WO1999058560A2 - Gene et proteine prostapine ainsi que leurs applications - Google Patents

Gene et proteine prostapine ainsi que leurs applications Download PDF

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Publication number
WO1999058560A2
WO1999058560A2 PCT/US1999/007123 US9907123W WO9958560A2 WO 1999058560 A2 WO1999058560 A2 WO 1999058560A2 US 9907123 W US9907123 W US 9907123W WO 9958560 A2 WO9958560 A2 WO 9958560A2
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Prior art keywords
prostapin
protein
prostate cancer
prostate
sample
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WO1999058560A3 (fr
Inventor
Daniel E. Afar
Rene S. Hubert
Kahan Leong
Arthur B. Raitano
Douglas C. Saffran
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Agensys Inc
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Urogenesys Inc
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Priority to EP99915178A priority Critical patent/EP1068320A2/fr
Priority to CA002324206A priority patent/CA2324206A1/fr
Priority to AU33760/99A priority patent/AU3376099A/en
Publication of WO1999058560A2 publication Critical patent/WO1999058560A2/fr
Publication of WO1999058560A3 publication Critical patent/WO1999058560A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the invention described herein relates to a novel gene, PROSTAPIN, and its expression product; to the expression of PROSTAPIN in normal and prostate cancer cells; and to diagnostic, prognostic, and therapeutic compositions and methods useful in the management of prostate cancer.
  • Prostate cancer is the most frequently diagnosed cancer and second leading cause of cancer death in men. Some 45,000 men die annually of this disease and only lung cancer has a higher mortality rate. In the United States, the chance of a man developing invasive prostate cancer during his lifetime is approximately 1 in 6 or greater. Numerous fundamental limitations in the presently available methods used for the treatment, diagnosis and prognosis of prostate cancer have rendered it impossible to effectively manage.
  • Surgical prostatectomy, radiation therapy, hormone ablation therapy, and chemotherapy are the main components in the current arsenal for treating prostate cancer.
  • these treatments are ineffective for the 45,000 prostate cancer patients who die of this disease every year. While some advances in the treatment of locally confined tumors have been achieved, prostate cancer is presently incurable once it has metastasized.
  • Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
  • the primary sites of prostate cancer metastasis are the regional lymph nodes and bone. Bone metastases occur in sites of hematopoietically active red bone marrow, including lumbar vertebral column, ribs, pelvis, proximal long bones, sternum and skull. Bony metastases of prostate cancer differ from those of other tumors commonly colonizing bone in that they are characterized by a net gain in bone formation (osteoblastic) rather than resorption predominant in bone metastases of breast cancer and melanoma.
  • Prostate cancer cells have been detected in the peripheral blood and bone marrow of patients with advanced stage disease using RT-PCR assays for PSA mRNA (Ghossein et al., 1995; Seiden et al., 1994; Wood et al., 194; Katz et al., 1994) or immunomagnetic bead selection for PSA protein (Brandt et al., 1996). When positive, these tests show that prostate cancer cells represent about 0.1-1.0% of the circulating blood cells.
  • a another factor complicating the management of prostate cancer is that reliable diagnostic and prognostic markers capable of accurately detecting early-stage tumors and/or predicting which patients will progress to advanced stages do not exist.
  • Early detection and diagnosis of prostate cancer currently relies on digital rectal examinations (DRE), prostate specific antigen (PSA) measurements, transrectal ultrasonography, and transrectal needle biopsy.
  • DRE digital rectal examinations
  • PSA prostate specific antigen
  • transrectal ultrasonography transrectal ultrasonography
  • transrectal needle biopsy Serum PSA measurements in combination with DRE represent the leading diagnostic approach at present.
  • PSA prostate specific antigen
  • Tumor suppressors are proteins which regulate cell growth. Absence of tumor suppressors by mutation, deletion, or loss of expression results in the malignant phenotype. Numerous tumor suppressor genes have been identified. Two of the more well known and studied tumor suppressor genes are the retinoblastoma (Rb) gene and the p53 gene, both of which are directly involved in influencing the cell cycle machinery. The expression of Rb inhibits cell cycle progression from G, into S phase. The p53 gene is the most frequently mutated gene in human cancers, with approximately half of all tumors containing abnormal p53 genes. p53 participates in a cell cycle checkpoint signal transduction pathway that causes either G 1 arrest or apoptosis following DNA damage. Loss of p53 function during tumorigenesis can result in progression through the cell cycle in the face of DNA damage and survival of a cell otherwise destined for death.
  • Rb retinoblastoma
  • a class of serine protease inhibitors known as the serpins includes a protein, maspin, which may function as a tumor suppressor in both breast and prostate cancer.
  • Maspin has been shown to be down-regulated in breast and prostate carcinoma (Zou et al., 1994, Science 263:526), and overexpression of maspin and/or exogenous addition of maspin has been shown to dramatically reduce the tumorigenic properties and metastatic potential of breast cancer ceils (Zou et al., 1994, Science 263:526).
  • LEI is converted to L-DNase II by digestion with elastase, whereupon it functions as an endonuclease in DNA degradation during apoptosis (Torriglia et al., 1998, MCB 18:3612).
  • serpins appear to have a role in apoptosis.
  • the present invention relates to a novel member of the serpin family, termed PROSTAPIN, which is expressed almost exclusively in the prostate.
  • PROSTAPIN expression is greatly attenuated or completely lost in cells of advanced prostate tumors and metastases, while its expression is maintained at or near normal levels in locally confined prostate cancers.
  • PROSTAPIN may function as a prostate-specific tumor suppressor, apoptosis-inducer or apoptosis-modulator.
  • PROSTAPIN gene and protein, as well as factors capable of activating PROSTAPIN expression may be useful as therapeutic agents capable of restoring critical tumor suppressor activity lost in advanced prostate cancer.
  • PROSTAPIN may represent an ideal marker for predicting and identifying progression to advanced stage and metastatic prostate cancer, and may also be useful for determining susceptibility to advanced disease and for gauging prostate tumor aggressiveness.
  • the invention provides polynucleotides corresponding or complementary to all or part of the PROSTAPIN gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding PROSTAPIN proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to the PROSTAPIN gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides which hybridize to the PROSTAPIN gene, mRNA, or to PROSTAPIN-encoding polynucleotides.
  • PROSTAPIN proteins and polypeptide fragments thereof.
  • the invention further provides antibodies that bind to PROSTAPIN proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker.
  • the invention further provides methods for detecting the presence of PROSTAPIN polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express PROSTAPIN.
  • the invention further provides methods and assays for determining PROSTAPIN expression status, diagnosing advanced prostate cancer, gauging tumor aggressiveness, and predicting susceptibility to advanced prostate cancer.
  • the invention further provides various therapeutic compositions and strategies for treating prostate cancer by restoring functional PROSTAPIN to prostate tumor cells.
  • FIG. 1 Nucleotide and amino acid sequences of PROSTAPIN (SEQ ID NOS. XX and XX, respectively), derived from the overlapping nucleotide sequences of SSH fragment cDNA clone 11P2A6 (5' 471 bp) (SEQ ID NO. XX) and cDNA clone 103 (SEQ ID NO. XX). Clones 11 P2A6 and 03 overlap across 304 bp beginning at position 168. The 5' untranslated region indicates two translational STOP signals (indicated by asterisks) upstream of the START ATG.
  • the sequence surrounding the start ATG (AAA ATG G) exhibits a Kozak sequence (A at position -3, and G at position +1).
  • the underlined sequence in the carboxyl region indicates the putative highly exposed reactive site loop that is characteristic of the serpin family.
  • FIG. 2 Amino acid sequence alignment of PROSTAPIN with several other Serpin family members. The alignment was performed using the PIMA1.4 alignment program of the Baylor College of Medicine Search Launcher Web site. The RSL sites are indicated in bold. The protease cleavage site, indicated by the P1 and P1' residues, is boxed.
  • FIG. 3 Semi-quantitative RT-PCR analysis of PROSTAPIN expression in normal human tissue, prostate cancer xenograft tissue, and cell lines using primers derived from clone 11 P2A6 cDNA (SEQ ID NO. XX). First strand cDNAs were prepared from 16 normal tissues, the LAPC xenografts (4AD, 4AI and 9AD) and HeLa cells. Normalization was performed by PCR using primers to actin and GAPDH. Expression of PROSTAPIN is detected only in normal prostate and in the LAPC-9 AD xenograft.
  • FIG. 4 Northern blot analyses of PROSTAPIN expression in various normal human tissues and prostate cancer xenografts.
  • a and B Multiple tissue northern blots probed with full length PROSTAPIN cDNA clone 103 (SEQ ID NO. XX). Size standards in kilobases (kb) are indicated.
  • C Multiple tissue RNA dot blot (Clontech, Human Master Blot cat# 7770-1) probed with PROSTAPIN CLONE 103 cDNA probe (SEQ ID NO. XX).
  • D Normal prostate and various prostate cancer xenograft Northern blot, showing lack of expression in the LAPC-4 prostate cancer xenografts and down-regulated expression in the LAPC-9 xenograft relative to normal prostate expression levels.
  • FIG. 5 Loss of PROSTAPIN expression in metastatic prostate cancer.
  • the prostate pool (lane 3) comprises several normal prostate cDNAs and was obtained from Clontech (Palo Alto, California).
  • Prostate 25 and Prostate 32 were derived from 25 and 32 year old individuals, respectively (BioChain). Gleason Grade and TMN Stage of the human prostate tumor specimens analyzed are indicated. For details, see Example 4.
  • FIG. 6 Mutant PROSTAPIN gene generated from prostate cancer xenograft LAPC-9 AD: LAPC-9 AD PROSTAPIN cDNA clone 2 nucleotide (SEQ ID NO. XX) and deduced amino acid (SEQ ID NO. XX) sequences. Point mutations in the nucleotide sequence and any resulting amino acid changes relative to the wild-type PROSTAPIN sequence of FIG. 1 (SEQ ID NO. XX) are indicated in boldface type and are underlined. A large insertion sequence (relative to wild-type PROSTAPIN) is indicated in bold and is underlined.
  • FIG. 7 Detection of PROSTAPIN protein in cell membrane fraction. The results show that PROSTAPIN is predominantly expressed in the light membrane fraction. See Example 6 for experimental details.
  • FIG. 8 Chromosomal mapping of human PROSTAPIN, showing position within serpin gene cluster on chromosome 18q21.3.
  • A Zooblot: Genomic DNAs prepared from several different organisms including human, monkey, dog, mouse, chicken and Drosophila.
  • B Human BACs 2002H14, 2074J2, 2100H19, and PAC 152i22, containing the PROSTAPIN gene, a non-specific BAC (2116L1 ), and a non-specific PAC (40P22) (lanes 4 and 5, respectively).
  • C Mouse BACs 74e14 (lanes 3 and 5), 213i3 (lanes 4 and 6) containing the mouse PROSTAPIN gene probed together with human positive (BAC 2074J2) and negative (BAC 40P22) controls.
  • FIG. 10 Intron/exon boundaries of the human wild-type PROSTAPIN gene. Sequence in capital letters designate exonic sequences (with the translation below) and sequence in lower case letters designate intronic sequences. A total of 6 introns and 7 exons were identified within the PROSTAPIN coding region.
  • FIG. 11 Amplification of PROSTAPIN exons from human genomic DNA: An example of the PCR products obtained from human genomic DNA using the primers described in Example 8. Human BAC DNA containing the PROSTAPIN gene was used as a positive control for PCR amplification.
  • FIG. 12 Western blot analysis of PROSTAPIN expression in lysates of cells transfected or transduced with PROSTAPIN using purified polyclonal antibody generated against a PROSTAPIN-GST fusion (see Example 9).
  • FIG. 13 Western blot analysis of PROSTAPIN expression in lysates derived from LAPC xenografts (LAPC-4 AD, 9AD, and 9AI), prostate cancer cell lines (TsuPrl , LNCaP, PC-3) and a prostate tumor-normal matched patient sample cells (see Example 10).
  • the terms "advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers which have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1 - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system.
  • AUA American Urological Association
  • stage C1 - C2 disease under the Whitmore-Jewett system
  • TNM tumor, node, metastasis
  • 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 presently 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 terms "metastatic prostate cancer” and “metastatic disease” mean prostate cancers which 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.
  • prostate cancer 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 the 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 thereafter.
  • the most common site for prostate cancer metastasis is bone.
  • Prostate cancer bone metastases are, on balance, characteristically osteobiastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain.
  • Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuciide scans, skeletal radiography, and/or bone lesion biopsy.
  • 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.
  • polypeptide means a polymer of at least 10 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used.
  • hybridize 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% formamide/6XSSC/0.1% SDS/100 ⁇ g/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSCt0.1% SDS are above 55 degrees C, and most preferably to stringent hybridization conditions.
  • One aspect of the invention provides polynucleotides corresponding or complementary to all or part of the PROSTAPIN gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding PROSTAPIN proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to the PROSTAPIN gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides which hybridize to the PROSTAPIN gene, mRNA, or to PROSTAPIN-encoding polynucleotides (collectively, "PROSTAPIN polynucleotides").
  • a PROSTAPIN polynucleotide may comprise a polynucleotide having the sequence shown in FIG. 1 (SEQ ID NO. XX), a sequence complementary thereto, or a polynucleotide fragment thereof.
  • Another embodiment comprises a polynucelotide which encodes the PROSTAPIN protein amino acid sequence shown in FIG. 1 (SEQ ID NO. XX) or a polynucleotide fragment thereof.
  • Another embodiment comprises a polynucleotide which is capable of hybridizing under stringent hybridization conditions to the PROSTAPIN cDNA shown in FIG. 1 (SEQ ID NO. XX) or to a polynucleotide fragment thereof.
  • the invention includes polypeptides derived from PROSTAPIN mutants, such as the LAPC-9 mutant PROSTAPIN described herein.
  • Such mutant PROSTAPIN polynucleotides may comprise the sequence of the LAPC-9 PROSTAPIN mutant, as shown in FIG. 6 (SEQ ID NO. X), or a polypeptide fragment thereof.
  • a related embodiment comprises a polynucleotide which is capable of hybridizing under stringent hybridization conditions to the PROSTAPIN mutant cDNA shown in FIG. 6 (SEQ ID NO. XX) or to a polynucleotide fragment thereof.
  • antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner.
  • PNAs peptide nucleic acids
  • non-nucleic acid molecules such as phosphorothioate derivatives
  • probes and primer pairs which allow the specific amplification of the polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof.
  • Probes may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioiuminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • a detectable marker such as, for example, a radioisotope, fluorescent compound, bioiuminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • Such probes and primers can be used to detect the presence of a PROSTAPIN polynucleotide in a sample and as a means for detecting a cell expressing a PROSTAPIN protein.
  • probes include polypeptides comprising all or part of the cDNA sequence shown in FIG. 1 (SEQ ID NO. XX).
  • primer pairs capable of specifically amplifying PROSTAPIN mRNA are described in the examples which follow.
  • a great many different primers and probes may be prepared based on the sequences provided in FIG. 1 (SEQ ID NO. XX) and used effectively to amplify and/or detect PROSTAPIN.
  • a polynucleotide is said to be "isolated” when it is substantially separated from contaminant polynucleotides which correspond or are complementary to genes other than the PROSTAPIN gene or which encode polypeptides other than PROSTAPIN gene product or fragments thereof.
  • a skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated PROSTAPIN polynucleotide.
  • PROSTAPIN polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the PROSTAPIN gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer; as coding sequences capable of directing the expression of PROSTAPIN polypeptides; as tools for modulating or inhibiting the expression of the PROSTAPIN gene(s) and/or translation of the PROSTAPIN transcript(s); and as therapeutic agents.
  • PROSTAPIN cDNA sequences described herein enables the isolation of other polynucleotides encoding the PROSTAPIN gene product(s), as well as the isolation of polynucleotides encoding PROSTAPIN gene product homologues, alternatively sliced isoforms, allelic variants, and mutant forms of the PROSTAPIN gene product.
  • Various molecular cloning methods that can be employed to isolate full length cDNAs encoding the PROSTAPIN gene are well known (See, for example, Sambrook, J.
  • cDNA libraries may be generated from normal testis tissue, placental tissue, prostate cancer cell lines, prostate cancer xenografts or another PROSTAPIN-expressing source. Phage clones containing PROSTAPIN gene cDNAs may be identified by probing with labeled PROSTAPIN cDNA or a fragment thereof.
  • the PROSTAPIN cDNA of FIG. 1 or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full length cDNAs corresponding to the PROSTAPIN-1 gene.
  • the PROSTAPIN gene itself may be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with PROSTAPIN DNA probes or primers.
  • the invention also provides recombinant DNA or RNA molecules containing a PROSTAPIN polynucleotide, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules.
  • a recombinant DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro. Methods for generating such molecules are well known (see, for example, Sambrook et al, 1989, supra).
  • the invention further provides a host-vector system comprising a recombinant DNA molecule containing a PROSTAPIN polynucleotide within a suitable prokaryotic or eukaryotic host cell.
  • suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell.
  • suitable mammalian cells include various prostate cancer cell lines such LnCaP, PC-3, DU145, LAPC-4, TsuPrl , other transfectable or transducible prostate cancer cell lines, as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of PROSTAPIN may be used to generate PROSTAPIN proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
  • PROSTAPIN may be preferably expressed in several prostate cancer cell lines, including for example PC-3, LNCaP and TsuPrl .
  • the host-vector systems of the invention are useful for the production of a PROSTAPIN protein or fragment thereof. Such host-vector systems may be employed to study the functional properties of PROSTAPIN and PROSTAPIN mutations.
  • Proteins encoded by the PROSTAPIN gene, or by fragments thereof, will have a variety of uses, including but not limited to generating antibodies, as therapeutic agents, and in methods for identifying ligands and other agents and cellular constituents that bind to a PROSTAPIN gene product.
  • Antibodies raised against PROSTAPIN proteins or fragments thereof may be useful in diagnostic and prognostic assays, imaging methodologies, and therapeutic methods in the management of prostate cancer.
  • Various immunological assays useful for the detection of PROSTAPIN proteins are contemplated, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like.
  • Such antibodies may be labeled and used as immunological imaging reagents capable of detecting prostate cells (e.g., in radioscintigraphic imaging methods).
  • PROSTAPIN refers to a protein that has or includes the amino acid sequence of human PROSTAPIN as provided in FIG. 1 (SEQ ID NO. XX), the amino acid sequence of other mammalian PROSTAPIN homologues, as well as allelic variants and conservative substitution mutants of these proteins that have PROSTAPIN activity.
  • the PROSTAPIN 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 below.
  • PROSTAPIN proteins will be collectively referred to as the PROSTAPIN proteins, the proteins of the invention, or PROSTAPIN.
  • PROSTAPIN polypeptide refers to a polypeptide fragment or a PROSTAPIN protein of at least 10 amino acids, preferably at least 15 amino acids, and more preferably at least 20 amino acids.
  • a specific embodiment of a PROSTAPIN protein comprises a polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO. XX).
  • wild-type PROSTAPIN is meant to refer to a protein having the amino acid sequence depicted in FIG. 1 (SEQ ID NO. XX).
  • allelic variants of human PROSTAPIN will share significant homology (e.g., 70 - 90%) to the PROSTAPIN amino acid sequence provided in FIG. 1.
  • allelic variants of the PROSTAPIN protein will contain conservative amino acid substitutions from the PROSTAPIN sequence herein described or will contain a substitution of an amino acid from a corresponding position in a PROSTAPIN homologue.
  • PROSTAPIN allelic variants will be proteins that share a high degree of homology with at least a small region of the PROSTAPIN amino acid sequence, but will further contain a radical departure form the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. Such alleles are termed mutant alleles of PROSTAPIN and represent proteins that typically do not perform the same biological functions. Mutant PROSTAPIN proteins having altered biological function are also included within the scope of the invention.
  • the term "functional mutant" when used to modify the term PROSTAPIN, is meant to refer to a PROSTAPIN polypeptide which contains one or more mutations that alter or eliminate PROSTAPIN biological activity, including the LAPC-9 mutant described herein (FIG.
  • mutant PROSTAPIN proteins and mutant PROSTAPIN polypeptides such as those corresponding to the amino acid sequences encoded by the LAPC-9 PROSTAPIN mutant as shown in FIG. 6 (SEQ ID NO. XX).
  • Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Such changes include substituting any of isoleucine (I), vaiine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for giutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (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 in the three-dimensional structure of the protein.
  • glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and vaiine (V).
  • Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with vaiine.
  • Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments.
  • PROSTAPIN proteins may be embodied in many forms, preferably in isolated form. As used herein, a protein is said to be "isolated” when physical, mechanical or chemical methods are employed to remove the PROSTAPIN protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated PROSTAPIN protein. A purified PROSTAPIN protein molecule will be substantially free of other proteins or molecules which impair the binding of PROSTAPIN to antibody or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of the PROSTAPIN protein include a purified PROSTAPIN protein and a functional, soluble PROSTAPIN protein. In one form, such functional, soluble PROSTAPIN proteins or fragments thereof retain the ability to bind antibody or other ligand.
  • the invention also provides PROSTAPIN polypeptides comprising biologically active fragments of the PROSTAPIN amino acid sequence, such as a polypeptide corresponding to part of the amino acid sequence shown in FIG. 1 (SEQ ID NO. XX).
  • Such polypeptides of the invention exhibit properties of PROSTAPIN, such as the ability to elicit the generation of antibodies which specifically bind an epitope associated with PROSTAPIN.
  • PROSTAPIN polypeptides can be generated using standard peptide synthesis technology and the amino acid sequences of the human PROSTAPIN protein disclosed herein. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a polypeptide fragment of the PROSTAPIN protein. In this regard, the PROSTAPIN- encoding nucleic acid molecules described herein provide means for generating defined fragments of PROSTAPIN. PROSTAPIN polypeptides are particularly useful in generating domain specific antibodies, identifying agents or cellular factors that bind to PROSTAPIN or a PROSTAPIN domain, and in prostate cancer therapeutic strategies which comprise the restoration of PROSTAPIN functionality.
  • PROSTAPIN polypeptides containing particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments containing such structures are particularly useful in generating subunit specific anti-PROSTAPIN antibodies or in identifying cellular factors that bind to PROSTAPIN.
  • Another aspect of the invention provides antibodies that bind to PROSTAPIN proteins and polypeptides.
  • the most preferred antibodies will selectively bind to PROSTAPIN and will not bind (or will bind weakly) to non-PROSTAPIN proteins and polypeptides.
  • Anti- PROSTAPIN antibodies that are particularly contemplated include monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more complement determining regions of these antibodies.
  • an antibody fragment is defined as at least a portion of the variable region of the immunoglobulin molecule which binds to its target, i.e., the antigen binding region.
  • a PROSTAPIN antibody is an antibody which (1 ) was raised against a preparation comprising a PROSTAPIN protein, a PROSTAPIN polypeptide, a mutant PROSTAPIN protein or polypeptide, a fusion protein comprising any of the foregoing, a cell preparation containing PROSTAPIN protein or polypeptide, a cell engineered to express a PROSTAPIN protein or polypeptide, or a similar PROSTAPIN immunogen, and/or (2) binds to a PROSTAPIN and/or mutant PROSTAPIN protein or polypeptide.
  • PROSTAPIN antibodies of the invention may be particularly useful in prostate cancer diagnostic and prognostic assays, imaging methodologies, and therapeutic strategies.
  • the invention provides various immunological assays useful for the detection and quantification of PROSTAPIN and mutant PROSTAPIN proteins and polypeptides.
  • Such assays generally comprise one or more PROSTAPIN antibodies capable of recognizing and binding a PROSTAPIN or mutant PROSTAPIN protein, as appropriate, and may be performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
  • immunological imaging methods capable of detecting prostate cancer are also provided by the invention, including but limited to radioscintigraphic imaging methods using labeled PROSTAPIN antibodies.
  • Such assays may be clinically useful in the detection, monitoring, and prognosis of prostate cancer, particularly advanced prostate cancer.
  • PROSTAPIN antibodies may also be used in methods for purifying PROSTAPIN and mutant PROSTAPIN proteins and polypeptides and for isolating PROSTAPIN homologues and related molecules.
  • the method of purifying a PROSTAPIN protein comprises incubating a PROSTAPIN antibody, which has been coupled to a solid matrix, with a lysate or other solution containing PROSTAPIN under conditions which permit the PROSTAPIN antibody to bind to PROSTAPIN; washing the solid matrix to eliminate impurities; and eluting the PROSTAPIN from the coupled antibody.
  • Other uses of the PROSTAPIN antibodies of the invention include generating anti-idiotypic antibodies that mimic the PROSTAPIN protein.
  • antibodies may be prepared by immunizing a suitable mammalian host using a PROSTAPIN protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
  • fusion proteins of PROSTAPIN may also be used, such as a PROSTAPIN GST-fusion protein.
  • a GST fusion protein comprising all or most of the open reading frame amino acid sequence of FIG. 1 may be produced and used as an immunogen to generate appropriate antibodies.
  • Cells expressing or overexpressing PROSTAPIN may also be used for immunizations. Similarly, any cell engineered to express PROSTAPIN may be used. This strategy may result in the production of monoclonal antibodies with enhanced capacities for recognizing endogenous PROSTAPIN.
  • the amino acid sequence of PROSTAPIN as shown in FIG. 1 may be used to select specific regions of the PROSTAPIN protein for generating antibodies.
  • hydrophobicity and hydrophilicity analyses of the PROSTAPIN amino acid sequence may be used to identify hydrophilic regions in the PROSTAPIN structure.
  • Regions of the PROSTAPIN protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.
  • amino acid sequences unique to the mutant are preferable.
  • the inserted or unique amino acid sequences shown in FIG. 6 may be used to select specific regions.
  • Methods for preparing a protein or polypeptide for use as an immunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be effective.
  • Administration of a PROSTAPIN immunogen is conducted generally by injection over a suitable time period and with use of a suitable adjuvant, as is generally understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
  • PROSTAPIN monoclonal antibodies are preferred and may be produced by various means well known in the art.
  • immortalized cell lines which secrete a desired monoclonal antibody may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the PROSTAPIN protein or PROSTAPIN fragment.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antiserum which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Scfy, or F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the PROSTAPIN protein can also be produced in the context of chimeric or CDR grafted antibodies of multiple species origin. Humanized or human PROSTAPIN antibodies may also be produced and are preferred for use in therapeutic contexts. Various approaches for producing such humanized antibodies are known, and include chimeric and CDR grafting methods; methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539).
  • Fully human PROSTAPIN monoclonal antibodies may be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display)(Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. in: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Jd., pp 65-82).
  • Fully human PROSTAPIN monoclonal antibodies may also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Jakobovits et al., published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
  • Reactivity of PROSTAPIN antibodies with PROSTAPIN protein or mutant PROSTAPIN protein may be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, PROSTAPIN proteins, peptides, PROSTAPIN-expressing cells or extracts thereof.
  • a PROSTAPIN antibody or a fragment thereof may be labeled with a detectable marker and used for targeting the detectable marker to a PROSTAPIN positive cell (Vitetta, E.S. et al., 1993, Immunotoxin therapy, in DeVita, Jr., V.T. et al., eds, Cancer: Principles and Practice of Oncology, 4th ed., J.B. Lippincott Co., Philadelphia, 2624-2636).
  • Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioiuminescent compound, chemiluminescent compound, a metal chelator or an enzyme.
  • Another aspect of the present invention relates to methods for detecting PROSTAPIN polynucleotides and PROSTAPIN proteins, as well as methods for identifying a cell which expresses PROSTAPIN.
  • the invention provides assays for the detection of PROSTAPIN polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, cell preparations, and the like.
  • Detectable PROSTAPIN polynucleotides include, for example, a PROSTAPIN gene or fragments thereof, PROSTAPIN mRNA, alternative splice variant PROSTAPIN mRNAs, and recombinant DNA or RNA molecules containing a PROSTAPIN polynucleotide.
  • a number of methods for amplifying and/or detecting the presence of PROSTAPIN polynucleotides are well known in the art and may be employed in the practice of this aspect of the invention.
  • a method for detecting PROSTAPIN mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using PROSTAPIN polynucleotides as sense and antisense primers to amplify PROSTAPIN cDNAs therein; and detecting the presence of the amplified PROSTAPIN cDNA.
  • a method of detecting the PROSTAPIN gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using PROSTAPIN polynucleotides as sense and antisense primers to amplify the PROSTAPIN gene therein; and detecting the presence of the amplified PROSTAPIN gene. Any number of appropriate sense and antisense probe combinations may be designed from the nucleotide sequence provided in FIG. 1 (SEQ ID NO. XX) and used for this purpose, as will be understood by those skilled in the art.
  • the invention also provides assays for detecting the presence of a PROSTAPIN protein in a tissue of other biological sample such as serum, bone, prostate, and other tissues, urine, cell preparations, and the like.
  • Methods for detecting a PROSTAPIN protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western Blot analysis, molecular binding assays, ELISA, ELIFA and the like.
  • a method of detecting the presence of a PROSTAPIN protein in a biological sample comprises first contacting the sample with a PROSTAPIN antibody, a PROSTAPIN-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a PROSTAPIN antibody; and then detecting the binding of PROSTAPIN protein in the sample thereto.
  • an assay for identifying a cell which expresses a PROSTAPIN gene comprises detecting the presence of PROSTAPIN mRNA in the cell.
  • Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled PROSTAPIN riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for PROSTAPIN, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
  • an assay for identifying a cell which expresses a PROSTAPIN gene comprises detecting the presence of PROSTAPIN protein in the cell or secreted by the cell.
  • Various methods for the detection of proteins are well known in the art and may be employed for the detection of PROSTAPIN proteins and PROSTAPIN expressing cells.
  • PROSTAPIN expression analysis may also be useful as a tool for identifying and evaluating agents which modulate PROSTAPIN gene expression.
  • PROSTAPIN expression is significantly reduced in prostate cancer samples, prostate cancer xenografts and cell lines. The mechanism of inactivation is unclear, since southern blotting of DNA derived from the xenografts (LAPC-4 AD, 4AI, 9AD), prostate cancer cell lines (PC-3, DU145, LNCaP) and normal human DNA show no remarkable differences in intensity or banding pattern. Similar observations were made for the tumor suppressor gene PTEN/MMAC1 , which encodes a dual-specificity phosphatase (Whang et al., 1998, PNAS 95: 5246).
  • PTEN/MMAC1 mRNA expression was restored in nonexpressing prostate cancer cells by in vitro treatment with the demethylating agent 5-azadeoxycytidine (Whang et al., 1998, PNAS 95: 5246). This suggests that methylation was responsible for silencing of the PTEN/MMAC1 gene.
  • a similar mechanism of transcriptionai inactivation may explain loss of PROSTAPIN expression in some of the prostate cancer specimens. Identification of a molecule or biological agent that could reactivate PROSTAPIN expression may be of therapeutic value in the treatment of prostate cancer. Such an agent may be identified by using a screen that allows for recognizing the acquisition of PROSTAPIN expression by RT-PCR, nucleic acid hybridization or antibody binding.
  • mutant PROSTAPIN polynucleotides and proteins may be applied to the detection of mutant PROSTAPIN polynucleotides and proteins using, as appropriate, probes, primers, antibodies and other binding agents capable of detecting such mutant forms.
  • PROSTAPIN gene expression appears to be lost or greatly attenuated in advanced prostate cancers.
  • determining the status of PROSTAPIN expression in an individual may be used to diagnose advanced stage prostate cancer as well as provide prognostic information useful in defining appropriate therapeutic options.
  • the expression status of PROSTAPIN may provide information useful for predicting susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness.
  • the invention provides methods and assays for determining PROSTAPIN expression status, diagnosing advanced prostate cancer, and predicting susceptibility to advanced prostate cancer.
  • PROSTAPIN expression status is meant to include quantitative and/or qualitative aspects, i.e., the level of wild type PROSTAPIN expression as well as the presence of functional PROSTAPIN mutations.
  • the invention provides assays useful in determining the presence of advanced stage prostate cancer in an individual.
  • advanced stage prostate cancer is commonly diagnosed by pathological examination of prostate and surrounding tissues surgically removed during radical prostatectomy.
  • surgery is not desirable, but is generally performed because there is no method of reliably distinguishing between advanced and localized prostate cancer other than pathological examination of surgically removed tissues.
  • the undesirable surgical option has already been performed.
  • the invention provides a means of distinguishing between advanced prostate cancer and locally confined prostate cancer by assaying for PROSTAPIN expression.
  • the means comprises detecting a marked loss or absence of wild type PROSTAPIN expression in prostate tumor tissues and ceils relative to expression levels in normal prostate tissue and cells.
  • PROSTAPIN mRNA is expressed at easily detectable levels in ail normal prostate tissues and all locally confined prostate cancer tissues tested.
  • wild type PROSTAPIN expression is either completely undetectable or greatly attenuated in all advanced stage prostate tumor specimens, cell lines derived from prostate cancer metastases, and SCID mouse xenografts derived from human prostate cancer metastases.
  • a method or assay for identifying the presence of advanced prostate cancer comprises determining the level of PROSTAPIN mRNA expressed by cells in a test sample, preferably a prostate, prostate tumor, lymph, bone or peripheral blood sample; and comparing the level so determined to the level of PROSTAPIN expressed in normal prostate, preferably a comparable known normal prostate tissue sample.
  • the absence or substantial attenuation of PROSTAPIN mRNA expression in the test sample relative to normal prostate indicates the presence of advanced prostate cancer.
  • Attenuation of PROSTAPIN mRNA expression is "substantial" when expression is reduced by at least about 10%, and preferably by about 30-50% or more, relative to PROSTAPIN mRNA expression levels detectable in normal prostate.
  • PROSTAPIN expression status may be determined at the protein level rather than at the nucleic acid level.
  • a method or assay would comprise determining the level of PROSTAPIN protein expressed by cells in a test sample, preferably a prostate, prostate tumor, lymph, bone or peripheral blood sample; and comparing the level so determined to the level of PROSTAPIN expressed in normal prostate, preferably a comparable known normal prostate tissue sample.
  • the absence or substantial attenuation of PROSTAPIN protein expression in the test sample relative to normal prostate indicates the presence of advanced prostate cancer.
  • PROSTAPIN antibodies or binding partners capable of detecting PROSTAPIN protein expression may be used in a variety of assay formats well known in the art for this purpose.
  • a specific, preferred embodiment comprises determining the expression status of a patient's PROSTAPIN mRNA or PROTEIN in the cells of a known prostate tumor sample and comparing the level of PROSTAPIN expression so determined to the level expressed by normal prostate cells, the presence of comparable expression levels being indicative of a locally confined or less advanced stage.
  • prostate tumor cells may be "known" by virtue of their origin, e.g., biopsied from a tumor mass, or by the presence of one or more molecular markers of prostate cancer cells. A number of such molecular markers are known, including for example PSCA and PSMA.
  • Assaying the expression status of a prostate cancer marker and PROSTAPIN in the same tissue sample may be particularly useful where tumor origin of the sample cannot be assured.
  • the presence of a known prostate cancer molecular marker in the sample can be used to identify the sample as prostate cancer, while the level of PROSTAPIN expressed in the same sample may be used as a tool for determining the presence of advanced prostate cancer (as well as susceptibility to advanced prostate cancer and tumor aggressiveness).
  • expression of PSCA and PROSTAPIN in a sample tissue are assayed together. PSCA is widely over-expressed across all stages and grades of prostate cancer. Thus the presence of PSCA over-expression relative to expression levels in normal prostate may be used to reliably identify samples which comprise prostate cancer cells.
  • a single sample may be heterogeneous for the expression of the known tumor marker.
  • a sample may be shown to contain some regions of cells expressing (or over-expressing, as appropriate) the marker while other regions do not express the marker (or expressing normal levels of the marker).
  • it may be most appropriate to use the level of PROSTAPIN expression in the regions showing expression or over-expression of the marker in order to reliably determine that patient's prostate cancer stage.
  • the tissue sampled is inherently heterogeneous for a number of cell types, such as, for example, blood.
  • the presence of the known prostate cancer marker may be used to identify and/or isolate the prostate cancer cells from other cells present in the sample.
  • the PROSTAPIN expression status in the known prostate cancer marker positive cells should be used for staging purposes. This type of combined analysis may be used not only for determining locally confined cancers, but also for determining advanced stage cancers, aggressiveness and susceptibility to advanced stage cancer, by assaying PROSTAPIN expression as described above together with a known prostate cancer marker.
  • Peripheral blood may be conveniently assayed by the combined analysis described above using RT-PCR to detect and quantify the expression of PROSTAPIN and known prostate tumor marker mRNAs.
  • RT-PCR amplification of a known tumor marker mRNA combined with the absence or attenuation of RT-PCR amplifiable PROSTAPIN mRNA (relative to normal prostate expression levels) provides an indication of the presence of advanced prostate cancer and may provide information concerning the aggressiveness of the originating tumor.
  • RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors.
  • RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25: 373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston et al., 1995, Clin. Chem. 41 : 1687-1688).
  • RT- PCR assays are well known in the art. Semi-quantitative RT-PCR assays for PROSTAPIN expression are described in greater detail by way of the examples which follow. Such assays may also be employed for the detection (and quantitation) of a known prostate tumor marker.
  • a related aspect of the invention is directed to predicting susceptibility to developing advanced prostate cancer in an individual.
  • a method for predicting susceptibility to advanced prostate cancer comprises determining the level of PROSTAPIN mRNA or PROSTAPIN protein expressed by cells in a first prostate or prostate tumor sample, comparing the level so determined to the level of PROSTAPIN mRNA or PROSTAPIN protein expressed in a second normal prostate tissue, the absence or substantial attenuation of PROSTAPIN mRNA or PROSTAPIN protein expression in the first sample relative to the second sample indicating susceptibility to advanced prostate cancer, wherein the degree of attenuated PROSTAPIN expression relative to normal prostate is proportional to the degree of susceptibility to advanced prostate cancer.
  • a method for gauging aggressiveness of a prostate tumor comprises determining the level of PROSTAPIN mRNA or PROSTAPIN protein expressed by cells in a sample of the prostate tumor, comparing the level so determined to the level of PROSTAPIN mRNA or PROSTAPIN protein expressed in a normal prostate tissue taken from the same individual or a normal prostate tissue reference sample, wherein the degree of PROSTAPIN mRNA or PROSTAPIN protein expression loss in the prostate tumor sample relative to the normal prostate sample proportionally indicating degree of aggressiveness.
  • Standard methods for the detection and quantification of PROSTAPIN mRNA include in situ hybridization using labeled PROSTAPIN riboprobes, Northern blot and related techniques using PROSTAPIN polynucleotide probes, RT-PCR analysis using primers specific for PROSTAPIN, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like.
  • semi-quantitative RT-PCR may be used to detect and quantify PROSTAPIN mRNA expression as described in the Examples which follow.
  • primers capable of amplifying PROSTAPIN may be used for this purpose, including but not limited to the various primer sets specifically described herein. Standard methods for the detection and quantification of protein may be used for this purpose.
  • polyclonal or monoclonal antibodies specifically reactive with the wild-type PROSTAPIN protein may be used in an immunohistochemical assay of biopsied tissue.
  • the invention provides methods and assays for identifying locally confined prostate cancer comprising determining the expression status of a patient's PROSTAPIN mRNA or PROTEIN in the cells of a known prostate tumor sample and comparing the level of PROSTAPIN expression so determined to the level expressed by normal prostate cells, the presence of comparable expression levels being indicative of a locally confined or less advanced stage.
  • the invention also provides methods and assays capable of detecting functional mutations of the PROSTAPIN gene. Similar to the loss or attenuation of PROSTAPIN expression, the presence of a functional PROSTAPIN mutation also correlates with advanced prostate cancer and may be used to distinguish advanced from locally confined prostate cancers, predict aggressiveness, and determine susceptibility to advanced prostate cancer.
  • the general molecular diagnostic methods described above may be used for this purpose, provided that the means used to detect expression are capable of specifically identifying a PROSTAPIN mutation expected to result in a loss of PROSTAPIN function.
  • molecular probes or primers specifically designed to hybridize to or amplify the mutant PROSTAPIN, but not wild-type PROSTAPIN are used.
  • a hybridization probe comprising the nucleotide sequence of the LAPC-9 PROSTAPIN mutant as shown in FIG. 6 (SEQ ID NO. XX) may be used.
  • a probe comprising a fragment of the sequence shown in FIG. 6 (SEQ ID NO. XX) which contains enough of the mutant sequence to render it capable of specifically hybridizing to mutant but not wild type PROSTAPIN may be used.
  • primers designed to PCR amplify polynucleotides containing the sequences specific to the LAPC-9 mutant PROSTAPIN sequence shown in FIG. 6 may be used to detect the expression of a functional PROSTAPIN mutant.
  • primers designed to amplify polynucleotides corresponding to either wild type or mutant PROSTAPIN may be used to amplify PROSTAPIN sequences which may then be sequenced and analyzed for the presence of mutations.
  • Functional PROSTAPIN mutants may also be identified at the genomic level, by direct sequencing or by SSCP analysis of genomic DNA to identity PROSTAPIN mutations or polymorphisms that correlate with prostate cancer. Mutant or polymorphic exons can be sequenced and compared to wild type PROSTAPIN using standard technologies, in one embodiment, the primer pairs described in Example 8 may be used to sequence particular PROSTAPIN exons.
  • PROSTAPIN Loss of wild type PROSTAPIN expression or the expression of functionally mutant PROSTAPIN correlates with advanced and metastatic prostate cancer.
  • PROSTAPIN is a member of a family of proteins which contain both tumor suppressors (e.g., maspin) and proteins involved in apoptosis (e.g., LEI). Accordingly, the PROSTAPIN protein may function as a prostate-specific tumor suppressor, apoptosis-inducer or apoptosis-modulator, or may have another biological activity involved in modulating prostate cancer progression.
  • Therapeutic strategies which restore functional PROSTAPIN to prostate tumor cells may result in inhibition of primary prostate tumors and prostate cancer metastasis, tumor regression, and/or an inhibition in the rate or extent of disease progression.
  • a vector comprising a polynucleotide encoding wild type PROSTAPIN or a peptide mimetic with PROSTAPIN biological activity may be administered to the prostate cancer patient such that the vector makes contact with the prostate tumor cells.
  • the vector will be capable of integrating the PROSTAPIN gene into the patient's tumor cells (e.g., retroviral vectors) and/or is capable of highly efficient in vivo transduction (e.g., adenoviral vectors).
  • the vector may be delivered via any route which results in the vector making contact with the tumor cells.
  • a preferred route of administration is by intraprostatic injection.
  • compositions comprising the wild type PROSTAPIN protein or a peptide mimetic or a small molecule mimetic may be administered to a patient such that the composition makes contact with the tumor cells.
  • methods capable of inducing transcription of functional PROSTAPIN in vivo may be employed.
  • PROSTAPIN restoration is accomplished via gene transfer methods, such as those further described below.
  • gene transfer methods such as those further described below.
  • PROSTAPIN functions as an apoptosis-inducing gene
  • gene therapy transfer of PROSTAPIN into prostate tumor cells may be used to trigger apoptosis of the tumor cells.
  • PROSTAPIN functions as a prostate-specific tumor suppressor gene
  • in vivo PROSTAPIN gene restoration therapy may be useful to slow or reverse prostate cancer cell growth.
  • a PROSTAPIN polynucleotide encoding wild type PROSTAPIN may be operably linked to a promoter capable of driving the expression of functional PROSTAPIN within the cells of the target tumor and utilized for gene therapy.
  • expression of the PROSTAPIN gene will be regulated by a prostate-specific promoter is utilized.
  • An example of a preferred promoter is the PSA promoter.
  • Various gene therapy vectors may be used to deliver the PROSTAPIN gene into the cells of the target tissue (e.g., prostate, prostate tumors, prostate metastasis), wherein PROSTAPIN protein is expressed and exerts PROSTAPIN functionality.
  • the target tissue e.g., prostate, prostate tumors, prostate metastasis
  • viral vectors well known in the gene therapy field including but not limited to adenoviral, retroviral, and vaccinia vectors. See, for example, Jolly, D. Cancer Gene Therapy, vol. 1 , pages 51-64 (1994).
  • Preferred viral vectors include adenovirus, more preferably in non-replicating or replication defective forms.
  • replication defective adenovirus vectors in which the E1A and E1B regions of the adenovirus genome have been deleted may be used.
  • Adenovirus type 5 of subgroup C is most preferred for generating replication- defective adenovirus vectors for PROSTAPIN gene therapy, although adenoviruses of any of the 42 different serotypes or subgroups A-F may be employed.
  • various cell lines may be used to propagate recombinant adenoviruses, so long as they complement any replication defect which may be present.
  • a preferred cell line is the human 293 cell line, although other replication permissive cell lines may be employed as appropriate.
  • Other complementary combinations of viruses and host cells may be employed in connection with the present invention; for example adenovirus lacking functional E2 in combination with E2-expressing cells, adenovirus lacking functional E4 in combination with E4-expressing cells, and the like.
  • the PROSTAPIN clone 103 cDNA (SEQ ID NO. XX) is inserted into a replication defective adenovirus in which the E1A and E1B regions have been deleted.
  • Recombinant adenovirus containing the PROSTAPIN cDNA is then propagated in 293 cells and purified according to standard methods. Purified recombinant adenovirus may then be delivered to the target tissue via an appropriate route which will result in delivery of the recombinant adenovirus to the cells of the target tissue.
  • the target tissue is the prostate or a locally confined primary prostate tumor, recombinant adenovirus may be injected intraprostatically, preferably in multiple doses.
  • tissue target is one or more tumors in an individual with advanced prostate cancer
  • a more systemic route of administration either alone or in combination with a direct delivery method (e.g., intraprostatic injection) may be used.
  • a direct delivery method e.g., intraprostatic injection
  • recombinant adenovirus may be injected directly into the lymph and/or vascular system in order to target tumors within lymphatic system or bone marrow as appropriate.
  • a polynucleotide encoding a PROSTAPIN protein in which the RSL site is deleted may be used to construct an adenovirus.
  • the resulting recombinant adenovirus may be used to study PROSTAPIN function and, specifically, the function of the RSL, by comparing the activities of the RSL-deleted PROSTAPIN and wild type PROSTAPIN proteins expressed in prostate cancer and other cell lines or in appropriate animal models.
  • adenoviruses encoding wild type PROSTAPIN and RSL- deleted PROSTAPIN may be used to compare the effects of the encoded proteins on tumor cell growth by expressing these two forms of PROSTAPIN in prostate cancer xenograft models.
  • prostate cancer cells into which these forms of PROSTAPIN may be introduced by the recombinant adenoviruses include LAPC-4, LAPC- 9, LnCap, PC-3.
  • Xenograft tumors may be conveniently generated by subcutaneous, orthotopic or intraosseous injection of the vector-transduced cells into SCID or other immune deficient mice.
  • retroviral vectors in which the PROSTAPIN gene may be inserted include, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • retroviral vectors may be engineered to include a polynucleotide encoding a protein which is specifically reactive with prostate cancer cells, such as, for example, polynucleotides encoding prostate cancer ceil specific antibodies or fragments thereof.
  • PROSTAPIN polynucleotides may be delivered to target tumor and surrounding tissue via liposomes.
  • liposomes comprised of DOTMA, such as the LipofectinTM products available from Vical, Inc. (San Diego, CA) may be used.
  • a variety of transfection techniques are known and may be used.
  • injection into the site of the target tumor or systemic injection methods may be used.
  • the target tumor is a primary prostate tumor
  • direct injection int the prostate is preferred.
  • the target comprises lymph and/or bone metastases
  • injection into the lymphatic system and/or arterial system, respectively may be preferred.
  • Liposomes may be enhanced to increase their tissue specificity by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the liposome may be couples to a monoclonal antibody which recognizes a cell surface prostate tumor antigen, such as PSCA.
  • PSCA cell surface prostate tumor antigen
  • PROSTAPIN TARGET PROTEASE The target of PROSTAPIN is likely to be a protease that plays a functional role in prostate cancer metastasis.
  • the PROSTAPIN gene and/or protein may be used as tools to identify this protease.
  • One method involves screening a yeast two-hybrid cDNA library with the prostapin gene as a bait, or by screening a cDNA expression library using prostapin protein as a probe.
  • prostapin protein may be used to study the biochemical interaction with a panel of known proteases, such as: Prostate Specific Antigen, human Kallikrein 2, urokinase type plasminogen activator, tissue plasminogen activator, plasmin, granzyme B, thrombin, cathepsins B, L and D, and human neutrophil elastase.
  • proteases such as: Prostate Specific Antigen, human Kallikrein 2, urokinase type plasminogen activator, tissue plasminogen activator, plasmin, granzyme B, thrombin, cathepsins B, L and D, and human neutrophil elastase.
  • kits are also provided by the invention.
  • Such kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a probe which is or can be detectably labeled.
  • probe may be an antibody or polynucleotide specific for a PROSTAPIN protein or a PROSTAPIN gene or message, respectively.
  • the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radionucieotide label.
  • a reporter-means such as a biotin-binding protein, such as avidin or streptavidin
  • EXAMPLE 1 ISOLATION OF PROSTAPIN cDNA FRAGMENT AND EXPRESSION ANALYSIS
  • LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated as described (Klein et al, 1997, Nature Med. 3: 402-408). Androgen dependent and independent LAPC-4 xenografts LAPC-4 AD and Al, respectively) and LAPC-9 AD xenografts were grown in male SCID mice and were passaged as small tissue chunks in recipient males.
  • LAPC-4 Al xenografts were derived from LAPC-4 AD tumors. Male mice bearing LAPC-4 AD tumors were castrated and maintained for 2-3 months. After the LAPC-4 tumors re-grew, the tumors were harvested and passaged in castrated males or in female SCID mice.
  • the human cell lines HeLa (cervical carcinoma), 293 (embryonic kidney), A431 (epidermoid carcinoma), Colo205 (colon carcinoma), KCL22 (lymphoid blast crisis of chronic myelogenous leukemia), LnCaP (prostate cancer), DU145 (prostate cancer) and PC-3 (prostate cancer) were obtained from the ATCC.
  • Tumor tissue and cell lines were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/ g tissue or 10 ml/ 10 8 cells to isolate total RNA.
  • Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
  • RSACDN (cDNA synthesis primer): 5'TrTTGTACAAGCTT 30 3'
  • Adaptor 1 5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT3'
  • Nested primer (NP)1 5 CGAGCGGCCGCCCGGGCAGGT3'
  • Nested primer (NP)2 5 ⁇ GCGTGGTCGCGGCCGAGGT3'
  • Suppression Subtractive Hybridization Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes which may be down-regulated in androgen independent prostate cancer compared to androgen dependent prostate cancer.
  • Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester) and the LAPC-4 Al xenograft (driver) were synthesized from 2 ⁇ g of poiy(A) + RNA isolated from xenograft tissue, as described above, using CLONTECH 's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide RSACDN 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 Rsa I for 3 hrs. at 37°C. Digested cDNA was extracted with phenol/chloroform (1 :1 ) and ethanol precipitated.
  • LAPC-4 Al Driver cDNA
  • Tester cDNA (LAPC-4 AD) was generated by diluting 1 ⁇ l of Rsa I digested LAPC-4 AD cDNA (400 ng) in 5 ⁇ l of water. The diluted cDNA (2 ⁇ l, 160 ng) was then ligated to 2 ⁇ l of adaptor 1 and adaptor 2 (10 ⁇ M), in separate ligation reactions, in a total volume of 10 ⁇ l at 16°C overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 ⁇ l of 0.2 M EDTA and heating at 72°C for 5 min.
  • the first hybridization was performed by adding 1.5 ⁇ l (600 ng) of driver cDNA to each of two tubes containing 1.5 ⁇ l (20 ng) adaptor 1- and adaptor 2- ligated tester cDNA. In a final volume of 4 ⁇ l, the samples were overlayed with mineral oil, denatured in an MJ Research thermal cycler at 98°C for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68"C. The two hybridizations were then mixed together with an additional 1 ⁇ l of fresh denatured driver cDNA and were allowed to hybridize overnight at 68°C. The second hybridization was then diluted in 200 ⁇ l of 20 mM Hepes, pH 8.3, 50 mM NaCI, 0.2 mM EDTA, heated at 70°C for 7 min. and stored at -20°C.
  • PCR Amplification Cloning and Seguencing of Gene Fragments Generated from SSH: To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 ⁇ l of the diluted final hybridization mix was added to 1 ⁇ l of PCR primer 1 (10 ⁇ M), 0.5 ⁇ l dNTP mix (10 ⁇ M), 2.5 ⁇ l 10 x reaction buffer (CLONTECH) and 0.5 ⁇ l 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 ⁇ l.
  • PCR 1 was conducted using the following conditions: 75°C for 5 min., 94°C for 25 sec, then 27 cycles of 94°C for 10 sec, 66°C for 30 sec, 72°C for 1.5 min. 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 ⁇ l 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 ⁇ M) were used instead of PCR primer 1.
  • PCR 2 was performed using 10-12 cycles of 94°C for 10 sec, 68°C for 30 sec, 72°C for .5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
  • PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
  • Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
  • First strand cDNAs were generated from 1 ⁇ g of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturers protocol was used and included an incubation for 50 min at 42°C with reverse transcriptase followed by RNAse H treatment at 37°C for 20 min. After completing the reaction, the volume was increased to 200 ⁇ l with water prior to normalization. First strand cDNAs from 16 different normal human tissues were obtained from Clontech.
  • First strand cDNA (5 ⁇ l) was amplified in a total volume of 50 ⁇ l containing 0.4 ⁇ M primers, 0.2 ⁇ M each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCI 2 , 50 mM KCI, pH8.3) and 1X Klentaq DNA polymerase (Clontech).
  • PCR Five ⁇ l of the PCR reaction was removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis.
  • PCR was performed using an MJ Research thermal cycler under the following conditions: initial denaturation was at 94°C for 15 sec, followed 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 was carried out for 2 min.
  • the band intensities of the 283 bp ⁇ -actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal ⁇ -actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization were required to achieve equal band intensities in all tissues after 22 cycles of PCR.
  • 11 P2A6 One of the gene fragment cDNA clones showing no homology to any known gene or EST sequence was designated 11 P2A6.
  • the isolated 11 P2A6 cDNA (SEQ ID NO. XX) was 471 bp in length and has the nucleotide sequence of nucleotide residues 1 through 471 in the PROSTAPIN cDNA sequence shown in FIG. 1 (SEQ ID NO. XX).
  • RT-PCR expression analysis showed that the 11 P2A6 (PROSTAPIN) gene is expressed at approximately equal levels in the LAPC-9 AD xenograft and in normal prostate tissue, but at greatly reduced levels in the LAPC-4 Al xenograft and at undetectable levels in the LAPC-4 AD xenograft (FIG. 3, panel A).
  • RT-PCR expression analysis of first strand cDNAs from 16 normal tissues detected expression of the 11 P2A6 (PROSTAPIN) gene only in prostate tissue after 30 cycles of PCR amplification, while lower level expression was detected in lung and placenta after 35 cycles (FIG. 3, panels B and C).
  • the full length cDNA encoding the gene corresponding to the 11 P2A6 clone was isolated as follows.
  • a normal human prostate cDNA library (Clontech) was screened with a probe comprising the 11 P2A6 cDNA (SEQ ID NO. XX).
  • SEQ ID NO. XX 11 P2A6 cDNA
  • Several positive clones were identified, and the largest of these, clone 103, was sequenced.
  • Clone 103 (SEQ ID NO XX) contains an open reading frame encoding a 379 amino acid protein (see FIG. 1). Amino acid homology analysis of the clone 103 sequence revealed 30- 40% homology to a class of serine protease inhibitors known as serpins.
  • PROSTAPIN PROSTAte serine Protease INhibitor
  • the 5' untranslated region contains two translational stop signals (indicated by asterisks in FIG. 1A) upstream of the start ATG, which falls within the Kozak sequence 5'-AAA ATG G-3'.
  • a highly conserved reactive site loop characteristic of the serpin family is located in the carboxy-terminal region of PROSTAPIN (underlined sequence in FIG. 1).
  • the serpin protease inhibitory domain is known as the reactive-site loop (RSL) and is located 30-50 residues from the carboxyl-terminus.
  • the RSL is about 15-20 amino acids in length and contains a hinge region and a variable region.
  • the hinge region confers stability to the serpin-protease complex.
  • the RSL hinge region is highly conserved among the serpins.
  • the RSL variable region contains the reactive site amino acid P1 , which determines specificity of inhibition. During inhibition, the RSL binds to the protease active site, undergoes nucleophillic attack by the catalytic serine residue, resulting in cleavage of the serpin at P1-P1'.
  • PROSTAPIN RSL hinge region amino acid sequence (GTEAAAATG) is highly homologous to all other serpins analyzed, with the exception of maspin (FIG. 2).
  • the PROSTAPIN RSL variable region amino acid sequence is distinct, with a lysine at P1 and a serine at P1'. This indicates that PROSTAPIN most likely targets a different protease than do the other serpins.
  • PROSTAPIN clone 103 (SEQ ID NO. XX) has been deposited with the American Type Culture Collection (“ATCC”) (Mannassas, VA) as plasmid pProstapin on May 15, 1998 as ATCC Accession Number 98757.
  • ATCC American Type Culture Collection
  • VA canssas, VA
  • EXAMPLE 3 NORTHERN BLOT ANALYSIS OF PROSTAPIN EXPRESSION
  • Example 4 Northern blot analysis on panels of normal human and prostate tumor xenograft tissues using a labeled PROSTAPIN clone 103 (SEQ ID NO. XX) probe were conducted to confirm the prostate specificity of PROSTAPIN expression initially established by RT-PCR expression analysis (see Example 1). Further Northern blot analysis of PROSTAPIN expression is described in Example 4.
  • FIG. 4 Two panels of normal human tissues were evaluated. The results from one of the panels, which contained 16 normal human tissues, are shown in FIG. 4 (Panels A & B). in this first panel, PROSTAPIN RNA was only detected in prostate, expressed as two distinct transcripts of about 2.3 and 3.0 kb. To extend this analysis, the clone 103 probe was used to analyze a second normal tissue panel, comprising an RNA dot blot matrix of 37 normal human tissues (Clontech, Palo Alto, CA; Human Master BlotTM). The results, shown in FIG. 4 (Panel C), show PROSTAPIN expression only in prostate and trachea.
  • No expression signal was detected in brain, spinal chord, heart, aorta, skeletal muscle, colon, bladder, uterus, stomach, testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland, salivary gland, mammary gland, kidney, liver, small intestine, spleen, thymus, peripheral leukocytes, lymph node, bone marrow, appendix, lung, placenta, fetal brain, fetal heart, fetal kidney, fetal liver, fetal spleen, fetal thymus, or fetal lung.
  • PROSTAPIN expression in various normal and prostate cancer clinical tissue samples was conducted by semi-quantitative RT-PCR in order to further examine the question of whether loss of PROSTAPIN expression correlates with advanced prostate cancer grade and clinical stage.
  • Concurrent analysis of PROSTAPIN expression in the LAPC xenografts and several prostate cancer cell lines was conducted.
  • TMN staging of the human prostate tumor tissues used in this analysis was as follows: (1) Stage T2c, tumor is confined to the prostate and involves both lobes; (2) Stage T3a, tumor extends through the prostate capsule; (3) Stage T3c, extracapsular extension with invasion of seminal vesicles.
  • the human tissue samples included normal prostate, prostate cancer, and 4 examples of prostate cancer together with their matched normal controls (i.e., from the same patient).
  • First strand cDNA was prepared from these tissues as well as the LAPC xenografts, prostate cancer cell lines (LNCaP, PC3, DU145) and normal prostate mRNA (obtained from Clontech and BioChain).
  • PCR analysis was performed on the normalized cDNAs using the primers described in Example 1 (i.e., SEQ ID NOS. XX and XX).
  • results show that PROSTAPIN is expressed in all normal prostate tissues and the prostate tumor specimens derived from patients with localized disease, but that PROSTAPIN expression is lost or dramatically attenuated in all tumor specimens from patients with extracapsular extension and advanced disease (FIG. 5, lanes 20 and 22).
  • results show complete absence of PROSTAPIN expression in all of the prostate cancer cell lines and the LAPC-4 xenografts (FIG. 5, lanes 6, 7, 9, 10, 1). All three of the prostate cancer cell lines originated from advanced prostate cancer patient metastasis tumors.
  • LnCaP was derived from a prostate cancer lymph node metastasis
  • PC-3 was derived from a prostate cancer bone metastasis
  • DU-145 was derived from a prostate cancer brain metastasis.
  • the LAPC-4 xenograft was derived from a lymph node metastasis. Consistent with the RT-PCR and Northern blot results obtained in Examples 1 and 3, respectively, a PROSTAPIN transcript was detected in the LAPC-9 xenograft.
  • the PROSTAPIN transcript expressed in the LAPC-9 xenograft is a substantially mutated, partially unspliced variant which includes point mutations, a stop codon in what would be the center of the wild type PROSTAPIN sequence, and an unspliced intron.
  • this PROSTAPIN mutant if any has not been characterized, it is likely that wild type PROSTAPIN functionality is either substantially altered or, more likely, completely lost.
  • LAPC-9 also represents advanced stage disease, as it was generated from a bone tumor biopsy of a patient with hormone-refractory metastatic prostate cancer.
  • the data obtained from this combined analysis indicates that loss of wild type PROSTAPIN expression and/or expression of a functional mutation correlates with advanced stage and metastatic prostate cancer. All prostate cancer cell lines, xenografts and patient samples derived from advanced stage tumors or metastasis show a complete lack or sharp attenuation of wild type PROSTAPIN expression. Moreover, it appears that loss of PROSTAPIN expression may coincide with the development of metastatic disease, since expression is lost even at the lower grade/stage of extracapsular disease. Loss of functional PROSTAPIN may be one of the first molecular events coinciding with and/or leading to the development of metastasis.
  • Clone 2 cDNA (SEQ ID NO XX) comprises 2472 bp and has the nucleotide and deduced amino acid sequences shown in (FIG. 6). Further analysis determined that clone 2 represents a partially unspliced version of the PROSTAPIN message with an intron of 714 bp and point mutations at positions 298 (G/T), 474 (TIC), 572 (A/G), 593 (AU), 1567 (MC), 1584 (T/C), 1613 (C/T), 1822 (G/T), 2085 (C/T) in FIG. 6 (clone 2 - SEQ ID NO. XX).
  • clone 2 contains several point mutations, one of which results in a stop codon at amino acid residue 90 in the open reading frame (FIG. 6). All point mutations were confirmed by sequencing of RT-PCR products of first strand cDNA derived from LAPC-9 AD.
  • cDNA clone 103 (SEQ ID NO. XX) was cloned into the pcDNA 3.1 Myc-His plasmid (Invitrogen) (which encodes a 6His tag at the carboxyl-terminus), transfected into 293T cells, and analyzed by subcellular fractionation. More specifically, the sequence encoding the PROSTAPIN ORF from PROSTAPIN clone 103 cDNA (SEQ ID NO. XX) was tagged with a 6His tag at the carboxyl- terminus and was transfected into 293T cells. Cell lysates were prepared by dounce homogenization of cells in a hypotonic buffer.
  • Cellular debris was removed by low speed centrifugation (10,000 X g) and the supernatant containing the cytosol and light membrane fraction was separated by a 100,000 X g centrifugation. Equal amounts of protein from each fraction were analyzed using an anti-His antibody (Santa Cruz) that recognizes recombinant PROSTAPIN or an anti-SV 40 large T antibody (Santa Cruz). Cell conditioned media (Cell sup) was also analyzed to identify any secreted PROSTAPIN protein.
  • PROSTAPIN The chromosomal localization of PROSTAPIN was determined using the GeneBridge 4 Human/Hamster radiation hybrid (RH) panel (Walter et al., 1994, Nat. Genetics 7:22) (Research Genetics, Huntsville Al). The following PCR primers, which amplify a 148 bp PROSTAPIN product, were employed:
  • the PROSTAPIN gene co-localizes to the 500 kb 18q21.3 region along with 6 other serpin family members, including maspin, leupin and bomapin (Bartuski et al., 1997, Genomics 43:321).
  • the region of 18q21.3 has been associated with loss of heterozygosity (LOH) in advanced metastatic prostate cancer and in recurrent disease (Brothman et al., 1999, The Prostate 38:303).
  • Loss of expression of PROSTAPIN alone or in combination with another serpin may contribute to the growth characteristics and invasiveness of aggressive prostate cancer. It is interesting to note that maspin expression in the prostate and in the LAPC xenografts (determined by RT-PCR) mimics the expression of PROSTAPIN. It may be that some of the genes in this serpin cluster at 18q21 may exhibit coordinate regulation of expression.
  • EXAMPLE 8 IDENTIFICATION OF INTRON-EXON BOUNDARIES OF PROSTAPIN GENE
  • Genomic PROSTAPIN clones were isolated by screening the human BAC library CID (Cal. Tech. library D, purchased from Research Genetics) and a human PAC library (release I, Peter deJong, University of New York, Buffalo). Four positive clones were obtained: 2062H14, 2074J2, 2100K19 from the BAC library; 152i22 from the PAC library. These clones were confirmed by Southern blot analysis (FIG. 9) using PROSTAPIN clone 103 cDNA probe (SEQ ID NO. XX).
  • PROSTAPIN gene was found to contain seven exons and six introns within the region of the coding sequence (FIG 10).
  • ProsEi atgggttctctcagcacagctaacg (SEQ ID NO. XX) (within exon, begins at start site)
  • Pros2 aattaattttgctgacccagagcg (SEQ ID NO. XX)
  • Prosl 2 aatcaatgactacgctaatgtcatgag (SEQ ID NO. XX)
  • Sheep polyclonal anti-PROSTAPIN antibodies were generated using a purified GST- PROSTAPIN fusion protein.
  • the fusion protein contains amino acid residues 1-106 from the PROSTAPIN sequence and was generated by PCR using the following primers:
  • GST-pros5' gtggatccatgggttctctcagca (underlined sequence: BamHI site)
  • GST-pros3 atacccgggtggcaatgctgagg (underlined sequence: Smal site)
  • the PCR product was inserted directionally into a pGEX-4T (GST-fusion) vector (Pharmacia). Fusion protein was produced in bacteria and purified using a Glutathione- Sepharose column (Pharmacia).
  • Immune serum was affinity purified using a GST-PROSTAPIN column. Purified antibody was used to probe western blots of lysates from several cell lines including: 293T cells transfected with pcDNA vector with or without His-tagged PROSTAPIN, TsuPrl cells infected with retrovirus generated with the retroviral expression vector pSR ⁇ tkneo with or without PROSTAPIN (FIG. 12). The results show that the anti-PROSTAPIN antibody recognizes His-tagged PROSTAPIN with similar intensity as an anti-His antibody. In addition, untagged PROSTAPIN expressed in TsuPrl cells is also recognized with high efficiency and specificity.

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Abstract

L'invention concerne un nouveau gène et une nouvelle protéine exprimés dans une prostate normale et dans la prostate atteinte d'un cancer confiné localement et appelés PROSTAPINE. Dans les cas de cancers de prostate avancés, l'expression de la PROSTAPINE est perdue ou atténuée. Font aussi l'objet de cette invention des séquences d'acides aminés et d'ADN complémentaire codant pour la PROSTAPINE, des vecteurs et des cellules hôtes contenant de polynucléotides de PROSTAPINE, des anticorps réagissant spécifiquement à la PROSTAPINE et différentes compositions associées utiles pour la détection, le diagnostic, le pronostic, le phasage, la surveillance, le traitement et la détermination de la sensibilité au cancer de la prostate, plus particulièrement au cancer de la prostate métastatique à un stade avancé.
PCT/US1999/007123 1998-03-31 1999-03-31 Gene et proteine prostapine ainsi que leurs applications Ceased WO1999058560A2 (fr)

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
26757: "Molecular cloning of Bomapin (protease inhibitor 10), a novel human Serpin that is expressed specifically in the bone marrow" JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 270, no. 45, 10 November 1995 (1995-11-10), page 26754 XP002120374 AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD., US ISSN: 0021-9258 cited in the application *
A.J. BARTUSKI ET AL: "cytoplasmic antiproteinase 2 (PI8) and Bomapin (PI10) map to the serpin cluster at 18q21.3" GENOMICS, vol. 43, no. 3, 1 August 1997 (1997-08-01), pages 321-328, XP002120372 cited in the application *
G.G. MAHAIRAS ET AL: EMBL DATABASE ENTRY D44876, ACCESSION NUMBER B44876, 31 October 1997 (1997-10-31), XP002120371 & UNPUBLISHED, *
R.S. HUBERT ET AL: "Identification of differentially expressed genes using prostate cancer xenograft models" INTERNATIONAL SYMPOSIUM ON BIOLOGY OF PROSTATE GROWTH, 15 - 18 March 1998, XP002120376 bethesda, Maryland, USA & THE PROSTATE, vol. 38, no. 4, March 1999 (1999-03), page 343 *
VERKAIK N S ET AL: "CLINICAL USEFULNESS OF RT-PCR DETECTION OF HEMATOGENOUS PROSTATE CANCER SPREAD" UROLOGICAL RESEARCH, vol. 25, no. 6, 1 December 1997 (1997-12-01), pages 373-384, XP002910109 ISSN: 0300-5623 cited in the application *
Z. ZOU ET AL: "Maspin, a Serpin with Tumor-suppressing activity in human mammary epithelial cells" SCIENCE., vol. 263, 28 January 1994 (1994-01-28), pages 526-529, XP002120373 AAAS. LANCASTER, PA., US cited in the application *

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