than 0.5 in the Beta-turn profile of Figure 9;
{XXXvll) a polynucleofide that is fully complementary to a poiynudeotide of any one of (I)-(XXXVI}; "(XXXVltlj a poiynudeotide that is fully complementary to a poiynudeotide of any one of (I)-(XXXVII); (XXXIX) apepfidethatisencodedbyanyof(i}to(XXXVIll);and;
(XL) a composition compn'sing a pdynudeotide of any of (l)-(XXXVlll) or peptide of pOOGX) together with a pharmaceutical exdpient and/or in a human unit dose form;
(XLI) a method of using a poiynudeotide of any (I)-{XXXV1I I) or peptide of (XXXIX) or a composition of (XL) in a method to modulate a cell expressing STEAP-1;
(XLJI) a method of using a poiynudeotide of any (I)-(XXXVII!) or peptide of (XXXIX) or a composition of (XL) in a method to diagnose, pivphylax, prognose, or treat an Individual who bears a cell expressing STEAP-1;
(XLIII) a method of using a poiynudeotide of .any (IHXXXVIII) or peptide of (XXXIX) or a composiBon of (XL) in 'i a method to diagnose, prophylax, prognose, or treat an Individual who bears a oeO expressing STEAP-1, said cell from a cancer of a fissue listed in Table I; .
(XLJV) amethodofusingapor/nudeotiderfany(l)-{XX^ a method to diagnose, prophylax, prognose, or treat a cancer;
(XLV) a method of using a poiynudeotide of any (IHXXXVIII} or peptide of (XXXIX) or a composition of (XL) in a method to diagnose, prophylax, prognose, or treat a cancer of a tissue listed hi Table I; and;
(XLVT) a method of using a polynudeolide of any (IHXXXVIII) or peptide of (XXXIX) or a composition of (XL) in a method to Identify or characterize a modulator of a ceB expressing STEAP-1.
As used herein, a range is understood to disclose speoificaBy an whote unit positions thereof.
Typical embodiments of the invention disclosed herein indude STEAP-1 polynudeofides (hat encode specific portions of STEAP-1 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example: .
(a) 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40.45.50,55,60,65.70. 75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165.170,175.180.185,190.195.200. 225,250,275,300,325,330,335,339 or more contiguous amino adds of STEAP-1 variant 1; the maximal lengths relevant . for other variants are: variant 2,258 amino adds; variant 3,282 amino adds, and variant 4,258 amino adds.
For example, representative embodiments of the Invention disclosed herein indude: polynudeotides and heir
encoJedpepfideslhefnselves encoding abort amirw a^ 10of the STEAP-1 protein shown ki Figure
2 or Figure 3, polynudeotides encoding about amino add 10 to about amino acid 20 of the STEAP-1 protein shown ki Figure 2 or Figure 3, polynudeotides encoding about amino add 20 to abort amino add 30 of Ihe STEAP-1 protein shown in Figure
2>o

or Figure 3, polynudeotides encoding about amino acid 30 to about amino add 40 of the STEAP-1 protein shown in Figure 2 cr Figure 3, polynudeotides encoding about amino add 40 to about a«v.ino add 60 of the STEAP-1 protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino acid 50 to about amino acid 60 of the STEAP-1 protein shown in Figure 2 or Figure 3, polynudeofides encoding about amino acid 60 to about ammo add 70 of the STEAP-1 protein shown in Figure 2 or Figure 3, polynudeotides encoding about amino add 70 to about amino add 80 of the STEAP-1 protein shown in Figure 2-or Figure 3, potynudeotides encoding about amino acid 80 to about amino add 90 of he STEAP-1 protein shown In Figure 2 or Figure 3, polynudeotides encoding about amino acid 90 to about amino add 100 of the STEAP-1 protein shown fo • Figure 2 or Figure 3, in increments of about 10 amino adds, ending at the carboxyi terminal amino acid set forth in Figure 2 or Figure 3. Accordingly, polynudeotides encoding portions of the amino acid sequence (of about 10 amino adds), of amino acids, 100 through the carboxyi terminal amino add of the STEAP-1 protein are embodiments of the invention. Wherein it is understood that each particular amino add position disdoses that position plus or minus five amino acid residues.
Polynudeotides encoding relatively long portions of a STEAP-1 protein are also within the scope of the invention.. For example, polynudeotides encoding from about amino add 1 (or 20 or 30 or 40 etc.) to about amino add 20, (or 30, or 40 or 50 etc.) of the CTEAP-1 protein 'or variant" shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art These porynudeotide fragments can indude any portion of the STEAP-1 sequence as shown in Figure 2.
Additional illustrative embodiments of the invention disclosed herein indude STEAP-1 polyn"deotide fragments encoding one or more of the biological motifs contained within a STEAP-1 p,otein 'or variant" sequence, including one or more of the motif-bearing subsequences of a STEAP-1 protein "or Volant* set forth in Tables V-XVIII and XXIf-LI. In another embodiment, typical polynudeotide fragments of the invention encode one or more of the regions of STEAP-1 protein or variant that exhibit homotogy to a known molecule. In another embodiment of the invention, typical polynudeotide fragments can encode one or more of the STEAP-1 "protein or variant N-glyeosylafion sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylaCon sites or N-mynstoytation site and amidatibn sites.
Note that to determine the startiny position of any peptide set forth in Tables V-XVIII and Table: XXII to U (collectively HLA Pepb'de Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to tfiree factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Pepfides listed in Table III. Generally, a unique Search Pepb'de Is used to obtain HLA peptfdes for a particular variant .The position of each Search reptkte relative to its respective parent molecule is listed in Table III. Accordingly, if a Search Peptide begins at position "X", one must add the value -A minus 1* to each position in Tables V-XVlll and Tables XXII-LI to obtain (he actual position of the HLA peptides in their parental molecule. For example if a particular Search Pepfide begins at posiSon 1 50 of its parental molecule, one must add 150 - 1, i.e., 149 to each HLA peptide amino acid position to calculate (he position of that amino acid in the parent molecule.
UsesefSTEAP-IPoinucteotides
IIJU.) Monitoring of Genetic Abnormalities
The polynudeofides of the preceding paragraphs have a number of different specific uses. The human STEAP-1 gene maps to the chromosomal location set forth in the Example entitled "Chromosomal Mapping of STEAP-1.' For example, because the STEAP-1 gene maps to this chromosome, polynudeotides that encode different regions of the STEAP-1 proteins are used to characterize cytogenefic abnormalities of this chromosomal locale, such as abnormafifies that are Identified as being associated with various cancers. to certain genes, a variety of chrorrKScm^abrKX^ rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g, Krajinovic etal., Mutat Res. 382(3-4): 81-83 (1998); Johansson ef a/., Blood 86(10}: 3905-3914 (1 995) and Finger etaL, P.NAS. 85(23): 9158-9162 (1988)). Thus, polynudeotides encoding specific regions of the STEAP-1 proteins provide new

that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region lhat encodes STEAP-1 that may contribute to the malignant phenotyoe. in this context, these polynudeotides satisfy a need in the art for expanding the sensitivity of ci«c«HOSOirwJ screening in order to identify subfle and less common chromosomal abnormalities (see e.g. Evans ef a,'., Am. J. Obstet Gynecol 171(4): 1055-1057 (1994)).
Furthermore, as STEAFM was shown to be highly expressed in prostate and other cancers, STEAP-1 polynudeotides are used in methods assessing the status of STEAP-1 gene products in normal versus cancerous tissues. Typically, polynudeotides that encode spedfic regions of the STEAP-1 proteins are used to assess the presence of ; perturbations (such as deletions, Insertions, point mutations, or alterations resul8nginatossofanantigenetc.)insp^dfc regions of the STEAP-1 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR- -assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi ef a/., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynudeotides encoding specific regions of a protein to examine these regions within the protein.
IIA2.) Antisense Embodiments
Other spedficaBy contemplated nudeic add related embodiments of the kwenSon disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nudeic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and indude molecules capable of inhfoifing the RNAor protein expression of STEAP-1. For example, antisense molecules can be RNAs or other molecules, induding peplide nudeic adds (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these dasses of nudeic acid molecules using the STEAP-1 porynttdeofides and potynucteoSde sequences cfisdosed herein.
Anfisense technology entails the administration of exogenous digonudeofides that bind to a target pdynudeofide located within the cells. The term "anfisense" refers to toe fact that such ofigonudeotides are complementary to their intracellular targets, e.g., STEAP-1. See for example, Jack Cohen, Ofigodeoxynudeotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The STEAP-1 antisense ofigonudeotides of the present invention indude derivaSves such as S-oligonudeotides (phosphorothioate derivatives or S-oSgos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth Inhibitory acfion. S-oligos (nudeoskJe.^hosphorothioates) are isoelectronfe -analogs of an wJigonucteofide (O-ofigp) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present Invention can be prepared by treatment of the corresponding O-ofigos with SH-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent See, e.g., Iyer, R. P. et a/., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. ef a/., J. Am. Chem.Soc. 112:1253-1254 (1990). Additional STEAP-1 antisense oKgonudeotides of the present invention Indude morpholino antisense oBgonudeofides known in the art (see, e,g.,,Partridge ef a/., 1996, Antisense & Nucleic Add Drug Development 6:169-175).
The STEAP-1 antisense oligonudeofides of the present Invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5' codons orlast 100 3' codons of a STEAP-1 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Useofancfiponudeotideoornplernentarytotn^
and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, STEAP-1 antisense digonudeotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule hat have a sequence that hybridizes to STEAP-1 mRNA. Optionally, STEAP-1 antisense oligonudeotide is a 30-mer oGgonudeotide that is complementary to a region in the first 10 51 codons or last 10 3' codons of STEAP-1. Alternatively, the antisense molecules

are modified to employ ribozymes hi the inhibition of STEAP-1 expression, see, e.g., L A. Couture & D. T. Stinchcomb; Trends Genet 12:510515 (1996).
II A3.) Primers and Primer Pairs
Further specific embodiments of these nudeotides of the Invention include primers and primer pairs, which atow the specific amplification of pdynudeotjdes of the invention or of any specific parts thereof, and probes that selectively or * specifically hybridize to nucleic add molecules of the invention or to any part thereof. Probes can be labeled with a
-. ,
detectable marker, such as, for example, a radfofsotope, fluorescent compound, bfoluminescent compound, a
chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a
-,~ STEAP-1 po[ynucteo^inasiarn$ea^ " .---«-
Examples of such probes include polypeptides comprising all or part of the human STEAP-1 cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying STEAP-1 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a STEAP-1 mRNA
The STEAP1 polynudeotLes of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the STEAP-1 gene(s), mRNA(s), or fragments thereof, as reagents for the diagnosis and/or piognosis of prostate cancer and other cancers; as coding sequences capable of dirediiig the expression of STEAP-1 polypeptides; as tools for modulating or inhibiting the expression of the STEAP-1 gene(s) and/or translation of the STEAP-1 transcripts); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a STEAP-1 or STEAP-1 related nucleic add sequence torn a naturally ocourrhgsciirce, such as humaris or others nudete acid sequence per se, which wouldcomprise all or most of the sequences found hfhe probe used. IIA4.) Isolation of STEAP-1-Encoding Nucleic Acid Molecules
The STEAP-1 cONA sequences described herein enable the isolation of other polynudeotides encoding STEAP-1 gene produces), 8° well as the isolation of porynudeotjdes encoding STEAP-1 gene product homologs, alternatively spliced isoforms, alteRc variants, and mutant forms of a STEAP-1 gene product as well as polynudeoSdes that encode analogs of STEAP-1-related proteins. Various molecular doning methods that can be employed to isolate fuIIenghcDNAseixxxling a STEAP-1 gene are wen known (see, for example, Sambrook, J. era/., Molecular Cloning: A Laboratory Manual, 2d coition, Cold Spring Harbor Press, New York, 1989; Current Protocols In Mofecuter Biology. Ausubelef a/., Eds., Wiley and Sons, 1995). For example, lambda phage doning methodologies can be conveniently employed, using commercially available doning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing STEAP-1 gene cDNAs can be identified by probing with a labeled STEAP-1 cDNA or a fragment thereof. For example, in one embodiment, a STEAP-1 cDNA(e.g., Figure 2) or aporfion thereof can be synftestoed and used as a probe to retrieve overlapping and RiHengfhcDNAscoirespoiKfing to a STEAP-1 gene. A STEAP-1 gene Itself can be isolated by screening genomfc DNA fibrarfo,bacMa! artificial chtonwsoene I crraixisonieHbraries(YACs), and the Bke,wKh STEAP-1 DNA probes or primers.
v HAS.) Recombinant Nudeic Acid Molecules and Host-Vector Systems
The hvenSon also provides recornbinantf^ analog or rnmotogtje (hereof, jnckKfing rxit not Im
viral and non-viral vectors we! known in (he art, and cells transfon^ or trarafectedwHh such reoontf^ molecules. Methodsforgenerating suchmotecutes are weflknc^ (see, forexanipie,Sambiookef at, 1989,supra).
The invention further provides a host-vector system comprising a recombinant DNA molecule containing a STEAP-1 polynudeotide, fragment, analog or homotogue hereof within a suitable prokaryofic or eukaryoGc host eel. Examples of
.
suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian eel or an Insect ce«
(e.g., a baculovtms-lnfedible cell such as an Sf9 or HighFive cell). Examples of suitable mansnaTian coBs include various
prostate cancer cell lines such as DU145 and TsuPrl, other transferable or transdudbte prostate cancer eel Cries, primary
cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS,
CHO, 293,293T cells). More particularly, a polynudeotide comprising the coding sequence of STEAP-I or a fragment, analog
or homotog thereof can be used to generate STEAP-1 proteins or fragments thereof usir^ any nimjberrfrwst-vector systems
routinely used and widely known in the art :!
A wide range of host-vector systems suitable for the expression of STEAP-I proteins orfragments thereof are avaBaWe, see for example, Samtoookef a/., 1989, supra; Current Protocols in Molecular Biology,T995,^upra}n^ferM vectors for mammalian expression indude but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and fte retroviral vector pSRotkneo (Muller el al., 1991, MCB11:1785). Using these expression vectors, STEAP-1 can be expressed in several prostate cancer and non-prostate cell fines, induding for example 293,293T, rat-1, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of a STEAP-1 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of STEAP-1 and STEAP-1 mutations or analogs.
Recombinant human STEAP-1 protein or an analog or homolog or fragment thereof can be produced by mammafian cells transfected with a construct encoding a STEAP-1-relaled nudeofide. For example, 293T cells can be transfected with an expression ptasmid encoding STEAP-1 or fragment, analog or homolog thereof, a STEAP-1-related protein is expressed In the 293T cells, and the recombinant STEAP-1 protein is isolated using standard purification methods (e.g., affinity purification using anG-STEAP-1 antibodies). In another embodiment, a STEAP-1 coding sequence is subdoned into foe nftoviral vector pSRoMSVtkneo and used to infect various mammalian ceO fines, such as NIH 3T3, TsuPrt, 293 and rat-1 in order to establish STEAP-1 expressing cell fines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a STEAP-1 coding sequence can be used for the generation of a secreted form of recombinant STEAP-1 protein.
As discussed herein, redundancy in the genetic code permits variation in STEAP-1 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host For example, preferred analog codon sequences typicaHy have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific spedes are calculated, for example, by ufiEzing codon usage tables avanabfeonthelNTERNETsuchasatUFldna.affirc.go.jp/-flakamura/codon.htrnl.
Additional sequence modifications are known to enhance protein expression in a ceMar host These indude eSmination of sequences encoding spurious polyadenylafion signals, exon/intron splice site sJgnals.-transposon-fike repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host as calculated by reference to known genes expressed in the host cefl. Where possible, the sequence fe modified to avoid predk^ hairpin secondary mR^strndures. OfteruseynwoTficafions indude the addition of a translafonal initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mo/. CeffB/b/., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryofic ribosomes initiate translation exdusively at the 5 proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2CG2-2666, (1995) and Kozak NAR15(20): 8125-8148 (1987)).

!IL1 STEAP-l-retatedPratdns
Another aspect of the present invention provides STEAP-1-reiated proteins. Spedfc cmixxiti iients of STEAP-1 proteins comprise a polypeptide having alt or part of the amino acid sequence of human STEAP-1 as shown in Figure 2 or Rgure 3, preferably Figure 2A. Alternatively, embodiments of STEAP-1 proteins comprise variant, homolog or analog polypeptides ttialhave alterations In the amino acid sequence of STEAP-1 shown In Figure 2 or Figure 3.
Embodiments of a STEAP-1 polypeptide include: a STEAP-1 polypeptide having a sequence shown in Rgure 2, a
peptide sequence of a STEAP-1 as shown in Figure 2 wherein T is U; at least 10 contiguous nudeotides of a polypeptide
having the sequence as shown in Rgure 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as
shown in Figure 2 where TisJ^ comprise, without limitation:
(I) a protein comprising, consisting essentially of, or consisting of an amino add sequence as shown in
Figure 2A-Q or Figure 3A-D;
(II) a STEAP-1-related protein that is at feast 90,91,92,93,94,95,96,97,98,99 or 100% homologous to
an entire amino acid sequence shown In Rgure 2A-Q or 3A-D;
(ill) a STEAP-1-related protein that is at teasl 90,91,92,93,94,95,96,97,98,99 or 100% Identical to an entire amino add sequence shown in Figure 2A-Q or 3A-D;
(IV) a protein (hat comprises at least one peptkle set forth in Tables V to U as set forth in United States
patent application 10/236,878 filed 06-September-2002 the specific contents of which are fully incorporated by
reference herein, optionally with a proviso that it is not an entire protein of Rgure 2;
!• V
(V) a protein that comprises at least one peptide set forth in Tables V-XVlll, coitecGvely, which peptide is
also set forth in Tables XXII to LI, collectively, optionally with a proviso that it is not an entire protein of Figure 2;
(VI) a protein that comprises at least two pepfietes selected from the pepfides set forth in Tables V-U,
optionally with a proviso that it Is not an entire protein of Rgure 2;
(VII) a protein that comprises at least two peptktes selected from fte pepfides set forth in Tables V to U
collectively, with a proviso that the protein is not a contiguous sequence from an amino add sequence of Rgure 2;
(VIII} a protein that comprises at feast one pepfide selected fromlhepepfides set forth h Tables V-XVHI;and at least one peptide selected from the peptides set forth In Tables XXII to U with a proviso that the protein Is not a
contiguous sequence from an amino add sequence of Figure 2;
%
(IX) a polypeptide comprising at least 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 amino adds of a protein of Figure 3A in any whole number Increment up
to 339 respectively that includes at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14.15,16.17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add positions) having a value greater than 0.5 in the
HydropWfidty profile of Figure 5;
(X) a polypeptide comprising at feast 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 amino adds of a protein of Rgure 3A in eny whole number increment up
to 339 respectively that includes at least at least 1,2,3,4,5,6,7,8,9,10,11,12,13.14.15,16.17,18,19,20,
21,22,23,24.25,26,27,28,29.30,31,32,33,34,35 amino add position(s) having a value less than 0.5 in the
Hydropathidty profile of Rgure 6;

(XI) a polypepGde comprising at least 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 amino acids of a protein of Figure 3A in any whole p»mber increment up
to 339 respectively that indudes at least at least 1,2,3,4,5,6,7,8,9.10,11,12,13, H, 15,16,17,18.19,20,
21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino acid position(s) having a value greater than 0.5 in
the Percent Accessible Residues profile of Figure 7;
(XII) a polypepfkie comprising at least 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 amino acids of a protein of Rgure 3A in any whole number increment up
to 339 respectively that includes at least at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,
21,22,23,24.25,26,27,28,29,30,31,32,33,34,35 amino acid position(s)"havingrjaf value greater thanfO.5 In
the Average Flexibility profile of Rgure 8;
(XIII) apdypepfidecomprisingatteast5,6,7,8,9, 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, amino acids of a protein of Rgure 3A in any whole number Increment up to
339 respectively that includes at least at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,
22,23,24,25,26.27,28,29,30,31,32,33,34,35 amino add positions) having a value greater than 0.5 h the
Beta-turn profile of Rgure 9;
(XIV) a polypepGde comprising at least 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 amino acids of a protein of Rgure 3B or 3D, in any whole number
increment up to 258 respectively that includes at teasM. 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 amino acid position® having a value greater than 0.5
in the HydrophiDcity profile of figure 5;
(XV) a pdypepBde comprising at leasts, 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 amino adds of a protein of Figure 3B or 3D, in any whole number
Increment up to 258 respectively that indudes at least at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,
18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position® having a value less than
0.5 in the Hydropathldty profile of Figure 6; %»
(XVI) a potypeptkJe comprising at leasts, 6,7,8,9,1C, 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 enino adds of a protein of Figure 3B or 3D, in any whole number
Increment up to 258 respectively that includes at least at least 1,2,3,4,5,6,7,8,9,10.11,12,13.14,15,16,17,
18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position® having a value greater
than 0.5 in the Percent Accessible Residues profile of Figure 7; *
(XVII) a polypeptide comprising at least 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 amino adds of a protein of Figure 3B or 3D, in any whote number
increment up to 258 respectively that indudes al least at teast 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17.
18,19,20,21,22,23,24,25, 26,27,28,29,30.31,32.33,34,35 amino add posi5on(s) having a value greater
than 0.5 in the Average Flexibility profile of Figure 8;
(XVIII) a polypepfide comprising at feast 5,6,7,8,9,10,11,12,13,14,15,16,17,16,19.20,21,22,23,24,
25,26,27,28.29,30,31,32,33,34, amino adds of a protesi of Rgure 3B or 3D in any whote number increment
Upb258respedivdythatinckjdesatleastatleast1.2l3,4>5,6,7,8>9l10>11l12,13,14,15,16l17l18,19,
.

20,21,22,23,24,25,26.27,28,29,30,31,32,33,34,35 amino add position® having a value greater than 0.5 in the Beta-turn profile of Figure 9;
(XIX) a polypepGde comprising at least 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 amino acids of a protein of Figure 3C, in any whole number increment up
to 282 respectively that indudes at least 1,2,3,4,5,6.7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,
23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position® having a value greater than 0.5 in the
Hydrophificity profile of Figure 5;
(XX) a polypepfide comprising at leasts, 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,
25,26,27,28,29;=30r31 r32,33,34,35 amino adds of a protein of Figure 3C, in any whole number increment up
to 282 respectively that indudes at least at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,
21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino add position(s) having a value less than 0.5 in the
Hydropathicity profile of Figure 6;
(XXI} a potypeptide comprising at least 5,6,7,«, 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 amino adds of a protein of Figure 3C, in any whole number increment up to 282 respectively that indudes at least at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21,22.23,24,25,26,27,28,29,30,31,32, oj, 04,35 amino add positions) having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 7;
(XXII) a polypepfide comprising at least 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 amino adds of a protein of Figure 3C, in any whole number increment up
to 282 respectively that includes at least at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,
21,22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino add posifion(s) having a value greater than 0.5 in
the Average Flexibility profile of Figure 8;
(XXIII) a polypepfide comprising at least 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, amino adds of a protein of Figure 3C in any whole number increment up to
282 respectively that indudes at least at least 1,2,3,4,5,6,7,8,9,10/11,12,13,14,15,16,17,18,19,20,21,
22,23,24,25,26,27,28,29,30,31,32,33,34,35 amino add positions) having a value greater than 0.5 in the
Beta-turn profile of Figure 9;
(XXIV) a pepfide that occurs at least twice in Tables V-Xvlll and XXII to U, collectively;
(XXV) a peptide that occurs at least three limes in Tables V1-XVIII and XXII to U, cottecfivdy,
(XXVI) a peptide that occurs at teast four times in Tables V-XXVIII and XXII to U, coBecfively;
(XXVII) a peptide that occurs at least five times in Tables V-XV1H and XXII to U, cottecSvely;
(XXVIII) a peptide that occurs at teast once in Tables V-Xvlll, and at least once in tables XXII to U;
(XXIX) a pepBde foal occurs at least once in Tables V-Xvlll, and at least twice in tables XXII to U;
(XXX) a peptide that occurs at least twice in Tables V-Xvlll, and at least once in tables XXII to U;
(XXXI) a pepfide that occurs at least twice in Tables V-XY1II, and at least twice in tables XXII to U;

(XXXII) a peptide wWch comprises one two, three, four, or five of the following characteristics, or an
ofigonudeotide encoding such peptide:
0 a region of at least 5 amino adds of a particular peptide of Rgure 3, in any whole number Increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to ex-greater than 0.5,0.6,0.7,0.8,0.9, or having a value equal to 1.0, in the Hydrophffidty profile of Figure 5;
i) a region of at least 5 amino acids of a particular peptide of Figure 3, in any whole number increment up to the full length of that protein in Rgure 3, that includes an amino acid position having a value equal to or less thai 0.5,0.4,0.3,0.2.0.1, or having a value equal to 0.0, in the Hydropathidty profile of Figure 6;
HO a region of at least 5 amino acids of a parted
up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5,0.6,0.7,0.8,0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of Rgure 7;
iv) a region of at least 5 amino acids of a particular pepb'de of Figure 3, in any whole number increment up to the full length of that protein in Figure 3, that includes an amino acid position having a value equal to or greater than 0.5,0.6,0.7,0.8,0.9, or having a value equal to 1.0, in the Average Flexibility profile of Rgure 8; or,
v) a region of at least 5 amino acids of a pailicub-- peptide of Rgure 3, to any whole number Increment up to the full length of that protein in Figure 3, that includes an amino add position hMig a value equal to or greater ttian 0.5,0.6,0.7,0.8,0.9, or having a value equal to 1.0, in the Beta-turn profile of Figure 9;;
(XXXIII) a composition comprising a peptide of (IJ-(XXXH) or an antibody or binding region thereof together with a
' • pharmaceutical exdpient and/or In a human unit dose form.
(XXXIV) a method of using a pepb'de of (IHXXXII), or an antibody or binding region thereof or a composition of
(XXXIII) in a method to modulate a cell expressing STEAP-1,;
(XXXV) amethodofusingapeptidecf(l}-(XXXII)cfanantibodyorbindingreg!onthereoforacompo
(XXXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a eel expressing STEAP-
1; "r
(XXXVI) arre(haiofusmgapepfideof(IHXXXII)wana
(XXXIII) In a method to diagnose, prophylax, prognose, or treat an Individual who bears a cefl expressing STEAP-1, said cell from a cancer of a tissue fisted in Table I;
(XXXVII) a method of using a peptic's of (I)-(XXXII) or an antibody or binding region ftereof or a composition of
(XXXIII) in a method to diagnose, prophylax, prognose, or treat a cancer;
(XXXVIII) a method of using a peptide of (I)-(XXXII} or an anfibody or binding region thereof or a composition of
(XXXIII) In a method to diagnose, prophylax, prognose, or treat a cancer of a tissue listed in Table I; and;
pOOdX)*a method of using a peptkJeof (IHXXXII) or an antibody or binding region (hereof or a composition (XXXIII) in a method to Identify or characterize a modulator of a cell expressing STEAP-1;
As used herein, a range is understood to spedficaity dfectose all whole unit positions thereof.
Typical embodiments of the Invention disclosed herein include STEAP-1 potynudeofides that encode specific
portions of STEAP-1 mRNA sequences (and those which are complementary to such sequences) such as flwse hat encode
the proteins and/or fragments thereof, for example: _.

(a) 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25.30,35,40,45,50,55, SO. 65,70,
' 75,80,85,90.95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,
225,250,275,300,325,330,335,339 or more contiguous amino acids of STEAP-1 variant 1; the maximal lengths relevant for other variants are: variant 2,258 amino acids; variant 3,282 amino acids, variant 4,258 amino adds..
In general, naturally occurring aJtelic variants of human STEAP-1 share a Wgh degree of structural Henfiy and
• homology (e.g., 90% or more homology). TypfcaBy, altelic variants.of a STEAP-1 protein contain conservative amino acid
substitutions within the STEAP-1 sequences described herein or contain a substitution of an amino add from a corresponding"' position in a homotogue of STEAP-1. One dass of STEAP-1 altefic variants are prote&is that share a high degree of homology with at least a small region of a particular STEAP-1 amino add sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orihology and paralogy can be important concepts describing (he relationship of members of a given protein family in one organism to the members of the same family in other organisms.
Amino add abbreviations are provided in Table II. -Conservative am'mo add substitutions can frequently be made In a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1,2, 3,4,5,6,7,8,9,10,11,12,13,14, Embodiments of the invention disclosed herein indude a wide variety of art-accepted variants or analogs of STEAP-1 proteins such as potypepfides having amino add insertions, deletions and substitutions. STEAP-1 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter ef a/., Hud. Adds Res., 13:4331 (1986); Zoller et a/., Nucl. Acids Res., 10:6487 (1987)},
cassette mutagenesis (Wells et a/., Gene,'34:315 (1985)), restriction selection mutagenesis (Wells et a/., Phitos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the ctoned DMA to produce the STEAP-1 variant DMA.
Scanning amino add analysis can also be employed to identify one or more amino acids along a contiguous
•X
sequence that is involved In a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino adds are relatively small, neutral amino adds. Such amino adds Indude alanine, glycine, serine, and cystelne. Alanine fe typically a preferred scanning amino add among this group because ft eliminates Ihe side-chain beyond toe bete-carbon and is less likely to alter the main-chain conformation of the variant Alanine Is also typically preferred because it Is the most common amino add. Further, it is frequently found in both buried and exposed posifions jCrekjhton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. MoL Bio!., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an Isosteric amino add can be used.
As defined herein, STEAP-1 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that Is "cross reactive' with a STEAP-1 protein having an amino add sequence of Figure 3. As used in this sentence, 'cross reactive' means that an antibody or T cell that specifically binds to a STEAP-1 variant also specifically binds to a STEAP-1 protein having an amino add sequence set forth in Figure 3. A polypeptide ceases to be a variant of a protein shown in Figure 3, when It no longer contains any epitope capable of being recognized by an antibody or T ceS that specifically binds to the starting STEAP-1 protein. Those skilled in the art understand that antibodies that recognize proteins , IW to epitopes of varying size, ami a grouping of ttiecfder of a^
as a typical number of amino acids in a minimal epitope. See, e.g., Nair etaL, J. Immunol 2000165(12): 6949-6955; Hebbes eta!., Mo! Immunol (1989) 26(9):eS5-73;Schwarbeia/., J Immunol (1985) 135{4):2598-608.

Other classes of STEAP-1-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino add sequence of Figure 3, or a fragment thereof. Another specific dass of STEAP-1 protein variants or analogs comprises one or more of the STEAP-1 biological motifs described herein or presently known in the art Thus, encompassed by the present invenlion are analogs of STEAP-1 fragments (nudeic or amino add) that have altered functional (e.g. immunogenic) properties relative to the starting fragment It is to be appreciated that motifs now or which become part of the art are to be applied to the nudeic or amino acid sequences of Figure 2 or Figure 3.
As discussed herein, embodiments of the claimed invention include polypeptides containing less than the fufl amino acid sequence of a STEAP-1 protein shown in Rgure 2 or Figure 3. For example, representadve embodiments of the invention comprise peptides/proteins having any 4,5,6,7,8,9,10,11,12,13,14,15 or more contiguous amino acids of a STEAP-1 protein shown in Figure 2 or Figure 3.
Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino add 1 to about amino add 10 of a STEAP-1 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino add 10 to about amino add 20 of a STEAP-1 protein shown hi Figure 2 or Figure 3, polypeptides consisting of about amino add 20 to about amino acid 30 of a STEAP-1 protein shown in Figure 2 or Figure 3, polypeptides.consisfing cf about amino add 30 to about amino add 40 of a STEAP-1 protein shown in Figure 2 or Figure 3, polypeptides consisting of about a-rlno add 40 to about amino add 50 of a STE&P-1 protein shown in Figure 2 or Rgure 3, polypeptides consisting of about amino add 50 to about amino add 60 of a STEAP-1 protein shown in Figure 2 or Rgure 3, polypeptides consisting of about amino add 60 to about amino acid 70 of a STEAP-1 protein shown In Figure 2 or Rgure 3, pcdypepfides consisting of about amino add 70 to about amino add 80 of a STEAP-1 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino add 80 to about amino add 90 of a STEAP-1 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino add 90 to about amino add 100 of a STEAP-1 protein shown in Figure 2 or Figure 3, etc. throughout (he entirety of a STEAP-1 amino add sequence. Moreover, polypeptides consisting of about amino add 1 (or 20 or 30 or 40 etc.) to about amino add 20, (or 130, or 140 or 150 etc.) of a STEAP-1 protein shown in Figure 2 or Figure 3 are embodiments of the invention. It is to be dppredated that the starting and stopping pooifons in this paragraph refer to the specified position as weB as that position plus or minus 5 residues.
STEAP-1-related proteins are generated using standard pepfide synthesis technology or using chemical cleavage methods wen known in the art Altemafively.recanbinant methods can be used to generate nudeic add moteades fiat encode a STEAP-1-related protein, in one embodiment, nudeic acid molecules provide a means to generate defined fragments of a . STEAP-1 protein (or variants, homotogsor analogs (hereof).
IILA.) Motif-bearing Protein Embodiments
Additional illustrative embodiments of the Invention disclosed herein indude STEAP-1 polypepfides comprising the
amino add residues of one or more of the biological motifs contained within a STEAP-1 polypeptkle sequence set forth in
Figure 2 or Figure 3. Various motifs are known in the art, and a protein can be evaluated for (he presence of such mofifs by
a number of pubfidy available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; searchlauncher.bcm.trnc.edu/seq-
searcn/struc-pre^cthtml; psortims.u-iokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scanJitml; expasy.cn/tools/scnpsit14itrnJ;
Epimatrix™ and Epimer™, Brown University, bromedu^Research/TB4W_U^^ and BIMAS,
bimas.dcrtnih.gov/.).
Motif bearing subsequences of aO STEAP-1 variant proteins are set forth and identified hi Tables V-XV1II and XXII-U.
Table IV(h) sets for* several frequently occurring motifs based on pfam searches (see URL address pfam.wusU.edu/). The columns of Table IV(h) fist (1) motif name abbreviation, (2) percent identity found amongst he

.1

different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
Pofypepfides comprising one or more of the STEAP-1 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the STEAP-1 motifs discussed above are associated with growth dysregufafion and because STEAP-1 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of foe malignant phenotype (see e.g. Chen ef at; Lab Invest, 78(2): 165-174 (1998); Gaiddon ef at. Endocrinology 136(10): 43314338 (1995); Hall ef al., Nucteic Acids Research 24(6): 1119-1126 (1996); Peterael ef at; ~ Oncogene 18(46): 6322-6329 (1999) and Q'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and ' myristoylafion are protein modifications also associated with cancer and cancer progression (see e.g. Dennis ef al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Ce8 Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston ef at, J. Natl. Cancer Inst Monogr. (13): 169-175(1992)).
In another embodiment, proteins of the invention comprise one or more of Ihe immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XVtl! and XXII-U. CTL epitopes can be determined using specific algorithms to identify peptides within a STEAP-1 prote'n that are capable of opfimaSy binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimef™ Brown University, URL brown.edu/Research/TB-HlV.Ub/epirrmtrix/epenatrochtrnl; and BIMAS, URL bimas.clcrLnih.gov/.) Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are weH known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are weB known in the arL'and are carried out without undue experimentation either in vftro or in vivo.
Also known ki the art are principles for creating analogs of such epitopes In order to modulate Jmmunogenldty. For example, one begins with an epitope that bears a CTL or! JTL motif (see, e.g., Ihe HLA Class I and HLA Class II -motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino add specified for that position. For example, on the basis' of residues defined in Table IV, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less-preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions In a peptide; see, e.g., Table IV.
A variety of references reflect the art regarding the identification and generation of epitopes to a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut ef at; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette ef at, J. Immunol. 2001166(2): 1389-1397; Sidney c? a/., Hum. Immunol. 1997 58(1)^12-20; Kondo ef at. knmunogenetics 1997 45(4): 249-258; Sidney ef al., J. Immunof. 1996157(8): 3480-90; and Falk ef at, Nature 351:290-6 (1991); Hunt ef at, Science 255:1261-3 (1992); Parker ef at, J. Immunol. 149:3580-7 (1992); Parker ef a/., J. Immunol 151-163-75 (1994)); Kastef at, 1994152(8): 3904-12; Borras-Cuesta ef at, Hum. Immunol. 200061(3): 266-278; Alexander ef a/., J. hnrnunol. 2000164(3); 164(3): 1625-1633; Alexander ef at, PMID: 7895164, Ul: 95202582; 0-Suffivan ef at, X Immunol. 1991147(8): 2663-2669; Alexander ef at, Immunity 19941(9): 751-761 and Alexander ef at, Immunol Res. 1998 18(2): 79-92.
Related embodiments of Ihe invention include polypeptides comprising combinations of the different motifs set forth in Table® IV(c), rV(b). IV(c), rV(d), and IV(h), and/or, one or more of the predicted CTL eprtopes of Tables V-XVII! and XXII-U and/or, one or more of toe predicted HTL epitopes of Tables XLVill-U, and/or, one or more of toe T cell binding motifs known in the art. Preferred embodirnents contain no Insertions, deletions or substitutes eifiwwMita fee motifis or

-
within the intervening sequences of the pdypepfides. In addition, embodiments which include a number of either N-terminal
and/or C-termlnal amino acid residues en either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the moBf is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino add residues.
STEAP-1-related proteins are embodied in many forms, preferably in isolated form. A purified STEAP-1 protein molecule win be substantially free of other proteins or molecules that impair the binding of STEAP-1 to antibody, T eel or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a STEAP-1-related proteins include purified STEAP-1-related proteins and functional, soluble STEAP-1-related proteins. In one
. * •" -•.--—•-*•=> :•---•• -----••,^-rr-s."**.- .--:,.--;.—.- - (,-,.-.«-'»,^:;1?r-™^._...-- ,^,.. ,-a.s-rt.-.^..,;*. 7 s..*.:..— ,- .•...>^>^^-v,.--. . ..-, .
embodiment, a functional, soluble STEAP-1 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides STEAP-1 proteins comprising biologically active fragments of a STEAP-1 amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the starting STEAP-1 protein, such as the ability to elicit the generation of antibodies thai specifically bind an epitope associated with the starting STEAP-1 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starling protein.
STEAP-1-related porypsDtides frat contain particularly interesting structures can be preceded and/or identified using vanbus analytic^ techniques wefltowwn in the art, incfcjdngjw
DooDttJe, Eisenberg, Karplus-Schufeor Jameson-Wolf anarysis, or based on immunogenictty. Fragments Biat contain such structures are partkailarty useful h generating su^
that bind to STEAP-1. For example, hydrjophiiicity profiles can be generated, and Immunogenicpepfide fragments idenKed, using the method of Hopp, T.P. and Woods, K.FL, 1931, Proc. Nat!. Acad. Scl U.SA 78:3824-3828. Hydropathidty profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doofittte, R.F., 1982, J. MoL Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogente peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments Identified, using (he method cf Shaskaran R., Ponnuswamy P.K., 1988, kit J. PepL Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deteage, G., Roux B., 1967, Protein Engineering 1:289-294.
CTL epitopescan be determined using spedfc algorithms fa icfenfifypepfides within a STEAP-1 protein that are capable ofoptimaDy binding to specified HLAaBeles (ag., by using IheSYTTCITHI site at WwWVVIde Web URL syfpeithlArni-heloelberg.com/; the listings in Table !V(A)-{E}; Epimatrix™ and Epimer™, Brown University, URL (browaedu^esearcfvTB-HIV_Lab/epimatrix/epimatri)di&TiI); and BiMAS, URL bimas.dcrtnih.gov/). IBustrafing this, peptide epifopes from STEAP-1 that are presented in the context of human MHC Class I molecules, e.g., HLAA1, A2, A3, A11, A24, B7 and B35 were predfcted (see, e.g., Tables V-XVIII, XXII-U). Specfficaliy, the complete amino acid sequence of the STEAP-1 protein and relevant portions of other variants, La, for HLA Class I predictions 9 flanking resHues on either sio^trf a point imitation or exon jucfion, and for HLA Class II predictions 14 Banking residues on either skte of a po^t mutatis or exoijuricfion correspondBig to that variant, were entered into the^^HLA Peptide Mofif Search algOTtfimfc The HLA peptide mofif search algoriflim was developed by Dr. Ken Parker based on binding of specific pepfide se


Hunt eta/., Sdence 255:1261-3 (1992); Parker ef a/., J. Immunol. 149:3580-7 (1992); Parker ef a/., J. bnmunoL 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as wefi as nur.ierous other HLA Class i molecules. Many HLA dass I binding peptides are 8-, 9-, 10 or 11 -fliers. For example, for Class IHLA-A2, the epitopes preferably contain a leucine (L) or methtonine (M) at position 2 and a valine (V) or leucine (L) at toe C-terminus (see, e.g., Parker ef a/., J. immunoL 149:3580-7 (1992)). Selected results of STEAP-1 predicted binding peptides are shown in Tables V-XVtl! and XXII-LI herein. In Tables V-XV1II and XXII-XLVIII, selected candidates, 9-mers and 10-mers, for each family member are shown along with; their location, the amino add sequence of each specific peptide, and an estimated binding score. In Tables XLVIH4J, selected candidates, 15-mers, for each family member are shown along with their location, the arnino addjsequence of each spedfie-peptidejand-an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37°C at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et a/., Prostate 30:73-8 (1997) and Peshwa et a/., Prostate 36:129-38 (1998)). Immunogenidty of specific peptides can be evaluated m vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cell"
It is to be appreciated 'Jiat every epitcpe predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA dass I or dass II motifs available h.the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeifhi.bmi-heidelberg.com/, or BIMAS, btmas.dcrtnih.gov/} are to be "applied" to a STEAP-1 protein in accordance wjth toe invention. As used in this context "applied" means that a STEAP-1 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skffi in the relevant art Every subsequence of a STEAP-1 protein of 8,9,10, or 11 amino add residues that bears a" HLA Class I motif, or a subsequence of 9 or more amino add residues that bear an HLA Class II mctif are within toe scope of toe invention.
III.B.) Expression of STEAP-1-related Proteins
In an embodiment described to toe examples that follow, STEAP-1 can be conveniently expressed in cells (such as 293T cells) transacted with a commerdaUy available expression vector such as a CMV-driven expression vector encoding STEAP-1 with a C-terminat 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen orTagS, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate toe production of a secreted STEaP-1 protein in transfected cells. The secreted HIS-tagged STEAP-1 in toe culture media can be purified, e.g., using a nickel column using standard techniques.
HI.C.) Modifications of STEAP-1-related Proteins
Modifications of STEAP-1-celated proteins such as covalent modifications are included within he scope of this invention. One type of covalent modification indudes reacting targeted amino add residues of a STEAP-1 poiypepdde with an organic derivatizing agent that Is capable of reacting with selected side chains or the N-or C-terminal residues of a STEAP-1 protein. Another type of covalent modification of a STEAP-1 poiypeptide included within the scope of Ws invention comprises altering toe native flrycosyiation pattern of a protein of toe bivenfion. Another type of covalent modification of STEAP-1 comprises finking a STEAP-1 poiypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene

glycol (PEG), polypropylene glycol. orpdyoxyalkytenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791 ,ia* or 4, i 79,337.
The STEAP-1-relaied proteins of the present Invention can also be modified to form a chimerfc molecule comprising STEAP-1 fused to another, heterologous polypeptide or amino add sequence. Such a chimeric molecule can be synfliesteed chemically or recombinantly. A chimeric mdecute can have a protein of foe invention fused to another tomor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invenBon can comprise a fusion of fragments of a STEAP-1 sequence (amino or nucleic add) such that a molecule is created that is riot, through its length, directly homologous to (he amino or nucleic acid sequences shown in Figure 2 or Figure 3. Such a chimeric molecule can "ooniprise multiples of the same subsequence of STEAP-1. A chimeric mdecutecarhGempriseaMonjpfaSTEAEsUelaM protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of a STEAP-1 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a STEAP-1-related protein with an immunoglobufin or a particular region of an immunoglobufin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The tg fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a STEAP-1 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, (he imrnunoglobuGn fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of tmmunoglobulin fusions see, e.g., U.S. Patent No. 5,428,130 issued June 27,1995.
III.D.) Uses of STEAP-1-related Proteins
The proteins of the Invention have a number of different specific uses. As STEAP-1 Js highly expressed in prostate
and other cancers, STEAP-1-related proteins are used in methods that assess the status of STEAP-1 gene products in
normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptjdes from specific regions
of a STEAP-1 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in
those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting
STEAP-1-retated proteins comprising the amino acid residues of one or more of the biological motifs contained within a
STEAP-1 potypepfide sequence in order to evaluate the characteristics of this region in normal versus cancerous (issues or
to elicit an Immune response to the epitope. Alternatively, STEAP-1-related proteins that contain (he amino acid residues of
one or more of the biological motifs in a STEAP-1 protein are used to screen for factors that interact with (hat region of
STEAP-1. '
STEAP-1 protein fragments/subsequences are particularly useful in generafing and characterizing domain-specific antibodies (e.g., anObodies recognizing an extracellular or kitraceBular epitope of a STEAP-1 protein), for identifying agents or celMar factors that bind to STEAP-1 or a particular structural domahlhereof, and hvar^ including but not fmited to dtagnostfc assays, cancer vaeones arc! methods of preparing
Proteins encoded by the STEAP-1 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and h methods for identifying igands and otoer agents and cefUar constituents that bind to a STEAP-1 gene product Antibodies raised against a STEAP-1 protein or fragment thereof are useful bi diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of STEAP-1 protein, such as those listed ki Table I. Such antibodies can be expressed mtraceflularty and used in methods of treating patients with such cancers. STEAP-1-related nucleic acids or proteins are also used in generafing HTL or CTL responses.

Various irnmunotogical assays useful for he detection of STEAP-1 prc4eirKareused,lnciudr^bulrKrflmitedtovark)Us
types of radoHTimunoassays, erc^i«™ikeu Bimiunosorbent assays (EUSA), enzyme-inked tmnumofhwrescent assays (EUFA),
krawnocytocnetrdcalinefh^ Antaxxfiescanbelafo^ardusedasinTOin^
detecting STEAP-1-expresshg cells (e.g., In radrasc&itigraphfc imaging methods). STEAP-1 proteins are also parfcularry useful in generafing cancer vaccines, as further descrbed herein.
IV.l STEAP-1 Antibodies ' •
Another aspect of the invention provides antibodies that bind to STEAP-1-felated proteins. Preferred antibodies - s^fk^y bind to a ST^-1-reiatedfrot^
related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25mM Tris and 150 mM Nad; or normal saline (0.9% NaCI); 4) animal serum such as human serum; or, 5) a combination of any of 1) through 4); these reactions preferably taking place atpH 7.5, altemaSvely in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4°C to 37°C. For example, anfibodies that bind STFAP-1 can bind STEAP-1-related proteins such as the homotogs or analogs thereof.
STEAP-1 antibodies of the invention are particularly useful in cancer (see, e.g., Table 0 diagnostic and prognostic
assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of
prostate and other cancers, to the extent STEAP-1 is also expressed or overexpressed In these other cancers. Moreover,
bitraceUularry expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the
expression.of STEAP-1 is Involved, such as advanced or metastatic prostate cancers or other advanced or metastatfc
.cacners.' vThe hvenfion also provides various irrrniiirrokigical assays iiseful fa he detection arriquan mutant STEAP-1-felated proteins. Such assays can comprise one or more STEAP-1 anfibodies capable of recognizing and binding a STEAP-1-related protein, as appropriate. These assays are performed withfo various imrrajrKAxj^ known in the art, inducing but not Smiled to various types of racloirnnwnoassays, enzyme-faked immure
Irrununotogfcal non-anSxxly assays of toe hvenfion ateo (x>mpriseTcenirr»™ricgertoty assays stimulatory) as well as major histocornpatibuity complex (MHO) binding assays.
h ackfition, brfflurK^fcal imaging rrat^ STEAP-1 are also yowled by ttehvenfico,irK^ 1 antibodies. Such assays are cfinfcafly useful in flwfeiedion,inonltQrtig,ardpro^ as prostate cancer.
STEAP-1 anfibodfes are also used h mehods for purifying a STEAP-1 related protein and for feolafing STEAP-1 rKirrokigues and relatedrrwlecuJes. Faexarr^, am STEAP-1 antibody, v»hkiir^ been coipled to a soTdn^ urKterixmfiOonsfriat permit he STEAP-1 anObocJy to Wnd to tte
impurities; and efcrlJng fw STEAP-1-related protein from the coupled anffixxty. Other uses of STEAP-1 anGbodfes in accordance with five Invention include generafing anHcBotypfc antibodies that mimic a STEAP-1 protein.
VartousmethocfefbrftepreparaGontfantiboc^ For example, anftxxfes can be prepared
by immunizing a suitable marnrnafan host us^aSTEAP-1-relatedprotehlpepSo^,aft^o^ientlinlsolaledor krvminooonjugated farm (Ant3x>ies: A Lat>xatory Manual, (^P^

Spring Harbor Press, NY (1989)). In addition, fusion proteins of STEAP-1 can also bs used, such as a STEAP-1 GST-fusion protein. !a a particular embodiment, a GST foster, protein caiipn^aH or nrost of ttteamino
3 is produced, then used as an trnmunogen to generate appropriate antibodies. In another embodiment, a STEAP-1-related protein is synthesized and used as an immunogen.
In addffion, naked DMA immunization techniques known In the art are used (wift or without purified STEAP-1-related protein or STEAP-1 expressing cells) to generate an immune response to the encoded knmunogen (for review, see Donnelly ef a/., 1997, Ann. Rev. Immunol. 15:617-648).
The amano acid sequence of a STEAP-1 protein as shown in Figure 2 or Figure 3 can be analyzed to select specifc. regions of the STEAP-1 protein for generating antibodies; For exampte/hydropHobldly and hytfophificity analyses of a STEAP-1 amino acid sequence are used to identify hydrophflic regions in the STEAP-1 structure. Regions of a STEAP-1 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Gamier-Robson, Kyte-Dooliffle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Nafl. Acad. Soi. U.SA 78:3824-3828. Hydropathiciiy profiles can be generated using the method of Kyte, J. and DooGttle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P.K., 1988, InL J. PepL Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is wffiiin the scope of the present invention. Preferred methods for the generation of STEAP-1 antflxxOes arefurther illustrated by way of the examples provided herein. Methods fbrpreparing a proteiqcrpdypeptide for use as an Jmmurwgen are vieB known h the art Also wen known in the art are methods for preparing immunogehfo conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, drect conjugation using, for example, carbodinide reagents are used; in other instances linking reagents such as Ihosesuppfied by Pferce Chemical Co., Rodkfbrd, IL, areeffecfive. AdmMistrationofaSTB^P-llrrnwnogenisoflen conducted by injecfiondv^r a sultabfe fimeperiod and with use rfasu'tabte adjuvant as feunotestood in the art. During the immunization schedule, filers of antibodies can be taken to o^temiine adequacy of antibody formafioa
STEAP-1 monoclonal antibodies can be produced by various means weB known in the art. For example, himortafized ceB ines that secrete a desired monoclonal antfcody are prepared using ttesta^aidhybridomalecnnologyofKbhlerand fvfflstelncirniodrficafjoflsfcatirnrnoftafoanf^
(he desired anfibodies are screened by immunoassay in whi* the anfigenfe a STEAP-1-felated protein. When the appropriate
Immortalized ceB culture is identified, the cefls can be expand and anfflxxfies produced eflher from hvirocutorescr^
asdtes fluid. »
The antibooles or fragments of the invention can also be pfockx^ by iBcombinaitnreans. Regions lhal bind specifically to the desired regions of a STEAP-1 protein can also be produced in the context of diimeric or complementarity-determining region (COR) grafted anfibodfes of mirftipte species origin. Humanized or human STIEAP-lanaxxfies can aiso be produced, and are ptefenied for use in SierapeuBc oonle^ subsfitutingc^cfnx)reoftherKxi4iurr^antibcdyCOf^forc^^
example, Jones ef a/., 1986, Nature 321:522-525; Rechmannef el. ,1988, Nature 332:323-327; Verhoeyenef at, 1988, Science 239:1534-1536). See also, Carter ef aL, 1993. Proc. Matt. Acad Sd USA 89:4285 and Sims ef at, 1993, J. bnmunoL 151:2296.
fvtetfaxls for proc*H^fu«y human n^
see Vaughanefa/., 1998, Nature Biotechnology 16:535^39). FuByhuman STEAP-1 moncdonalanindes can be generated using doning technologies employing large human Ig gene combinatorial ixan^

Building an fa vflro Immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Mars, Clark, M. (Ed.), Nottingham Academic, pp 4564 (1993); Burton and Barbas, Human Antibodies torn combinatorial fibraries. Jd., pp 65-82). FuBy human STEAP-1 monoclonal anffixxfies can also be produced ushgtransgenic mice engineered to cont^ W098/24893,Kucherlapafia^
Drugs 7(4): 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 ^l()vember2000; and, 6,114598 issued 5 September 2000). This method avoids the to v*o manipufafion required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of STEAP-1 antibodies witha STEAP-1-related protein can be established by a number of weH known:'"~ means, including Western blot, immunoprecipitation, EUSA, and FACS analyses using, as appropriate, STEAP-1-related proteins, STEAP-1-expressing cells or extracts Biereof. A STEAP-1 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal dictator or an enzyme. Further, bi-spectfic antibodies spedfic for two or more STEAP-1 epitopes are generated using methods generally known in the art Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al, Cancer Res. 53:2560-2565).
In one ernbodurt&nt the invention provides for monoclonal antibodies identified as mouse hybrtdoma X92.130.1.1(1) and mouse hybridoma X120.545.1.1 deposited with he American Type Culture Collection, located at 10801 University Blvd. Manassas, VA 20110-2209 on 06-FebruafY-2004 and assigned ATCC Accession numbers PTA-5803 and PTA-5803 respectively.
.! V
V.) STEAP-1 Cellular Inimune Responses
The mechanism by which T cells recognize anugens has been delineated. EfRcadous pepfide epitope vaccine compositions of the bivenfion Induce a therapeutic or prophylacSc Immune responses in very broad segments of the worldwide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of Immunology-related technology Is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by KLA-restricted T cells (Buus, S. et at, Ceff 47: i071,1986; BabbitCB. P. el a/., Wafum 317:359,1985; Townsend, A. and Bodmer, H., Aim. Rev. Immund 7:601,1989; Germain, R N., Annu. Rev. Immunol. 11:403,1993). Through the study of single amho add substituted antigen analogs and the seqcendng of endogenously bound, naturally processed pepfides, critical residues that correspond to motifs required for spedfic binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et a/., J. Immunol. 160:3363,1998; Rammensee, ef al., Immunogenefcs 41:178,1995; Rammensee ef a/., SYFPEfTHI, access via Worid Wide Web at URL (134.2.96.221/scripls.hlasefver.(Jlrtxxne.htm); Sette, A. and Sidney, J. Curr. Opin. Imtnunol. 10:478,1998; Engelhard, V. H., Curr. Opin. Immunol 6:13,1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79,1992; SinigagBa, F. and Hammer, J. Curr. Biot. 6:52,1994; Ruppertef at, CeJ74.-929-937, 1993; Kondo et al.. JJmmunol. 155:4307-4312,1995; Sidney ef al., J. ftncnunot 157:3480-3490,1996; Sidney erf a/., Human Immunol. 45:79-93,1996; Sette, A. and Sidney, J. Immunogenettes 1999 Nov. 50(34)201-12, Review).
Furthermore, x-ray crystallographic analyses of HLA-pep6de complexes have revealed pockets within he pepfide Wndtagctefi/grooveofHlAriTOleculeswhkto
these residues hi turn determine the HLA binding capacity of the pepfides in which they are present (See, «.£. Madden, D.R. Annu. Rev. immunoi. 13:587,1995; Smith, et a/., ImmuntytMl, 1996; Fremont ef a/.. /mmarory8:305,1998rStem et

a/., StructuK 2:245,1994; Jones. E.Y. Curr. Opln. Immunol. 9:75,1997; Brown. J. H. ef al., Nature 364:33,1993; Guo, H. C. etat.,Proc. Natl. Acad. Sti. USA90:6053,1993; Guo, H. C. etal, Natun360:364,1992; Silver, M. L eta!., Natum360:367, 1992; Matsumura, M. et at., Scfence 257:927,1992; Madden et al, CeB 70:1035,1992; Fremont, D. H. et at., Science 257:919,1992; Saper, M. A., Bjoricman, P. J. and WJley, D. C., J. Mol Biol. 219:277,1991.)
Accordingly, the definition of class I and class 1! aflete-spedfic HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).
Thus, by a process of HLA motif identification, candidates for epitope4>ased vaccines have been identified; such candidates can be further evaluated by HLA-pepfide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can.be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
Various strategies can be utilized to evaluate cellular immunogenicity, including:
1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wenhorth, P. A. era/., Mol. Immunol.
32:603,1995; Celis, E. et al., Pmc. Natl. Acad. Sci. USA 912105,1994; Tsai, V. eiai., J. Immunol. 158:1796,1997;
Kawashima, I. et at., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral blood lymphocytes
(PBL) from normal subjects with a test pepfide in the presence of antigen presenting cells in vitro over a period of several
weeks. T cells specific for the peptio'o become activated during this time and are detected using, e.g., a lymphokine- or
51Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P, A. et a/., J, Inmunol. 26:97,1996; Wentworth, P.
A. ef a/., frrf. Immunol. 8:651,1996; Alexander, J. etal., J. Immunol. 159:4753,1997). For example, in such methods
pepfides In incomplete Freund*s adjuvant are administered subcutaneously to HLA iransgente mice. Several weeks following
immunization, splenocytes are removed and cultured hi v*o in foe presence of test peptide for approximately one week.
Peptide-spedficT cells are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
3) Demonstration of recall T cell responses from immune Individuals who have been either effectively vaccinated
and/or from chronically HI patients (see, e.g., Rehermann, B. ef a/., J. Exp. Ued. 18T:1047,1995; Doolan, D. L et.el.,
Irmun!ty7$7t 1997; Bertonl, R. ef al., J. Clin. invest 100:503,1997; ThreBceld, S. C. ef al., J. Immunol. 159:1648,1997;
Diepokter, H.M.efa/.,J. Viroi 71:6011,1997). Accordingly, recall responsesare detected by culturing PBL from subjects
(hat have been exposed to the antigen due to disease and thus have generated an immune response "naturally", or from
patients who were vaccinated against the antigen. PBL from subjects are cultured In vitro for 1 -2 weeks in the presence of
test peptide ptus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells. At
the end of the culture period, T ceB activity is detected using assays including 51Cr release Involving pepDde-sensitized targets, T cell proliferation, or lymphokine release.
VI.) *STEAP«1 Transgenic Animals
Nucleic adds that encode a STEAP-1-related protein can also be used to generate either transgenic animals or "knock out" animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cONA encoding STEAP-1 can be used to done genomic DMA 8iat encodes STEAP-1. The doned genomic sequences can then be used to generate transgenic animals containing cefls that express DMA that encode STEAP-1. Methods for generating transgenic animate, particularly animals such as mfce or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 issued 12 April 1988. and

'4,870,009 Issued 26 September 1989. Typically, particular cells would be targeted for STEAP-i transgene incorporation with tissue-specific enhancers.
Transgente animals that include a copy of a transgene encoding STEAP-I can be used to examine the effect of increased expression of DMA that encodes STEAP-1. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpressfon. In accordance with this ' aspect of the Invention, an animal is'treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals fliat bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homdogues of STEAP-1 can be used to construct a STEAP-1 'knock our animal foat has a defective or altered gene encoding STEAP-1 as a result of homologous recombination between the endogenous gene encoding STEAP-1 and altered genomic DNA encoding STEAP-1 introduced into an embryonic cell of the animal. For example, cDNA that encodes STEAP-1 can be used to done genomic DNA encoding STEAP-1 in accordance with established techniques. A portion of the genomic DNA encoding STEAP-1 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typicafy, several kilobases of unaltered flanking DNA (both at the ff and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi, Ceff. §1:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cefi line (e.g., by electroporation) and cells in which the introduced DNA has hcmologously recombined with the endogenous DNA are selected (see, e.g., U et al, Cell 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Terafocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a "knock out" animal. Progeny
harboring the homologously recombined CNA in their germ cells can be identified by standard techniques and used to breed
x animals in which all cells of the animal contain the homologously recombined DNA Knock out animals can be characterized,
for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a STEAP-1 polypepfide.
VII.) Methods for the Detection of STEAP-1
Another aspect of (he present invention relates to methods for detecting s> fEAP-1 pofynucteotides and STEAP-1-relafed proteins, as wen as meflrods for identifying a eel that expresses STEAP-1 The expression profile of STEAP-1 makes R a diagnostic marker for metastasized disease. Accordingly, the status of STEAP-1 gene products provides information useful for predcBng a variety of factors hckxfing suscepfibifity to advanced stap^ disease, rate of progress^ arn^ aggressiveness. As discussed in detai herein, the status of STEAP-1 gene products hi patient sampies^an be analyzed by a vanety protocol that are weB known in foe art to^
including In stu hybridization, RT-PCR analysis (for example on laser rapture mtaotfissectedsan^), Western bW and tissue array analysts.
More particularly, Bve invention provides assays for the detection etf STEAP-1 poryrwcleoidesh a bictoo^sarnpte,
such as serum, bone, prostate, and other Issues, urine, semen, eel preparations, and the ike. Detectable STEAP-1

rxHvnudeoticles include, for exarnpte, a STEAP-1 gene or fragment hereof, STEAP-1 mRNA, atemafive spice variant STEAP-1
mRr^aralrecorribiriantDNAorRNAr^ AmmterofrreBrodsforarnpfifying
andfor detecting toe presence of STEAP-1 porynudeotides are well known in tr^ art arvj can be employed in flw practice of flvs aspect of foe invention.

In one embodiment, a melhod for detecting a STEAP-1 mRNA In a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a STEAP-1 pdynudeotides as sense and antisense primers to amplify STEAP-1 cDNAs therein; and detecting (he presence of the amplified STEAP-1 cDNA. Optionally, the sequence of (he amplified STEAP-1 cDNA can be determined.
In another embodiment, a method of defecting a STEAP-1 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the Isolated genomic DNA using STEAP-1 polynucleotides as sense and anBsense primers; and detecting the presence of the amplified STEAP-1 gene. Any number of appropriate sense and antisense probe combinations can be designed from a STEAP-1 nudeotide sequence (see, e.g.. Figure 2) and used for this purpose.
The invention also provides assays for detecting the presence of a STEAP-1 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cefl preparafions, and the fike. Methods for detecting a STEAP-1-related protein are also wed known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELJSA, EUFA and the fike. For example, a method of detecting the presence of a STEAP-1-reiated protein In a biological sample comprises first contacting the sar.ple with a STEAP-1 antibody, a STEAP-1-reactive fragment .thereof, or a recombinant protein containing an antigen-binding region of a STEAP-1 antibody; and then detecting the binding of STEAP-1-relaied protein in the sample.
Methods for identifying a cell that expresses STEAP-1 are also within fhe scope of the invention. In one embodiment, an assay for identifying a ceH that expresses a STEAP-1 gene comprises detecting fhe presence of STEAP-1 mRNA in (he eel Methods for the detection of particular mRNAs in cells are wefl known and include, for example, hybrideaBon assays using complementary DNA probes (such as In situ hybrkSzafwnusSrglabeN STEAP-1 rtwprobes^
techniques) and various nudefc acid amplification assays (such as RT-PCR using complementary primers specific for STEAP-1, and other amptificaGon type detection methods, such as, for example, branched DMA, SISBA, TMA and the ike). Alternatively, an assay for identifying a cell that expresses a STEAP-1 gene comprises detectir^ the present of STEAP-1-related protein h the cell or secreted by the eel Various methods for the detection of proteins are wd known in Bie art and are employed for Bie detection of STEAP-1-related proteins and cells that express STEAP-1-related proteins.
STEAP-1 expression analysis is also useful as a tool for Identifying and evafuafing agents ftat modulate STEAP-1 gene expression. For example, STEAP-1 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits STEAP-1 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that
quantifies STEAP-1 expression by RT-PCR, nucleic add hybridization or antibody binding.
«
VIII.) Methods for Monitoring the Status of STEAP-1-related Genes and Their Products Oncogenesisis known tobe a multistep process where cellular growth becomes progressively dysregutaled and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Aters ef a/., Lab Invest 77(5): 437-438 (1997) and Isaacs etal., Cancer Sun/. 23:19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant STEAP-1 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage Biat therapeutic options are more Broiled and or the prognosis is wrse. In such examinations, the status of STEAP-1 in a biological sample of Interest can be compared, for example, to the status of STEAP-1 in a corresponding nomial sample (e^. a sampfe from that individual or alternatively another individual that is not affected by a pathology). An alteration in lie status of STEAP-1 in the biological sample (as compared to the normal sample) provides evidence of dysregulated ceMar growtu In addjfion to

L.

using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Graver tt a/., J. Conip. weuroi. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501} to compare STEAP-1 status in a sample.
The term 'status' in Ws context is used according to its art accepted ineaningarKlreferstothecondifonorstateofa gene and Its products. Typfcaly, skied artisans use a number of parametersto evaluate the condition or state of a gene and its products. These irduoV},l)ut are not Med to the l^^
expressing cells) as we! as the level, and biological activity of expressed gene products (such as STEAfM mRNA, polynudeotides and polypepfides). Typically, an alteration in the status of STEAP-1 comprises a change in (he location of STEAP-1 and/or STEAP-1 expressing cells and/or an increase in STEAP-1 mRNA and/or profein expression.
STEAP-1 status in a sample can be analyzed by a number of means weB known in the art, including without Bmitation, imiminohistochemical analysis, « s/fu hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a STEAP-1 gene and gene products are found, for example in Ausubel ef a/, eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (ImmunobtotGng) and 18 (PCR Analysis). Thus, the status of STEAP-1 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genornic Southern analysis (to examine, for example perturbations In a STEAP-1 gene), Northern analysis and/or PCR analysis of STEAP-1 mRNA (to examine, for example alterations in the poiynudeotide sequences or expression levels of STEAP-1 mRNAs), and, Western and/or immunohistodiemical analysis (to examine, for example alterations in porypeptjde sequences, alterations in pdypepBde localization within a sample, alterations in expression levels of STEAP-1 proteins and/or associations of STEAP-1 proteins with potypeptide binding partners). Detectable STEAP-1 porynudeofides include, for example, a STEAP-1 gene defragment thereof, STEAP-1 mRNA, alternative spfice variants, STEAP-1 mRNAs, and recombinant DMA or RNA molecules containing a STEAP-1 potynucteotkte.
The expression profile of STEAP-1 makes it a diagnostic marker for local and/or metastasized disease, and provides information on (he grow&i or "vcogenic potential of a biological sample. In particular, the status of STEAP-1 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining STEAP-1 status and diagnosing cancers that express STEAP-1, such as cancers of the fissueoSsted in Table L For example, because STEAP-1 mRNA Is so highly expressed in prostate and ofoer cancers relative to normal prostate fissue, assays mat evaluate fte levels of STEAP-1n^R^IAIranso^^aproteirBhabtologlcal sample can be used to diagnose a disease associated with STEAP-1 dysregulafion, and can provide prognosfc hformafion useful in defining appropriate therapeutic options.
The expression status of STEAP-1 provides Information including the presence, stage and kyafion of dyspiasfic, precancerous ami carKxrotJsajte, predict^
aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of Ihe hvenfion is directed to the various molecular pogrwsticarKidiagrwsfornelfK^ status of STEAP-1 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized fay dysregulated cellular growth, such as cancer.
As described above, foe status of STEAP-1 in a biological sample can be examined by a number of weHcnown procedures in Ihe art. For example, (he status of STEAP-1 in a biological sample taken from a specific focafion in Ihe body can be examined by evaluating the sample for the presence or absence of STEAP-1 expressing cefc (e.g. hose hat express STEAP-1 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growft, for example, when STEAP-l^xpressir^ceflsarefourKJhaWolcgk^

because such alterations In the status of STEAP-1 in a biological sample are often associated wiih dysregulated cellular growth. SpedficaBy, one Indicator of dysregulated cellular growBi Is toe metastases of cancer cells fmm =»n organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated ceflular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et a!., Prostate 42(4): 31W17 (2000);Su el a/., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman el a/., J Uroi 1995 Aug 154(2 Pt 1):474-8).
In one aspect, the Invention provides methods for monitoring STEAP-1 gene products by determining the status of STEAP-1 gene products expressed by cells from an individual suspected of having a disease associated wtti.dysregulated __. cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of STEAP-1 gene products in a corresponding normal sample. The presence of aberrant STEAP-1 gene products in the test sample relative to the normal sample provides an Indication of the presence of dysregulated cell growth within the cells of the individual.
In another aspect, the invention provides assays useful in determining foe presence of cancer in an individual, comprising detecting a significant increase in STEAP-1 mRNA or p.otetn expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of STEAP-1 mRNA can, for example, be evaluated in fissues including but not limited to those listed in Table I. The presence of significant STEAP-1 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express STEAP-1 mRNA or express it at lower levels.
In a related embodiment, STEAP-1 status Is determined at the protein level rather thai at the nucleic add level. For exampte,fsuch a rr«^ comprises determining the tev^
. i %
compariig the level so determined to the level of STEAP-1 expressed to a conesponding normal sample, in one embodiment the presence of STEAP-1 protein is evaluated, for example, using immunohistochemical methods. STEAP-1 antibodies or binding partners capable of detecBng STEAP-1 protein expression are used ha variety of assay fonnabweflknc^m in ttw art for this purpose.
In a further embodiment, one can evaluate the status of STEAP-1 nudeofide and amino add sequences h a biological sample in order to identify perturbafons in the structure of these molecules. These petturtafions can include insertions, deletions, subsCtufions and the like. Such evaluations are useful because perturbafions in the nucteotide and amino add sequences ae observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et a/., 1999, J. Cutan. Pathol. 26(8)369-378}. For example, a mutation in the sequence of STEAP-1 may be indicaBve of the presence or promotion of a tumor. Such assays therefore have diagnostic arrfpiedk^ value wr^re a mutafon in STEAP-1 hdkates a potential loss of function or hcrease hi tumor growth.
A wide variety of assays for observing perturbafions in rHK^Sdearriarnho add sequence are wefi known in ttie art For example, the size and structure of nudefc add or amino acW sequences of STEAP-1 gene proclucts are observed Northern, Southern, Western, PCR and DM sequeremg protocols dtsoissed herein. In addition, other methods for observing perturbation in nucteofictearKlarnirw acid in (he art (see, e.g., U.S. Patent Nos. 5,382,510 Issued 7 September 1999, and 5,952,170 issued 17 January 1995).
Additionally, one can exatrine the methylalion status of a STEAP-1 gene in a biological sampte. Aberrant demethyla&marKl/orhypem^ylatta
transformed eels, and can result in altered expression of various genes. For example, promoter hyperrnehyfafion of to pt-dass glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently sOence transcripUon of this gene and is the most frequently detected genomfc alterafion in prostate

carcinomas (De Marco et at, Am. J. Patiwl. 155(6): 1985-1992 (1999)). In addition, this alteration is present hi at feast 70% of cases of high-grade jj«usiaiic iniraepithetial neoplasia (PIN) (Brooks et a/., Cancer Epidemioi. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate waicers) is induced by deoxy-azacyfidine In lymphoblastoid cells, suggesfing that tumorat expression is due to demethylaaon (Lethe et a/., Int J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining melhylation status of a gene are weH known in the art For example, one can utilize, in Southern hybridization , approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methyiation status of CpG islands, in addition, MSP (methyiaiion specific PCR) can rapidly profile the mettiylafion status of a« the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DMA by sodium bisulfite (which will convert all unmethylated cytosines to uracii) followed by amplification using primers specific for methylated versus unmethylated DMA. Protocol involving methyiaiion interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
Gene amplification is an additional method for assessing the status of STEAP-1. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quanfitate the transcription of mRNA (Thomas, 1980, Proc. Nad. Acad. Sci. USA, 77:5201-5205), dot blotting (DMA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DMA duplexes, RNA duplexes, and DMA-RNA hybrid duplexes or DMA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Biopsied Bssue or perpheral blood can be convenien try assayed for foe presence of cancer cefe using for example,
Norttiem, dot blot or RT-PCRanatysis to'deted STEAP-1 expression. The presence of RT-PCRampfifiable STEAP-1 mRNA
provides an indication of the presence of cancer. RT-PCR assays are wel known in the art RT^CR detection assays for tumor
cells in peripheral blood are currenfly being evaluated for use in Iheo^cisisandrnanagemertofanumberofhurnansolid
tumors. In fie prostate cancer field, toese include RT-PCR assays for the detection of ceils expressing PSA and PSI« (Verkatk et
a/., 1997, Uroi. Res. 25:373-384; Ghossein eiai, 1995, J. din. Oncd. 13:1195-2000; Heston et at, 1995, din. Chem. 41:1687-
1688). ;
Afurtr^aspedofltelnvenfonisanassessmentofth^ In
one embodiment, a method for predfcfing suscepfibility to canracorrpisesdetecfirg STEAP-1 rrf^ a fissue sample, its presence indkafingsuscepfibBay to cancer, wherein the degree of STEAP-1 mRNA expression correlates to the degree of susceptibity. In a specific embodiment, the presence of STEAP-1 In prostate or other issue fs examined, with tie presence of STEAP-1 in fte sampte providing an hdfcatfon of prostate car«^«jscep8)Ety (or treeme|gen orte to Wenfifyperturbafons in toe structured The
presence cf one or mcceperturbaforis in STEAP-1 gene prod^
emergence or existence of a tumor).
* The hvenfion also comprises methods for gauging tumor aggressiveness, In one ernboSrnent a metrKxl for gauging
aggressiveness of a tumor comprises detenriWng ^tevel of STEAP-1 mRNA wSTCAP-lprotw expressed by iumorcete,
comparing foe level so determined to toe level of STEAP-1 mRNA cc STEAP-1 proi^ expressed h a coniesporicAigiKmnal
tissue taken from Ihe same individual or a normal fissue referem*sampte,whereh fie degree of STEAP-1 rr^^
protein expresstonh the tumor sarnpterelafive to biaspecffic
embodiment, aggressiveness of a tumor is evaluated fay (fetermHng Ihe extent to wNdiSl&\M

with higher expression levels indicating more aggressive tumors. Another ernbocfiment is Ihe evaluation of the integrity of STEAP-
Inudeoflcte 3rd arrtfnoai& sequences in a bido^
suchasirwerfibns.ffefeCaB.sutetitutiorisandtheHce. The presence of one or more perfarbafioris hdkates more aggressws
tumors.
Another embodiment of the invention is directed to methods for ctserving the progressfon of a malignancy hi an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over fime comprise determining the level of STEAP-1 mRNAor STEAP-1 proNn expressed by The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art For example, another embodiment of the invention is Directed to methods for observing a coincidence between the expression of STEAP-1 gene and GTEAP-1 gene products (or perturbations in STEAP-1 gene and STEAP-1 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating Ihe status of a fissue sample. A wide variety of factors associated with malignancy can be utilized, such as tie expression of genes associated wffli malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer eta) as we! as gross cytotagka! observations (see, e.g., Becking ja/., 1984, Anal. Quant Cytd. 6{2):74-88; Epstein, 1995, Hum. Pathol. 26(2)^23-9; Thorson er a/., 1998, Mod. Patfiol. 11(6):543-51;Baisdene/a/, 1999, Am. J. Surg. Pattid. 23(8):916-24). Melhods (or observing a coincidence between the expression of STEAP-1 gene and STEAP-1 gene products (or perturbafcrvs in STEAP-1 gene and STEAP-1 gene products) and another fector fliat is associated with malignancy are useful, for example, beousetrw presence of a set of specifcfectors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
In one embodiment methods for observing a cofoddence between tie e>cpfiesstonc)f STEAP-1 o^Te ami STEAP-I gene products (or perturbations in STEAP-1 gene and STEAP-1 gene products) aralarwfoer facto assodated with entails detecfing the overexpression of STEAP-1 mRNA or protein in a fissue sanpte, detecfing fteoverexpressbn of PSA mRNA or protein in a fissue sample (or PSCA or PSM expression), and observing a coinddere» of STEAP-1 mRNA or proteh and PSA ,-aRNAor protein overexpression (or PSCA or PSM expression). In a specific embcdirrKnt, tie expressiori of STEAP-1 and PSA mRNA In prostate fissue is exanwied, where the coincidence of STHAP-1andPSAmRN.\overexpressJpnlnliesanvteindKafes the existence of prostate cancer, prostate cancer susoepGbflity or the enwiigerKje or status of a prostate lurr^
Methods for detecfing and quanfifytng the expression of STEAP-1 mRM or prc^ are o^scrbedhereh, and standard
nucleic add and protein detecttoi and quanGtotion technologies are well k^^ Standard methods for the detection
arxJquantHkafon of STEAM lechnicfJesusirgSTE^P-lpolyrurieotid
type detecfion methods, such as, for example, branched ONA,SISBA,TMA and the Ska. tnaspedfcemt»a«nent,semi-
quanfitafiveRT-PCR Is used to detect and quantify STEAP-1 mRNA expression. Any number of primers capable of ampiying
STEAP-1 can be used for the purpose, kxducBngb^ In a
specific emtocftrientporyckxTal a rt™^ an Jmmundiistochernlca! assay of biopsted tissue.

IX.) Identification of Molecules That tnte«rf With STEAP-1
The STEAP-1 protein and nucleic add sequences disdosed herein aDow a skBled artisan to identify proteins, small molecules and other agents that interact with STEAP-1, as well as pathways activated by STEAP-1 via any one of a variety of art accepted protocols. For example, one can utifize one of the so-cafled interaction trap systems (also referred to as the two-hybrid assay"). In such systems, molecules interact and reconstitute a banscription factor which directs expression of a reporter gene, whereupon the expression of the reporter gone is assayed. Oiher systems identify protein-protein interactions in vivo through reconstitufon of a eukaryofic transcriptional activator, see, e.g., U.S. Patent Nos. 5,955,280 issued 21 September 1999,5,925,523 issued 20 July 1999,5,846,722 issued 8 December 1998 and 6,004,746 issued 21 December 1999. Algorithms are also available in the art for genome-based predictions of protein fundon (see, e.g., Marcotte, ef a/., Nature 402:4 November 1999,83-86).
Alternatively one c?n screen peptide libraries to identify molecules that interact with STEAP-1 protein sequences. In such methods, peptides that bind to STEAP-1 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the STEAP-1 protein(s).
Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior Information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to Identify molecules that interact with STEAP-1 protein sequences are disdosed for example in U.S. Patent Nos. 5,723,286 Issued 3 March 1998 and 5,733.731 Issued 31 March 1998.
Alternatively, cell lines that express STEAP-1 are used to identify protein-protein hteracfions mediated by STEAP-1. Such Interactions can be examined using Immunopredpitatjon techniques (see, e.g., Handton B.J., ef a/. Bfochem. Bfoohys. Res. Commun. 1999,261:646-51). STEAP-1 protein can be immunoprecipHated from STEAP-1-expressing cell fines using anG-STEAP-1 antibodies. Alternatively, antibodies against His-tag can be used in a cell One engineered to express fusions of STEAP-1 and a His-tag (vectors mentioned above). The Immunopredjpitated complex can be examined for protein association by procedures such as Western blotting, ^S-methtonine labeling of proteins, protein „ jJcrosequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and tigands that interact with STEAP-1 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, Including molecules that interfere with STEAP-1 's cbility to mediate phosphorylation and de-phosphoryiaSon, interaction with DMA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesJs. Similarly, small molecules that modulate STEAP-1-related ion channel, protein pump, or cell communtcafion functions are identified and used to treat patients that have a cancer that expresses STEAP-1 (see, e.g., Hide, B., ionic Channels of Excitable Membranes 2* Ed., Sinauer Assoc., Sunderland, MA, 1992). Moreover, Bgands hat regulate STEAP-1 function can be Identified based on thelrabffity to bind STEAP-1 and activate a reporter construct Typicat methods are discussed for example in U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods for forming hybrid Bgands in which at least one ligand is a smaB molecule. In an illustrative embodiment, cells engineered to express a fusion protein of STEAP-1 and a DNA-binding protein are used to co-express a fusion protein of a hybrid flgand/smafl motecute and a cDNA fibrary transcriptional activator protein. TheceDs further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that ocxxirson^fflwhybnVifigand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and lie unknown smafl rnotecute or the unknown ligand is identified. This method provides a means of Identifying modulators, which activate or Inhibit STEAP-1.

An embodiment of this invention comprises a method of screening for a molecule that interacts with a STEAP-1 amino acid sequence shown hi Figure ? or Figure 3, comprising the step? of contacting a population of molecules with a STEAP-1 amino acid sequence, allowing the population of molecules and the STEAP-1 amino acid sequence to interact under conditions that facilitate an Interaction, determining the presence of a molecule that interacts with the STEAP-1 amino add sequence, and then separating molecules that do not Interact with the STEAP-1 amino acid sequence from molecules Biatdo. In a specific embodiment, the method further comprises purifying, charactertzmg and HentWymg a molecule fliat interacts with the STEAP-1 amino acid sequence. The identified molecule can be used to modulate a function performed by STEAP-1. In a preferred embodiment, the Sf EAP-1 amino acid sequence is contacted with a library of peph'des.
JQ Therapeutic Methods and Compositions
The identification of STEAP-1 as a protein that is normally expressed In a restricted set of (issues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.
Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ Is one that is necessary to sustain fife, such as the heart or colon. A non-vital oryan is one that can be removed whereupon the Individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.
For example, Hercepfin® is an FOA approved pharmaceutical that consists of an antibody which Is imrnunoreactive with the protein variously known as HER2, HER2/neu. and erb-b-2. It Is marketed by Genentech and has been a commercially successful antitumor agent Hercept'n® sales reached almost $400 million in 2001 Herceptin® is a treatment for HER2 positive metastab'c breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal fissues. in particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu In normal kidney is also confirmed by iafif, Z., et al., B.J. U. International (2002) 89:5-9. As shown to this article (which evaluated whether renal ceB carcinoma should be a preferred indication for anti-HER2 antibodies such as HercepGn) both protein and rriRNA are
produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue.
•;-.r • .
Despite the fact that HER2/neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, Le., 'cardiotoxiclty,' has merely been a side effect to treatment When patients were treated with Hercepfin alone, significant cardiotoxicfty occurred in a very low percentage of patients. To minimize cariotoxictty there !s a more stringent entry requirement for he treatment with
HER2/heu. Factors such as predisposition to heart condition are evaluated before treatment can occur.
»
Of particular note, although kidney tissue is indicated to exhibit normal expression, possfcly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of foe diverse array of normal tissues in which HER2 is expressed, there is very fittte occurrence of any skte effect Only cardiac tissue has manifested any appreciable side effect at afi. A tissue such as kidney, where HER2/neu expression is espedaiy notable, has not been the basis for any side effect
Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth factor receptor (EGFR); Erbitux (Imdone). EGFR is also expressed in numerous normal fissues. There have been very fimltedskte effects in normal fissues following use of anfi-EGFR therapeutics. A general skte effect mat occurs wffli the EGFR treatment is a severe skin rash observed in 100% of the pafents undergoing treatment .

1

Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat Hie utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed. For example, expression in vital organs is not in and of itself detrimental. In addition, organs regarded as dispensfcte, such as the prostate and ovary, can be removed without affecting mortality. Finally, some vital organs are not affected by normal organ expression because of an immunoprivilage. tnmunoprivilaged organs are organs fhat are protected from blood by a Wood-organ barrier and thus are not accessible to immunotherapy. Examples of immunoprivilaged organs are the brain and tesfis.
Accordingly, therapeutic approaches that inhibit the activity of a STEAP-1 protein are useful for patients suffering from a cancer that expresses STEAP-1. These therapeutic approaches generally faU Wo three classes. The first class modulates STEAP-1 function as it relates to tumor cell growth leading to inhibition or retardation of tumor cell growth or inducing its killing. The second class comprises various methods for inhibiting the binding or association of a STEAP-1 protein with its binding partner or with other proteins. The third dass comprises a variety of methods for inhibiting the transcription of a STEAP-1 gene or translation of STEAP-1 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a STEAP-1-related protein or STEAP-1-related nucleic acid. In view of the expression of STEAP-1, cancer vacdnes prevent andfor treat STEAP-1-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor anb'gen in a vaccine that generates cell-mediated humoral immune responses as anti-cancer therapy is wefl known in fce art and has been e immunogens (Hodge era/., 1995, bit J. Cancer 63:231-237; Fongef al, 1997, J. bnmunoL 159:3113*3117):
Such methods can be readily practiced by employing a STEAP-1-related protein, or a STEAP-1-«woding nuctefc acid molecule and recombinarit vector^ capable of expressing and presenting the STEAP-1 bnmunogen (which typically comprises a number of T-cell epitopes or antibody). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreacfive epitopes are known in the art (see, e.g., Heryin et a/., Ann Med 1999 Feb 31(1}:66-78; Maruyama et al., Cancer bnmunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods of generating an immune response (e.g. cell-mediated and/or humoral) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a STEAP-1 protein shown in Figure 3 or analog or homuiog thereof) so
>, *-
that the mamma! generates an immune response that is specific for fhat epitope (e.g. generates anfibodes (hat specifically recognize that epitope). In a preferred method, a STEAP-1 himunogen contains a biological motif, see e.g., Tables V-XVIII and XXII-LI, or a peptide of a size range from STEAP-1 indicated in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9.
The entire STEAP-1 protein, immunogenlc regions or epitopes thereof can be combined and delivered by various
means. Such vaccine compositions can include, for example, fipopepfides (e.g., Vlliello, A. et at, J. C&i Invest 95:341,
^
1995), peptide compositions encapsulated in poly(DL-lactide-co-glycoIide) ("PLG") microspheres (see, e.g., EUrtdge, et a/., Molec. Immunol. 28:287-294,1991: Alonso era/., Vaccine 12299-306,1994; Jones et a/., Vaccine 13:67^681,1995), peptJde compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et at, Mature 344:873-875.1990; Hu et al., CBn Exp Immunot. 113:235-243,1998), multiple anfjgen peptide systems (MAPs) (see e.g.. Tarn. J. P., flroc. Waft Mad. ScL U.SA 85:5409-5413,1988; Tam, J.P., J. ImmunoL Methods 196:17-32,1996), pepfides formulated as muifivalent peptides; pepGdes for use in ballistic delivery systems, typically crystallized pepfides, viral defvery vectors (Perkus, M. E eta/., In: Concepts In vaccine development, Kaufmann, S. H. E., ed., p. 379,1996; Chakrabarti, S. ef at, Nature 320:535,1986; Hu, S. Let at., Nature 320:537,1986; Kteny, M.-P. et at, AIDS BMrechnohgytm.1986; Top. F. H. etal., J. Infect Dis. 124:148,1971; Chanda, P. K. etal., Virology 175:535,1990), partjdes of viral w synthetic origin (e.g., Kofler, N. et a!.,J. ImmunoL Methods, 192:25,1996; Eklridge, J. H. ef at, Sem. HematoL 30:16.1993; Fafo. L D., Jr. er at,

Nature Med. 7:649,1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L A. Annu. Rev. Immunol. 4:369,1986; Gupia, R. K.etal.,Vacdne 11:293,1993), liposomes(Reddy,R.ef at., J.fcimunof. 148:1585,1992; Rock,K.L, /mrouno/. Today 17:131,1996), or, naked or particle absorbed cDNA (Ulmer, J. B. ef at, Sefence 259:1745,1993; Robinson, H. L, Hunt, L A., and Webster, R. G., Vaccine 11:957,1993; Shiver, J. W. ef af., In: Concepts in vaccine devetopmenf, Kaufmann, S. H. E, ed., p. 423,1996; Cease, K. B., and Berzofsky, J. A., Anna. Rev. ImmunoL 12:923,1994 and Eldridge, J H. ef at, $em. Hematot 30:16,1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant bnmunotherapeufics, Inc. (Needham, Massachusetts) may also be used.
In patients with STEAP-1-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.
Cellular Vaccines:
CTL epitopes can be determined using specific algorithms to idenfify peplides within STEAP-1 protein that bind corresponding HLA alteles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL brown.edu/ResearciVTB-HIVJ^^epimatrU^imatrix.html); and, BIMAS, (URL bimas.dcrtnih.gov/; SYFPEJTHI at URL syfpeithi.bmi-heidelberg.conV). In a preferred embodiment, a STEAP-1 knmunogen contains one or more amino acid sequences identified using techniques welt known in the art, such as tr,a sequences shown in Tables V-Xvlll and XXII-LI or a peptide of 8,9,10 or 11 amino acids specified by an HLA Cfass I molif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)} and/or a pepfide of at least 9 amino adds that comprises an HLA Class II motif/supermo" (e.g., Table IV (B) or Table IV (C)). As is appreciated in (he art, the HLA Class I binding groove Is essentially closed ended so that peptkJes of only a particular size range can fit into the groove and be bound, generally HLA Class I epilopes are 8,9,10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino adds can be bound by an HLA Class it molecule. Due to (he binding groove differences between HLA Class 1 and H, HLA Class I motifs are length specific, i.e., position two of a Class I motif is ihe second amino add In an amino to carboxyl direction of the peptide. The amino add positions in a Class tl motif are relative only to each other, not the overall peptide, i.e., additional amino adds can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class If epitopes are often 9,10,11,12,13, 14,15,16,17,18,19,20,21,22,23,24, ur 25 amino adds long, or longer than 25 amino adds.
A wide variety of methods for generating an immune response In a mammal are knew in the art (for example as the first step in the generafion of hybridomas). Methods of generafing an immune response in a mammal comprise exposing he mammal's immune system to an immunogenic epitope on a protein (e.g. a STEAP-1 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to STEAP-1 in a host, by contacting the host with a sufficient amount of at least one STEAP-1 B celt or cytotoxic T-ceH epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the STEAP-1 B cell or cytotoxic T-cefl epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a STEAP-1-refated protein or a man-made mulfiepitopic peptide comprising: administering STEAP-1 immunogen (e.g. a STEAP-1 protein or a peptide fragment thereof, a STEAP-1 fusion protein or analog etc.) in a vacdne preparation to a human or another mammal!' Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Patent No. 6,146,635) or a universal helper epitope such as a PADRE™ pepfide (Epimmune Inc., San Diego, CA; see, e.g., Alexander ef at, J. Immunol. 2000164(3); 164(3): 1625-1633; Alexander ef af., Immunity 19941(9): 751-761 and Alexander ef at, Immunoi. Res. 199818(2): 79-92). An alternative method comprises generafing an immune response in an hdrvklual against a STEAP-1 immunogen by. administering in vivo to musde or skin of the individual's body a DMA motecute that comprises a DMA sequence that encodes a STEAP-1 immunogen, Ihe DMA sequent* operalivelyBrfced to regulatory

sequent which ccmtrol toe expresswn of Ihe ON A sequence; wher^^^
sequence is expressed in toe cells and an immune response is generated against fie immunogen (see, e.g., U.S. Patent No. 5,962,428). Optionally a genetic vacdne facilitator such as antonfc fipWs; saponins; tecfins; estnogenic compounds; hydroxylated lower atkyis; dimethyl sulfoxide; and urea is also administered. In addition, an anffidiotypfc antibody can be administered that mimics STEAP-1, In order lo generate a response to the target antigen.
rVucfeic Acid Vaccines:
Vaccine compositions of Die invention include nucleic acid-mediated modalities. DMA or RNA that encode protein(s) of the invention can be administered to a patient Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing STEAP-1. Constructs comprising DNA encoding a STEAP-1 -related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded STEAP-1 protein/immunogen. Alternatively, a vaccine comprises a STEAP-1-related protein. Expression of the STEAP-1-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells (hat bear a STEAP-1 protein. Various prophylactic and therapeutic genetic Immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et »L, Science 247:1465 (1990) as wen as U.S. Patent No«. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DMA-based delivery technologies include "naked DMA', facilitated (buprvicalrie, polymers, peptide-mediated} delivery, cationte Epid complexes, and particle-mediated ('gene gun1) or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
For therapeutic or prophylactic Immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene deEtey systems foal can be used in the pracficeof the hvenfion Include, but are. not Irrtted to, vaccinia, fowlpox, canarypox, adenovlrus, Influenza, poliovirus, adeno-assoa'aled virus, lenCvirus, and sindbts virus (see, e.g., Restrfb, 1996, Curr. Optn. kranunoL 8:658-663; Tsang etal. J.Nafl. Cancer Inst 87:982-990 (1995)). Non-vral delivery systems
f f > .
can also be employed by infroducfog naked DNA encoding a STEAP-1-related protein into Ihe patient (e.y.,inIrarmisculanV or • htradermafly) to induce an anti-tumor response.
Vaccinia virus is used, for example, as a vector to express nudeofide sequences that encode the pepfides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein knmunogenic pepfide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in Immunization protocols are described in, e.g., U.S. Patent No. 4,722,848. Another vector is BC6 (Bacflle Calmette Guerin). BCG vectors are described in Stover ef a/., A/aft/re 351:456-460 (1991). A wide variety of other vectors useful for therapeufic administration or knmunizaBon of the peptkJesoftteinventk)n,e.g.adenoarKJaderK>-ass^^ detoxified anthrax toxin vectors, and Ihe like, will be apparent to (hose sklUed in the art from the description herein.
Thus, gene delivery systems are used to deEver a STEAP-1-retet^nudefcacWiTKjtecufe. honeetnbodiment,triefuB-tengtti human STEAP-1 cDNA is employed. In another embedment STEAP-1 nucleic add nrctecutes encoding specfccytotoxfc Tlymphoc^(CTL)andi'oranffiKxlyepitQpesareempto
Ex Vivo Vaccines
Various ex vAostrategies can also be employed to generate an immune response. One approach involves the use of antigen presenfing ceils (APCs) such as dendrifccells (DC) to present STEAP-1 anfigen to a patients iniruie system. DerxJrffic ceHs express MHC dass I and II molecules, B7 co-sGmulator, and L-12, and are ftushio^yspeolaSzBd arisen prwenfingceb. In prostate cancer, autologous dendritic cells pulsed with peptktes of the prostate-spedfic membrane anfigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate
69; Murphy Bl a/., 1996, Prostate 29:371-380). Thus, dendritic cells can be usod to present STEAP-1 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with STEAP-1 pepOdes capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic ceils are pulsed with the complete STEAP-1 protein. Yet another embodiment Involves engineering the overexpressfon of a STEAP-1 gene in . dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur ef a/., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et a/., 1996, Cancer Res. 56:3763-3770}, lenlivirus, adeno-assodated virus, DMA transfecfion (Ribas et a/., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et at., 1997, J. Exp. Med. 186:1177-1182). Cells that express STEAP-1 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) STEAP-1 as a Target for Antibody-based Therapy
STEAP-1 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and inlracellular molecules (see, e.g., complement and ADCC mediated killing as wefl as the use of intrabodies). Because STEAP-1 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of STEAP-1-imnuinoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused .by binding of the immurtoreacfive composition to non-target organs and tissues. Antibodies specifically reactive with domains of STEAP-1 are useful to treat STEAP-1-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibifing cell proliferation or function.
i1 STEAP-1 antibodies can be introduced into a patient such that the antibody binds to STEAP-1 and modulates a
function, such as an Interaction with a binding partner, and consequently mediates destruction of the tumor ceBs and/or
inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include
complement-mediated cytofysis, antibody-dependent cellular cytotoxidty, modulation of the physiological function of STEAP-
1, Inhibition of ligand binding or signal transduction pathways, modulation of tumor ceil differentiation, alteration of tumor
angiogenests factor profiles, and/or apoptosis. Examples include Rituxan® for Non-Hodgkins Lymphoma, Hercepfin® for
metastatic breast cancer, and Erbitux® for colorectal cancer. . f
Those skilled in the art understand that antibodies can be used to specifically target and bind knmunogenic molecules such as an immunogenic region of a STEAP-1 sequence shown In Figure 2 or Figure 3. to adilion, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Stevers ef A Blood 93:113678-3684 (June 1,1999)). When cytotoxic and/or therapeutic agents are delivered directly to cefls, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. STEAP-1), the cytotoxic agent wffl exert Hs known biological effect (i.e. cytotoxkaty} on those cells.
A wide variety of compositions and methods tor using antibody-cvtotoxfc agent conjugates to kfll cefls are known in the art In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targefing agent (e.g. an anfi-STEAP-1 antibody) that binds to a marker (e.g. STEAP-1) expressed, accessible to binding or tocafized on the cefl surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a eel expressing STEAP-1, comprising conjugating the cytotoxic agent to an antibody that immunospedfically binds to a STEAP-1 epitope, and, exposing the ceB to the antibody-agent conjugate. Another Ikistrafive embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenteraBy to said individual a

pharmaceutical composition comprising a therapeub'cally effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent
Cancer immunoBierapy using anti-STEAP-1 antibodies car. be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arien et el, 1998, Crit Rev. Immunol. 18:133-138), multiple myeloma (OzaW et at, 1997, Blood 90:3179-3186, Tsunenari ef al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et at, 1992, Cancer Res. 52:2771-2776), B-ceB lymphoma (Funakoshi et at., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong etai, 1996, Leuk. Res. 20:581-589), colorecta) cancer (Moun ef a/., 1994, Cancer Res. 54:6160-6166; Vetders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et a/., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or racfioisotope, such as the conjugation of Y91 or I131 to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals) respectively, while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzuMAb) with paditaxe! (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent To treat prostate cancer, for example, STEAP-1 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamictn (e.g., Mylotarg™, Wyem-Ayerst, Madison, NJ, a recombinant humanized lgG4 kappa antibody conjugated to antitumor an'Jbiolic caticheamidn) or a maytanslnokl (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA, also see e.g., US Patent 5,416,064) or Auristab'n E (Geattle Genefics).
Although STEAP-1 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastafic cancers. Treatment with the anfibody therapy of the Invention is Indicated for patients who have received one or more rounds^f chemotherapy. Alternatively, antibody therapy of the in venfion is combined with a chemotherapeutic or radiafion regimen for patients who have not received chemoflierapeufic treatment Additionally, anSbody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxidty of the chemotherapeutic agent very weft. Fan et al. (Cancer Res. 53:4637-4642,1993), Prewett et al. (International J. of Onco. 9:217-224.1996), and Hancock et at. (Cancer Res. 51:4575-4580,1991) describe the use of various antibodies together with chemotherapeutic agents.
Although STEAP-1 antibody therapy is • >seful for all stages of cancer, antibody therapy can be particularly aporopriate in advanced or metastafic cancers. Treatment with the antibody therapy of the invention is indicated for parents who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention Is combined with a chemotherapeutic or radiafion regimen for patients who have not received chemotherapeutic treatment Addifionally,
antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not
» tolerate the toxidty of the chemotherapeutic agent very well.
Cancer patients can be evaluated for the presence and level of STEAP-1 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative STEAP-1 imaging, or other techniques hat reliably indicate the presence and degree of STEAP-1 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose, Mettwxis for immunohlstafcemk^ analysis of tun^
Anfi-STEAP-1 monoclonal antibodies that treat prostate and other cancers include those hat initiate a potent immune response against the tumor or those that are directly cytotoxic. hi this regard, anti-STEAP-1 monoclonal antibodies (MAbs) can elicit tumor cefl lysis by either complement-mediated or antibody-dependent cell cytotoxidty (ADCC) mechanisms, both of which require an intact Fc portion of (he irrmuinoglobulln molecule for interaction wHh effector ceBFc receptor sites on complement proteins. In addition, anfi-STEAP-1 MAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express STEAP-1. Mechanisms by which directly cytotoxic MAbs act include: inhfiRton of cefl

growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a parficufar anfi-STEAF-i iviAb o/erts an anfi-tumor effect is evaluaied using any number of In vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the ait
In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric MAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response jean lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target STEAP-1 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-STEAP-1 MAbs as wed as combinations, or cocktails, of different MAbs. Such MAb cocktails can have certain advantages inasmuch as they contain MAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic MAbs with MAbs tttat rely on immune effector functionality. Such MAbs in combination can exhibit synergistic therapeutic effects. In addition, anfi-STEAP-1 MAbs can be administered concomitantiy with other therapeutic modalities, Including but not limited to various chemotherapeutic agents, androgen-btockers, immune modulators (e.g., IL-2, GM-Cr^), surgery or radiation. The anti-STEAP-1 MAbs are administered in their "naked* or unconjugated form, or can have a therapeutic agent(s) conjugated to them,
Anti-STEAP-1 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anB-STEAP-1 antibody preparation, via an acceptable route of administration such as intravenous injection ((V), typically at a dose 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 mgAg bodyweight In ^::eral, doses In the range of 10-1000 mg MAb per week are effective and weB tolerated.
Based on clinical experience with the Herceptin™ MAb in the treatment of melastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anfi-STEAP-1 MAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose Is administered as a 90-minute or longer infusion. The periodic maintenance dose Is administered as a 30 minute or longer infusion, provided he initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors hdude, for example, ihe binding affinity and half life of the Ab or MAbs used, Ihe degree of STEAP-1 expression in the patent, the extent of circulating shed STEAP-1 antigen, the desired steady-state antibody concentration level, frequency of teatment, and the influence of chemoBierapeutic or other agents used in combination with (he treatment method of the invention, as well as (he health status of a particular patient
Optionally, patients should be evaluated for (he levels of STEAP-1 in a given sample (e.g. the levels of circulating STEAP-1 antigen and/or STEAP-1 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels to bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
AnHdiotypic anB-STEAP-1 antibodies can also be used in anti-cancer therapy as a vaccine for Inducing an immune response to cells expressing a STEAP-1-felated protein. In particular, (he generation of anfi-kfiotypic anSbodfes is weB known in the art; this methodology can readily be adapted to generate anWdfotypfc anti-STEAP-1 antibodies that mimic

t
/

an epitope on a STEAP-1-related protein {see, for example, Wagner et a/., 1997, Hybridoma 16:33-40; Foon et a/., 1995, J. Clin. Invest 96:334-342; Herlyn ef a/., 1996, Cancer Immuno!. Immunotner. 43:65-76). Such an antHdfotypjc antibody can be used in cancer vaccine strategies.
X.C.) STEAP-1 as a Target for Cellular Immune Responses
Vacdnes and methods of preparing vaccines that contain an ImmunogenicaHy effective amount of one or more HLA-Wnding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with Bie invention encompass compositions of one or more of the daimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various pepfides. Polymers have the advantage of increased immundogical reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantiy or by chemical synthesis.
Carriers that can be used with vaccines of the invention are well known bt the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino adds such as poly my sine, poly L-g!utamic acid, Influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable {/.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant Adjuvants such as Incomplete Freunrfs adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials; well known in the art Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripafrnitoyVS-glycerylcysteinlyseryl- serine (PaCSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothtolated-guanine-containing (CpG) oligonudeoBdes has been found to Increase CTL responses 10- to 100-fold, (see, e.g. Davfla and Celis, J. Immunol. 165:539-547 (2000)}
Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially «nmune to later development of cells that express or overexpress STEAP-1 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
In some embodiments, it may be desirable to combine the class I peptide components with components ftat induce or facilitate neutralizing anfibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the Invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, CA) molecule (described e.g., In U.S. Patent Number 5,736,142).
A vactine*of the invention can also include antigen-presenting ceils (ARC), such as dendritic ceHs (DC), as a vehicle to present peptides of (he InvenGon. Vacdriecomposifions can be created irJvto.fclcw^derKiriticceB mobilization and harvesting, whereby loading of dendritic cells occurs in vftro. For example, dendritic ceb are transfected, e.g., with a minigene in accordance wfflr the invention, or are pulsed with pepfides. The dendritic cell can then be administered to a patient to eficitimmune responses to vivo. Vaccine compositions, either DNA- or pepfide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.

Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a potyepltoplc composition for use in a vaca'p*. or for setecfing discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the Mowing principles be balanced Tn order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous In sequence In the native antigen from which the epitopes are derived.
• 1.} Epitopes are selected which, upon administration, mimic immune responses mat have been observed to " be correlated with tumor clearance. For HLA Class I this indudes 34 epitopes that come from at teast'one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 34 epitopes are selected from at least one TAA (see, e.g., Rosenberg ef a/., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenlcity. for HLA Class I an ICso of 500 nM or less, often 200 nM or less; and for Class It an ICso of 1000 nM or less.
3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
4.) When selecting epitopes from cancer-related antigens It is often useful to select analogs because the patient may have developed tolerance to the native epitope.
5.) Of particular relevance are epitopes referred to as "nested epitopes." Nested epitopes occur where at least two epitopes overlap In a given pep^de sequence. A nested peptide sequence can comprise B ceo, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect Is to avoid providing a peptide that 3s any longer than the amino terminus of the amlno terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in (he peptide. When provkfing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence In order to insure that it does not have pathological or other deleterious biological properties.
6.) If a polyepitopic protein is created, or when creating a minigene/an objective is to generate the smallest peptide that encompass the epitopes of interest This principle is similar, if not the same as hat employed when selecting a peptide comprising nested epitopes. However, With an artificial polyepitopic peptide, the size minimization objective is balanced against the need to Integrate any spacer sequences between epitopes in the potyepitoptc protein. Spacer amino acid residues can, for example, be introduced to avoid junctionai epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Juncfional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a juncfonal epitope that is a 'dominant epitope.' A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
7.) Where the sequences of multiple variants of the same target protein are present potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA dass I binding peptide or the entire 9-mer core of a class II bhdngpepfide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.

t
*

XC.1. Minigene Vaccines
A number of different approaches are available which allow simultaneous delivery of multiple epitcpes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the Invention uses minigene constructs encoding a pepBde comprising one or multiple epitopes of the invention.;
The use of multi-epitope minigenes is described below and in, Ishioka eta!., J. Immunol. 162:3915-3925,1999; An, L and Whitton, J. L, J. Virol. 71:2292,1997; Thomson, S. A. ef a/., J. lirmunol. 157:822,1996; Whitton, J. L ef a/., J. Virol. 67:348,1993; Hanke, R. ef a/., Vaccine 16:426,1998. For example, a mulb'-epitope DNA plasmid encoding supermofif-
- .. *
and/or motif-bearing epitopes derived STEAP-1, the PADRE® universal helper T cell epitope or multiple HTL epitopes from STEAP-1 (see e.g., Tables V-XVIII and XXII to LI), and an endoplasmic reh'culum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DMA-encoded epitopes in vivo can be correlated with the /n vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized ceils expressing the encoded epitopes.
For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression, in human cells, the amlno add sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon tjhoice for each amlno acid. Th^ese epitope-encoding DMA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitinatjon signal sequence, and/or an endoplasmic reticulu-, targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
The minigene sequence may be converted to DNA by assembling ofigonudeotides that encode the plus and minus strands of the minigene. Overlapping ofigonudeotides (30-100 bases long) may be synthesized, phosphoryiated, purified and annealed under appropriate conditions using well known techniques. The ends of the ofigonudeofides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope pdypeptide, can then be cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are preferably hduded in the vector to ensure expression in the target cells. Several vedor elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenyiatjon signal for efficient transcription termination; an £ coff origin of replication; and an £ co//selectable marker (e.g. arnpicillin or kanamydn resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Patent No*. 5,580,859 and 5,589,466 for other suitable promoter sequences.
Additional vector modifications may be desired to optimize minigene expression and immunogenicity. in some
cases, folrons are required for efficient gene expression, and one or more synthetic wnaturaByKiocurnngintrons could be
incorporated into the transcribed region of the minigene. The inclusion of mRNAstabffization sequences and sequences for
replication in mammalian cells may also be considered for increasing minigene expression. -.

Once an expression vector Is selected, the minigene is cloned into the pdylinker region downstream of the promoter. This plasmid is transformed into an appropriate £ co//strain, and DMA is prepared using standard techniques. The orientation and DMA sequence of Bie minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
In addition, immunosfimulatory sequences (ISSs or CpGs) appear to play, a role in the Immunogenicity of DNA -vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to ennance immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, CA). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby Improving HTL induction. In contrast lo HTL or CTL induction, specifically deorecsing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-p) may be beneficial In certain diseases.
Therapeutic quantities of plasmid DNA can be produced for example, by fermentafion in £ coff, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or i bforeactor according to weMmown techniques. Plasmid DNA can be purified using standard Woseparatjon technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitufion of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as 'naked DNA,' is currently being used for intramuscular (IM) administration in clinic9' trials. To maximize the immunotherapeufic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cafonic lipids, glycolipids, and fusogenio liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BtoTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, et a/., Proc. NattAcad. Sd. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to Influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensifeafjon can be used as a functional assay for expression and HLA class I presentation of
minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cen fine ttiat is suitable as
a target for standardCTL chromium release assays. The transfecfion method used win be dependent on foe final
formulation. Electroporafion can be used for 'naked* DNA, whereas catkxiic lipkls allow direct In vitro transfecfion. A '
plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected ceBs using
fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target ceBs for
epitope-specific CTL fines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of,
minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to
assess HTL activity. ~
41

'**"'• * In vivo immunogenidty is a second approach for functional testing of minigene DNA formulafions. Transgenic mice
expressing appropriate human I !LA proteins are immunized with the DNA product The dose and route of administration are
formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipld-complexed DNA). Twenty-one days after
immunization, splenocytes are harvested and resGmulated for one week hi the presence of pepttdes encoding each epitope
being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptkJe-toaded, "Cr-labeled target
cells using standard techniques. Lysis of target cells that were sensitized by KLA loaded with peptkie epitopes,
; ; corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs.
Immunogenidty of HTL epitopes is confirmed in transgenic mice in an analogous manner.
Alternatively, the nucleic adds can be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.
Minigenes can also be delivered using other bacterial or viral delivery systems well known In the art, e.g., an expression construct encoding epitopes of the Invention can be incorporated into a viral vector such as vaccinia.
X.C.2. Combinations of CTL Peptides with Helper Peptides
Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenidty.
For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contain at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively smaD, neutral molecules, such as amino acids or amino add mimefics, which are substantially uncharged under physiddg^al conditions. The spacers are typically selected from, e.g., Ala, G!y, or ofter neutral spacers of nonpolar amino adds or neutral polar amino adds. It will be understood that the opGonally present spacer need not be comprised of the same residues and thus may be a hctero- or nomo-ofigomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic pepfide or the T helper peptide may be acylated.
| hi certain embodiments, the T helper pepfide is one that Is recognized by T helper ceBs present In a majority of a
! genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or ad of the HLA
| dass II molecules. Examples of such amino acid bind many HLA Class II^^nxrfea^irKiudeseo^erwesiromao^ensaich
1 as tetanus toxoid at positions 830-843 QYIKAMSKFIGITE; (SEQ ID NO: 64), Piasmcf. protein at positions 378-398 DIEKKIAKMEKASSVFNWNS; (SEQ ID NO: 65), and Streptococcus 18kD protein at positions 116-131GAVDSILGGVATYGAA; (SEQ ID NO: 66). Other examples indude peptides bearing a DR14-7 supermofif, or either of the DR3 motifs.
Alternatively, it is possible to prepare synthetic pepGdes capable of stimulating T helper lymphocytes, h a looser/ HLA-restricted fashion, using amino add sequences not found in nature (see, e.g., PCT puUcafon WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, CA) are designed, most preferably, to bind most HLA-OR (human HLA dass II) molecules. For instance, a pan-DR-bfodsng epffope pepUe having theformjla:XKXVMWTLKAAX(SEQIDNO: 67), where X is either cycbhexvlalar^ either o-alanine or L-alanine, has been found to bind to mostHLA-DRalteles, and to stimulate the response of T helper

lymphocytes from most ftvdividuals, regardless of their HLA type. Anal^afiveofaparhORbkidingepitopecomfm'sesan V natural amfno adds and can be provided in the form of nucleic acids that encode foe epitope.
HTL peptide epitopes can also be modified to after Ihesr biological properties. For example, they can be modified to include D-atnino acids la increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other moteaiies such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper pepfide can be conjugated to one or more palmitic acid chains at either the amino or carboxyt termini.
XC.3. Combinations of CTLPeptides with T Cell Priming Agents
In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes, Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic add residues can be attached to the e-and a- amino groups of a lysine residue and then finked, e.g., via one or more Dnking residues such as Gly, Gty-Gly-, Ser, Ser-Ser, or the like, to an Immunogenfe pepfide. The fipWafed pepSde can then be administered either directly in a micelle or particle, incorporated Into a liposome, or emulsified in an adjuvant, e.g., incomplete Freunrfs adjuvant In a preferred embodiment, a particularly effective fenmunogenic composition comprises palmitic add attached to e- and a- amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the fmmunogenic pepfide.
As another example of lipid priming of CTL responses, £ coff fipoprcJofo*. such as tripafmitoyl-S-• gtyoerytcysteEnlyoeryf- serirte (PsCSS) can be used to prime virus specific CTL when covalenBy attached to an appropriate pepfide (see, e.g., Deres, ef a!a Nature 342:561,1989). Pepfides of the invenfion can be coupled to PaCSS, for example, and the Gpopapfide administered to an hvfvidual to prime spedficafly en immune response to ttte target antigen. Moreover, because tfte induction of neutralizing anybodies can also be primed with PsCSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and celt-mediated responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Pepttdes
An embodiment of a vaccine composition In accordance with the Invention comprises ex vivo administration of a cocktail of epitope-baaring pepfa'des to PBMC, or isolated DC therefrom, from flte paSenFs blood. A pharmaceutical to facilitate harvesting of DC can be used, such as P"vjenipoiefin™ (Pharmada-Monsanto, SL Louis, MO) or GM-CSF/IL-4. After pulsing the DC with pepfides and prior to reinfusion kilo patients, (he DC are washed to remove unbound pepttdes. In th?? embodiment, a vaccine comprises pepRde-pulsed DCs which present the pulsed pepfide epitopes complexed with HLA molecules on their surfaces.
TheDCranbeptifeedexvtow'thacocktaHofpepGdesrs^^
Optionally, a helper T ceO (HTL) peptide, such as a natural or artificial loosely restricted HLA Class tl peptide, can be Included to facilitate the CTL response. Thus, a vaccine in accordance with the fcwenSon is used toVeat a cancer which expresses or overexpresses STEAP-1.
XD. Adoptive immunotherapy
Anfigenfc STEAP-1-related pepfides are used to eDcft a CTL and/or HTL response ex vivo, as weB. The resulting
CTL or HTL cells, can be used to treat tumors in paBents thatdo not respwd to ofter conventional forms of fterapy, orwiB
rwtrespcfldtoatterapeufcvacdnepeptite BtiAoCTLorHTL
responses to a parBcular antigen are induced by incubating in tissue culture the paSenfs, or genetically compatible, CTL or HTL precursor cefe together with a source ofanfigen-presenfing ceEs (ARC), such as derrfrfe cells, and Irieeippropriate hnmunogenic peptide. After an appropriate incubaSon time (typically about 7-28 days), in which tie precursor ceBs are activated and expanded Into effector cete, the cefls are infused back Into ftepaOenL where they wffl destroy (CTL) or

facilitate destruction (HTL) of their specific target cell (e.g., a tumor celO- Transfected dendritic cells may also be used as antigen presenting cells.
ftp Administration of Vaccines for Therapeutic or Prophylactic Purposes
Pharmaceutical and vaccine compositions of the Invention are typically used to treat and/or prevent a cancer that expresses or overexpresses STEAP-1. lij therapeutic applications, peptide and/or nucleic add compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or stow symptoms and/or complications. An amount adequate to accomplish this is defined as Therapeutically effective dose." Amounts effective for this use will depend on. e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
For pharmaceutical compositions, the immunogenic peptides of the invention, or ONA encoding them, are generally administered to an individual already bearing a tumor that expresses STEAP-1. The peptides or DNA encoding them can be administered Individually or as fusions of one or more pepfjde sequences. Patients can be treated with foe immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
For therapeutic use, administration should generally begin at the first diagnosis of STEAP-1-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, pdyepitopic polypeptldes, minigenes, or TAA-spedfic CTLs or pulsed dendritic cells) delivered to toe patient may vary according to Vie stage of the disease or the patient's health status. For example, In a patient with a tumor that expresses STEAP-1, a vaccine comprising STEAp-1-specific CTL may be more efficacious in killing tumor cells in patient with' advanced disease than alternative embodiments.
It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a oytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
The dosage for an initial therapeutic Immunization generally occurs in « unit dosage range where the lower value Is about 1,5,50,500, or 1,000 pg and the higher value is about 10,000; 20,000; 30,000; or 50,000 pg. Dosage values for a human typically range from about 500 pg to about 50,000 pg per 70 kilogram patient Boosting dosages of between about 1.0 ng to about 50,000 ng of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that toe neoplasla, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known In the ait
In certain embodiments, the peptides and compositions of the present Invention are employed in serious disease states, that is, fife-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of Ihe pepfides in preferred compositions of Vie invention, ft is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions
The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally Ihe dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1,5,50,500, or 1000 pg and the higher value is about 10,000; 20,000; 30,000; or 50,000 pg. Dosage values for a human typical/ range


from about 500 pg to about 50,000 pg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 ng to about 50,000 pg of peptide administered at defined intervals from about four weeks to six months after thft initial administration of vaccine. The bnmunogenicity of the vaccine can be assessed by measuring the specific activity of CTL. and HTL obtained from a sample of the patient's blood.
The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, fotrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are
administered parentafly, e,g., intravenously, subcutaneously, totradermslly, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a soluBon of the immunogenic pepfides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glydne, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophifized, the lyophilized preparation being combined with a sterile solution prior to administration.
The compositions may contain pharmaceutioally acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, lonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactale, sodium chloride, potassium chloride, calcium chloride, sorbttan monolaurate, triethanolamine oleate, etc.
The concentraHon of peptides of the invention In flie pharmaceufical formulations can vary widely, ia, from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and wi be selected primarily by fluid volumes, viscosities, efc.t in accordance with the particular mode of administration selected.
A human unit dose form otfc composition is typically Included In a phairoaeeuficalajmposftktt that comprises a human unit dose of an acceptable carrier, In one embodiment an aqueous carrier, and is administered in a vofume/quanfity that is known by Ihose of skfllin the art to be used for administration of sumpositk)(w to humans (see, 6.51., Remtagton's Pharmaceutical Sciences, 1?"- Edition. A. Gennaro, Editor, Mack Publishing Co., Easlon, Pennsylvania, 1985}. For example a pepfide dose for initial immunization can be from about 1 to about 50,000 pg, generally 100-5,000 fig, for a 70 kg patient For example, for nucleic adds an Initial Immunization may be performed using an expression vector in the form of naked nucleic add administered IM (or SO or ID) In the amounts of 0.5-5 mg at multiple sites. The nucleic add (0.1 to 1000 \ig) can also be administered using a gene gun. Following an Incubafion period of 3-4 weeks, a booster dose » then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5x109 pfu.
For antibodies, a treatment generally involves repeated administration of (he anfr-STEAP-1 anfibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dqse in the range of about 0.1 to about 10 mg/kg body weight In general, doses hi therange of 10-500 mgMAb per week are effecfive and wefl tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anfi- STEAP-1 MAfa preparation represents an acceptable dosing regimen. As appreciated by those of skfll In the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half tie of a composition, (he binding affinity or an Ab, the Immunogenidty of a substance, the degree of STEAP-1 expression in the patient, the extent of circulating shed STEAP-1 anfigen, the desired steady-state cwwentra^ level, l^tjency of treatment, and the Influence of chemofherapeutic or other agents used in combination with Ihe treatment method of he invention, as well as (he healtti status of a particular patient Non-limiting preferred human unit doses are. lor example, SOOug - 1mg, 1mg -50mg,50mg-10()mgl1(){)mg-200nigl200mg-3W)mg,40^-50 BOPmg, 800mg-900mg, 900mg - 1g, or 1mg-700mg. In certain embodiments, flie dose is in a range of 2-5 mg/kg body

I

weight, e.g., with (bliow on weekly doses of 1-3 mg/kg; O.Smg, 1,2,3,4,5,6,7,8,9,10mg/kg body weight followed, e.g., in two, (hree or four weeks by weekly doses; 0.5 - 10mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225,250,275,300,325,350,375,400mg ro* of body area weekly; 1 -600mg m2 of body area weekly; 225400mg m2 of body area weekly; these does can be followed by weekly doses for 2,3,4,5,6,7,8,9,19,11,12 or more weeks.
In one embodiment, human unit dose forms of pdynudeotides comprise a suitable dosage range or effective amount that provides any therapeutic effect As appreciated by one of ordinary skill In (he art a therapeutic effect depends on a number of factors, including the sequence of the polynudeotide, molecular weight of the polynudeotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a ' variety of parameters known in the art, such as severity of symptoms, history of (he patient and the like. Generally, for a polynudeotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1,0.25,0.5,1,2,5,10,20,30,40,50,60,70,80,90,100,200,300,400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60,80,100,200,300,400,500,750,1000,1500, 2000,3000,4000,5000,6000,7000,8000,9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynudeoiide compared to more direct application to the nudeolida to diseased tissue, as do polynudeotides of increasing length.
In one embodiment, human unit dose fo"ns of T-cefls comprise a suitable dosage range or effective amount that provides any therapeutic effect As appredated by one of ordinary skin In (he art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the Hke. A dose may be about 104 cells to about 10* cells, about 10* cells to about 10» cetfs, about 10« to about lOHceOs, or about 10»toabout5x10"cells. A dose may also about 10s cetls/m2 to about 1010 cells/m2, or about 10s ceNs/m2 to about 10* cells/m2.
Proteins(s) of the Invention, and/or nudefc adds encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular (issue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluhift monolavers, liquid crystals, phospholipid dispersions, lamellar layers and the Eke. In these preparations, (he peptide to be defivered is incorporated as part of a iposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 anfigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invenfon can be directed to the site of lymphoid cells, where (he liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phosphoGpids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, 6.5., Iposome size, add lability and stabHityof the Bposomes In the Wood stream. A variety of methods are available for preparing fposomes, as described In, e.g., Szoka, et at., Ann. Rev. Biophys, Bheng. 9:467 (I960), and U.S. Patent Nos. 4,235,871,4,501,728,4,837,028, and 5,019,369.
For targeting cells of (he immune system, a Ogand to be incorporated into (he fiposome can indude, e.g., antibodies or fragments thereof spedfic for cell surface determinants of (he desired immune system ceGs. A fiposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, infer afia, the manner of administration, (he peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxfc soBd carriers may be used which fodude, for example,
• pnarniaceuiioal grades of mannitol, lactose, stanch, magnesium stearate, sodiurii saccharin, talcum, ceButose, glucose,.
sucrose, magnesium catbonate, and the like. For oral administration, a pharmaceuficaliy acceptable nontoxte composition is formed by incorporating any of the normally employed exdpients, such as those carriers previously listed, and generaBy 10-95% of active Ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant Typical percentages of pepfides are about 0.01%-20% by weight, preferably about 1%-10%. .The' surfactant must, of course, be nonloxic, and preferably soluble in the propellant Representative of such agents are Ihe F- -««, a .. ^esters or partial esters of fatty adds containing from about 6 to 22 carbon atoms, such ascaprofc, octanoic, lauric, palmitic, stearic, finoleic, linolenic, olesteric and oleic adds with an aliphatic polyhydric alcohol or its cydic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1 %-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant A carrier can also be induded, as desired, as with, e.g., lecithin for intranasal delivery.
ff.) Diagnostic and Prognostic Embodiments of STEAP-1.
As disclosed herein, STEAP-1 pdynudeotides, polypeptides, reactive cytotoxic T ceBs (CTL), reacfive helper T cells (HTL) and anti-polypepfide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions assodated with dysregulated cell growth such as cancer, in parficuiar the cancers fisted in Table I (see, e.g., both its specific pattern of fissue expression as well as its overexpression in certain cancers as described for example In the Example entitled 'Expression analysis of STEAP-1 in normal tissues, and patient specimens"}.
STEAP-1 can be analogized to a prostate assodated antigen PSA, fte archetypal marker tirat has been used by medical practitioners for years to Identify and monitor the presence of prostate cancer (see, e.g., Merrill ef a/., J. UroL 163(2): / 503-5120 (2000); Pctescik ef a/., J. Uro!. Aug; 162(2):293-306 (1999) and Forfier ef a/., J. Nat Cancer hist 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in simHar contexts kiduding p53 and K-ras (see, e.g., Tulchinsky et a/., int J Mol Med 1999 Jul 4(1):99-102 and Minirnoto ef a/., Cancer Detect Prev 2000;24{1):1-12). Therefore, (his disclosure of STEAP-1 polynudeotides and polypepfides (as well as STEAP-1 polynudeofide probes and anti-STEAP-1 antibodies used to Identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules In methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions assodated with cancer.
Typical embodiments of diagnostic methods which ufflke the STEAP-1 poiynudeofides, polypepfides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynudeotides, polypeptides, reactive T cells and antibodies. For example, Just as PSA potynudeotides are used as probes (for example In Northern analysts, see, e.g., Sharief ef a/., Biochem. Mol. But Int 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa ef a/., J. UroL 163(4): 1189-1190 (2000)) to observe (he presence and/or the
level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the STEAP-1
* __
polynudeotides described herein can be utilized In the same way to detect STEAP-1 overexpression or the metastasis of
prostate and other cancers expressing this gene. Alternatively, just as PSA polypepfides are used to generate antibodies specific for PSA which can fcen be used to observe fte presence and/or Ihe level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan ef a/., Urology 55(4}:560-3 (2000)) or the metastasis of prostate eels (see, e.g., Alanen ef a/., Pathol. Res. Pract 192{3):233-7 (1996}), the STEAP-1 polypepOdes described herein can be utilized to

generate antibodies for use in detecting STEAP-1 overexpresston of the metastasis of prostate cells and cells of other cancers expressing this gene.
Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing STEAP-1 polynudeotices and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain STEAP-1-expresslng cells (lymph node) is found to contain STEAP-1-expressing cells such as the STEAP-1 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively STEAP-1 pojynudept'des and/orpolypepfides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express STEAP-1 or express STEAP-1 at a different level are found to express STEAP-1 or have an increased expression of STEAP-1 (see, e.g., the STEAP-1 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may lurther wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to STEAP-1) such as PSA, PSCA eta (see, e.g., Alanen et a/., Pathol. Res. Pract 192(3): 233-237(1996)).
The use of immunohistochemistry to identify the presence of a STEAP-1 pdypeptide within a (issue section can indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypepfide that is expressed In cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered disfrlbutton of the porypepttdewHMn the cancer cells, as compared to corresponding non-malignant tissue.
! The STEAP-1 polypepGde arid.tomunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, eel membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein In a non-polar manner over the whole cell surface.
The phenomenon of altered subcellular protein localization in a disease state has been demonstrated wilh MUC1
*:s
and Her2 protein expression by use of Immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in add-on to some supranudear localization of the glycoprotein, whereas malignant lesions often demonstrate an apolar staining pattern (Diaz et a/, The Breast Journal, 7; 40-45 (2001); Zhang et a!, Clinical Cancer Research, 4; 2669-2676 (1S08): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45:1547-1557 (1997)}. In addition, normal breast
epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant ceOs can express
»
the protein over the whole cell surface (Oe Potter, el a/, International Journal of Cancer, 44; 969-974 (1989): McCormk*, et al, 117;935-943(2002)). Alternatively, oTstrfoufion of the protein may be altered from a surface only locaCzaQon to indude diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, ef af, The Breast Journal, 7:4045 (2001)).
Alteration*] the Iccafization/dJstribution of a protein in the cell, as detected by immunohistochenvcal methods, can also provide valuable information concerning the favorabitity of certain treatment modalities. This last pofrit is illustrated by a situation where a protein may be intracellular in normal tissue, but cell surface in malignant cells; the cefl surface location makes the cells favorably amenable to antibody-based diagnostic and treatment regimens. When such an alteration of protein localization occurs for STEAP-1, the STEAP-1 protein and immune responses related Ihereto are very useful Accordingly, the ability to determine whether alteration of subcelfular protein localization occurred for 24P4C12 make lie

STEAP-1 protein and immune responses related thereto very useful. Use of the STEAP-1 compositions allows those sidled in the art to make important diagnostic and therapeutic decisions.
Immunohistochemical reagents specific to STEAP-I are also useful to detect metastases of tumors expressing STEAP-1 when the polypeptide appears in tissues where STEAP-1 is not normally produced.
Thus, STEAP-1 pdypepfides and antibodies resulting from immune responses thereto are useful in a variety of Important contexts such as diagnostic, prognostic, preventafjve and/or therapeutic purposes known to those skied to toe art
'• Just as PSA polynudeotide fragments and polynudeotide variants are employed by skied artisans for use in methods of monitoring PSA, STEAP-1 pdynudeofide fragments and pdynucteofide variants are used in an analogous manner. In particular, typical PSA pdynudeolides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynudeotide must indude less than the whole PSA sequence to function In the polymerase chain reaction. In the context of such PCR reactions, skiBed artisans generally create a variety of different polynudeotide fragments that can be used as primers in order to amplify different portions of a polynudeotide of interest or to optimize amplification reactions (see, e.g., Caetano-AnoBes, G. Blotedniques 25(3): 472-476.478-480 (1998); Robertson et a/., Methods Mol. Bio). 98:121-154 {1998}). An additional Illustration of the use of such fragments Is provided in the Example entitled 'Expression analysis of STEAP-1 In normal tissues, and patient specimens," where a STEAP-1 polynudeotide fragment is used as a probe to show the expression cf STEAP-1 RNAs in cancer cells. In addition, variant polynudeotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et si., Fetal Diagn. Ther. 1996 Nov-Dec 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2. Unit 2, Frederick M. Ausubei et at eds., 1995)). Polynudeqtide fragments and variants are useful in this context where they are capable of binding to a target potynudeoSde sequence (e.g., a STEAP-1 potynudeotida'shown in Figure 2 or variant thereof) under conditions of high stringency.
Furthermore, PSA pofypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. STEAP-1 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T ceBs) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubet et a/, eds., 1995). In this context, each epftope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typicany, skilled artisans create a variety of different polypeptide tragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to utilize a polypepfide comprfsfog one of the STEAP-1 biological moGfs discussed herein or a motif-bearing subsequence which is readily identified uy one of skifl in the art based on motifs available in the art PdypepGde fragments, variants or analogs are typically useful in this context as long as Brey comprise an epitope capable of generating an antibody orTcell spedfic for a target polypeptide sequence (e.g. a STEAP-1 polypeptide shown in Figure 3).
As shown herein, (he STEAP-1 poiynudeotides and polypepfides (as well as Ihe STEAP-1 porynucteofide probes andanti-STEAP-1anlfbodiesorT make them useful in diagnosing cancers such as those Bsted in Table L Diagnostic assays fliat measure (he presence of STEAP-1 gene products, in order to evaluate the presence or onset of a disease condition described hereh, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in toe art for molecules having s^aracomplernentary characterisfics to PSA in situations where, for example, a definite dagnc^ of metastasis of prostafc origin catwot be made

the basis of a test for PSA alone (see, e.g., Alanen el at, Pathol. Res. Pract 192(3): 233-237 (1996)), aid consequently, materials such as STEAP-1 pdynudaotides and polypeptktes (as wed as the STEAP-1 polynucleotide probes and anfi-STEAP-1 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
Finally, in addition to their use in diagnostic assays, the STEAP-1 polynudeoBdes disclosed herein have a number of other utilities such as their use in the identification of oncogenetfc associated chromosomal abnormalities in (he chromosomal region to which the STEAP-1 gene maps (see the Example entitled "Chromosomal Mapping of STEAP-1'
i
below). Moreover, in addition to their use in diagnostic assays, the STEAP-1-related proteins and polynudeotides disclosed herein have other utilitiessuch as theiruse to the forensic analysis of fissues of unkrwwn origin (see, e.g.,TakahamaK Forensic Sd Int 1996 Jun 28;80(1-2): 63-9).
Additionally, STEAP-1-related proteins or polynudeotides of the invention can be used to treat a pathologic condition characterized by the over-expression of STEAP-1. For example, (he amino add or nucleic acid sequence of Figure 2 or Figure 3, or fragments of either, can be used to generate an immune response to a STEAP-1 anflgen. Antibodies or • other molecules that react with STEAP-1 can be used to modulate flie function of this molecule, and thereby provide a therapeutic benefit
Xll.l Inhibition of STEAP-1 Protein Function
The Invention Includes various methods and oppositions for inhJbli^ the bincfing of ^ partner or its association with other protein(s) as wed as methods for Inhibiting STEAP-1 function.
XJLA.) Inhibition of STEAM With Intracellular Antibodies
\ In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to STEAP-1 are
introduced Into STEAP-1 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-STEAP-1 antibody is expressed intracellularly, binds to STEAP-1 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are wdl known. Such Intracellular antibodies, also known as Intrabodies', are specifically targeted to a particular compartment within the cell, providing control over where the Inhibitory activity of the treatment is focused. This technology has been successfully applied In the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proa NaB. Acad. Sd. USA 92:3137-3141; Beedi et al., 1994, J. Bid. Chem. 289:23931-23336; Deshane et al., 1994, Gene Ther. 1:332-337).
Single chain antibodies comprise the variable domains of the heavy and fight chain joined byyi flexible linker pdypeptide, and are expressed as a single pdypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment Joined to the fight chain constant region. Well-known intracellular trafficking signals are engineered into recombinant pdynudeotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader pbptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino add motif. Intrabodies intended to exert activity in the nudeus are engineered to indude a nudear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, inlrabodies are used to capture STEAP-1 In the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such STEAP-1 intrabodies in order to achieve the desired targeting. Such STEAP-1 intrabodies are designed to bind specifically to a particular STEAP-1 domain. In another embodiment cytosofic intrabodies fiiat specifically bind to a STEAP-1 protein are used to prevent STEAP-1 from gaining access to (he nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing STEAP-1 from forming transcription complexes with other factors).
In order to specifically direct the expression of such intrabodtes to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be uflized (See, for example, U.S. Patent No. 5.919,652 issued 6 July 1999).
XI1.B.1 Inhibition of STEAP-1 with Recombinant Proteins
In another approach, recombinant molecules bind to STEAP-1 and thereby inhibit STEAP-1 function. For example, ftese recornbinant molecules prevent or inhibit STEAP-1 from accessbig/binding to its binding partners) or associating with other protein{s). Such recombinant molecules can, for example, contain he reacfive part(s) of a STEAP-1 specific anfibody molecule. In a particular enrtxxfiment, toe STEAP-1 binding domain of a STEAP-1 binrfu^ partner is engSwered into a dimerfc ' fusion protein, whereby to fosfonpn^ comprises to
IgG, such as human IgG1. Such IgGporfion can contah, for example, the Qtf and Ctfdomahs and (he Ifrge reason, but not tie C«1 domain. Surtcfirnentfijskmproteirs are administer
fie expression of STEAP-1, whereby the dime* fusion protein speotk^bWs to STEAP-1 aralbtocksS^
with a birfding partner. Such d&neric fusion protehs are farihercombined into mulfitnericprotehs using tawmanfibo technologies. N
XII.C.) Inhibition of STEAP-1 Transcription or Translation
The present irtvenfion also comprises various methods and compositions for inhibiting fte transcription ot me
STEAP-1 gene. Similarly, the Invention also provides methods and composffions for inhibiting the translation of STEAP-1
mRNA into protein. "
In one approach, a method of inhibiting the transcription of the STEAP-1 gene comprises oontacSng the STEAP-1 gene with a STEAP-1 antisense porynudeotide. In another approach, a method of inhibiting STEAP-1 mRNA translation comprises contacting a STEAP-1 mRNA with an antisense potynudeofide. In another approach, a STEAP-1 specific nbozyme is used to cleave a STEAP-1 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of (he STEAP-1 gene, such as STEAP-1 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a STEAP-t gene transcription factor are used to MM. STEAP-1 mRNA transcription. The various polynucteorjdes and compositions useful in (he aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in he art
Other factors that inhibit the transcription of STEAP-1 by interfering with STEAP-1 tanscriptional acfivafion are also useful to treat cancers expressing STEAP-1. Similarly, factors that interfere with STEAP-1 processing are useful, to treat cancers that express STEAP-1. Cancer treatment methods utilizing such fanfors am also within the scope of the invention.

yil.D.) General Considerations for Therapeutic Strategics
Gene transfer and gene therapy technologies can be used to deBvcr flierapeuficpolynucteotide motecutes to tumor cells synthesizing STEAP-1 (i.e., anfisense, ribozyme, polynucteofietes encoding Mrabodies and other STEAP-1 Wtfatoy mctecutes). A number of gene therapy approaches are known in the art Recombinant vectors erwoo^ STEAP-1 anBsensepcJynucteofides, ribozymes, factors capable of Interfering with STEAP-1 iranscnpftm.awJ so forth, can be deSvered to target such gene therapy approaches.
The above therapeufic approaches can be combined with any (wclawktevar^ofsurgk^.dieniotherapyor radiation toerapy regimens. The therapeutic approaches of foe invention can enable Bie use of reduced dosages of chenrotierapy (or other therapies) anci/or less frequent adriwistration, an do not tolerate the toxicity of the cnemotherapeufc agent welL
The anfi-tumor activity of a particular composifion (e.g., anfisense, ribozyme, htrabody), or a comNnaSon of such composifions, can be evaluated using various fovfco and to vfo assay systems, Jfovtro assays that evaluate toerapeuflcacfivfty irKiKteceBgnrwMbass^j soft agar assays arri other assa^ determining fte extent to which a ffBrapeufc composifion wffl Inhibit ^Wre^rf STEAP-1 to a bindngparher, eta.
/nWw.trteef^ of a STEAP-1 therapetrfccomrxBilfcnc^te For example,
xenogerric prostate cancer models can be used, wherein human prostate ram^explants or passaged xanogtaft Issues are
htrodtK^intainmuTWCom^ For
example, PCT Patent Application W098/16628 and U.S. Patent 6,107,540 describe various xenogre?. models of human prostate cancer capable of recapltuiaflng the development of primary hm»rs,mlcfonwtastasIs,arHl^f«mafionof osteobiasfic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure Inhibition of tumor formation, tumor regression or metastasis, andtfieiike.
In mo assays that evaluate the promotion of apoptosis are useful fn evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeu&c composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptoGc foci are foundln the tumors of the treated mice provides an indication of the therapeutic efficacy of the composifion.
ccrrp)stocornrxisir$a(&rk?suita^ Suitable earners inckide any material fliat when
combined with the therapeutic composifion retains the anti-tumor func&on of the forapeufic composition and is generaBy non-reactive with the patient's Immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the Bke (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osai., Ed., 1980).
composition to the tumor site. Potenfially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, Infraorgan, orthotopfc, and foe Ike. A preferred formulation for intravenous injection comprises the therapeuSc composifion in a solution of preserved bacfedostafic water, sterile unpreserved water, and/or diluted in polyvinylrhloride orpolyefliyfene bags containwg 0.9% sterte Sodium Chloride for Injection, DSP. Therapeutic protein preparations can be fyophJKzed and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative} or in sterile water prior to injection.
Dosages and administration protocols for the Irealrnent of camsre using the fwegoi^ rneHwd and the target cancer, and wft^m^^

XIIH MentificatioaChanH^etfeatIonandUserfMc^atwsofSTEAP.1
Methods to Identify and Use Modulators
In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression proffle, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In anofter embodiment, having identified differentially expressed genes important fn a particular state; screens are performed to .Identify modulators that alter expression of Individual genes, either Increase or decrease. In arwlher embodiment, screening b perfwn^ to idOTfify modulators that alter a IM>^
Again, having identified the Importance of a gene in a particular state, screens are performed to identify agents (hat bind and/or modulate the biological activity of the gene product
In addition, screens are done for genes that are induced in response to a candidate agent After identifying a modulator (one that suppresses a cancer expression pattern leading to a norrr^ expression pattern, or a rmxlulator of a cancer gene that leads to expressionrof ^genl'aslTnormai fissuej a sareene performed to identify genes that are soecfficafly modulated hi response to the agent Comparing expression profiles between normal fissue and agent-treated cancer tissue reveab'genes that are not expressed in normal tissue or cancer tissue, but are expressed In agent treated tissue, and vice versa. 7neseagent-«^cci?i; sequences are idenbled a
genes or proteins. In particular these sequences and (he proteins they encode are used in marking or Identifying agent-treated ceBs. In addition, antibodies are raised against the agent-Induced proteins and used to target novel fterapeufcs to the treated cancer tissue sample.
! ' I Modulator-related Identification and Screening Assays:
Gene Expression-related Assays
Proteins, nucleic adds, and antibodies of the Invention are used in screening assays. The cancer-asscdated proteins, antibodies, nucleic adds, modified proteins and cells containing these sequences are used in screenhg assays, such as e"*1' "ating the effect of drug candidates on a 'gene expression profile,* expression profile of porypepfides or alteration of biological function. In one embodiment the expression profiles are used, preferably in conjunction with high
(e.g., Davis, GF, et al, J Biol Screen 7:69 (2002); ZIokamik, et at, Science 279:84-8 (1998); HekJ, Genome Res 6:986-94,1996).
The cancer proteins, antibodies, nucleic adds, modified proteins and cells containing the native or modified cancer proteins or genes are used hi screening assays. That Is, the present invention comprises methods forscreening for compositions which modulate me cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a "gene expression profile" or biological function. In one embodiment, expression profiles are used, preferably fn conjunction with high throughput screening techniques to aflow monitoring after treatment with a candidate agent, see ZIokamik, supra.
A variety of assays are executed directed to (he genes and proteins of the invention. Assays are run on an individual nucleic add or protein level That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. 'Modulation" hi this context indudes an increase or a decrease in gene expression. The preferred amount of modulation wffl depend on tie original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and hi some embodiments 300-1000% or greater. Thus, If a gene exnfbKs a44oW-

increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.
The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression. Expression Monitoring to Identify Compounds that Modify Gene Expression
In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such proffles wifl typically involve one or more of the genes of Figure 2. In fills embodiment, e.g., cancer nucleic add probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microb'ter plate, can be used with dispensed primers in desired weds. A PCR reaction can then be performed and analyzed for each wed.
Expression monitoring Is performed to identify compounds that nToalfy"oTe expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in Figure 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or Interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.
In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such 'combinatorial chemical libraries* are then screened hi one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional 'lead compounds,' as compounds for screening, or as therapeutics.
• In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by Identifying a chemical compound (called a 'lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.
If required, the target sequence is prepared using known techniques. For example, a sampteTs treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example^ an in vitro transcription with labels covatently attached to the nudeotides is performed. Generally, the nucleic adds are labefed with biotin-FfTC or PE, or with cy3 or cy5.
The target sequence can be labeled with, e.g., a fluorescent a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epttope tag or biotin which spedftcally binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above,

thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin fe typically removed prior to analysis.
As wBl be appreciated by those in the art, these assays can be direct hybridization assays or can cornprise •sandwich assays', which Include Ihe use of multiple probes, as is generally outlined in U.S. Patent Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this embodiment, In general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nudeic acid probes, under conditions that aflow the formation of a hybridization complex.
A variety of hybridizaSon conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label . probe hybridizatfon complex only hi the presence of target Stringency can be controlled by altering a step parameter that Is a thermodynamic variable, Including, but not limited to, temperature, fixmamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, efex-These parameters may also be used to control non-specific binding, as is generally outlined In U.S. Patent No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
TTiereactkxisoi^edr^ein can be acccfflplished^ a variety of ways. Components of the reacfion can be added simultaneously, or sequenfialiy, in diiierent orders, with preferred embodjments outlined below. In addition, the reaction may Include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background Interactions. Reagents that otherwise Improve the effidency.of the assay, such as protease inhibitors, nudease inhibitors, antHrtcrobial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of Ihe target The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between slates, forming a gene expression profile.
Biological Activity-related Assays
The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a eel comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of Vie Invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.
In one aspect, the assays are evaluated In the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokbies, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, cardnogenics, or other eels (i.e., eel-cell contacts). In another example, the determinations are made at different stages of the cell cycle process, hi this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once Identified, similar structures are evaluated to identify critical structural features of the compound.
In one embodiment, a method of modulating (e.g., inhibiting) cancer cell division is provided; (he method comprises administration of a cancer modulator. In another embodiment a method of modulating (e.g., inhibiting;) cancer is provided; Ihe method comprises administration of a cancer modulator. In a further embodiment, methods of treating eels or individuals with cancer are provided; the method comprises administration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, fancticx., apoptosis, senescence, location, enzymatic activity, signal transducfion, etc. of a cell. In one embodiment, a cancer Inhibitor Is an antibody as discussed above. In another embodiment the cancer inhibitor is an antisense motecute. A variety of cell growth, proliferation, and metastasis assays are known to those of skiH in the art, as described herein.
High Throughput Screening to Identify Modulators
t
The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or hhibffion of cancer gene transcription, inhibition or enhancement of potypeptide expression, and inhibition or enhancement of polypepfide activity.
in one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of protemaceous cellular extracts, are used. In thisway.Khrariesof proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are Hbraries of bacteria!, fungal, viral, and mammafian proteins, with the latter being preferred, and human proteins being especially prefaced. Particularly useful test compound wdl be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or figands and receptors. Use of Soft Aaar Growth and Colony Formation to Identify and Characterize Modulators Nwmal ceHs require a sofid substrate to attach and grow. When ceHs are transformed, fl>ey lose Wsphenotype
, and grow detached from he substrate. For example, transformed cells can grow in sftredsusoertston culture or suspended
i in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfecfed wfm tumor suppressor genes,
can regenerate normal phenotype aric(once again require a soBd substrate to attach to and grow. Soft agar growOi or colony formation in assays are used to identify modulators of cancer sequences, which when expressed hi hostceBs, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' abffity to grow
I suspended in solid or semtsofid media, such as agar.
f Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of
« Animal Cells a Manual of Basic Technique (^d ed., 1994). See also, me niethods section of Garkavtsevetal.(1996}( supra
I . •••'
I Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators
Normal cells typically grow in * flat and organized pattern in cell culture until they touch other ceOs. When (he cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities h disorganized foci. Thus, transformed cells grow to a higher saturation density (nan corresponding normal cells. This is detected morphologically by the formation of a disoriented monolaver of cefls or eels in foci. Alternatively, labeling index with ("HHriymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay wilt reveal proliferation capacity of cells and the ability of modulators to affect same. See Freshney (1994), supra. Transformed eels, when transfected wiliTtumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a tower density.
In this assay, labeling index with 3H)-thymidine at saturation density is a preferred method of measuring density . [imitation of growth. Transformed host cells are transfecled with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of ueiis labeling with {^HJ-tnymidine is determined by incorporated cpm.
*l

1

Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns Uie cells to a normal phenotype.
Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulator? Transformed ceDs have lower serum dependence fian their normal counterparts (see, e.g., Ten*, J. Nad Cancer InsL 37:167-175 (1966); Eagteetal., J:Exp.Med 131:836-879 (1970)); Freshney, supra TWs Is In part due to release of various growth factors by the transformed cells. The degree of grtwM factor cfseromdeperxtence erf tran^ can be compared with that of control. For example, jgrowth factor or serum dependence of a ceB is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention,
Use of Tumor-specific Marker Levels to Identify and Characterize Modulators
Tumor cells release an increased amount of certain factors (hereinafter tumor specific markers') than their normal counterparts. For example, plasmlnogen activator (PA) Is released from human gfioma at a higher level than from normal brain ceils (see, e.g., GuDino, Angiogenesis, Tumor Vasculartzaflon, and Potential Interference wifh Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed) 1985)}. Similarly, Tumor Angiogenesis Factor (TAP) te released at a higher level In tumor cefe than their normal counterparts. See, e.g., FoJkman, Angiogenesis and Cancer, Cam Cancer Bid. (1992)), while bFGF is released from endotheEal tumors (EnsoB, B et al).
Various techniques which measure the release of these factors are described In Freshney (1994), supra Also, see, Unktess et al., J. BioL Chem. 249:4295-4305 (1974); Strickland & Beers, J. Bid. Chem. 251:5694-5702 (1976); Whuret at., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potenfial Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anficancer Res. 5:111-130 (1985). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds hat modulate cancer-associated sequences of the invention.
Invasiveness Into Matriael to Identify and Characterize Modulators
The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that rnodulate cancer associated sequences. Tumor rails exhibit a positive correlation between malignancy and Invasiveness of cells into Matrigel or some other exfraceBularmaWx constituent In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host ceils would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side cf the filter, is rated as invasiveness. and rated historically by number of cells and distance moved, or by prelabeling the cells with "M and counting the radfoacfivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
Effects of cancer-associated sequences on cell growth are tested in transgente cHTmmune-suppressed organisms. Transgente organisms are prepared In a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such a$ mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterotogous gene into the endogenous cancer gene site in liie mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of Die cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.



To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells cony ning the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice fart possess germ cefls some of which are derived from the mutant cell Brie. Therefore, by breeding the chimeric mice itfe possfte to obtain a new line of mice containing the Introduced genetic lesion (see, e.g., CapeccM et aL, Science 244:1288 (1989)). Chimeric mice can be derived according to US Patent 6,365,797, issued 2 April 2002; US Patent 6,107,540 issued 22 August 2000; Hogan et at, Manipulafing the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Terafccarcinornas and Embryonic Stem Cefls: A Practical Approach, Robertson, ed., IRL Press, Washington. O.C., (1987).
Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic "nude" mouse (see, e.g., Giovanefla et at., J. Nafl. Cancer Inst 52321 (1974)), a SCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradley etal., Br. J. Cancer38563 (1978); Setoyeta)., Br. J. Cancer 41:52 (1980)) can be used as a host Transplantabte tumor cefls (typlcaUyabc^W cells) Injected into isogenfc hosts produce invasive tumors in a high proportion of .cases, whIerwrmal(»Bscfsim|ar origin wiB not hi hosts which developed invasive tumors, cells expressing cancer-associated sequence are injected subcutaneously or orthotopicaay. Mice are then separated Into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumorgrowfh Js measured (e.g., by volume or by Hs two largest dimensions, or weight) and compared to the control Tumors lhat have statistically significant reducfibn (using, e.g., Students T test) are said to have inhibited growth.
In vitro Assays to Identify and Characterize Modulators
Assays to iden%ccmp^tKils">^ modulating acfivfty can be performed to vitro. For example, a cancer poiypepfide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours, ki one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. Jhelevel of protein Is measured using immunoassays such as Western Wotting, EUSA and the like with an antibody that^ selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e. g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fkrorescenSy or radioactivefy labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, a reporter gene system can be devised using a cancer protein promoter operably inked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a eel. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis GF, supra; Gonzalez, J. & Negutescu, P. Curr. Opin. Biotechnol. 1998:9:624).
As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important In a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.
In one embodiment, screening for modulators of expression of specific gene(s} is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the abity to modulate differentiaty expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.

5
f Binding Assays to Identify and Characterize Modidators
In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally
used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the
I amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.
Thus, the methods comprise combining a cancer'protein of the invention and a candidate compound such as a : ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utflize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skin in the art Moreover, in some embodiments variant or derivative cancer proteins are used.
Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support The support can, e.g., be one having Isolated sample receiving areas (a mtcrotiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, Is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.
Examples of suitable kisolubte supports indu*m&TOfiter plates, anays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™, etc. Mfcrofiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of Ihe composition to the support is not crucial so long as it fe compatible with the reagents and overall methods of toe invention, maintains the activity of the composition and is nortdiffusabie. Preferred methods of binding include toe use of antibodies which do not sterically block either the Bgand binding site or activation sequence when attaching the protein to the support, direct binding to 'sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, eta Following binding of the protein or figand/binding agent to fie support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with boyjne serum albumin (BSA), casein or other innocuous protein or other mofety. Once a cancer protein of the invention is bound b the support, and a test compound Is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, normaiura! binding agents identified in screens of chemical libraries, pepfide analoos, etc.
Of particular interest are assays to identify agents that have a tow toxkaty for human cells. A wide variety of
assays can be used for this purpose; including proliferation assays, cAMP assays, labeled m tftro protein-protein binding
assays, eledrophoreGc mobility shift assays, immunoassays for protein binding, functional assays (phosphoryJatton assays,
etc.) and the tike. •
A determination of binding of the test compound (Jigand, binding agent, modulator, etc.) to a cancer protein of the invention can b»done in a number of ways. The test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., \ a fluorescent label), washing off excess reaqent, and determining whether the label is present on the solid support Various blocking and washing steps can be utilized as appropriate.
In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled wifti different labels, e.g., I125, for the proteins and a fluorophor for the compound. Proximity reagents, e.g.. quenching or energy transfer reagents are also useful.

I

Competitive Binding to Identify and Characterize Modulators
In one embodiment the binding of the "test compound" is determined by competitive binding assay with a •competitor." The competitor Is a binding moiety that binds to the target molecule {e.g., a cancer protein of the invention}. Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain cvcumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Ether the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. IncubaGons are performed at a temperature that facilitates optimal activity, typically between four and 40°C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient Excess reagent is generally removed or washed away. The second component is then added, and Hie presence or absence of the labeled component is followed, to indicate binding.
In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor
modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if Hie competitor is labeled, (he presence of label In the post-test compound wash solution indicates displacement by the test compound. Altemafiveh/, If the test compound is labeled, the presence of the label on the support indicates displacement
• In an alternative embodiment Ihe test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indfcates that flie test o>mpoundbin ! i •»
of the Invention. Accordingly, the competitive binding methods comprise differentia! screening to identity agents that are capable of modulating the activity of flie cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor In a first sample. A second sarpte comprises a test compound, the cancer protein, and a competitor. The binding of the competitor Is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the earner protein and potentiaBy modulating its activity. That is, if the binding of the compefitor is different hi the second sample relative to he first sample, the agent is capable of binding to the cancer protein.
Alternatively, differential screening is used to identify drug candidates that bind to he native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind tcxfte-modified proteins. Moreover, such drug candidates that affect (he activity of a native cancer protein are also kfenfified by screening drugs for the ability to either enhance.or reduce the activity of such proteins.
Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a 8me sufficient to
V
allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radbiabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents can be included in Hie screening assays. These include reagents fike salts, neutral proteins, e.g. albumin, detergents, eta which are used to faciitate optimal protein-protein binding and/or reduce non-specific or background Interactions. Abo reagents that otherwise improve the efficiency of the assay, such as protease inhtrftors,

nuclease inhibitors, anlMnicrobial agents, etc., can be used. The mixture of components is added in an order (hat provides for the requisite binding.
Use of Polwmdeotides to Down-regulate or Inhibit a Protein of the Invention.
Polynudeotide modulators of cancer can be introduced into a cell containing the target nudeotide sequence by formation of a conjugate with a Ggand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not (muted to, ceil surface 'receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the figand binding molecule does not substantially interfere with the ability of the fgartd binding molecule to bind to Its corresponding molecule or receptor, or block entry of the sense or antisense oligonudeotiae or its conjugated version Into the cell. Alternatively, a polynudeotide modulator of cancer can be introduced into a celt containing Ibe target nucleic add sequence, e.g., by formation of a poiynudeotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment Inhibitory and Anfisense Nudeotides
hi certain embouirnerus, She activity- cf a cancer-associated protein is down-regulated, or entirely Inhibited, by the use of antisense polynudeofide or inhibitory small nudear RNA (snRNA), i.e., a nucleic add complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic add sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynudeotide to the mRNA reduces the translation and/or stability of the mRNA.
In the context of this invention, antisense polynudeotides can comprise naturally occurring nudeofides, or synthetic spedes formed from naturally occurring subunits or their dose homologs. Antisense polynudeotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing spedes which are known for use in the art Analogs are comprised by (his invention so long as they function effectively to hybridize with nudeotides of the Invention. See, e.g., Isis Pharmaceuticals, Carlsbad, CA; Sequftor, Inc., Na8ck,MA.
Such antisense polynudeotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is cold by several vendors, including Applied Biosystems. The preparation of other oligonudeob'des such as phosphorothioates and alkylated derivative is also well known to those of ski in Bie art.
Antisense molecules as used herein indude antisense or sense oligonudeotides. Sense ofigonudepfides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense ofigonudeolide comprise a single stranded nucteic add sequence (either RNA or DNA) capable of binding to target mRNA (sense) or ONA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to (he present invention, comprise a fragment generally at least about 12 nudeotides, preferably from about 12 to 30 nudeotides. The ability to derive an antisense or a sense ofigonudeotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein iCohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)). Ribozymes
In addition to antisense polynudeotides, ribozymes can be used to target and inhibit transcription of cancer-associated nudeotide sequences. A ribozyme is an RNA moteculu that calalytically deaves other RNA molecules. Different kinds of ribozymes have been described, induding group I ribozynics, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25:289-317 (1994) for a general review of the properties of different ribozymes).

s The general features of hairpin ribozymes are described, e.g., in Hampeletal.,Nucl. Adds Res. 18:299-304
| (1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678. Methods of preparing are wen known to
• those of skill in the art (see, e.g., WO 94/26877; Ojwang et aL, Proc. NaU. Acad. Sci. USA 90:6340-6344 (1993); Yamada et
aL, Human Gene Therapy 13945 (1994); Leavitt et al., Proc. Nal Acad Sci. USA 92699- 703 (1995); Leavitt et tL, Human
Gene Therapy 5:1151-120 (1994); and Yamada et aL, Virology 205:121-126 (1994)).
|
Use of Modulators in Phenotypic Screening
In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profae. By 'administration' or 'contacting' herein ismeantthat the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and infracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to Hie ceo, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatabte gene therapy systems can also be used. Once (he modulator has been administered to Ihe ceils, the cells are washed if desired and are .slowed to incubate under preferably physSctogteal conditions for some period. The ceils are then harvested and a new gora expression profile is generated. Thus, e.g., cancer tissue is screened foragents that modulate, e.g., induce or suppress, the cancer phenotype, A change In at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By ticfining such a signature for the cancer phenotype, screens tor new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need riot be represented in the orfgta^ transcript for he target protein need fo.change. The modulator inhibifing function will serve as a surrogate marker
%
As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.
Use of Modulators to Affect Peplides of the Invention
Measurements of cancer pofypepfide activity, or of (he cancer pr^otype are performed using a vanety of assays. For example, the effects of modulators upon the function of a cancer poiype^defs) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the pdypepSdes of this invention. When (he functional outcomes are determined using intact ceBs or animals, a variety of
effects can be assesses such as, in Ihe case of a cancer associated with solid tumors, tumor growth, tumor metastasis,
%
neovascularization, hormone release, Iranscriptional changes to both known and uncharacterized genetic markers (e.g., by
Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in kitracellular second
messengers such as cGNIP. 7"
MelhodsV Identifying Characterizing Cancer-associated Sequences
Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying eels containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an indivkJuat, e.g., determining aO or part of fte sequence of at least one gene of the invention in the

individual. This is general!/ done in at least one tissue of the individual, e.g., a (issue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of foe gene can then be compared to the sequence of a known cancer gene to determine if any differences exist This is done using any number of known homotogy programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene In the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocafions, and the like are Identified in the cancer gene locus.
i
XIV.) RNAI and Therapeutic Use of Small Interfering RNA fslRNAsl The present invention Is also directed towards siRNA oligonucfeofldes, particularly double stranded RNAs encompassing at least a fragment of the STEAP-1 coding i^kmorS'UTRregions.orcompiernent, or any anfisense ofigonucteotkte specific tc the STEAP-1 sequence. In one embodiment such digonudeofides are used to elucidate a funcfion of STEAP-1, or are used to screen for or evaluate modulators of STEAP-1 function or expression. In another embodiment, gene expression of STEAP-1 is reduced by using siRNA transfecfion and results bi significantly diminished prodferative capacity of transformed cancer celts (hat endogenously express the antigen; cefis treated with specific STEAP-1
siRNAs show reduced survival as measured, e.g., by a metabolic readout of cell viability, correlating to the reduced
( *
proliferative capacity. Thus, STEAP-1 s'RNA compositions comprise siRNA (double stranded RNA) that correspond to the
nucleic add ORF sequence of the STEAP-1 protein or subsequences thereof; these subsequences are generally 5,6,7,8, 9,10,11,12,13,14,15,16,17,18,19/20,21,22,23,24,25,26,27,28,29,30,31.32,33,34,35 or more than 35 contiguous RNA nudeotides in length and contain sequences that are complementary and non-complementary to at feast a portion of the mRNA coding sequence In a preferred embodiment, the subsequences are 19-25 nudeotides in length, most preferably 21-23 nudeotides in length.
RNA interference is a novel approach to silencing genes in vitro and in vivo, thus small double stranded RNAs (siRNAs) are valuable therapeutic agents. The power of siRNAs to silence specific gene activities has now been brought to animal models of disease and is used in humans as well. For example, hydrodynamic infusion of a solution of siRNA into a mouse with a siRNA against a particular target has been proven to be therapeutfcaHy effective.
The pioneering work by Song ef a/, indicates that one type of entirely natural nudeic acid, small interfering RNAs (siRNAs), served as therapeutic agents even without further chemical modification (Song, E., et at. "RNA interference targeting Fas protects mice from fulminant hepatitis' NatMed. 9(3): 347-51 (2003)). This work provided the first hi vivo evidence that infusion of siRNAs Into an animal could alleviate disease. In thatcase, the authors gave mice injections of siRNA designed to silence the FAS protein (a cell death receptor that when over-activated during inflammatory response Induces hepatocytes and other cells to die). The next day, the animals were given an antibody specific to Fas. Control mice died of acute h'ver failure within a few days, while over 80% of the siRNA-treated mice remained free from serious disease and survived. About 80% to 90% of their fiver cells incorporated the naked siKNA ohgonucleotides. hurthermore, the RNA molecules functioned for 10 days before losing effect after 3 weeks.
For use in human therapy, siRNA is delivered by efficient systems that induce long-lasting RNAi activity. A major caveat for clinical use is delivering siRNAs to the appropriate cells. Hepatocytes seem to be particularly receptive to

exogenous RNA. Today, targets located in the fiver are attractive because liver is an organ that can be readily targeted by nucleic acid molecules and viral vectors. However, ether tissue and organs targets are preferred as weB.
Formulations of siRNAs with compounds that promote transit across cell membranes are used to improve administration of siRNAs in therapy. Chemically modified synthetic siRNA, that are resistant to nudeases and have serum stability have concomitant enhanced duration of RNAi effects, are an additional embodiment
Thus, siRNA technology is a therapeutic for human malignancy by delivery of siRNA molecules directed to STEAP-1 to individuate with the cancers, such as those listed in Table 1. Such administration of siRNAs leads to reduced growth of cancer cells expressing STEAP-1, and provides an anti-tumor therapy, lessening the morbidity and/or mortality associated with malignancy.
The effectiveness of this modality of gene product knockdown is significant when measured in vitro or in Wro. Effectiveness in vitro is readily demonstrable through application of siRNAs to cells in culture (as described above) or to aliquots of cancer patient biopsies when in vitro methods are used to detect the reduced expression of STEAP-1 protein.
XV.l Kits/Articles of Manufacture
For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits
are within the scope of (he invention. Such kits can comprise a earner, package, or container that is compartmentalized to
receive one or more containers such as vials, tubes,iand.the like, each of the containers) wmpristag one of the separate
elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described
herein. For example, the containers) can comprise a probe that Is or can be detectaWy labeled. Such probe can be an
antibody or polynudeoBde specific for a protein or a gene or message of the invention, respectively. Where the method
utilizes nucleic add hybridization to defect the target nucleic add, the kit can also have containers containing nudeofide(s)
for amplification of the target nudeic add sequence. Kits can comprise a container comprising a reporter, such as a bioBn-
binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or
radiolsotope label; such a reporter can be used with, e.g., a nucleic add or antibody. The kit can indude all or part of the
amino add sequences in Figure 2 or Figure 3 or analogs thereof, or a nudeic add molecule that encodes such amino add
sequences. ~ ,,,-
The kB of toe kwenfion wffl typically comprise flie contafoer (Jescribedab^ve and oie a moreover containers associated there"/tth fiat comprise materials desirable from a commercial and user standpoint, including buffers, diluents, liters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
A label can be present on or with the container to indicate ttiat the compc^twn is used for a spectfc therapy flierapeuficappficafon,suchasapfognsfc^
either to vivo or mv&o use, such as those described herein. Directions and or other information can ateo be included on an inserts) or labels) which Is included with or on toe kit The label can be on or assodated with the container. A label a can be on a container when tetters, numbers or other characters forming the label are molded or etched into the container itself; a label can be assodated with a container when it is present within a receptade or carrier that also nokfe the container, e.g., as a package insert The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table i.
The terms "kit" and "artide of manufadure" can be used as synonyms.
In another embocfimenl of the invention, an artide{s) of manufacture containing compositions, such as amino add sequence(s), small motecute(s), nucleic add sequence(s), and/or antibody(s), e.g., materials useful for the diagnose,

prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. SuiWULtcontainers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s). small molecuie(s), nucleic acid sequence^), cell population® and/or anlibody(s). In one embodiment, the container holds a poiynudeotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of STEAP-1 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other composiBons or toots used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune 'response;" together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive bnmunotfierapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated Indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.
The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophyiaxlng a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents In the composition can be an antibody capable of specifically binding STEAP-1 and modulating the function of STEAP-1.
The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further Include other materials desirable from a commercial and user standpoint, Including other buffers, diluents, filters, stirrers, needles, syringes, and/or package Inserts with Indications and/or Instructions for use.
EXAMPLES:
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which is intended to limit the scope of the invention.
Example 1; SSH-Generated Isolation w cDNA Fragment of the STEAP-1 Gene Materials and Methods LAPC Xenografts:
LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated as described (Klein et ai, 1997, Nature Med. 3:402-408; Craft et al., 1999, Cancer Res. 59:5030-5036). Androgen dependent and independent LAPCX xenografts (LAPC-4 AD and Al, respectively) and LAPC-9 xenografts (LAPC-9 AD and Al, respectively) were grown in intact male SCID mice or in castrated males, respectively, and were passaged as small tissue chunks h recipient males. LAPC-4 Al xenografts were derived from LAPC-4 AD tumors and LAPC-9 Al xenografts were derived from LAPC-9 AD tumors. To generate the Al xerjpgrafts, male mice bearing LAPC AD tumors were castrated arid maintained for 2-3 months. After the LAPC tumors re-grew, the tumors were harvested and passaged in castrated males or in female SCID mice.
LAPC-4 AD xenografts were grown intratibiaSy as follows. LAPC-4 AD xenograft tumor tissue grown subcutaneously was minced into 1-2 mm3 sections while the tissue was bathed in IX iscoves iiKaiium, minced tissue %.v2? then centrifuged at 1.3K rpm for 4 minutes, the supernatant was resuspended in 10 ml ice cold 1X Iscoves medium and centrifuged at 1.3K rpm for 4 minutes. The pellet was then resuspended in 1X Iscoves with 1 % pronase E and incubated lor 20 minutes at room temperature with mild cocking agitation followed by incubation on ice for 2-4 minutes. Filtrate was

centrifuged at 1.3K rpm for 4 minutes, and the pronase was removed from the aspirated pellet by resuspending in 10 ml Iscoves and re-centrifuging. Clumps of cells were then plated in PrEGM medium and grown ovemighl The ceils were (hen harvested, filtered, washed 2X RPMI, and counted. Approximately 50,000 ceHs were mixed with and equal volume of ice-cold Mafrige! on tee, and surgically injected into the proximal tibial metaphyses of SCIO mice via a 27 gauge needle. After 10-12 weeks, LAPC-4 tumors growing in bone marrow were recovered.
Ceil Lines and Tissues'.
Human cell Ones (e.g., HeLa) were obtained from the ATCC and were maintained in DMEM with 5% fetal calf serum. Human tissues for RNA and protein analyses were obtained from the Human Tissue Resource Center (HTRC) at the UCLA (Los Angeles, CA) and from QualTek, Inc. (Santa Barbara, CA). .
RNA Isolation:
Tumorlissue and ce| lines were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 mi/ g fissww 10 nil/IP cells to Isolate total R^
and Midi kits. Total and mRNA were quantified by spectrophotometrte analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
OiioonudeoMes:
The foQowing HPLC purified ofigonudeofides were used.
DPNCDN fcDNA synthesis printer):
STnTGATCAAGCTTaoS' (SEQIDNO: 68)
Adaptor 1:
FCTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 69)
S'GGCCCGTCCTAGS' (SEQ ID NO: 70)
Adaptor 2: •
S'GTAATACGACTCACTATAGGGCAGCXSTGGTCGCGGCCGAGS'(SEQ ID NO: 71) S'CGGCTCCTAGS (SEQ ID Na 72) PCR primer 1: " yCTAATACGACTCACTATAGGGCS1 (SEQIDNO: 73)
Nested primer fNPM: • » . -
5TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 74)
Nested primer fNPte
ffAGCGTGGTCGCGGCCGAGGAS' (SEQ ID NO: 75)
Suppression Subtractive Hybridization:
Suppression Subtracfive Hybridization (SSH) was used to identify cDNAs corresporxfing to genes, which may be up-regulated in androgen dependent prostate cancer compared to benign prostafohyperplasia(BPH).
Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester) and the BPHfesue (driver) were synthesized from 2 jig of poly(A)+ RNA isolated from xenograft and BPH tissue, as described above, using CLONTECH's PCR-SetedcDNASubtracticflKitarKllngofdigcinudeoUdeRSAOTN

carried oul as described in the Kit's user manual |nofoool (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Rsa I for 3 hrs. nl 37*0. Digested cDNA was extracted with phenol/chloroform (1:1) and efhanol precipitated.
Driver cDNA (BPH) was generated by combining in a 4 to 1 ratio Rsa I digested BPH cDNA with digested cDNA from mouse fiver, in order to ensure that murine gortos were subtracted from the tester cDNA (LAPC-4 AD).
Tester cONA (LAPC-4 AD) was generated by diluting 1 pi of Rsa I digested LAPC-4 AD cDNA (400 ng) in 5 pi of water. The diluted cDNA (2 pi, 160 ng) was then looted to 2 pi of adaptor 1 and adaptor 2 (10 pM), in separate figatfon ; reactions, In a total volume of 10 pi at 16°C overnight, using 400 u of T4 DNA ligase (CLONTECH). LlgaBon was terminated with 1 pi of 0.2 M EDTA and heating at 72eC for 5 min.
The first hybridization was performed by adding 1.5 pi (600 ng) of driver cDNA to each of two tubes containing 1.5 pi (20 ng) adaptor 1- and adaptor 2- ligated tester cDNA. In a final volume of 4 pi, the samples were overiayed with mineral 08, denatured In an MJ Research thermal cycler at 98°C for 1.5 minutes, aruJ then were altowed to hybridize for 8 hrs at 68*C. The two hyt)rkfiza^s-weceAerui«xeri.too«ther.wltti an additional 1 pi of fresh denatured driver cDNA and were allowed to hybridize overnight at 68'C. The second hybridization was then (Muted in 200 pi of 20 wM Hepes, pH 8.3,50 mM Nad, 0.2 mM EDTA, heated at 70eC for 7 min. and stored at -20°C.
PCR Amplification. Ctonino and Seouendng of Gena Fragments federated from SSH:
To ampWy gene fragments resulting from SSH reactions, two PC^ampfitkafkm were performed In the primary PCR reaction 1 pi of the diluted final hybridization mix was added to 1 pi of PCR primer 1 (10 pM), 0.5 pi dNTP mix (10 pM), 2.5 pi 10 x reacfion buffer (CLQNTECH) and 0.5 pi 50 x Advantage cDNA polymerase Mix (CLONTECH) In a final volume of 25 pi. PCR 1 was conducted using the following conditions: 75°C for 5 min., 94°C for 25 sec., then 27 cyctes of 940Cfor10secI660Cfor30sec,72°Cfor1.5rnin. Five separate primary PCR reactions were performed for each experiment The products were pooled and diluted 1:10 with water. For the secondary PCR reacfion, 1 pi from fte pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 pM) were used instead of PCR primer 1. PCR2wasperfornTedusing10-12^cyclesof94X;fbr10sec,68»Cfor30sec, 72°C for 1.5 minutes. The PCR products were analyzed using 2% agarose gel etedrophoresis.
The PCR products were inserted Into pCR2.1 using the T/A vector duiing kit (hvttrogen). Transformed E. col were subjected to blue/white and ampidUin selection. White colonies were picked and arrayed Into 96 wefl plates and were grown in liquid culture-overnight To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCRjModucts were analyzed using 2% agarcse gel etedrophoresis.
Bacterial dones were stored in 20% glycero) in a 96 well format Plasmid DNA was prepared, sequenced, and subjected to nudeic add homdogy searches of the GenBank, dbEST, and NCI-CGAP databases.
RT-PCR Expression Analysis:
First strand cDNAs were generated from 1 pgofrnRh^witholigo(aT)12-18priniir^usir«theGibco-BRL
Superscript PreampGfication system. The manufacturer's protocol was used and inducted an Incubation for 50 min at 42°C x
with reverse Iranscriplase followed by RNase H treatment at 37°C for 20 mm. After completing the reaction, the volume was increased to 200 pi with water prior to normalization. First strand cDNAs from 16 different normal human tissues were obtained from Ctontech.

Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5'atatcqccgcgctcgtcgtogacaa3' (SEQ ID NO: 76) rnd 5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 77) to amplify p-actio. First strand cDNA (5 jil) was amplified in a total volume of 50 (J oxrtaining 0.4 jiM primers, 0.2 \M each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCb, 50 mM KCI, pH8.3) and 1X Wentaq DNA polymerase (Ctontech). Rve jJ of the PCR reaction was removed at 18,20, and 22 cycles and used for agarose gel etedrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: initial denaturation was at 94°C fix 15 sec, followed by a18I20,and22cydesof94°Cfor15,650C for2min,72°C forSsec. A final extension at 72°C was carried out for 2 min. After agarose gel etedrophoresis, the band intensities of the 283 bp f>actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal p-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization were required to achieve equal band intensities in all tissues after22cydesofPCR.
To determine expression levels of the STEAP-1 gene, 5 pi of normalized first strand cDNA was analyzed by PCR using 25,30, and 35 cycles of amplification using BiefoSowiiig-primerpalrsT
51 ACT TTG TTG ATG ACC AGG ATT GGA 3' (SEQ ID NO: 78) 51 CAG MC TTC AGC ACA CAC AGG MC 3'(SEQ ID NO: 79)
Semi quantitative expression analysis was achieved by comparing the PCR products at cycle numbers that give Bght band
intensities. .
Several SSH experiments were conduced as described in the Materials and Methods, supra, and ted to the isolation of numerous candidate gene fragment dones. Ail candidate dones were sequenced and subjected to homology analysis against all sequences in the major public gene and EST databases in order to provide information on the identify of the corresponding gene and to help guide the decision to analyze a particular gene for differential expression. In general, gene fragments which had no homotogy to any known sequence in any of the searched databases, and thus considered to represent novel genes, as well as gene fragments showing homotogy to previously sequenced expressed sequence tags (ESTs), were subjected to differential expression analysis by RT-PCR and/or Northern analysis.
One of the cDNA clones, designated STEAP-1, was 436 bp in length and showed homology to an EST sequence in the NCI-CGAP tumor gene database. The full length cDNA encoding the STEAP-1 gene was subsequently isolated using this cDNA and re-named STEAP-1. The STEAP-1 cDNA nudeotide sequence correspondsto nudeofjde residues 150 through 585 in the STEAP-1 cDNA sequence as shown in FIG. 1. Another done, designated 28P3E1, 561 bp in length r showed homology to a number of EST sequences in the NCI-CGAP tumor gene database or in other databases. Part of the 28P3E1 sequence (356 bp) is identical to an EST derived from human fetal tissue. After the full-length STEAP-1 cDNA was obtained and sequenced, it became apparent that this done also corresponds to STEAP-1 (more spedficafly, to residues 622 through the 3' end of the STEAP-1 nudeotide sequence as shown in FIG. 1).
Example 2: Isolation of Full Length STEAP-1 Encoding cDNA
The 436 bp STEAP-1 gene fragment (See Example Entitled, "SSH-Generated Isolation of cONA Fragment of the STEAP-1 Gene*) was used to isolate additional cDNAs encoding the 8P1D4/STEAP-1 gene. Briefly, a normal human prostate cDNA library (Ctontech) was screened with a labeled probe generated from the 436 bp STEAP-1 cDNA^ One of the
H3

positive clones, done 10, is 1195 bp in length and encodes a 339 amino acid protein having nudeotide and add sequences bearing no significant homology to any known human genes or proteins (homotogy to a rat KMncy injury Protein described in International ApplicaBon W098/53071). The encoded protein contains at feast fi \H Subsequent identification of additional "STEAP" proteins ted to the re-designation of the STEAP-1 gena proAx.1 as "STEAP-r. The STEAP-1 cONA and encoded amino add sequences are shown in FIG. 2A-Q. STEAP-1 cONA dorio 10 was deposited with the American Type Culture Collection ("ATCC") (10801 University Blvd., Manassas, VA 20110-2209 USA) as plasmtd STEAP-1 done 10.1 on August 26,1998 as ATCC Accession Number 98849. The STEAP-1 cONA done can be exdsed therefrom using EcoRl/Xbal double digest (EcoRI atthe ffend, Xbal at the 3'end).
Examples: Chromosomal Mapping of STEAP-1
Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are avattabte hdudtng fluorescent In s#u hybridization (FISH), humari/hamsierradiatiwrliybiw (RHJijartefe {Wafer (A&rfQM-Nature GeneBcs 7:22; Research Genetics, Huntsvfe At), human-rodent somafic oen hybrid panels such as is avaflaWe from (he Coriell Institute (Camden, New Jersey), and genomic viewers ub'fizing BLAST homoJogles to sequenced and mapped gonomic clones (NCBI, Bethesda, Maryland).
STEAP-1 maps to chromosome 7q21 using STEAP-1 sequence and the NCBf BLAST toot: (located on the World Wide Web at (.ncbijilm.nih.o^v/genome/seq/page.cgi?F=HsBlasLhtrrd&&ORG=Hs)).
Example 4: Expression Analysis of STEAP-1
•"•^"^™™^*^™™p— ^^^™ ^~™
Expression of STEAP-1 in stomach cancer patient specimens is shown In Figure 14(a}-(e). Figure 14(a) RNA was extracted from normal stomach (N) and from 10 different stomach cancer patient spedmens (T). Northern Wot with 10 ng of total RNA/lane was probed with STEAP-1 sequence. Results show strong expression of an approximately 1.6kb STEAP-1 in the stomach tumor tissues. The lower panel represents ethidium bromide staining of the blot showing quality of the RNA samples.
Figure 14(b) shows that STEAP-1 was expressed in rectum cancer patient tissues. RNA was extracted from normal rectum (N), rectum cancer patient tumors (T), and rectum cancer metastasis (M). Northern blots with 10 fig of total RNA were probed with the STEAP-1 sequence. Results show strong expression of STEAP-1 in the rectum cancer patient tissues. The tower panel represents ethidium bromide staining of the-bfof showing quality of the RNA samples.
Expression of STEAP-1 by RT-PCR demonstrated that STEAP-1 is strongly expressed in human umbilical vein endolrtelial cells (HUVEC) (Figure 14(c)}. First strand cONA was prepared from HUVEC cefisT LAPC-4AD and LAPC-9AD prostate cancer xenografts, as well as from human brain tissues. Normalization was performed by PCR using primers to acfin and GAPDHrSerni-quantitaSve PCR, using primers to STEAP-1, was performed at 27 and 30 cydes of amplification. As a control, PCR using primers to acfin is shown. Results show strong expression of STEAP-1 in HUVEC cells simlar to the expression*detected in prostate cancer xenograft tissues. Expression of STEAP-1 in HUVEC ceHs indicates that targeting STEAP-1 may also target endothelial cells of the neovasculature of the tumors. In F^ure14(d) picture of the RT- \ PCR agarosc gei is suown. in Figure 14(e) PCR products were quantitated using the Alphalmager software. Results show strong of expression of STEAP-1 in normal prostate amongst all the normal tissues tested. Upregulation of STEAP-1 expression was delected in prostate cancer pod, bladder cancer pool, kidney cancer pool, colon cancer pod, king cancer

pool, ovary cancer pod, and breast cancer pool. Strong expression of STEAfM was detected h cancer metastasis pool, prostate cancer xenograft pool, and prostate metastasis to lymph node.
STEAP-1 Expression in lymphoma patient specimens (Figure 14(f)}. First strand cDNA was prepared from a panel of lymphoma patient specimens. Normalization was performed by PCR using primers to acfin. Semi-quanb'taOve PCR. using primers to STEAP-1, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Expression was recorded as strong or medium, if signal is detected as 26 or 30 cydes of amplification respectively, and absent if no signal is detected even at 30 cycles of amplification. Results show expression of STEAP-1 In 8 of 11 (72.7%) tumor specimens tested.
Example 5: Splice Variants of STEAP-1
Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. . Splice variants are mRNA variants spliced dfferenfly from the same transcript In eukaryotes, when e muie-exoR-fSft^k transcribed from genomfc DMA, the initial RNA Is spliced to produce funeral inRNA,whfch has only exons and bused for translation inb an amino add sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have Afferent coding and/or non-coding (5* or 3' end) portions, from the original transcript Transcript variants can code for similar or different proteins with flie same or a sfmflar function or can encode proteins with dnerentfuncfions, and can be expressed in he same fissue at the same b'me, or in different tissues at (he same time, or in the same fissue at different times, or in
different tissues at different times. Proteins encoded by transcript variants can have sfcnflar or different cellular or
t ' %
extracellular localizations, e.g., secreted versus intracellular.
Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length doning experiment, or by use of fuB-tength transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-dusters and assembled into a consensus sequence. The original gene sequence is compared to (he consensus sequences) or other full-length sequences. Each consensus sequence is a potential spfice variant for that gene (see, e.g., Kan, L, et a/., Gene structure prediction and alternative splicing analyse using genomicaUy afigned ESTs, Genome Research, 2001 May, 11(5):889-900.) Even when a variant is identified lhat is not a full-length done, that portion of the variant is very useful for antigen generation and for further cloning of the fuHength splice variant, using techniques kr.own in the art
Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs indude FgenesH (A. Salamov and V. Solovyev, *Ab initio gene finding in Drosophila genomic DNA," Genome Research. 2000 Aoril;10(4):51&-22); Grafl (URL compbfo.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mitedu/GENXAN.html). For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett 2001 Jun 8; 498(2-3):2l2-6; de Souza, S.J., et a/., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl Acad Sd U S A. 2000 Nov 7; 97(23): 12690-3.
To further confirm the parameters of a transcript variant, a variety of techniques are avaSabte in the art such as full-length doning, proteomic validation, PCR-based validation, and 5' RACE validation, etc. (seee.g., Proteomic Validation: Brennan, S.O., et at, Albumin banks peninsula: a new termination variant characterized by etectrospray mass spectromelry, BiochemBiophysActa. 1999 Aug 17;1433(1-2):321-6;FerranUP,ef a/., Differential spldiigc^Dfe^nessenger RNA produces

forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1a97 Oct 1;249(1):1-7. For PCR-based Validation: Welfmann S, ef a/., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by UghtCyder technology, Clin Chem. 2001 Apr;47(4):654-60; Jia, HP., et a/., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan 24; 263(1-2):2m. For PCR-based and 5' RACE Validation: Brigle, K.E., ef a/., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta 1997 Aug 7; 1353(2): 191-8).
, ft is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps Is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that STEAP-1 has a particular expression profile related to cancer. Alternative transcripts and splice variants of STEAP-1 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
The exon composition of the original transcript, designated as STEAP-1 v.1, is shown in Table Ull. Using the full-
length gene and EST sequences, two transcript variants were identified, designated as STEAP-1 v.2 and v.3. Compared with
STEAP-1 v.1, franscript variant STEAP-1 v.2 did not spice out fatron 4 of STEAP-1 v.1 and variant STEAP-1 v.3 spliced out
one additional exon from inlron 4 of STEAP-1 v.1, as shown in Figure 11. Theoretically, each different combinaSon of exons
in spafial order, e.g. exons 2 and 3, is a potential spice variant Figure 11 shows the schematic alignment of exons of the
transcript variants. .
Example 6: Single ftlucleotide Polymorphisms of STEAP-1
A Single Nudeofide Polymorphism (SNP) is a single base pair variafion in a nudeofjde sequence at a specific location. At any given point of the genome, there are four possible nudsotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in me genome of an individual. Hapblype refers to the base pair sequence of more than one location oh (he same DMA molecule (or the same chromosome in higher organisms),
/•
often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino adds of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause Inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haptotypes) have many applications, inducing diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the geneSc relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, * SNP analysis to dissect human traits,' Curr. Opin. NeurobioL 2001 Oct; 11(5):637-641; M. Pirmohamed and B. K Park, 'Genetic susceptibility to adverse drug reactions,' Trends Pharmacol. Sd. 2001 Jun; 22(6}:298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses,' The use of single nucteofide polymorphisms fn the isolation of common disease genes,* Pharmacogenomics. 2000 Feb; 1{1):39-47; R. Judson, J. C. Stephens and A. Windemuth, The predictive power of haplotypes in cJinkal response," Pharmacogenomics. 2000 Feb.; 1(1):15-25),
SNPs are identified by a variety of art-accepted methods (P. Bean, The promising voyage of SNP target discovery," Am. Clirt Lab. 2001 Oct-Nov; 20(9}: 18-20; K. M. Weiss, 'In search of human variation,' Genome Res. 1998 Jut; 8{7):691-697; M. M. She, "Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and ywioiyping technologies," Cltn. Chem. 2001 Feb; 47{2}:164-172). For example, SNPs are identified by sequencing DNA fragments (hat show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence
96

data In public and private databases, one can discover SNPs by comparing sequences using computer programs (Z. Gu, L Hillier and P. Y. Kwok, "Single nudeotide polymorphism hunting in cyberspace," Hum. MufaL 1998; 12(4):221-225). SNPs can be verified and genotype or haptotype of an individual can be determined by a variety of methods Including direct sequencing and high throughput rrfeoarrays (P. Y. Kwok, "Mehods for genotyping single nucteotide polymorphisms," Annu. Rev. Genorwcs Hum. Genet 2001; 2235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathls, B. Erwfo, P. Grass, B. Nines and A Duesterhoeft, 'High-throughput SNP genotyping with toe Masscode system,* Mot. Kagn. 2000 Dec; 5(4}:329-340)."
Using the methods described above, fourteen SNPs were identified in the transcript from done GTH9, designated as STEAP-i v.2, at positions 602 (C/G), 386 (C/I), 1087 fT/G), 1447 (T/C), 1621 (AT), 1625 (Off), 1716 (C/A), 2358 (C/T), 2646 (T/G), 2859 (T/G), 2908 (AH), 3006 (G/C), 3107 (C/T), and 3180 (ATT). T$f transcripts or.proteins with attemative aHeles were designated as variants STEAP-1 v.4, v.5, v.6, v.7, v.8, v.9, v.10, v.11, v.12, v.13, v.14, v.15, v.16 and v.17, respectively. Figure 10 shows the schematic alignment of the SNP variants. Figure 12 shows the schematic aBgnment of protein variants, corresponding to nudeotide variants. These alteles of the SNPs, though shown separately here, can occur in different combinafons (haptotypes) and in any one of the transcript variants (such as STEAP-1 v.1 and v.3) that contains
the sequence context of (he SNPs. Eg., the first two SNP were also on STEAP-1 v.3 at foe same positions, but at 572 and
356, respectively, on STEAP-1 v.1.
Example 7: Production of Recomfamant STEAP-1 In Prokaiyotic Systems
To express recombinant STEAP-1 and STEAP-1 variants in prokaryotfc cells, the fun or partial length STEAP-1 and STEAP-1 variantcDNA sequences are donedhto any one of a variety (rf expression vectors known in 8ie art Thefufl length cONA, or any 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 or more contiguous amino acids from STEAP-1, variants, x>r analogs thereof are used.
A. In vitro transcription and franslafion constructs:
pCRII: To generate STEAP-1 sense and anti-sense RNA probes for RNA In situ investigations, pCRIi constructs {Invitrogen, Carlsbad CA) are generated encoding either aH or fragments of the STEAP-1 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of STEAP-1 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the eel and iissue_expression of STEAP-1 at foe RNA level. Transcribed
"•X
STEAP-1 RNA representing (he cDNA amino acid coding region of the STEAP-1 gene Is used In to vitro translafic" systems such as the TnT™ Coupled Reficulolysate System (Promega, Corp., Madison, VW) to synthesize STEAP-1 protein.
B. Bacterial Constructs:
pGEX Constructs: To generate recombinant STEAP-1 proteins in bacteria that are fused to foe Glutathione S-transferase (GST) protein, afl or parts of the STEAP-1 cDNA or variants are cloned kite the GST- ftision vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of recombinant STEAP-1 protein sequences with GST fused at the amino-terminus and a six histidine epilope (6X His) at the carboxyl-terminus. The GST and 6X His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the ftision protein with anti-GST and anfi-His antibodies. The 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3* end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreSdsston™ recognition site in pGEX-6P-1, may be employed such that it permits deavage of the GST tag from STEAP-1-celaled protein Thft amnWIlin resistance gene and p6R322 origin permits setectkxi and maintenance of the pGEX piasmids in £ coK.
pMAL Constructs: To generate, in bacteria, recombinant STEAP-1 proteins that are fused to maltose-binding protein (M8P), all or parts of the STEAP-1 cDNA protein coding sequence are fused to the MBP gene by cloning Into flie

pMAL-c2X and PMAL-p2X vectors {New England Btolabs. Beverly, MA). These constructs allow controlled expression of recombinant STEAM protein sequences with MBP fused al the amino-terminus and a 6X His epitope tag at In* carluXyJ-lerminas. The MBP and 6X His tags permit purification of Ihe recombinant protein from induced bacteria with toe appropriate affinity matrix and allow recognition of the fusion protein with anfi-MBP and anti-His antibodies. The 6X His epitope tag is generated by adding 6 Msfidine codons to the 3' cloning primer. A Factor Xa recognition site permits cteavage of the pMAL tag from STEAP-1. The pMAL-c2X and pMAL-p£X vectors are optimized to express the recombinant protein in toe cytoplasm or periplasm respec8vely. Periplasm expression enhances foldmg of proteins with disulfide bonds.
pET Constructs: To express STEAP-1 in bacterial cells, all or parts of the STEAP-1 cONA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wl). These vectors aBow tightly controlled expression of recombinant STEAP-1 protein In bacteria with and without fusion to proteins that enhance sdubtBty, such as NusA and Ihioredoxin (Trx), and epitope tags, such as 6X His and S-Tagw that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 411 such ftat regions of the -SllAF^-pft^efri^re 6>qjrassed-ss amino-terminal fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express STEAP-1 in toe yeast species Saccharomyces cer&visiae for generafion of recombinant protein 2nd funcfional studies, aH or parts of the STEAP-1 cDNA protein coding sequence are cloned into the pESCfamSh/of vectors each of which contain 1 of4selecJable markers, HJS3,TRP1,l£U2, and URA3{Stratagene, La Joda,CA). These vectors atow controlled expression from the same plasmW of up to 2 diferent genes w cloned sequences containing either Flag™ or Myc epftope tags in the same yeast cell. This system Is useful to confirm protein-protein
interactions of STEAP-1. In addition, expression in yeast yields similar posHranslab'onal ratifications, such as
\ glycosylations and phosphorylafions, that are found when expressed in eukaryofjc celts.
pESP Constructs: To express STEAP-1 Ni the yeast species Saccharomyces pombe, al or parts of toe STEAP-1 cDNA protein coding sequence are cloned Into the pESP family of vectors. These vectors allow controBed high level of expression of a STEAP-1 protein sequei ice that is fused at either toe amino terminus or at the carboxyl terminus to GST which aids purification of toe recombinant protein. A Flag™ epitope tag aBows detection of toe recombinant protein with anti-Flag™ antibody.
Example 8; Production of Recombinant STEAP-1 In Higher Eukatyotic Systems
A. Mammalian Constructs:
To express recombinant STEAP-1 in eukaryotic ceHs, the full or partial length STEAP-1 cDNA sequences can be cloned into any one of a variety of expression vectors known in toe art One or more of the fcfowing regions of STEAP-1 are expressed in these constructs, amino acids 1 to 339 of STEAP-1 v.1, v.4, amino acids 1 to 258 of v.2, amino acids 1 to 282 of vA or any 8.9,10,11.12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37, 38,39,40,41,42,43,44.45,46,47,48,49,50 or more contiguous amino acids from STEAP-1, variants, or analogs thereof, in certain embodiments a region of a specific variant of STEAP-1 is expressed that encodes an amino acid at a specific position which differs from toe amino acid of any other variant found at that position, in other embodiments, a region of a variant of STEAP-1 is expressed that lies partly or entirely within a sequence that is unique to (hat variant
The constructs can be transfected info any one of a wide variety of mammalian cefls such as 293T eels. Transfected 293T cell lysates can be probed with toe anti-STEAP-1 poiydonal serum, described herein.
pcDNA4/HisMax Constructs; To express STEAP-1 in mammalian cells, a STEAP-1 ORF, or portions thereof, of STEAP-1 are doned into pcDNA4/HfeMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven tomjhe

cytomegatowus (CMV) promoter and the SP16 translation^ enhancer. The recombinant protein has Xpress™ and six htsfidine (6X His) epilopes fused to Ihe amino-terminus. The pcDNA4frysMax vector also contains the bovine growtn hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with iiie SV4Q origin for episomal replication and simple vector rescue in cell fines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampidllin resistance gene and CdE1 origin permits selection and maintenance of the plasmkl in £ co/7.
pcDNA3.1/MvcHis Constructs; To express STEAP-1 in mammalian cells, a STEAP-1 ORF, or portions (hereof, of STEAP-1 with a consensus Kbzak translation initiation site was cloned into pcDNA3.1/MycHis Version A (InvBrogen, Carlsbad, CA). Protein expression is driven from the cytomegaloviais (CMV) promoter. The recombinant proteins have the myc epitope and 6X His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains Ihe bovine growth hormone (BGH) polyadenylation signal and transcription terminafion sequence to enhance mRNA stabffify, along with the SV40 origin for episomal replication and simple vector rescue In cell fines expressing the large T antigen. The Neomydh resistance gene was used, as ft allows for selection of mammalian cefis expressing the protein and the ampidin resistance gene and CdE1 origin permits selection and maintenance of the plasmid In £ coS.
pcONAS.ifCT-GFP-TQPO Construct; To express STEAP-1 h mammalian cells and to allow detection of tie recombinant proteins usinri fluorescence, a STEAP-1 ORF, or portions thereof, with a consensus Kbzak translation Initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression Is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyHermkius • facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains tie bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stabity along with the SV40 origin forepisomafrepGcation and simple vector rescue in eel ines expressing the large T anfigen. The Neomydn resistance gene allows for selection of mammalian cells that express the protein, and the ampkalin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in £ coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/N7-GFP-TOPO spanning the entire length of a STEAP-1 profebi.
PAPtag: A STEAP-1 ORF, or portions thereof, is doned into pAPtag-5 (GenHunter Corp. Nashvifle, TN). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a STEAP-1 protein while fusing the IgGic signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an aminc-terminal IgGs signal sequence is fused to the amino-terminus of a STEAP-1 protein. The resulting recombinant STEAP-1 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as Rgands or receptors that interact with STEAP-1 proteins. Protein expression is driven from the CMV promoter aid Die recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl-terminus that fadStates detecfion and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian eels expressing (he recombinant protein and the arnpidlrTn resistance gene permits selection of the plasmid in £ coS.
PtaoS: A STEAP-1 ORF, or portions thereof, was cloned into pTag-5. This vector is simflar to pAPtag but without
the alkaline phosphatase fusion. This construct generated STEAP-1 protein with an amino-terminal IgGic signal sequence
and myc and 6X His epitope tags at the carboxyl-lerminus that facilitate detecfion and affinity purification. The resulting recombinant STEAP-1 protein was optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with Ihe STEAP-1 proteins. Protein expression was driven from Ihe CMV promoter. The Zeocin resistance gene present in the vector allowed for selection of mammalian cells expressing the'protein, and the ampidllin resistance gene permits selection of the plasmid ki £ cofc

PsecFc: A STEAP-1ORF, or portions (hereof, was also doned into psecFc. The psecFc vector was assembled by cloning the human immunogtobufin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generated an lgG1 Fc fusion at the carboxyl-terminus of the STEAP-1 proteins, while fusing the IgGK signal sequence to N-terminus. STEAP-1 fusions utilizing the murine lgG1 Fc region are also used. The resulting recombmant STEAP-1 proteins were optimized for secretion into the media of Iransfected mammalian cells, and can were used as Immunogens or to identify proteins such as figands or receptors that interact with STEAP-1 protein. Protein expression is driven from the CMV promoter. The hygromydn resistance gene present in the vector allowed for selection of mammalian cells that express me recomfainant protein, and the ampicillin resistance gene permits selection of the plasmid in £ coli.
pSRa Constructs; To generatermammalian cell fines that express STEAP-1 consfitufively. STEAP-1 ORF. or.... portions mereof, of STEAP-1 were cloned into pSRa constructs. Amphotropic and ecotropfc retroviruses were generated by transfecSon of pSRa constructs Into the 293T-10A1 packaging fine or co-lransfection of pSRa and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus was used to infect a variety of mammafian cell Bnes, resulting in Gie integrationof Sie^onsd gsns, STEAP-1, Into ttie host ceB-lnes. Protein expression was driven from a long terminal repeat (LTR), The Neomytin resistance gene present In the vector allowed for setecfion of mammalian cells that express toe protein, and the ampiciilin resistance gene and ColE1 origin permit selection and maintenance of the piasmid in £cc,7. The retroviral vectors-were thereafter be used for infecfion and generatJon of various cell Bnes using, for example, PC3, NiH 3T3, TsuPrl, 293 or rat-1 cefls.
Additional pSRa constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyWerminus of STEAP-1 sequences to allow detection using anti-Rag antibodies. For example, the FLAG111 sequences' gat tac aag gat gacgacgataag3'(SEQ!DNO: 80) is added to doming primer at the 3" end of the ORF. Additional pSRa constructs were made to produce both amino-terminal and carboxyl-tefminal GFP and myc/6X His fusion proteins of me full-length STEAP-1 proteins.
Additional Viral Vectors; Additionalronstrncts are mate for virakned^^
High virus t'ter leading to high level expression of STEAP-1 is achieved in viral delivery systems such as adenovirat vectors and hbtpes amplicon vectors. A STEAP-1 coding sequences or fragments thereof are amplified by PCR and subdoned into (he AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, STEAP-1 coding sequences or fragments thereof are doned into the HSV-1 vector (Imgenex) to generate herpes vira! vectors. The viral vectors are thereafter used for bifecGon of various cell lines such as PC3, NIK 3T3,293 or rat-1 cells.
Regulated Expression Systems: To control expression of STEAP-1 in mammalian celts, coding sequences of STEAP-1, or portions thereof, are cloned into regulated mammalian expression systems such as-the T-Rex System (Invitrogen), the GeneSwtteh System (Invitrogen) and me tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant STEAP-1. These vectors are thereafter used to control expression of STEAP-1 in various ceil fines such as PCS, NIH 3T3,293 or rat-1 cefls. B. Baiulovirus Expression Systems
To generate recombinant STEAP-1 proteins in a baculovirus expression system, STEAP-1 ORF, or portions (hereof, are doned into the baculovirus transfer vector pBfueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-STEAP-1 is co-transfected with helper plasmid pBac-N-B!ue (Invitrogen) into SF9 (Spodoptera fnigiperda} insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for delate). Baculovirus is then collected from cell supernatant and purified by plaque assay.
100

Recombinant STEAP-1 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant STEAP-1 protein can be detected using anti-STEAP-1 or anWiis-tag antibody. STEAP-1 prolein can be purified and used in various cell-based assays or as immunogen to generate polydonal and monoclonal antibodies specific for STEAP-1.
Example 9: Antiaenicltv Profiles and Secondary Structure
Figures 5{a)-9(a) and 5(b)-9(b) depict graphically five amino acid profiles of the STEAP-1 variants 1 and 3 respectively, each assessment available by accessing the ProtScale website located on the World Wide Web at (URLexpasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.
These profite^Figure 5(a) and (b), Hydrophilictty, (Hopp T.P., Woods K.R., 1981. Proc. Nad. Acad. Set U.SA 78:3824-3828); Rgure 6(a) and (b), Hydropafhidty, (Kyte J., Doolittle RJ., 1982. J. Mol. Bfol. 157:105-132); Figure 7{a) and (b), Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); Figure 8(a) and (b), Average Ftexfefflty, (Bhaskaran R, and Ponmiswamy P.K., 1988. tot J. Pepl Protein Res. 32:242-255); Rgure 9{a) and (b). Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify anfigenfc regions cf the STEAP-1 protein. Each of foe above amino acid profiles of STEAP-1 were generated using fte fallowing ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino add profile values normalized to Se between 0 and 1.
, Hydrophifidty (Figure 5(a) and (b)), Hydropathidty (Figure 6(a) and (b)) and Percentage Accessible Residues (Figure 7(a) and (b)) profiles were used to determine stretches of hydrophlfic amino adds (i.e., values greater than 0.5 on the Hydrophilidty and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathidty profile). Such regions are Dkely to be exposed to the aqueous environment, be present on (he surface of the protein, and thus avaflable for immune recognition, such as by antibodies.
Average Flexibility (Figure 8(a) and (b)) and Beta-turn (figure 9(a) and (b)) profiles determine stretches of amino adds (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures su^ as beta sheets and alpha helices. Such regions are also more likely to be exposed on the prolein and thus accessible to immune recognition, such as by antibodies.
Ant/genie sequences of (he STEAP-1 protein and of the variant proteins indicated, e.g., by (he profiles set forth in Figure 5(a) and (b), Figure 6(a) and (b), Figure 7(a) and (b), Figure 8(a) and (b), and/or Figure 9(a) and {b) are used to prepare bnmunogens, either pepttdes or nucleic acids (hat encode them, to generate therapeutic and diagnostic anfi-STEAP-
1 antibodies. The immunogen can be any 5,6.7,-8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,
40,45,50 or more than 50 contiguous amino adds, or the corresponding nudefc adds (hat encode ftem, from (he STEAP-1
protein variants listed in Figures 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region
of at least 5 amino adds of Figures 2 and 3 in any whole number increment that includes an amino acid position having a
. 4 2 and 3 in any whole number increment that includes an amino add position having a value less than 0.5 in the
Hydropathidty profile of Figure 6(a) and (b); a peptide region of at least 5 amino adds of Figures 2 and 3 in any whole
V
number increment that indodes an amino acid position having a value greater than 0.5 in (he Percent Accessible Residues profile of Figi ire 7(a) and (b); a peptide region of at least 5 amino adds of Figures 2 and 3 in any whole number increment that indudes an amino add position having a value greater than 0.5 in the Average Flexibility pt«file on Figure 8(a) and (b); and, a peptide region of at least 5 amino acids of Figures 2 and 3 in any whole number increment that indudes an amino

add position having a value greater than 0.5 in the Beta-turn profile of Figure 9(a) and (b). Peptide immunogens of the invention can ateo comprise nucleic acids that encode any of the foiyoiiiy.
All immunogens of the invention, peptide or nucleic ackl, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excjpient compatible with human physiology.
The secondary structures of STEAP-1 variant 1 and variant 3, namely the predicted presence and location of alpha
- helices, extended strands, and random coils, are predicted from the respective primary amino acid sequences using the
HNN - Hierarchical Neural Network method (Guermeur, 1997, http^/pfail.ibcp.fr/cgi- ;
bin/npsa_automatpl?page=npsa_nn.htm!)l accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.cn/tooteO. The analysis indicates that STEAP-1 variant 1 is composed of 64.60% alpha helix, 4.72% extended strand, and 30.68% random coil (Figure 13a). STEAP-1 variant 2 Is composed of 62.79% alpha helix, 3.10% extended strand, and 34.11% random coil (Figure 13b). STEAP-i variants is composed of 58.87% alpha helix, 5.32% extended strand, and 35.82% random coil (Figure 13c).
Analysis for the potential presence of transmembrane domain; in STFM^l variants _were canied out using a variety of transmembrane prediction algorithms accessed from (he ExPasy molecular biology server located on the World Wide Web at (.expasy.cn/tools/). Shown graphically are he results of analysis of variant 1 depfcfing the presence and location of 6 transmembrane domains using the TJMpred program (Figure 13d) and TMHMM program (Figure 13e). Also shown are the results of analysis of variant 2 depicting the presence and location of 4 transmembrane domains using TMpred (Figure 13f) and 3 transmembrane domains using TMHMM (Figure 13g). Analysis of variant 3 predicts the presence of 4 transmembrane domains using the TMpred (Figure 13h) and 3 transmembrane domains with TMHMM (Figure 13i). The results of each program, namely the, amino acids encoding the transmembrane domains are summarized In Table XX
Example 10: Generation of STEAP-1 Polydonal Antibodies
Polyclonal antibodies can be raised In a mammal, for example, by one or more Injections of an immunizing agent
and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant win be injected in the mammal by mulfiple
subcutaneous or intraperitoneal injections. In addition to immunizing with a full length STEAP-1 protein variant, computer
algorithms are employed in design of immunogens that based on amino add sequence analysis contain characteristics of
being antigenic and available for recognition by the immune system of the Immunized host (see the Example entitled
'Antigenidty Profiles and Secondary Structure*). Such regions would be predicted to be hydrophific, flexible, in beta-turn
conformations, and be exposed on the surface of the protein (see, e.g., Figure 6(a) and (b), Figure 6(a) and (b), Figure 7(a)
and (b), Figure 8(a) and (b), and/or Figure 9{a) and (b) for amino acid profiles that indicate such regions of STEAP-1 protein
variants 1 and 3). „
For example, recombinant bacterial fusion proteins or peptides containing hydrophiftc, flexible, beta-turn regions of STEAP-1 protein uarianls are used as antigens to generate porydonal antibodies in New Zealand White rabbits or monodonal antibodies as described in example entitled ('Generation of STEAP-1 Monoclonal Antibodies (MAbs). For example, such regions include, but are not limited to, amino acids 1-40, amino acids 143-165, amino acids 180-220, of STEAP-1 variants 1,2, and 3, amino acids 312-339 of STEAP-1 variant 1. and amino acids 250-282 of STEAP-1 variant 3. It is useful to conjugate the immunizing agent to a protein known to be imrmmogenic in the mammal being immunized. Examples of such immunogenic proteins include, but sre not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 250-282 of STEAP-1 variant 3 is conjugated to KLH. This peplide is then used as jmmunogen. Alternatively the immunizing agent may include all or portions of the STEAP-1 variant proteins, analogs or fusion proteins thereof. For example, the STEAP-1 variant 1

I

amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. In another embodiment, amino acids 250-282 of STEAP-1 variant 1 is fused to GST using recombinant techniques and the pGEX expression vector, expressed, purified and used to immunize a rabbit Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
Other recombfnant bacterial fusion proteins (hat may be employed include maltose binding protein, LacZ,
thioredoxin, NusA, or an immunogiobulin constant region (see Hie section entitled "Production of STEAP-1 in ProkaryoBc
Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Unstey,
P.S., Brady, W., Umes, M., Grosmaire, L, Damte, N., and Ledfaetter, L(1991)J.Exp. Med. 174,561-566). . _
In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-ftisfon vectors (see the secfjon entitled 'Production of Recombinant STEAP-1 In Eukaryolic Systems'), and retain post-translalional modifications such as glycosylafions found in native protein. In one embodiment amino acids 185-218 of STEAP-1 varianM were doned into toe_ _ TagS mammalian secretion vector, and expressed In 293T cells. The recombinant proton was purified by metal chelate chromatography from tissue culture supematants of 293T usis stably expressing the recombinant vector. The purified Tag5 STEAP-1 variant 1 protein is then used as immunogen.
During the immunization protocol, ft is useful to mix or emulsify (he antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete FreuncTs adjuvant (CFA) and MFt-TDM adjuvant (monophosphoryi Upid A, synthetic trehalose dicorynomycolate).
In a typical protocol, rabbits are initially immunized subcutaneous!/ with up to 200 ng, typically 100-200 fig, of fusion protein or peptide conjugate^to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 jig, typically 100-200 jig, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the filer of the antiserum by ELISA.
To test reactivity and specificity of immune serum, such as (he rabbit serum derived from immunization with the GST-fusion of STEAP-1 variant 1 protein, the fuHengtti STEAP-1 variant 1 cDNA is doned into pCDNA 3.1 myc-hb
'expression vector (Invitrogen, see the Example entitled 'Production of Recombinant STEAP-1 in Eukaryofic Systems'). After transfecfion of the constructs into 293T cells, cell I/sates are probed with the anti-STEAP-1 serum and with an B-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured STEAP-1 protein using the Western blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant STEAP-1-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenous!/ express STEAP-1 are also carried out to test reactivity and specificity.
Anti-serum from rabbits immunizecTwith STEAP-1 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-STEAP-1 variant 1 fusion protein is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-STEAP-1 fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbtts as wefi as

fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free pepGde.
Example 11: Generation of STEAP-1 Monoclonal Antibodies fMAbs)
In one embodiment, therapeutic MAbs to STEAP-1 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would bind, internalize, disrupt or modulate the biological function of the STEAP-1 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such MAbs Include those designed to encode or contain the extracellular domain or the entire STEAP-1 protein variant sequence, regions predicted to contain functional motifs, and regions of the STEAP-1 protein variants predicted to be antigenic from computer analysis of (he amino acid sequence (see, e.g., Ffeure 6{aHb), Figure 6{aHb), Figure 7(a}-(b), Figure 8(a)-(b), or Figure 9{aHb}, and the Example entitled 'Anfigeniclry Profiles and Secondary Structure*). Immunogens include pepBdes, recomblnant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murtne IgG FC fusion proteins, in addition, pTAGS protein, DMA vectors encoding the pTAGS cells engineered to express high levels of a respective STEAP-1 variant wch as 293T-STEAP-1 variant 1 or 3T3, RAT, or 300.19-STEAP-1 variant Imurine Pre-B cete, are used to immunize mice.
To generate MAbs to STEAP-1 variants, mice are first immunized totraperttoneaBy (IP) or in the foot pad with, typicaHy, 10-50 pg of protein Immunogen or 107 STEAP-1-expressing cells mixed In complete Freund1 s adjuvant Examples of other adjuvants used are Titermax (Sigma) and Immuneasy (Qiagen). Mice are then subsequently Immunized IP every 2-4 weeks with, typically, 10-50 ng of protein Immunogen or 107 During the immunization protocol, test bleeds are taken J-10 days following an injection to monitor liter and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determmed by ELJSA, Western blotting, immunopredpitation, fluorescence microscopy, and flow cytomelric analyses, fusion 'and hybridoma generation is then carried out with established procedures well known m the art (see, e.g., Hariow and Lane, 1988).
The binding affinity of STEAP-1 variant 1 specific monoclonal antibodies was determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which STEAP-1 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of ski h flie art The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Wetford K. 1991, Opt Quant Elect 23:1; Morton and Myszka, 1998, Methods in

Enzymology 295:268) to monitor Womolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
To generate monodonal antibodies specific for other STEAP-1 variants, immunogens are designed to encode
amino acid sequences unique to the variants. In one embodiment, a peptide encoding amino adds unique to STEAP-1
variants are synthesized, coupled to fOH, and used as immunogen. In another ernbodnnent, peptjdes or bacterial fusion
proteins are made that encompass (he unique sequence generated by alternative splicing in the variants. Hybridomas are
then selected that recognize the respective variant specific antigen and afeo recognize the fun length variant protein
expressed in cells. Such selection utilizes immunoassays described above such as Western blotting, immunopredpltatton,
and flowcytometry. - ..,^,- In one embodiment the invention provides for monoclonal antibodies designated X9Z1.30.1.1(1) (a.k.a. M2/92.30) and X120.545.1.1 (a.k.a M2.120.545). M2/92.30 and M2/120.545 were idenfified and are shown to react and bind with cell surface STEAP-1 (See, Figures 15 and 18). Rgure 16 shows that the anfi-STEAP-1 MAb M2/3Z30 binds endogenous ceH surface STEAP-1 expressed in bladder and prostate cancer xenograft cells. Additionally, M2/92.30 reacts and binds with marine STEAP-1 as shown in Rgure 17.
The anti-bodies designated X92.1.30.1.1(1) (a.lca M2/92.30) and X120.545.1.1 (a.lca. MZ120.545) were sent (via Federal Express} to the American Type Culture Coflecfion (ATCC), P.O. Box 1549, Manassas, VA 20106 on OS-February-20Q4 and assigned Accession numbers PTA-S802 and PTA-5803 respectivety.
To done the M2/Xa2.30 and M2/X120.545 antibodies the following protocols were used. Hybridoma ceds were rysed with Trizo) reagent (Us Technologies, Gibco BRL). Total RNA was purified and quantified. First strand cDNAs was generated from total RNA with ofigo (dT)12-18 priming using the Gibco-BRL Superscript PreampfiScafion system. PCR products were cloned into the pCRScript vector (Stratagene, La Jolla). Several dones were sequenced and (he variable heavy ("VH") and variable light (*VL") chain regions determined. The nudeic add and amino add sequences of M2/X92.30 and M2/X120.545 variable heavy and light chain regions are fisted in Figure 19(a)-19(d) and Figure 20{a)-{e).
Example 12: Characterization of STEAP-1 Antibodies
A. Cell Surface Binding
Reactivity of STEAP-1 antibodies with a STEAP-1-related protein can be established by a number of weO known means, including Western blot, immunopredpitafion, EUSA, and FACS analyses using, as appropriate, STEAP-1-related proteins, STEAP-1-expressing cells or extracts thereof. As shown in Figure 15 FACS analysis of recombinant 3T3 and Rat-1 cells stably expressing either STEAP-1 or a control stained with anli-STEAP MAb M2/92.30 (10 ug/ml) and cell surface .bound MAb was detected with a goat anti-mouse IgG-PE conjugate secondary reagent TJie stained cefls were then subjected to FACS analysis. As indicated by the fluorescent shift of the Rat1-STEAP1 and 3T3-STEAP1 eels compared to the respective control cefls, M2/92.30 specifically binds to eel surface STEAP1.
. *r
in addition, when UGB1 bladder cancer cells and LAPC9 prostate cancer cells were stained with 10 ug/ml of either MAb M2/92.30 orj/vith a control anti-KLH MAb. Surface bound MAb 92.30 was detected with goat-anti-mouse IgG-PE conjugated secondary Ab. Stained cells were then subjected to FACS analysis. These results demonstrate that the anti-STEAP1 MAb M2/92.30 spedficaily binds endogenous cell surface STEAP1 expressed in bladder and prostate cancer xenograft cells (Figure 21).,
STEAP-1 M2/92.30 is also shown to bind to murine STEAP-1 protein (See Figure 17). In this experiment 293T cells were transiently transfected with either pCDMA3.1 encoding the murine STEAP1 cONA or with an empty vector. 48 hours later, the cells were harvested and stained with antj*STEAP1 MAb M2/92.30 (10 ug/ml} and ceH surface bound MAb


92.30 was detected with a goat anti-mouse IgG-PE conjugate secondary reagent Cells wero then subjected to FAGS analysis. STEAP-1 M2/92.30 was shown to bind specifically to STEAP-1-expressed 293T ceils.
STEAP-1 M2/120.545 is also shown to specifically bind to STEAP-1 (See Rgure 18). 3T3-neo (Panel A. filled histograms) and 3T3-STEAP1 cells (Panel A, no fill histograms) and Ratt-neo (Panel B, filled histograms) and Ratl-STEAP cells (Panel B, no fill histograms) were stained with MAb M2/120.545 (10 ug/ml) and surface bound MAb was detected with goat antt-mouse IgG-PE conjugated secondary Ab. Cells were then subjected to FACS analysis. As indicated by the fluorescence shift of the 3T3-STEAP1 and Rat1-STEAP1 cells compared to their respective neo controls, MAb M2/120.545 specifically binds cell surface STEAP1. In Panel C, LNCaP cells were stained with either MAb M2/120.545 or a control anti-KLH MAb and subjected to FACS analysis as above. In Panel 0, Fluorescence microscopy of the M2/120.545 stained LNCaP cells showing bn'ght cell surface fluorescence.
Reactivity and specificity of M2/92.30 and M2/120.545 were also determined by immunoprecipitation. Figure 25 shows 3T3-STEAP1 and 3T3-neo cells were lysed to RIPA buffer (25mM Tris-CI pH7.4; 150 mM Nad, 0.5mM EDTA, 1% Triton X-100, 0.5% deoxychdic add, 0.1% SOS, and protease inhibitor cocktail). The ceil lysates were predeared with protein 6 sepharose beads and then Incubated with 5 ug of either MAb M2/92.30 or M2/120.545 for 2 hours at room temperature. ProteinG beads were added and the mixture was ftirflier incubated for 1 hour. The immune complexes were washed and sofubflized in SOS-PAGE sample buffer. The solublBzed samples were then subjected to SOS-PAGE and Western blot analysis using a rabbit anti-STEAP pAb. Whole cell lysates of 293T cells transfected with STEAP1 was also run as a positive oontrol. Arf immuncrsacfive band of -37 kO was seen only in samples derived from 3T3-STEAP1 ceDs indicative of specific immunoprecipitation of STEAP1 by both M2/92.30 and M2/120.545 MAbs.
i B. STEAP-1 Antibody Intemateation
Immunotherapy based on the delivery of towns towards specific ceil targets using monoclonal antibodies is considered a modality in the therapy of malignancies. The general principle is the delivery of toxins or anfineoplastfc drugs to cancer cells with molecules that bind to antigens or receptors that are either uniquely expressed or.overexpressed on tie target cells relative to normal tissues.
Immunotoxins consist of cell selective figands (usually monoclonal anfibodies or cytokines) linked covalc^y to toxins. The Interaction of antibody or ligand with ceO surface receptors triggers intemafizatJon. hi defined intracellular vesicle compartments, the toxin moiety escapes to the cytosol, where it catalyGcally alters critical cell functions leading to eel death. See, Frankel AE., increased Sophistication of Immunotoxins, Clinical cancer research 8:942-944, (2002) and Allen TM, Ugand-Targeted Therapeutics in Anti-cancer Therapy; Nature Reviews. 2:750-760, (2002).
Sapprin is a ribosome-inacfivating protein (RIP) that catalyzes the in vitro depurinarjon of a specific adenine residue
»
in large ribosomal RNAs. EndoY, et. a/., Mechanism of Action of the Toxin Lectin Ridn on Eukaryotic Cells; The Site and Characteristics of the Modification in 28S RNA Caused by the Toxin, J. Biol. Chem. 262,5908-5912, (1987). It usually cannot enter cefls unless complexed to an appropriate carrier molecule. Covalenl conjugation of saporin to monoclonal antibodies that-recognize tumor antigens produces immunotoxins that possess both cancer cell selectivity and are internalized. See, Raved, DJ, Sapoin Immunotoxins, din. Top. Mxrobiol. Immunol. 234:51-61, (1998) and Ravell DJ, et at., Therapy of Human T-cell Acute Lymphoblasfic Leukemia with a Combination of Anti-CD? and An(i-CD38-Saporin Immunotoxins is Significantly Better than Therapy with Each Individual Immunotoxins. Br. J. Cancer 84:571-578. (2001). These mofcc;!cs have recently entered phase I clinical trails for leukemia and multiple myeloma. Foon KA. Monoclonal Antibody Therapies for Lymohomas..Cancer J. 6:273-278, (2000).

The intemaiization of STEAP-1 M2/92.30 is shown in Figure 22. In this experiment, PC3-STEAP1 cells were stained at 4 degrees C with M2/120.545 MAb (10 ug/ml), washed, then incubated with goat anti-mouse IgG-PE conjugate secondary Ab. One-half of the cells were moved to 37 degrees C for 30 minutes and the other half remained at 4 degrees C. Cells from each treatment were then subjected to fluorescent microscopy. Cells that remained at 4 degrees C showed bright staining on the circumference of the cell surface. Cells that were moved to 37 degrees C showed loss of the staining on the cell circumference and the appearance of punctate and aggregated fluorescence indicative of capping and intemaiization.
STEAP-1 intemaiization by STEAP1 M2/120.545 MAb is shown in Rgure 23. PC3-STEAP1 cells were stained at 4C with M2/120.545 MAb (10 ug/ml), washed, then incubated with goat anti-mouse IgG-PE conjugate secondary Ab. One-half of the cells were moved to 37C for 30 minutes and the other half remained at 4C. Cefls from each treatment were then subjected to fluorescent microscopy. Cells that remained at 4C showed bright "ring-like' cell surface fluorescence. Cells that were moved to 37C showed loss of the "ring-like" cell surface fluorescence and the appearance of punctate and aggregated fluorescence indicative of capping and intemaiization.
One approach for selecting appropriate antibody candidates forimmunotoxin delivery employs killing with a secondary antibody conjugated with a drug or toxin molecule. The secondary conjugated antibody piggybacks onto the primary antibody allowing the evaluation of the primary antibody to internalize and traffic to appropriate intracellular compartments. Once the conjugate is internalized, sapodn breaks away from the targeting agent and inacSvaies the ribosomes to eliminate target cells. Kohls MD and Lappi DA. MAb-ZAP: A Tool for Evaluating AnBbody Efficacy for Use In an immunotoxin. Bib Techniques. 28(1): 162-165 (2000).
Tb select the appropriate antibody candidate using the above approach, a secondary immunotoxin, anti-mouse IgG - saporin conjugates (Advanced Targeting Systems, San Diego, CA) was used to demonstrate that murine Steap-1 M2/120.S45 enters target cells via expression of Steap-1 on the ceil surface of LNCaP cell. The following protocols were used. LNCap cells were plated at 5000 cells/90 \il /well in 96-well plate and incubated overnight Second immunotoxin conjugates (anti-mouse kjG-saporine and anti-goat IgG-saporin) and anti-mouse IgG were made in cell medium containing the final concentration at 100 ng/ml. 10 pi were added to each well. The primary antibody L added at the concentration from 1-1000 ng/ml. The plates were incubated 72 hours and the viability was determined by MTT assay. The results in Figure 24 show that LNCaP cells were killed in the presence of anfj-mouse IgG-saporin. No effects were defected with either the secondary antfcody alone (anti-mouse IgG) or nonspecific secondary antibody conjugates (anti-goat IgG saporin). No toxkaty was observed with the primary antibody (M2/120.545) alone tested up to 1 pg/ml.
Example 13: HLA Class I and Class H Binding Assays
HLA class I and class II binding assays using purified HLA molecules are performed in^ccordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney ef a/., Current Protocols «Immunology 18.3.1 (1998); Sidney, ef a/., J, Immunol. 154:247 (1995); Sette, ef a/., Mot. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various untabeled peplide inhibitors and 1-10 nM «3-radfolabeled probe peplkJes as described. Following incubation, MHC-peplide complexes are separated from free pepttde by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.
Since under these conditions [label][HLAJ, the measured ICw values are reasonable approximations of the true Ko values. Peplide inhibitors are typically tested at concentrations ranging from 120 ng/ml to 1.2

l, and are tested in two to four comptetely independent experiments. To allow comparison of (he data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the ICso of a positive control for inhibition by the ICso for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into ICso nM values by dividing the ICso nM of the positive controls for inhibition by the relative binding of the peptide of interest This method of data compilation is accurate and consistent for comparing peptides that have been (esied on different days, or with different tots of purified MHC.
Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).
Construction of "Minlaene" Murti-Eoitope DNA Plasmids This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epftope analogs as described herein.
A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA A1 and -A24 motif-bearing pepSde epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived STEAP-1 , are selected such that mulfipte supermofifs/rnotrfs are vaprssented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from STEAP-1 to provide broad popufafion coverage, i.e, both HLA DR-1-4-7 supermofif-bearing .epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct The selected CTL and HTLepHopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may 'additionally include sequences that direct the HTL epitopes to fte endoplasmic reUculurn. For example, the li protein may be fused to one or more HTL epitopes as described in the art, wherein the CUP sequence of the li protein is removed and replaced with an HLA class II epitope sequence so that HLA class il epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene cc ^positions are available and known to those of skill in the ait
The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-ligh t chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc andTiis antibody epitope tag coded for by the pcDNA 3,1 Myc-His vector.
Overlapping oligonudeotides that can, for example, average about 70 nudeotides in length with 15 nucteotide overlaps, are synthesized and HPLC-purified. The ofigonucteofides encode the selected peptide epitopes as well as appropriate tinker nudeotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping ofigonudeofides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C for 15 sec, anneafing temperature (5* below the lowest calculated Tm of each primer pair) for 30 sec, and 72°C for 1 min.
For example, a minigene is prepared as follows. For a first PCR reaction, 5 fig of each of two oligonucteofides are . \ annealed and extended: In an example using eight ofigonudeuiides, i.e., four paii» uf pri;»cr3, oligcr.udcciidcs 1+2, 3+4, 5+6, and 7+8 are combined in 1 00 j«l reactions containing Pfu polymerase buffer {1x= 10 mM KCL, 10 mM (NI-MJzSCx, 20 mM Tris-chloride, pH 8.75, 2 mM MgSQ«, 0.1 % Triton X-100, 100 ng/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pto polymerase. The fuB-tertglh dimer products are gel-purled, and two reactions containing the product of 1 +2 and 3+4, and

the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are Ihen mixed, and 5 cycles of annealing and extension earned out before flanking primers are added to amplify the fa!! length product The fu8-length product is gel-purified and cloned into pCR-b!unt (Invitrogen) and individual dones are screened by sequencing.
Example 15; The Plasmld Construct and the Degree to Which It Induces Immunooenlcitv.
The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vftro by determining epitope presentation by APC following transducfion or transfecfion of foe APC with an epitope-expressing nucleic acid construct Such a study determines 'antigenicity" and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quanlitafion can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts era/., J. /mmuno/. 156:683-692,1996; Demote ef a/., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be esfimated by measuring foe amount of lysisorl^phokinejetease induced by diseased^transfected target cote, and then determining the concentration of pepflde necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama ef a/., J. Immunol. 154:567-576,1995).
Alternatively, immunogenicity is confirmed through 'm vivo injections into mice and subsequent In vSro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferaBort assays, respectively, as detailed e.g.. In AJexanderef a/., Immunity 1:751-761,1994.
For example, to confirm the capacity of a DMA minigene construct containing at least one HLA-A2 supermofif pepfidfc to induce CTLs m ww^HIAA/LI/K6 transgenic mice, for example, are immunized intramuscularly wift 100 ng of naked cDN A. As a means of comparing the level of CTLs Induced by cDN A Immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single porypeptide as they would be encoded by the minigene.
SpJenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide "~ """ epitopes encoded in the minigene or the pofyepftopfc peptide), then assayed for peptkte-specjfic cytotoxfc activity in a 51Cr release assay. The results hdteate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene va^ne and polyepitopic vaccine.
It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermoBf pepfide ' epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermoff epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.
To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for r those epitopes that cross react with the appropriate mouse MHC molecule, i-ANestricted mice, for example,, are Immunized intramuscularly with 100 ng of plasmid DNA. As a means of comparing the level of HTLs induced by ONA Immunization, a group of control animals is also immunized with an actual peptide composition emulsified h complete Freuntfs adjuvant CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated wi(h each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-lhymidine incorporation proliferation assay, (see, e.g., Alexander ef a/. Immunity 1:751-761,1994). The results mdkate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.
DMA mtnigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombtnantprotein

(e.g., Bamett ef ai, Aids Res. and Human flefrowruses 14, Supplement 3:3299-3309,1998) or recombinant vaccinia, for example, expressing a rranigene or DNA encoding the comolete protein of interest (see, e.g., Hanke et a/., Vaccine 16:439-445,1998; Sedegah et a/., Proc. Nail Acad. Scl USA 95:7648-63,1S98; Hanke and McMfchaei, Immunol. Letters G6:177-181,1999; and Robinson et a/., A/ah/re Wed. 5:526-34,1999).
For example, the efficacy of the DNA minigene used in a prune boost protocol is initially evaluated in transgenic mice. In this example, A2.1/KI» transgenic mice are immunized JM with 100 yg of a DNA minigene encoding the immunogenic pepfktes including at least one HLA-A2 supermotif-bearing peptide. After an hcubafion period (ranging from 3-9 weeks), the mice are boosted IP with 1Q7 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 ^g of ON A or reccHT^ant vaccinia vw'th^ minigene sequence, or with DNA encoding the mtnigene, but without the vaccinia boost After an additional incubation period of two weeks, sptenocytes from the mice are immediately assayed for peptide-specffic activity in an HJSPOT assay. Additionally, sptenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specnlc activity in an alpha, beta and/or gjamrna IFN EUSA.
It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward toe HLA-A2 supermotif peptktes than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols In humans is described below in the Example entitled "Induction of CTL Responses Using a Prime Boost Protocol."
Example 16: Potycpitopic Vaccine Compositions from Multiple Antigens
Example 17: Use of peotides to evaluate an Immune response
Peptides of foe invention may be used to analyze an immune response for the presence of specific anybodies, CTL or HTL directed to STEAP-1. Such an analysis can be performed In a manner described by Ogg ef a/., Scfenee 279:2103-2106,1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
In this example highly sensitive human leukocyte antigen tetrameric complexes jptelramers") are used for a cross-sectional analysis of, for example, STEAP-1 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages^ disease or following immunization comprising a STEAP-1 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey ef at., N. Engl. J. Med. 337:1267,1997). Briefly, purified HLA heavy chain (A*0201 in this example) and p2-microglobulin are synthesized by means of a prokaryofic expression system. The heavy chain is modified by deletion of the transmembrane-cvtosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biolinylalion site. The heavy chain, p2-microglobulin, and peptide are refolded by dflution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotmylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5' triphosphate and magnesium. Streptawdin-phycoerythrin conjugate is added in a
HO

1:4 molar ratio, and liie ieirameric product is concentrated to 1 rag/ml. The resulting product is referred to as tetramer-phycoerythrin.
For the analysis of pafient blood samples, approximately one million PBMCs are cenlrifuged at 300g for 5 minutes and resuspended in 50 nl of cold phosphate-buffered saline. Tri-cotor analysis Is performed with the telramer-phycoerythrin, along with anS-CDfJ-Tricotor. and anti-CD38. The PBMCs are incubated With tetramer and antibodies on Ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples, ^gpjitals for
, .«,,iK ,:»,-!"-•?: ^~IirI"-~"
the letramers indude both A*0201-negative individuals and A*0201*positive non^seasedtJoTiors. The percentage of cells . stained with the teframer is then determined by flow cytometry. The resulls indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating tie extent of immune response to the STEAP-1 epitope, and thus (he status of exposure to STEAP-1, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 18; Induction of Immune Responses Using a Prime Boost Protocol
A prime boostprotocol similar in its underlying principle to ftatused toconfirm flieefficacy of a DMA vaccine in transgenic mice, such as described above in the Example entitled The Plasmid Construct and the Degree to Which It Induces Immunogenia'ty,' can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DMA foBowed by a boost using recombinant virus encoding the vaccine, or recombinant proteflT/polypeptkte or a peptide mixture administered in an adjuvant
For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled "Construction of "^mlgene" Mulfi-Epitope DNA Plasmids" in the form of naked nucleic acid administered IM {or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 ng) can also be administered using a gene gun. Following an incubation period of 34 weeks, z booster dose is then administered. The booster can be -recombinant fowlpox virus administered at a dose of 5-107 to 5x109 pfu. An alternative recombinant virus, such as an MVA, -canarypox, adenovirus, or adeno-associated virus, can also be used for foe booster, or the poiyepitopic protein or a mixture of the peptides can be administered For e valuaSon of vaccine efficacy, pafient blood samples are obtained before immunization as welt as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononudear cells are isolated from fresh heparinized Wood by FtcolI-Hypaque density gradient centrifugatfon, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL acSvity.
Analysis of the results indicates that a magnitude of response sufficient to achieve a fterapeufic or protective
immunity against STEAP-1 is generated. *
Example 19: Complementary Polynucleotides
Sequences complementary to fe? STEAP-1 -encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring STEAP-1. Although use of ofigpnudeotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonudeotides are designed using, e.g., OUGO 4.06 software (National Biosciences) and the coding sequence of STEAP-1. To inhibit transcription, a complementary oiigonudeotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oiigonudeotide is designed to prevent ribosomal binding to a ST6AP-1 -encoding transcript.
in

Example 20; Purification of Naturally-occurring or Recombinant STEAP-1 Using STEAP-1 -Specific Antibodies Naturally occurring or recombiiiant STEAP-1 is substantially purifjed by immunoaffinity chromatography using
anlibodies specific for STEAP-1. An immunoaffirnty column is constructed by covalendy coupling anti-STEAP-1 antibody to
an activated chromatographic resin, such as CNBr-acdVated SEPHAROSE (Amersham Pharmacia Biotech). After the
coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing STEAP-1 are passed over the irnmunoaffinity column, and the column is washed under
conditions that allow the preferential absortance of STEAP-1 (e.g.. high ionic strength buffers in the presence of detergent).
The column is eiuted under conditions that disrupt antibody/STEAP-1 binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such asureacf thiocyanateion), amiGCRPiscdlected.^^,^^ .....
Example 21: Identification of Molecules Which Interact with STEAP-1
STEAP-1, or biologically active fragments thereof, are labeled with 1211 Bdton-Hunter reagent (See, e.g., Bolton ef al. (1973) Bfochem. J. 133:529.) Candidate molecules previously arrayed in the wefls of a muffi-weB plate are incubated with the labeled STEAP-1, washed, and any wells with labeled STEAP-1 complex are assayed. Data obtained using different concentrations of STEAP-1 are used to calculate values for the number, affinity, and association of STEAP-1 wEft the candidate molecules.
Example 22: In Vivo Assay for STEAP-1 Tumor Growth Promotion
The effect of the STEAP-1 protein on tumor cell growth Is evaluated 'm vivo by evaluaSng tumor development and growth of cells expressing or lacking STEAP-1. For example, SCID mice are injected subcutaneously on each flank with 1 x 10s of either 3T3, or prostate cancer ceil lines (e.g. PCS cells) containing tkNeo empty vector or STEAP-1. At feast two strategies may be used: (1) Constitutive STEAP-1 expression under regulation of a promoter such as a consfituBve promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 pubfished 5 July 1989), adenovirus (such as Adenovirus 2), bovine papllloma virus, avian sarcoma virus, cytomegatovirus, a retrovirus, hepatitis-IB virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immune-globulin promoter, provided such promoters are compatible with (he host cefl systems, and (2) Regulated expression
"under control of an inducible.vector system, such as ecdysone, tetracycFme, eta, provided such promoters are compatfcfe with the host cell systems. Tumor volume is then monitored by caliper measurement at the appearance of palpable tumors and Mowed over time to determine if STEAP-1-expressing cells grow at a faster rate and whether tumors produced by STEAP-1-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascutarization,
reduced responsiveness to chemotherapeutic drugs).
»
Additionally, mice can be implanted with 1 x 10s of the same cells orihoiopically to determine if STEAP-1 has an effect on local growth in the prostate, and whether STEAP-1 affects the ability of the cells to metastasize, specificaBy to lymph nodes, and bone (Miki T et al, Oncol Res. 2001:12:209,^ X et al, Int J Cancer. 1991,49538). The effect of STEAP on bone tumor formation and growth may be assessed by injecting prostate tumor cells intratibiafly.
The assay is also useful to determine (he STEAP-1 inhibitory effect of candidate therapeutic compositions, such as for example, STEAP-1 intrabodies, STEAP-1 antisense molecules and ribozymes.
Example 23: STEAP-1 Monoclonal Antibody-mediated Inhibition of Tumors to Vivo
The significant expression of STEAP-1 in cancer tissues and surface localization, together with its restrictive expression in normal tissues makes STEAP-1 a good target for antibody therapy. Similarly, STEAP-1 is a target tor T ceU-

based immunotherapy. Thus, the therapeutic efficacy of anfi-STEAP-1 MAbs in human prostate cancer xenograft mouse models is evaluated by using recombtnant ce!! fines such as PC3-STEAP-1, and 3T3-STEAP-1 (see, e.g., Kaighn, M.E, et al., Invest Urol, 1979.17(1): 16-23), as weS as human prostate xenograft models such as LAPC SAO {Saffran et al PNAS 1999,10:1073-1078).
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse ortholopfc prostate cancer xenograft models. The antibodies can be unconjugated, as dTscussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art Anfi-STEAP-1 MAbs inhibit formation of both king and prostate xenografts. Anti-STEAP-1 MAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of ariti-STEAP-1 MAbs in the treatment of local and advanced stages prostate cancer. (See, e.g., Saffran, D., et at., PNAS 10:1073-1078 or world wide web URL pnas.org/cgi/doi/10.1073/pnas.051624698).
Administration of the anti-STEAP-1 MAbs fed to retardation of established orthotopic tumor growth and inhibitor! of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate hat STEAP-1 as an attractive target for imtnunofherapy and demonstrate (he therapeutic potential of anfi-STEAP-1 MAbs for the treatment of local and metastatic prostate cancer. This example demonstrates that unconjugated STEAP-1 monoctona! antibodies are effective to inhibit the growth dhun^ prostate tumor xenograflsgrowV to SCiDnrice; accordingly a combination of such efficacious monoclonal antibodies is also effective. Tumor Inhibition using multiple unconjugated STEAP-1 MAbs Materials and methods STEAP-1 Monoclonal Antibodies:
, Monoclonal antibodies are raised against STEAP-1 as described in the Example enfifted ^Generation of STEAP-1
Monoclonal Antibodies (MAbs)!* The antibodies are characterized by ELISA, Western Hot, FACS, and faummopreoipftafibn
for their capacity to bind STEAP-1. Epitope mapping data for the anti-STEAP-1 MAbs, as determined by EUSA and Western
analysis, recognize epitopes on the STEAP-1 protein. Immunohistochemical analysis of prostate cancer tissues and cells
with these antibodies is performed. •
The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protetn-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at -20°C. Protein determinations are performed by a Bradford assay (Bto-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktaB comprising a mixture of Individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of UM-UC3 and CaLul tumor xenografts. Cell Lines and Xenografts
The prostate cancer ceB lines, PC3 and LNCaP ceB line as well as the fibroblast line MI^STS (American Type Culture Collection) are maintained in RPMI and DMEM respectively, supplemented with L-glutamine and 10% FBS.
PC3-STEAP-1and 3T3-STEAP-1 cell populations are generated by retroviral gene transfer as described in Hubert,
R.S., et al., Proc Nati Acad Sd U S A. 1999.96(25}: 14523. ' T
The LAPg-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCIO) mice (laconic Farms) by s.c. trocar implant (Craft, N., et al., Nat Med. 1999,5:280). Single-cell suspensions of LAPC-9 tumor cefls are prepared as described in Craft, et al.
Xenograft Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1 x 10 s cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCIO mice. To test antibody efficacy on tumor formation, i.e. antibody

injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (1CN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as length x width x height Mice with Subcutaneous tumors greater fiian 1.5 cm m diameter are sacrificed.
Orthotopic injections are performed under anesthesia by using ketamine&ylazine. For prostate orthotopic studies, an incision is made through the abdomen to expose the prostate and LAPC or PC3 tumor cells (5 x 10s) mixed with Matrigel are injected into the prostate capsule in a 10-pl volume. To monitor tumor growth, mice are palpated and blood Is collected on a weekly basis to measure PSA levels. The mice are segregated inte'poiips^a'tfieappropriate treatments, with an6-STEAP-1 or control MAbs being injected i.p.
Anti-STEAP-1 MAbs Inhibit Growth of STEAP-1-Ext>ressino Xenoaraft-Cancer Tumors The effect of anfl-STEAP-1 MAbs oh tumor formation is tested by using LNCaP and LAPC9 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor ceb directly In the mouse prostate, respectively, results in a local tumor growth, development of metastasis hi distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et a!., PNAS supra). The features make the orthotopic model more . representative of human disease progression and allowed us to follow the therapeutic effect of MAbs on cfinically relevant end points.
Accordingly, tumor cells are Injected Into the mouse prostate, and 2 days later, the mfce are segregated into two groups and treated with either a) 200-500pg, of anti-STEAP-1 Ab, or b) PBS three tunes per week for two to five weeks.
A major advantage of the orthotopic cancer models is the ability to study the development of metastases. Formation of metastasis In mice bearing established orthotopic tumors is studies by IHC analysis on lung secfions using an antibody against a tumor-specific cell-surface protein such as anfi-CK20 forprostate cancer (IJn S et al, Cancer Detect Prev. 2Qu1;25:2Q2).
Another advantage of xenograft cancer models Is the ability to study neovascularizaSon and angiogenesis. Tumor growth is partly dependent on new blood vessel development Although the capillary system and developing blood network is of host origin, the initiation and architecture of the neovasculature Is regulated by the xenograft tumor (Davidoff AM et al, din Cancer Res. 2001:7:2870; Solesvft Oet at,, EurJ Cancer CKnOncol. 1984,20:1295). The effect of antibody and smaC mofecule on nccvascularizaSon is studied in accordance with procedures known In the art, such as by IHC analysis of tumor tissues and their surrounding microenvironment
Mice bearing established orthotopic tumors are administered 1000pg injections of either an8-STEAP-1 MAb or PBS over-a 4-week period. Mice In both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and kings are analyzed for the presence of tumor cells by IHC analysis. These studies demonstrate a broad anti-tumor efficacy of anti-STEAP-1 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Ant>STEAP-1 anfibodies inhibit tumor formation of tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-STEAP-1 MAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-STEAP-1 MAbs are efficacious on major dinicaly relevant end points (tumor growth), prolongation of survival, and health.
Effect of STEAP-1 MAbs on the Growth of Human Prostate Cancer Xenogralts in mice Male ICR-SCID mice; 5-6 weeks old (Charles River Laboratory, Wilmington, MA were used. The mice were maintained in a controlled environment using the protocols set forth in the NIH Guide for the Care and Use of Laboratory

Animals. A LAPC-9AD androgeiKlependent human prostate cancer tumor was used to establish xenograft models. Stock tumors regular!/ maintained in SCID mice were sterile!/ dissected, minced, and digested using Pronase (CaJbfochem, San Diego, CA). Cefl suspensions generated were incubated overnight at 37 degrees C to obtain a homogeneous singte-cefl
suspension.
STEAP-1 M2/92.30 and M2/120.545 were tested at two different doses of 100 pg and 500 pg. PBS and anfi-KLH MAb were used as controls. The study cohort consisted of 6 groups with 10 mice in each group. MAbs were dosed IP twice a week for a total of 12 doses, starting the same day as tumor ceB injection.
Tumor size was monitored through caliper measurements twice a week. The longest dimension (L) and toe dimension perpendicular to it (W) were taksn to calculate tumor volume using the formula WZxL/2. SerumPSA .,-..,-,,,,,,,. concentration at treatment day 40 for each animal was measured using commercial HJSA kit The Kruskaf-Waffis test and the Mann-Whitney U test were used to evaluate differences of tumor growth and PSA level among groups. All tests were two-sided with 6=0.05.
The results of the experiment set forth In Figure 26 and Figure 27 show that STEAP-1 M2/9Z30 and M2/120.545 significantly retard the growth of human prostate xenograft in a dose-dependent manner.
Example 24; Therapeutic and Diagnostic use of Anti-STEAP-1 Antibodies. In Humans.
Anti-STEAP-i monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or Shsrapeutic purposes in humans. Western blot and immunohistochemicai analysis of cancer tissues and cancer xenografts with anti-STEAP-1 MAb show strong extensive staining in carcinoma but significantly lower or undefectabte levels in normal tissues. Detection of STEAP-1 in carcinoma and in metastatjc disease demonstrates the usefulness of the MAb as a diagnosfic and/or prognostic indicator. Anti-STEAP-1 antibodies are therefore used in diagnostic applications such as
immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
As determined by flow cytometry, anti-STEAP-1 MAb specifically binds to carcinoma cells. Thus, anti-STEAP-1 antibodies are used in diagnosfic whole body imaging applications, such as radioJmmunosdnfigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et al. AnBcancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of STEAP-1. Shedding or release of an extracellular domain of STEAP-1 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatotogy 27:563-568 (1998)), allows diagnostic detection of STEAP-1 by anfi-STEAP-1 antibodjes in serum and/or urine samples from suspect patients.
Anti-STEAP-1 antibodies that specifically bind STEAP-1 are used in therapeutic appBcafions for the treatment of cancers that express STEAP-1. Anti-STEAP-1 antibodies are used as an unconjugated modaljty and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known HI the art, such as a prodrugs, enzymes or radioisotopes. In predinical studies, unconjugated and conjugated anti-STEAP-1 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g.. the Example entitled "STEAP-1 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo"). Either conjugated and unconjugated anti-STEAP-1 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.

Efcmiplc 25: Human Clinical Trials for tlio Treatment and Diagnosis of Human Carcinomas through use of Hunfan AntJ-STEAP-1 Antibodies In vivo
Antibodies are used in accordance with the present invention which recognize an epitope on STEAP-1, and are used in the treatment of certain tumors such as those fisted in Table I. Based upon a number of factors, including STEAP-1 expression levels, tumors such as those listed in Table I are presently preferred indications, in connection with each of these indications, three-clinical approaches are successfully pursued.
I.) Adjuncfive therapy: In adjunctive therapy, patients are treated with anti-STEAP-1 antibodies in combination with a chemotherapeufic or anfineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-STEAP-1 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxia'ty of the chemotherapeutic agent Anti-STEAP-1 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or anSneoplastic agents adriamytin (advanced prostrate carcinoma), dsplafin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubtem (precCnicaT).
11.) Monotherapy: In connection with the use of the anti-STEAP-1 antibodies in monotherapy of tumors, the anybodies are administered to patients without a chemotherapeutic or anfineoplastic agent In one embodiment, monotherapy is conducted cfinicafly in end stage cancer patients with extensive rrictas'atic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.
III.) Imaging Agent Through binding a radkxiucfide (e.g., iodine or yttrium (I131, Y") to anti-STEAP-1 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent in such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of ceils expressing STEAP-1. In connection with the use of the anti-STEAP-1 antibodies as Imaging agents, Hie antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgica! screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (111 ln)-STEAP-1 antibody is used as an imaging agent in a Phase I human cfinical trial in patients having a carcinoma that expresses STEAP-1 (by analogy see, e.g., Divgi et al. J, Waft Cancer Inst o3:97-104 (1991)). Patients are followed with standard anterior and postcnor gamma^amera. The results indicate that primary lesions and metastatic lesions are identified.
Pose and Route of Administration
As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-STEAP-1 antibodies can be administered with doses in the range of 5 to 400 mg/m2, with the lower doses used, e.g., in connection with safety studies. The affinity of anti-STEAP-1 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of 3WII in trie art for determining analogous dose regimens. Further, anti-STEAP-1 antibodies that are fully human anltoodies, as compared to the chimerlc antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-STEAP-1 anfibodies can be lower, perhaps in the range of 50 to 300 mg/m2, and still reman efficacious. Dosing in mg/m2, as opposed to (he conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.
Three distinct delivery approaches are usefu! for delivery of ant: STEAP-1 antibodies. Conwntinnal intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavily, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid

tumors possess vasculaturc that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.
Cfinical Development Plan (GDP)
Overview: The CDP follows and develops treatments of anti-STEAP-1 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent Trials initially demonstrate safety and aiereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anfi-STEAP-1 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is STEAP-1 expression tevels in their tumors as determined by biopsy.
As with any protein or anfibpdy infusion-based therapeutic, safety concerns are related primarily to (I) cyfokine release syndrome, i.e., hypotension, fever, shaking, chills; (tQ the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (Bi) toxidry to normal cells that express STEAP-1. Standard tests and follow-up are utilized to monitor.each of these safety concerns. AnG-STEAP-1 antibodies are found to be safe upon human administration.
Example 28: Human Glintesi Trial: Manothorapy v.'Sth Human Anti-STEAP-1 Antibody
Anfi-STEAP-1 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with me exception being that patients do not receive chemotherapy concurrency with the receipt of doses of anti-STEAP-1 antibodies.
Example 27; Human Clinicaf Trial: Diagnostic Imaging with Anti-STEAP-1 Antibody
Once again, as the adjuncfive therapy discussed above is safe within the safety criteria discussed above, a human cfinical trial is conducted concerning the use of anfi-STEAP-1 antibodies as a diagnostic imaging agent The protocol Is designed in a substantially sbniar manner to those described in the art, such as in Oivgi ef a/. J. Naff. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.
•-;
Example 28: Human Clinical Trial Adjunctive Therapy with Human Anti-STEAP-1 Antibody and Chemotherapeutic. radiation, and/or hormone ablation therapy.
A phase I human clinical (rial is initiated to assess the safety of six intravenous doses of a human anti-STEAP-1 antibody in connection with die treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-STEAP-1 antibodies when utilized as an adjunctive therapy to an antneopjasfic or Chemotherapeutic or hormone ablation agent as defined herein, such as, without limitation: dsplatin, topotecan, doxorubioh, adriamycai, taxoi, Lupron, Zoladex, Eulexin, Casodex, Anandron or the like, is assessed. The trial design includes delivery of six single doses of an anti-STEAP-1 antibody with dosage of antibody escalating from approximately about 25 mg/m * to about 275 mg/m2 over he course of the treatment in accordance with the following schedule:
II?-

DayO Day? Day 14 Day21 Day28 Day35
MAbDose 25 75 125 175 225 275
mg/m2 mg/m2 mg/mz mg/m2 mg/m2 mg/m2
Chemotherapy + + + + + +
(standard dose)
Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytdkirie release^syndrome, i.e., hypotension, fever, shaking, chilis; pi} the development of an immunogenic response to the material (i.e., development of human antibodies by the patent to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express STEAP-1. Standard tests and foBow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly redurt'onjn_tumqrm^ as evidenced by MR) or other imaging.
The anfi-STEAP-1 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 25: Identification and Confirmation of Potential Signal Transduction Pathways
Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). Fibronecfin in parficular has been associated wift the MAPK signaling cascade that control.cell mitogenesis (Jiang F, Jia Y, Cohen I. Blood. 2002,99:3579). In addition, the STEAP-1 protein contains several phosphoryiation sites (see Table XXI) indicating an association with specific signaling cascades. Using immunoprecipiiation and Western blotting techniques, proteins are identified that associate with STEAP-1 and mediate
/
signaling events. Several paflways known to play a role in cancer biology can be regulated by STEAP-1, including
phospholipid pathways such as PI3K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, tOcatenin, etc,
as weB as mitogenic/survivaJ cascades sue* as ERK^ 1999,
274:801; Oncogene. 2000,19:3003, J CellBiol. 1997,138:913.).). In order to determine whether expression of STEAP-1
is sufficient to regulate specific signaling pathways not otherwise active in resting PCS cells, the effect of these genes on the
activation of the p38 MAPK cascade was investigated in the prostate cancer cefl fine PC3. Activation of the p38 kinase is
dependent on its phosphorylation on tyrosine and serine residues. Phosphorylated p38 can be distinguished from the non-
phosphorylated state by a Phospho-p38 MAb. This phospho-specific Ab was used to study the phosphorylation state of p38
in engineered PCS cefl lines. " >
PCS cells were transfected with neomycin resistance gene alone or with STEAP-1 in pSRa vector. Cells were grown overnight in 0.5% FBS, then stimulated with 10% FBS for 5 minutes with or without 10 pg/ml MEK inhibitor PD98058. Cell lysates were resolved by 12.5% SDS-PAGE and Western blotted with anfi-phospho-ERK (Cell Signaling) and anti-ERK(Zymed). NIH*3T3 cells were transfected with neomycin resistance gene alone or with STEAP-1 in pSRa vector. Cells were treated as above but without the MEK inhibitor. In addition, NIH-3T3-Neo cells were treated with 10mg/ml Na satycilate. Expression of STEAP-1 induces the plwsphorylation of ERK-1 and ERK-2 in serum and was inhibited by (he upstream MEK kinase inhibitor PD98058.
In another set of experiments, the sufficiency of expression of STEAP-1 in the prostate cancer cell line PC3 to activate the mitogente MAPK pathway, namely the ERK cascade, was examined. Activation of ERK is dependent on its phosphorylation on tyrosine and serine residues. Ptiosphorylated ERK can be distinguished from the non-phosQhorytated

slate by a Phospho-ERK MAb. This phospho-spedfic Ab was used to study the phosphorytation state of ERK in engineered PC3 cell Bnes. PCS cetls, expressing an activated form of Ras, were used as a positive control.
The results show that while expression of the control neo gene has no effect on ERK phosphoryfation, expression of STEAP-1 in PC3 cells fe sufficient to Induce an increase hi ERK phosphorylation (Figure 28). These results were verified using anti-ERK western blotting and confirm the activation of the ERK pathway by STEAP-1.
Since FBS contains several components that may contribute to receptor-mediated ERK activation, we examined 9ie effect of STEAP-1 in tow and optimal levels of FBS. PC3 ceils expressing neo or STEAP-1 were grown in either 0.1% or 10% FBS overnight The cells were analyzed by anfi-Phospho-ERK western blotting. This experiment shows that STEAP-1 induces the phosphorylation of ERK in 0.1 % FBS, and confirms that expression of STEAP-1 is sufficient to induce acfivalton of the ERK signaling cascade hi the absence of additional stimuli.
To confirm that STEAP-1 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that fie downstream of well-characterized signal transductfon pathways. The reporters and examples of these assodated trans^ption factcfs, signal Iransducfion palhways, and . activation stimuli are listed below.
1. NFkB-Juc, NFkB/ReJ; 'k-idnase/SAPK.'gfowth/apoptosis/stress
2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation
3. AP-1-luc, FOS/JUN; MAPK/SAPWPKC;grc*tti/apoptosis/stress
4. ARE-luc, androgen receptor; steroids/MAPK; growtWdirferentiation/apoptosis
!
5. p53-kic, p53; SAPK; growth/differentiation/apoptosis
6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
7. TCF-luc, TCF/Lef, G-catenin, Adhesion/invasion
Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by Rpid-mediated transfecfion (TFX-50, Promega). Luciferase activity, an indicator of relaSve transcriptional activity, is measured by incubation of cell extracts with tua'ferm substrate and luminescence of the reaction is monitored in a luminometer.
Signaling pathways activated by STEAP-1 are mapped and used for the identificaBon and validation of therapeufc targets. When STEAP-1 is involved in cell signaling, it is used as target for diagnostic, prognosfr, preventatfve and/or
therapeutic purposes.
»
Example 30; Involvement of STEAP-1 in smalt molecule transport and cell-cell communication.
Cell-cell communication is essential in maintaining organ integrity and homeostasfe, both of which become deregulated during tumor formation and progression. Intercellular communications can be measured using two types of assays (J. Biol. diem. 2000,275:25207). In the first assay, cells loaded with a fluorescent dye are incubated in the presence of uruabeled recipient cells and the cell populations are examined under fluorescent microscopy. This qualitative assay measures the exchange of dye between adjacent cells. In the second assay system, donor and recipient cell populations are treated as above and quantitative measurements of the recipient ceB population are performed by FACS analysis. Using these two assay systems, cells expressing STEAP-1 are compared to controls that do not express STEAP-1, and it is found that STEAP-1 enhances cefl communications. Figure 29 demonstrate that STEAP-1 mediates 8w transfer

of the small molecute calcein between adjacent cells, and thereby regulates cell-celt communication in prostate cancer cells. In this experiment, recipient PCS cells were labeled with dextran-Texas Red and doner PCS cells were labeled with cal The results show that co-culturing of control PCS and PCS cells faff to mediate calcein transfer. Similarly, co-
Incubation of control PCS and PC3-STEAP-1 does, not altow the transfer of calcein. However, co-culturing PC3-STEAP-1
donor and PC3-STEAP-1 recipient cells mediates small molecule transfer as depicted by co-localization of green and red
pigments In the same oefis. Taken together, (he data shown In figures 29 and 30 demonstrate that STEAP-1 mediates small
molecute transfer and regulates cell-cell communication by forming biter-cellular communication channels (hat are similar in
function to gap junctions. Additionally, STEAP-1 M2/120.545 effect on Gap junction was confirmed (See, Figure 31). In this experiment, PCS cells were transfected with neomycin resistance gene alone or with STEAP-1 in pSRa vector. Recipient cells were labeled with 1 mg/m! dextran-Texas Red and donor cells were labeled with 2.5 fjg/ml calcein AM. The donor (green) and recipient (red) eels were co-cultured at 37°C for 18-24 hours and analyzed by microscopy for the co-localization of fluorescent dyes. In aft experiments, the same cells were used as donor and acceptor. Cells were incubated with the indicated amounts of STEAP-1/120.545 MAb for 10 minutes prior to plating andMAb was maintained in Ihe culture for 24 hours prior to analysis. STEAP1/120.545 reduces STEAP-1 mediated gap junction in a dose-dependent manner. The results show that STEAP-1/1 20.545 reduces STEAP-1 mediated gap junction in a dose-dependent manner.
Thus, because STEAP-1 functions in cell-cell communication and small molecule transport, it is used as a target or
marker for diagnostic, prognostic, preventative and/or therapeutic purposes. -^
Example 31: RNA interference (RNAi)
RNA interference (RNAi) technology is implemented to a variety of cell assays relevant to oncology. RNAi is a post-transcriptional gene silencing mechanism activated by double-stranded RNA (dsRNA). RNAi induces specific mRNA
V
degradation leading to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs called short interfering RNA (siRNA) have the correct composition to activate the RNAi pathway targeting for degradation, specifically some mKNAs. See, Elbashir S.M., ct. al., Duplexes of 21-mideotide RNAs Mediate RNA interference in Cultured Mammalian Cells. Nature 411(6836):494-8 (2001). Titus, RNAi technology is used successfully in mammalian cells to silence targeted genes.
2-o

Loss of cell proliferation control is a hallmark of cancerous cells; thus,, assessing the role of STEAP-1 in ceH
is relevant Accordingly, RNAi was used to investigate the function of Hie STEAP-1 antigen. I., generate siRNA for STEAP-1, algorithms were used that predict digonudeotides that exhibit the critical molecular parameters (G:C content, melting temperature, etc.) and have the ability to significantly reduce the expression levels of tot STEAP-1 protein when introduced into cells. Accordingly, one targeted sequence for the STEAP-1 siRNA is: 5* AAGCTCATTCTAGCGGGAMT 3' (SEQ ID NO: 81). In accordance with this Example, STEAP-1 siRNA compositions aft used that comprise siRNA (double stranded, short interfering RNA) that correspond to the nucleic add ORF sequence of tU STEAP-1 protein or subsequences thereof. Thus, siRNA subsequences are used in this manner are generally 5,6,7,8, S, 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 configuc** RNA nudeoGdes in length. These siRNA sequences are complementary and non-complementary to at least a portion of (fa" mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nudeoGdes in length, most preferably 2; 23 nudeotides In length. In preferred embodiments, these siRNA achieve knockdown of STEAP-1 anfigen in cells expressing the protein and have functional effects as described belov:.
The selected siRNA {STEAP-l.b oOgo) was tested in numerous cell fines in the survival/proEferafion MTS assay (measures ceBular metabolic activity). Tetrazoflum-based coforirnetrfc assays (i.e., MTS) detect viable cefls exclusively, since Sving cells are metabdically active and therefore can reduce tetrazolium salts to colored formazan compounds; dead cefls, however do not Moreover, this STEAP-1 .b ofigo achieved knockdown of STEAP-1 antigen in cefls expressing the protein and had functional effects as described below using the following protocols.
Mammalian siRNA transfections: The day before a'RNA transfecfion. the different cefl fines ware plated in media (RPM11640 with 10% FBSw/o antibiotics) at 2x103 cells/well in 80 nl (96 wefl plate format) forthesurvival/MTS assay. Jn parallel with the STEAP-1 specific siRNA ofigo, the following sequences were Inducted in every experiment as controls: a) Mock transfected cells with Lipofectamine 2000 {Invitrogen, Carlsbad, CA) and annealing buffer (no siRNA); b; Luciferase-4 specific siRNA (targeted sequer.oo: 5--MGGGACGAAGACGAACACUUCTT-31) (SEQ 10 NO: 82); and, c) Egt specific siRNA (targeted sequence: S'-MCTGAAGAfJCTGAAGACAATAA-S1) (SEQ ID NO: 83). SiRNAs were used at 10nM and 1 fig/ml Lipofectamine 2000 final concentration.
The procedure was as follows: The siRNAs were first diluted in OPTIMEM (serum-free transaction media, Invitrogen) at 0.1uM nM (10-fold concentrated) and incubated 5-10 min RT. Lipofectamine 2000 was diluted at 10 pg/mf (10-fold concentrated) for the total number transfections and incubated 5-10 minutes at room temperature (RT). Appropriate amounts of diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 with diluted 10-fold concentrated siRNA and Incubated at RT for 20-30* (5-fold concentrated transfect'on solution). 20 pis of the 5-fotd concentrated transfedion solutions were added to the respective samples and incubated at 3Z°C for 96 hours before analysis.
MTS assay; The MTS assay is a cotorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays based on a tetrazolium compound [3-{4(5-dimethylthia2ol-2-yl)-5-{3- . carfaoxyrnethoxyphenyt)-2-(4-sulfophenyQ-2H-tetrazo{aim, inner salt; MTS(b}J and an electron coupling reagent (phenazine ethosulfate; PES), Assays were performed by adding a small-amount of the Solution Reagent directly to culture wells, incubating for 1-4 hours and then recording absorbance at 490nm with a 96-well plate reader. The quantity of colored formazan product as measured by the amount of 490nm absorbance is directly proportional to the mitochondria! activity arid/or the number of fiving cells in culture.
In order to address the function of STEAP-1 h cells, STEAP-1 was silenced by (ransfeding (he endogenously expressing STEAP-1 ceH fines.- As shown in Figure 32, ERK-1 and ERK-2 phosphorylation were both Induced by 10% serum, and were inhibitedby M2/92.30 MAb and siRNA to STEAP-1. In this experiment, PC3 cefls were transfecjed with
'2-1

neomycin resistance geito alone or with STEAP-1 and MAb in pSHa vector. For RNAi knockdown. PCE-STEAP-1 cells were stably transacted with a pPUR-U6-27-STEAP-1 vector containing siRNA to STEAP-1: Cells were starved in 0.1 % FBS for 18 hours at 37°C, placed on ice for 10 minutes without or with 10 M9/ml M2/92.30 MAb, brought to RT for 3 minutes then stimulated with 10% FBS for 5 minutes. Cells were lysed in RIPA buffer, whole cell fysates resolved by 12.5% SDS-PAGE and proteins detected by Western blotting. Phospho-ERK was detected with rabbit antiserum (Cell Signaling) and ERK was detected with rabbit anh'-ERK (Zymed). STEAP-1 was detected with sheep anti-STEAP-1 'and acfin was detected with antf-actirt MAb (Santa Cruz).
Additionally, As shown in Rgure 33, Specific STEAP-1 RNAi stably expressed in PC3-STEAP-1 cells reduces the STEAP-1 induced cell-cell communication. In this experiment, PC3 cells were transfected with neomycin resistance gene atone or with STEAP-1 in pSRa vector. For RNAi knockdown, PCE-STEAP-1 cells were stably transfected with a pPUR-U6-27-STEAP-1 vector containing siRNA to STEAP-1 or an empty vector. Recipient cells were labeled with 1 mg/ml dextran-Texas Red and donor cells were labeled with 2.5 pg/ml calcein AM. The donor (green) and recipient (red) ceUs were co-cultured at 37° C for 18-24 hours and analyzed by microscopy for the co-localization of fluorescent dyes. In an experiments, ihe same cells were used as donor and acceptor.
Another embodiment of ihe invention is a method to analyze STEAP-1 related celt proGferafion is Ihe measurement of DNA synthesis as a marker for proliferation. Labeled DNA precursors (i.e. ^-ThymJdine) are used and their incorporation to DNA is quantified. Incorporation of the labeled precursor into DNA is directly proportional to the amount of cefl division occurring in the culture. Another rwthod used to measure cefl proliferation is performing donogenic assays. In these assays, a defined number of cells are plated onto the appropriate matrix and the number of colonies formed after a period of growth following siRNA treatment is counted.
; In STEAP-1 cancer target validation, complementing the cell survival/proliferation analysis wift apoptosis and cefl cyde profiling studies are considered. The biochemical hallmark of the apoptotic process is genomic DNA fragmentation, an irreversible event that commits the cell to die. A method to observe fragmented DNA in cells is the immunotogical detection of histone-complexed DNA fragments by an immunoassay (i.e. cell death detection ELJSA) which measures the enrichment of histone-complexed DNA fragments (mono- and oligo-nucfeosomes) in the cytoplasm of apoptotic ceils. This assay does not require pre-Jabeling of the cells and can detect DNA degradation in cells that do not proliferate hi vitro (i.e. freshly isolated tumor cells).
The most important effector molecules for triggering apoptofic cell death are caspases. Caspases are proteases
that when activated cleave numerous substrates at the carboxy-terminal site of an aspartata residue mediating very early
stages of apoptosis upon activation. All caspases are synthesized as pro-enzymes and activation involves cleavage at
aspartate residues. In particular, caspase 3 seems to play a central role in the initiation of cellularwents of apoptosis.
Assays for determination of caspase 3 activation detect early events of apoptosis. Following RNAi treatments, Western blot
detection of active caspase 3 presence or proieolytic cleavage of products (i.e. PARP) found in apoptotic ceHs further
! support an active induction of apoptosis. Because the cellular mechanisms that result in apoptosis are complex, each has its
I advantages and limitations. Consideration of other criteria/endpoints such as cellular morphology, chromatin condensation,
| membrane Webbing", apoptoUc bodies help to further support cell death as apoptotic. Since not all the gene targets that
• regulate cell growth are anti-apoptotic, the DNA content of permeabilized cells is measured to obtain Ihe profile of DNA
j content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due to the leaking out to the cytoplasm (sub-G1
1 population). In addition, the use of DNA stains (i.e., propidium iodide) also differentiate between Ihe different phases of the
' cell cyde in the cell population due to the presence of different quantities of DNA in GQ/G1, S and G2/M. hi these studies the
• subpopulations can be quantified. -
i
For the STEAP-1 gene, RNAi studies facilitate the understanding of the contribution of the gene product in cancer pathways. Such active RNAi molecules have use in identifying assays to screen for MAbs that are active anil-tumor therapeutics. Further, siRNA are administered as therapeutics to cancer patients for reducing the malignant growth of several cancer types, including those listed in Table 1. When STEAP-1 plays a rote in cell survival, cefl proliferation, tumorigenesis, or apoptosis, R is used as a target for diagnostic, prognostic, preventafive and/or therapeutic purposes.
Example 32: Modulation of fffEAP-1 function.
Ion transport plays an important role regulating cell growth intracellular permeability, molecular trafficking and signal transduction {Minke B. Cell Mol Neurobiol. 2001,21:629; Gbtovina et at, Am J Physiol Heart Circ Physiol. 2001,280:H746) these are functions that are especiaily relevant to the neoplastic condition. Cell-cell communication regulates homeostasis, cefl proliferation and cefl death (Evans WH, Martin PE Mol Membr Biol. 200219:121; Carruba G, et d, Ann N Y Acad Sci. 2002,963:156) these functions too are especially relevant to the neoplastic condition.
Using control cell lines and cell lines exptessin^TEAP-l.inhibibfs.of.STEAP-1 function are identified. For example, PCS and PC3-STEAP-1 cefls can be incubated in the presence and absence of MAb or small molecule inhibitors. The effect of these MAb or small molecule inhibitors are investigated using the Ion flux, cell communication, proliferation and signaling assays described above
Signal transduction and biological output mediated by transporters can be modulated through various mechanisms, including Inhibition of receptor and Sgand binding, ion antagonists, protein interactions, regulation of ion and small molecule transport, etc (Tang W et al, Front Biosoi 2002,7:1583). Using control ceH fines and cell fines expressing STEAP-1, modulators (inhibitors or enhancers) of STEAP-1 function are identified. For example, PC3 and PC3-STEAP-1 ceQs are incubated in the presence and .absence of MAb or small molecule modulators. In view of the functions of STEAP-1 disclosed herein, modulators that are ton channel Wockers used in the context of the present invention include such compounds as amlodipine, azulene, dihydropyridines, thiantnes, nifedine, verapamil and their derivatives (Tanaka Y, Shigenobu K. Cardiovasc Drug Rev. 2001,19:297; Djuric D, Mifrovic V, Jakovtjevte V. Arzheirnittefforschurtg. 2002,52:365; Kourie Jl, Wood HB. Prog Biophys Mol Biol. 2000;73:91); and, modulators that are inhibitors of cell communication used hi the context of the present invention include such compounds as beta-glycyrrhetinic add, rednoids, TPA (Krutovskikh VA et al, Oncogene. 2002,21:1989; Rudkin et al, J Surg Res. 2002,103:183; Ruch J et al, J Cell Biochem. 2001.83:163). Accordingly, the effect(s) of MAb or small molecule inhibitors are investigated using the ton flux, cell communication, proliferation and signaOng assays described Examples above.
When MAb and small molecules modulate, e.g., inhibit, the transport and tumorigenic function of STEAP-1, they
are used for preventad've, prognostic, diagnostic and/or therapeutic purposes.
* ^ Throughout this application, various website data content publications, patent applications and patents are ^referenced. (Websites are referenced by Uieir Uniform Resource Locator, or URL, addresses on Ihe World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as
single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to (he models and methods of Ihe invention, in addition to those described herein, wilf become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention. •

TABLES:
TABLE I: Tissues that Express STEAP-1 when malignant-Prostate
Bladder
Kidney
Colon
Lung
Pancreas
Ovary
Breast
Stomach
Rectum
Lymphoma TABLE II: Amlno Acid Abbreviations

SINGLE LETTER THREE LETTER FULL NAME
.
F Pne phenytatenme
L Leu teucine
S Ser serine
Y Tyr tyrosine
, c Cys cysteJne
w Trp tryptophan
P Pro profine
H His histidine
Q Gin pjutamine
R Arg arginine
I lie isofeucine
M Met mdhionine
T Thr threonine
N Asn asparagine
K Lys lysine
V Val vafine
A Ala alanine
D Asp asparficacid •
E Glu gfutamic aa'd
G Gty gWne

TABLE III(a): Amino Add Substitution Matrix
Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more fikely a substitution is found in related, natural proteins. (See world wide web URL ikp.unibe.ch/manual/btosum62.htrnl)
ACDEFGHIKLMNPQRSTVWY.
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 -20 -3 1-3 -2 0-1 2 0 0-1-2 -3 -2 E
6-3-1 0-3 0 0-3 -4 .-3 -3 -2-2-1 1 3 F
6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G
8 -3 -1 -3-2 1-2 0 0 -1 -2 -3-2 2 H
4-3 2 1-3-3 -3 -3 -2 -1 3-3-11
5-2-1 0-1 1 2 0 -1 -2 -3 -2 K
4 2-3-3 -2 -2 -2 -1 1 -2 -1 L
5-2-2 0 -1 -1 -1 1~~=1—-sl.H
6-20013 -3 -4 -2 N
7 -1 -2 -1 -1 -2 -4 -3 P
5 1 0 -1 -2 -2 -1 Q
5 -1 -1 -3-3 -2 R
4 1 -2 -3 -2 S
5 0 -2 -2 T
4 -3 -1 V
11 2 W
7 Y

TABLE lii(u)

Original residue Conservative substitution
Ala (A) Gly; Ser; Val
Arg(R) Lys
Asn(N) Gin; His
Asp(D) Glu
Cys(C) Ser
Gln(Q) Asn, His
Glu (E) Asp
Gly(G) Ala; Pro
His(H) Asn; Gin
lle(l) Leu; Val
Leu(L) lie; Val
Lys{K) Arg; Gin; Glu
Met(M) Leu; Tyn He; Ve!
Phe(F) MetLeu;Tyr
Ser(S) Thr
ThrfT) Ser
' Trp(W) Tyr
Tyr(Y) Trp; Phe
Val(V) He; Leu; Ala

TABLE IV:
HLA Class l/ll Motifs/Supem.vuis
TABLE IV (A): HLA Class I SupcrmotffsfMotifs

SUPERMOTIF POSITION POSITION POSITION
2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Anchor)
A1 TJLVMS | FWY
A2 LMI/WrQ IVAMTL
A3 VSMA7I/ RK
A24 YFWIVLMT FIVWLM
B7 ' P \filFMWYA
B27 RHK FfLWMIVA
B44 ED FWYLIMVA
B58 ATS FWYL/VMA
B62 QL/VMP RVYW/MJ

MOTIFS
A1 TSM Y
A1 DBAS V
A2.1 IMVQIAT VUMAT
A3 LMVISATFCGD KfKHFA
A11 VTMUSAGMCOF KPYH
A24 YFWM FUW
A*3101 WTAUS we
A*3301 MVALF/Sr RK
A*6801 AVTWSU RK
B*0702 P UAFWYAIV
B*3501 P LMFWY/V/4
B51 P LNfWYAM
B*5301 P MFWYALV
B*5401 P ATWLWFVW
Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary .anchor position for a motif or supermoSf as specified In the above table.
TABLE IV (B): HLA Class II Supermotif

1 6 9

W.F.Y.V.U ArV.I.L.P.C.S.T A.V.I.L.e^S.T.M.Y

TABLE IV (C): HLA Class II Motifs

8 9
1" anchor 6
MOTIFS
r anchor 1 234
DR4 preferred deleterious FMYL/VW M 1 W 1 VSTCR4L/M MH R MH WDE
DR1 preferred
deleterious MWVWY C CH PAMQ FD VMATSR/C CWD M GDE AVM D
DR7 preferred deleterious MFUVWY M C W A G IVMSAC7R
i M GRD IV N G
DR3 Motif a preferred Motif b preferred MOTIFS 1° anchor 1 UVMFY UVMFAY 2 3 Tanchor4 D DNQEST 5 1° anchors KRH
DR Supermotif MFLIVWY VMSTACFU
Italicized residues indicate less preferred or tolerated" residues
TABLE IV (D): HLA Class I Supermotifs


SUPER-MOTIFS POSITION: 1 2345 678 C-terminus
A1 rAnchor 1-Anchor
TILVMS FWY
A2 rAnchor 1'Anchor
UVKMTO UVMAT
A3 Preferred rAnchor YFW YFW YFW P rAnchor
; deleterious • DE (3/5); VSMA7U (4/5) DE (4/5) (3/5) (4/5) (4/5) RK
A24 rAnchor rAnchor
VFWLMT FlYWIAf
B7 Preferred FWY (5/5) 1'Anchor FWY FWY 1-Anchor
UVM(3/5) deleterious DE(3/5); P(5/5);
QN(3/5) P (4/5) DE
_ . . OS) (3/5) VllfMWYA G ON DE (4/5) (4/5) (4/5)
B27 1* Anchor . RHK 1'Anchor FYLWWA
B44 rAnchor rAnchor
ED FWYL»i!VA
B58 rAnchor 1' Anchor
ATS FWYUMIM
B62 rAnchor rAnchor
OUVMP FWYW/VM
Italicized residues indicate less preferred or toierated* residues

TABLE IV (E): HLA Class I Motifs


POSITION 1 2 3 4. 5 6 7 8 9 C-
termhus
P A
or C-termkuJs
DEQN YFW 1"Anchor ' Y
DEA YFW
RHKLiVMP A
A1 preferred GFYW 1'Anc
9^ner STM
deleterious DE
A1 preferred GRHK ASTCUVM rAnchor 6STC ASTC UVM DE rAnchor
9-mer DE4S Y
deleterious A RHKDEPYFW DE PQN RHK PG GP
A1 ' 10- preferred YFW I'Anchor STM DEAQN A YFWQN PASTC GDE P rAnchor Y
mer
deleterious GP RHKGLIVM DE RHK QNA RHKYFWRHK A
A1 10-mer preferred YFW STCLIVM TAnchor -DEAS A YFW PG G YFW 1'Ancha-Y








deleterious RHK RHKDEPYFW P G PRHK ON
STC YFW
rAnchor VUMAT
A2.1 preferred YFW rAnchor YFW
9-mer LMIVQAT
RKH DERKH
A3 preferred RHK deleterious DEP
defeferiousDEP DERKH
POSITION:! 2 3 4 5 6 7 8 9 C-
Termhus
A£1
10- prefenred AYFW rAnchor IMIVQAT LViM G G FYWL VIM rAnchor VOUAT
mer deleterious DEP \ DE 'RKHA p RKH DERKHRKH
YFW
P rAnchor KYRHf*
PRHKYF A W
A11 preferred A deleterious DEP
rAnchor YFW LMVISATFCGD
DE
YFW
YFW YFW
rAnchor KRYH
YFW
VTLMISAGNCD F

A24 preferred YFWRHK rAnchor STC YFW YFW rAnchor
9-mer YFWW FUW
deleterious DEG DE G QNP DERHKG AQN
A6801 Preferred YFWSTC rAnchor * AVTMSU YFWUV
M YFW P 1'Anchor RK
deleterious GP DEG RHK A
A24 Preferred
10-
mer
Deleterious
A3101 Preferred RHK Deleterious DEP
A3301 Preferred
Deleterious GP

rAnchor YFWM
1'Anchor MVTAL/S
1°Anchor MVALF/Sf

GDE
YFW
DE
YFW
DE

ON

YFWP
ON DEA
RHK DE
YFW YR/\£ AP rAnchor
" WC
ADE DE DE DE
AYFW
rAnchor RK

rAnchor FUW





DE
B0702Preferred RHKFWY 1°Anchor RHK . P
deleterious DEQNP DEP

DE

RHK RHK RHK
GDE ON

PA rAnchor IMFWYAI V
DE



IZH

POSITION 1
A1 preferred GFYW r/jd,'/
9-mer MM
deleterious DE
A1 preferred GRHK AMur/i, 9-mer
deleterious A B3501 Preferred FWYLIVI/l
deleterious AGP
B51 Preferred LIVMFV/C r/.,
deletefousAGPDER
HKSTC
B5301 preferred UVMFWY J
f-
deleterious AGPQN B5401 preferred FWY T
deleterious GPQNDE



3 4 5 6 7 8 9 C-terminus
or C-lemwius
DEA RHKLIVMP YFW A G P A DEQN YFW TAnchor Y
DE4S W GSTC DE PON ASTC RHK UVM DE PG GP rAnchor Y
FWY G G FWY 1°Anchor LMFWY/V A
FWY STC FWY
.._ -g ~ S G FWY DEQN GUtr 1'Anchor UVFIV/4 M
FWY STC FWY G UVMFWYFWY RHKQN DE 1'Anchor IMFWY/4L V
FWYLIVM GDESTC UVM RHKDE DE AUVM FWYA P
QNDGE DE TAnchor ATIVLMF WY

TABLE tV{F):

Summary of HLA-supertypes
3veraH^nenotvDfc frequencies of HLA-supertypes in different ethnic populations
Spedfidty Phenotyplc frequency
Supertype ^osition 2 >Terminus Caucasian MA Black Japanese Chinese Hispanic Average
37 3 MLMVFWY *3.2 i5.1 57.1 43.0 49.3 49.5
A3 AILMVST *K 37.5 42.1 45.8 52.7 43.1 44.2
A2 AILMVT MLMVT 45.8 39.0 424 45.9 43.0 4Z2
A24 YFfWIVLMT -|{YWLM) 23.9 38.9 58.6 40.1 38.3 . 40.0
344 EfD) :WYLIMVA 43.0 21.2 429 39.1 39.0 37.0
A1 TlfLVMS) W 47.1 16.1 21.8 14.7 26.3 25.2
327 RHK FYLfWMl) 28.4 26.1 13.3 13.9 35.3 23.4
362 QL(IVMP) FWYCMIV) 12.6 4.8 36.5 25.4 1.1 18.1
358 ATS FWYflJV) 10.0 25.1 1.6 9.0 5.9 10.3

TABLE IV (GJ:
Salcuiated population coverage afforded by different HLA-supertype combinations
HLA-supertypes
Phenotypfc frequency

Japanese
>U Blacks
Chinese
Caucasian
Average
37.5
38.4
36.1
86.2
•Ospante
B3.0
36.3
99.5
99.3
100.0
42, A3 and B7
39.6
99.9
100.0
99.8
99.8
A2,A3,B7,A24,B44
andA1
A2,A3,B7,A24,
344,A1,B27,B62,
and B 58
Motifs indicate the residues defining supertype specifidtes. The motifs incorporate residues determined on the basis of MJbHshed data to be recognized by multiple alletes within (he supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.

Fable IV(h): Frequently Occurring Motifs
Name avrg. % dentity Description 'otential FuncBon
zf-C2H2 '•' 34% 3nc finger, C2H2 type todete acid-binding protein functions as transcription factor, nuclear location )robable
cvtochrome b N 58% Cytochrome b(N-termina!)/b6/petB membrane bound oxidase, generate superoxkJe
Ip 19% Imrmmoglobulin domain domains are one hundred amino acids ong and include a conserved ntradomain disulfide bond.
WD40 18% \ND domain, G-bela repea tandem repeats of about 40 residues, 3ach containing a Trp-Asp motif. Function HI signal transduction and >rotein Interaction
>DZ 23% 3DZ domain nay function in targeting signding TTOlecules to sub-membranous sites
.RR 28% jeudne Rich Repeat short sequence moSfs involved In Hnteln-protein Interactions
Tdnase 23% 'rotefn kinase domain conserved catalytic core common to both serine/threonine and tyrosine )rotein kinases containing an ATP rinding site and a catalytic site
PH ' ' \ 16% PH domain riecksbfn homotogy involved in nfracellular signaling or as constituents Dfthecytoskeieton
EOF 34% EGF-fike domain 30-40 amho-acW tang found In the extracellular domain of membrane-x>und proteins or in secreted proteins
Rvt 49% Reverse transcn'ptase [RNA-dependentDNA x>lymerase)
tak 25% Ank repeat Cytoplasmic protein, associates integral membrane proteins to the cyloskeleton
Oxidored_q1 2% MDH-Jbiquinone/plastoquinone complex 1), various chains membrane associated Involved in proton translocation across the nembrane
Efhand 4% Fhand ^Icium-blndtfig domain, consists of a12 residue loop flanked on boSi sides by a 2 residue.alpha-heTical domain
*vp 9% Relroviral aspartyl rotease ^partyl or add proteases, centered on catalytic aspartyl residue
Collagen * 2% ^ollagen tn'pte helix repeat 20 copies) xtracellular structural proteins involved formafioirof connective tissue. Trie equence consists of the G-X-Y and the )olypeptide chains forms a triple hefix.
Fn3 0% bronectin type ill domain jocated in the extracellular Bgand-nding region of receptors and is about 00 amino acid residues long with two airs of cysteincs fcvGlved in bisulfide wnds
7»m_1 9% transmembrane receptor rhodopsin family) even hydrophobic transmembrane egtons, with the N-terminus located xtracellulariywhJe BieC-terminusts ytoplasmtc. Signal throuqh G proteins
132-

Description of use
See Thorium-229 (Th-229)
Table IV(I): Examples of Medical Isotopes: Isotope
Aclinium-225
(AC-225)
Actinhim-227 (AC-227)
See Thorfum-228 (Th-228) See Thorium-229 (Th-229)
Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the skeleton resulting from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy
Bismuth-212 (Bi-212)
•g*!y*» Bismuth-213 (BI-213)

Cobalt-60 (Cc-60)
Copper-64 (Cu-64)
Copper-67 (Cu-67)
lodine-131 (1-131)
lridium-192 (lr-192)
Lutetium-177 (Lu-177)
Radiation source for radiotherapy of cancer, for food irradiators, and for sterifization of medical supplies
A positron emitter used for cancer therapy and SPECT imaging
Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic studies (i.e.. breast and colon cancers, and lymphoma)
' Cancer radiofrnmunotherapy
Erbium-169 (Er-169)
Europium-152 (Eu-152)
Europium-154 (Eu-154)
Gadofinium-153 (Gd-153)
Gold-198 (Au-198)
Holmium-166 (Ho-166)
lodine-125 (M25)
Rheumatoid arthritis treatment particularly for the small joints associated wifli fingers and toes
Radiation source for food Irradiation and for sterifization of medical supplies
r V
Radiation source for food irradiation and for sterilization of medical supplies Osteoporosis detection and nuclear medical quality assurance devices
Implant and intracaviry therapy of ovarian, prostate, and brain cancers
Multiple myeloma treatment In targeted skeletal therapy, cancer radioimmunotherapy, bone marrow ablation, and rheumatoid arthritis treatment
Osteoporosis detection, diagnostic imaging, tracer orugs, brain cancer treatment, radfotabefing, iumor imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for treatment of prostate cancer, determination of gjomeruiar filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs
Thyroid function evaluation, thyroid disease detection, treatment of thyroid cancer as weD as oner non-malignant thyroid diseases (i.e., Graves disease, goiters, and hypefthyroidism). treatment of leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy
Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and Implants for breast and prostate tumors
t Cancer radioimmunotherapy and treatment of blocked arteries (i.e., arteriosclerosis and restenosis)

Parent of Technetium-99m (Tc-99m) which is used for imaging ttie brain, Iver, lungs, heart,
Molybdenum-99 and other organs. Currently. Tc-99m Is the most widely used radtoisotope used for diagnostic
(Mo-99) imaging of various cancers and diseases involving the brain, heart, liver, lungs; also u*ed in
detection of deep vein thrombosis of the legs
(Sw)194 Cancer raowimrrwmMesrapy Pallacfium-103 Prostate cancer treatment

(Pd-103)
Ut195m studies on biod'stribution and metabolism of cispialin, a chemcJhcrape'j'Jc drug

Pdycythemia rubra vera (Wood cell disease) and leukemia treatment, bone cancer
Phosphorus-32 diagnosis/treatment; colon, pancreatic, and fiver cancer treatment; radratebefing nucleic acids
(P-32) for in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e.,
arteriosclerosis and restenosis), and Intracavity therapy
blocked arteries (i.e., arteriosclerosis and restenosis) See Acfinium-227 (Ac-227)
Phosphorus-33 Leukemia treatment bone disease diagnosis/treatment, radtolabeling, and Ireatment of
(P-33) ..............
Radium-223 (Ra-223)
Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of (Re-1 86) lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy
Rhenium-1 88 Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pah relief, (Re-186) treatment of rheumatoid arthritis; and treatment of prostate cancer

(W-188)
Xenon-127 (Xe-127)
Ytterbium-175 (Yb-175)
Yttrium-90 (Y-90)
Yttrium-91
Riiodmm-105 (Rh-105)
Samarium-145 (Sro-145)
Samarium-153 (Sm-153)
Scandium-47 (Sc-47)
Sefenium-75 (Se-75)
Strontium-85 (Sr-85)
Strontium-89 (Sr-89)
Technetium-99m, (Tc-99m)
Thorium-228 {TTi-228)
Thorium-229 fHi-229)
Thulfum-170 (Tm-170)
Ttn-117m (Sn-117m)
Cancer radioimmunotherapy
Ocular cancer treatment
Cancer radfoimmunotnerapy and bone cancer pain relief
Cancer radioimmunotherapy and bone cancer pain relief
Radio-tracer used in brain studies, imaging of adrenal cortex by gamma-scWigraphy, lateral locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of bile acid loss from the endogenous poof
Bone cancer detection and brain scans
Bone cancer pain relief, multiple myeloma treatment, and osfeoblastic therapy
'See Molybdenum-99 (Mo-99)
Parent of Bismuih-212 (Bi-212) which is an alpha emitter used in cancer rafioimmunotherapy
Parent of Aclinium-225 (Ac-225) and grandparent of Bismutn-213 (Bi-213) which are alpha emitters used in cancer radioimmunotherapy
Gamma source tor blood irradiators, energy source for implanted medical devices
-**
%
Cancer immunotherapy and bone cancer pain relief
Parent for Rhenium-168 (Re-188) which is used for cancer diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries (i.e., arteriosclerosis and restenosis)
Neuroimagtng of brain disorders, high resolution SPEC! studies, pulmonary function tests, and cerebral blood (low studies
Cancer radioimmujioUierapy
Microseeds obtained from irradiating Ytlrium-89 (Y-89) for liver cancer treatment
A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer radwimmunotherapy

(Y-91) (i.e., lymphoma. breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable
liver cancers)
Tables V-XVUI: Set forth in United States patent appticafion number 10/236,878; filed 06-September-2002, the spea'fic contents are fully incorporated by reference herein.
Table XDC Frequently Occurring Motifs

Name avrg. % identity Description Potential Function '
zf-C2H2 34% 2nc finger, C2H2 type Nucleic acid-binding protein functions as transcription factor, nuclear location probable
cytochrome b N 38% Cytochrome b(N-terrnina!)/b6/petB membrane bound oxidase, generate superoxide
Iq 19% mmunoglobulin domain domains are one hundred amino acids long and nchide a conserved Mradomain disufSde bond.
VVD40 18% m) domain, G-beia repeat tandem repeats of about 40 residues, each xmtaining a Trp-Asp mofif. Function in signal ransducfion and protein interaction
PDZ 23% 5DZ domain nay function in targeting signaling molecules to sub-membranous sites
LRR 28% .eua'ne Rich Repeat short sequence mofife involved in protein-protein interactions
3kinase 23% totein kinase domain conserved catalytic core common to both serine/threonine and tyrosine protein kinases containing an ATP binding site and a catalytic
sfte
3H 16% 'H domain )leckstrin homology involved in intracellular ignaling or as constituents of the cytoskeleton
EGF 34% EGF-fike domain (MO amfrio-acid long found in the extracellular domain of membrane-bound proteins or in secreted proteins
Rvt 9% Reverse transcriptase (RNA-dependent DMA pdymerase)
tak 5% ^nk repeat "ytoplasmic protein, associates integral nembrane proteins to the cytoskeleton -
Oxidored_q1 2% ADH-Ubiquinone/plastoquinone complex 1), various chains nembrane associated brvoived in proton anslocafon across the membrane

Table XIX, continued: Frequently Occurnng Motifs
Name avrg. % denBty Description Potential Function
Efhand 24% EFhand calcium-binding domain, consists of a1 2 residue oop flanked on both sides by a 12 residue alpha-lelicaJ domain
fyp 79% tetroviral asparty! protease Aspartyl or acid proteases, centered on a catalytic aspartyl residue
Collagen 42% Collagen triple helix repeat (20 copies) extracellular structural proteins involved in formation of connective tissue. The sequence consists of the G-X-Y and (he pdypeptide chains forms a triple helix.
fn3 20% HbronecSn type III domain Located In the extracellular Sgand-binding region of receptors and is about 200 amino add esidues long with two pairs of cystelnes nvoivad in disulfide bonds
7\m 1 9% 7 transmembrane receptor rhodopsin family) . seven hydrophobic transmembrane regions, with le N-terminus located extraceOuiarh/ white the D-terminus is cytoplasmic. Signal through G proteins
Table XX: Motifs «,id Post-translational Modifications of STEAP-1:
N-glycosylation site
143 -146 NGTK (SEQ ID NO: 84) 331 - 334 NKTE (SEQ ID NO: 85)
i •
Protein kinase C phosphorylafion site
3-5 SrK
160-162TrK
,187-189 SyR
246-248TwR
Casein kinase II pnosphorylatkm site
3-6 SricD {SEQ ID NO: 86)
8-11 TnqE (SEQIDNtt 87)
240 - 243 SvsD (SEQ ID NO: 88)
246-249 T-.vrE (SEQ ID NO: 89)
Tyrosine kinase phosphorylation site
19 • 27 RRNLEEDDY (SEQ ID NO: 90)
N-myristoylation site
133 -138 GVIAAI (SEQ ID NQ 91) 265-270 GTIHAL (SEQ ID NO: 92)
Bipartite nudear targeting sequence
4-20 RKDITNQEELWKMKPRR (SEQ ID NO: 93)
Table XXI: Protein Characteristics of STEAP-1

Bioinformatic URL (Located on the World Wide Web Outcome Program at)
ORF ORF finder Protein length 1193 bp 339 aa

Transmembrane region TM Pred HMMTop Sosui
Signal Peptide
Pi
Molecular weight
Localization
Signal P
pl/MWtoo) pl/MWtod PSORT
Motifs
Pfam Prints
i Blocks"
Bfoinformatic URL (Located on Ihe World Wide Web
Program at)
(.ch.embnet.org/)
(.enam.hu/hmmtop/)
(.genome.adLJp/SOSui/)
TMHMM
(. (.cbs.dfei.dk/senfces/SignalP/)
(.expasy.ch/lools/) {.expasy.cn/toote/) http://psortnibb.ac.jp/
PSQRTII
http://psoft.nibb.ac.jp/
(.sanger.ac.uk/Pfam/) (.biochem.ud.ac.uk/)
{.bfocks.fticrc.org/)

Outcome
6 TM at aa 73-91, 120-141,
163-181, 218-236, 253-274,
286-304
6 TM at aa73-90,117-139,
164-182,220-238,257-274,
291-309
6 TM at aa70-92,114-136,
163-184,219-241,255-273,
292-313
6 TM at 3373-95,117-139,
164-182, 218-240, 252-274.
289-311
potential cleavage between aa 136 and 137 9.2 pi
60% plama membrane, 40%
go!ai,30%sndoplasmte
reticutum
66% endoplasmic reficulum,
11% mitochondria, 11%
plasma membrane
none
Transforming protein P21 ras
signature, Rbronecfin type ill
repeat signature
Ha!f-A-TPR repeat, Arsenical
pump membrane protein
signature, M protein repeat

Tables XXII - LI: Set forth in United Slates patent application number 10/236,878; fifed 06-September-2002, the specific contents are fully incorporated by reference herein.
Table III: Search PepWes

STEAP1 Variant 1:
nonamers, decamers and 15-mers:
MESRKDITNQ EELHKMKPRR LQHTQELFPQ WHLPIKIAAI VSITLLALVY LPGVIAAIVQ SYPMRRSYRY KLLNWAYQQV VSDSLTWREF HYIQSKLGIV VLIFKSILFL PCLRKKILKI

aa 1-339 (SEQ ID NO: 94) NLEEDDYLHK DTGETSMLKR IASLTFLYTL LREVIHPLAT LHNGTKYKKF PHWLDKWMLT QQNKEDAWIE HDVWRMEIYV SLLLGTIHAL IFAWNKWIDI RHGWEDVTKI NKTEICSQL

PVI..LHLHQTA SHQQYFYKIP RKQFGLLSFF SLGIVGLAIL KQFVWYTPPT

HADEFDCPSE ILVINKVLPM JAVLHAIYSL ALLAVTSIPS FMIAVFLPIV

60 120 180 240 300 339

Variant2:
9-mers aa 247-258 (SEQ ID NO: 95)
WREFHYiQVfoNI
10-mers aa 246-258 (SEQ ID NO: 96)
TWREFHYiQVNNI
15-mers aa 241-258 (SEQ !D NO: 37)
VSDSLTvVREFHYIQVNNI ..
Variants:
9-mers aa 247-(SEQ ID NO: 98)
WREFHYIQIIHKKSDVPESLWDPaTRFKGLNUQS
10-mers aa 246- (SEQ ID NO; 99)
TWREFHY^QUHKKSOVPESLWDPaTRFKGL^yQS

15-mersaa241- {SEQ ID NO: 100) VSD&TWREFHYIQUKKKSDWESLWDPCLTRFKGLNLIQS
Variant*
9-mers aa 160-176 (SEQ ID NO: 101)
RKQFGLLSLFFAVLHAI
10^ers aa 159-177 (SEQ ID NO: 102)
TRKQFGLLSLFFAVLHA1Y
15-mers aa 154-182 (SEQ ID NO: 103)
DKVWMLTRKQFGLLSLFFAVLHAIYSLSYP
Table LIU: Exon Composfion of STEAP-1 (6P1D4) variant 1.

Exon number Start End
1 1 34
2 35 149
3 150 662
4 663 827
5 828 1176
An antibody or fragment thereof comprising an antigen binding site that binds specifically to STEAP-1 protein (SEQ ID NO: )








We Claim:
1. An antibody or fragment thereof comprising
(a) a complementarity determining region (CDR) from an antibody designated X92.1.30.1.1(l) (ATCC Accession number PTA-5802) or X120.545.1.1 (ATCC Accession number PTA-5803);
(b) the heavy chain variable region sequence of SEQ ID NO:50; or
(c) a light chain variable region sequence selected from the group consisting of SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56,
wherein the antibody or antibody fragment of (a), (b), or (c) binds to the STEAP-1 proteinofSEQIDNO:3.
2. The antibody or fragment of claim 1, wherein the antibody is a monoclonal antibody.
3. The antibody or fragment of claim 2, wherein the antibody is designated X92.1.30.1.1(1) and assigned A.T.C.C. Accession No.: PTA-5802.
4. The antibody or fragment of claim 2, wherein the antibody is designated X120.545.1.1 and assigned A.T.C.C. Accession No.: PTA-5803.
5. The antibody or fragment of claim 2, wherein the monoclonal antibody is a humanized antibody.
6. The antibody or fragment of claim 1, wherein the fragment is an Fab, F(ab')2, Fv or SfV fragment.
7. An antibody or fragment thereof which is a humanized or modified form of the monoclonal antibody designated X92.1.30.1.1(1) and assigned A.T.C.C. Accession No.: PTA-5802, wherein the antibody or fragment specifically binds STEAP-1 and/or exhibits the biological activity of X92.1.30.1.1(1).
8. An antibody or fragment thereof which is a humanized or modified form of the monoclonal antibody designated X120.545.1.1 and assigned A.T.C.C. Accession No.: PTA-5803, wherein the antibody or fragment specifically binds STEAP-1 and/or exhibits the biological activity of X120.545.1.1.
9. An antibody or fragment thereof that binds to the same epitope as an antibody selected from the antibody designated X92.1.30.1.1(l) (ATCC Accession number PTA-5802) and the antibody designated X120.545.1.1 (ATCC Accession number PTA-5803).

10. An antibody or fragment thereof comprising all heavy and light chain complementarity determining regions (CDRs) from an antibody designated X92.1.30.1.1(l) (ATCC Accession number PTA-5802) or XI20.545.1.1 (ATCC Accession number PTA-5803).
11. An antibody or fragment thereof comprising the heavy chain variable region sequence of SEQ ID NO:50 and a light chain variable region sequence selected from the group consisting of SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56.
12. A binding agent capable of binding to STEAP-1, wherein the antibody of claim 3 or 4 displaces the binding agent in a competitive binding assay.
13. A binding agent capable of binding to STEAP-1, wherein the binding agent displaces the antibody of claim 3 or 4 in a competitive binding assay.
14. The binding agent of claim 12, wherein the binding agent is an antibody, or a fragment thereof.
15. The binding agent of claim 13, wherein the binding agent is an antibody, or a fragment thereof.
16. A recombinant protein comprising the antigen binding region of an antibody of claims 1-4.
17. The antibody or fragment of claims 1-11, 14 or 15, wherein the antibody or fragment is coupled to a detectable marker, a toxin, or a therapeutic agent.
18. The antibody or fragment of claim 17, wherein the antibody or fragment is coupled to a detectable marker selected from a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound and a chemiluminescent compound.
19. The antibody or fragment of claim 18, wherein the antibody or fragment is coupled to a radioisotope selected from 212Bi, 13111 131In,90Y, 186Re,211At, 1251, 188Re, 153Sm, 213Bi,32P,andLu.
20. The antibody or fragment of claim 17, wherein the antibody or fragment is coupled to a toxin.
21. The antibody or fragment of claim 20, wherein the antibody or fragment is coupled to a toxin selected from ricin, ricin A-chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl065, and a cisplatin.
22. The antibody or fragment of claim 17, wherein the antibody or fragment is coupled to an auristatin.
23. A transgenic animal that produces the monoclonal antibody of claim 2.
24. A hybridoma that produces the monoclonal antibody of claim 2.
25. A vector comprising a polynucleotide encoding the antibody or fragment thereof of claims 1-11,14 or 15.
26. The vector of claim 18 that encodes said antibody or fragment as a single-chain.
27. A pharmaceutical composition that comprises the antibody or fragment of claims 1-11, 14 or 15 in a human unit dose form.
28. An assay for detecting the presence of a STEAP-1 protein in a biological sample comprising contacting the sample with an antibody or fragment of claims 1-11, 14 or 15, and detecting the binding of STEAP-1 protein in the sample thereto.
29. The assay of claim 28, wherein the biological sample is from a patient who has or is suspected of having a cancer listed in Table I.
30. The assay of claim 29, wherein the biological sample is serum.
31. The assay of claim 29, wherein the biological sample is prostate.
32. The assay of claim 31, wherein the biological sample is peripheral blood.
33. The assay of claim 32, wherein the peripheral blood comprises prostate cancer cells.
34. A method of inhibiting growth of cancer cells that express STEAP-1 in a subject, comprising: administering to said subject the vector of claim 19-wherein the single chain monoclonal antibody is expressed in the cells.
35. A method of inhibiting growth of cancer cells that express STEAP-1, the method comprising exposing the cells to the antibody or fragment of claims 1-11, 14 or 15.
36. The method of claim 35, wherein the cancer cells are from a cancer set forth in Table 1.
37. The method of claim 36, wherein the cancer cells are from malignant prostate.
38. A method for treating a cancer in a subject comprising administering to the subject a pharmaceutical composition comprising the antibody of claims 1-11, 14 or 15.
39. The method of claim 38, wherein the cancer is prostate cancer.
40. A method of inhibiting growth of cancer cells that express STEAP-1, the method comprising exposing the cells to the antibody or fragment of claim 20.
41. The method of claim 40, wherein the antibody or fragment is coupled to a toxin selected from ricin, ricin A-chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl065, and a cisplatin.
42. The method of claim 41, wherein the toxin is an auristatin.
43. The method of claim 40, wherein the cancer cells are from a cancer set forth in Table 1.
44. The method of claim 43, wherein the cancer cells are from malignant prostate.
45. A method for treating a cancer in a subject comprising administering to the subject a pharmaceutical composition comprising the antibody or fragment of claim 20.
46. The method of claim 45, wherein the antibody or fragment is coupled to a toxin selected from ricin, ricin A-chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl065, and a cisplatin.
47. The method of claim 46, wherein the toxin is an auristatin.
48. The method of claim 46, wherein the cancer is prostate cancer.
49. A method of delivering a cytotoxic agent or a diagnostic agent to a cell that expresses a STEAP-1 protein comprising: providing a cytotoxic agent or a diagnostic agent conjugated to the antibody or fragment of claims 1-11, 14 or 15 to form an antibody-agent or fragment-agent conjugate; and exposing the cell to the antibody-agent or fragment-agent conjugate.
50. The method of claim 49, wherein the cytotoxic agent or the diagnostic agent is selected from the group consisting of a detectable marker, a toxin, and a therapeutic agent.
51. The method of claim 50, wherein the diagnostic agent is delivered, and wherein the diagnostic agent is a detectable marker selected from a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound and a chemiluminescent compound.
52. The method of claim 51, wherein the diagnostic agent is a radioisotope selected from 212Bi, 13111 131In,90Y,™Re,211At, 1251,188Re, 153Sm,213Bil 32P,andLu.
53. The method of claim 50, wherein the cytotoxic agent is delivered, and wherein the cytotoxic agent is a toxin selected from ricin, ricin A-chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl065, and a cisplatin.
54. The method of claim 53, wherein the toxin is an auristatin.
55. A method for detecting STEAP-1 in a test sample, comprising contacting a test sample obtained from a subject having or suspected of having a cancer cell expressing STEAP-1 with the antibody or fragment of claims 1-11, 14 or 15; and determining an amount of STEAP-1 from the test sample bound to the antibody or fragment.
56. The method of claim 55, wherein the test sample is peripheral blood.
57. The method of claim 56, further comprising comparing the amount of STEAP-1 bound to the antibody or antibody fragment from the test sample with the amount of STEAP-1 from a control sample.
58. The method of claim 57 wherein the test sample is sample is blood, urine, semen, prostate, colon, bladder, pancreas, ovary, cervix, testis, breast, bone, lymph node, lung, liver, brain, serum, or a cell preparation.
59. The method of claim 58 wherein the test sample is peripheral blood.
60. The method of claim 59, wherein the peripheral blood comprises prostate cancer cells.
61. The method of claim 57 further comprising: taking the test sample and the control sample from a patient who has or who is suspected of having a cancer listed in Table I.
62. Antibodies and molecules derived therefrom that bind to STEAP-1 Proteins as claimed in any of the above claims substantially as described in the specification and illustrated in the accompanying drawings and sequence listing.

Documents:

6301-delnp-2006-1-Abstract-(06-09-2012).pdf

6301-delnp-2006-1-Claims-(06-09-2012).pdf

6301-delnp-2006-1-Correspondence Others-(06-09-2012).pdf

6301-delnp-2006-1-Correspondence Others-(15-02-2013).pdf

6301-delnp-2006-1-Form-3-(15-02-2013).pdf

6301-delnp-2006-abstract.pdf

6301-delnp-2006-Assignment-(13-08-2012).pdf

6301-delnp-2006-assignment.pdf

6301-DELNP-2006-Claims-(05-02-2010).pdf

6301-delnp-2006-Claims-(15-02-2013).pdf

6301-delnp-2006-claims.pdf

6301-delnp-2006-Correspendence Others-(15-02-2013)-.pdf

6301-delnp-2006-Correspondence Others-(06-09-2012).pdf

6301-delnp-2006-Correspondence Others-(13-08-2012).pdf

6301-delnp-2006-Correspondence Others-(15-02-2013).pdf

6301-delnp-2006-Correspondence Others-(21-02-2013).pdf

6301-delnp-2006-Correspondence Others-(31-12-2012).pdf

6301-DELNP-2006-Correspondence-Others (05-02-2010).pdf

6301-DELNP-2006-Correspondence-Others (05-02-2010).pdf

6301-delnp-2006-Correspondence-Others-(27-02-2013).pdf

6301-delnp-2006-correspondence-others.pdf

6301-delnp-2006-description (complete).pdf

6301-delnp-2006-drawings.pdf

6301-delnp-2006-form-1.pdf

6301-delnp-2006-form-2.pdf

6301-delnp-2006-form-26.pdf

6301-delnp-2006-Form-3-(27-02-2013).pdf

6301-delnp-2006-form-3.pdf

6301-delnp-2006-form-5.pdf

6301-delnp-2006-GPA-(13-08-2012).pdf

6301-delnp-2006-pct-237.pdf

6301-delnp-2006-pct-326.pdf

6301-delnp-2006-pct-373.pdf

6301-delnp-2006-Petition-137-(06-09-2012).pdf


Patent Number 257304
Indian Patent Application Number 6301/DELNP/2006
PG Journal Number 39/2013
Publication Date 27-Sep-2013
Grant Date 23-Sep-2013
Date of Filing 26-Oct-2006
Name of Patentee AGENSYS, INC
Applicant Address 1545-17TH STREET, SANTA MONICA, CA 90404 (US)
Inventors:
# Inventor's Name Inventor's Address
1 CHALLITA-EID, PIA, M 15745 MORRISON STREET, ENCINO, CA 91436 (US)
2 JAKOBOVITS, AYA 3135 HUTTON DRIVE, BEVERLY HILLS, CA 90210 (US)
3 ETESSAMI, SOUDABEH 18336 COLLINS ST., TARZANA, CA 91358 (US)
4 PEREZ-VILLAR,JUAN, J 12424 TEXAS AVENUE, LOS ANGELES, CA 90025 (US)
5 MORRISON, KAREN, J 1044 YALE STREET, SANTA MONICA, CA 90403 (US)`
6 JIA, XIAO-CHI 2262 BEVERLY DRIVE, LOS ANGELES, CA 90034 (US)
7 FARIS, MARY 2538 ALMADEN COURT, LOS ANGELES, CA 90077 (US)
8 GUDAS, JEAN 17157 PALISADES CIRCLE, PACIFIC PALISADES, CA 90272 (US)
9 RAITANO, ARTHUR, B 12685 ROSE AVENUE, LOS ANGELES, CA 90066 (US)
10 NA NA
PCT International Classification Number C07K 16/30
PCT International Application Number PCT/US2004/012625
PCT International Filing date 2004-04-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 PCT/US2004/012625 2004-04-22 U.S.A.