Prostate-specific polypeptide pamp and encoding nucleic acid molecules Number:7,393,921 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to novel prostate specific nucleic acid molecules and polypeptides and related methods for diagnosing or predicting susceptibility to a prostate neoplastic condition.<H polypeptide, molecules, Prostate, specif, 7393921.html, Patent, pto, 7393921

Title: Prostate-specific polypeptide pamp and encoding nucleic acid molecules

Abstract: The present invention relates to novel prostate specific nucleic acid molecules and polypeptides and related methods for diagnosing or predicting susceptibility to a prostate neoplastic condition.

Patent Number: 7,393,921 Issued on 07/01/2008 to Lin

Inventors: Lin; Biaoyang (Bothell, WA)
Assignee: Institute for Systems Biology (Seattle, WA)

Appl. No.: 09/729,653

Filed: December 4, 2000

Current U.S. Class: 530/350 ; 424/185.1; 424/277.1; 530/300; 530/828

Current International Class: C07K 14/00 (20060101)

Field of Search: 530/350,300,828 435/387.1 424/185.1,277.1

References Cited [Referenced By]

U.S. Patent Documents


5861248	January 1999	Russell et al.

Foreign Patent Documents


WO 00/65067	Nov., 2000	WO

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Primary Examiner: Helms; Larry R.
Assistant Examiner: Davis; Minh-Tam
Attorney, Agent or Firm: McDermott Will & Emery LLP

Claims

What is claimed is:

1. An isolated PAMP polypeptide comprising the amino acid sequence shown as SEQ ID NO:2.

2. An isolated PAMP polypeptide consisting of the amino acid sequence shown as SEQ ID NO:2.

Description

BACKGROUND OF THE INVENTION

This invention relates generally to cancer and, more specifically, to a prostate-specific gene that can be used to diagnose and treat prostate cancer, including advanced or metastatic prostate cancer.

Cancer is currently the second leading cause of mortality in the United States. However, it is estimated that by the year 2000 cancer will surpass heart disease and become the leading cause of death in the United States. Prostate cancer is the most common non-cutaneous cancer in the United States and the second leading cause of male cancer mortality.

Cancerous tumors result when a cell escapes from its normal growth regulatory mechanisms and proliferates in an uncontrolled fashion. As a result of such uncontrolled proliferation, cancerous tumors usually invade neighboring tissues and spread by lymph or blood stream to create secondary or metastatic growths in other tissues. If untreated, cancerous tumors follow a fatal course. Prostate cancer, due to its slow growth profile, is an excellent candidate for early detection and therapeutic intervention.

During the last decade, most advances in prostate cancer research have focused on prostate specific antigen (PSA), a member of the serine protease family that exhibits a prostate-specific expression profile. Serum PSA remains the most widely used tumor marker for monitoring prostate cancer, but its specificity is limited by a high frequency of falsely elevated values in men with benign prostatic hyperplasia (BPH). Other biomarkers of prostate cancer progression have proven to be of limited clinical use in recent surveys because they are not uniformly elevated in men with advanced prostate cancer. Due to the limitations of currently available biomarkers, the identification and characterization of prostate specific genes is essential to the development of more accurate diagnostic methods and therapeutic targets. In many cases, the clinical potential of novel tumor markers can be optimized by utilizing them in combination with other tumor markers in the development of diagnostic and treatment modalities.

Normal prostate tissue consists of three distinct non-stromal cell populations, luminal secretory cells, basal cells, and endocrine paracrine cells. Phenotypic similarities between normal luminal cells and prostate cancer cells, including the expression of PSA, have suggested that prostate adenocarcinomas derive from luminal cells. However, a number of recent studies suggest that at least some prostate cancers can arise from the transformation of basal cells and report the expression of various genes in normal prostate basal cells as well as in prostate carcinoma cells. These genes include prostate stem cell antigen (PSCA), c-met and Bcl-2. Because none of these genes is universally expressed in all basal cells and prostate carcinomas, the utility of these genes as diagnostic markers is limited. Likewise, because PSA is expressed in luminal secretory cells in normal prostate tissue, this antigen has limited utility as a marker for basal cell derived carcinomas.

Thus, there exists a need for the identification of additional prostate specific genes that can be used as diagnostic markers and therapeutic targets for prostate cancer. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a PAMP nucleic acid molecule containing a nucleic acid sequence encoding substantially a PAMP polypeptide. A PAMP nucleic acid molecule of the invention encodes substantially the amino acid sequence shown as SEQ ID NO:2. A PAMP nucleic acid molecule can encode, for example, the amino acid sequence shown as SEQ ID NO:2 and, in one embodiment, contains the nucleotide sequence shown as SEQ ID NO:1.

Further provided by the invention is a substantially pure PAMP nucleic acid probe which contains substantially the nucleotide sequence of nucleotides 1 to 3221 of SEQ ID NO:1, or a fragment thereof, provided that the probe does not have the nucleotide sequence of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780.

The invention also provides a substantially pure PAMP nucleic acid probe which contains at least 10 contiguous nucleotides of SEQ ID NO:1, where the contiguous nucleotides include at least one nucleotide of the nucleotide sequence shown as position 1 to position 3221 of SEQ ID NO:1, provided that the probe does not have the nucleotide sequence of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780. Such a PAMP nucleic acid probe can contain, for example, at least 15 contiguous nucleotides of SEQ ID NO:1, and can be, for example, 15 to 18 nucleotides in length. If desired, a substantially pure PAMP nucleic acid probe of the invention can further include a detectable label.

The invention also provides a substantially pure PAMP polypeptide which contains substantially the amino acid sequence shown as SEQ ID NO:2. In one embodiment, a substantially pure PAMP polypeptide of the invention has the amino acid sequence SEQ ID NO:2.

In addition, the invention provides a substantially pure PAMP polypeptide fragment, which includes at least eight contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2. Such a PAMP polypeptide fragment can include, for example, at least ten contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2.

The present invention provides a method of diagnosing or predicting susceptibility to a prostate neoplastic condition in an individual. The method is practiced by obtaining a sample from the individual; measuring a test expression level of PAMP RNA by hybridization with a PAMP nucleic acid probe comprising at least 10 contiguous nucleotides of SEQ ID NO:1, the contiguous nucleotides including at least one nucleotide of the nucleotide sequence shown as position 1 to position 3221 of SEQ ID NO:1 in the sample; and comparing the test expression level of PAMP RNA to a control expression level of PAMP RNA, where a test expression level 2-fold or more greater than the control expression level indicates the presence of a prostate neoplastic condition. In a method of the invention, the sample can contain a prostate cell or a prostate tissue, and the control expression level can be determined using a normal prostate cell or an androgen-dependent cell line. The sample can be, for example, a fluid such as blood, serum, urine or semen. In one embodiment, the PAMP nucleic acid probe contains at least 10 contiguous nucleotides of SEQ ID NO:1, the contiguous nucleotides including at least one nucleotide of the nucleotide sequence shown as position 1 to position 3221 of SEQ ID NO:1, provided the probe does not have the nucleotide sequence of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780. A PAMP nucleic acid probe useful in a method of the invention can be, for example, 15 to 18 nucleotides in length and can contain, if desired, a detectable label.

The invention also provides a method of diagnosing or predicting susceptibility to a prostate neoplastic condition in an individual by obtaining a sample from the individual; measuring a test expression level of PAMP polypeptide by contacting a cell, a cell lysate, or fractionated sample thereof, from the individual with a binding agent selective for PAMP polypeptide residues 1 to 1074 of SEQ ID NO:2, and determining the amount of selective binding of the agent; and comparing the test expression level of PAMP polypeptide to a control expression level of PAMP polypeptide, where a test expression level 2-fold or more greater than the control expression level indicates the presence of a prostate neoplastic condition. In a method of the invention, the binding agent selective for the PAMP polypeptide residues 1 to 1074 of SEQ ID NO:2 can include, for example, an antibody, and can further include, if desired, a detectable label.

Further provided by the invention is a method of diagnosing metastatic prostate cancer in an individual by obtaining a sample from the individual, wherein the sample is not a prostate sample; measuring a test expression level of PAMP RNA by hybridization with a PAMP nucleic acid probe comprising at least 10 contiguous nucleotides of SEQ ID NO:1, the contiguous nucleotides including at least one nucleotide of the nucleotide sequence shown as position 1 to position 3221 of SEQ ID NO:1 in the sample; and comparing the test expression level of PAMP RNA to a control expression level of PAMP RNA, where a significant test expression level as compared to the control expression level indicates the presence of metastatic prostate cancer.

In addition, the invention provides a method of diagnosing metastatic prostate cancer in an individual by obtaining a sample from the individual, where the sample is not a prostate sample; measuring a test expression level of PAMP polypeptide by contacting a cell, a cell lysate, or fractionated sample thereof, from the individual with a binding agent selective for PAMP polypeptide residues 1 to 1074 of SEQ ID NO:2, and determining the amount of selective binding of the agent; and comparing the test expression level of PAMP polypeptide to a control expression level of PAMP polypeptide, where a significant test expression level as compared to the control expression level indicates the presence of metastatic prostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the full-length nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequence of PAMP. Predicted transmembrane domains are underlined.

FIG. 2 shows northern analysis of PAMP expression in androgen-stimulated cells. Left panel: "+" indicates androgen-stimulated RNA; "-" indicates androgen-starved RNA. Right panel: A time course northern blot showing PAMP expression at 4, 8, 12, 16, 24, 36, 48 hours after androgen stimulation.

FIG. 3 shows analysis of PAMP expression using two multiple tissue northern blots (MTN) from ClonTech (Palo Alto, Calif.).

FIG. 4 shows hybridization of PAMP to a multiple tissue expression (MTE) array (ClonTech) containing 50 human tissues. The RNAs are as follows: A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3; substantia nigra; B4, temporal lobe; B5, thalamus; B6, acumens; B7, spinal cord; C1, heart; C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; D1, testis; D2, ovary; D3, pancreas; D4, pituitary gland; D5, adrenal gland; D6, thyroid gland; D7, salivary gland; D8, mammary gland; E1, kidney; E2, liver; E3, small intestine; E4, spleen; E5, thymus; E6, peripheral leukocyte; E7, lymph node; E8, bone marrow; F1, appendix; F2, lung; F3, trachea; F4, placenta; G1, fetal brain; G2, fetal heart; G3, fetal kidney; G4, fetal liver; G5, fetal spleen; G6, fetal thymus; G7, fetal lung; H1, yeast total RNA; H2, yeast tRNA; H3, E. Coli rRNA; H4, E. Coli DNA; H5, poly r(A); H6, human C.sub.0t 1 DNA; H7, human DNA; H8, human DNA; B8, F5-F8, G8 contain no RNAs.

FIG. 5 shows RNA in situ hybridization with PAMP sense and antisense probes. A: Anti-sense probe of PAMP hybridized to a section of prostate cancer tissue sample. B: Sense probe of PAMP hybridized to prostate cancer section. C: Anti-sense probe of PAMP hybridized to normal prostate gland section. D: Sense probe of PAMP hybridized to normal prostate gland section.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to the discovery of the full-length coding sequence for PAMP, a gene with several transcripts expressed specifically in the prostate. The prostate-specific nucleic acid sequence and encoded gene product are useful as both diagnostic markers for neoplastic conditions of the prostate and as targets for therapy.

As disclosed herein in Example I, the PAMP cDNA contains 4485 nucleotides and is predicted to encode a protein of 1382 amino acids with at least 4 transmembrane domains (see FIG. 1). As further disclosed herein, PAMP expression is induced by androgen in the prostate carcinoma cell line LNCaP. Expression of the PAMP transcript was induced by 4 hours and maintained at least to 48 hours following androgen treatment (see FIG. 2). As further disclosed herein, expression of the 2.0 and 3.2 kb PAMP transcripts was specific to prostate among 16 adult human tissues assayed by northern analysis (FIG. 3), while the 5.0 and 6.5 kb forms were expressed in prostate, ovary and testis. Furthermore, among 50 human fetal and adult tissues assayed, significant expression was only detected in the prostate (see FIG. 4). As further disclosed herein in Example III, RNA in situ analysis demonstrated that PAMP was expressed in epithelial cells in normal prostate and prostate cancer cells. These results demonstrate that PAMP is an androgen-regulated prostate-specific gene product.

Based on these results, the invention provides methods for diagnosing prostate neoplastic conditions. As discussed above, a PAMP gene of the invention is primarily expressed in prostate cells and becomes elevated in response to androgens. As such, a PAMP nucleic acid molecule or polypeptide of the invention can be used alone or in combination with other molecules as a specific marker for prostate cells and prostate neoplastic conditions.

PAMP Nucleic Acid Molecules

The present invention provides a PAMP nucleic acid molecule containing a nucleic acid sequence encoding substantially the amino acid sequence shown as SEQ ID NO:2. A PAMP nucleic acid molecule can encode, for example, the amino acid sequence shown as SEQ ID NO:2 and, in one embodiment, contains the nucleotide sequence shown as SEQ ID NO:1.

The nucleic acid sequence of the PAMP cDNA (SEQ ID NO:1) and the deduced amino acid sequence (SEQ ID NO: 2) were determined as disclosed in Example I. As shown in FIG. 1, the PAMP cDNA contains 4485 nucleotides and is predicted to encode a protein of 1382 amino acids.

The nucleic acid molecules of the invention and short oligonucleotide probes corresponding to unique sequences are useful in a variety of diagnostic procedures which employ probe hybridization methods. One advantage of employing nucleic acid hybridization in diagnostic procedures is that very small amounts of sample can be used because the analyte nucleic acid molecule can be amplified to many copies by, for example, polymerase chain reaction (PCR) or other well known methods for nucleic acid molecule amplification and synthesis.

As used herein, the term "nucleic acid molecule" is intended to mean a single- or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. The term is intended to include nucleic acid molecules of both synthetic and natural origin. A nucleic acid molecule of natural origin can be derived from any animal, such as a human, non-human primate, mouse, rat, rabbit, bovine, porcine, ovine, canine, feline, or amphibian, or from a lower eukaryote. A nucleic acid molecule of the invention can be of linear, circular or branched configuration, and can represent either the sense or antisense strand, or both, of a native nucleic acid molecule. A nucleic acid molecule of the invention can further incorporate a detectable moiety such as a radiolabel, a fluorochrome, a ferromagnetic substance, a luminescent tag or a detectable binding agent such as biotin.

As used herein, the term "substantially pure nucleic acid molecule" is intended to mean a nucleic acid molecule that is substantially free from cellular components or other contaminants that are not the desired molecule. A substantially pure nucleic acid molecule can also be sufficiently homogeneous so as to resolve as a band by gel electrophoresis, and generate a nucleotide sequence profile consistent with a predominant species.

Nucleic Acid Probes

A nucleic acid probe of the invention can contain substantially the nucleotide sequence of a portion of nucleotides 1 to 3221 of SEQ ID NO:1. The term "probe," as used herein in reference to a substantially pure nucleic acid molecule of the invention, is intended to refer to a portion of the nucleic acid molecule having the ability to selectively hybridize with the parent nucleic acid molecule. The term "selectively hybridize" refers to an ability to bind the parent nucleic acid molecule without substantial cross-reactivity with a molecule that is not the parent nucleic acid molecule. Therefore, the term includes specific hybridization where there is little or no detectable cross-reactivity with other nucleic acid molecules. The term also includes minor cross-reactivity with other molecules provided hybridization to the subject nucleic acid molecule is distinguishable from hybridization to the cross-reactive species. Thus, a probe of the invention can be used, for example, as a PCR primer to selectively amplify a nucleic acid molecule of the invention; as a selective primer for 5' or 3' RACE to determine additional 5' or 3' sequence of a nucleic acid molecule of the invention; as a selective probe to identify or isolate a nucleic acid molecule of the invention on a RNA or DNA blot, or genomic or cDNA library; or as a selective inhibitor of transcription or translation of PAMP in a tissue, cell or cell extract. In one embodiment, the following sequences are excluded as nucleic acid probes of the invention: one or any combination of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780. In another embodiment, one or any combination of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201, nucleotides 1 to 1530 of AK026780, or nucleotides 1531 to 2000 of AK026780 is excluded from a probe of the invention. In a further embodiment, one or any combination of AA363808, AW959484, BE165930, BE893201, or AK026780, or a subsequence thereof containing at least ten contiguous nucleotides of any of these five sequences, is excluded from a probe of the invention.

Thus, the invention provides a substantially pure PAMP nucleic acid probe which contains substantially the nucleotide sequence of nucleotides 1 to 3221 of SEQ ID NO:1, or a fragment thereof, provided that the probe does not have the nucleotide sequence of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780.

In one embodiment, the invention provides a substantially pure PAMP nucleic acid probe which contains at least 10 contiguous nucleotides of SEQ ID NO:1, where the contiguous nucleotides include at least one nucleotide of the nucleotide sequence shown as position 1 to position 3221 of SEQ ID NO:1, provided that the probe does not have the nucleotide sequence of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780. Such a PAMP nucleic acid probe can contain, for example, at least 15 contiguous nucleotides of SEQ ID NO:1, and can be, for example, 15 to 18 nucleotides in length. If desired, a substantially pure PAMP nucleic acid probe of the invention can further include a detectable label.

As used herein, the term "probe" refers to a portion of a subject nucleic acid molecule having at least 10 nucleotides.

A probe of the invention includes at least 10 contiguous nucleotides corresponding to the reference nucleic acid molecule, and can include at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or at least 25 nucleotides and, if desired, can include at least 30, 40, 50, 100, 300 or 500 nucleotides, and can include up to the full length of the reference nucleic acid molecule minus one nucleotide. Probes of such lengths are able to selectively hybridize with the subject nucleic acid molecule in a variety of detection formats described herein.

As used herein, the term "substantially the nucleotide sequence" in reference to a nucleic acid molecule or nucleic acid probe of the invention includes sequences having one or more additions, deletions or substitutions with respect to the reference sequence, so long as the nucleic acid molecule retains its ability to selectively hybridize with the subject nucleic acid molecule.

Nucleic acid molecules and probes of the invention are useful as hybridization probes in diagnostic procedures. The probes can be as long as the full length transcript or as short as about 10-15 nucleotides, and preferably about 15-18 nucleotides. A probe of the invention can correspond to coding region or untranslated region sequence. The particular application and degree of desired specificity will be one consideration well known to those skilled in the art in selecting a probe. For example, if it is desired to detect PAMP and other related species, the probe can correspond to a coding sequence and be used in low stringency hybridization conditions. Alternatively, using high stringency conditions with a probe of the invention will select a PAMP nucleic acid molecule having substantially the nucleotide sequence shown as SEQ ID NO: 1. Untranslated region sequences corresponding to a PAMP transcript can also be used to construct probes since there is little evolutionary pressure to conserve non-coding domains. Probes as small as 15 nucleotides are statistically unique sequences within the human genome. Therefore, fragments of the PAMP sequences of 15 nucleotides or more in length can be constructed from essentially any region of a PAMP cDNA, mRNA or promoter/regulatory region and be capable of uniquely hybridizing to PAMP DNA or RNA.

Nucleic acid probes can be produced recombinantly or chemically synthesized using methods well known in the art. Additionally, PAMP hybridization probes can be labeled with a variety of detectable labels including, for example, radioisotopes, fluorescent tags, reporter enzymes, biotin and other ligands. Such detectable labels can additionally be coupled with, for example, colorimetric or photometric indicator substrate for spectrophotometric detection. Methods for labeling and detecting such probes are well known in the art and can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

The nucleic acid probes of the invention can be hybridized under various stringency conditions readily determined by one skilled in the art. Depending on the particular assay, one skilled in the art can readily vary the stringency conditions to optimize detection of a PAMP nucleic acid molecule.

In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. The hybridized nucleic acid molecules generally have at least 60% identity, at least 75% identity, at least 85% identity; or at least 90% identity. Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree. C., followed by washing in 0.2.times.SSPE, 0.2% SDS, at 42.degree. C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree. C., followed by washing in 0.1.times.SSPE, and 0.1% SDS at 65.degree. C.

The phrase "low stringency hybridization" refers to conditions equivalent to hybridization in 10% formamide, 5.times. Denhart's solution, 6.times.SSPE, 0.2% SDS at 22.degree. C., followed by washing in 1.times.SSPE, 0.2% SDS, at 37.degree. C. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20.times.SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al., supra, 1999). Nucleic acid molecules encoding polypeptides hybridize under moderately stringent or high stringency conditions to substantially the entire sequence, or substantial portions, for example, typically at least 15-30 nucleotides of the nucleic acid sequence set forth in SEQ ID NO:1.

The invention also provides a modification of a PAMP nucleotide sequence that hybridizes to a PAMP nucleic acid molecule, for example, a nucleic acid molecule referenced as SEQ ID NO:1, under moderately stringent conditions. Modifications of PAMP nucleotide sequences, where the modification has at least 60% identity to a PAMP nucleotide sequence, are also provided. The invention also provides modification of a PAMP nucleotide sequence having at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity.

Identity of any two nucleic acid sequences can be determined by those skilled in the art based, for example, on a BLAST 2.0 computer alignment, using default parameters. BLAST 2.0 searching is available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html., as described by Tatiana et al., FEMS Microbiol Lett. 174:247-250 (1999); Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997).

PAMP Polypeptides

The invention also provides a substantially pure PAMP polypeptide which contains substantially the amino acid sequence shown as SEQ ID NO:2. In one embodiment, a substantially pure PAMP polypeptide of the invention has the amino acid sequence SEQ ID NO:2.

In addition, the invention provides a substantially pure PAMP polypeptide fragment, which includes at least eight contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2. Such a PAMP polypeptide fragment can include, for example, at least ten contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2.

In one embodiment, the invention provides a PAMP polypeptide fragment that includes at least eight contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2, provided that the fragment does not contain eight or more contiguous amino acids of AK026780. In a further embodiment, the invention provides a PAMP polypeptide fragment that includes at least eight contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2, provided that the fragment does not contain eight or more contiguous amino acids of AK026780, or eight or more contiguous amino acids encoded by any of the six reading frames of AA363808, AW959484, BE165930, BE893201 or AK026780.

Polypeptide fragments of the invention include peptides that can function as antigenic determinants to generate antibodies that are selective for a PAMP polypeptide encoded by substantially residues 1 to 1074 of SEQ ID NO:2.

Exemplary polypeptide fragments include those fragments having amino acids 1 to 8, 2 to 9, 3 to 10, etc. Other polypeptide fragments of residues 1 to 1074 shown in FIG. 1 are also included as peptides that are potential antigenic fragments capable of eliciting an immune response to generate antibodies selective for PAMP polypeptide residues 1 to 1074. It is understood that, while eight residues is the minimum length of a polypeptide fragment of the invention, a fragment can be longer and can include 9, 10, 11, 12, 13, 14, 15, 18, 20, 25, 30, 35, 40, 45 or more contiguous amino acids of residues 1 to 1074 of the PAMP polypeptide shown as SEQ ID NO:2.

The nucleic acid molecules and polypeptides of the invention encode a PAMP polypeptide. The term "PAMP polypeptide" as used herein, means a polypeptide that is structurally similar to human PAMP and that at least one biological activity of PAMP. Such a PAMP polypeptide has 50% or more sequence identity to SEQ ID NO:2, and can have 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to human PAMP (SEQ ID NO:2). Percent amino acid identity can be determined using Clustal W version 1.7 (Thompson et al., Nucleic Acids Res. 22:4673-4680 (1994))).

The present invention is directed to the full-length PAMP polypeptide sequence or large fragments of this full-length sequence. Thus, the term "PAMP polypeptide," as used herein, refers to a polypeptide corresponding to at least 350 of the 1382 residues of human PAMP. In view of the above, it is understood that a fragment containing, for example, residues 1075 to 1382 of SEQ ID NO:2 is not a "PAMP polypeptide" as defined herein.

Thus, it is clear to the skilled person that the term "PAMP polypeptide" encompasses polypeptides with one or more naturally occurring or non-naturally occurring amino acid substitutions, deletions or insertions as compared to SEQ ID NO: 2, provided that the peptide has at least 50% amino acid identity with SEQ ID NO: 2 and corresponds to at least 350 residues of full-length PAMP. A PAMP polypeptide can be, for example, a naturally occurring variant of human PAMP (SEQ ID NO: 2), a species homolog including mammalian and non-mammalian homologs and murine, bovine, and primate homologs, a PAMP mutated by recombinant techniques, and the like. In view of the above, it is clear to the skilled person that the Drosophila polypeptide encoded by AAF57545.1 (CG11237) and the C. elegans polypeptide encoded by ZK520 (T27880), which each share 44% amino acid identity with human PAMP (SEQ ID NO:2) are not encompassed by the invention.

Modifications to SEQ ID NO: 2 that are encompassed within the invention include, for example, an addition, deletion, or substitution of one or more conservative or non-conservative amino acid residues; substitution of a compound that mimics amino acid structure or function; or addition of chemical moieties such as amino or acetyl groups.

In one embodiment, the invention provides a PAMP polypeptide having an amino acid sequence corresponding to at least 350 of the 1382 residues of human PAMP, provided that the polypeptide does not contain the sequence of AK026780. In another embodiment, the invention provides a PAMP polypeptides having an amino acid sequence corresponding to at least 350 of the 1382 residues of human PAMP, provided that the polypeptide does not contain the sequence of AK026780 or the amino acid sequence encoded by any of the six reading frames of AA363808, AW959484, BE165930, BE893201 or AK026780.

The invention also provides antibodies that specifically bind a PAMP polypeptide. In one embodiment, the invention provides an antibody selective for PAMP polypeptide residues 1 to 1074, or a PAMP polypeptide fragment containing at least eight contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2.

As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. With regard to an antibody of the invention which is selective for PAMP polypeptide residues 1 to 1074 or a polypeptide fragment thereof, the term "antigen" means a native or synthesized fragment of PAMP residues 1 to 1074. Such an antibody of the invention, or antigen binding fragment of such an antibody, is characterized by having specific binding activity for PAMP polypeptide residues 1 to 1074, or a PAMP polypeptide fragment, of at least about 1.times.10.sup.5 M.sup.-1. Thus, Fab, F(ab').sub.2, Fd and Fv fragments of an anti-PAMP antibody, which retain specific binding activity for PAMP polypeptide residues 1 to 1074, or a polypeptide fragment thereof, are included within the definition of an antibody. Specific binding activity can be readily determined by one skilled in the art, for example, by comparing the binding activity of the antibody to PAMP polypeptide residues 1 to 1074 or a polypeptide fragment thereof versus a control polypeptide that does not include PAMP polypeptide residues 1 to 1074. Methods of preparing polyclonal or monoclonal antibodies are well known to those skilled in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)).

In addition, the term "antibody" as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al. (Science 246:1275-1281 (1989)). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995)).

An antibody of the invention can be prepared using as an immunogen a PAMP polypeptide, which can be prepared from natural sources or produced recombinantly, or a PAMP polypeptide fragment of the invention, which contains at least 8 contiguous amino acids of residues 1 to 1074 of SEQ ID NO:2. Such polypeptide fragments are functional antigenic fragments if the antigenic peptides can be used to generate an antibody selective for PAMP polypeptide residues 1 to 1074 of SEQ ID NO:2. As is well known in the art, a non-immunogenic or weakly immunogenic PAMP polypeptide or polypeptide fragment can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (see, for example, Harlow and Lane, supra, 1988). An immunogenic PAMP polypeptide fragment can also be generated by expressing the peptide portion as a fusion protein, for example, to glutathione S transferase (GST), polyHis or the like. Methods for expressing peptide fusions are well known to those skilled in the art (Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999)).

Diagnostic Methods

Methods of diagnosing or predicting susceptibility to a prostate neoplastic condition in an individual further are provided by the invention. The methods of the invention are practiced by obtaining a sample from an individual; measuring a test expression level of PAMP in the sample; and comparing the test expression level of PAMP to a control expression level of PAMP, where a test expression level 2-fold or more greater than the control expression level indicates the presence of a prostate neoplastic condition. In a method of the invention, the sample can contain, for example, a prostate cell or prostate tissue and, in one embodiment, is a fluid such as blood, serum, urine or semen. The control expression level can be determined, for example, using a normal prostate cell or an androgen-dependent cell line.

In a diagnostic method of the invention, a test expression level can be determined, for example, by measuring the amount of PAMP RNA and, in one embodiment, the amount of PAMP RNA is determined by hybridization with a PAMP nucleic acid probe containing at least 10 contiguous nucleotides of SEQ ID NO:1 and also including at least one nucleotide of the nucleotide sequence shown as position 1 to position 3221 of SEQ ID NO:1, provided that the probe does not have the nucleotide sequence of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780. A nucleic acid probe useful in a method of the invention can be, for example, 15 to 18 nucleotides in length, and, if desired, can further include a detectable label.

In a diagnostic method of the invention, a test expression level also can be determined, for example, by measuring the amount of PAMP polypeptide. In one embodiment, a diagnostic method of the invention is practiced by determining an amount of PAMP polypeptide by contacting a cell, a cell lysate, or fractionated sample thereof from the individual to be diagnosed with a binding agent selective for PAMP polypeptide residues 1 to 1074 of SEQ ID NO:2, and determining the amount of selective binding of the agent. A binding agent selective for PAMP polypeptide residues 1 to 1074 of SEQ ID NO:2 can be, for example, an antibody, and, if desired, can further include a detectable label.

As disclosed herein in FIG. 4, PAMP was most highly expressed in prostate among 50 human tissues analyzed. These results indicate that PAMP expression outside of the prostate can be indicative of advanced prostate cancer, in which cancerous prostate cells have metastasized. Thus, the invention also provides a method of diagnosing metastatic prostate cancer in an individual by obtaining a sample from the individual, where the sample is not a prostate sample; measuring a test expression level of PAMP in the sample; and comparing the test expression level of PAMP to a control expression level of PAMP, where a significant test expression level as compared to the control expression level indicates the presence of metastatic prostate cancer.

As described herein, the term "prostate neoplastic condition" is intended to refer to a benign or malignant and metastatic prostate lesion of proliferating cells. For example, primary prostate tumors are classified into stages TX, T0, T1, T2, T3, and T4. Metastatic prostate cancer is classified into stages D1, D2, and D3. The term is also intended to include prostate neoplasm.

As used herein, the term "sample" is intended to mean any biological fluid, cell, tissue, organ or portion thereof, that includes or potentially includes nucleic acid molecules and polypeptides of the invention. The term includes samples present in an individual as well as samples obtained or derived from the individual. For example, a sample can be a histologic section of a specimen obtained by biopsy, or cells that are placed in or adapted to tissue culture. A sample further can be a subcellular fraction or extract, or a crude or substantially pure nucleic acid molecule or protein preparation. A sample can be prepared by methods known in the art suitable for the particular format of the detection method.

As used herein, the term "detectable label" refers to a molecule that renders a nucleic acid molecule of the invention detectable by an analytical method. An appropriate detectable label depends on the particular assay format; such labels are well known by those skilled in the art. For example, a detectable label selective for a nucleic acid molecule can be a complementary nucleic acid molecule, such as a hybridization probe, that selectively hybridizes to the nucleic acid molecule. A hybridization probe can be labeled with a measurable moiety, such as a radioisotope, fluorochrome, chemiluminescent marker, biotin, or other moiety known in the art that is measurable by analytical methods. A detectable label also can be a nucleic acid molecule without a measurable moiety. For example, PCR or RT-PCR primers can be used without conjugation to selectively amplify all or a desired portion of the nucleic acid molecule. The amplified nucleic acid molecules can then be detected by methods known in the art.

As used herein, the term "binding agent" when used in reference to a PAMP polypeptide, is intended to mean a compound, including a simple or complex organic molecule, a metal containing compound, carbohydrate, peptide, protein, peptidomimetic, glycoprotein, lipoprotein, lipid, nucleic acid molecule, antibody, or the like that selectively binds a PAMP polypeptide or PAMP polypeptide fragment, or to a PAMP gene regulatory sequence such as a promoter or enhancer element. For example, a binding agent can be a polypeptide that selectively binds with high affinity or avidity to a PAMP polypeptide, without substantial cross-reactivity with other polypeptides that are unrelated to a PAMP polypeptide. The affinity of a binding agent that selectively binds a PAMP polypeptide will generally be greater than about 10.sup.5 M.sup.-1 and more preferably greater than about 10.sup.6 M.sup.-1. High affinity interactions can be preferred, and will generally be greater than about 10.sup.8 M.sup.-1 to 10.sup.9 M.sup.-1. Specific examples of such selective binding agents include a polyclonal or monoclonal antibody selective for a PAMP polypeptide or peptide fragment thereof, nucleic acid molecule, nucleic acid analog, or small organic molecule, identified, for example, by affinity screening of a library. For certain applications, a binding agent can be utilized that preferentially recognizes a particular conformational or post-translationally modified state of a PAMP polypeptide. The binding agent can be labeled with a detectable moiety, if desired, or rendered detectable by specific binding to a detectable secondary binding agent.

As used herein, the term "test expression level" is used in reference to a PAMP mRNA or polypeptide expression and refers to the extent, amount or rate of synthesis of the nucleic acid sequence shown as SEQ ID NO: 1 or the PAMP polypeptide shown as SEQ ID NO: 2. The amount or rate of synthesis can be determined by measuring the accumulation or synthesis of PAMP RNA, PAMP polypeptide or by measuring an activity associated with a PAMP polypeptide.

In methods of the invention, the sample can be, for example, a prostate cell or prostate tissue such as a tissue biopsy. A sample can also be a fluid sample, for example, blood, serum, urine or semen. A normal sample can be, for example, a normal prostate cell or an androgen-dependent cell line.

In methods of the invention, a test expression level can be determined by measuring the amount of PAMP RNA, for example, by hybridization with a nucleic acid probe comprising substantially the nucleotide sequence of SEQ ID NO:1, or a fragment thereof. The probe can be, for example, an oligonucleotide of 15 to 18 nucleotides in length and, if desired, can contain a detectable label.

In one embodiment, a test expression level is determined by measuring the amount of PAMP RNA by hybridization with a PAMP nucleic acid probe that contains at least 10 contiguous nucleotides of SEQ ID NO:1, where the contiguous nucleotides include at least one nucleotide of the nucleotide sequence shown as position 1 to position 3221 of SEQ ID NO:1, provided that the probe does not have the nucleotide sequence of AA363808, AW959484, BE165930, nucleotides 1 to 614 of BE893201 or nucleotides 1 to 1530 of AK026780.

Alternatively, a test expression level can be determined by measuring the amount of PAMP polypeptide. The amount of PAMP polypeptide can be determined, for example, by contacting a cell, a cell lysate or fractionated sample thereof, from an individual with a binding agent selective for a PAMP polypeptide and determining the amount of selective binding of the agent. The selective binding agent can be, for example, an antibody or other molecule identified as a PAMP polypeptide binding agent by the methods disclosed herein and, if desired, can contain a detectable label.

In one embodiment, a test expression is determined by measuring the amount of PAMP polypeptide using a binding agent selective for PAMP polypeptide residues 1 to 1074 of SEQ ID NO:2.

A prostate neoplastic condition is a benign or malignant prostate lesion of proliferating cells. Prostate neoplastic conditions include, for example, prostate interepithelial neoplasia (PIN) and prostate cancer. Prostate cancer is an uncontrolled proliferation of prostate cells which can invade and destroy adjacent tissues as well as metastasize. Primary prostate tumors can be classified into stages TX, T0, T1, T2, T3, and T4 and metastatic tumors can be classified into stages D1, D2 and D3. Similarly, there are classifications known by those skilled in the art for the progressive stages of precancerous lesions or PIN. The methods herein are applicable for the diagnosis or treatment of any or all stages of prostate neoplastic conditions.

The methods of the invention are also applicable to prostate pathologies other than neoplastic conditions. Such other pathologies include, for example, benign prostatic hyperplasia (BPH) and prostatitis. BPH is one of the most common diseases in adult males. Histological evidence of BPH has been found in more than 40% of men in their fifties and almost 90% of men in their eighties. The disease results from the accumulation of non-malignant nodules arising in a small region around the proximal segment of the prostatic urethra which leads to an increase in prostate volume. If left untreated, BPH can result in acute and chronic retention of urine, renal failure secondary to obstructive uropathy, serious urinary tract infection and irreversible bladder decompensation. Prostatitis is an infection of the prostate. Other prostate pathologies known to those skilled in the art exist as well and are similarly applicable for diagnosis or treatment using the methods of the invention. Various neoplastic conditions of the prostate as well as prostate pathologies can be found described in, for example, Campbell's Urology, Seventh Edition, W. B. Saunders Company, Philadelphia (1998). Therefore, the methods of the invention are applicable to both prostate neoplastic conditions and prostate pathologies.

Therefore, the invention provides a method for both diagnosing and prognosing a prostate neoplastic condition including prostate cancer and prostate interepithelial neoplasia as well as other prostate pathologies such as BPH and prostatitis.

The invention also provides diagnostic methods relating to liver cancer. While PAMP expression is not observed in normal liver, expressed sequences corresponding to portions of PAMP have been isolated from tumor tissues such as hepatocellular carcinoma, and, to a lesser extent, invasive ovarian tumor, genitourinary tract tumors and endometrial adenocarcinoma. Furthermore, the 5.0 and 6.5 kb PAMP transcripts were expressed and testis and ovary tissues (see FIG. 3). These results indicate that elevated PAMP expression can be used to diagnose hepatocellular carcinoma, genitourinary tract tumors, endometrial adenocarcinoma, ovarian cancer and testicular cancer. One skilled in the art understands that the methods are practiced as described herein for prostate neoplastic conditions, with the sample chosen appropriately. For example, a sample containing liver cells is assayed according to a method of the invention for diagnosing or predicting susceptibility to liver cancer such as hepatocellular carcinoma.

Thus, the invention provides a method of diagnosing or predicting susceptibility to hepatocellular carcinoma in an individual by obtaining a sample from said individual; measuring a test expression level of PAMP in the sample; and comparing the test expression level of PAMP to a control expression level of PAMP, where a test expression level 2-fold or more greater than the control expression level indicates the presence of hepatocellular carcinoma.

The invention also provides a method of diagnosing or predicting susceptibility to ovarian cancer in an individual by obtaining a sample from said individual; measuring a test expression level of PAMP in the sample; and comparing the test expression level of PAMP to a control expression level of PAMP, where a test expression level 2-fold or more greater than the control expression level indicates the presence of ovarian cancer.

Thus, the invention provides a method of diagnosing or predicting susceptibility to testicular cancer in an individual by obtaining a sample from said individual; measuring a test expression level of PAMP in the sample; and comparing the test expression level of PAMP to a control expression level of PAMP, where a test expression level 2-fold or more greater than the control expression level indicates the presence of testicular cancer.

The invention provides a method of diagnosing or predicting prostate neoplastic conditions based on a finding of a positive correlation between a test expression level of PAMP in neoplastic cells of the prostate and the degree or extent of the neoplastic condition or pathology. The diagnostic methods of the invention are applicable to numerous prostate neoplastic conditions and pathologies as described above. One consequence of progression into these neoplastic and pathological conditions can be increased expression of PAMP in prostate tissue. The increase in PAMP expression in individuals suffering from a prostate neoplastic condition can be measured by comparing the amount of PAMP to that found, for example, in normal prostate tissue samples or in normal blood or serum samples. A two-fold or more increase in a test expression level in a prostate cell sample relative to a control expression sample obtained, for example, from normal prostate cells or from an androgen-dependent cell line is indicative of a prostate neoplastic condition or pathology. Similarly, an increase in PAMP expression leading to two-fold or more secretion into the blood or other circulatory fluids of the individual compared to control blood or fluid samples also can be indicative of a prostate neoplastic condition or pathology.

As a diagnostic indicator, PAMP can be used qualitatively to positively identify a prostate neoplastic condition or pathology as described above. Alternatively, PAMP also can be used quantitatively to determine the degree or susceptibility of a prostate neoplastic condition or pathology. For example, successive increases in the expression levels of PAMP can be used as a predictive indicator of the degree or severity of a prostate neoplastic condition or pathology. For example, increased expression can lead to a rise in accumulated levels and can be positively correlated with increased severity of a neoplastic condition of the prostate. A higher level of PAMP expression can be correlated with a later stage of a prostate neoplastic condition or pathology. For example, increases in expression levels of two-fold or more compared to a normal sample can be indicative of at least prostate neoplasia. PAMP also can be used quantitatively to distinguish between pathologies and neoplastic conditions as well as to distinguish between the different types of neoplastic conditions.

Correlative increases can be determined by comparison of PAMP expression from the individual having, or suspected of having, a neoplastic condition of the prostate to expression levels of PAMP from known samples determined to exhibit a prostate neoplastic condition. Alternatively, correlative increases also can be determined by comparison of a test expression level of PAMP expression to expression levels of other known markers of prostate cancer such as prostate specific antigen (PSA), glandular kallikrein 2 (hK2) and prostase/PRSS18. These other known markers can be used, for example, as an internal or external standard for correlation of stage-specific expression with increases in PAMP expression and severity of the neoplastic or pathological condition. Conversely, a regression in the severity of a prostate neoplastic condition or pathology can be followed by a corresponding decrease in PAMP expression levels and can similarly be assessed using the methods described herein.

Given the teachings and guidance provided herein, those skilled in the art will know or can determine the stage or severity of a prostate neoplastic condition or pathology based on a determination of PAMP expression and correlation with a prostate neoplastic condition or pathology. A correlation can be determined using known procedures and marker comparisons as described herein. For a review of recognized values for such other marker in normal versus pathological tissues, see, for example, Campbell's Urology, Seventh Edition, W. B. Saunders Company, Philadelphia (1998).

The use of PAMP expression levels in prostate cells, the circulatory system and urine as a diagnostic indicator of a prostate pathology allows for early diagnosis as a predictive indicator when no physiological or pathological symptoms are apparent. The methods are particularly applicable to any males over age 50, African-American males and males with familial history of prostate neoplastic conditions or pathologies. The diagnostic methods of the invention also are particularly applicable to individuals predicted to be at risk for prostate neoplastic conditions or pathologies by reliable prognostic indicators prior to onset of overt clinical symptoms. All that is necessary is to determine the PAMP prostate tissue or circulatory or bodily fluid expression levels to determine whether there is an increase in these PAMP levels in the individual suspected of having a prostate pathology compared to a control expression level such as the level observed in normal individuals. Those skilled in the art will know by using routine examinations and practices in the field of medicine those individuals who are applicable candidates for diagnosis by the methods of the invention.

For example, individuals suspected of having a prostate neoplastic condition or pathology can be identified by exhibiting presenting signs of prostate cancer which include, for example, a palpable nodule (>50% of the cases), dysuria, cystitis and prostatitis, frequency, urinary retention, or decreased urine stream. Signs of advanced disease include pain, uremia, weight loss and systemic bleeding. Prognostic methods of this invention are applicable to individuals after diagnosis of a prostate neoplastic condition, for example, to monitor improvements or identify residual neoplastic prostate cells using, for example, imaging methods known in the art and which target PAMP.

Therefore, the invention also provides a method of predicting the onset of a prostate neoplastic condition or pathology. The method consists