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WO2000020584A2 - Gene human 36p1a6 exprime dans le cancer de la prostate, de la vessie, du pancreas et du colon - Google Patents

Gene human 36p1a6 exprime dans le cancer de la prostate, de la vessie, du pancreas et du colon Download PDF

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WO2000020584A2
WO2000020584A2 PCT/US1999/022576 US9922576W WO0020584A2 WO 2000020584 A2 WO2000020584 A2 WO 2000020584A2 US 9922576 W US9922576 W US 9922576W WO 0020584 A2 WO0020584 A2 WO 0020584A2
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cancer
protein
polynucleotide
mrna
prostate
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WO2000020584A9 (fr
WO2000020584A3 (fr
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Daniel E. Afar
Rene S. Hubert
Stephen Chappell Mitchell
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Agensys Inc
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Urogenesys Inc
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Priority to CA002344552A priority patent/CA2344552A1/fr
Priority to IL14231099A priority patent/IL142310A0/xx
Publication of WO2000020584A2 publication Critical patent/WO2000020584A2/fr
Publication of WO2000020584A3 publication Critical patent/WO2000020584A3/fr
Publication of WO2000020584A9 publication Critical patent/WO2000020584A9/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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention described herein relates to a novel gene and its encoded protein, termed 36P1 A6, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers which express 36P1A6, particularly including prostate, bladder, pancreatic, colon, cervical and ovarian cancers.
  • Cancer is the second leading cause of human death next to coronary disease.
  • millions of people die from cancer every year.
  • cancer causes the death of well over a half-million people annually, with some 1.4 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise.
  • cancer is predicted to become the leading cause of death.
  • carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Many cancer patients experience a recurrence.
  • prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common male cancer and is the second leading cause of cancer death in men. In the United States alone, well over 40,000 men die annually of this disease - second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
  • the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the management of this disease.
  • the serum PSA assay has been a very useful tool, its specificity and general utility is widely regarded as lacking in several important respects.
  • DRE digital rectal examinations
  • PSA prostate specific antigen
  • TRUS transrectal ultrasonography
  • TRNB transrectal needle biopsy
  • PSA is the most widely used tumor marker for screening, diagnosis, and monitoring prostate cancer today.
  • several immunoassays for the detection of serum PSA are in widespread clinical use.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • PSA is not a disease-specific marker, as elevated levels of PSA are detectable in a large percentage of patients with BPH and prostatitis (25-86%)(Gao et al., 1997, Prostate 31 : 264-281), as well as in other nonmalignant disorders and in some normal men, a factor which significantly limits the diagnostic specificity of this marker.
  • PSA diagnostics have sensitivities of between 57-79% (Cupp & Osterling, 1993, Mayo Clin Proc 68:297-306), and thus miss identifying prostate cancer in a significant population of men with the disease.
  • PSM prostate specific membrane antigen
  • PSCA a GPI-linked cell surface molecule
  • the present invention relates to a novel gene, designated 36P1 A6, which is expressed or over-expressed in a number of human cancers.
  • This gene appears to be expressed in several normal tissues, including particularly, prostate and colon, with lower levels detected in pancreas, kidney, lung and small intestine.
  • Expression or overexpression of the 36P1A6 gene occurs in cancers of the prostate, bladder, pancreas, colon, cervix and ovary, and perhaps in other cancers. More specifically, high level expression of this gene is detected in a number of prostate cancer xenograft tumors as well as in several pancreatic, colon, ovary and bladder carcinoma cells. Expression is also detected in other tumor cell lines.
  • 36P1 A6 may encode the human homolog of the murine EHF gene (Bochert et al., 1998, Biochem Biophys Res Commun 246(1 ):176-81), a member of the ETS family of transcription factors.
  • the nucleotide and deduced amino acid sequences of the full length 36P1 A6 cDNA are shown in FIG. 1A.
  • the isolated 36P1A6 gene contains an ETS homology region (which corresponds to its DNA binding domain), and a pointed domain (which probably functions as a protein-protein interaction domain).
  • ESX an epithelial specific ETS family member that is up-regulated in breast cancer (Chang et al., 1997,Oncogene 14(13):1617-22).
  • the highest homology to ESX lies in the ETS homology region.
  • 36P1A6 Aberrant expression of 36P1A6 in cancer may result in modulated transcription of genes involved in the development and/or progression of cancers expressing 36P1 A6.
  • a number of potential approaches to the diagnosis and treatment of cancers expressing 36P1A6 involving the inhibition of 36P1A6 function are therefore possible.
  • the 36P1A6 transcript, protein and/or factors which bind to or modulate the expression or function of this gene or its encoded protein may represent a useful diagnostic marker and/or therapeutic target for a number of different cancers.
  • the invention provides polynucleotides corresponding or complementary to all or part of the 36P1A6 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 36P1A6 proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to the 36P1A6 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides which hybridize to the 36P1A6 genes, mRNAs, or to 36P1 A6-encoding polynucleotides.
  • Recombinant DNA molecules containing 36P1A6 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 36P1 A6 gene products are also provided.
  • the invention further provides 36P1A6 proteins and polypeptide fragments thereof.
  • the invention further provides antibodies that bind to 36P1A6 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, antibodies labeled with a detectable marker.
  • the invention further provides methods for detecting the presence of 36P1A6 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 36P1A6.
  • the invention further provides various therapeutic compositions and strategies for treating cancers which express 36P1A6 such as prostate, bladder, colon, pancreatic, cervical and ovarian cancers, including therapies aimed at inhibiting the transcription, translation, processing or function of 36P1A6 and cancer vaccines.
  • FIG. 1A Nucleotide and deduced amino acid sequences of a full length cDNA encoding the 36P1A6 gene. The start methionine and putative Kozak sequence is indicated in bold, the ETS homology region is boxed, and the pointed domain is underlined.
  • FIG. 1 B Nucleotide sequence of SSH-isolated 36P1A6 cDN A fragment.
  • FIG. 2 RT-PCR analysis of 36P1A6 gene expression in prostate cancer xenografts, normal prostate, and other tissues and cell lines, showing expression in prostate cancer xenografts and normal prostate at approximately equal levels (Panel A); and showing expression in prostate, lung and pancreas in normal tissues at 30 cycles of PCR amplification, and lower level expression in several other tissues at 35 cycles (Panels B and C).
  • FIG. 3 Northern blot analyses of 36P1 A6 expression in various normal human tissues and prostate cancer xenografts, showing predominant expression of 36P1A6 in prostate and colon, with lower levels detected in pancreas, kidney, lung and small intestine, and showing upregulated expression of 36P1A6 in the LAPC-9 and LAPC-4 xenografts (AD, Al, grown subcutaneously and intra-tibially), when compared to normal prostate.
  • AD Al
  • FIG. 4 Expression of 36P1A6 in prostate and multiple cancer cell lines and prostate cancer xenografts.
  • Xenograft and cell line filters were prepared with 10 ⁇ g of total RNA per lane.
  • the blots were analyzed using the full length 36P1A6 cDNA as probe. All RNA samples were normalized by ethidium bromide staining and subsequent analysis with a ⁇ -actin probe. Lanes are as indicated on the figure.
  • FIG. 5 Amino acid sequence alignments of the 36P1 A6 ORF with murine EHF (A) and the human ESX pointed domain (B) and ETS homology domain (C).
  • 36P1A6 shows 93.7% identity with murine EHF across a 300 residue overlap (Score: 1556.0; Gap frequency: 0.0%) and shows 81.8% identity to the hESX ETS homology domain across a 99 residue overlap (Score: 453.0; Gap frequency: 0.0%).
  • the present invention relates to a novel gene and protein, designated 36P1A6.
  • the invention is based, in part, on the identification of the 36P1A6 gene and on the characterization of the 36P1 A6 gene expression patterns in various cancers.
  • FIG. 1A A full length cDNA encoding the 36P1A6 gene has been isolated.
  • the nucleotide and deduced amino acid sequences of this 36P1A6 cDNA are shown in FIG. 1A.
  • the nucleotide sequence of the initially isolated cD A fragment corresponding to and identifying the 36P1A6 gene is provided in FIG. 1B.
  • 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 osteoblastic 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 radionuclide 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 0.1XSSC/0.1% SDS are above 55 degrees C, and most preferably to stringent hybridization conditions.
  • identity is used to express the percentage of amino acid residues at the same relative position which are the same.
  • homology is used to express the percentage of amino acid residues at the same relative positions which are either identical or are similar, using the conserved amino acid criteria of BLAST analysis, as is generally understood in the art. Further details regarding amino acid substitutions, which are considered conservative under such criteria, are provided below.
  • One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 36P1A6 gene, mRNA, and/or coding sequence. preferably in isolated form, including polynucleotides encoding a 36P1A6 protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 36P1A6 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides which hybridize to a 36P1 A6 gene, mRNA, or to a 36P1 A6-encoding polynucleotide (collectively, "36P1 A6 polynucleotides").
  • the 36P1A6 gene and protein is meant to include the 36P1 A6 gene and protein specifically described herein and the genes and proteins corresponding to other 36P1A6 proteins and structurally similar variants of the foregoing.
  • Such other 36P1A6 proteins and variants will generally have coding sequences which are highly homologous to the 36P1 A6 coding sequence.
  • a 36P1A6 polynucleotide may comprise a polynucleotide having the nucleotide sequence of human 36P1A6 as shown in FIG. 1 A, wherein T can also be U; a polynucleotide which encodes all or part of the 36P1A6 protein show in FIG. 1A; a sequence complementary to the foregoing; or a polynucleotide fragment of any of the foregoing.
  • Another embodiment comprises a polynucleotide having the sequence as shown in FIG. 1A, from nucleotide residue number 113 through nucleotide residue number 1012, wherein T can also be U.
  • Another embodiment comprises a polynucleotide encoding a 36P1A6 polypeptide whose sequence is encoded by the cDNA contained in the plasmid as deposited with American Type Culture Collection as Accession No. 207198.
  • Another embodiment comprises a polynucleotide which is capable of hybridizing under stringent hybridization conditions to the human 36P1A6 cDN A shown in FIG. 1 A or to a polynucleotide fragment thereof.
  • genomic DNA e.g., genomic DNA, cDNAs, ribozymes, and antisense molecules
  • nucleic acid molecules based on an alternative backbone or including alternative bases, whether derived from natural sources or synthesized.
  • 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 may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • a detectable marker such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • Such probes and primers can be used to detect the presence of a 36P1 A6 polynucleotide in a sample and as a means for detecting a cell expressing a 36P1A6 protein.
  • probes include polypeptides comprising all or part of the human 36P1 A6 cDNA sequence shown in FIG. 1 A.
  • primer pairs capable of specifically amplifying 36P1A6 mRNAs are also described in the Examples which follow.
  • primers and probes may be prepared based on the sequences provided in herein and used effectively to amplify and/or detect a 36P1 A6 mRNA.
  • 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 36P1A6 gene or which encode polypeptides other than 36P1A6 gene product or fragments thereof.
  • a skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 36P1 A6 polynucleotide.
  • the 36P1A6 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 36P1A6 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 36P1A6 polypeptides; as tools for modulating or inhibiting the expression of the 36P1 A6 gene(s) and/or translation of the 36P1 A6 transcript(s); and as therapeutic agents.
  • the 36P1A6 cDNA sequences described herein enable the isolation of other polynucleotides encoding 36P1A6 gene product(s), as well as the isolation of polynucleotides encoding 36P1A6 gene product homologues, alternatively spliced isoforms, allelic variants, and mutant forms of the 36P1A6 gene product.
  • Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 36P1 A6 gene are well known (See, for example, Sambrook, J.
  • the 36P1A6 gene itself may be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 36P1 A6 DNA probes or primers.
  • BACs bacterial artificial chromosome libraries
  • YACs yeast artificial chromosome libraries
  • the invention also provides recombinant DNA or RNA molecules containing a 36P1A6 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 36P1A6 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 or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive 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 a 36P1 A6 may be used to generate 36P1 A6 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
  • a wide range of host-vector systems suitable for the expression of 36P1A6 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra).
  • Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSR tkneo (Muller et al., 1991 , MCB 11 :1785).
  • 36P1A6 may be preferably expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1 , 3T3, PC-3, LNCaP and TsuPrl .
  • the host-vector systems of the invention are useful for the production of a 36P1 A6 protein or fragment thereof. Such host-vector systems may be employed to study the functional properties of 36P1 A6 and 36P1 A6 mutations.
  • Recombinant human 36P1A6 protein may be produced by mammalian cells transfected with a construct encoding 36P1 A6.
  • 293T cells are transfected with an expression plasmid encoding 36P1A6, the 36P1 A6 protein is expressed in the 293T cells, and the recombinant 36P1A6 protein is isolated using standard purification methods (e.g., affinity purification using anti-36P1A6 antibodies).
  • the 36P1A6 coding sequence is subcloned into the retroviral vector pSR ⁇ MSVtkneo and used to infect various mammalian cell lines, including 3T3CL7, PC3 and LnCaP in order to establish 36P1A6 expressing cell lines.
  • various mammalian cell lines including 3T3CL7, PC3 and LnCaP
  • Various other expression systems well known in the art may also be employed.
  • Expression constructs encoding a leader peptide joined in frame to the 36P1 A6 coding sequence may be used for the generation of a secreted form of recombinant 36P1 A6 protein.
  • Proteins encoded by the 36P1 A6 genes, or by fragments thereof, will have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents (i.e., other ETS family members) and cellular constituents that bind to a 36P1A6 gene product.
  • Antibodies raised against a 36P1A6 protein or fragment thereof may be useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 36P1 A6 protein, including but not limited to cancers of the prostate, bladder, ovary, cervix, pancreas and colon. Such antibodies may be expressed intracellularly and used in methods of treating patients with such cancers.
  • 36P1 A6 proteins Various immunological assays useful for the detection of 36P1 A6 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.
  • ELISA enzyme-linked immunosorbent assays
  • ELIFA enzyme-linked immunofluorescent assays
  • Such antibodies may be labeled and used as immunological imaging reagents capable of detecting 36P1A6 expressing cells (e.g., in radioscintigraphic imaging methods).
  • 36P1A6 proteins may also be particularly useful in generating cancer vaccines, as further described below.
  • 36P1A6 PROTEINS Another aspect of the present invention provides 36P1A6 proteins and polypeptide fragments thereof.
  • the 36P1A6 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. Fusion proteins which combine parts of different 36P1 A6 proteins or fragments thereof, as well as fusion proteins of a 36P1A6 protein and a heterologous polypeptide are also included.
  • Such 36P1A6 proteins will be collectively referred to as the 36P1A6 proteins, the proteins of the invention, or 36P1A6.
  • 36P1A6 polypeptide refers to a polypeptide fragment or a 36P1A6 protein of at least 10 amino acids, preferably at least 15 amino acids.
  • a specific embodiment of a 36P1A6 protein comprises a polypeptide having the amino acid sequence of human 36P1 A6 as shown in FIG. 1 A.
  • allelic variants of human 36P1 A6 will share a high degree of structural identity and homology (e.g., 90% or more identity).
  • allelic variants of the 36P1A6 proteins will contain conservative amino acid substitutions within the 36P1 A6 sequences described herein or will contain a substitution of an amino acid from a corresponding position in a 36P1A6 homologue.
  • One class of 36P1A6 allelic variants will be proteins that share a high degree of homology with at least a small region of a particular 36P1 A6 amino acid sequence, but will further contain a radical departure form the sequence, such as a non-conservative substitution, truncation, insertion or frame shift.
  • 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), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for 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 valine (V).
  • Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine.
  • 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.
  • 36P1 A6 proteins may be embodied in many forms, preferably in isolated form.
  • a protein is said to be "isolated” when physical, mechanical or chemical methods are employed to remove the 36P1 A6 protein from cellular constituents that are normally associated with the protein.
  • a skilled artisan can readily employ standard purification methods to obtain an isolated 36P1A6 protein.
  • a purified 36P1 A6 protein molecule will be substantially free of other proteins or molecules which impair the binding of 36P1 A6 to antibody or other ligand. The nature and degree of isolation and purification will depend on the intended use.
  • Embodiments of a 36P1A6 protein include a purified 36P1 A6 protein and a functional, soluble 36P1 A6 protein. In one form, such functional, soluble 36P1A6 proteins or fragments thereof retain the ability to bind antibody or other ligand.
  • the invention also provides 36P1A6 polypeptides comprising biologically active fragments of the 36P1 A6 amino acid sequence, such as a polypeptide corresponding to part of the amino acid sequence of 36P1 A6 as shown in FIG. 1 A.
  • Such polypeptides of the invention exhibit properties of the 36P1A6 protein, such as the ability to elicit the generation of antibodies which specifically bind an epitope associated with the 36P1A6 protein.
  • 36P1A6 polypeptides can be generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art based on the amino acid sequences of the human 36P1A6 proteins disclosed herein. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a polypeptide fragment of a 36P1A6 protein. In this regard, the 36P1A6-encoding nucleic acid molecules described herein provide means for generating defined fragments of 36P1 A6 proteins.
  • 36P1A6 polypeptides are particularly useful in generating and characterizing domain specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 36P1A6 protein), in identifying agents or cellular factors that bind to 36P1 A6 or a particular structural domain thereof, and in various therapeutic contexts, including but not limited to cancer vaccines.
  • domain specific antibodies e.g., antibodies recognizing an extracellular or intracellular epitope of a 36P1A6 protein
  • 36P1A6 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-36P1A6 antibodies or in identifying cellular factors that bind to 36P1A6.
  • Another aspect of the invention provides antibodies that bind to 36P1A6 proteins and polypeptides.
  • the most preferred antibodies will specifically bind to a 36P1A6 protein and will not bind (or will bind weakly) to non-36P1A6 proteins and polypeptides.
  • Anti- 36P1A6 antibodies that are particularly contemplated include monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more complementarity 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.
  • 36P1A6 antibodies of the invention may be particularly useful in cancer diagnostic and prognostic assays, and imaging methodologies.
  • Intracellularly expressed antibodies e.g., single chain antibodies
  • 36P1A6 antibodies may be therapeutically useful in treating cancers in which the expression of 36P1A6 is involved, such as prostate, bladder, pancreatic, colon, cervical and ovarian cancers.
  • such antibodies may be useful in diagnosis and/or prognosis of prostate, bladder, pancreatic, colon, cervical and ovarian cancers.
  • the invention also provides various immunological assays useful for the detection and quantification of 36P1A6 and mutant 36P1A6 proteins and polypeptides.
  • Such assays generally comprise one or more 36P1 A6 antibodies capable of recognizing and binding a 36P1A6 protein 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 cancers expressing 36P1A6 (e.g., cancers of the prostate, bladder, ovary, cervix, pancreas, colon) are also provided by the invention, including but limited to radioscintigraphic imaging methods using labeled 36P1 A6 antibodies. Such assays may be clinically useful in the detection, monitoring, and prognosis of 36P1A6 expressing cancers.
  • 36P1A6 antibodies may also be used in methods for purifying 36P1A6 and mutant 36P1A6 proteins and polypeptides and for isolating 36P1A6 homologues and related molecules.
  • the method of purifying a 36P1A6 protein comprises incubating a 36P1 A6 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing 36P1 A6 under conditions which permit the 36P1 A6 antibody to bind to 36P1A6; washing the solid matrix to eliminate impurities; and eluting the 36P1A6 from the coupled antibody.
  • Other uses of the 36P1A6 antibodies of the invention include generating anti-idiotypic antibodies that mimic the 36P1 A6 protein.
  • antibodies may be prepared by immunizing a suitable mammalian host using a 36P1A6 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 36P1 A6 may also be used, such as a 36P1A6 GST-fusion protein.
  • a GST fusion protein comprising all or most of the open reading frame amino acid sequence of FIG. 1A may be produced and used as an immunogen to generate appropriate antibodies.
  • a 36P1 A6 peptide may be synthesized and used as an immunogen.
  • naked DNA immunization techniques known in the art may be used (with or without purified 36P1A6 protein or 36P1A6 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).
  • the amino acid sequence of 36P1 A6 as shown in FIG. 1 A may be used to select specific regions of the 36P1A6 protein for generating antibodies.
  • hydrophobicity and hydrophilicity analyses of the 36P1A6 amino acid sequence may be used to identify hydrophilic regions in the 36P1A6 structure.
  • Regions of the 36P1 A6 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.
  • Methods for the generation of 36P1 A6 antibodies are further illustrated by way of the examples provided herein.
  • 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 36P1A6 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.
  • 36P1A6 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 hybridoma technology of Kohler and Milstein or modifications which immortalize producing B cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the 36P1A6 protein or a 36P1A6 fragment.
  • the cells may be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the 36P1 A6 protein can also be produced in the context of chimeric or CDR grafted antibodies of multiple species origin. Humanized or human 36P1A6 antibodies may also be produced and are preferred for use in therapeutic contexts.
  • Fully human 36P1A6 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, id., pp 65- 82).
  • large human Ig gene combinatorial libraries i.e., phage display
  • Fully human 36P1A6 monoclonal antibodies may also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Kucherlapati and 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 36P1A6 antibodies with a 36P1A6 protein may be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 36P1A6 proteins, peptides, 36P1A6- expressing cells or extracts thereof.
  • a 36P1A6 antibody or fragment thereof of the invention may be labeled with a detectable marker or conjugated to a second molecule.
  • detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme.
  • bi-specific antibodies specific for two or more 36P1A6 epitopes may be generated using methods generally known in the art. Homodimeric antibodies may also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).
  • Another aspect of the present invention relates to methods for detecting 36P1A6 polynucleotides and 36P1A6 proteins, as well as methods for identifying a cell which expresses 36P1A6.
  • the invention provides assays for the detection of 36P1A6 polynucleotides in a biological sample, such as serum, bone, prostate, colon, ovary, cervix, bladder, pancreas, and other tissues, urine, semen, cell preparations, and the like.
  • Detectable 36P1A6 polynucleotides include, for example, a 36P1A6 gene or fragments thereof, 36P1A6 mRNA, alternative splice variant 36P1A6 mRNAs, and recombinant DNA or RNA molecules containing a 36P1A6 polynucleotide.
  • a number of methods for amplifying and/or detecting the presence of 36P1A6 polynucleotides are well known in the art and may be employed in the practice of this aspect of the invention.
  • a method for detecting a 36P1A6 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cD A so produced using a 36P1A6 polynucleotides as sense and antisense primers to amplify 36P1A6 cDNAs therein; and detecting the presence of the amplified 36P1A6 cDNA.
  • a method of detecting a 36P1 A6 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 36P1A6 polynucleotides as sense and antisense primers to amplify the 36P1A6 gene therein; and detecting the presence of the amplified 36P1A6 gene.
  • Any number of appropriate sense and antisense probe combinations may be designed from the nucleotide sequence provided for 36P1 A6 (FIG. 1 A) and used for this purpose.
  • the invention also provides assays for detecting the presence of a 36P1A6 protein in a tissue of other biological sample such as serum, bone, prostate, colon, ovary, cervix, bladder, pancreas, and other tissues, urine, cell preparations, and the like.
  • Methods for detecting a 36P1A6 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 36P1A6 protein in a biological sample comprises first contacting the sample with a 36P1A6 antibody, a 36P1A6-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 36P1A6 antibody; and then detecting the binding of 36P1 A6 protein in the sample thereto.
  • an assay for identifying a cell which expresses a 36P1A6 gene comprises detecting the presence of 36P1A6 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 36P1A6 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 36P1A6, 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 36P1A6 gene comprises detecting the presence of 36P1 A6 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 36P1 A6 proteins and 36P1 A6 expressing cells.
  • 36P1A6 expression analysis may also be useful as a tool for identifying and evaluating agents which modulate 36P1A6 gene expression.
  • 36P1 A6-1 expression is significantly upregulated in prostate cancer and other cancers. Identification of a molecule or biological agent that could inhibit 36P1A6-1 over-expression may be of therapeutic value in the treatment of such cancers. Such an agent may be identified by using a screen that quantifies 36P1A6 expression by RT-PCR, nucleic acid hybridization or antibody binding.
  • Determining the status of 36P1A6 expression patterns in an individual may be used to diagnose cancer and may provide prognostic information useful in defining appropriate therapeutic options.
  • the expression status of 36P1 A6 may provide information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness.
  • the invention provides methods and assays for determining 36P1A6 expression status and diagnosing cancers which express 36P1A6, such as cancers of the prostate, bladder, ovary, colon, cervix and pancreas.
  • 36P1A6 expression status in patient samples may be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, western blot analysis of clinical samples and cell lines, and tissue array analysis.
  • the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 36P1A6 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue.
  • the presence of 36P1A6 mRNA may, for example, be evaluated in tissue samples including but not limited to colon, lung, prostate, pancreas, bladder, breast, ovary, cervix, testis, head and neck, brain, stomach, etc.
  • the presence of significant 36P1 A6 expression in any of these tissues may be useful to indicate the emergence, presence and/or severity of these cancers, since the corresponding normal tissues do not express 36P1 A6 mRNA or express it at lower levels.
  • 36P1A6 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 36P1A6 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 36P1A6 expressed in a corresponding normal sample.
  • the presence of 36P1 A6 protein is evaluated, for example, using immunohistochemical methods.
  • 36P1A6 antibodies or binding partners capable of detecting 36P1A6 protein expression may be used in a variety of assay formats well known in the art for this purpose.
  • peripheral blood may be conveniently assayed for the presence of cancer cells, including but not limited to prostate, bladder, colon, pancreatic, cervical and ovarian cancers, using RT-PCR to detect 36P1A6 expression.
  • cancer cells including but not limited to prostate, bladder, colon, pancreatic, cervical and ovarian cancers
  • 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. In the prostate cancer field, these include 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.
  • a related aspect of the invention is directed to predicting susceptibility to developing cancer in an individual.
  • a method for predicting susceptibility to cancer comprises detecting above normal levels of 36P1A6 mRNA or 36P1A6 protein in , a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 36P1A6 mRNA expression present is proportional to the degree of susceptibility.
  • the presence of elevated levels of 36P1A6 in prostate tissue may provide an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor).
  • the presence of elevated levels of 36P1 A6 in ovary tissue may provide an indication of ovarian cancer susceptibility (or the emergence or existence of an ovarian tumor).
  • a method for gauging aggressiveness of a tumor comprises determining the level of 36P1A6 mRNA or 36P1 A6 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 36P1A6 mRNA or 36P1A6 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 36P1A6 mRNA or 36P1A6 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness.
  • aggressiveness of prostate, bladder, ovarian, pancreatic or colon tumors is evaluated by determining the extent to which 36P1A6 is expressed in the tumor cells, with higher expression levels relative to corresponding normal samples indicating more aggressive tumors.
  • Standard methods for the detection and quantification of 36P1A6 mRNA include in situ hybridization using labeled 36P1A6 riboprobes, Northern blot and related techniques using 36P1A6 polynucleotide probes, RT-PCR analysis using primers specific for 36P1A6, 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 36P1A6 mRNA expression as described in the Examples which follow.
  • primers capable of amplifying 36P1A6 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 36P1 A6 protein may be used in an immunohistochemical assay of biopsied tissue.
  • 36P1A6 as a putative transcription factor which is expressed in a number of human cancers (e.g., cancers of the prostate, colon, bladder, pancreas, cervix, and ovary), opens a number of therapeutic approaches to the treatment of these cancers.
  • Therapeutic approaches aimed at inhibiting the activity of the 36P1 A6 protein may be useful for patients suffering from cancers which express this protein. It is possible that 36P1A6 is involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.
  • therapeutic approaches aimed at inhibiting the activity of the 36P1A6 protein are expected to be useful for patients suffering from cancers expressing 36P1A6.
  • These therapeutic approaches generally fall into two classes.
  • One class comprises various methods for inhibiting the binding or association of the 36P1A6 protein with DNA or with other proteins.
  • Another class comprises a variety of methods for inhibiting the transcription of the 36P1 A6 gene or translation of 36P1 A6 mRN A.
  • 36P1A6 may be introduced into 36P1A6 expressing cells via gene transfer technologies, wherein the encoded single chain anti-36P1A6 antibody is expressed intracellularly, binds to 36P1A6 protein, and thereby inhibits its function.
  • Methods for engineering such intracellular single chain antibodies are well known.
  • intracellular antibodies also known as "intrabodies”
  • intracellular antibodies may be specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment will be focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).
  • Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors. See, for example, Richardson et al., 1995, Proc. Natl.
  • Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide.
  • single chain antibodies may be expressed as a single chain variable region fragment joined to the light chain constant region.
  • Well known intracellular trafficking signals may be engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the expressed intrabody to the desired intracellular compartment.
  • intrabodies targeted to the endoplasmic reticulum (ER) may be engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif.
  • Intrabodies intended to exert activity in the nucleus may be engineered to include a nuclear localization signal. Lipid moieties may be joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies may also be targeted to exert function in the cytosol. For example, cytosolic intrabodies may be used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
  • intrabodies may be used to capture 36P1A6 in the nucleus, thereby preventing its activity within the nucleus.
  • Nuclear targeting signals may be engineered into such 36P1A6 intrabodies in order to achieve the desired targeting.
  • Such 36P1A6 intrabodies may be designed to bind specifically to a particular 36P1A6 domain, such as, for example, the ETS homology region or the pointed domain of the 36P1 A6 protein.
  • cytosolic intrabodies which specifically bind to the 36P1A6 protein may be used to prevent 36P1A6 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 36P1A6 from forming transcription complexes with other factors).
  • the transcription of the intrabody may be placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer.
  • the PSA promoter and/or promoter/enhancer may be utilized (See, for example, U.S. Patent No. 5,919,652).
  • a method of inhibiting the transcription of the 36P1A6 gene comprises contacting the 36P1A6 gene with a 36P1A6 antisense polynucleotide.
  • a method of inhibiting 36P1A6 mRNA translation comprises contacting the 36P1A6 mRNA with an antisense polynucleotide.
  • a 36P1A6 specific ribozyme may be used to cleave the 36P1A6 message, thereby inhibiting translation.
  • Such antisense and ribozyme based methods may also be directed to the regulatory regions of the 36P1 A6 gene, such as the 36P1 A6 promoter and/or enhancer elements.
  • proteins capable of inhibiting a 36P1A6 gene transcription factor may be used to inhibit 36P1A6 mRNA transcription.
  • the various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
  • Gene transfer and gene therapy technologies may be used for delivering therapeutic polynucleotide molecules to tumor cells synthesizing 36P1A6 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 36P1A6 inhibitory molecules).
  • 36P1A6 i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 36P1A6 inhibitory molecules.
  • a number of gene therapy approaches are known in the art.
  • Recombinant vectors encoding 36P1A6 antisense polynucleotides, ribozymes, factors capable of interfering with 36P1A6 transcription, and so forth, may be delivered to target tumor cells using such gene therapy approaches.
  • the above therapeutic approaches may be combined with chemotherapy or radiation therapy regimens. These therapeutic approaches may also enable the use of reduced dosages of chemotherapy and/or less frequent administration, particularly in patients that do not tolerate the toxicity of the chemotherapeutic agent well.
  • the anti-tumor activity of a particular composition may be evaluated using various in vitro and in vivo assay systems.
  • In vitro assays for evaluating therapeutic potential include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 36P1A6 to a binding partner, etc.
  • a 36P1 A6 therapeutic composition may be evaluated in a suitable animal model.
  • xenogenic prostate cancer models wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice, are appropriate in relation to prostate cancer and have been described (Klein et al., 1997, Nature Medicine 3: 402- 408).
  • PCT Patent Application W098/16628, Sawyers et al., published April 23, 1998 describes various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease.
  • xenografts from bearing mice treated with the therapeutic composition may be examined for the presence of apoptotic foci and compared to un-treated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
  • compositions used in the practice of the foregoing methods may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method.
  • Suitable carriers include any material which when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16 th Edition, A. Osal., Ed., 1980).
  • Therapeutic formulations may be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site.
  • Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like.
  • a preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP.
  • Therapeutic protein preparations may be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water containing, for example, benzyl alcohol preservative, or in sterile water prior to injection.
  • Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer and will generally depend on a number of other factors appreciated in the art.
  • the invention further provides prostate cancer vaccines comprising a 36P1 A6 protein or fragment thereof, as well as DNA based vaccines.
  • a tumor antigen in a vaccine for generating humoral and cell-mediated immunity for use in anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159: 3113-3117).
  • Such methods can be readily practiced by employing a 36P1A6 protein, or fragment thereof, or a 36P1A6-encoding nucleic acid molecule and recombinant vectors capable of expressing and appropriately presenting the 36P1A6 immunogen.
  • viral gene delivery systems may be used to deliver a 36P1A6-encoding nucleic acid molecule.
  • Various viral gene delivery systems which can be used in the practice of this aspect of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and Sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).
  • Non-viral delivery systems may also be employed by using naked DNA encoding a 36P1A6 protein or fragment thereof introduced into the patient (e.g., intramuscularly) to induce an anti- tumor response.
  • the full-length human 36P1A6 cDNA may be employed.
  • 36P1A6 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) epitopes may be employed.
  • CTL epitopes can be determined using specific algorithms (e.g., Epimer, Brown University) to identify peptides within a 36P1A6 protein which are capable of optimally binding to specified HLA alleles.
  • Various ex vivo strategies may also be employed.
  • One approach involves the use of dendritic cells to present 36P1A6 antigen to a patient's immune system. Dendritic cells express MHC class I and II, B7 costimulator, and IL-12, and are thus highly specialized antigen presenting cells.
  • autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al., 1996, Prostate 29: 371-380).
  • Dendritic cells can be used to present 36P1A6 peptides to T cells in the context of MHC class I and II molecules.
  • autologous dendritic cells are pulsed with 36P1A6 peptides capable of binding to MHC molecules.
  • dendritic cells are pulsed with the complete 36P1A6 protein.
  • Yet another embodiment involves engineering the overexpression of the 36P1A6 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56: 3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869), and tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186: 1177-1182).
  • Cells expressing 36P1A6 may also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
  • Anti-idiotypic anti-36P1A6 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 36P1A6 protein.
  • the generation of anti-idiotypic antibodies is well known in the art and can readily be adapted to generate anti-idiotypic anti-36P1A6 antibodies that mimic an epitope on a 36P1 A6 protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33- 40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al., 1996, Cancer Immunol Immunother 43: 65-76).
  • Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
  • Genetic immunization methods may be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 36P1A6.
  • Constructs comprising DNA encoding a 36P1 A6 protein/immunogen and appropriate regulatory sequences may be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 36P1A6 protein/immunogen.
  • Expression of the 36P1A6 protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against prostate cancer.
  • Various prophylactic and therapeutic genetic immunization techniques known in the art may be used (for review, see information and references published at Internet address www.genweb.com). KITS
  • 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 36P1A6 protein or a 36P1A6 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 radioisotope label.
  • a reporter-means such as a biotin-binding protein, such as avidin or streptavidin
  • 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.
  • LAPC-4 AD xenografts were grown intratibially as follows.
  • LAPC-4 AD xenograft tumor tissue grown subcutaneously was minced into 1-2 mm 3 sections while the tissue was bathed in 1X Iscoves medium, minced tissue was then centrifuged at 1.3K rpm for 4 minutes, the supernatant was resuspended in 10 ml ice cold 1X Iscoves medium and centrifuged at 1.3K rpm for 4 minutes. The pellet was then resuspended in 1 X Iscoves with 1% pronase E and incubated for 20 minutes at room temperature with mild rocking agitation followed by incubation on ice for 2-4 minutes.
  • Human cell lines e.g., HeLa
  • DMEM fetal calf serum
  • 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.
  • Adaptor 2 5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3'
  • Nested primer (NP)1 5'CTAATACGACTCACTATAGGGC3' Nested primer (NP)1 : 5 CGAGCGGCCGCCCGGGCAGGA3'
  • SSH Suppression Subtractive Hybridization
  • Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 ⁇ g of poly(A) + RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1 , Catalog No. K1804-1 ). The resulting cDNA was digested with Dpn II for 3 hrs. at 37°C. Digested cDNA was extracted with phenol/chloroform (1 :1 ) and ethanol precipitated.
  • Driver cDNA was generated by combining in a 1 :1 ratio Dpn II digested cDNA from the relevant xenograft source (see above) with a mix of digested cDNAs derived from human benign prostatic hyperplasia (BPH), the human cell lines HeLa, 293, A431 , Colo205, and mouse liver.
  • BPH human benign prostatic hyperplasia
  • Tester cDNA was generated by diluting 1 ⁇ l of Dpn II digested cDNA from the relevant xenograft source (see above) (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 overlaid 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 Sequencing 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 1.5 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.
  • 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.
  • SSH clones candidate gene fragment clones
  • All candidate clones were sequenced and subjected to homology analysis against all sequences in the major public gene and EST databases in order to provide information on the identity of the corresponding gene and to help guide the decision to analyze a particular gene for differential expression.
  • One of the SHH clones comprising about 269 bp, showed significant homology to an endometrial tumor library-derived EST (AA337475) but no homology to any known gene, and was designated 36P1A6.
  • the nucleotide sequence of this SHH clone is shown in FIG. 1 B.
  • Differential expression analysis by RT-PCR showed expression in all LAPC xenografts and in normal prostate at approximately equal levels (FIG. 2, Panel A).
  • further RT-PCR expression analysis of first strand cDNAs from 16 normal tissues detected expression in prostate, lung and pancreas after 30 cycles of PCR amplification (FIG. 2, panels B and C). After 35 cycles of amplification this gene was detectable in several other tissues.
  • the isolated 36P1 A6 gene fragment of 269 bp was used as a probe to identify the full length cDNA for 36P1A6, resulting in the isolation of clone 13, which is approximately 5 kb in size. Sequencing of the 5' 3160 bp of clone 13 revealed an ORF of 300 amino acids with strong homology to murine EHF (Bochert et al., 1998, Biochem Biophys Res Commun 246(1 ):176-81), a member of the ETS family of transcription factors. The nucleotide and deduced amino acid sequences of this cDNA are shown in FIG. 1 A.
  • the isolated 36P1A6 gene which is likely to be human EHF, contains an ETS homology region (which corresponds to its DNA binding domain) and a pointed domain (which probably functions as a protein-protein interaction domain).
  • the nearest human homolog is ESX, an epithelial specific ETS family member that is up-regulated in breast cancer (Chang et al., 1997,Oncogene 14(13):1617-22).
  • the highest homology to ESX lies in the ETS homology region.
  • Various amino acid sequence alignments of the 36P1 A6 protein with murine EHF and human ESX sequences are provided in FIG. 5.
  • RNAs derived from human prostate cancer xenografts and an extensive panel of prostate and non-prostate cancer cell lines were analyzed by Northern blot using 36P1A6 cDNA as probe. All RNA samples were quantitatively normalized by ethiduim bromide staining and subsequent analysis with a labeled ⁇ -actin probe.
  • 36P1A6/hEHF may be a marker and/or therapeutic target in prostate, bladder, colon, pancreatic, cervical and ovarian cancer, as well as potentially other cancers.
  • Another method of expressing 36P1A6 utilizes the retroviral expression vector pSR ⁇ MSVtkneo.
  • the 36P1A6 coding sequence (from translation initiation ATG to the termination codons) is amplified by PCR using ds cDNA template from 36P1A6 cDNA.
  • the PCR product is subcloned into pSR ⁇ MSVtkneo via the EcoR1 (blunt-ended) and Xba 1 restriction sites on the vector and transformed into DH5 ⁇ competent cells. Colonies are picked to screen for clones with unique internal restriction sites on the cDNA. The positive clone is confirmed by sequencing of the cDNA insert.
  • Retroviruses are then made and used for infection and generation of the various cell lines expressing 36P1 A6 (e.g., 3T3CL7, PC3, and LnCap).
  • 36P1 A6 cDNA is cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen) which provides a His-tag at the N-terminus
  • pBlueBac-36P1A6 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details).
  • Baculovirus is then collected from cell supernatant and purified by plaque assay.
  • Recombinant 36P1A6 protein is then generated by infection of HighFive insect cells (InVitrogen) with the purified baculovirus. Recombinant 36P1A6 protein may be detected using 36P1A6-specific antibody. 36P1A6 protein may be purified and used in various cell based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 36P1 A6.
  • GST glutathione-S-transferase
  • Reactivity of serum from immunized mice to full length 36P1A6 protein is monitored by ELISA using a partially purified preparation of HIS-tagged 36P1A6 protein expressed from 293T cells (Example 6). Mice showing the strongest reactivity are rested for 3 weeks and given a final injection of fusion protein in PBS and then sacrificed 4 days later. The spleens of the sacrificed mice are then harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from growth wells following HAT selection are screened by ELISA and Western blot to identify 36P1A6 specific antibody producing clones. The binding affinity of a 36P1A6 monoclonal antibody may be determined using standard technology.
  • Affinity measurements quantify the strength of antibody to epitope binding and may be used to help define which 36P1A6 monoclonal antibodies are preferred for diagnostic or therapeutic use.
  • the BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity.
  • the BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991 , Opt. Quant. Elect. 23:1 ; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time.
  • BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
  • luciferase (luc) based transcriptional reporter assays are carried out in cells expressing 36P1A6. These transcriptional reporters contain consensus binding sites for known transcription factors which lie downstream of well characterized signal transduction pathways. The reporters and examples of there associated transcription factors, signal transduction pathways, and activation stimuli are listed below.
  • NFkB-luc NFkB/Rel
  • Ik-kinase SAPK
  • 36P1A6-mediated effects may be assayed in cells showing mRNA expression, such as the 36P1A6-expressing cancer cell lines shown in FIG. 4.
  • Luciferase reporter plasmids may be introduced by lipid mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cells extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.
  • TFX-50 lipid mediated transfection
  • 36P1 A6 in prostate cancer and other cancers suggests a functional role in tumor progression. It is possible that 36P1A6 functions as a transcription factor involved in activating genes involved in tumorigenesis or repressing genes that block tumorigenesis. 36P1A6 function can be assessed in mammalian cells using in vitro approaches. For mammalian expression, 36P1A6 can be cloned into a number of appropriate vectors, including pcDNA 3.1 myc-His-tag and the retroviral vector pSR ⁇ tkneo (Muller et al., 1991 , MCB 11 :1785). Using such expression vectors, 36P1A6 can be expressed in several cell lines, including PC-3, NIH 3T3, LNCaP and 293T. Expression of 36P1 A6 can be monitored using anti-36P1 A6 antibodies.
  • Mammalian cell lines expressing 36P1A6 can be tested in several in vitro and in vivo assays, including cell proliferation in tissue culture, activation of apoptotic signals, tumor formation in SCID mice, and in vitro invasion using a membrane invasion culture system (MICS) (Welch et al. ,lnt. J. Cancer 43: 449-457). 36P1A6 cell phenotype is compared to the phenotype of cells that lack expression of 36P1 A6.
  • MIMS membrane invasion culture system
  • Cell lines expressing 36P1A6 can also be assayed for alteration of invasive and migratory properties by measuring passage of cells through a matrigel coated porous membrane chamber (Becton Dickinson). Passage of cells through the membrane to the opposite side is monitored using a fluorescent assay (Becton Dickinson Technical Bulletin #428) using calcein-Am (Molecular Probes) loaded indicator cells. Cell lines analyzed include parental and 36P1A6 overexpressing PC3, 3T3 and LNCaP cells. To assay whether 36P1A6 has chemoattractant properties, parental indicator cells are monitored for passage through the porous membrane toward a gradient of 36P1A6 conditioned media compared to control media. This assay may also be used to qualify and quantify specific neutralization of the 36P1 A6 induced effect by candidate cancer therapeutic compositions.
  • the effect of the 36P1A6 protein on tumor cell growth may be evaluated in vivo by gene overexpression in tumor-bearing mice.
  • SCID mice can be injected SQ on each flank with 1 x 10 6 of either PC3, TSUPR1 , or DU145 cells containing tkNeo empty vector or 36P1A6.
  • At least two strategies may be used: (1 ) Constitutive 36P1A6 expression under regulation of an LTR promoter, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc.
  • Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if 36P1 A6 expressing cells grow at a faster rate.
  • mice may be implanted with 1 x 10 5 of the same cells orthotopically to determine if 36P1A6 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.
  • the assay is also useful to determine the 36P1A6 inhibitory effect of candidate therapeutic compositions, such as for example, 36P1 A6 intrabodies, 36P1 A6 antisense molecules and ribozymes.

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Abstract

L'invention concerne un nouveau gène humain exprimé dans le cancer de la prostate, de la vessie, du pancréas, du colon, du col et des ovaires. L'invention concerne en outre de l'ADNc entier qui code un facteur de transcription présumée, avec un degré d'homologie élevé par rapport au gène EHF murin et à la protéine ESX humaine.
PCT/US1999/022576 1998-10-02 1999-10-02 Gene human 36p1a6 exprime dans le cancer de la prostate, de la vessie, du pancreas et du colon Ceased WO2000020584A2 (fr)

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EP99954686A EP1117794A2 (fr) 1998-10-02 1999-10-02 Gene human 36p1a6 exprime dans le cancer de la prostate, de la vessie, du pancreas et du colon
AU10975/00A AU1097500A (en) 1998-10-02 1999-10-02 Human gene expressed in cancers of prostate, bladder, pancreas and colon, 36p1a6
CA002344552A CA2344552A1 (fr) 1998-10-02 1999-10-02 Gene human 36p1a6 exprime dans le cancer de la prostate, de la vessie, du pancreas et du colon
IL14231099A IL142310A0 (en) 1998-10-02 1999-10-02 Human gene expressed in cancers of prostate, bladder, pancreas and colon, 36p1a6

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EP0972201B1 (fr) * 1997-02-25 2005-08-17 Corixa Corporation Composes servant au diagnostic immunologique du cancer de la prostate et leurs procedes d'utilisation
US6262245B1 (en) * 1997-02-25 2001-07-17 Corixa Corporation Compounds for immunotherapy of prostate cancer and methods for their use
EP1049800A4 (fr) * 1998-01-21 2002-09-25 Axys Pharm Inc Genes associes a l'asthme
IL139988A0 (en) * 1998-06-01 2002-02-10 Urogenesys Inc Tumor antigen useful in diagnosis and therapy of prostate and colon cancer
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EP1117794A2 (fr) 2001-07-25
AU1097500A (en) 2000-04-26

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