AU2001237973A1 - 84p2a9: a prostate and testis specific protein highly expressed in prostate cancer - Google Patents

Description

84P2A9: A PROSTATE AND TESTIS SPECIFIC PROTEIN HIGHLY EXPRESSED IN PROSTATE CANCER

This application claims the benefit of United States provisional patent application number 60/178,560, filed January 26, 2000, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The invention described herein relates to a novel gene and its encoded protein, termed 84P2A9, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express 84P2A9, particularly prostate cancers.

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, cancer causes the death of well over a half-million people annually, with some 1.4 million new cases diagnosed per year. While deaths from heart disease have been declining significandy, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence. Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North

America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men In the United States alone, well over 40,000 men die annually of this disease - second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities Unfortunately, these treatments are ineffective for many and are often associated with undesuable consequences

On the diagnostic front, the lack of a prostate tumor marker that can accurate!) detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med.3:402) More recendy identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc Nad. Acad. Sci. USA 93. 7252), prostate-specific membrane (PSM) antigen (Pinto et al , Clin Cancer Res 1996 Sep;2(9):1445-51), STEAP (Proc Nad Acad Sci U S A. 1999 Dec 7;96(25):14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Nad Acad. Sci USA 95: 1735).

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

SUMMARY OF THE INVENTION

The present invention relates to a novel, largely prostate and testis-related gene, designated 84P2A9, that is over-expressed in multiple cancers including prostate, testis, kidney, brain, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung cancers Northern blot expression analysis of 84P2A9 gene expression in normal tissues shows a highly prostate and testis-related expression pattern in adult tissues. Analysis of 84P2A9 expression in normal prostate and prostate tumor xenografts shows over-expression in LAPC-4 and LAPC-9 prostate tumor xenografts, with the highest expression in LAPC-9. The nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences of 84P2A9 are shown in FIG. 2. Portions of the 84P2A9 amino acid sequence show some homologies to ESTs in the dbEST database. The prostate and testis-related expression profile of 84P2A9 in normal adult tissues, combined with the over-expression observed in prostate tumor xenografts, shows that 84P2A9 is aberrandy over-expressed in at least some cancers, and thus serves as a useful diagnostic and/or therapeutic target for cancers such as prostate, testis, kidney, brain, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung cancers (see, e.g , FIGS. 4-8).

The invention provides polynucleotides corresponding or complementary to all or part of the 84P2A9 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 84P2A9 proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids, DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 84P2A9 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 84P2A9 genes, mRNAs, or to 84P2A9-encodιng polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 84P2A9. Recombinant DNA molecules containing 84P2A9 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 84P2A9 gene products are also provided. The invention further provides antibodies that bind to 84P2A9 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammahan antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker.

The invention further provides methods for detecting the presence and status of 84P2A9 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 84P2A9. A typical embodiment of this invention provides methods for monitoring 84P2A9 gene products in a tissue or hematology sample having or suspected of having some form of growth disregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 84P2A9 such as prostate cancers, including therapies aimed at inhibiting the transcription, translation, processing or function of 84P2A9 as well as cancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. shows the 84P2A9 suppression subtractive hybridization (SSH) DNA sequence of about 425 nucleotides in length (SEQ ID NO: 3) This sequence was identified in comparisons of cDNAs from various androgen dependent and androgen independent LAPC xenografts.

FIG. 2. shows the nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences of 84P2A9. See Example 2, infra. The sequence surrounding the start ATG

(AAC ATG G) (SEQ ID NO: 4) exhibits a Kozak sequence (A at position -3, and G at position +1). The start methionine with Kozak sequence is indicated in bold, the nuclear localization signals are boxed

FIGS. 3A and 3B. show the amino acid sequence alignment of 84P2A9 (SEQ ID

NO: 2) with KIAA1552 (SEQ ID NO: 5) and LUCA15 (SEQ ID NO: 6). FIG. 3A shows that the 84P2A9 protein sequence (bottom line) has some homology to the human brain protein KIAA1152 (39.5% identity over a 337 amino acid region, Score: 407.0; Gap frequency: 5.9%). FIG. 3B shows that the 84P2A9 protein sequence (bottom line) contains a domain that is homologous to a portion of the LUCA15 tumor suppressor protein (64.3% identity over a 42 amino acid region, Score: 138.0; Gap frequency: 0.0%).

FIGS. 4A-4C. show the Northern blot analysis of the restricted 84P2A9 expression in various normal human tissues (using the 84P2A9 SSH fragment as a probe) and LAPC xenografts. Two multiple tissue northern blots (Clontech) (FIGS. 4A and 4B) and a xenograft northern blot (FIG. 4C) were probed with the 84P2A9 SSH fragment. Lanes 1-8 in FIG. 4A consist of mRNA from heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas respectively. Lanes 1-8 in FIG. 4B consist of total RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and leukocytes respectively. Lanes 1-5 in FIG. 4C consist of mRNA from prostate, LAPC-4 AD, LAPC-4 Al, LAPC-9 AD and LAPC-9 Al respectively. Size standards in lαlobases (kb) are indicated on the side. Each lane contains 2 μg of mRNA for the normal tissues and 10 μg of total RNA for the xenograft tissues. The results show the expression of 84P2A9 in testis and prostate and the LAPC xenografts

FIG. 5. shows the Northern blot analysis of 84P2A9 expression in prostate and multiple cancer cell lines. Lanes 1-56 show expression in LAPC-4 AD, LAPC-4 Al, LAPC-9 AD, LAPC-9 Al, LNCaP, PC-3, DU145, TsuPrl, LAPC-4 CL, HT1197, SCaBER, UM-UC-3, TCCSUP, J82, 5637, 293T, RD-ES, PANC-1, BxPC-3, HPAC, Capan-1, SK-CO-1, CaCo-2, LoVo, T84, Colo-205, KCL 22, PFSK-1 , T98G, SK-ES-1, HOS, U2-OS, RD-ES, CALU-1, A427, NCI-H82, NCI-H146, 769-P, A498, CAKI-1, SW839, BT20, CAMA-1, DU4475, MCF-7, MDA-MB-435s, NTERRA-2, NCCIT, TERA-1, TERA-2, A431, HeLa, OV-1063, PA-1, SW626 and CAOV-3 respectively. High levels of 84P2A9 expression were detected in brain (PFSK-1, T98G), bone (HOS, U2-OS), lung (CALU-1, NCI-H82, NCI-H146), and kidney (769-P, A498, CAKI-1, SW839) cancer cell lines. Moderate expression levels were detected in several pancreatic (PANC-1, BxPC-3, HPAC, CAPAN-1), colon (SK-CO-1, CACO-2, LOVO, COLO- 205), bone (SK-ES-1, RD-ES), breast (MCF-7, MDA-MB-435s) and testicular cancer (NCCIT) cell lines.

FIG. 6. shows the Northern blot analysis of 84P2A9 expression in prostate cancer patient samples. Prostate cancer patient samples show expression of 84P2A9 in both the normal and the tumor part of the prostate tissues Lanes 1-7 show Normal prostate, Patient 1 normal adjacent tissue, Patient 1 Gleason 9 tumor, Patient 2 normal adjacent tissue, Patient 2 Gleason 7 tumor and Patient 3 Gleason 7 tumor respectively. These results provide evidence that 84P2A9 is a very testis specific gene that is up- regulated in prostate cancer and potentially other cancers Similar to the MAGE antigens, 84P2A9 may thus qualify as a cancer-testis antigen (Van den Eynde and Boon, Int J Ckn Lab Res. 27:81-86, 1997).

FIG. 7. shows RNA was isolated from kidney cancers (T) and their adjacent normal tissues (N) obtained from kidney cancer patients. Lanes 1-15 show 769-P- clear cell type; A498 - clear cell type; SW839 - clear cell type; Normal Kidney; Patient 1, N; Patient 1 , tumor; Patient 2, N; Patient 2, tumor, clear cell type, grade III; Patient 3, N; Patient 3, tumor, clear cell type, grade II/IV; Patient 4, N; Patient 4, tumor, clear cell type, grade II/IV; Patient 5, N, Patient 5, tumor, clear cell type, grade II; and Patient 6, tumor, metastasis to chest wall respectively (N ---•-normal adjacent tissue and CL=cell line) Northern analysis was performed using 10μg of total RNA for each sample. Expression of 84P2A9 was seen in all 6 tumor samples tested as well as in the three kidney cell lines, 769-P, A498 and SW839.

FIG. 8. shows RNA was isolated from colon cancers (T) and their adjacent normal tissues (N) obtained from colon cancer patients. Lanes 1-11 show Colo 205, LoVo; T84; Caco-2; Patient 1, N; Patient 1, tumor, grade 2, T3NlMx (positive for lymph node metastasis); Patient 2, N; Patient 2, tumor, grade 1, T2N0Mx; Patient 3, N; Patient 3, tumor, grade 1 , T2NlMx (positive for lymph node metastasis); and Patient 4, tumor, grade 2, T3 Nl MX (positive for lymph node metastasis); respectively (N=normal adjacent tissue and CL=cell line). Northern analysis was performed using 10μg of total RNA for each sample. Expression of 84P2A9 was seen in all 4 tumor samples tested as well as in the 4 colon cancer cell lines Colo 205, LoVo, T84 and Caco-2.

FIG. 9. Shows expression of 84P2A9 assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots. Cancer cell lines from left to right are HeLa (cervical carcinoma), Daudi (Burkitt's lymphoma), K562 (CML), HL-60 (PML), G361 (melanoma), A549 (lung carcinoma), MOLT-4

(lymphoblastic leuk.), SW480 (colorectal carcinoma) and Raji (Burkitt's lymphoma). 84P2A9 expression was seen in kidney cancers, breast cancers, prostate cancers, lung cancers, stomach cancers, colon cancers, cervical cancers and rectum cancers 84P2A9 was also found to be highly expressed in a panel of cancer cell lines, specially the MOLT- 4 lymphoblastic leukemia and the A549 lung carcinoma cell lines The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues, isolated from healthy donors, can indicate that these tissues are not fully normal and that 84P2A9 can be expressed in early stage tumors.

FIG. 10 shows the expression of 84P2A9 in bladder cancer patient specimens. Expression of 84P2A9 was seen in 4 bladder cancer patient specimens tested and in three bladder cell lines (CL), UM-UC-3 (lane 1), J82 (lane 2) and SCABER (lane 3) RNA was isolated from normal bladder (Nb), bladder tumors (T) and their adjacent normal tissues (N) obtained from 6 bladder cancer patients (P). Tumor from PI is transitional carcinoma, grade 4; P2 is invasive squamous carcinoma; P3 is transitional carcinoma, grade 3; P4 is non-invasive papillary carcinoma, grade 1 /3, P5 is papillary carcinoma, grade 3/3; and P6 is transitional carcinoma, grade 3/2. Northern analysis was performed using lOμg of total RNA for each sample.

FIG. 11 shows the expression of 84P2A9 protein in 293T cells. 293T cells were transiendy transfected with either pCDNA3.1 V5-HIS epitope tagged 84P2A9 plasmid or with empty control vector and harvested 2 days later. Cells were lysed in SDS-PAGE sample buffer and lysates were separated on a 10-20% SDS-PAGE gel and then transferred to nitrocellulose. The blot was blocked in Tns-buffered saline (TBS)+ 2% non-fat milk and then probed with a 1 :3,000 dilution of murine anti-V5 monoclonal Ab (Invitrogen) in TBS+0.15% Tween-20+ 1% milk The blot was washed and then incubated with a 1:4,000 dilution of anti-mouse IgG-HRP conjugate secondary antibody Following washing, anti-V5 epitope unmunoreactive bands were developed by enhanced chemiluminescence and visualized by exposure to autoradiographic film. Indicated by arrow is a specific anti-V5 lmmunoreactive band of approximately 87 Kd that corresponds to expression of the epitope-tagged 84P2A9 protein in the transfected cells DETAILED DESCRIPTION OF THE INVENTION

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

DEFINITIONS:

As used herein, the terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and "locally advanced disease" mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage Cl - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presendy diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles. The term "antibody" is used in the broadest sense. Therefore an "antibody" can be naturally occurring or man made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-84P2A9 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/ or one or more complementaπty determining regions of these antibodies. As used herein, an antibody fragment is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen binding region. In one embodiment it specifically covers single anti-84P2A9 antibody (including agonist, antagonist and neutralizing antibodies) and anti-84P2A9 antibody compositions with polyepitopic specificity The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ι.e., the antibodies comprising the population are identical except for possible naturally-occurπng mutations that are present in minor amounts

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. As used herein, the terms "hybridize", "hybridizing", "hybridizes" and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamιde/6XSSC/0.1% SDS/ 100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in O.lXSSC/0.1% SDS are above 55 degrees C. As used herein, a polynucleotide is said to be "isolated" when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 84P2A9 gene or that encode polypeptides other than 84P2A9 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 84P2A9 polynucleotide. As used herein, a protein is said to be "isolated" when physical, mechanical or chemical methods are employed to remove the 84P2A9 protein from cellular constituents that are normally associated with the protein A skilled artisan can readily employ standard purification methods to obtain an isolated 84P2A9 protein.

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

As used herein, the terms "metastatic prostate cancer" and "metastatic disease" mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM+ under the TNM system As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation, and approximately half of these patients die within 6 months after developing androgen refractory status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often characteristically osteoblasttc rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequendy in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

"Moderately stringent conditions" are described by, identified but not limited to, those in Sambrook et al., Molecular Cloning. A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM tπsodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

As used herein "motif as in biological motif of an 84P2A9-realted protein, refers to any set of amino acids forming part of the primary sequence of a protein, either contiguous or capable of being aligned to certain positions that are generally invariant or conserved, that is associated with a particular function or modification (e.g. that is phosphorylated, glycosylated or amidated).

As used herein, the term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/ or RNA. In the art, this term if often used interchangeably with "oligonucleotide" As discussed herein, an polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T) (as shown for example in SEQ ID NO: 1) can also be uracil (U). This description 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).

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

"Stringency" of hybridization reactions is readily determtnable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybndizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Stringent conditions" or "high stringency conditions", as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chlonde/0.0015 M sodium cιtrate/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 albumιn/0.1% Ficoll/0.1% ρolyvιnylpyrrohdone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (PH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium, citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.

A "transgenic animal" (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A "transgene" is a DNA that is integrated into the genome of a cell from which a transgenic animal develops. As used herein, the 84P2A9 gene and protein is meant to include the 84P2A9 genes and proteins specifically described herein and the genes and proteins corresponding to other 84P2A9 encoded proteins or peptides and structurally similar variants of the foregoing. Such other 84P2A9 peptides and variants will generally have coding sequences that are highly homologous to the 84P2A9 coding sequence, and preferably share at least about 50% amino acid homology (using BLAST criteria) and preferably 50%, 60%, 70%, 80%, 90% or more nucleic acid homology, and at least about 60% amino acid homology (using BLAST criteria), more preferably sharing 70% or greater homology (using BLAST criteria).

The 84P2A9-related proteins of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or are readily available in the art. Fusion proteins that combine parts of different 84P2A9 proteins or fragments thereof, as well as fusion proteins of an 84P2A9 protein and a heterologous polypeptide are also included. Such 84P2A9 proteins are collectively referred to as the 84P2A9-related proteins, the proteins of the invention, or 84P2A9. As used herein, the term "84P2A9-related polypeptide" refers to a polypeptide fragment or an 84P2A9 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids

STRUCTURE AND EXPRESSION OF 84P2A9 As discussed in detail below, experiments with the LAPC-4 AD xenograft in male SCID mice have resulted in the identification of genes that are involved in the progression of androgen dependent (AD) prostate cancer to androgen independent (Al) cancer Briefly, mice that harbored LAPC-4 AD xenografts were castrated when the tumors reached a size of 1 cm in diameter. The tumors regressed in size and temporarily stopped producing the androgen dependent protein PSA. Seven to fourteen days post- castration, PSA levels were detectable again in the blood of the mice. Eventually such tumors develop an Al phenotype and start growing again in the castrated males. Tumors were harvested at different time points after castration to identify genes that are turned on or off during the transition to androgen independence. Suppression subtractive hybridization (SSH) (Diatchenko et al., 1996, PNAS

93:6025) was then used to identify novel genes, such as those that are overexpressed in prostate cancer, by comparing cDNAs from various androgen dependent and androgen independent LAPC xenografts This strategy resulted in the identification of novel genes exhibiting tissue and cancer specific expression. One of these genes, designated 84P2A9, was identified from a subtraction where cDNA derived from an LAPC-4 AD tumor, 3 days post-castration, was subtracted from cDNA derived from an LAPC-4 AD tumor grown in an intact male. The SSH DNA sequence of about 425 bp (Fig. 1) is novel and exhibits homology only to expressed sequence tags (ESTs) in the dbEST database

84P2A9, encodes a putative nuclear protein that exhibits prostate and testis- related expression. The initial characterization of 84P2A9 indicates that it is aberrandy expressed multiple cancers including prostate, testis, kidney, brain, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung cancers. The expression of 84P2A9 in prostate cancer provides evidence that this protein has a functional role in tumor progression It is possible that 84P2A9 functions as a transcription factor involved in activating genes involved in tumongenesis or repressing genes that block tumongenesis. As is further described in the Examples that follow, the 84P2A9 genes and proteins have been characterized using a number of analytical approaches. For example, analyses of nucleotide coding and amino acid sequences were conducted in order to identify potentially related molecules, as well as recognizable structural domains, topological features, and other elements within the 84P2A9 mRNA and protein structures Northern blot analyses of 84P2A9 mRNA expression were conducted in order to establish the range of normal and cancerous tissues expressing 84P2A9 message

A full length 84P2A9 cDNA clone (clone 1) of 2345 base pairs (SEQ ID NO: 1) was cloned from an LAPC-4 AD cDNA library (Lambda ZAP Express, Stratagene) (Fig

2). The cDNA encodes an open reading frame (ORF) of 504 amino acids (SEQ ID NO: 2) Sequence analysis revealed the presence of six potential nuclear localization signals and is predicted to be nuclear using the PSORT program (http.//psort.nιbb.ac.jp:8800/form.html). The protein sequence has some homology to a human brain protein KIAA1152 (SEQ ID NO: 5) (39.5% identity over a 337 amino acid region), and contains a domain that is homologous to the LUCA15 tumor suppressor protein (SEQ ID NO: 6) (64.3% identity over a 42 amino acid region) (GenBank Accession #P52756)(Fιg. 3).

84P2A9 expression is prostate and testis-related in normal adult human tissues, but is also expressed in certain cancers, including prostate, testis, kidney, bram, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung cancers, (see, e.g., FIGS. 4- 8). Human prostate tumor xenografts originally derived from a patient with high grade metastatic prostate cancer express high levels of 84P2A9 (FIG. 4).

As disclosed herein, 84P2A9 exhibits specific properties that are analogous to those found in a family of genes whose polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic assays directed to examining conditions associated with disregulated cell growth such as cancer, in particular prostate cancer (see, e.g., both its highly specific pattern of tissue expression as well as its over expression in prostate cancers as described for example in Example 3) The best known member of this class is PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol 163(2): 503-5120 (2000); Polascik et al., J. Urol. Aug;162(2).293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91 (19): 1635-1640(1999)). A variety of other diagnostic markers are also used in this context including p53 and K-ras (see, e.g , Tulchinsky et al., Int J Mol Med 1999 Jul;4(l):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12) Therefore, this disclosure of the 84P2A9 polynucleotides and polypeptides (as well as the 84P2A9 polynucleotide probes and anti-84P2A9 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 84P2A9 polynucleotides, polypeptides and antibodies described herein are analogous to those methods from well established diagnostic assays which employ PSA polynucleotides, polypeptides and antibodies For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Shanef et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al, J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 84P2A9 polynucleotides described herein can be utilized in the same way to detect 84P2A9 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods of monitoring PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 84P2A9 polypeptides described herein can be utilized to generate antibodies for use in detecting 84P2A9 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene. Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the testis 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 84P2A9 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 84P2A9 expressing cells (lymph node) is found to contain 84P2A9 expressing cells such as the 84P2A9 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis Alternatively 84P2A9 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when a cells in biological sample that do not normally express 84P2A9 or express 84P2A9 at a different level are found to express 84P2A9 or have an increased expression of 84P2A9 (see, e.g., the 84P2A9 expression in kidney, lung and colon cancer cells and in patient samples etc. shown in Figures 4-10). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 84P2A9) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 84P2A9 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechmques 25(3): 472- 476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in Example 3, where an 84P2A9 polynucleotide fragment is used as a probe to show the overexpression of 84P2A9 mRNAs in cancer cells. In addition, in order to_ facilitate their use by medical practitioners, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al, Fetal Diagn. Ther. 1996 Nov-Dec;l l (6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds , 1995)). Polynucleotide fragments and variants are typically useful in this context as long as they have the common attribute or characteristic of being capable of binding to a target polynucleotide sequence (e.g. the 84P2A9 polynucleotide shown in SEQ ID NO: 1) under conditions of high stringency.

Just as PSA polypeptide fragments and polypeptide variants are employed by skilled artisans for use in methods of monitoring the PSA molecule, 84P2A9 polypeptide fragments and polypeptide variants can also be used in an analogous manner In particular, typical PSA polypeptides used in methods of monitoring PSA are fragments of the PSA protein which contain an antibody epitope that can be recognized by an antibody or T cell that specifically binds to the PSA protein. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995). In this context, each epιtope(s) in a protein of interest functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans generally create a variety of different polypeptide fragments that can be used in order to generate antibodies specific for different portions of a polypeptide of interest (see, e.g., U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 84P2A9 biological motifs discussed herein or available in the art. Polypeptide fragments and variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. the 84P2A9 polypeptide shown in SEQ ID NO: 2).

As shown herein, the 84P2A9 polynucleotides and polypeptides (as well as the 84P2A9 polynucleotide probes and anti-84P2A9 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers of the prostate. Diagnostic assays that measure the presence of 84P2A9 gene products, in order to evaluate the presence or onset of the particular disease conditions described herein such as prostate cancer are particularly useful in identifying patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a testing for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequentiy, materials such as 84P2A9 polynucleotides and polypeptides (as well as the 84P2A9 polynucleotide probes and anti-84P2A9 antibodies used to identify the presence of these molecules) must be employed to confirm metastases of prostatic origin

Finally, in addition to their use in diagnostic assays, the 84P2A9 polynucleotides disclosed herein have a number of other specific utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in lq32.3. Moreover, in addition to their use in diagnostic assays, the 84P2A9-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun 28;80(l-2): 63-9).

84P2A9 POLYNUCLEOTIDES

One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 84P2A9 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 84P2A9 protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or ohgonucleotides complementary to an 84P2A9 gene or mRNA sequence or a part thereof, and polynucleotides or ohgonucleotides that hybridize to an 84P2A9 gene, mRNA, or to an 84P2A9 encoding polynucleotide (collectively, "84P2A9 polynucleotides") .

One embodiment of an 84P2A9 polynucleotide is an 84P2A9 polynucleotide having the sequence shown in SEQ ID NO: 1. An 84P2A9 polynucleotide can comprise a polynucleotide having the nucleotide sequence of human 84P2A9 as shown in SEQ ID NO: 1, wherein T can also be U; a polynucleotide that encodes all or part of the 84P2A9 protem; a sequence complementary to the foregomg; or a polynucleotide fragment of any of the foregomg. Another embodiment comprises a polynucleotide havmg the sequence as shown in SEQ ID NO: 1, from nucleotide residue number 163 through nucleotide residue number 1674, or from residue number 718 through residue number 1390, wherein T can also be U Another embodiment comprises a polynucleotide encoding an 84P2A9 polypeptide whose sequence is encoded by the cDNA contained in the plasmid as deposited with American Type Culture Collection as Accession No. PTA-1151 Another embodiment comprises a polynucleotide that is capable of hybridizing under stringent hybridization conditions to the human 84P2A9 cDNA shown m SEQ ID NO: 1 or to a polynucleotide fragment thereof.

Typical embodiments of the invention disclosed herein include 84P2A9 polynucleotides encodmg specific portions of the 84P2A9 mRNA sequence (and those which are complementary to such sequences) such as those that encode the protem and fragments thereof, for example of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. For example, representative embodiments of the invention disclosed herein include: polynucleotides encoding about ammo acid position 1 to about amino acid 10 of the 84P2A9 protem shown in Fig. 2 (SEQ ID NO: 2), polynucleotides encoding about ammo acid 10 to about amino acid 20 of the 84P2A9 protein shown in Fig. 2, polynucleotides encodmg about ammo acid 20 to about ammo acid 30 of the 84P2A9 protem shown in Fig. 2, polynucleotides encoding about amino acid 30 to about ammo acid 40 of the 84P2A9 protem shown in Fig. 2, polynucleotides encoding about am o acid 40 to about ammo acid 50 of the 84P2A9 protem shown m Fig. 2, polynucleotides encoding about ammo acid 50 to about ammo acid 60 of the 84P2A9 protem shown in Fig. 2, polynucleotides encodmg about ammo acid 60 to about ammo acid 70 of the 84P2A9 protem shown in Fig. 2, polynucleotides encoding about amino acid 70 to about ammo acid 80 of the 84P2A9 protem shown in Fig. 2, polynucleotides encoding about ammo acid 80 to about ammo acid 90 of the 84P2A9 protem shown m Fig. 2 and polynucleotides encodmg about amino acid 90 to about amino acid 100 of the 84P2A9 protem shown m Fig. 2, etc. Following this scheme, polynucleotides (of at least 10 nucleic acids) encoding portions of the ammo acid seqμence of amino acids 100-504 of the 84P2A9 protem are typical embodiments of the invention.

Polynucleotides encoding larger portions of the 84P2A9 protem are also contemplated. For example polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about ammo acid 20, (or 30, or 40 or 50 etc ) of the 84P2A9 protem shown m Fig. 2 can be generated by a variety of techniques well known m the art An illustrative embodiment of such a polynucleotide consists of a polynucleotide havmg the sequence as shown in FIG. 2, from nucleotide residue number 718 through nucleotide residue number 1390 Additional illustrative embodiments of the invention disclosed herem include

84P2A9 polynucleotide fragments encodmg one or more of the biological motifs contained within the 84P2A9 protem sequence. In one embodiment, typical polynucleotide fragments of the invention can encode one or more of the nuclear localization sequences disclosed here . In another embodiment, typical polynucleotide fragments of the invention can encode one or more of the regions of 84P2A9 that exhibit homology to LUCA 15 and/or KIAA1152 and/or NY-Lu-12 lung cancer antigen (AF 042857), which exhibits Zinc finger and RNA binding motifs (see, e.g., Gure et al, Cancer Res. 58(5)- 1034-1041 (1998) In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 84P2A9 N-glycosylation sites, cAMP and cCMP-dependent protem kmase phosphorylation sites, casern kmase II phosphorylation sites or N-myπstoylation site and amidation sites as disclosed m greater detail m the text discussing the 84P2A9 protem and polypeptides herem. In yet another embodiment of the invention, typical polynucleotide fragments can encode sequences that are unique to one or more 84P2A9 alternative splicing variants, such as the splice variant that generates the 4.5 KB transcript that is overexpressed in prostate cancers shown in FIG. 4.

The polynucleotides of the preceding paragraphs have a number of different specific uses. For example, because the human 84P2A9 gene maps to chromosome lq32.3, polynucleotides encoding different regions of the 84P2A9 protem can be used to characterize cytogenetic abnormakties on chromosome 1, band q32 that have been identified as bemg associated with various cancers. In particular, a variety of chromosomal abnormakties in lq32 mcluding translocations and deletions have been identified as frequent cytogenetic abnormakties m a number of different cancers (see, e.g., Bieche et al., Genes Chromosomes Cancer, 24(3): 255-263 (1999); Gorunova et al., Genes Chromosomes Cancer, 26(4): 312-321 (1999); Reid et al , Cancer Res. (22): 5415- 5423 (1995)). Consequentiy, polynucleotides encodmg specific regions of the 84P2A9 protem provide new tools that can be used to delineate with a greater precision than previously possible, the specific nature of the cytogenetic abnormalities in this region of chromosome 1 that can contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening m order to identify more subde and less common chromosomal abnormalities (see, e g., Evans et al., Am. J. Obstet. Gynecol 171 (4)- 1055-1057 (1994)).

Alternatively, as 84P2A9 is shown to be highly expressed m prostate cancers (Fig 4), these polynucleotides can be used m methods assessmg the status of 84P2A9 gene products m normal versus cancerous tissues Typically, polynucleotides encoding specific regions of the 84P2A9 protem can be used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting m a loss of an antigen etc.) m specific regions (such regions containing a nuclear localization signal) of the 84P2A9 gene products. Exemplary assays mclude both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protem to examine these regions within the protem.

Other specifically contemplated nucleic acid related embodiments of the invention disclosed herem are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone or including alternative bases, whether derived from natural sources or synthesized. For example, antisense molecules can be RNAs or other molecules, mcluding peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bmd DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules usmg the 84P2A9 polynucleotides and polynucleotide sequences disclosed herem Antisense technology entails the administration of exogenous ohgonucleotides that bind to a target polynucleotide located within the cells. The term "antisense" refers to the fact that such ohgonucleotides are complementary to their intracellular targets, e.g , 84P2A9 See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1 :1-5 (1988) The 84P2A9 antisense ohgonucleotides of the present mvention mclude derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-ohgos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-ohgos (nucleoside phosphorothioates) are lsoelectronic analogs of an okgonucleotide (O-okgo) in which a nonbπdging oxygen atom of the phosphate group is replaced by a sulfur atom. The S- okgos of the present mvention can be prepared by treatment of the corresponding O- okgos with 3H-l,2-benzodιthιol-3-one-l,l-dιoxιde, which is a sulfur transfer reagent See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 84P2A9 antisense ohgonucleotides of the present mvention mclude moφhohno antisense ohgonucleotides known m the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6. 169-175).

The 84P2A9 antisense ohgonucleotides of the present mvention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 N- terrmnal codons or last 100 C-terminal codons of the 84P2A9 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an okgonucleotide complementary to this region allows for the selective hybridization to 84P2A9 mRNA and not to mRNA specifying other regulatory subumts of protein kmase. Preferably, the 84P2A9 antisense ohgonucleotides of the present mvention are a 15 to 30-mer fragment of the antisense DNA molecule havmg a sequence that hybridizes to 84P2A9 mRNA. Optionally, 84P2A9 antisense okgonucleotide is a 30-mer okgonucleotide that is complementary to a region in the first 10 N-terminal codons or last 10 C-terminal codons of 84P2A9. Alternatively, the antisense molecules are modified to employ ribozymes m the inhibition of 84P2A9 expression. L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996). Further specific embodiments of this aspect of the mvention mclude primers and primer pairs, which allow the specific ampkfication of the polynucleotides of the mvention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the mvention or to any part thereof Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers can be used to detect the presence of an 84P2A9 polynucleotide in a sample and as a means for detecting a cell expressing an 84P2A9 protem. Examples of such probes mclude polypeptides compαsmg all or part of the human

84P2A9 cDNA sequences shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 84P2A9 mRNAs are also described m the Examples that follow. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herem and used effectively to ampkfy and/or detect an 84P2A9 mRNA.

The 84P2A9 polynucleotides of the mvention are useful for a variety of purposes, mcluding but not limited to their use as probes and primers for the amplification and/or detection of the 84P2A9 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 84P2A9 polypeptides; as tools for modulating or inhibiting the expression of the 84P2A9 gene(s) and/or translation of the 84P2A9 transcπpt(s); and as therapeutic agents.

ISOLATION OF 84P2A9-ENCODING NUCLEIC ACID MOLECULES The 84P2A9 cDNA sequences described herem enable the isolation of other polynucleotides encoding 84P2A9 gene product(s), as well as the isolation of polynucleotides encoding 84P2A9 gene product homologs, alternatively spkced isoforms, allekc variants, and mutant forms of the 84P2A9 gene product. Various molecular cloning methods that can be employed to isolate full length cDNAs encodmg an 84P2A9 gene are well known (See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition., Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds , Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniendy employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 84P2A9 gene cDNAs can be identified by probing with a labeled 84P2A9 cDNA or a fragment thereof. For example, m one embodiment, the 84P2A9 cDNA (FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full length cDNAs corresponding to an 84P2A9 gene. The 84P2A9 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome kbranes (BACs), yeast artificial chromosome libraries (YACs), and the like, with 84P2A9 DNA probes or primers.

RECOMBINANT DNA MOLECULES AND HOST-VECTOR SYSTEMS

The mvention also provides recombinant DNA or RNA molecules containing an 84P2A9 polynucleotide or a fragment or analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as vaπous viral and non-viral vectors well known m the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. As used herem, a recombinant DNA or RNA molecule is a DNA or RNA molecule that has been sub_jected to molecular manipulation in vitro. Methods for generating such molecules are well known (see, for example, Sambrook et al, 1989, supra).

The mvention further provides a host-vector system comprismg a recombinant DNA molecule containing an 84P2A9 polynucleotide or fragment or analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells mclude a yeast cell, a plant cell, or an animal cell, such as a mammahan cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammahan cells mclude vanous prostate cancer cell lines such as DU145 and TsuPrl, other tiransfectable or transducible prostate cancer cell lines, as well as a number of mammalian cells routinely used for the expression of recombinant protems (e.g., COS, CHO, 293, 293T cells) More particularly, a polynucleotide comprismg the cod g sequence of 84P2A9 or a fragment or analog or homolog thereof can be used to generate 84P2A9 protems or fragments thereof using any number of host-vector systems routinely used and widely known m the art

A wide range of host-vector systems suitable for the expression of 84P2A9 protems or fragments thereof are available, see for example, Sambrook et al , 1989, supra, Current Protocols m Molecular Biology, 1995, supra) Preferred vectors for mammalian expression mclude but are not limited to pcDNA 3 1 myc-His-tag (Invitrogen) and the retrovrral vector pSRαtkneo (Muller et al , 1991, MCB 11 1785) Usmg these expression vectors, 84P2A9 may be preferably expressed m several prostate cancer and non-prostate cell lines, mcluding for example 293, 293T, rat-1, NIH 3T3 and TsuPrl The host- vector systems of the mvention are useful for the production of an 84P2A9 protem or fragment thereof Such host-vector systems can be employed to study the functional properties of 84P2A9 and 84P2A9 mutations or analogs

Recombinant human 84P2A9 protem or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding 84P2A9 In an illustrative embodiment described m the Examples, 293T cells can be transfected with an expression plasmid encoding 84P2A9 or fragment or analog or homolog thereof, the 84P2A9 or related protem is expressed m the 293T cells, and the recombmant 84P2A9 protem can be isolated usmg standard purification methods (e g , affinity purification usmg anti-84P2A9 antibodies) In another embodiment, also described m the Examples herein, the 84P2A9 coding sequence is subcloned into the retrovrral vector pSRαMSVtkneo and used to fect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 m order to estabhsh 84P2A9 expressmg cell lines Vanous other expression systems well known m the art can also be employed Expression constructs encoding a leader peptide jomed in frame to the 84P2A9 codmg sequence can be used for the generation of a secreted form of recombmant 84P2A9 protem

Protems encoded by the 84P2A9 genes, or by analogs or homologs or fragments thereof, will have a vaπety of uses, mcluding but not limited to generating antibodies and m methods for identifying ligands and other agents and cellular constituents that bmd to an 84P2A9 gene product Antibodies raised agamst an 84P2A9 protem or fragment thereof can be useful m diagnostic and prognostic assays, and imaging methodologies m the management of human cancers characterized by expression of 84P2A9 protem, including but not limited to cancers of the prostate and testis. Such antibodies can be expressed lntracellularly and used m methods of treating patients with such cancers. Vanous immunological assays useful for the detection of 84P2A9 protems are contemplated, mcludmg but not limited to vanous types of radioimmunoassays, enzyme- linked immunosorbent assays (ELISA), enzyme-linked lmmunofluorescent assays (ELIFA), lmmunocytochemical methods, and the like. Such antibodies can be labeled and used as immunological imaging reagents capable of detecting 84P2A9 expressmg cells (e g., in radioscintigraphic imaging methods). 84P2A9 protems can also be particularly useful in generating cancer vaccines, as further descnbed below.

84P2A9 POLYPEPTIDES

Another aspect of the present invention provides 84P2A9-related protems and polypeptide fragments thereof Specific embodiments of 84P2A9 proteins comprise a polypeptide havmg all or part of the amino acid sequence of human 84P2A9 as shown m FIG. 2. Alternatively, embodiments of 84P2A9 protems comprise variant polypeptides havmg alterations m the ammo acid sequence of human 84P2A9 shown m FIG. 2.

In general, naturally occurring allekc vaπants of human 84P2A9 share a high degree of structural identity and homology (e.g., 90% or more identity). Typically, allelic vanants of the 84P2A9-related protems contam conservative amino acid substitutions within the 84P2A9 sequences descnbed herem or contam a substitution of an amino acid from a corresponding position m a homologue of 84P2A9. One class of 84P2A9 allelic vanants are protems that share a high degree of homology with at least a small region of a particular 84P2A9 ammo acid sequence, but further contam a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In compansons of protem sequences, the terms, Similanty, identity, and Homology each have a distinct meaning. Moreover, Orthology and Paralogy are important concepts descnbing the relationship of members of a given protem family m one organism to the members of the same family m other organisms. Conservative amino acid substitutions can frequendy be made m a protem without altering either the conformation or the function of the protem. Such changes mclude substituting any of isoleucme (I), vakne (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagme (N) and vice versa; and serme (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role m the three-dimensional structure of the protem. For example, glycme (G) and alanme (A) can frequendy be mterchangeable, as can alanme (A) and va ne (V) Methionine (M), which is relatively hydrophobic, can frequendy be mterchanged with leucine and isoleucme, and sometimes with vahne. Lysine (K) and arginine (R) are frequendy mterchangeable in locations m which the significant feature of the amino acid residue is its charge and the differing pK's of these two ammo acid residues are not significant. Still other changes can be considered "conservative" m particular environments (see, e.g. Table 2 herem; pages 13- 15 "Biochemistry" 2^nd ED. Lubert Stryer ed (Stanford Umversity); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al, J Biol Chem 1995 May 19; 270(20):11882-6).

Embodiments of the mvention disclosed herem mclude a wide variety of art accepted variants of 84P2A9 protems such as polypeptides havmg ammo acid insertions, deletions and substitutions. 84P2A9 variants can be made usmg methods known m the art such as site-directed mutagenesis, alanme scanning, and PCR mutagenesis. Site- directed mutagenesis [Carter et al, Nucl. Acids Res., /i/4331 (1986); Zoller et al, Nucl. A ds Res., /0:6487 (1987)], cassette mutagenesis [Wells et al. Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al, Philos. Trans. R Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the 84P2A9 vanant DNA.

Scanning ammo acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is mvolved m a specific biological activity such as a protein-protein interaction. Among the preferred scanning ammo acids are relatively small, neutral ammo acids. Such ammo acids include alanme, glvcine, sertne, and cysteme Alanme is typically a preferred scanning amino acid among this group because it eliminates the side-cham beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanme is also typically preferred because it is the most common ammo acid Further, it is frequendy found m both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co, N.Y.); Chothia, J. Mol. Biol, 150:1 (1976)]. If alanme substitution does not yield adequate amounts of vanant, an lsosteπc ammo acid can be used.

As defined herem, 84P2A9 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope m common with an 84P2A9 protem havmg the amino acid sequence of SEQ ID NO. 2, such that an antibody or T cell that specifically binds to an 84P2A9 variant will also specifically bmd to the 84P2A9 protem havmg the amino acid sequence of SEQ ID NO: 2. A polypeptide ceases to be a of the protem shown m SEQ ID NO. 2 when it no longer contams an epitope capable of bemg recognized by an antibody or T cell that specifically bmds to an 84P2A9 protem. Those skilled m the art understand that antibodies that recognize protems bmd to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of ammo acids in a minimal epitope. See, e.g., Nair et al, J. Immunol 2000 165(12): 6949-6955; Hebbes et al, Mol Immunol (1989) 26(9):865-73; Schwartz et al, J Immunol (1985) 135(4):2598-608. Another specific class of 84P2A9-related protem variants shares 90% or more identity with the ammo acid sequence of SEQ ID NO. 2 or a fragment thereof Another specific class of 84P2A9 protem variants or analogs comprise one or more of the 84P2A9 biological motifs described below or presendy known in the art. Thus, encompassed by the present mvention are analogs of 84P2A9 fragments (nucleic or ammo acid) that 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 apphed to the nucleic or amino acid sequences of FIG. 2.

As discussed herem, embodiments of the claimed invention include polypeptides containing less than the 504 amino acid sequence of the 84P2A9 protem shown m FIG. 2. For example, representative embodiments of the mvention compnse peptides/proteins havmg any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous ammo acids of the 84P2A9 protem shown m Fig. 2 (SEQ ID NO: 2) Moreover, representative embodiments of the invention disclosed herem mclude polypeptides consisting of about amino acid 1 to about amino acid 10 of the 84P2A9 protem shown in Fig. 2, polypeptides consisting of about ammo acid 10 to about amino acid 20 of the 84P2A9 protem shown m Fig. 2, polypeptides consisting of about amino acid 20 to about amino acid 30 of the 84P2A9 protem shown m Fig. 2, polypeptides consisting of about am o acid 30 to about ammo acid 40 of the 84P2A9 protem shown in Fig. 2, polypeptides consisting of about ammo acid 40 to about ammo acid 50 of the 84P2A9 protem shown in Fig. 2, polypeptides consisting of about ammo acid 50 to about ammo acid 60 of the 84P2A9 protem shown in Fig. 2, polypeptides consisting of about ammo acid 60 to about am o acid 70 of the 84P2A9 protem shown m Fig. 2, polypeptides consisting of about ammo acid 70 to about amino acid 80 of the 84P2A9 protem shown m Fig. 2, polypeptides consisting of about amino acid 80 to about ammo acid 90 of the 84P2A9 protem shown in Fig. 2 and polypeptides consisting of about amino acid 90 to about ammo acid 100 of the 84P2A9 protem shown m Fig. 2, etc. throughout the entirety of the 84P2A9 sequence. Following this scheme, polypeptides consisting of portions of the ammo acid sequence of ammo acids 100-504 of the 84P2A9 protem are typical embodiments of the mvention. Polypeptides consisting of larger portions of the 84P2A9 protem are also contemplated. For example polypeptides consisting of about ammo acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 84P2A9 protem shown in Fig. 2 can be generated by a variety of techniques well known m the art. It is to be apprecited that the starting and stoppmg positions m this paragraph refer to the specified position as well as that position plus or minus 5 residues.

Additional illustrative embodiments of the mvention disclosed herem mclude

84P2A9-related protems containing the ammo acid residues of one or more of the biological motifs contamed within the 84P2A9-related protem sequence as shown in Figure 2. In one embodiment, protems of the mvention comprise one or more of the 84P2A9 nuclear localization sequences such as RKRR at residues 42-45 of SEQ ID NO: 2, RKRR at residues 47-50 of SEQ ID NO: 2, KRRP at residues 101-104 of SEQ ID NO: 2, RRRRRK at residues 135-139 of SEQ ID NO: 2 and/or KKRK at residues 186- 189 of SEQ ID NO: 2. In another embodiment, protems of the mvention comprise one or more of the 84P2A9 N-glycosylation sites such as NRTL at residues 131-134 of SEQ ID NO: 2, NQTN at residues 212-215 of SEQ ID NO: 2 and/or NCSV at residues 394- 397 of SEQ ID NO: 2. In another embodiment, protems of the mvention comprise one or more of the regions of 84P2A9 that exhibit homology to LUCA 15 and/or KIAA1152. In another embodiment, protems of the mvention comprise one or more of the 84P2A9 cAMP and cGMP-dependent protem kinase phosphorylation sites such as KRRS at residues 48-51 of SEQ ID NO- 2 and/or RRPS at residues 102-105 of SEQ ID NO. 2. In another embodiment, protems of the invention compnse one or more of the 84P2A9 Protem Kinase C phosphorylation sites such as TLR at residues 133-135 of SEQ ID NO: 2, SNK at residues 152-154 of SEQ ID NO: 2, SDR at residues 171-173 of SEQ ID NO. 2, TNK at residues 214-216 of SEQ ID NO: 2, SRR at residues 313-315 of SEQ ID NO. 2, SSK at residues 328-330 of SEQ ID NO: 2 and/or SVR at residues 396-398 of SEQ ID NO: 2. In another embodiment, protems of the mvention compnse one or more of the 84P2A9 casern kinase II phosphorylation sites such as SALE at residues 10-13 of SEQ ID NO. 2, SSLE at residues 70-73 of SEQ ID NO: 2, SLEE at residues 71-74 of SEQ ID NO: 2, SDSD at residues 91-94 of SEQ ID NO: 2, TNKD at residues 214-217 of SEQ ID NO. 2, SESD at residues 232-235 of SEQ ID NO: 2, SSTD at residues 240-243 of SEQ ID NO. 2, TNDE at residues 248-251 of SEQ ID NO: 2, TELD at residues 287-290 of SEQ ID NO: 2 and/or TEHD at residues 374-377 of SEQ ID NO: 2. In another embodiment, protems of the mvention comprise one or more of the N-myπstoylation sites such as GSDSSL at residues 67-72 of SEQ ID NO: 2, GLFTND at residues 245-250 of SEQ ID NO: 2, GGACGI at residues 269-274 of SEQ ID NO: 2, GGTPTS at residues 336-341 of SEQ ID NO: 2, GTPTSM at residues 337- 342 of SEQ ID NO. 2, GSLCTG at residues 409-414 of SEQ ID NO: 2, GSGLGR at residues 459-464 of SEQ ID NO: 2 and/or GLGLGF at residues 481-486 of SEQ ID NO: 2 In another embodiment, protems of the invention compnse one or more amidation sites such as RGRK at residues 45-48 of SEQ ID NO: 2 and/or RGKR at residues 113-116 of SEQ ID NO: 2. An illustrative embodiment of such a polypeptide mcludes two or more ammo acid sequences selected from the group consisting of KKRK, NQTN, NCSV, TNK, SRR, SSK, SVR, GLFTND, GGACGI, GGTPTS, GTPTSM and GSLCTG (as identified above in SEQ ID NO- 2). In a preferred embodiment, the polypeptide comprises three or four or five or six or more am o acid sequences KKRK, NQTN, NCSV, TNK, SRR, SSK, SVR, GLFTND, GGACGI, GGTPTS, GTPTSM and GSLCTG (as identified above m SEQ ID NO: 2).

In another embodiment, proteins of the mvention comprise one or more of the unmunoreactive epitopes identified by a process described herem such as such as those shown in Table 1. Processes for identifying peptides and analogues havmg affinities for HLA molecules and which are correlated as immunogenic epitopes, are well known m the art. Also disclosed are principles for creating analogs of such epitopes in order to modulate lmmunogemcity. A variety of references are useful in the identification of such molecules. See, for example, WO 9733602 to Chestnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al, J. Immunol. 2001 166(2): 1389-1397; Alexander et al, Immunol. Res. 18(2): 79-92; Sidney et al , Hum Immunol. 1997 58(1): 12-20; Kondo et al, Immunogenetics 1997 45(4): 249-258; Sidney et al, J. Immunol. 1996 157(8): 3480- 90; and Falk et al. Nature 351: 290-6 (1991), Hunt et al. Science 255:1261-3 (1992); Parker et al, J. Immunol. 149:3580-7 (1992); Parker et al, J. Immunol. 152:163-75 (1994)); Kast et al, 1994 152(8). 3904-12; Borras-Cuesta et al , Hum. Immunol. 2000 61 (3). 266-278; Alexander et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al, PMID: 7895164, UI: 95202582; O'Sulkvan et al, J. Immunol. 1991 147(8): 2663- 2669; Alexander et al. Immunity 1994 1 (9): 751-761 and Alexander et al, Immunol. Res. 1998 18(2): 79-92. The disclosures of these publications are hereby incoφorated by reference herem m their entireties Related embodiments of the mvention comprise polypeptides containing combinations of the different motifs discussed herem, where certam embodiments contam no insertions, deletions or substitutions either within the motifs or the intervening sequences of these polypeptides. In addition, embodiments which mclude a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, mclude a greater portion of the polypeptide architecture m which the motif is located). Typically the number of N- terminal and/or C-terminal ammo acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

In another embodiment of the mvention, proteins of the mvention comprise amino acid sequences that are unique to one or more 84P2A9 alternative sphcmg variants, such as the splice variant encoded by the 4.5 KB transcript that is overexpressed in prostate cancers and shown m FIG. 4. The monitoring of alternative splice variants of 84P2A9 is useful because changes in the alternative sphcmg of protems is suggested as one of the steps m a senes of events that lead to the progression of cancers (see, e.g., Carstens et al , Oncogene 15(250: 3059-3065 (1997)) Consequendy, momtormg of alternative splice variants of 84P2A9 provides an additional means to evaluate syndromes associated with perturbations m 84P2A9 gene products such as cancers.

Polypeptides comprising one or more of the 84P2A9 motifs discussed herem are useful m elucidating the specific characteristics of a malignant phenotype in view of the observation that the 84P2A9 motifs discussed herem are associated with growth disregulation and because 84P2A9 is overexpressed m cancers (FIG. 4) Thus, the presence in a protem of motifs related to these enzymes or molecules is relevant. For example, Casern kmase II, cAMP and cCMP-dependent protem kmase and Protem Kmase C for example are enzymes known to be associated with the development of the malignant phenotype (see, e.g., Chen et al. Lab Invest , 78(2): 165-174 (1998); Gaiddon et al, Endocrmology 136(10): 4331-4338 (1995); HaU et al. Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al, Oncogene 18(46): 6322-6329 (1999) and O'Bπan, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristylation are protem modifications also associated with cancer and cancer progression (see, e.g., Dennis et al, Biochim. Biophys. Acta 1473(l):21-34 (1999); Raju et al, Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protem modification associated with cancer and cancer progression (see, e.g., Treston et al, J. Nad. Cancer Inst. Monogr. (13): 169-175 (1992)) In addition, nuclear localization sequences are beheved to influence the malignant potential of a cell (see, e.g., Mirski et al. Cancer Res. 55(10): 2129-2134 (1995)). The protems of the mvention have a number of different specific uses. As

84P2A9 is shown to be highly expressed m prostate cancers (Fig 4), these peptides/proteins are used m methods assessmg the status of 84P2A9 gene products m normal versus cancerous tissues and elucidating the malignant phenotype Typically, polypeptides encodmg specific regions of the 84P2A9 protem are used to assess the presence of perturbations (such as deletions, msertions, pomt mutations etc.) m specific regions (such regions containing a nuclear localization signal) of the 84P2A9 gene products. Exemplary assays utilize antibodies or T cells targeting 84P2A9-related protems comprising the amino acid residues of one or more of the biological motifs contamed within the 84P2A9 polypeptide sequence m order to evaluate the characteristics of this region m normal versus cancerous tissues. Alternatively, 84P2A9 polypeptides containing the amino acid residues of one or more of the biological motifs contamed within the 84P2A9 protems are used to screen for factors that mteract with that region of 84P2A9.

As discussed herem, redundancy in the genetic code permits variation in 84P2A9 gene sequences. In particular, one skilled m the art will recognize specific codon preferences by a specific host species, and can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons havmg a useage frequency of less than about 20% m known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as: htφ://www.dna.affrc.go.jp/~nakamura/codon.html Nucleotide sequences that have been optimized for a particular host species by replacmg any codons havmg a useage frequency of less than about 20% are referred to herem as "codon optimized sequences."

Additional sequence modifications are known to enhance protem expression in a cellular host. These mclude ehmmation of sequences encoding spurious polyadenylation signals, exon/mtron splice site signals, transposon-hke 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 m the host cell. Where possible, the sequence is modified to avoid predicted haiφin secondary mRNA structures. Other useful modifications mclude the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol, 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic πbosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7). 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)) Nucleotide sequences that have been optimized for expression m a given host species by ehmmation of spurious polyadenylation sequences, elimination of . exon/intron sphcmg signals, ehmmation of transposon-hke repeats and/or optimization of GC content in addition to codon optimization are referred to herem as an "expression enhanced sequence."

84P2A9 protems are embodied m many forms, preferably m isolated form. A purified 84P2A9 protem molecule will be substantially free of other protems or molecules that impair the binding of 84P2A9 to antibody or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of an 84P2A9 protem mclude a purified 84P2A9 protem and a functional, soluble 84P2A9 protem In one embodiment, a functional, soluble 84P2A9 protein or fragment thereof retains the ability to be bound by antibody, T cell or other hgand.

The invention also provides 84P2A9 protems comprismg biologically active fragments of the 84P2A9 am o acid sequence corresponding to part of the 84P2A9 amino acid sequence shown m FIG. 2. Such protems of the mvention exhibit properties of the 84P2A9 protem, such as the ability to ehcit the generation of antibodies that specifically bmd an epitope associated with the 84P2A9 protem; to be bound by such antibodies; to ehcit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL.

84P2A9-related protems are generated usmg standard peptide synthesis technology or usmg chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode an 84P2A9-related protem. In one embodiment, the 84P2A9-encodιng nucleic acid molecules descnbed herem provide means for generating defined fragments of 84P2A9 protems. 84P2A9 protem fragments/subsequences are particularly useful m generating and characterizing domain specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of an 84P2A9 protem), m identifymg agents or cellular factors that bmd to 84P2A9 or a particular structural domain thereof, and m vanous therapeutic contexts, mcludmg but not limited to cancer vaccmes or methods of preparing such vaccmes.

84P2A9 polypeptides containing particularly interesting structures can be predicted and/or identified usmg vanous analytical techniques well known m the art, mcludmg, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doohttle, Eisenberg, Kaφlus-Schultz or Jameson- Wolf analysis, or on the basis of lmmunogenicity. Fragments containing such structures are particularly useful m generating subunit specific anti-84P2A9 antibodies, or T cells or m identifymg cellular factors that bmd to 84P2A9.

Illustrating this, the binding of peptides from 84P2A9 protems to the human MHC class I molecule HLA-A2 were predicted. Specifically, the complete ammo acid sequence of the 84P2A9 protem was entered mto the HLA Peptide Motif Search algorithm found m the Bioinformatics and Molecular Analysis Section (BIMAS) Web site (http://bimas.dcrt.nih.gov/). The HLA Peptide Motif Search algoπthm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules and specifically HLA-A2 (see, e.g., Falk et al. Nature 351: 290-6 (1991); Hunt et al. Science 255:1261-3 (1992); Parker et al, J. Immunol. 149:3580-7 (1992); Parker et al, J. Immunol. 152:163-75 (1994)). This algoπthm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protem sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I bmdtng peptides are 8-, 9-, 10 or 11-mers. For example, for class I HLA- A2, the epitopes preferably contam a leucme (L) or methionine (M) at position 2 and a vahne (V) or leucme (L) at the C-terminus (see, e.g., Parker et al, J. Immunol. 149:3580-7 (1992)). Selected results of 84P2A9 predicted binding peptides are shown in Table 1 below. It is to be appreciated that every epitope predicted by the DIMAS site, or specified by the HLA class I or class I motifs available m the art are to be applied (e.g., visually or by computer based methods, or appreciated by those of skill m the relevant art) or which become part of the art are within the scope of the mvention. In Table 1, the top 10 ranking candidates for each family member are shown along with their location, the ammo acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half-time of dissociation of complexes containing the peptide at 37°C at pH 6.5. Peptides with the highest bmdtng score (ι.e. 63.04 for 84P2A9) are predicted to be the most tighdy bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition. Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al. Prostate 30:73-8 (1997) and Peshwa et al , Prostate 36:129-38 (1998)). lmmunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) _ in the presence of antigen presenting cells such as dendritic cells.

In an embodiment described m the examples that follow, 84P2A9 can be conveniendy expressed m cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-dnven expression vector encodmg 84P2A9 with a C-termmal 6XHιs and MYC tag (pcDNA3.1 /mycHIS, Invitrogen or Tag5, GenHunter Coφoration, Nashville TN) The Tag5 vector provides an IgGK secretion signal that can be used to faciktate the production of a secreted 84P2A9 protem m transfected cells. The secreted HIS-tagged 84P2A9 in the culture media can be puπfied, e g , usmg a nickel column usmg standard techniques.

Modifications of 84P2A9-related protems such as covalent modifications are mcluded within the scope of this mvention. One type of covalent modification mcludes reacting targeted ammo acid residues of an 84P2A9 polypeptide with an orgamc deπvatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the 84P2A9 Another type of covalent modification of the 84P2A9 polypeptide mcluded within the scope of this mvention comprises altermg the native glycosylation pattern of a protem of the mvention. "Altermg the native glycosylation pattern" is mtended for puφoses herem to mean deleting one or more carbohydrate moieties found m native sequence 84P2A9 (either by removing the underlymg glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present m the native sequence 84P2A9. In addition, the phrase mcludes qualitative changes in the glycosylation of the native protems, mvolvmg a change m the nature and proportions of the various carbohydrate moieties present Another type of covalent modification of 84P2A9 comprises knkmg the 84P2A9 polypeptide to one of a variety of nonprotemaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, m the manner set forth m U.S. Patent Nos. 4,640,835, 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 84P2A9 of the present mvention can also be modified in a way to form a chimeric molecule comprising 84P2A9 fused to another, heterologous polypeptide or amino acid sequence Such a chimeric molecule can be synthesized chemically or recombinandy. A chimeric molecule can have a protem of the invention fused to another tumor-associated antigen or fragment thereof, or can comprise fusion of fragments of the 84P2A9 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, direcdy homologous to the amino or nucleic acid sequences respectively of FIG. 2 (SEQ ID NO. 2); such a chimeric molecule can comprise multiples of the same subsequence of 84P2A9. A chimeric molecule can comprise a fusion of an 84P2A9-related protem with a polyhistidtne epitope tag, which provides an epitope to which immobilized nickel can selectively bmd. The epitope tag is generally placed at the ammo- or carboxyl- terminus of the 84P2A9. In an alternative embodiment, the chimeric molecule can comprise a fusion of an 84P2A9-related protem with an immunoglobulin or a particular region of an immunoglobulin For a bivalent form of the chimeric molecule (also referred to as an "lirrmunoadhesin"), such a fusion could be to the Fc region of an IgG molecule The lg fusions preferably mclude the substitution of a soluble (transmembrane domain deleted or inactivated) form of an 84P2A9 polypeptide m place of at least one variable region within an lg molecule. In a particularly preferred embodiment, the immunoglobulin fusion mcludes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27, 1995

84P2A9 ANTIBODIES

Another aspect of the mvention provides antibodies that bmd to 84P2A9-related protems and polypeptides. Preferred antibodies specifically bmd to an 84P2A9-related protem and will not bmd (or will bmd weakly) to non-84P2A9 protems. In another embodiment, antibodies bmd 84P2A9-related protems as well as the homologs thereof.

84P2A9 antibodies of the mvention are particularly useful m prostate cancer diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful m the treatment, diagnosis, and/or prognosis of other cancers, to the extent

84P2A9 is also expressed or overexpressed m other types of cancer Moreover, mtracellularly expressed antibodies (e g, smgle cham antibodies) are therapeutically useful in treating cancers m which the expression of 84P2A9 is involved, such as for example advanced and metastatic prostate cancers.

The mvention also provides vaπous immunological assays useful for the detection and quantification of 84P2A9 and mutant 84P2A9-related protems. Such assays can compnse one or more 84P2A9 antibodies capable of recognizing and binding an 84P2A9 or mutant 84P2A9 protem, as appropnate, and are performed within vanous immunological assay formats well known m the art, mcludmg but not limited to vanous types of radioimmunoassays, enzyme-linked lmmunosorbent assays (ELISA), enzyme- linked immunofluorescent assays (ELIFA), and the like. Related linmunological but non-antibody assays of the mvention also compnse T cell lmmunogemcity assays (inhibitory or stimulatory) as well as major histocompatibikty complex (MHC) bmdtng assays. In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressmg 84P2A9 are also provided by the mvention, mcluding but limited to radioscintigraphic imaging methods usmg labeled 84P2A9 antibodies. Such assays are clinically useful in the detection, momtormg, and prognosis of 84P2A9 expressmg cancers such as prostate cancer.

84P2A9 antibodies can also be used m methods for purifying 84P2A9 and mutant 84P2A9 protems and polypeptides and for isolating 84P2A9 homologues and related molecules. For example, in one embodiment, the method of punfying an 84P2A9 protem compnses mcubating an 84P2A9 antibody, which has been coupled to a sohd matπx, with a lysate or other solution containing 84P2A9 under conditions that permit the 84P2A9 antibody to bmd to 84P2A9; washing the sohd matπx to eliminate lmpunties; and eluting the 84P2A9 from the coupled antibody. Other uses of the 84P2A9 antibodies of the mvention mclude generating anti-idiotypic antibodies that mimic the 84P2A9 protem. Vanous methods for the preparation of antibodies are well known m the art. For example, antibodies can be prepared by immunizing a suitable mammahan host usmg an 84P2A9-related protem, peptide, or fragment, in isolated or rmmunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds, Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion protems of 84P2A9 can also be used, such as an 84P2A9 GST-fusion protem In a particular embodiment, a GST fusion protem compnsing all or most of the open reading frame ammo acid sequence of FIG. 2 is produced and used as an immunogen to generate appropnate antibodies In another embodiment, an 84P2A9 peptide is synthesized and used as an immunogen

In addition, naked DNA immunization techmques known in the art are used (with or without punfied 84P2A9 protem or 84P2A9 expressmg cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al, 1997, Ann. Rev Immunol. 15: 617-648).

The amino acid sequence of 84P2A9 as shown m FIG. 2 can be used to select specific regions of the 84P2A9 protem for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of the 84P2A9 ammo acid sequence are used to identify hydrophikc regions in the 84P2A9 structure. Regions of the 84P2A9 protem that show immunogenic structure, as well as other regions and domams, can readily be identified usmg vanous other methods known m the art, such as Chou-Fasman, Garmer- Robson, Kyte-Dookttle, Eisenberg, Kaφlus-Schultz or Jameson-Wolf analysis. Thus, each region identified by any of these programs/methods is within the scope of the present mvention. Methods for the generation of 84P2A9 antibodies are further illustrated by way of the examples provided herem.

Methods for preparing a protem or polypeptide for use as an immunogen and for preparing immunogenic conjugates of a protem with a earner such as BSA, KLH, or other earner protems are well known m the art. In some circumstances, direct conjugation usmg, for example, carbodiimide reagents are used; m other mstances linking reagents such as those suppked by Pierce Chemical Co, Rockford, IL, are effective Administration of an 84P2A9 immunogen is conducted generally by injection over a suitable time penod and with use of a suitable adjuvant, as is generally understood m the art. During the immunization schedule, liters of antibodies can be taken to determine adequacy of antibody formation.

84P2A9 monoclonal antibodies can be produced by vanous means well known m the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared usmg the standard hybndoma technology of Kohler and Milstein or modifications that immortalize producmg B cells, as is generally known. The immortalized cell lines that secrete the desired antibodies are screened by immunoassay m which the antigen is an 84P2A9-related protem. When the appropnate immortalized cell culture secreting the desired antibody is identified, the cells can be expanded and antibodies produced either from m vitro cultures or from ascites fluid

The antibodies or fragments can also be produced, usmg current technology, by recombmant means. Regions that bmd specifically to the desired regions of the 84P2A9 protem can also be produced m the context of chimenc or complementanty determining region (CDR) grafted antibodies of multiple species oπgm. Humanized or human 84P2A9 antibodies can also be produced and are preferred for use m therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al, 1986, Nature 321: 522-525; Riechmnan et al, 1988, Nature 332: 323-327; Verhoeyen et al, 1988, Science 239: 1534-1536). See also, Carter et al, 1993, Proc. Nad Acad. Sci. USA 89. 4285 and Sims et al, 1993, J. Immunol. 151 : 2296 Methods for producmg fully human monoclonal antibodies mclude phage display and transgenic methods (for review, see Vaughan et al, 1998, Nature Biotechnology 16: 535- 539).

Fully human 84P2A9 monoclonal antibodies can be generated usmg clomng technologies employing large human lg gene combinatorial kbranes (ι.e, phage display) (Gnffiths and Hoogenboom, Building an m vitro immune system: human antibodies from phage display kbranes. In: Protem Engineering of Antibody Molecules for Prophylactic and Therapeutic Apphcations in Man. Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatonal kbranes. Id, pp 65-82). Fully human 84P2A9 monoclonal antibodies can also be produced usmg transgenic mice engineered to contam human immunoglobulin gene loci as descnbed m PCT Patent Appkcation W098/24893, Kucherlapati and Jakobovits et al, published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opm. Invest. Drugs 7(4): 607-614). This method avoids the in vitro manipulation required with phage display technology and efficiendy produces high affinity authentic human antibodies

Reactivity of 84P2A9 antibodies with an 84P2A9-related protem can be estabhshed by a number of well known means, mcludmg Western blot, immunoprecipitation, ELISA, and FACS analyses usmg, as appropnate, 84P2A9-related protems, peptides, 84P2A9-expressιng cells or extracts thereof An 84P2A9 antibody or fragment thereof of the invention is labeled with a detectable marker or conjugated to a second molecule Suitable detectable markers mclude, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi- specific antibodies specific for two or more 84P2A9 epitopes are generated usmg methods generally known in the art. Homodimeπc antibodies can also be generated by cross-linking techniques known m the art (e.g., Wolff et al. Cancer Res. 53. 2560-2565).

84P2A9 TRANSGENIC ANIMALS Nucleic acids that encode 84P2A9 or its modified forms can also be used to generate either transgemc animals or "knock out" animals which, m turn, are useful m the development and screening of therapeutically useful reagents. In accordance with estabhshed techniques, cDNA encoding 84P2A9 can be used to clone genomic DNA encoding 84P2A9 and the genomic sequences used to generate transgenic animals that contam cells that express DNA encodmg 84P2A9. Methods for generating transgemc animals, particularly animals such as mice or rats, have become conventional m the art and are descnbed, for example, m U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for 84P2A9 transgene incoφoration with tissue-specific enhancers. Transgemc animals that mclude a copy of a transgene encoding 84P2A9 can be used to examine the effect of increased expression of DNA encodmg 84P2A9. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the mvention, an animal is treated with a reagent and a reduced mcidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of 84P2A9 can be used to construct an 84P2A9 "knock out" animal that has a defective or altered gene encodmg 84P2A9 as a result of homologous recombination between the endogenous gene encoding 84P2A9 and altered genomic DNA encoding 84P2A9 introduced mto an embryonic cell of the animal For example, cDNA encoding 84P2A9 can be used to clone genomic DNA encoding 84P2A9 m accordance with estabhshed techniques. A portion of the genomic DNA encoding 84P2A9 can be deleted or replaced with another gene, such as a gene encodmg a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are mcluded m the vector [see, e.g.,, Thomas and Capecchi, Cell. 5J,:503 (1987) for a description of homologous recombination vectors] The vector is introduced mto an embryonic stem cell line (e.g., by electrop oration) and cells m which the introduced DNA has homologously recombined with the endogenous DNA are selected [see, e.g.,, Li et al. Cell. 69:915 (1992)]. The selected cells are then mjected mto a blastocyst of an animal (e*g, a mouse or rat) to form aggregation chimeras [see, e g„ Bradley, in Teratocaranomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted mto a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA m their germ cells can be identified by standard techniques and used to breed animals m which all cells of the animal contam the homologously recombined DNA. Knock out animals can be characterized for instance, for their ability to defend agamst certain pathological conditions and for their development of pathological conditions due to absence of the 84P2A9 polypeptide.

METHODS FOR THE DETECTION OF 84P2A9

Another aspect of the present mvention relates to methods for detecting 84P2A9 polynucleotides and 84P2A9-related protems and vanants thereof, as well as methods for identifymg a cell that expresses 84P2A9. 84P2A9 appears to be expressed m the LAPC xenografts that are denved from lymph-node and bone metastasis of prostate cancer. The expression profile of 84P2A9 makes it a potential diagnostic marker for metastasized disease. In this context, the status of 84P2A9 gene products provide information useful for predicting a vanety of factors mcludmg susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness As discussed m detail below, the status of 84P2A9 gene products m patient samples can be analyzed by a vaπety protocols that are well known m the art mcludmg lmmunohistochemical analysis, the vaπety of Northern blotting techniques mcluding m situ hybnckzation, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue anay analysis.

More particularly, the mvention provides assays for the detection of 84P2A9 polynucleotides m a biological sample, such as serum, bone, prostate, and other tissues, uπne, semen, cell preparations, and the like. Detectable 84P2A9 polynucleotides mclude, for example, an 84P2A9 gene or fragments thereof, 84P2A9 mRNA, alternative sphce vanant 84P2A9 mRNAs, and recombmant DNA or RNA molecules containing an 84P2A9 polynucleotide. A number of methods for amplifying and/or detecting the presence of 84P2A9 polynucleotides are well known m the art and can be employed m the practice of this aspect of the mvention.

In one embodiment, a method for detecting an 84P2A9 mRNA m a biological sample compnses producmg cDNA from the sample by reverse transcription usmg at least one primer; amplifying the cDNA so produced usmg an 84P2A9 polynucleotides as sense and antisense primers to amphfy 84P2A9 cDNAs therein; and detecting the presence of the amplified 84P2A9 cDNA Optionally, the sequence of the amplified 84P2A9 cDNA can be determined.

In another embodiment, a method of detecting an 84P2A9 gene m a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA usmg 84P2A9 polynucleotides as sense and antisense primers to amphfy the 84P2A9 gene therein; and detecting the presence of the amplified 84P2A9 gene. Any number of appropriate sense and antisense probe combinations can be designed from the nucleotide sequences provided for the 84P2A9 (FIG. 2) and used for this puφose.

The mvention also provides assays for detecting the presence of an 84P2A9 protem m a tissue of other biological sample such as serum, bone, prostate, and other tissues, urine, cell preparations, and the like. Methods for detecting an 84P2A9 protem are also well known and mclude, for example, immunoprecipitation, lmmunohistochemical analysis, Western Blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, m one embodiment, a method of detecting the presence of an 84P2A9 protem m a biological sample comprises first contacting the sample with an 84P2A9 antibody, an 84P2A9-reactive fragment thereof, or a recombinant protem containing an antigen binding region of an 84P2A9 antibody; and then deteςting the binding of 84P2A9 protem m the sample thereto.

Methods for identifying a cell that expresses 84P2A9 are also provided. In one embodiment, an assay for identifymg a cell that expresses an 84P2A9 gene compnses detecting the presence of 84P2A9 mRNA in the cell. Methods for the detection of particular mRNAs m cells are well known and mclude, for example, hybndization assays usmg complementary DNA probes (such as m situ hybndization usmg labeled 84P2A9 nboprobes, Northern blot and related techmques) and vanous nucleic acid ampkfication assays (such as RT-PCR usmg complementary primers specific for 84P2A9, and other ampkfication type detection methods, such as, for example, branched DNA, SISBA, TMA and die like). Alternatively, an assay for identifying a cell that expresses an 84P2A9 gene compnses detecting the presence of 84P2A9 protem in the cell or secreted by the cell Vanous methods for the detection of protems are well known m the art and are employed for the detection of 84P2A9 protems and 84P2A9 expressmg cells. 84P2A9 expression analysis is also useful as a tool for identifymg and evaluating agents that modulate 84P2A9 gene expression. For example, 84P2A9 expression is significantiy upregulated m prostate cancer, and is also expressed m other cancers mcludmg prostate, testis, kidney, bram, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung cancers. Identification of a molecule or biological agent that could inhibit 84P2A9 expression or over-expression m cancer cells is of therapeutic value. For example, such an agent can be identified by usmg a screen that quantifies 84P2A9 expression by RT-PCR, nucleic acid hybridization or antibody binding.

MONITORING THE STATUS OF 84P2A9 AND ITS PRODUCTS

Assays that evaluate the status of the 84P2A9 gene and 84P2A9 gene products in an individual can provide information on the growth or oncogenic potential of a biological sample from this individual. For example, because 84P2A9 mRNA is so highly expressed in prostate cancers (as well as the other cancer tissues shown for example in FIGS. 4-8) as compared to normal prostate tissue, assays that evaluate the relative levels of 84P2A9 mRNA transcripts or protems m a biological sample can be used to diagnose a disease associated with 84P2A9 deregulation such as cancer and can provide prognostic information useful in defining appropnate therapeutic options.

Because 84P2A9 is expressed, for example, m vanous prostate cancer xenograft tissues and cancer cell lines, and cancer patient samples, the expression status of 84P2A9 can provide information useful for determining information mcludmg the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to vaπous stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequendy, an important aspect of the mvention is directed to the vanous molecular prognostic and diagnostic methods for examining the status of 84P2A9 m biological samples such as those from mdtviduals suffermg from, or suspected of suffermg from a pathology characterized by disregulated cellular growth such as cancer

Oncogenesis is known to be a multistep process where cellular growth becomes progressively disregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al. Lab Invest. 77(5): 437-438 (1997) and Isaacs et al. Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of disregulated cell growth (such as aberrant 84P2A9 expression m prostate cancers) can allow the early detection of such aberrant cellular physiology before a pathology such as cancer has progressed to a stage at which therapeutic options are more limited In such exammations, the status of 84P2A9 m a biological sample of mterest (such as one suspected of having disregulated cell growth) can be compared, for example, to the status of 84P2A9 m a corresponding normal sample (e.g. a sample from that individual (or alternatively another mdividual) that is not effected by a pathology, for example one not suspected of having disregulated cell growth). Alterations in the status of 84P2A9 m the biological sample of mterest (as compared to the normal sample) provides evidence of disregulated cellular growth. In addition to usmg a biological sample that is not effected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g , Grever et al, J. Comp. Neurol. 1996 Dec 9;376(2):306-14 and U.S. patent No. 5,837,501) to compare 84P2A9 m normal versus suspect samples. The term "status" m this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These mclude, but are not limited to the location of expressed gene products (mcludmg the location of 84P2A9 expressmg cells) as well as the, level, and biological activity of expressed gene products (such as 84P2A9 mRNA polynucleotides and polypeptides). Alterations in the status of 84P2A9 can be evaluated by a wide variety of methodologies well known m the art, typically those discussed herem Typically an alteration m the status of 84P2A9 comprises a change m the location of 84P2A9 and/or 84P2A9 expressmg cells and/or an increase m 84P2A9 mRNA and/or protem expression.

As discussed m detail herem, in order to identify a condition or phenomenon associated with disregulated cell growth, the status of 84P2A9 m a biological sample is evaluated by a number of methods utilized by skilled artisans mcluding, but not hmited to genomic Southern analysis (to examine, for example perturbations in the 84P2A9 gene), Northern analysis and/or PCR analysis of 84P2A9 mRNA (to examine, for example alterations m the polynucleotide sequences or expression levels of 84P2A9 mRNAs), and Western and/or lirrmunohistochernical analysis (to examine, for example alterations m polypeptide sequences, alterations m polypeptide localization within a sample, alterations m expression levels of 84P2A9 protems and/or associations of 84P2A9 protems with polypeptide bmdtng partaers). Detectable 84P2A9 polynucleotides mclude, for example, an 84P2A9 gene or fragments thereof, 84P2A9 mRNA, alternative splice vanants 84P2A9 mRNAs, and recombmant DNA or RNA molecules containing an 84P2A9 polynucleotide.

The expression profile of 84P2A9 makes it a diagnostic marker for local and/or metastasized disease. In particular, the stams of 84P2A9 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The mvention provides methods and assays for determining 84P2A9 status and diagnosing cancers that express 84P2A9, such as cancers of the prostate, bladder, testis, ovaries, breast, pancreas, colon and lung. 84P2A9 status in patient samples can be analyzed by a number of means well known m the art, mcludmg without limitation, lmmunohistochemical analysis, m situ hybndization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis of clinical samples and cell lines, and tissue array analysis. Typical protocols for evaluating the status of the 84P2A9 gene and gene products can be found, for example m Ausubul et al. eds, 1995, Current Protocols In Molecular Biology, Umts 2 [Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting] and 18 [PCR Analysis] .

As described above, the stams of 84P2A9 m a biological sample can be examined by a number of well known procedures m the art. For example, the status of 84P2A9 m a biological sample taken from a specific location m the body can be examined by evaluating the sample for the presence or absence of 84P2A9 expressmg cells (e.g those that express 84P2A9 mRNAs or proteins). This examination can provide evidence of disregulated cellular growth, for example, when 84P2A9 expressmg cells are found in a biological sample that does not normally contam such cells (such as a lymph node). Such alterations in the status of 84P2A9 in a biological sample are often associated with disregulated cellular growth. Specifically, one indicator of disregulated cellular growth is the metastases of cancer cells from an organ of oπgm (such as the testis or prostate gland) to a different area of the body (such as a lymph node). In this context, evidence of disregulated cellular growth is important for example because occult lymph node metastases can be detected m a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g, Muφhy et al. Prostate 42(4): 315-317 (2000);Su et al, Semin. Surg. Oncol. 18(1): 17- 28 (2000) and Freeman et al, J Urol 1995 Aug;154(2 Pt l):474-8).

In one aspect, the mvention provides methods for momtormg 84P2A9 gene products by determining the status of 84P2A9 gene products expressed by cells m a test tissue sample from an individual suspected of having a disease associated with disregulated cell growth (such as hypeφlasia or cancer) and then comparmg the stams so determined to the status of 84P2A9 gene products m a corresponding normal sample, the presence of aberrant 84P2A9 gene products m the test sample relative to the normal sample providing an indication of the presence of disregulated cell growth within the cells of the mdividual. In another aspect, the mvention provides assays useful m determining the presence of cancer m an individual, comprismg detecting a significant mcrease m 84P2A9 mRNA or protein expression m a test cell or tissue sample relative to expression levels m the corresponding normal cell or tissue. The presence of 84P2A9 mRNA can, for example, be evaluated m tissue samples mcludmg but not limited to prostate, testis, kidney, brain, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung tissues (see, e.g , FIGS. 4-8). The presence of significant 84P2A9 expression m any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, smce the corresponding normal tissues do not express 84P2A9 mRNA or express it at lower levels.

In a related embodiment, 84P2A9 status is determined at the protem level rather than at the nucleic acid level For example, such a method or assay compnses determining the level of 84P2A9 protem expressed by cells in a test tissue sample and comparmg the level so determined to the level of 84P2A9 expressed m a corresponding normal sample In one embodiment, the presence of 84P2A9 protem is evaluated, for example, usmg lmmunohistochemical methods 84P2A9 antibodies or binding partaers capable of detecting 84P2A9 protem expression are used m a vanety of assay formats well known m the art for this puφose.

In other related embodiments, one can evaluate the status 84P2A9 nucleotide and ammo acid sequences m a biological sample in order to identify perturbations in the structure of these molecules such as msertions, deletions, substitutions and the like. Such embodiments are useful because perturbations m the nucleotide and ammo acid sequences are observed in a large number of protems associated with a growth disregulated phenotype (see, e.g, Marrogi et al, 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation is the sequence of 84P2A9 may be indicative of the presence or promotion of a tumor. Such assays can therefore have diagnostic and predictive value where a mutation m 84P2A9 indicates a potential loss of function or increase in tumor growth.

A wide vaπety of assays for observing perturbations in nucleotide and amino acid sequences are well known m the art. For example, the size and structure of nucleic acid or ammo acid sequences of 84P2A9 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herem In addition, other methods for observing perturbations m nucleotide and amino acid sequences such as smgle strand conformation polymoφhism analysis are well known in the art (see, e.g, U.S. Patent Nos. 5,382,510 and 5,952,170).

In another embodiment, one can examine the methylation statas of the 84P2A9 gene m a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequendy occurs m immortalized and transformed cells and can result m altered expression of vanous genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protem expressed m normal prostate but not expressed in >90% of prostate carcmomas) appears to permanendy silence transcription of this gene and is the most frequendy detected genomic alteration m prostate carcmomas (De Marzo et al. Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present m at least 70% of cases of high-grade prostatic lntraepithekal neoplasm (PIN) (Brooks et al, Cancer Epidemiol. Biomarkers Prev, 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed m normal prostate but is expressed m 25-50% of prostate cancers) is mduced by deoxy-azacytidine m lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al. Int. J. Cancer 76(6): 903-908 (1998)). In this context, a vanety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, m Southern hybndization approaches, methylation- sensitive restriction enzymes which can not cleave sequences that contam methylated CpG sites, m order to assess the overall methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present m a CpG island of a given gene This procedure mvolves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosmes to uracil) followed by ampkfication usmg primers specific for methylated versus unmethylated DNA Protocols mvolvmg methylation mterference can also be found for example m Current Protocols In Molecular Biology, Units 12, Frederick M. Ausubul et al. eds, 1995.

Gene ampkfication provides an additional method of assessmg the status of 84P2A9, a locus that maps to lq32.3, a region shown to be perturbed m a variety of cancers. Gene amplification is measured in a sample direcdy, for example, by conventional Southern blotting or Northern blotting to quantitate the ttanscnption of mRNA (Thomas, 1980, Proc. Nad Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybndization, usmg an appropriately labeled probe, based on the sequences provided herem. Alternatively, antibodies are employed that recognize specific duplexes, mcludmg DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protem duplexes The antibodies m 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.

In addition to the tissues discussed herem, biopsied tissue or penpheral blood can be convemendy assayed for the presence of cancer cells, mcluding but not limited to prostate, testis, kidney, bram, bone, skin, ovarian, breast, pancreas, colon, lymphocytic and lung cancers usmg for example, Northern, dot blot or RT-PCR analysis to detect 84P2A9 expression (see, e g, FIGS 4-8) The presence of RT-PCR ampkfiable 84P2A9 mRNA provides an indication of the presence of the cancer. RT-PCR detection assays for tumor cells m penpheral blood are currendy being evaluated for use m the diagnosis and management of a number of human sohd tumors. In the prostate cancer field, these mclude RT-PCR assays for the detection of cells expressmg PSA and PSM (Verkaik et al, 1997, Urol. Res. 25:373-384; Ghossein et al, 1995, J. Chn. Oncol. 13:1195-2000; Heston et al, 1995, Chn. Chem. 41:1687-1688). RT-PCR assays are well known m the art.

A related aspect of the mvention is directed to predicting susceptibikty to developmg cancer m an mdividual In one embodiment, a method for predicting susceptibikty to cancer compnses detecting 84P2A9 mRNA or 84P2A9 protem m a tissue sample, its presence indicating susceptibikty to cancer, wherein the degree of 84P2A9 mRNA expression present correlates to the degree of susceptibikty. In a specific embodiment, the presence of 84P2A9 in prostate or other tissue is examined, with the presence of 84P2A9 m the sample providing an indication of prostate cancer susceptibikty (or the emergence or existence of a prostate tumor). In a closely related embodiment, one can evaluate the mtegnty 84P2A9 nucleotide and ammo acid sequences m a biological sample m order to identity perturbations in the structure of these molecules such as msertions, deletions, substitutions and the like, with the presence of one or more perturbations m 84P2A9 gene products m the sample providing an indication of cancer susceptibikty (or the emergence or existence of a tumor). Another related aspect of the mvention is directed to methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor compnses determining the level of 84P2A9 mRNA or 84P2A9 protem expressed by cells in a sample of the tumor, comparmg the level so determined to the level of 84P2A9 mRNA or 84P2A9 protem expressed m a corresponding normal tissue taken from the same mdividual or a normal tissue reference sample, wherein the degree of 84P2A9 mRNA or 84P2A9 protem expression m the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 84P2A9 is expressed in the tumor cells, with higher expression levels mdicatmg more aggressive tumors. In a closely related embodiment, one can evaluate the mtegnty of 84P2A9 nucleotide and ammo acid sequences in a biological sample m order to identify perturbations m the structure of these molecules such as msertions, deletions, substitutions and the like, with the presence of one or more perturbations mdicatmg more aggressive tumors. Yet another related aspect of the mvention is directed to methods for observing the progression of a makgnancy in an mdividual over time In one embodiment, methods for observing the progression of a malignancy m an mdividual over time compnse determining the level of 84P2A9 mRNA or 84P2A9 protem expressed by cells m a sample of the tumor, comparmg the level so determined to the level of 84P2A9 mRNA or 84P2A9 protem expressed in an equivalent tissue sample taken from the same mdividual at a different time, wherein the degree of 84P2A9 mRNA or 84P2A9 protem expression m the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining the extent to which 84P2A9 expression m the tumor cells alters over time, with higher expression levels mdicatmg a progression of the cancer. Also, one can evaluate the mtegnty 84P2A9 nucleotide and ammo acid sequences m a biological sample in order to identify perturbations m the structure of these molecules such as msertions, deletions, substitutions and the like, with the presence of one or more perturbations mdicatmg a progression of the cancer. The above diagnostic approaches can be combined with any one of a wide vaπety of prognostic and diagnostic protocols known m the art For example, another embodiment of the mvention disclosed herem is directed to methods for observmg a comcidence between the expression of 84P2A9 gene and 84P2A9 gene products (or perturbations m 84P2A9 gene and 84P2A9 gene products) and a factor that is associated with makgnancy as a means of diagnosing and prognosticating the status of a tissue sample. In this context, a wide vanety of factors associated with makgnancy can be utilized such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g, Boclαng et al, 1984, Anal. Quant. Cytol. 6(2):74-88; Eptse , 1995, Hum. Pathol. 26(2):223-9; Thorson et al, 1998, Mod. Pathol 11 (6):543-51 ; Baisden et al, 1999, Am. J. Surg Pathol. 23(8):918-24). Methods for observmg a comcidence between the expression of 84P2A9 gene and 84P2A9 gene products (or perturbations m 84P2A9 gene and 84P2A9 gene products) and an additional factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

In a typical embodiment, methods for observmg a comcidence between the expression of 84P2A9 gene and 84P2A9 gene products (or perturbations m 84P2A9 gene and 84P2A9 gene products) and a factor that is associated with malignancy entails detecting the overexpression of 84P2A9 mRNA or protem m a tissue sample, detecting the overexpression of PSA mRNA or protem in a tissue sample, and observing a comcidence of 84P2A9 mRNA or protem and PSA mRNA or protem overexpression. In a specific embodiment, the expression of 84P2A9 and PSA mRNA in prostate tissue is examined. In a preferred embodiment, the comcidence of 84P2A9 and PSA mRNA overexpression in the sample provides an indication of prostate cancer, prostate cancer susceptibikty or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 84P2A9 mRNA or protem are descnbed herem and use of standard nucleic acid and protem detection and quantification technologies is well known m the art. Standard methods for the detection and quantification of 84P2A9 mRNA mclude m situ hybndization usmg labeled 84P2A9 πboprobes, Northern blot and related techmques usmg 84P2A9 polynucleotide probes, RT-PCR analysis usmg primers specific for 84P2A9, and other ampkfication type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 84P2A9 mRNA expression as descnbed in the Examples that follow. Any number of primers capable of amplifying 84P2A9 can be used for this puφose, mcludmg but not limited to the vanous pnmer sets specifically descnbed herem. Standard methods for the detection and quantification of protem are used for this puφose. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 84P2A9 protem can be used m an lmmunohistochemical assay of biopsied tissue.

IDENTIFYING MOLECULES THAT INTERACT WITH 84P2A9

The 84P2A9 protem sequences disclosed herein allow the skilled artisan to identify protems, small molecules and other agents that mteract with 84P2A9 and pathways activated by 84P2A9 via any one of a variety of art accepted protocols For example, one can utilize one of the variety of so-called interaction trap systems (also referred to as the "two-hybrid assay"). In such systems, molecules that mteract reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Typical systems identify protem-protem interactions m vivo through reconstitution of a eukaryotic transcriptional activator and are disclosed for example m U.S. Patent Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.

Alternatively one can identify molecules that mteract with 84P2A9 protem sequences by screening peptide libraries. In such methods, peptides that bind to selected receptor molecules such as 84P2A9 are identified by screening kbranes that encode a random or controlled collection of ammo acids. Peptides encoded by the hbranes are expressed as fusion protems of bactenophage coat proteins, the bactenophage particles are then screened agamst the receptors of interest.

Accordingly, peptides havmg a wide variety of uses, such as therapeutic or diagnostic reagents, can thus be identified without any pnor information on the structure of the expected kgand or receptor molecule Typical peptide hbranes and screening methods that can be used to identify molecules that interact with 84P2A9 protem sequences are disclosed for example m U S Patent Nos. 5,723,286 and 5,733,731.

Alternatively, cell lines that express 84P2A9 are used to identify protein-protein interactions mediated by 84P2A9 Such interactions can be examined usmg lrnrnunoprecipitation techniques as shown by others (Hamilton BJ, et al Biochem. Biophys Res Commun 1999, 261:646-51). Typically 84P2A9 protem can be lmmunoprecipitated from 84P2A9 expressmg prostate cancer cell lines usmg anti- 84P2A9 antibodies. Alternatively, antibodies agamst His-tag can be used m a cell line engmeered to express 84P2A9 (vectors mentioned above). The lmmunoprecipitated complex can be examined for protem association by procedures such as Western blotting, ³⁵S-methionine labeling of protems, protem microsequencing, silver staining and two dimensional gel electrophoresis.

Small molecules that mteract with 84P2A9 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that mterfere with protem function, mcludmg molecules that interfere with 84P2A9's abikty to mediate phosphorylation and de -phosphorylation, second messenger signaling and tumongenesis. Typical methods are discussed for example m U.S. Patent No. 5,928,868 and mclude methods for forming hybrid hgands m which at least one hgand is a small molecule. In an illustrative embodiment, the hybrid hgand is introduced mto cells that m turn contam a first and a second expression vector. Each expression vector mcludes DNA for expressing a hybrid protem that encodes a target protem linked to a codmg sequence for a transcriptional module. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second hybrid protems to each other, an event that occurs only if the hybrid hgand binds to target sites on both hybrid protems. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown hybrid protem is identified.

A typical embodiment of this invention consists of a method of screening for a molecule that interacts with an 84P2A9 ammo acid sequence shown in FIG. 1 (SEQ ID NO: 2), comprising the steps of contacting a population of molecules with the 84P2A9 amino acid sequence, allowing the population of molecules and the 84P2A9 ammo acid sequence to mteract under conditions that facilitate an interaction, determining the presence of a molecule that mteracts with the 84P2A9 ammo acid sequence and then separating molecules that do not mteract with the 84P2A9 ammo acid sequence from molecules that do mteract with the 84P2A9 amino acid sequence. In a specific embodiment, the method further mcludes purifying a molecule that mteracts with the 84P2A9 amino acid sequence. In a preferred embodiment, the 84P2A9 ammo acid sequence is contacted with a kbrary of peptides.

THERAPEUTIC METHODS AND COMPOSITIONS

The identification of 84P2A9 as a protem that is normally prostate and testis- related and which is also expressed m cancers of the prostate (and other cancers), opens a number of therapeutic approaches to the treatment of such cancers. As discussed herem, it is possible that 84P2A9 functions as a transcription factor mvolved m activating tumor-promoting genes or repressing genes that block tumongenesis.

The expression profile of 84P2A9 is reminiscent of the Cancer-Testis (CT) antigens or MAGE antigens, which are testis-related genes that are up-regulated m melanomas and other cancers (Van den Eynde and Boon, Int J Ckn Lab Res. 27-81-86, 1997). Due to their tissue-specific expression and high expression levels m cancer, the MAGE antigens are currendy bemg investigated as targets for cancer vaccmes (Durrant, Anticancer Drugs 8:727-733, 1997; Reynolds et al, Int J Cancer 72.972-976, 1997). The expression pattern of 84P2A9 provides evidence that it is likewise an ideal target for a cancer vaccme approach to prostate cancer. Its structural featares indicate that it may be a transcription factor, and provide evidence that 84P2A9 is a small molecule target.

Accordingly, therapeutic approaches a med at inhibiting the activity of the 84P2A9 protem are expected to be useful for patients suffermg from prostate cancer, testicular cancer, and other cancers expressmg 84P2A9. These therapeutic approaches generaUy faU into two classes. One class comprises various methods for mhibiting the binding or association of the 84P2A9 protem with its binding partner or with others protems. Another class comprises a vaπety of methods for inhibiting the transcription of the 84P2A9 gene or translation of 84P2A9 mRNA.

84P2A9 as a Target for Antibody-Based Therapy The structural featares of 84P2A9 indicate that this molecule is an attractive target for antibody-based therapeutic strategies A number of typical antibody strategies are known in the art for targeting both exttaceUular and lntraceUular molecules (see, e g , complement and ADCC mediated killing as weU as the use of lntrabodies discussed herein) Because 84P2A9 is expressed by cancer ceUs of various lineages and not by corresponding normal ceUs, systemic administration of 84P2A9-immunoreactive compositions would be expected to exhibit exceUent sensitivity without toxic, nonspecific and/ or non-target effects caused by binding of the lmmunotherapeutic molecule to non-target organs and tissues Antibodies specificaUy reactive with domains of 84P2A9 can be useful to treat 84P2A9-exρressιng cancers systemicaU) , either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of mhibiting ceU proliferation or function

84P2A9 antibodies can be introduced mto a patient such that the antibody bmds to 84P2A9 and modulates or perturbs a function such as an interaction with a binding partner and consequendy mediates growth mhibition and/or destruction of the tumor ceUs and/or inhibits the growth of the tumor ceUs Mechamsms by which such antibodies exert a therapeutic effect can mclude complement-mediated cytolysis, antibody-dependent ceUular cytotoxicity, modulating the physiological function of 84P2A9, mhibiting hgand binding or signal transduction pathways, modulating tumor ceU differentiation, altering tumor angiogenesis factor profiles, and/or by inducing apoptosis

Those skiUed m the art understand that antibodies can be used to specificaUy target and bmd immunogenic molecules such as an immunogenic region of the 84P2A9 sequence shown m Figure 1 In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents In this context, skilled artisans understand that when cytotoxic and/or therapeutic agents are dekvered direcdy to ceUs by conjugating them to antibodies specific for a molecule expressed by that ceU (e g 84P2A9), it is reasonable to expect that the cytotoxic agent wiU exert its known biological effect (e g cytotoxicity) on those ceUs

A wide variety of compositions and methods for usmg antibodies conjugated to cytotoxic agents to kiU ceUs are known m the art In the context of cancers, typical methods entail administering to an animal havmg a tumor a biologicaUy effective amount of a conjugate comprismg a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-84P2A9 antibody) that bmds to a marker (e.g. 84P2A9) expressed, accessible to binding or locakzed on the ceU surfaces. A typical embodiment consists of a method of dekvenng a cytotoxic and/or therapeutic agent to a ceU expressmg 84P2A9 comprismg conjugating the cytotoxic agent to an antibody that lmmunospecificaUy bmds to an 84P2A9 epitope and exposmg the ceU to the antibody- agent conjugate. Another specific illustrative embodiment consists of a method of treating an individual suspected of suffering from metastasized cancer comprismg the step of administering parenteraUy to said mdividual a pharmaceutical composition comprising a therapeuticaUy effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent

Cancer lmmunotherapy usmg anti-84P2A9 antibodies may foUow the teachings generated from various approaches that have been successfuUy employed m the treatment of other types of cancer, mcludmg but not limited to colon cancer (Arlen et al, 1998, Cπt. Rev. Immunol. 18:133-138), multiple myeloma (Ozalu et al, 1997, Blood 90:3179-3186; Tsunenaπ et al, 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al, 1992, Cancer Res. 52:2771-2776), B-ceU lymphoma (Funakoshi et al, 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al, 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al, 1994, Cancer Res. 54:6160-6166; Velders et al, 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al, 1991, J. Chn. Immunol. 11 :117-127). Some therapeutic approaches mvolve conjugation of naked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20 antibodies (e.g, Ritaxan™, IDEC Pharmaceuticals Coφ.), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastazumab) with packtaxel (Genentech, Inc.). For treatment of prostate cancer, for example, 84P2A9 antibodies can be administered m conjunction with radiation, chemotherapy or hormone ablation.

Although 84P2A9 antibody therapy is useful for aU stages of cancer, antibody therapy is particularly appropriate m advanced or metastatic cancers. Treatment with the antibody therapy of the mvention is indicated for patients who have received one or more rounds of chemotherapy, while combining the antibody therapy of the mvention with a chemotherapeutic or radiation regimen is preferred for patients who have not received chemotherapeutic treatment. AdditionaUy, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very weU. It is desirable for some cancer patients to be evaluated for the presence and level of 84P2A9 expression, preferably usmg lmmunohistochemical assessments of tumor tissue, quantitative 84P2A9 imaging, or other techmques capable of reliably indicating the presence and degree of 84P2A9 expression. lmmunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this puφose. Methods for lmmunohistochemical analysis of tumor tissues are weU known in the art.

Anti-84P2A9 monoclonal antibodies useful in treating prostate and other cancers mclude those that are capable of initiating a potent immune response agamst the tumor or those that are direcdy cytotoxic. In this regard, anti-84P2A9 monoclonal antibodies (mAbs) can ehcit tumor ceU lysis by either complement-mediated or antibody-dependent ceU cytotoxicity (ADCC) mechanisms, both of which require an mtact Fc portion of the immunoglobulin molecule for interaction with effector ceU Fc receptor sites or complement protems. In addition, anti-84P2A9 mAbs that exert a direct biological effect on tumor growth are useful in the practice of the mvention. Potential mechamsms by which such direcdy cytotoxic mAbs can act include mhibition of ceU growth, modulation of ceUular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism by which a particular anti-84P2A9 mAb exerts an anti-tumor effect is evaluated usmg any number of in vitro assays designed to determine ceU death such as ADCC, ADMMC, complement-mediated ceU lysis, and so forth, as is generaUy known m the art. The use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses m some patients. In some cases, this wiU result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentiaUy, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the practice of the therapeutic methods of the mvention are those that are either fully human or humanized and that bmd specificaUy to the target 84P2A9 antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic methods of the mvention contemplate the administration of smgle anti-84P2A9 mAbs as weU as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certam advantages masmuch as they contam mAbs that target different epitopes, exploit different effector mechanisms or combme direcdy cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs m combination can exhibit synergistic therapeutic effects. In addition, the administration of anti-84P2A9 mAbs can be combined with other therapeutic agents, mcludmg but not limited to vaπous chemotherapeutic agents, androgen-blockers, and immune modulators (e g, IL-2, GM-CSF). The antι-84P2A9 mAbs are admimstered m their "naked" or unconjugated form, or can have therapeutic agents conjugated to them.

The anti-84P2A9 antibody formulations are administered via any route capable of dehvermg the antibodies to the tumor site. PotentiaUy effective routes of admmistration mclude, but are not limited to, intravenous, intraperitoneal, intramuscular, mttatamor, intradermal, and the like. Treatment wiU generaUy involve the repeated administration of the anti-84P2A9 antibody preparation via an acceptable route of admimstration such as intravenous injection (IV), typicaUy at a dose in the range of about 0.1 to about 10 mg/kg body weight. Doses m the range of 10-500 mg mAb per week are effective and weU tolerated.

Based on clinical experience with the Herceptin mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV foUowed by weekly doses of about 2 mg/kg IV of the anti- 84P2A9 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer mfusion. The periodic maintenance dose is admimstered as a 30 minute or longer mfusion, provided the initial dose was weU tolerated However, as one of skill in the art will understand, various factors wiU influence the ideal dose regimen m a particular case. Such factors can mclude, for example, the binding affinity and half hfe of the Ab or mAbs used, the degree of 84P2A9 expression m the patient, the extent of circulating shed 84P2A9 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic agents used in combination with the_ treatment method of the mvention, as weU as the health status of a particular patient.

OptionaUy, patients should be evaluated for the levels of 84P2A9 m a given sample (e.g. the levels of circulating 84P2A9 antigen and/or 84P2A9 expressmg ceUs) in order to assist m the determination of the most effective dosmg regimen and related factors. Such evaluations are also be used for monitoring puφoses throughout therapy, and are useful to gauge therapeutic success m combination with evaluating other parameters (such as serum PSA levels m prostate cancer therapy).

Inhibition of 84P2A9 Protein Function

Within the first class of therapeutic approaches, the mvention mcludes various methods and compositions for mhibiting the binding of 84P2A9 to its binding partner or its association with other protem(s) as weU as methods for mhibiting 84P2A9 function.

Inhibition of84P2A9 With Intracellular Antibodies

In one approach, recombinant vectors encoding smgle cham antibodies that specificaUy bmd to 84P2A9 are introduced mto 84P2A9 expressmg ceUs via gene transfer technologies, wherem the encoded single cham anti-84P2A9 antibody is expressed mtraceUularly, bmds to 84P2A9 protem, and thereby inhibits its function. Methods for engmeermg such mttaceUular smgle cham antibodies are weU known. Such mttaceUular antibodies, also known as "lnttabodies", are specificaUy targeted to a particular compartment within the ceU, providing control over where the inhibitory activity of the treatment wiU be focused. This technology has been successfully apphed m the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). lnttabodies have been shown to virtaaUy eliminate the expression of otherwise abundant ceU surface receptors. See, for example, Richardson et al, 1995, Proc. Nad. Acad. Sci. USA 92: 3137-3141; Beerh et al, 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al, 1994, Gene Ther. 1 : 332-337.

Smgle cham antibodies comprise the variable domains of the heavy and light cham joined by a flexible hnker polypeptide, and are expressed as a smgle polypeptide. OptionaUy, single cham antibodies are expressed as a smgle cham variable region fragment joined to the kght cham constant region WeU known mttaceUular trafficking signals are engineered mto recombinant polynucleotide vectors encoding such smgle cham antibodies m order to precisely target the expressed lnttabody to the desired mttaceUular compartment For example, lntrabodtes targeted to the endoplasmtc reticulum (ER) are engmeered to incoφorate a leader peptide and, optionaUy, a C- terminal ER retention signal, such as the KDEL ammo acid motif. lnttabodies mtended to exert activity in the nucleus are engmeered to mclude a nuclear localization signal. Lipid moieties are joined to lnttabodies in order to tether the lnttabody to the cytosohc side of the plasma membrane. lnttabodies can also be targeted to exert function m the cytosol For example, cytosohc lnttabodies are used to sequester factors within the cytosol, thereby preventing them from be g transported to their natural ceUular destination.

In one embodiment, lnttabodies are used to capture 84P2A9 m the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engmeered into such 84P2A9 lnttabodies m order to achieve the desired targeting Such 84P2A9 lnttabodies are designed to bmd specificaUy to a particular 84P2A9 domain. In another embodiment, cytosohc lnttabodies that specificaUy bmd to the 84P2A9 protem are used to prevent 84P2A9 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g , preventing 84P2A9 from forming ttanscnption complexes with other factors).

In order to specificaUy direct the expression of such lnttabodies to particular ceUs, the transcription of the lnttabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer In order to target lnttabody expression specificaUy to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S Patent No. 5,919,652)

Inhibition of ^"84P2A9 With Recombinant Proteins

In another approach, recombinant molecules that bmd to 84P2A9 thereby prevent or inhibit 84P2A9 from accessing/binding to its bmdmg partner(s) or associating with other protem(s) are used to inhibit 84P2A9 function _ Such recombinant molecules can, for example, contam the reactive part(s) of an 84P2A9 specific antibody molecule. In a particular embodiment, the 84P2A9 bmding domain of an 84P2A9 bmdtng partner is engmeered mto a dimenc fusion protem compnsing two 84P2A9 hgand bmding domains linked to the Fc portion of a human IgG, such as human IgGl . Such IgG portion can contam, for example, the Cn2 and CH3 domains and the hinge region, but not the CH domain Such dimenc fusion protems are adrnmistered m soluble form to patients suffermg from a cancer associated with the expression of 84P2A9, mcludmg but not limited to prostate and testicular cancers, where the dimenc fusion protem specificaUy bmds to 84P2A9 thereby blocking 84P2A9 mteraction with a bmding partner. Such dimenc fusion protems are further combined mto multimenc protems usmg known antibody knkmg technologies

Inhibition of 84P2A9 Transcription or Translation Within the second class of therapeutic approaches, the mvention provides various methods and compositions for mhibiting the transcription of the 84P2A9 gene. Similarly, the mvention also provides methods and compositions for mhibiting the translation of 84P2A9 mRNA mto protem

In one approach, a method of inhibiting the transcription of the 84P2A9 gene comprises contacting the 84P2A9 gene with an 84P2A9 antisense polynucleotide. In another approach, a method of mhibiting 84P2A9 mRNA translation comprises contacting the 84P2A9 mRNA with an antisense polynucleotide. In another approach, an 84P2A9 specific ribozyme is used to cleave the 84P2A9 message, thereby mhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 84P2A9 gene, such as the 84P2A9 promoter and/or enhancer elements. Similarly, protems capable of mhibiting an 84P2A9 gene transcription factor can be used to inhibit 84P2A9 mRNA ttanscnption. The various polynucleotides and compositions useful in the aforementioned methods have been described above The use of antisense and ribozyme molecules to inhibit transcription and translation is weU known m the art. Other factors that inhibit the transcription of 84P2A9 through interfering with

84P2A9 transcriptional activation are also useful for the treatment of cancers expressmg

84P2A9. Similarly, factors that are capable of mterfermg with 84P2A9 processmg are useful for the treatment of cancers expressmg 84P2A9. Cancer treatment methods utilizing such factors are also within the scope of the mvention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used for dekveπng therapeutic polynucleotide molecules to tumor ceUs synthesizing 84P2A9 (i.e , antisense, nbozyme, polynucleotides encodmg lnttabodies and other 84P2A9 inhibitory molecules). A number of gene therapy approaches are known m the art. Recombmant vectors encoding 84P2A9 antisense polynucleotides, nbozymes, factors capable of mterfermg with 84P2A9 ttanscnption, and so forth, can be dekvered to target tumor ceUs usmg such gene therapy approaches. The above therapeutic approaches can be combmed with any one of a wide vanety of surgical, chemotherapy or radiation therapy regi ens. These therapeutic approaches can enable the use of reduced dosages of chemotherapy and/or less frequent administration, an advantage for aU patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent weU. The anti-tumor activity of a particular composition (e.g, antisense, nbozyme, lnttabody), or a combination of such compositions, can be evaluated usmg vanous m vitro and in vivo assay systems. In vitro assays for evaluating therapeutic potential mclude ceU growth assays, soft agar assays and other assays indicative of tumor promoting activity, bmdmg assays capable of determinmg the extent to which a therapeutic composition wiU inhibit the bmding of 84P2A9 to a bmdmg partner, etc.

In vivo, the effect of an 84P2A9 therapeutic composition can be evaluated m a suitable animal model. For example, xenogenic prostate cancer models wherein human prostate cancer explants or passaged xenograft tissues are introduced mto immune compromised animals, such as nude or SCID mice, are appropnate in relation to prostate cancer and have been descnbed (Klem et al, 1997, Nature Medicme 3: 402-408). For example, PCT Patent Application W098/16628, Sawyers et al, published April 23, 1998, descnbes various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted usmg assays that measure mhibition of tumor formation, tumor regression or metastasis, and the like. See, also, the Examples below.

In vivo assays that evaluate the promotion of apoptosis are useful m evaluating therapeutic compositions In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untteated conttol xenograft-bearmg mice. The extent to which apoptotic foci are found m the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used m the practice of the foregomg methods can be formulated mto pharmaceutical compositions compnsmg a earner suitable for the desired dekvery method. Suitable carriers mclude any material that when combmed with the therapeutic composition retams the anti-tumor function of the therapeutic composition and is generaUy non-reactive with the patient's immune system. Examples mclude, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bactenostatic water, and the like (see, generaUy, Remington's Pharmaceutical Sciences 16^th Edition, A. Osal, Ed, 1980)

Therapeutic formulations can be solubihzed and administered via any route capable of dehvermg the therapeutic composition to the tumor site. PotentiaUy effective routes of administration mclude, but are not limited to, mttavenous, parenteral, intraperitoneal, intramuscular, lnttatumor, lnttadermal, lntøaorgan, orthotopic, and the like. A preferred formulation for mttavenous mjection compnses the therapeutic composition in a solution of preserved bactenostatic water, sterile unpreserved water, and/or ckluted m polyvmylchloπde or polyethylene bags containing 0.9% steπle Sodium Chloride for Injection, USP. Therapeutic protem preparations can be lyophihzed and stored as sterile powders, preferably under vacuum, and then reconstituted m bactenostatic water containing, for example, benzyl alcohol preservative, or m sterile water prior to injection. Dosages and administration protocols for the treatment of cancers usmg the foregomg methods wiU vary with the method and the target cancer, and wiU generaUy depend on a number of other factors appreciated m the art.

CANCER VACCINES

As noted above, the expression profile of 84P2A9 shows that it is highly expressed m advanced and metastasized prostate cancer. This expression pattern is reminiscent of the Cancer-Testis (CT) antigens or MAGE antigens, which are testis- specific genes that are up-regulated m melanomas and other cancers (Van den Eynde and Boon, Int J Chn Lab Res. 27-81 -86, 1997). Due to their tissue-specific expression and high expression levels m cancer, the MAGE antigens are currendy bemg investigated as targets for cancer vaccmes (Durrant, Anticancer Drugs 8 727-733, 1997; Reynolds et al, Int J Cancer 72:972-976, 1997).

The mvention further provides cancer vaccmes compnsmg an 84P2A9-related protem or fragment as weU as DNA based vaccmes. In view of the expression of 84P2A9, cancer vaccmes are effective at specificaUy preventing and/or treating 84P2A9 expressmg cancers without creating non-specific effects on non-target tissues. The use of a tumor antigen m a vaccme for generating humoral and ceU-mediated immunity for use m anti- cancer therapy is weU known in the art and has been employed in prostate cancer usmg human PSMA and rodent PAP lmmunogens (Hodge et al, 1995, Int. J. Cancer 63:231-237; Fong et al, 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing an 84P2A9 protem, or fragment thereof, or an 84P2A9-encodιng nucleic acid molecule and recombinant vectors capable of expressmg and appropriately presenting the 84P2A9 immunogen (which typicaUy comprises a number of humoral or T ceU epitopes lmmunoreactive epitopes). In this context, skiUed artisans understand that a wide vaπety of different vaccme systems for delivery of lmmunoreactive epitopes are known m the art (see, e.g, Heryln et al, Ann Med 1999 Feb;31 (l):66-78; Maruyama et al. Cancer Immunol Immunother 2000 Jun;49 (3): 123-32) Briefly, such techmques consists of methods of generating an immune response (e.g. a humoral and/or ceU mediated response) m a mammal comprising the steps exposmg the mammal's immune system to an lmmunoreactive epitope (e g. an epitope of the 84P2A9 protem shown m SEQ ID NO: 2) so that the mammal generates an immune response that is specific for that epitope (e.g generates antibodies that specificaUy recognize that epitope). In a preferred method, the 84P2A9 immunogen contains a biological motif. In a highly preferred embodiment, the 84P2A9 immunogen contains one or more amino acid sequences identified usmg one of the pertinent analytical techmques weU known in the art such as the sequences shown m Table 1.

A wide variety of methods for generating an immune response in a mammal are weU known in the art (for example as the first step m the generation of hybridomas). Methods of generating an immune response m a mammal comprise exposmg the mammal's immune system to an exogenous immunogenic epitope on a protem (e.g the 84P2A9 protem of SEQ ID NO: 2) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 84P2A9 m a host, by contacting the host with a sufficient amount of 84P2A9 or a B ceU or cytotoxic T-ceU eliciting epitope or analog thereof; and at least one periodic interval thereafter contacting the host with additional 84P2A9 or a B ceU or cytotoxic T-ceU eliciting epitope or analog thereof. A specific embodiment consists of a method of generating an immune response agamst an 84P2A9 protem or a multiepitopic peptide compnsmg admmistenng 84P2A9 immunogen (e.g. the 84P2A9 protem or a peptide fragment thereof, an 84P2A9 fusion protem etc.) m a vaccme preparation to humans or animals. TypicaUy, such vaccme preparations further contam a suitable adjuvant, (see, e.g, U.S Patent No. 6,146,635). A representative variation on these methods consists of a method of generating an immune response in an mdividual agamst an 84P2A9 immunogen compnsmg administering in vivo to muscle or sk of the individual's body a genetic vaccine facilitator such as one selected from the group consisting of: anionic hpids; saponins; lectins; esttogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea; and a DNA molecule that is dissociated from an infectious agent and comprises a DNA sequence that encodes the 84P2A9 immunogen, the DNA sequence operatively hnked to regulatory sequences which control the expression of the DNA sequence; wherem the DNA molecule is taken up by ceUs, the DNA sequence is expressed in the ceUs and an immune response is generated agamst the immunogen. (see, e.g, U S. Patent No. 5,962,428)

In an illustrative example of a specific method for generating an immune response, viral gene delivery systems are used to dehver an 84P2A9-encodιng nucleic acid molecule. λ^anous viral gene delivery systems that can be used m the practice of this aspect of the mvention mclude, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, pokovirus, adeno-associated virus, lentivirus, and smdbus virus (Restifo, 1996, Curr Opm. Immunol. 8:658-663). Non-viral dekvery systems can also be employed by usmg naked DNA encodmg an 84P2A9 protem or fragment thereof introduced mto the patient (e g , mttamuscularly or mttadermaUy) to mduce an anti-tumor response In one embodiment, the fuU-length human 84P2A9 cDNA is employed. In another embodiment, 84P2A9 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) epitopes can be employed. CTL epitopes can be determined usmg specific algonthms (e.g, Eprmer, Brown Umversity) to identify peptides within an 84P2A9 protem that are capable of optimaUy bmding to specified HLA aUeles.

Vanous ex vivo strategies can also be employed. One approach involves the use of dendntic ceUs to present 84P2A9 antigen to a patient's immune system. Dendntic ceUs express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting ceUs. In prostate cancer, autologous dendntic ceUs pulsed with peptides of the prostate-specific membrane antigen (PSMA) are bemg used m a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al, 1996, Prostate 28:65-69; Muφhy et al , 1996, Prostate 29:371-380). Thus, dendntic ceUs can be used to present 84P2A9 peptides to T ceUs m the context of MHC class I and II molecules. In one embodiment, autologous dendntic ceUs are pulsed with 84P2A9 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendntic ceUs are pulsed with the complete 84P2A9 protem. Yet another embodiment mvolves engmeermg the overexpression of the 84P2A9 gene in dendntic ceUs usmg various implementing vectors known m the art, such as adenovirus (Arthur et al, 1997, Cancer Gene Ther. 4:17-25), rettovirus (Henderson et al, 1996, Cancer Res 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al, 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al, 1997, J. Exp Med 186.1177-1182). CeUs expressmg 84P2A9 can also be engmeered to express immune modulators, such as GM-CSF, and used as immunizing agents.

Anti-idiotypic anti-84P2A9 antibodies can also be used m anti-cancer therapy as a vaccme for mducmg an immune response to ceUs expressmg an 84P2A9 protem. SpecificaUy, the generation of anti-idiotypic antibodies is weU known m the art and can readily be adapted to generate anti-idiotypic anti-84P2A9 antibodies that mimic an epitope on an 84P2A9 protem (see, for example, Wagner et al, 1997, Hybndoma 16: 33-40; Foon et al, 1995, J. Clin. Invest 96:334-342; Herlyn et al, 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccme strategies. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and ceUular immune responses directed agamst cancer ceUs expressmg 84P2A9 Constructs compnsmg DNA encodmg an 84P2A9-related protem/immunogen and appropnate regulatory sequences can be mjected direcdy mto muscle or skin of an mdividual, such that the ceUs of the muscle or skm take-up the construct and express the encoded 84P2A9 protem/immunogen. Alternatively, a vaccme compnses an 84P2A9- related protem. Expression of the 84P2A9 protem immunogen results m the generation of prophylactic or therapeutic humoral and ceUular immunity against bone, colon, pancreatic, testicular, cervical and ovarian cancers. Vaπous prophylactic and therapeutic genetic immunization techniques known m the art can be used (for review, see information and references pubhshed at Internet address www.genweb.com)

KITS

For use m the diagnostic and therapeutic apphcations described herem, kits are also provided by the mvention. Such kits can comprise a carrier bemg compartmentakzed to receive in close confinement one or more containers such as vials, tabes, and the like, each of the contaιner(s) compnsmg one of the separate elements to be used m the method. For example, the contamer(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for an 84P2A9-realted protem or an 84P2A9 gene or message, respectively. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have contamers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protem, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.

The kit of the mvention wiU typicaUy compnse the container descnbed above and one or more other containers compnsmg matenals desirable from a commercial and user standpoint, mcludmg buffers, diluents, filters, needles, syringes, and package mserts with instructions for use. A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic appkcation, and can also indicate directions for either m vivo or in vitro use, such as those descnbed above.

ρ84P2A9-l has been deposited under the requirements of the Budapest Treaty on January 6, 2000 with the American Type Culture CoUection (ATCC), 10801 Umversity Blvd., Manassas, VA 20110-2209 USA, and have been identified as ATCC Accession No. PTA-1151.

EXAMPLES

Various aspects of the invention are further descnbed and lUusttated by way of the several examples that foUow, none of which are mtended to limit the scope of the mvention.

Example 1: SSH-Generated Isolation of cDNA Fragment of the 84P2A9 Gene

Materials and Methods

LAPC Xenografts and Human Tissues: LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated as described (Klem et al, 1997, Natare Med. 3: 402-408). Androgen dependent and mdependent LAPC-4 xenografts LAPC-4 AD and Al, respectively) and LAPC-9 AD and Al xenografts were grown m male SCID mice and were passaged as smaU tissue chunks m recipient males. LAPC-4 and —9 Al xenografts were derived from LAPC-4 or -9 AD tumors, respectively Male mice bearmg AD tumors were castrated and maintained for 2-3 months. After the tumors re -grew, the tumors were harvested and passaged in castrated males or m female SCID mice. Human tissues for RNA and protem analyses were obtained from the Human Tissue Resource Center (HTRC) at the UCLA (Los Angeles, CA) and from QualTek, Inc. (Santa Barbara, CA). A bemgn prostatic hypeφlasia tissue sample was patient-derived.

CeU Lmes

Human ceU lines (e.g , HeLa) were obtained from the ATCC and were mamtamed m DMEM with 5% fetal calf serum.

RNA Isolation.

Tumor tissue and ceU lines were homogenized m Tnzol reagent (Life

Technologies, Gibco BRL) usmg 10 ml/ g tissue or 10 ml/ 10⁸ ceUs to isolate total RNA. Poly A RNA was purified from total RNA usmg Qiagen's Ohgotex mRNA Mini and

Midi kits. Total and mRNA were quantified by specttophotometnc analysis (O.D.

260/280 nm) and analyzed by gel electrophoresis.

Ohgonucleotides: The foUowmg HPLC puπfied ohgonucleotides were used.

DPNCDN (cDNA synthesis pnmer)- 5'TTTTGATCAAGCTT₃₀3' (SEQ ID NO: 7)

Adaptor 1 :

5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO

8)

3'GGCCCGTCCTAG5' (SEQ ID NO- 9) Adaptor 2- 5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO.10) 3'CGGCTCCTAG5' (SEQ ID NO. 11) PCR primer 1 5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO. 12)

Nested primer (NP)1 :

5 CGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 13)

Nested primer (NP)2:

5ΑGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 14)

Suppression Subttactive Hybridization:

Suppression Subttactive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentiaUy expressed m prostate cancer. The SSH reaction utilized cDNA from two LAPC-4 AD xenografts. SpecificaUy, the 84P2A9 SSH sequence was identified from a subtraction where cDNA derived from an LAPC-4 AD tamor, 3 days post-casttation, was subttacted from cDNA derived from an LAPC-4 AD tamor grown m an mtact male. The LAPC-4 AD xenograft tamor grown m an mtact male was used as the source of the "tester" cDNA, while the cDNA from the LAPC-4 AD tamor, 3 days post-casttation, was used as the source of the "driver" cDNA.

Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly (A) ⁺ RNA isolated from the relevant xenograft tissue, as described above, usmg CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117- 1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs. at 37°C. Digested cDNA was extracted with phenol/chloroform (1 :1) and ethanol precipitated.

Driver cDNA was generated by combining m a 1 :1 ratio Dpn II digested cDNA from the relevant xenograft source (see above) with a mix of digested cDNAs derived from human bemgn prostatic hypeφlasia (BPH), the human ceU lines HeLa, 293, A431, Colo205, and mouse liver. Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant xenograft source (see above) (400 ng) m 5 μl of water The diluted cDNA (2 μl, 160 ng) was then hgated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), m separate kgation reactions, m a total volume of 10 μl at 16°C overnight, usmg 400 u of T4 DNA kgase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72°C for 5 mm.

The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tabes containing 1.5 μl (20 ng) Adaptor 1 - and Adaptor 2- hgated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral od, denatured m an MJ Research thermal cycler at 98°C for 1.5 minutes, and then were aUowed to hybridize for 8 hrs at 68°C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were aUowed to hybridize overnight at 68°C. The second hybridization was then diluted m 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70°C for 7 mm. and stored at -20°C.

PCR Amplification. Cloning and Sequencing of Gene Fragments Generated from SSH:

To amphfy gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10 x reaction buffer (CLONTECH) and 0.5 μl 50 x Advantage cDNA polymerase Mix (CLONTECH) m a final volume of 25 μl. PCR 1 was conducted usmg the foUowmg conditions: 75°C for 5 mm, 94°C for 25 sec, then 27 cycles of 94°C for 10 sec, 66°C for 30 sec, 72°C for 1.5 mm. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the 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 μM) were used mstead of PCR primer 1. PCR 2 was performed usmg 10-12 cycles of 94°C for 10 sec, 68°C for 30 sec, 72°C for 1.5 minutes The PCR products were analyzed usmg 2% agarose gel electrophoresis. The PCR products were inserted mto pCR2.1 usmg the T/A vector cloning kit (Invitrogen). Transformed E. cok were subjected to blue /white and ampicilhn selection. White colonies were picked and arrayed mto 96 weU plates and were grown m kquid culture overnight. To identify mserts, PCR amplification was performed on 1 ml of bacterial culture usmg the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed usmg 2% agarose gel electrophoresis.

Bacterial clones were stored m 20% glycerol m a 96 weU format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis

First sttand cDNAs can be generated from 1 μg of mRNA with ohgo (dT)12-18 priming using the Gibco-BRL Superscript Preamphfication system. The manufacturer's protocol can be used and mcluded an incubation for 50 mm at 42°C with reverse ttanscπptase foUowed by RNAse H tteatment at 37°C for 20 mm. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First sttand cDNAs from 16 different normal human tissues can be ob tamed from Clontech

Normalization of the first strand cDNAs from multiple tissues can be performed by usmg the primers 5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 15) and 5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 16) to amphfy β-actin. First strand cDNA (5 μl) can be amplified m a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1XPCR buffer (Clontech, 10 mM Tns-HCL, 1.5 mM MgCl₂, 50 mM KC1, pH8.3) and IX Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR can be performed usmg an MJ Research thermal cycler under the foUowmg conditions: Initial denaturation can be at 94°C for 15 sec, foUowed by a 18, 20, and 22 cycles of 94°C for 15, 65°C for 2 min, 72°C for 5 sec. A final extension at 72°C can be earned out for 2 mm After agarose gel electrophoresis, the band mtensities of the 283 bp β-actin bands from multiple tissues can be compared by visual inspection. DUution factors for the first sttand cDNAs can be calculated to result m equal β-actin band mtensities m aU tissues after 22 cycles of PCR Three rounds of normakzation can be required to achieve equal band mtensities in aU tissues after 22 cycles of PCR

To determine expression levels of the 84P2A9 gene, 5 μl of normalized first strand cDNA can be analyzed by PCR usmg 25, 30, and 35 cycles of ampkfication usmg primer pairs that can be designed with the assistance of (MIT; for details, see, www genome wi.mit.edu).

Semi quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band mtensities.

Results

Two SSH experiments described m the Materials and Methods, supra, led to the isolation of numerous candidate gene fragment clones (SSH clones). AU candidate clones were sequenced and subjected to homology analysis agamst aU sequences m the major pubhc gene and EST databases m order to provide information on the identity of the corresponding gene and to help guide the decision to analyze a particular gene for differential expression In general, gene fragments that had no homology to any known sequence m any of the searched databases, and thus considered to represent novel genes, as weU as gene fragments showmg homology to previously sequenced expressed sequence tags (ESTs), were subjected to differential expression analysis by RT-PCR and/or Northern analysis.

One of the SHH clones compnsmg about 425 bp, showed significant homology to several testis-denved ESTs but no homology to any known gene, and was designated 84P2A9.

Northern expression analysis of first sttand cDNAs from 16 normal tissues showed a highly prostate and testis-related expression pattern m adult tissues (FIG 4).

Example 2: Full Length Cloning of 84P2A9

A fuU length 84P2A9 cDNA clone (clone 1) of 2347 base pairs (bp) was cloned from an LAPC-4 AD cDNA library (Lambda ZAP Express, Stratagene) (Fig. 2) The cDNA encodes an open reading frame (ORF) of 504 ammo acids. Sequence analysis revealed the presence of six potential nuclear localization signals and is predicted to be nuclear usmg the PSORT program (htφ://psort.mbb.ac.jp:8800/form.html) The protem sequence is homologous to a human bram protem KIAA1152 (39.5% identity over a 337 amino acid region), and exhibits a domain that is homologous to the LUCA15 tumor suppressor protem (64.3% identity over a 42 amino acid region) (GenBank Accession #P52756)(Fιg. 3). The 84P2A9 cDNA was deposited on January 5, 2000 with the American Type Culture CoUection (ATCC; Manassas, VA) as plasmid p84P2A9-l, and has been assigned Accession No. PTA-1151.

The 84P2A9 protems have no homology to any known protems, but the sequence does overlap with several ESTs derived from testis.

Example 3: 84P2A9 Gene Expression Analysis

84P2A9 mRNA expression m normal human tissues was analyzed by Northern blotting of two multiple tissue blots (Clontech; Palo Alto, California), compnsmg a total of 16 different normal human tissues, usmg labeled 84P2A9 SSH fragment (Example 1) as a probe. RNA samples were quantitatively normalized with a β-actin probe. The results demonstrated expression of a 2.4 and 4.5 kb ttanscnpt m normal testis and prostate (Fig. 4).

To analyze 84P2A9 expression m prostate cancer tissues lines northern blotting was performed on RNA derived from the LAPC xenografts. The results show high levels of 84P2A9 expression m aU the xenografts, with the highest levels detected m LAPC-9 AD, LAPC-9 Al (Fig. 4 and Figure 5). These results provide evidence that 84P2A9 is up-regulated m prostate cancer.

In addition, high levels of expression were detected m bra (PFSK-1, T98G), bone (HOS, U2-OS), lung (CALU-1, NCI-H82, NCI-H146), and kidney (769-P, A498, CAKI-1, SW839) cancer ceU lines (Fig. 5). Moderate expression levels were detected m several pancreatic (PANC-1, BxPC-3, HPAC, CAPAN-1), colon (SK-CO-1, CACO-2, LOVO, COLO-205), bone (SK-ES-1, RD-ES), breast (MCF-7, MDA-MB-435s) and testicular cancer (NCCIT) ceU lines (Fig. 5). In addition, prostate cancer patient samples show expression of 84P2A9 m both the normal and the tumor part of the prostate tissues (Fig. 6). These results suggest that

84P2A9 is a very testis specific gene that is up-regulated in prostate cancer and potentiaUy other cancers Simύar to the MAGE antigens, 84P2A9 may thus qualify as a cancer-testis antigen (Van den Eynde and Boon, Int J Chn Lab Res. 27:81-86, 1997).

84P2A9 expression in normal tissues can be further analyzed usmg a multi-tissue RNA dot blot containing different samples (representing mamly normal tissues as weU as a few cancer ceU lines).

Example 4: Generation of 84P2A9 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one or more mjections of an immumzmg agent and, if desired, an adjuvant TypicaUy, the immumzmg agent and/or adjuvant wiU be mjected m the mammal by multiple subcutaneous or mttaperitoneal mjections. TypicaUy a peptide can be designed from a codmg region of 84P2A9. Alternatively the immunizing agent may mclude aU or portions of the 84P2A9 protem, or fusion protems thereof. For example, the 84P2A9 amino acid sequence can be fused to any one of a vaπety of known fusion protem partners that are weU known m the art such as maltose bmding protein, LacZ, thioredoxin or an immunoglobulin constant region (see, e.g , Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds, 1995; Lmsley, P.S, Brady, W, Urnes, M, Grosmaire, L, Damle, N, and Ledbetter, L.(1991) J.Exp. Med. 174, 561-566). Other such recombmant bacterial protems include glutathione-S-transferase (GST), and HIS tagged fusion protems of 84P2A9 (which can be purified from induced bacteria usmg the appropriate affinity matrix). It may be useful to conjugate the immunizing agent to a protein known to be immunogenic m the mammal bemg immunized. Examples of such immunogenic protems mclude but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobu n, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic ttehalose dicorynomycolate). In a typical protocol, rabbits can be initiaUy immunized subcutaneously with about 200 μg of fusion protem or KLH-peptide mixed m complete Freund's adjuvant Rabbits are then mjected subcutaneously every two weeks with about 200 μg of immunogen m incomplete Freund's adjuvant Test bleeds are taken approximately 7 10 days foUowing each immunization and used to momtor the liter of the antiserum b^ ELISA

Specificity of the antiserum is tested by Western blot and immunoprecipitation analyses using lysates of geneticaUy engmeered ceUs or ceUs expressmg endogenous 84P2A9 To geneticaUy engineer ceUs to express 84P2A9, the full length 84P2A9 cDNA can be cloned mto an expression vector that provides a 6Hιs tag at the carboxyl-termmus (pCDNA 3 1 myc-his, InVitrogen) After transfection of the constructs mto 293T ceUs, ceU lysates can be lmmunoprecipitated and Western blotted usmg anti-His or v5 anti- epitope antibody (Invitrogen) and the anti-84P2A9 serum (see, e g , FIG 11) Sera from His-tagged protem and peptide immunized rabbits as weU as depleted GST and MBP protem sera are purified by passage over an affinity column composed of the respective immunogen covalendy coupled to Affigel matrix (BioRad)

Example 5: Production of Recombinant 84P2A9 in Bacterial and Mammalian Systems BACTERIAL CONSTRUCTS

Production of Recombmant 84P2A9 usmg pGEX Constructs

To express 84P2A9 m bacterial ceUs, a portion of 84P2A9 was fused to the Glutathione S-ttansferase (GST) gene by cloning mto pGEX 6P 1 (Amersham Pharmacia Biotech, NJ) AU constructs were made to generate recombmant 84P2A9 protein sequences with GST fused at the N-terminus and a six histidine epitope at the C- terminus The six histidine epitope tag was generated by adding the histidine codons to the cloning primer at the 3' end of the ORF A PreScission™ recognition site permits cleavage of the GST tag from 84P2A9 The ampicillin resistance gene and pBR322 ongm permits selection and mamtenance of the plasmid m E cok In this construct, a fragment containing ammo acids 1 to 151 of 84P2A9 was cloned mto pGEX-6P-l Additional constructs can be made m pGEX-6P-l spanning regions of the 84P2A9 protem such as amino acids 1 to 504 and ammo acids 151 to 504.

MAMMALIAN CONSTRUCTS To express recombmant 84P2A9 m mammalian systems, the fuU length 84P2A9 cDNA can for example, be cloned mto an expression vector that provides a 6Hιs tag at the carboxyl-termmus (pCDNA 3.1 myc-his, InVittogen). The constructs can be transfected mto 293T ceUs. Transfected 293T ceU lysates can be probed with the anti- 84P2A9 polyclonal serum described in Example 4 above m a Western blot. The 84P2A9 genes can also be subcloned mto the retroviral expression vector pSRαMSVtkneo and used to establish 84P2A9 expressmg ceU lines as foUows. The 84P2A9 coding sequence (from translation initiation ATG to the termination codons) is amplified by PCR usmg ds cDNA template from 84P2A9 cDNA. The PCR product is subcloned mto pSRαMSVtkneo via the EcoRl (blunt-ended) and Xba 1 restriction sites on the vector and ttansformed mto DH5α competent ceUs. Colonies are picked to screen for clones with unique internal restriction sites on the cDNA. The positive clone is confirmed by sequencmg of the cDNA msert. Rettoviruses may thereafter be used for infection and generation of various ceU hnes usmg, for example, NIH 3T3, TsuPrl, 293 or rat-1 ceUs. Specific mammalian systems are discussed herem.

Production of Recombmant 84P2A9 usmg pcDNA3.1 /V5-Hιs-TOPO Constructs

To express 84P2A9 m mammahan ceUs, the 1512 bp (504 ammo acid) 84P2A9 ORF along with perfect ttanslational start Kozak consensus sequence was cloned mto pcDNA3.1 /V5-Hιs-TOPO (Invittogen, Carlsbad, CA). Protem expression is dπven from the cytomegalovirus (CMV) promoter. The recombmant protem has the V5 epitope and six histidines fused to the C-terminus. The pcDNA3.1 /V5-Hιs-TOPO vector also contams the bovme growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 oπgm for episomal rephcation and simple vector rescue m ceU lines expressmg the large T antigen The Neomycm resistance gene aUows for selection of mammalian ceUs expressing the protem and the ampicillin resistance gene and ColEl oπgm permits selection and maintenance of the plasmid in E. coh

pSRa Constructs To generate mammahan ceU hnes expressmg 84P2A9 constitutively, the 1551 bp

(517 amino acid) ORF is bemg cloned mto pSRa constructs. Amphottopic and ecottopic rettoviruses are generated by transfection of pSRa constructs mto the 293T-10A1 packagmg line or co-teansfection of pSRa and a helper plasmid (φD ) m 293 ceUs, respectively The retrovius can be used to mfect a variety of mammahan ceU lines, resulting in the mtegration of the cloned gene, 84P2A9, into the host ceU-hnes Protem expression is dπven from a long terminal repeat (LTR). The Neomycin resistance gene aUows for selection of mammalian ceUs expressmg the protem and the ampicillin resistance gene and ColEl origin permits selection and mamtenance of the plasmid m E. coh. Additional pSRa constructs are bemg made to produce both N-terminal and C- terminal GFP and myc/6 HIS fusion protems of the fuU-length 84P2A9 protem.

Example 6: Production of Recombinant 84P2A9 in a Baculovirus System

To generate a recombinant 84P2A9 protem m a baculovirus expression system, the 84P2A9 cDNA is cloned mto the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-termmus SpecificaUy, pBlueBac— 84P2A9 is co-ttansfected with helper plasmid pBac-N-Blue (Invitrogen) mto SF9 (Spodoptera frugiperda) msect ceUs to generate recombmant baculovirus (see Invittogen instruction manual for details). Baculovirus is then coUected from ceU supernatant and punfied by plaque assay. Recombmant 84P2A9 protem is then generated by infection of HighFive msect ceUs (InVittogen) with the purified baculovirus. Recombmant 84P2A9 protem may be detected usmg anti-84P2A9 antibody. 84P2A9 protem may be purified and used m various ceU based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 84P2A9. Example 7: Chromosomal Mapping of the 84P2A9 Gene

The chromosomal localization of 84P2A9 was determined usmg the GeneBπdge4 radiation hybrid panel (Walter et al, 1994, Nat. Genetics 7:22) (Research Genetics, HuntsviUe Al). The foUowmg PCR primers were used to locahze 84P2A9:

84P2A9.1 gacttcactgatgcgatggtaggt (SEQ ID NO: 17) 84P2A9.2 gtcaatactttccgatgctttgct (SEQ ID NO: 18)

The resulting mapping vector for the 93 radiation hybrid panel DNAs was: 0000100011001011001000001100010010010000100010100110001000000010010010010 10000000100000010000. This vector and the mapping program at http://www- genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl placed 84P2A9 on chromosome lq32.3 (D1S1602- D1S217).

Example 8: Identification of Potential Signal Transduction Pathways

To determine whether 84P2A9 direcdy or mdirecdy activates known signal ttansduction pathways m ceUs, luciferase (luc) based transcriptional reporter assays are carried out in ceUs expressmg 84P2A9 These transcriptional reporters contam consensus binding sites for known transcription factors that he downstteam of weU characterized signal ttansduction pathways The reporters and examples of there associated transcription factors, signal ttansduction pathways, and activation stimuk are ksted below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/sttess 2. SRE-luc, SRF/TCF/ELK1 ; MAPK/SAPK; growth/differentiation

3. AP-l-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/sttess

4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis 5 p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/sttess 84P2A9-medιated effects may be assayed m ceUs showmg mRNA expression.

Luciferase reporter plasmids may be introduced by hpid mediated transfection (TFX-50,

Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of ceUs extracts with lucifeπn substrate and luminescence of the reaction is monitored m a luminometer.

Example 9: Generation of 84P2A9 Monoclonal Antibodies

To generate MAbs to 84P2A9, typicaUy Balb C mice are immunized mttapentoneaUy with about 10-50 μg of protem immunogen mixed m complete Freund's adjuvant Protem immunogens mclude bacterial and baculovirus produced recombmant 84P2A9 proteins and mammalian expressed human IgG FC fusion protems. Mice are then subsequendy immunized every 2-4 weeks with 10-50 μg of antigen mixed in Freund's mcomplete adjuvant. Alternativel) , Ribi adjuvant is used for initial immunizations. In addition, a DNA-based immunization protocol is used m which a mammalian expression vector such as pCDNA 3.1 encoding the 84P2A9 cDNA alone or as an IgG FC fusion is used to immunize mice by direct injection of the plasmid DNA. This protocol is used alone and in combination with protem immunogens. Test bleeds are taken 7-10 foUowmg immunization to monitor liter and specificity of the immune response Once appropriate reactivity and specificity is obtamed as determined by ELISA, Western blotting, and immunoprecipitation analyses, fusion and hybridoma generation is then carried with estabkshed procedures weU known in the art (Harlow and Lane, 1988).

In a typical specific protocol, a glutathione-S-ttansferase (GST) fusion protem encompassmg an 84P2A9 protem is synthesized and used as immunogen. Balb C mice are lnittaUy immunized mttapentoneaUy with 10-50 μg of the GST-84P2A9 fusion protem mixed in complete Freund's adjuvant. Mice are subsequendy immunized every 2 weeks with 10-50 μg of GST-84P2A9 protem mixed in Freund's mcomplete adjuvant for a total of 3 immunizations. Reactivity of serum from immunized mice to fuU length 84P2A9 protem is monitored by ELISA using a partiaUy purified preparation of HIS- tagged 84P2A9 protem expressed from 293T ceUs (Example 5) Mice showmg the strongest reactivity are rested for 3 weeks and given a final mjection of fusion protem in PBS and then sacrificed 4 days later. The spleens of the sacrificed mice are then harvested and fused to SPO/2 myeloma ceUs usmg standard procedures (Harlow and Lane, 1988). Supernatants from growth weUs foUowmg HAT selection are screened by ELISA and Western blot to identify 84P2A9 specific antibody producmg clones.

The bmdmg affinity of an 84P2A9 monoclonal antibody may be determined usmg standard technology. Affinity measurements quantify the strength of antibody to epitope bmdtng and may be used to help define which 84P2A9 monoclonal antibodies are preferred for diagnostic or therapeutic use. The BIAcore system (Uppsala, Sweden) is a preferred method for determining bmdmg affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K 1991, Opt. Quant. Elect. 23:1 ; Morton and Myszka, 1998, Methods m Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniendy generates association rate constants, dissociation rate constants, equikbπum dissociation constants, and affinity constants.

Example 10: In Vitro Assays of 84P2A9 Function

The expression of 84P2A9 m prostate cancer provides evidence that this gene has a functional role m tumor progression. It is possible that 84P2A9 functions as a transcription factor mvolved m activating genes mvolved m tumongenesis or repressing genes that block tumongenesis. 84P2A9 function can be assessed m mammalian ceUs usmg m vitro approaches. For mammahan expression, 84P2A9 can be cloned mto a number of appropriate vectors, mcluding pcDNA 3.1 myc-His-tag (Example 5) and the rettoviral vector pSRαtkneo (MuUer et al, 1991, MCB 11:1785). Usmg such expression vectors, 84P2A9 can be expressed in several ceU lines, mcluding NIH 3T3, rat-1, TsuPrl and 293T. Expression of 84P2A9 can be momtored usmg anti-84P2A9 antibodies (see Examples 4 and 9).

Mammahan ceU hnes expressmg 84P2A9 can be tested m several in vitro and m vivo assays, mcluding ceU proliferation m tissue culture, activation of apoptotic signals, tamor formation m SCID mice, and m vitro mvasion usmg a membrane invasion culture system (MICS) (Welch et al. ,Int. J. Cancer 43: 449-457). 84P2A9 ceU phenotype is compared to the phenotype of ceUs that lack expression of 84P2A9 The transcriptional effect of 84P2A9 can be tested by evaluating the effect of 84P2A9 on gene expression usmg gene arrays (Clontech) and transcriptional reporter assays (Stratagene).

CeU hnes expressmg 84P2A9 can also be assayed for alteration of mvasive and migratory properties by measuring passage of ceUs through a mattigel coated porous membrane chamber (Becton Dickinson). Passage of ceUs through the membrane to the opposite side is momtored using a fluorescent assay (Becton Dickinson Technical BuUetin #428) usmg calcein-Am (Molecular Probes) loaded indicator ceUs CeU hnes analyzed mclude parental and 84P2A9 overexpressing PC3, NIH 3T3 and LNCaP ceUs To determine whether 84P2A9-exρressιng ceUs have chemoattractant properties, indicator ceUs are monitored for passage through the porous membrane toward a gradient of 84P2A9 conditioned media compared to control media. This assay may also be used to qualify and quantify specific neutralization of the 84P2A9 mduced effect by candidate cancer therapeutic compositions The function of 84P2A9 can be evaluated usmg anti-sense RNA technology coupled to the various functional assays described above, e.g. growth, mvasion and migration. Anti-sense RNA ohgonucleotides can be introduced mto 84P2A9 expressmg ceUs, thereby preventing the expression of 84P2A9. Control and anti-sense containing ceUs can be analyzed for proliferation, mvasion, migration, apoptotic and transcriptional potential. The local as weU as systemic effect of the loss of 84P2A9 expression can be evaluated

Example 11: In Vivo Assay for 84P2A9 Tumor Growth Promotion

The effect of the 84P2A9 protem on tamor ceU growth may be evaluated m vivo by gene overexpression m tumor-bearing mice. For example, SCID mice can be mjected SQ on each flank with 1 x 10⁶ of either PC3, TSUPR1, or DU145 ceUs containing tkNeo empty vector or 84P2A9. At least two strategies may be used. (1) Constitutive 84P2A9 expression under regulation of a promoter such as a constitutive promoter obtamed from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 pubhshed 5 July 1989), adenovirus (such as Adenovirus 2), bovme papiUoma virus, avian sarcoma virus, cytomegalo virus, a rettovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host ceU systems, and (2) Regulated expression under conttol of an inducible vector system, such as ecdysone, tet, etc, provided such promoters are compatible with the host ceU systems. Tumor volume is then monitored at the appearance of palpable tumors and foUowed over time to determine if 84P2A9 expressmg ceUs grow at a faster rate and whether tumors produced by 84P2A9-expressιng ceUs demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vasculaπzation, reduced responsiveness to chemotherapeutic drugs). AdditionaUy, mice may be implanted with 1 x 10⁵ of the same ceUs orthotopicaUy to determine if 84P2A9 has an effect on local growth in the prostate or on the ability of the ceUs to metastasize, specificaUy to lungs, lymph nodes, and bone marrow

The assay is also useful to determine the 84P2A9 inhibitory effect of candidate therapeutic compositions, such as for example, 84P2A9 lnteabodies, 84P2A9 antisense molecules and ribozymes.

Example 12: Western Analysis of 84P2A9 Expression in Subcellular Fractions

Sequence analysis of 84P2A9 revealed the presence of nuclear locakzation signal. The ceUular location of 84P2A9 can be assessed usmg subceUular fractionation techmques widely used m ceUular biology (Storrie B, et al. Methods Enzymol. 1990;182:203-25). Prostate or testis ceU lines can be separated into nuclear, cytosohc and membrane fractions. The expression of 84P2A9 m the different fractions can be tested usmg Western blotting techmques.

Alternatively, to determine the subceUular localization of 84P2A9, 293T ceUs can be ttansfected with an expression vector encodmg HIS-tagged 84P2A9 (PCDNA 3.1 MYC/HIS, Invittogen). The ttansfected ceUs can be harvested and subjected to a differential subceUular fractionation protocol as previously described (Pemberton, P.A. et al, 1997, J of Histochemistry and Cytochemistry, 45:1697-1706.) This protocol separates the ceU mto fractions enriched for nuclei, heavy membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma membrane and endoplasmic reticulum), and soluble proteins.

Throughout this appkcation, various pubkcations are referenced (within parentheses for example). The disclosures of these pubhcations are hereby incoφorated by reference herein m their entireties.

The present mvention is not to be hmited in scope by the embodiments disclosed herem, which are mtended as smgle illustrations of mdividual aspects of the mvention, and any that are functionaUy equivalent are within the scope of the mvention. Various modifications to the models and methods of the mvention, m addition to those described herem, wiU become apparent to those skiUed in the art from the foregoing description and teachings, and are simdarly mtended to faU within the scope of the mvention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the mvention.

TABLES

TABLE 1 : predicted bmdtng of peptides from 84P2A9 protems to the human MHC class I molecule HLA-A2

TABLES 3-16 provide additional analyses of the predicted bmdmg of peptides from 84P2A9 proteins to various HLA molecules. TABLE 2: AMINO ACID SUBSTITUTION MATRIX

Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitation matrix (block substitation mattix). The higher the value, the more likely a substitation is found in related, nataral proteins.

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

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

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

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

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

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

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

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

4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 I

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

4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L

5 -2 -2 0 -1 -1 1 -1 -1 M 6 -2 0 0 1 0 -3 -4 -2 N

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

5 1 0 -2 -2 -1 Q

5 -1 -3 -3 -2 _. R 4 -2 -3 -2 S 5 0 -2 -2 T 4 -3 -1 V 11 2 w

7 Y

HLA peptide motif search results

TABLE 3A

Echoed User Peptide Sequence (length = 504 residues)

1 MΞELVHD VS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQ LVA

101 KRRPSSNLNN NVRGKRPL H ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQP PEGCRDQD D SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGWLESEE TNQTN DKME CEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS D FYEKESGG ACGITGWPW WEKEDPTELD KNVPDPVFES 301 I TGSFP MS HPSRRGFQAR L3RLHGMSSK NIKKSGGTPT SMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA 401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA

TABLE 3B HLA peptide motif search results

TABLE 4A

Echoed User Peptide Sequence Oength = 504 residues)

1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMΛVDLPQD

151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGWPW WEKEDPTELD KNVPDPVFES

301 I TGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSA HLA peptide motif search results

TABLE 5A

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHOLVS ALEES≤EQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SD3DDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KPMAVDLPQD lbl ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSA

TABLE 5B 92 HLA peptide motif search results

TABLE 6A

Echoed User Peptide Sequence (length = 504 residues) i MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSΞLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP- GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSΛ

TABLE 6B 94 HLA peptide motif search results

TABLE 7A

Echoed User Peptide Sequence (length = 504 residues) i MEELVHDLVS ALEΞSSEQAR GGFAETGDHS RSISCPLKRO ARKRRGRKRR

51 SYNVHHPWET GHCLΞEG.SD5 SLEEPSKDYR ENHNNNKKDH SDSDDOHLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCRDQDMU SDRAYQYQEF TKNKVKKRKL K1IRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

251 EGRQGDDEQS DWF EKESGG ACGITGWPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSA

TABLE 7B 96 HLA peptide motif search results

TABLE 8A

Echoed User Peptide Sequence (length = 504 residues)

MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR fil SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD 151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND 251 EGRQGDDEQS DWFYEKΞSGG ACGITGWPW WSKEDPTELD KNVPDPVFEΞ 301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG 351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA 401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 501 GKSA

TABLE 8B HLA peptide motif search results

TABLE 9A

Echoed User Peptide Sequence (length = 504 residues)

MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLP0D

151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

?51 EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES

301 I TG3FPLM3 HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

531 GKSA

TABLE 9B BDLA peptide motif search results

TABLE 10A

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHDLVS ALEESΞEOAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGWPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

•351 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSA

TABLE 10B HLA peptide motif search results

TABLE 11 A

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDOMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCR30DMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

2U1 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGWPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP CARTEHDQHQ LLRDNRAERG HKKNCSVRTA

101 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA sm GKSA

TABLE 1 IB 104 HLA peptide motif search results

TABLE 12A

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA lϋl KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDS5SLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGWPW WEKEDPTELD KNVPDPVFES

301 I TGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT ΞMVPIPGPVG

.351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSA

TABLE 12B 106 HLA peptide motif search results

TABLEΪ3A

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKT, KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ΛCGI GVVPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSA

TABLE 13B 108 HLA peptide motif search results

| User Parameters and Scoring Information I

| method selected to limit number of results explicit number j

| number of results requested 30 ; j HLA molecule type selected B7 length selected for subsequences to be scored 10 echoing mode selected for input sequence Y echoing format - numbered lines j length of user's input peptide sequence | 504 j

| number of subsequence scores calculated 495 jnumber of top-scoring subsequences reported back in scoring output table: 30 {

TABLE 14A

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRO ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

101 ISNKRTMTOP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ 01 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSΞLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGVVPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

J51 NKRMVHFSPD SHHHDHWFSP "GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM 1 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA

501 GKSA

TABLE 14B 110 HLA peptide motif search results

TABLE 15A

474

^|i ²⁶ _.JL - QAMQRPKGL 3.000 1

!| 143 MAVDLPQDI 2.400 I

I 2δ 184^{'' '} ; KVKKRKLKI ; 2.400 j

. 29 293 1 VPDPVFESI 2.400 1

30 [ 90 ^"" HSDSDDQML '. 2.250 i

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR

51 SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDD0MLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCP.DQDMD SDRAYQYQEF TKNKVKKRKL KIIRQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDS5SLS STDAGLFTND

251 EGRQGDDEQS DWFYEKE3GC ACGITGWPW WEKEDPTELD KNVPDPVFES

301 ILTGSFPLMS HPSRRGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

401 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA liOl GKΞA

112

TABLE 15B HLA peptide motif search results

TABLE 16A

Echoed User Peptide Sequence (length = 504 residues)

1 MEELVHDLVS ALEESSEQAR GGFAETGDHS RSISCPLKRQ ARKRRGRKRR fll SYNVHHPWET GHCLSEGSDS SLEEPSKDYR ENHNNNKKDH SDSDDQMLVA

101 KRRPSSNLNN NVRGKRPLWH ESDFAVDNVG NRTLRRRRKV KRMAVDLPQD

151 ISNKRTMTQP PEGCRDQDMD SDRAYQYQEF TKNKVKKRKL KI1RQGPKIQ

201 DEGVVLESEE TNQTNKDKME CEEQKVSDEL MSESDSSSLS STDAGLFTND

251 EGRQGDDEQS DWFYEKESGG ACGITGWPW WEKEDPTELD KNVPDPVFES

301 ILTG3FPLMS HPS RGFQAR LSRLHGMSSK NIKKSGGTPT SMVPIPGPVG

351 NKRMVHFSPD SHHHDHWFSP GARTEHDQHQ LLRDNRAERG HKKNCSVRTA

101 SRQTSMHLGS LCTGDIKRRR KAAPLPGPTT AGFVGENAQP ILENNIGNRM

451 LQNMGWTPGS GLGRDGKGIS EPIQAMQRPK GLGLGFPLPK STSATTTPNA 01 GKSA

TABLE 16B 114

Claims (1)

1. CLAIMS-

   1. A polynucleotide that encodes an 84P2A9-related protein, wherem the polynucleotide is selected from the group consisting of

   (a) a polynucleotide havmg the sequence as shown in FIG. 2, wherem T can also be U;

   (b) a polynucleotide havmg the sequence as shown m FIG. 2, from nucleotide residue number 165 through nucleotide residue number 1676, wherein T can also be U;

   (c) a polynucleotide encoding an 84P2A9-related protem whose sequence is encoded by the cDNAs contained m the plasmids designated pi 29.1 -US- PI deposited with American Type Culture Collection as Accession No. PTA-1151 ;

   (d) a polynucleotide encoding an 84P2A9-related protem that is at least 90% identical to the ammo acid sequence shown in FIG. 2 over its entire length; and

   (e) a polynucleotide that is fully complementary to a polynucleotide of any one of (a)-(d).

   2. A polynucleotide of claim 1 that encodes the polypeptide sequence shown m FIG. 2.

   3. A fragment of a polynucleotide of claim 1 compnsmg:

   (a) a polynucleotide havmg the sequence as shown in FIG. 2, from nucleotide residue number 720 through nucleotide residue number 1392,

   (b) a polynucleotide that is a fragment of the polynucleotide of (a) that is at least 10 nucleotide bases m length; or (c) a polynucleotide that selectively hybridizes under stringent conditions to the polynucleotide of (a) or (b).

   4 A polynucleotide that encodes an 84P2A9-related protem, wherem the polypeptide mcludes an amino acid sequence selected from the group consisting of KKRK, NQTN, NCSV, TNK, SRR, SSK, SVR, GLFTND, GGACGI,

   GGTPTS, GTPTSM and GSLCTG

   5 A polynucleotide that encodes an 84P2A9-related protem, wherem the polypeptide comprises an HLA class I Al, A2, A3, A24, B7, B27, B58, B62 supermotif, or an HLA class II O'Sullrvan DR supermotif or an Alexander pan DR binding epitope supermotif or an HLA DR3 motif

   6 A polynucleotide of any one of claims 1 -4 that is labeled with a detectable marker

   7. A recombmant expression vector that contains a polynucleotide of any one of claims 1-4.

   8 A host cell that contains an expression vector of claim 7.

   9. A process for producmg an 84P2A9-related protem compnsmg culturmg a host cell of claim 8 under conditions sufficient for the production of the polypeptide

   10 The process of claim 9, further comprismg recovering the 84P2A9-related protein so produced.

   11. An 84P2A9-related protem produced by the process of claim 10.

   12 An isolated 84P2A9-related protem.

   13 The 84P2A9-related protem of claim 12, wherem 84P2A9-related protem has the amino acid sequence shown in SEQ ID NO. 2 An isolated 84P2A9-related protem that has an ammo acid sequence which is exacύy that of an amino acid sequence encoded by a polynucleotide selected from the group consisting of:

   (a) a polynucleotide havmg the sequence as shown m FIG. 2, wherem T can also be U;

   (b) a polynucleotide havmg the sequence as shown m FIG. 2, from nucleotide residue number 165 through nucleotide residue number 1676, wherem T can also be U;

   (c) a polynucleotide encoding an 84P2A9-related protem whose sequence is encoded by the cDNAs contamed in the plasmids designated pl29.1-US-

   Pl deposited with American Type Culture Collection as Accession No. PTA-1151;

   (d) a polynucleotide encodmg an 84P2A9-related protem havmg the ammo acid sequence shown in FIG. 2; and

   (e) a polynucleotide that is fully complementary to a polynucleotide of any one of (a) -(d).

   An isolated 84P2A9-related protem of claim 14 selected from the group consisting of:

   (a) a polynucleotide having the sequence as shown m FIG. 2, from nucleotide residue number 720 through nucleotide residue number 1392;

   (b) a polynucleotide that is a fragment of the polynucleotide of (a) that is at least 10 nucleotide bases m length; or

   (c) a polynucleotide that selectively hybridizes under stringent conditions to the polynucleotide of (a) or (b) 16 An antibody or fragment thereof that immunospecifically bmds to an 84P2A9- related protem.

   17 The antibody or fragment thereof of claim 16, which is monoclonal.

   18. A recombmant protein comprising the antigen bmding region of a monoclonal antibody of claiml 7.

   19. The antibody or fragment thereof of claim 17, which is labeled with a detectable marker.

   20. The recombinant protein of claim 18, which is labeled with a detectable marker

   21 The antibody fragment of claim 16, which is an Fab, F(ab')2, Fv or Sfv fragment

   22. The antibody of claim 16, which is a human antibody.

   23. The recombinant protem of claim 20, which comprises murme antigen bmding region residues and human constant region residues.

   24. A non-human transgenic animal that produces a monoclonal antibody of claim 20.

   25. A hybridoma that produces an antibody of claim 22.

   26. A smgle cham monoclonal antibody that comprises the variable domams of the heavy and light chains of a monoclonal antibody of claim 17.

   27. A vector compnsmg a polynucleotide encoding a single cham monoclonal antibody that immunospecifically bmds to an 84P2A9-related protem.

   28. An assay for detecting the presence of an 84P2A9-related protem or polynucleotide in a biological sample comprismg contacting the sample with an antibody or polynucleotide, respectively, that specifically binds to the 84P2A9- related protem or polynucleotide, respectively, and detecting the binding of 84P2A9-related protem or polynucleotide, respectively, in the sample thereto An assay for detecting the presence of an 84P2A9 related protem or polynucleotide comprising obtaining a sample, evaluating said sample m the presence of an 84P2A related protem or polynucleotide, whereby said evaluating means produces a result that indicates the presence or amount of 84P2A9-related protem or polynucleotide, respectively

   An assay of claim 29 for detecting the presence of an 84P2A9 polynucleotide m a biological sample, comprising

   (a) contacting the sample with a polynucleotide probe that specifically hybridizes to a polynucleotide encoding an 84P2A9-related protem havmg an ammo acid sequence shown in FIG 2, and

   (b) detecting the presence of a hybridization complex formed by the hybridization of the probe with 84P2A9 polynucleotide in the sample, wherein the presence of the hybridization complex indicates the presence of 84P2A9 polynucleotide within the sample

   An assay for detecting the presence of 84P2A9 mRNA m a biological sample compnsmg

   (a) producing cDNA from the sample by reverse transcnption usmg at least one primer,

   (b) amplifying the cDNA so produced usmg 84P2A9 polynucleotides as sense and antisense primers to amphfy 84P2A9 cDNAs therein,

   (c) detecting the presence of the amplified 84P2A9 cDNA,

   wherem the 84P2A9 polynucleotides used as the sense and antisense probes are capable of amplifying the 84P2A9 cDNA contamed within the plasmid as deposited with American Type Culture Collection as Accession No PTA-1151

   A method of claim 31 for monitoring 84P2A9 gene products compnsmg determinmg the status of 84P2A9 gene products expressed by cells in a test tissue sample from an mdividual;

   comparing the status so determined to the status of 84P2A9 gene products m a corresponding normal sample; and

   identifymg the presence of aberrant 84P2A9 gene products in the test sample relative to the normal sample.

   33. The method of claim 32, wherem the 84P2A9 gene products are monitored by comparmg the polynucleotide sequences of 84P2A9 gene products in the test tissue sample with the polynucleotide sequences of 84P2A9 gene products in a corresponding normal sample.

   34. The method of claim 32, wherem the 84P2A9 gene products are monitored by comparmg the levels 84P2A9 gene products m the test tissue sample with the levels of 84P2A9 gene products m the corresponding normal sample.

   35 A method of diagnosing the presence of cancer in an mdividual comprising:

   (a) determining the level of 84P2A9 mRNA or protem expressed m a test sample obtained from the individual; and

   (b) comparmg the level so determined to the level of 84P2A9 mRNA or protem expressed m a comparable known normal tissue sample,

   whereby the presence of elevated 84P2A9 mRNA or protem expression m the test sample relative to the normal tissue sample provides an indication of the presence of cancer.

   36. The method of claim 35, wherem the cancer is selected from the group consisting of leukemia and cancer of the prostate, testis, kidney, brain, bone, skin, ovary, breast, pancreas, colon, and lung, and the test and normal tissue samples are selected from the group consisting of serum, blood or urine and tissues of the prostate, testis, kidney, bram, bone, skm, ovary, breast, pancreas, colon, and lung. Use of an 84P2A9-related protem or an immunogenic portion thereof, a vector comprismg a polynucleotide encoding a smgle cham monoclonal antibody that immunospecifically binds to an 84P2A9-related protem, an antisense polynucleotide complementary to a polynucleotide havmg 84P2A9 coding sequences, or a ribozyme capable of cleavmg a polynucleotide havmg 84P2A9 codmg sequences, for the preparation of a composition for treating a patient with a cancer that expresses 84P2A9

   The use of claim 37, wherem the cancer is selected from the group consisting of leukemia and cancer of the prostate, testis, kidney, bram, bone, skin, ovary, breast, pancreas, colon, lymphocytic and lung

   A pharmaceutical composition compnsmg an 84P2A9-related protem, an antibody or fragment thereof that specifically binds to an 84P2A9-related protem, a vector comprising a polynucleotide encoding a smgle cham monoclonal antibody that immunospecifically bmds to an 84P2A9-related protem, a polynucleotide comprising an 84P2A9-related protem coding sequence, an antisense polynucleotide complementary to a polynucleotide havmg an 84P2A9 coding sequences or a nbozyme capable of cleavmg a polynucleotide havmg 84P2A9 coding sequences and, optionally, a physiologically acceptable carrier

   A method of treating a patient with a cancer that expresses 84P2A9 which comprises administering to said patient a vector comprismg a polynucleotide encoding a smgle cham monoclonal antibody that immunospecifically bmds to an 84P2A9-related protein, such that the vector dekvers the smgle cham monoclonal antibody codmg sequence to the cancer cells and the encoded single cham antibody is expressed mtracellularly therein

   A vaccme composition for the treatment of a cancer expressmg 84P2A9 comprising an immunogenic portion of an 84P2A9 -related protem and a physiologically acceptable carrier 42 A method of mhibiting the development of a cancer expressing 84P2A9 in a patient, comprising administering to the patient an effective amount of the vaccme composition of claim 41.

   43 A method of generating an immune response m a mammal comprismg exposmg the mammal's immune system to an immunogenic portion of an 84P2A9-related prote of claim 41, so that an immune response is generated to 84P2A9.

   44 A method of dehvermg a cytotoxic agent to a cell expressmg 84P2A9 comprismg conjugating the cytotoxic agent to an antibody or fragment thereof of claim 16 that specifically bmds to an 84P2A9 epitope and exposmg the cell to the antibody-agent conjugate

   45 A method of mducmg an immune response to an 84P2A9 protem, said method comprismg:

   providing an 84P2A9-realted protein epitope;

   contacting the epitope with an immune system T cell or B cell, whereby the immune system T cell or B cell is mduced

   46. The method of claim 45, wherem the immune system cell is a B cell, whereby the mduced B cell generates antibodies that specifically bmd to the 84P2A9-related protem.

   47. The method of claim 45, wherem the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the 84P2A9 protein.

   48 The method of claim 45, wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a CTL or the antibody producmg activity of a B cell.