WO2005100998A2 - Membrane markers for use in cancer diagnosis and therapy - Google Patents

Membrane markers for use in cancer diagnosis and therapy Download PDF

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Publication number
WO2005100998A2
WO2005100998A2 PCT/EP2005/004153 EP2005004153W WO2005100998A2 WO 2005100998 A2 WO2005100998 A2 WO 2005100998A2 EP 2005004153 W EP2005004153 W EP 2005004153W WO 2005100998 A2 WO2005100998 A2 WO 2005100998A2
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hla
protein
plasma membrane
polynucleotide sequence
slc30a5
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PCT/EP2005/004153
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German (de)
French (fr)
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WO2005100998A3 (en
Inventor
Silvia TÓRTOLA-PÉREZ
Stephanie Lamer
Steffen Heim
Miriam Fuchs
Thomas Buschmann
Volker Seibert
Nikoleta-Kyriaki Fotiadis
Katrin Stedronsky
Jörn Meuer
Kirsten Rothmann-Cosic
Tatiana Ilynia
Lüder FELS
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Europroteome Ag
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Publication of WO2005100998A3 publication Critical patent/WO2005100998A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the invention relates to agents and methods for the diagnosis, prognosis and treatment of non-steroid dependent cancer. Specifically, the invention relates to the use of nucleic and amino acid sequences encoding plasma membrane proteins of the invention that are differentially expressed in tumour tissues for the diagnosis of both early and late stage non-steroid specific cancers, cancer prognosis, as well as screening for therapeutic agents that regulate the gene expression and/or biological activity of such proteins. This invention further relates to the biological technologies designed to inhibit the gene expression and/or biological activity of the proteins plasma membrane proteins of the invention, including using agents identified in screening assays described herein, as well as molecules that are complementary to polynucleotide sequences encoding plasmas membrane proteins of the invention and antibody targeting of said proteins. In specific embodiments, the proteins are of human origin.
  • Examples of such diagnostic tests include alpha-fetoprotein and teratocarcinoma for the identification of primary liver cancer in humans (IZOTOP, Hungary) detection of gastrointestinal cancers using carcinoembryo ⁇ ic antigen (Abbot/Roche, Switzerland), chorionic gonadotropin for the detection of trophoblasts and germ cell cancers (IZOTOP, Hungary; BioCheck Inc., CA), and prostatic acid phosphatase or prostate specific antigens for prostate carcinomas.
  • carcinoembryo ⁇ ic antigen Abbot/Roche, Switzerland
  • chorionic gonadotropin for the detection of trophoblasts and germ cell cancers
  • prostatic acid phosphatase or prostate specific antigens for prostate carcinomas.
  • the present invention addresses these issues by providing tumour-specific plasma membrane proteins for the diagnosis and prognosis of non-steroid dependent cancers. Furthermore, the plasma membrane protein encoding amino acid sequences and their corresponding polynucleotide sequences are used to screen for agents that alter their biologically activity and/or gene expression for the purpose of identifying and developing a therapeutic agent for the treatment of non-steroid dependent cancers.
  • the present invention relates to the use of polynucleotide sequences encoding plasma membrane proteins of the invention, the amino acid sequences encoding such proteins, derivatives or fragments thereof, for the diagnosis, prognosis and treatment of non-steroid dependent cancers.
  • the given proteins are of human origin.
  • the present invention relates to the use of adenylate cyclase type III (ADCY3), alpha-2C adrenergic receptor (ADRA2C), aquaporin 5 (AQP5), asialoglycoprotein receptor 1 (ASGR1), beta-secretase (BACE), beta-amyloid peptide-binding protein (BBP), CD47, C-terminal tensin-like protein (CTEN), fractalkine (CX3CL1), deleted in malignant brain tumours (DMBT1), microsomal dipeptidase precursor (DPEP1), ectonucleoside triphosphate disphosphohydrolase 6 (ENTPD6), FCGR3A, gap junction beta-2 protein (GJB2), interferon-induced transmembrane protein 1 (IFITM1), integrin beta-5 protein (ITGB5), inositol 1 ,4,5-triphosphate receptor type 2 (ITPR2), low density lipoprotein receptor-related protein 8 (LRP8),
  • the amino acid sequences encoding said proteins also shown in Table 1 (pp. 64-66), and derivatives or fragments thereof, for the diagnosis, prognosis and treatment of non-steroid dependent cancers.
  • the use of said polynucleotide and amino acid sequences in methods for screening for gene and/or protein expression modulators, as well as modulators of biological activities of said proteins are included within the scope of this invention.
  • the given proteins are of human origin.
  • the present invention relates to a method for screening a library of test molecules or compounds to identify at least one therapeutic molecule or compound which specifically modulates the expression of a plasma membrane protein-encoding gene of the invention comprising, contacting a reporter construct under the control of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3C 1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3
  • the present invention also relates to a method for screening a library of test molecules or compounds to identify at least one therapeutic molecule or compound which specifically binds and/or interacts with a plasma membrane protein of the invention, a derivative or fragment thereof with a test molecule or compound, or a library of test molecules or compounds, under conditions that allow for specific binding and/or interaction, and detecting the level of specific binding and/or interaction.
  • a decrease in the level of binding and/or interaction relative to a control indicates a potential therapeutic activity.
  • the library of molecules or compounds is selected from the group consisting of DNA molecules, peptides, polypeptides, agonists, antagonists, polyclonal antibodies, monoclonal antibodies, immunoglobulins, pharmaceutical agents, and naturally occurring or synthetic molecules or compounds. Furthermore, the library of molecules or compounds may also be composed of molecules or compounds previously known to modulate the gene expression and/or the biological activity of an unrelated protein, or play a therapeutic role in a non-cancer related disease and/or disorder.
  • test molecules or compounds identified in the methods of the invention demonstrate the ability to decrease, suppress or inhibit the expression of a given plasma membrane protein-encoding gene of the invention, and/or the biological activity of a given plasma membrane protein of the invention.
  • Methods are also provided for treating a non-steroid dependent cancer resulting from the aberrant biological activity of a given plasma membrane protein, and/or the aberrant expression of a gene encoding a plasma membrane protein of the invention in a mammalian, preferably a human subject: comprising providing a composition that comprises a therapeutic agent identified to bind and/or interact with a given plasma membrane protein, or modulate the level of expression of a polynucleotide encoding a given plasma , membrane, and administering to a mammalian subject a therapeutically effective amount of said composition.
  • the invention provides an alternative method for treating a non-steroid dependent cancer resulting from the aberrant expression of a polynucleotide sequence encoding a given plasma membrane protein of the invention comprising: administering a therapeutically effective amount of a polynucleotide sequence complementary to an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, mud 3,
  • complementary polynucleotide molecules used according to the invention are selected from siRNA, mo ⁇ holinos, PNAs, triple-helix forming oligonucleotides, double and single stranded polynucleotide sequences.
  • a method for treating a non-steroid dependent cancer resulting from the aberrant biological activity of a plasma membrane of the invention comprising: administering a therapeutically effective amount of an antibody directed agaisnt an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29
  • complementary polynucleotide molecules and antibodies specific for a given plasma membrane protein of the invention decrease, suppress or inhibit gene expression or the biological activity of a given plasma membrane protein of the invention, respectively.
  • a further aspect of the invention is a method of modulating proliferation, differentiation and/or cell migration of target cells comprising administering a test molecule or compound identified in the screening methods of the invention to said target cells.
  • said target cells are neoplastic, epithelial or cancer cells.
  • One embodiment of the invention provides a method for determining whether a subject is at risk of developing or has a non-steroid dependent cancer caused by the aberrant expression and/or biological activity of a plasma membrane protein of the invention, comprising the means for measuring the level of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1
  • ADCY3, ADRA2C AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, TH
  • the invention provides a kit with instructions to use the kit based on the above-mentioned method, whereby the kit comprises an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2,
  • FIG. 1 Heat map of genes found to be up-regulated in colon and rectal cancer. Whereas rows represent individual nucleotide sequences spotted on the oiigo microarray employed, columns correspond to the individual patient samples analysed. Each square in the matrix represents the expression level of a single nucleotide sequence. Individual genes can be represented on the oligo microarray by more than one sequence. The genes shown are more than 1.5 fold up-regulated, with a frequency of at least 30% within the 25 experiments described herein. Squares that are represented by the colour red indicate an up-regulation of a given gene, whereas green squares signify gene down-regulation. Furthermore, black squares denote either no gene regulation or no data for the mentioned sequence in the indicated experiment.
  • Samples derived from cancer patients are labelled with an internal coding system: samples from rectal cancer patients begin with the letters RNC, whereas those samples commencing with the letters CNC signify samples collected from colon cancer patients.
  • a plasma membrane protein of the invention' refers to a protein of the invention listed in Table 1 (pp. 64-66) that contains one or more membrane-spanning ⁇ -helices, wherein the ⁇ -helices allow the protein to extend across the lipid bilayer of a plasma membrane.
  • the amino acid sequences of the protein can be typically divided into sequences that are localized within the cell cytosol, those spanning the lipid bilayer, or those extending into the extracellular space, depending on the number of membrane-spanning ⁇ -helices.
  • This definition also includes proteins that are attached to the non-cytoplasmic monolayer (extracellular) of a plasma membrane via a covalent linkage (via a specific oligosaccharide) to phosphatidylinositol, also referred to as a glycosylphosphatidylinositol (GPI) anchor. These proteins generally lack one or more membrane spanning ⁇ -helices.
  • Membrane-spanning and associated proteins of the invention include adenylate cyclase type III (ADCY3), alpha-2C adrenergic receptor (ADRA2C), aquaporin 5 (AQP5), asialoglycoprotein receptor 1 (ASGR1), beta-secretase (BACE), beta- amyloid peptide-binding protein (BBP), CD47, C-terminal tensin-like protein (CTEN), fractalkine (CX3CL1), deleted in malignant brain tumours (DMBT1), microsomal dipeptidase precursor (DPEP1), ectonucleoside triphosphate disphosphohydrolase 6 (ENTPD6), FCGR3A, gap junction beta-2 protein (GJB2), interferon-induced transmembrane protein 1 (IFITM1), integrin beta-5 protein (ITGB5), inositol 1 ,4,5-triphosphate receptor type 2 (ITPR2), low density lipoprotein receptor-related protein 8 (LRP8)
  • TM domains The corresponding accession numbers for the polynucleotide and amino acid sequences encoding these proteins are outlined in Table 1 (pp. 64-66).
  • Membrane-spanning ⁇ -helices are also commonly referred to as transmembrane (TM) domains.
  • the term 'derivative' refers to a modification of a polypeptide sequence, polynucleotide sequence, or a complementary polynucleotide sequence.
  • Derivatives of the proteins of the invention include polypeptides that have been modified chemically, and retain a biological activity of a plasma membrane protein listed in Table 1 (pp. 64-66).
  • Derivatives of a given plasma membrane protein of the invention also include polypeptides that have an altered length and/or amino acid sequence as compared to the wild-type amino acid sequence. Such derivatives are at least 60% identical or similar to an amino acid sequence encoding a plasma membrane protein of the invention selected from the amino acid sequences shown in Table 1 (pp.
  • Preferred derivatives are at least about 65%, 70%, and even more preferably at least 80%, 85%, 90%, 95%, or 98% identical or similar to an amino acid sequence encoding a plasma membrane protein of the invention selected from the amino acid sequences shown in Table 1.
  • modified polynucleotide sequences that encode a biologically active plasma membrane protein of the invention, and are selected from the polynucleotide sequences shown in Table 1 (pp. 64-66).
  • Modified nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from a polynucleotide sequence encoding a given polypeptide of the invention, due to the degeneracy of the genetic code.
  • chemical modifications of polynucleotide sequences include, but are not limited to, the replacement of hydrogen by an alkyl, acyl, or amino group.
  • fragment' refers to a portion of a polypeptide sequence that comprises at least 5 consecutive amino acid residues and retains the biological activity of a plasma membrane protein of the invention, or to those polynucleotide sequences which, when translated, produce a polypeptide retaining some functional characteristic of a plasma membrane protein of the invention, e.g. antigenicity, or structural domain characteristics.
  • 'biological activity' may be used interchangeably with the terms 'biologically active', 'bioactivity' or 'activity' and, for the purposes herein, means an effector or antigenic function that is directly or indirectly performed by a plasma membrane protein of the invention (whether in its native or denatured conformation), derivative or fragment thereof.
  • Effector functions include phosphorylation (kinase activity) or activation of other molecules, induction of differentiation, mitogenic or growth promoting activity, signal transduction, immune modulation, DNA regulatory functions and the like, whether presently known or inherent.
  • Antigenic functions include possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured, plasma membrane protein of the invention, derivative or fragment thereof.
  • a biological activity of such a protein can be that it functions as regulator of a signalling pathway of a target cell.
  • a signalling pathway can, for example, modulate cell differentiation, proliferation and/or migration of such a cell, as well as tissue invasion, tumour development and/or metastasis.
  • a target cell according to the invention can be an epithelial, neoplastic or cancer cell.
  • nucleotide sequence' refers to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to a peptide polynucleotide sequence (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide polynucleotide sequence
  • 'protein' and 'polypeptide' are used interchangeably and refer to a linear polymer of amino acids linked together by peptide bonds in a specific sequence. Such polymers are generally flexible and may take on a variety of shapes, however based on the backbones and side chains of the amino acids associated with one another, these molecules tend to fold into their correct confirmation.
  • genes for a particular protein may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitution, additions or deletions, all of which still code for polypeptides having substantially the same biological activity.
  • the phrase 'a polynucleotide sequence encoding a biologically active plasma membrane protein of the invention' may thus refer to one or more genes within a particular individual.
  • certain differences in nucleotide sequences- may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in the amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
  • non-steroid dependent cancer 1 refers to a cancer that arises from epithelial cell origin and may include, but is not limited to, breast, lung, gastrointestinal, prostate, ovarian, cervical, endometrial cancers, bladder and/or other cancers.
  • epithelial cancers may be of different stages, for example precancerous, early and/or late stage cancers. Cancers may also be of varying degrees in grading, wherein grading refers to the extent of histological differentiation the cancer has progressed. Guidelines to the staging and grading of cancer are known to those skilled in the art, and are described in the 'Cancer Staging Handbook' from the American Joint Committee on Cancer.
  • an epithelial cancer may be referred to as a neoplasm of epithelial origin.
  • gastrointestinal cancer 1 refers to a cancer state associated with the gastrointestinal tract of a given subject.
  • gastrointestinal cancers include, but are not limited to oesophageal, stomach, small intestine, colon, rectal, pancreatic, liver, gallbladder, and biliary tract cancers.
  • gastrointestinal cancers may be at different stages, as well as varying degrees of grading (see the 'Cancer Staging Handbook' from the American Joint Committee on Cancer).
  • 'neoplasm' refers to any new and abnormal growth, specifically a new growth of tissue in which the growth is uncontrolled and progressive.
  • 'biological sample' and 'test sample' refer to all biological fluids, excretions, tissues and cells isolated from any given subject.
  • samples include, but are not limited to, blood, blood serum, plasma, nipple aspirate, urine, semen, seminal fluid, seminal plasma, prostatic fluid, excreta, tears, saliva, sweat, biopsy, ascites, cerebrospinal fluid, milk, lymph, or tissue extract samples.
  • the phrase 'library of test molecules or compounds' includes, but is not limited to, libraries containing DNA molecules, peptides, antagonists, monoclonal antibodies, polyclonal antibodies, immunoglobulins and/or pharmaceutical agents. These may include new or already known molecules or compounds. Furthermore, natural or chemically synthesised molecules or compounds are also included within the scope of the invention. Furthermore, the terms 'monoclonal antibodies' and 'immunoglobulins' used herein include fragments or derivatives thereof.
  • biologically active surface refers to any two- or three-dimensional extension of a material that biomolecules can bind to, or interact with, due to the specific biochemical properties of this material and those of the biomolecules.
  • biochemical properties include, but are not limited to, ionic character (charge), hydrophobicity, or hydrophilicity.
  • adsorbent refers to any material that is capable of accumulating (binding) a biomolecule.
  • the adsorbent typically coats a biologically active surface and is composed of a single material or a plurality of different materials that are capable of binding a biomolecule.
  • materials include, but are not limited to, anion exchange materials, cation exchange materials, metal chelators, polynucleotides, oiigonucleotides, peptides, antibodies, metal chelators etc.
  • the term 'modulate' refers to an alteration in the gene expression and/or biological activity of a plasma protein of the invention as compared to wild-type gene expression and/or biological activity. Such alterations include an up-regulation, i.e. stimulation or increase, or a down-regulation, i.e. suppression, inhibition or decrease of said responses.
  • 'Cells', 'host cells', 'target cells' and 'recombinant host cells' can be used interchangeably and refer to a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence. It will be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in succeeding generations as a result of mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within this definition.
  • the phrase 'delivery complex' refers to a targeting means (e.g., a molecule that results in higher affinity binding of an antisense polynucleotide sequence, protein, polypeptide, or peptide to a target cell surface and/or increased cellular uptake by a target cell).
  • targeting means include, but are not limited to sterols (e.g. cholesterol), lipids (e.g. a cationic lipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associated virus, or retrovirus).
  • Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to intemalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell such that the polynucleotide, protein, polypeptide, or peptide is released in a functional form.
  • reporter construct refers to a target polynucleotide sequence linked in-frame to another polynucleotide sequence to provide a coding unit whose product is easily assayed.
  • reporter genes include, but are not limited to, ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein
  • GFP enhanced green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • Ds-Red fluorescent protein Ds-Red fluorescent protein
  • He-red far-red fluorescent protein
  • SEAP secreted alkaline phosphatase
  • SEAP secreted alkaline phosphatase
  • CAT chloramphenicol acetyltransferase
  • a complementary polynucleotide sequence' can be used interchangeably with the term "antisense polynucleotide sequence", and refers to a polynucleotide or anti- gene agent that is comprised of at least about 10 nucleotides and is capable of hybridising with its complementary nucleotide sequence in vivo and in vitro.
  • a complementary polynucleotide sequence is comprised of at least 15, 18, 20, 25, 30, 35, 40, or 50 nucleotides.
  • Such polynucleotide sequences are typically complementary to the 5' untranslated (UTR) region of the mRNA (up to and including the AUG translation initiation codon), the 3'UTR, or a non-coding region of a given polynucleotide. Specific binding of such sequences to a polynucleotide sequence encoding a given protein of the invention is effective in altering transcription or translation of the corresponding mRNA in a host cell expressing said mRNA.
  • UTR 5' untranslated
  • polynucleotide sequences may be linear, circular, or triple helix forming and are complementary to a plasma membrane protein-encoding polynucleotide sequence of the invention selected from the polynucleotide sequences shown in Table 1 (pp. 64-66).
  • polynucleotide sequences may be a single or double stranded nucleic acid (e.g., Mo ⁇ holinos, PNAs or siRNA) and comprise: a) at least one modified base moiety which is selected from the group of 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino- methyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-
  • RNA interference to inhibit expression of a given protein of known function
  • siRNAs Small interfering RNAs generated by ribonuclease III cleavage of longer dsRNAs are used to block translation, and are preferably between 20-25 nucleotides in length.
  • the invention also includes the use of RNA Lassos to inhibit the translation of a given protein of known function.
  • the term 'antagonist' refers to a molecule or compound which, when bound to or interacts with a plasma membrane protein of the invention, decreases the biological activity of such a protein.
  • Such an antagonist may interact with the ligand-binding domain of a given plasma membrane protein of the invention, thereby preventing the ligand-induced activation of such a protein.
  • An antagonist may also inhibit the interaction between a given plasma membrane protein of the invention and another molecule by stencally hindering a critical protein-protein interaction site e.g. a dimerisation domain.
  • an antagonist may decrease, suppress or inhibit the expression of a plasma membrane protein-encoding polynucleotide sequence of the invention selected from the polynucleotide sequences shown in Table 1 (pp.
  • an antagonist can be a molecule or compound which inhibits or decreases the expression or biological activity of a protein which is located downstream of a plasma membrane protein of the invention, or which interacts with a given protein of the invention.
  • An antagonist can also be a molecule or compound which decreases the amount or the duration of the effect of the biological or immunological activity of a plasma membrane protein of the invention.
  • Antagonists may include proteins, polynucleotide sequences, carbohydrates, antibodies or fragments thereof, or any other molecule that exerts such effects.
  • 'neoplastic cell' and 'neoplastic tissue' refer to a cell or tissue, respectively, that has undergone significant cellular changes (transformation). Such cellular changes are manifested by an escape from specific control mechanisms, increased growth potential, alteration in the cell surface, karyotypic abnormalities, morphological and biochemical deviations from the norm, and other attributes conferring the ability to invade, metastasise and kill.
  • the term 'antibody' refers to a polypeptide substantially encoded by an immunoglobulin gene or fragments thereof, which bind and recognise a specific antigen. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterised fragments produced by peptidase digestion.
  • antibody fragments that are produced either by modifying whole antibodies or synthesised using recombinant DNA methodologies.
  • an antibody will, under appropriate binding conditions, interact with its specific antigen or fragment thereof.
  • the antibodies used within the scope of the invention bind preferentially to a plasma membrane protein of the invention selected from the group of membrane proteins shown in Table 1 (pp. 64-66).
  • the invention provides methods for screening for therapeutic agents for the treatment of a non-steroid dependent cancer resulting from the aberrant expression and/or biological activity of a plasma membrane protein of the invention.
  • the methods identify candidates, test molecules or compounds, or agents (e.g. peptides, peptidomimetics, small molecules or other drugs) which decrease and/or inhibit the biological activity of a plasma membrane protein of the invention, derivative or fragment thereof, or have an inhibitory effect on, for example, the expression of a polynucleotide sequence encoding a plasma membrane protein of the invention.
  • Plasma membrane proteins of the invention include ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1 , CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TF
  • Preferred proteins of the invention include ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TF
  • the most preferred plasma membrane proteins of the invention are ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, and/or TMEM5.
  • Proteins or peptides capable of interacting directly or indirectly with a given plasma membrane protein of the invention, derivative or fragment thereof, can be identified by various methods. For example, such molecules can be identified using methods based on various binding assays (see references on: yeast-2-hybrid Bemis et al. (1995) Methods Cell Biol. 46, 139-151, Fields and Stemglanz (1994) Trends Genet. 10, 286-292, Topcu and Borden (2000) Pharm. Res. 17, 1049-1055; yeast 3 hybrid: Zhang et al. (1999) Methods Enzymol. 306, 93-113; GST pull-downs as in Palmer et al. (1998) EMBO J. 17, 5037-5047; and phage display as in Scott and Smith (1990) Science 249] 386-390).
  • the biological activity of a given plasma membrane protein of the invention is to modulate cell differentiation, proliferation, transformation, migration of specific target cells, e.g. epithelial or cancer cells, as well as tissue invasion, tumour development and/or metastasis. It is also understood that the biological activity of a protein is dependent upon several molecular aspects. including, but not limited to, the gene expression of a given polynucleotide sequence encoding a given protein; the translation of the corresponding polynucleotide sequence, post-translational modification, and/or protein folding, as well as interactions with other molecules.
  • An alteration in gene expression, translation, post-translation modification, or protein folding may affect the biological activity of a given protein of the invention such that cellular differentiation, proliferation, transformation, as well as tissue invasion, tumour development and/or metastasis will be affected.
  • Assays for determining whether a protein has at least one biological activity of a plasma membrane protein of the invention are described herein.
  • reporter assays are described herein for the determination of gene expression of polynucleotide sequence encoding a plasma membrane protein of the invention.
  • the invention provides assays for screening test molecules or compounds that bind to, interact with, or modulate the biologically active form of a plasma membrane protein of the invention, derivative or fragment thereof.
  • assays for screening test molecules or compounds that modulate the expression of a polynucleotide sequence encoding a plasma membrane of the invention are also provided.
  • the test compounds according to the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries, aptially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the One-bead-one-compound' library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Bindseil et al. (2001) Drug Discov. Today 6, 840-847; Grabley et al. (2000) Ernst Schering Res. Found. Workshop, pp. 217-252; Houghten et al. (2000) Drug Discov. Today 5, 276-285; Rader, C. (2001) Drug Discov. Today 6, 36-43).
  • the assay is a cell-based assay in which a cell expresses a biologically active plasma membrane protein of the invention, derivative or fragment thereof.
  • the expressed protein is contacted with a test molecule or compound and the ability of the test molecule or compound to bind to or interact with the protein is determined.
  • the cell can, for example, be a eukaryotic cell such as, but not limited to a yeast cell, an invertebrate cell (e.g. C. elegans), an insect cell, a teleost cell, an amphibian cell, or a cell of mammalian origin.
  • Determining the ability of the test molecule or compound to bind to or interact with a given protein can be accomplished, for example, by coupling the test molecule or compound with a radioisotope (e.g. 125 l, 35 S, 14 C, or 3 H) or enzymatic (e.g. horseradish peroxidase, alkaline phosphatase, or luciferase) label such that binding or interaction of the test molecule or compound to the biologically active polypeptide, derivative or fragment thereof, can be determined by detecting the labelled molecule or compound in the complex.
  • a radioisotope e.g. 125 l, 35 S, 14 C, or 3 H
  • enzymatic e.g. horseradish peroxidase, alkaline phosphatase, or luciferase
  • the assay comprises contacting a cell, which expresses a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a known molecule or compound which binds or interacts with the given protein to form an assay mixture, contacting the assay mixture with a test molecule or compound, and determining the ability of the test molecule or compound to bind to or interact with the given protein, wherein determining the ability of the test molecule or compound to bind or interact with a given plasma membrane protein of the invention is compared to a control.
  • the determination of the ability of the test molecule or compound to bind to or interact with a given protein is based on competitive binding/inhibition kinetics of the test molecule or compound and known target molecules or compounds for a given protein of the invention.
  • Methods of detecting competitive binding, or the interaction of two molecules for the same target, wherein the target is a plasma membrane protein of the invention, derivative or fragment thereof, are known to those skilled in the art.
  • the assay is a cell-based assay comprising contacting a cell expressing a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a test molecule or compound and determining the ability of the test molecule or compound to inhibit the biological activity of the given polypeptide. This can be accomplished, for example, by determining whether a given protein of the invention continues to bind to or interact with a known target molecule, or whether a specific cellular function has been abrogated.
  • a target molecule can be an extracellular molecule that activates a signal transduction pathway within a cell, a second intercellular protein that has a catalytic activity, a protein that regulates transcription of specific genes, or a protein that initiates protein translation.
  • Determining the ability of a biologically active plasma membrane protein of the invention, derivative or fragment thereof, to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target [e.g., intracellular Ca 2+ , diacylglycerol and inositol triphosphate IP3)], detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction (via a regulatory element that may be responsive to a given polypeptide) of a reporter gene operably linked to a polynucleotide encoding a detectable marker, e.g., ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase
  • the assay of the present invention is a cell-free assay comprising contacting a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a test molecule or compound, and determining the ability of the test molecule or compound to bind to, or interact with a given plasma membrane protein of the invention. Binding or interaction of the test molecule or compound to a given protein of the invention can be determined either directly or indirectly as described above.
  • the assay includes contacting a plasma membrane protein of the invention with a known target molecule or compound, which binds, or interacts with a given plasma membrane protein of the invention to form an assay mixture.
  • the assay mixture is contacted with a test molecule or compound, and the determination of the ability of the test molecule or compound to interact with a given plasma membrane protein of the invention is based on competitive binding/inhibition kinetics of the test molecule or compound and known molecules or compounds for a given plasma membrane protein of the invention.
  • Methods of detecting competitive binding, or interaction, of two molecules for the same target, wherein the target is a plasma membrane protein of the invention, derivative or fragment thereof, are known to those skilled in the art.
  • the assay is a cell-free assay comprising contacting a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a test molecule or compound, and determining the ability of the test molecule or compound to inhibit the activity of the plasma membrane protein. Determining the ability of the test molecule or compound to inhibit the activity of a given plasma membrane protein of the invention can be accomplished, for example, by determining the ability of a given plasma membrane protein of the invention to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test molecule or compound to modulate the biological activity of a given plasma membrane protein of the invention can be accomplished by determining the ability of the plasma membrane protein to further modulate a target molecule.
  • a plasma membrane protein of the invention derivative or fragment thereof, or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Binding of a test molecule or compound to a plasma membrane protein of the invention with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test molecule or compound and either the non- adsorbed target protein or a biologically active plasma membrane protein of the invention, derivative or fragment thereof.
  • the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of binding or activity of said polypeptide can be determined using standard techniques.
  • a biologically active plasma membrane protein of the invention, derivative or fragment thereof, or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • inhibitors of the expression of a plasma membrane protein of the invention are identified in a method in which cells are contacted with a candidate molecule or compound and the expression of the selected mRNA or protein [i.e., the mRNA or protein corresponding to a polynucleotide or a biologically active polypeptide] in the cell is determined.
  • the cell is an animal cell.
  • the cell can be derived from an insect, fish, amphibian, mouse, rat, or human.
  • the level of expression of the selected mRNA or protein in the presence of the candidate molecule or compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate molecule or compound.
  • the candidate molecule or compound can then be identified as a inhibitor of expression of a given plasma membrane protein of the invention based on this comparison. For example, when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate molecule or compound than in its absence, the candidate molecule or compound is identified as an inhibitor of the selected mRNA or protein expression.
  • the level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
  • a therapeutic agent specific for a plasma membrane protein of the invention can also be identified by using a reporter assay, in which the level of expression of a reporter construct, under the control of a gene promoter specific for a given protein of the invention, is measured in the presence or absence of a test molecule or compound or a library of test molecules are compounds.
  • a promoter can be isolated by screening a genomic library with a cDNA encoding a plasma membrane protein of the invention; preferably containing the 5' end of the cDNA.
  • a portion of said promoter typically from 20 to about 500 base pairs long is then cloned upstream of a reporter gene, e.g., a ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), neomycin gene, in a plasmid.
  • a reporter gene e.g., a ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT),
  • the transfected cells are distributed into wells of a multi-well plate and various concentrations of test molecules or compounds are added to the wells. After several hours of incubation, the level of expression of the reporter construct is determined according to methods known in the art. A difference in the level of expression of the reporter construct in transfected cells incubated with the test molecule or compound relative to transfected cells incubated without the test molecule or compound will indicate that the test molecule or compound is capable of modulating the gene expression of a given plasma membrane protein of the invention and is thus considered a therapeutic agent specific for a plasma membrane protein of the invention. Due to the cellular nature of this assay, it is considered according to the invention as a cell-based assay.
  • polynucleotide sequences used in screening for molecules or compounds that modulate the expression of a gene encoding a plasma membrane protein of the invention, derivative or fragment thereof are selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671 , NMJ12104, NM.032027, NM_001777, NM_032865, NM 02996, NM_007329, NMJ04413, NM_001247, NM.000569, NM.004004, NM_003641, NM_002213, NMJ02223, NM_004631, NM_005898, NMJ03953, NM.002538, NM_052932, NM_002845, NM_006511 , NM 00343, NM_003982, NM_022902, NMJ00232, NMJ06474, NM_003264,
  • polynucleotide sequences used in screening for molecules or compounds that bind to, interact with, or modulate the biologically active form of a plasma membrane protein of the invention are selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NMJ01671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NMJ07329, NMJ04413, NMJ01247, NM_000569, NM 04004, NM_003641, NM_002213, NM_002223, NMJ04631, NM.005898, NM.003953, NM_002538, NM.052932, NM_002845, NM_006511, NM_000343, NMJ03982, NM_022902, NM_000232, NM_006474, NM_003264, NM
  • polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM 04036, NM_000683, NM_001651, NM_001671 , NMJ12104, NM_032027, NMJ01777, NM_032865, NMJ02996, NM_007329, NM.004413, NM_001247, NMJ00569, NM_004004, NM.003641, NM_002213, NMJ02223, NM_004631, NM_005898, NM_003953, NMJ02538, NM_052932, NM_002845, NM_006511, NM.000343, NM_003982, NM.022902, NM_000232, NMJ06474, NM.003264, NM_020208, NM_005765, NM.012463, NM_002250, NMJ302856, NM__
  • polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NMJ32027, NMJ301777, NM_032865, NM_002996, NMJ07329, NM.004413, NMJ01247, NM_000569, NMJ04004, NM_003641, NM_002213, NM_002223, NMJ04631, NM_005898, NM_003953, NMJ02538, NM_052932, NMJ02845, NM_006511 , NM_000343, NMJ03982, NM.022902, NM_000232, NMJ06474, NMJ03264, NMJ320208, NMJ05765, NMJ12463, NMJ02250, NMJ02856, NM_015480, NMJ14255, NMJ
  • polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NM.032865, NM_002996, NM_007329, NMJ304413, NM_001247, NM_000569, NMJ04004, NM_003641, NM_002213, NM.002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NMJ00343, NM_003982, NM.022902, NM.000232, NM_006474, NM_003264, NM.020208, NMJ05765, NM_012463, NM_002250, NM.00
  • amino acid sequences used in screening for molecules or compounds that bind to, interact with, or modulate the biologically active form a plasma membrane protein of the invention, derivative or fragment thereof are selected from the amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NPJ01662, NP_036236, NPJ304416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NPJ01238, NP_000566, NP.003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP.000334, NP_003973, NP_075053, NPJ00223, NP_006465, NP_003255, NPJ6459
  • amino acid sequences used in screening for molecules or compounds that bind to, interact with, or modulate the biologically active form of a plasma membrane protein of the invention are selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP_001662, NP_036236, NP_004416, NP_001768, NP_006254, NPJ02987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NP_002204, NP.002214, NP.004622, NP_005889, NP.003944, NP.002529, NP_443164, NP_002836, NP_006502, NP_000334, NP.003973, NP_075053, NP_000223, NP_006465, NP_003255, NP.064593
  • amino acids sequences selected from the group of amino acid sequences shown in accession numbers NP.004027, NP_000674, NP_00162, NPJ01662, NP_036236, NP_004416, NP_001768, NPJ06254, NP_002987, NPJ15568, NP_004404, NPJ01238, NP_000566, NP_003995, NP_003632, NP.002204, NP_002214, NP_004622, NPJ05889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP_005756, NP_036595, NPJ02241, NP.002847, NP_056295, NP_05
  • amino acid sequence selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP.001662, NP_036236, NP_004416, NP_001768, NPJ06254, NP.002987, NPJ15568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP 303944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP.006465, NP_003255, NP_064593, NP_005756, NP_036595, NP_002241, NP_002847, NP.056295,
  • amino acid sequences selected from the group of NPJ04027, NP_000674, NPJ30162, NP_001662, NP_036236, NP_004416, NP_001768, NP_006254, NP_002987, NP_015568, NP.004404, NP_001238, NPJ300566, NP_003995, NPJ03632, NP_002204, NP_002214, NP.004622, NP_005889, NP_003944, NP_002529, NP_443164, NPJ02836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP_005756, NP_036595, NPJ02241 , NP_002847, NP.056295, NP_055070, and NP
  • test molecules or compounds identified in the above-described assays are considered within the context of the invention as specific plasma membrane protein therapeutic agents.
  • test molecules or compounds considered to be therapeutic agents specific for a plasma membrane protein of the invention are those molecules or compounds specific for ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B,
  • therapeutic agents of the invention are specific for ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN- 5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TF
  • Most preferred therapeutic agents of the invention are those that are specific for ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5 proteins.
  • a therapeutic agent specific for a plasma membrane protein of the invention can be used for treating a non-steroid dependent cancer, and may be applied to any patient in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, and primates. But most preferably, the patient in need of such therapy is a human.
  • Therapeutic agents of the invention used for treating non-steroid dependent cancer include those agents that are specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN- 5, CD83, mud 3, SCARB1 , SLC21A8, SLC29
  • therapeutic agents that are specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN- 5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A
  • therapeutic agents that are specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of specific for an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CL
  • therapeutic agents specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5 proteins for the treatment of non-steroid dependent cancer.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for the treatment of a non-steroid dependent cancer as described herein. Treating a non-steroid dependent cancer
  • a non-steroid dependent cancer resulting from aberrant gene expression and/or biological activity of a plasma membrane protein of the invention can be treated with a therapeutic agent specific for a given protein of the invention.
  • the aberrant gene expression of a polynucleotide sequence encoding a given plasma membrane protein of the invention may result in the altered level of its corresponding polypeptide.
  • increased polypeptide levels of a given plasma membrane protein, as a consequence of aberrant.gene expression can result in -abnormal cell proliferation, cell differentiation, cell migration, tumour development or metastasis within a given subject.
  • Subjects identified as having a non-steroid dependent cancer or abnormal cell proliferation, cell differentiation, cell migration, tumour development or metastasis can be treated by administering a therapeutic agent specific for a given plasma membrane protein of the invention which has been shown to decrease the level of expression of its respective gene, or has been shown to inhibit the biological activity of the given protein.
  • a non-steroid dependent cancer resulting from the aberrant expression of a polynucleotide sequence encoding a plasma membrane protein of the invention are, for example, using complementary polynucleotide sequences, siRNA, and/or triple helix molecules to inhibit gene expression at the polynucleotide level, or using therapeutic agents identified in the various screening methods provided by the invention.
  • antibodies specific for a given polypeptide may also be used to treat a non-steroid dependent cancer. Such antibodies are able to inhibit the biological activity of a plasma membrane protein of the invention.
  • Therapeutic agents identified as decreasing, suppressing, or inhibiting the gene expression or biological activity of a plasma membrane protein of the invention can be administered to a subject at a therapeutically effective dose to treat a non-steroid dependent cancer.
  • the invention also provides methods for preventing the formation and/or development of tumours.
  • a specific lesion such as a pre- neoplastic lesion, e.g., hype ⁇ lasia, metaplasia, and dysplasia.
  • a specific lesion such as a pre- neoplastic lesion, e.g., hype ⁇ lasia, metaplasia, and dysplasia.
  • such lesions can be found in epithelial tissue. Therefore, the invention provides a method for inhibiting the development of such a lesion into a neoplastic lesion, comprising administering to a subject having a pre-neoplastic lesion, a therapeutic amount of an agent specific for a given a plasma membrane protein of the invention.
  • a therapeutic amount of such an agent will effectively decrease, suppress or inhibit the development of a pre-neoplastic lesion into a neoplastic lesion with minimal side effects.
  • the invention provides a method for decreasing, suppressing or inhibiting epithelial cell proliferation, differentiation or migration, comprising contacting a tissue in which epithelial cells display an abnormally high proliferative rate, abnormal differentiation and/or migration, with a therapeutic agent specific for a given plasma membrane protein of the invention.
  • a tissue in which epithelial cells display an abnormally high proliferative rate, abnormal differentiation and/or migration with a therapeutic agent specific for a given plasma membrane protein of the invention.
  • the inhibition of the development of a non-steroid dependent cancer is anticipated by the anti-proliferative, anti-differentiating and/or anti-migrative effects of the given therapeutic agent, identified within the assays of the invention.
  • the abnormally proliferating, differentiating or migrating cells are epithelial cells that are present in breast, lung, oesophageal, stomach, small intestinal, colonic, rectal, pancreatic, liver, gallbladder, biliary, prostatic, ovarian, cervical, and endometrial tissues.
  • Another aspect of the present invention relates to diagnostic assays for determining the expression of a biologically active plasma membrane protein of the invention, or a polynucleotide sequence encoding a biologically active a plasma membrane protein of the invention in the context of a biological sample (e.g., blood, serum, cells, tissue) to determine whether an individual is afflicted with a non-steroid dependent • cancer, or is at risk of developing a non-steroid dependent cancer resulting from an aberrant expression or biological activity of a plasma membrane protein of the invention.
  • a biological sample e.g., blood, serum, cells, tissue
  • the level of gene expression can be assayed in a biological sample to determine if a given protein may be present in a biological sample at raised levels as compared to a standard.
  • Such assays can be used for prognostic or predictive pu ⁇ oses for the prophylactic treatment of an individual prior to the development of a non-steroid dependent cancer.
  • a method for detecting the presence or absence of a plasma membrane protein of the invention or a polynucleotide sequence encoding a plasma membrane protein of the invention, in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or agent capable of detecting a given polypeptide or polynucleotide (e.g., mRNA, genomic DNA) such that the presence of a given polypeptide or polynucleotide is directly labelled.
  • a compound or agent capable of detecting a given polypeptide or polynucleotide e.g., mRNA, genomic DNA
  • Examples include detection of a primary antibody using a fluorescently labelled secondary antibody and end labelling of a DNA probe with biotin such that it can be detected with fluorescently labelled streptavidin.
  • the detection method of the invention can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for the detection of mRNA include, but are not limited to, Northern hybridizations, in situ hybridizations, reverse transcription-polymerase chain reaction (RT-PCR), microarrays (chips) coated with a polynucleotide active surface (e.g. cDNA or oligonucleotide sequences).
  • In vitro techniques for the detection of a plasma membrane protein of the invention include, but are not limited to, various techniques known to those skilled in the art such as enzyme linked immunosorbent assays (ELISAs), Western blots, immuno-precipitations, immunofluorescence, tissue-array analysis using antibodies specific for a plasma membrane protein of the invention, mass spectroscopy analysis of microarrays (chips) coated with a biologically active surface (e.g. cationic, anionic, hydrophobic, metal ion or antibody surface etc.), or fragment thereof.
  • ELISAs enzyme linked immunosorbent assays
  • Western blots Western blots
  • immuno-precipitations immunofluorescence
  • tissue-array analysis using antibodies specific for a plasma membrane protein of the invention mass spectroscopy analysis of microarrays (chips) coated with a biologically active surface (e.g. cationic, anionic, hydrophobic, metal ion or antibody surface etc.), or fragment thereof.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample contains mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • Preferred biological samples include, but are not limited to, blood, blood serum, plasma, nipple aspirate, urine, semen, seminal fluid, seminal plasma, prostatic fluid, excreta, tears, saliva, sweat, biopsy, ascites, cerebrospinal fluid, milk, lymph, or tissue extract samples isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting said polypeptide of the invention, or mRNA, or genomic DNA encoding a given plasma protein of the invention, such that the presence of the given protein, or mRNA, or genomic DNA encoding the given polypeptide is detected in the biological sample, and comparing the presence of the given polypeptide of the invention, or mRNA or genomic DNA encoding a given protein of the invention in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the given protein of the invention in the test sample.
  • the polynucleotide sequence encoding a plasma membrane protein of the invention is used as a template for the generation of probes and/or primers designed for use in determining the level of expression of a polynucleotide sequence encoding a plasma membrane protein of the invention
  • a probe and/or primer comprising a substantially purified oligonucleotide is used in which the oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 15, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of sense or antisense sequences encoding a plasma membrane protein of the invention.
  • primers based on a polynucleotide sequence encoding a given plasma membrane protein of the invention can be used in PCR reactions to clone its corresponding homolog. Such primers are preferably selected in a region that does not share significant homology to other genes.
  • probes based on a polynucleotide sequence of the invention can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto and which can be detected, e.g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
  • probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which over-express a plasma membrane protein of the invention, such as by measuring the level of a polynucleotide sequence encoding a given plasma membrane protein of the invention in a sample of cells from a patient; e.g. detecting the mRNA level of a given gene encoding a plasma membrane protein of the invention.
  • specific nucleotide probes can be generated from a polynucleotide sequence encoding a plasma membrane protein of the invention that facilitates histological screening of intact tissue and tissue samples for the presence of a specific polynucleotide sequence that encodes a given plasma membrane protein of the invention.
  • the use of such probes can be for both predictive and therapeutic evaluation, wherein the subsequent expression or presence of a.given plasma membrane protein of the invention manifests, for example, unwanted cell growth, abnormal differentiation or the activation of cell migration within a tissue.
  • the oligonucleotide probes can help facilitate the determination of the molecular basis for cancer associated with the expression of a plasma membrane protein of the invention.
  • kits for determining whether a subject is at risk of developing a non-steroid dependent cancer caused by or contributed by an aberrant gene expression and/or biological activity of a plasma membrane protein of the invention are kits for determining whether a subject is at risk of developing a non-steroid dependent cancer caused by or contributed by an aberrant gene expression and/or biological activity of a plasma membrane protein of the invention.
  • Proteins from a biological sample can be isolated and/or detected using techniques known to those skilled in the art, including but not limited to western blot analysis, immunohistochemistry, or mass spectrometry, wherein mass spectrometry includes, but is not limited to matrix-assisted laser deso ⁇ tion ionisation/time of flight (MALDI-TOF), surface-enhanced laser desorption ionisation/time of flight (SELDI-TOF), MS-MS, or ESI-MS .
  • MALDI-TOF matrix-assisted laser deso ⁇ tion ionisation/time of flight
  • SELDI-TOF surface-enhanced laser desorption ionisation/time of flight
  • MS-MS MS-MS
  • ESI-MS ESI-MS
  • antibodies or fragments thereof may be utilised for the detection of a given plasma membrane protein of the invention in a biological sample comprising applying a labelled antibody directed against to a given plasma membrane protein of the invention to said sample under conditions that favour an interaction between the labelled antibody and its corresponding protein.
  • a biological sample comprising applying a labelled antibody directed against to a given plasma membrane protein of the invention to said sample under conditions that favour an interaction between the labelled antibody and its corresponding protein.
  • a plasma membrane protein of the invention it is possible to determine not only the presence of a plasma membrane protein of the invention, but also its cellular distribution.
  • a blood serum sample only the serum levels of a given protein can be detected, whereas its level of expression and cellular localisation can be detected in histological samples. It will be obvious to those skilled in the art, that a wide variety of methods can be modified in order to achieve such detection.
  • Such labelling methods include, but not limited to, linking the antibody to an enzyme as used in an enzyme linked immunosorbent assay (ELISA), tagging the antibody with a radioactive isotope as used in a radioimmunoassay (RIA), linking the antibody to a fluorescent compound, attaching fluorescence emitting metals such as .sup.152 Eu, or others of the lanthanide series via metal chelating groups such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) to the antibody, or coupling the antibody to a chemi- or bioluminescent compound.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • an antibody coupled to an enzyme is detected using a chromogenic substrate that is recognised and cleaved by the enzyme to produce a chemical moiety, which is readily detected using spectrometric, fluorimetric or visual means.
  • Enzymes used to for labelling include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Detection may also be accomplished by visual comparison of the extent of the enzymatic reaction of a substrate with that of similarly prepared standards.
  • radiolabelled antibodies can be detected using a gamma or a scintillation counter, or they can be detected using autoradiography.
  • fluorescently labelled antibodies are detected based on the level at which the attached compound fluoresces following exposure to a given wavelength. Fluorescent compounds typically used in antibody labelling include, but are not limited to, fluorescein isothiocynate, rhodamine, phycoerthyrin, phycocyanin, allophycocyani, o-phthaldehyde and fluorescamine.
  • antibodies coupled to a chemi- or bioluminescent compound can be detected by determining the presence of luminescence.
  • luminescence include, but are not limited to, luminal, isoluminal, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, luciferin, luciferase and aequorin.
  • in vivo techniques for the detection of a plasma membrane protein of the invention include introducing into a subject a labelled antibody directed against a given polypeptide or fragment thereof.
  • An alternative method for the detection of a given plasma membrane protein of the invention within a biological sample comprises contacting said sample with an adsorbent present on a biologically active surface under specific binding conditions such that a given protein within the sample is able to bind to the said adsorbent, and detecting the presence of bound protein using mass spectrometry.
  • the adsorbent present on a biologically active surface is composed of a single material or a plurality of different materials that are capable of binding a given plasma membrane protein of the invention.
  • Such materials include, but are not limited to, polynucleotides, oligonucleotides, peptides, antibodies directed against a given protein of the invention, known binding molecules and/or compounds of a given protein of the invention, as well as the therapeutic agents identified within specific embodiments of the invention.
  • such material may also include anion exchange materials, cation exchange materials, and metal chelators.
  • the above method may also be used to detect a polynucleotide sequence encoding a plasma membrane protein of the invention within a biological sample.
  • Mass spectrometric methods used to detect bound protein may include, but are not limited to, matrix-assisted laser deso ⁇ tion ionisation/time-of-flight (MALDI-TOF), surface-enhanced laser desorption ionisation/time-of-flight (SELDI-TOF), liquid chromatography coupled with MS, MSS, or ESI-MS.
  • MALDI-TOF matrix-assisted laser deso ⁇ tion ionisation/time-of-flight
  • SELDI-TOF surface-enhanced laser desorption ionisation/time-of-flight
  • the methods used to detect plasma membrane proteins of the invention and/or the polynucleotide sequences encoding them can also be used for determining whether a subject is at risk of developing a non-steroid dependent cancer or has developed a non-steroid dependent cancer.
  • Such methods may also be employed in the form of a diagnostic kit comprising at least one probe and/or primer, a plasma membrane protein specific antibody or a biologically active surface described herein, which may be conveniently used, for example, in clinical settings to diagnose patients exhibiting symptoms or a family history of a non-steroid dependent cancer.
  • Such diagnostic kits also include solutions and materials necessary for the detection of a polynucleotide or polypeptide encoding a plasma membrane protein of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A
  • the polynucleotide sequences detected in a biological sample are selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511 , NM_000343, NM_003982, NM_022902, NMJ00232, NM_006474, NM_003264, NM_020208, NM_005765,
  • polynucleotide sequences selected from the polynucleotide sequences shown in accession numbers NM.004036, NM_000683, NM_001651, NMJ301671, NM_012104, NMJ32027, NM_001777, NM_032865, NMJ02996, NMJ07329, NM_004413, NM_001247, NM_000569, NM_004004, NM_003641 , NM_002213, NM_002223, NM_004631, NM_005898, NM.003953, NM_002538, NMJ52932, NM_002845, NM_006511, NM.000343, NM_003982, NM_022902, NM_000232, NM.006474, NM.003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_
  • polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NMJ04036, NM_000683, NM_001651, NMJ301671, NMJ12104, NM_032027, NMJ01777, NM_032865, NM_002996, NM.007329, NM_004413, NMJ01247, NMJ00569, NM_004004, NMJ03641 , NM_002213, NMJ02223, NM.004631 , NM_005898, NMJ03953, NMJ02538, NM_052932, NM_002845, NM_006511, NMJ00343, NM.003982, NM_022902, NMJ00232, NMJ06474, NM_003264, NM_020208, NM.005765, NM 12463, NMJ02250, NM_002856, NMJ15480, NMJ14255,
  • polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NMJ300569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511 , NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NM_005765, NM_012463, NM_
  • polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NMJ32865, NM_002996, NM_007329, NM.004413, NM_001247, NM_000569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NMJ352932, NM_002845, NM_006511 , NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NMJ320208, NM_005765, NM.012463, NM_002250,
  • polypeptide sequences detected within a biological sample according to the methods of the invention are selected from the amino acid sequences shown in accession numbers, NP_004027, NP_000674, NP_00162, NP_001662, NP_036236, NP_004416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP.003632, NP.002204, NP.002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP_005756, NP_036595, NP
  • polypeptide sequences detected within a biological sample are selected from the amino acid sequences shown in accession numbers NP_004027, NP_000674, NP 0162, NP_001662, NP_036236, NP_004416, NP.001768, NP.006254, NP_002987, NP_015568, NP.004404, NP.001238, NP_000566, NP_003995, NP.003632, NP.002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NPJ02836, NP_006502, NP_000334, NPJ03973, NP_075053, NP_000223, NPJ06465, NP_003255, NP_064593, NP.005756, NP_036595, NP_002241 , NP_002847, NP_0562
  • amino acids sequences selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP.001662, NP_036236, NP.004416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP.000334, NP_003973, NP_075053, NP_000223, NPJ306465, NPJ03255, NP_064593, NP_005756, NP_036595, NP_002241, NP_002847, NP_0562
  • amino acid sequence selected from the group of amino acid sequences shown in accession numbers NPJ304027, NPJ300674, NP_00162, NP_001662, NP_036236, NPJ04416, NP_001768, NP_006254, NP_002987, NP_015568, NPJ04404, NP.001238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP.443164, NP_002836, NP_006502, NP_000334, NP.003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP.005756, NP_036595, NP_002241, NP_002847, NP_056295,
  • amino acid sequences selected from the group of NPJ04027, NP_000674, NP_00162, NP_001662, NP_036236, NP_004416, NP_001768, NP.006254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NPJ02204, NP_002214, NP_004622, NP_005889, NP.003944, NPJ02529, NP_443164, NP_002836, NP_006502, NPJ300334, NP_003973, NP.075053, NP_000223, NP_006465, NPJD03255, NP_064593, NP_005756, NP_036595, NP_002241 , NP_002847, NP_056295, NP_055070, and
  • a non-steroid dependent cancer resulting from aberrant gene expression of a polynucleotide sequence encoding a plasma membrane protein of the invention can be treated by the administration and/or in situ generation of complementary polynucleotide sequences or their derivatives which specifically hybridise (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a given plasma membrane protein of the invention, so as to inhibit the expression of that protein, e.g. by inhibiting transcription and/or translation.
  • the binding may be by classical base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • this form of treatment is know to those skilled in the art as 'antisense' therapy, and refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding of the complementary polynucleotide sequence to its target polynucleotide sequences.
  • Complementary polynucleotide sequences used in selected embodiments of the invention are capable of specifically hybridising (e.g.
  • polynucleotide sequences used in selected embodiments of the invention are capable of specifically hybridising (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a given plasma membrane protein selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NMJ01777, NM_032865, NM.002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NMJ03641, NMJ02213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NMJ02845, NM_006511 , NM.000343, NM 03982, NM_022902, NM_000232
  • polynucleotide sequences capable of specifically hybridising (e.g. bind) under cellular conditions, a polynucleotide sequence encoding a given protein of the invention selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NMJ01671, NMJ12104, NM_032027, NM.001777, NM.032865, NM_002996, NM 07329, NM_004413, NM.001247, NM_000569, NM_004004, NMJ03641, NMJ02213, NM_002223, NMJ04631, NM_005898, NMJ03953, NM_002538, NM_052932, NM 02845, NM_006511, NM_000343, NM.003982, NM_022902, NMJ00232, NM_006474, NM_003264, NM_
  • polynucleotide sequences that possess complementarity to a polynucleotide sequence encoding a given plasma membrane protein selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651 , NM_001671, NMJ12104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM.004413, NM_001247, NMJ300569, NM.004004, NM_003641, NM_002213, NMJ02223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NMJ22902, NM_000232, NM.006474, NM_003264, NM_020208,
  • complementary polynucleotide sequences are delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA encoding a plasma membrane protein of the invention.
  • the complementary polynucleotide sequence is a nucleic acid molecule that is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridising with the mRNA and/or genomic sequences of a gene encoding a plasma membrane protein of the invention.
  • Such polynucleotide sequences are preferably modified such that they are resistant to endogenous nucleases, e.g.
  • Molecules for use as complementary polynucleotide sequences are selected from the group of, but not limited to phosphodiamidate, phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA (Froehler et al. (1988) Nucleic Acids Res. 16, 4831-4839; Hyrup and Nielsen (1996) Bioorg. Med. Chem. 4, 5-23; Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451; Stein et al. (1988) Nucleic Acids Res.
  • oligonucleotides either DNA or RNA, or derivatives thereof
  • the complementary polynucleotide sequence will bind to said mRNA transcript and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence 'complementary' to a portion of an RNA means a sequence having sufficient complementarity to be able to hybridise with the RNA, forming a stable duplex; in the case of double-stranded antisense polynucleotide sequences, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridise will depend on both the degree of complementarity and the length of the complementary polynucleotide sequence. Generally, the longer the hybridising polynucleotide sequence, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridised complex.
  • polynucleotide sequences are complementary to the 5' end of the mRNA, e.g., the 5' UTR up to and including the AUG translation initiation codon, should work most efficiently at inhibiting translation.
  • sequences complementary to the 3' UTR of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well (Wagner, R. (1994) Nature 372, 333-335). Therefore, polynucleotide sequences complementary to either the 5' UTR, 3' UTR, or non-coding regions of a gene encoding a given plasma membrane protein of the invention could be used in an antisense approach to inhibit the translation of the endogenous mRNA of a given plasma membrane protein of the invention.
  • Polynucleotide sequences that are complementary to the 5' UTR of the mRNA should include the complement of the AUG start codon (e.g. -20 to +20 nucleotides). Polynucleotide sequences complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridise to the 5', 3' or non-coding region of an mRNA encoding a plasma membrane protein of the invention, complementary polynucleotide sequences should be at least 10 nucleotides in length, and are preferably polynucleotide sequences ranging from 15 to about 50 nucleotides in length.
  • the antisense polynucleotide sequence is at least 15 nucleotides, at least 18 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, or at least 50 nucleotides in length.
  • in vitro studies are first performed to quantitate the ability of the complementary polynucleotide sequence to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between specific gene inhibition via a given complementary polynucleotide sequence, and non-specific biological effects of such polynucleotide sequences. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using complementary polynucleotide sequences are compared with those obtained using control sequences.
  • control oligonucleotide sequence is of approximately the same length as the test complementary sequence and should have a similar nucleotide composition, molecular weight and melting temperature. However, these parameters should differ no more than necessary to prevent specific hybridisation to the target sequence. Such sequences may be of sense, inverse or of scrambled nature.
  • the complementary polynucleotide sequences can be DNA or RNA, or chimeric mixtures, or derivatives thereof, single-stranded or double-stranded, or triple helix-forming oligonucleotides.
  • Such polynucleotide sequences can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridisation, etc., or may include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6553-6556; Lemaitre et al. (1987) Proc.
  • the complementary polynucleotide sequences may be conjugated to another molecule, e.g., a peptide, hybridisation triggered cross-linking agent, transport agent, triggered cleavage agent, etc.
  • EPEI ethoxylated polyethylenimine
  • the complementary polynucleotide sequences may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannos
  • the complementary polynucleotide sequence may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. Furthermore, complementary polynucleotide sequences lacking a pentose sugar moiety are also within the scope of the invention.
  • the complementary polynucleotide sequence comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate (Stein and Cohen (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.97-117, J. Cohen, ed.
  • Oligonucleotides Antisense Inhibitors of Gene Expression, pp.82-85), a formacetal or analogue thereof, as well as ⁇ -DNA (Rayner et al. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression. pp.119-136, J. Cohen, ed. (Boca Raton, FL: CRC Press, Inc.)), 2'-0-methyl RNA (Shibahara et al. (1989) Nucleic Acids Res. 17, 239-252), a mo ⁇ holine backbone (Summerton and Weller (1997) Antisense polynucleotide sequences Drug Dev.
  • Complementary polynucleotides of the invention may be synthesized by standard methods known in the art.
  • phosphorothioate oligomers may be synthesized by the method of Stein et al. (1988) Nucleic Acids Res. 16, 3209-3021) and methylphosphonate oligomers can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451).
  • Mo ⁇ holino oligomers may be synthesized by the method of Summerton and Weller, U.S. Patent Nos. 5,217,866 (published in June 1993) and 5,185,444 (February 1993), etc.
  • RNA interference to alter post-transcriptional expression of a gene encoding a plasma membrane protein of the invention is also within the scope of the invention (Boutla et al. (2001) Curr. Bi . 11, 1776-1780; Moss, E.G. (2001) Curr. Biol. 7,R772-R775; Bernstein et al. (2001) RNA 7, 1509-1521).
  • RNAi is the process of sequence-specific, post-transcriptional gene silencing, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.
  • siRNAs small interfering RNAs
  • ribonuclease III cleavage from longer dsRNAs.
  • the length of siRNAs is between 20-25 nucleotides (Elbashir et al. (2001) Nature 411, 494-498).
  • Triple helix-forming oligonucleotides to modify expression of a gene encoding a plasma membrane protein of the invention are also within the scope of the invention. Triple helix formation is used to inhibit the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
  • the invention also includes the use of circular RNA (i.e. RNA Lassos) to inhibit the translation of a specific protein of known function.
  • Nucleic Acids Res. 16, 4831-4839 a phosphordiamidate, a methylphosphonate (Miller, P. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.85-92), an alkyl phosphotriester (Miller, P. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.82-85), a formacetal or analogue thereof, as well as ⁇ -DNA (Rayner et al.
  • Oligonucleotides Antisense Inhibitors of Gene Expression. pp.119-136, J. Cohen, ed. (Boca Raton, FL CRC Press, Inc.)), 2'-0-methyl RNA (Shibahara et al. (1989) Nucleic Acids Res. 17, 239-252), a mo ⁇ holine backbone (Summerton and Weller (1997) Antisense polynucleotide sequences Drug Dev. 7, 187-195), repeating N-(2-aminoethyl)-glycine units linked by peptide bonds (peptide nucleic acids; PNAs) (Hanvey et al. (1992) Science 258, 1481-1485; Nielsen et al.
  • Complementary polynucleotides of the invention may be synthesized by standard methods known in the art.
  • phosphorothioate oligomers may be synthesized by the method of Stein et al. (1988) Nucleic Acids Res. 16, 3209-3021) and methylphosphonate oligomers can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451).
  • Mo ⁇ holino oligomers may be synthesized by the method of Summerton and Weller, U.S. Patent Nos. 5,217,866 (published in June 1993) and 5,185,444 (February 1993), etc.
  • RNA interference RNA interference
  • RNAi is the process of sequence-specific, post-transcriptional gene silencing, initiated by double-stranded
  • dsRNA RNA that is homologous in sequence to the silenced gene.
  • the mediators of sequence-specific messenger RNA degradation are small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs.
  • siRNAs small interfering RNAs
  • the length of siRNAs is between 20-25 nucleotides (Elbashir et al. (2001)
  • Triple helix-forming oligonucleotides to modify expression of a gene encoding a plasma membrane protein of the invention are also within the scope of the invention. Triple helix formation is used to inhibit the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
  • RNA Lassos circular RNA
  • Preferred complementary polynucleotides sequences of the above embodiments of the present invention include silencing (RNAi) antisense polynucleotides, morpholino oligomers (Summerton and Weller (1999) Antisense polynucleotide sequences Drug Dev. 7, 187-195), peptide nucleic acids (PNAs) (Egholm et al. (1992) J. Am. Chem. Soc. 114, 1895-1897) and/or locked nucleic acids (LNAs) (Kumar et al. (1998) Bioorg. Med. Chem. Lett. 8, 2219-2222; Petersen et al. (2000) J. Mol. Recognit. 13, 44-53).
  • RNAi silencing
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • complementary polynucleotide sequences are delivered to cells that express a plasma membrane protein of the invention in vivo.
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense polynucleotide sequences, designed to target the desired cells e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface can be administered systematically.
  • polynucleotide sequences used in the screening for therapeutic agents, the design of probes and/or primers for the detection of polynucleotide levels, and as target sequences for complementary polynucleotide sequences for the treatment of a non-dependent steroid cancer are selected from the group of nucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM 01671, NM_012104, NM_032027, NM_001777, NMJ332865, NM_002996, NM_007329, NM_004413, NM 01247, NM.000569, NM_004004, NM 303641, NM.002213, NMJ02223, NMJ304631, NMJD05898, NMJ03953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NMJ03982,
  • polypeptides comprising of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP_001662, NP.036236, NP_004416, NP_001768, NPJ06254, NPJ02987, NPJ515568, NP_004404, NP_001238, NP_000566, NPJ03995, NP_003632, NPJ302204, NP_002214, NP_004622, NP_005889, NP.003944, NP_002529, NP.443164, NP_002836, NP.006502, NPJ00334, NP.003973, NP_075053, NP_000223, NP_006465, NP.003255, NP 64593, NP.005756, NP_036595, NP_002241, NP_002847, NP.056295, NP
  • the invention also includes polynucleotide variants having at least 50% polynucleotide sequence identity to a polynucleotide shown in accession numbers NM J04036, NM_000683, NM_001651, NM_001671 , NM_012104, NM_032027, NMJ01777, NM_032865, NM_002996, NM_007329, NM_004413, NMJ01247, NM_000569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631 , NM_005898, NMJ03953, NM_002538, NM_052932, NMJ02845, NM_006511, NMJ00343, NM.003982, NM_022902, NM.000232, NM_006474, NM_003264, NM.020208, NM_005765, NMJ12463
  • Preferred polynucleotides have a sequence at least about 50% homologous, more preferably at least about 60%, 65%, 70%, 75%, or 80%, and even more preferably at least about 85% homologous with, or a complement of a polynucleotide sequence shown in accession numbers NM_004036, NM_000683, NMJ01651, NM_001671 , NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NMJ07329, NM_004413, NM_001247, NM_000569, NM_004004, NM.003641, NM_002213, NM_002223, NMJ04631, NM_005898, NM_003953, NM_002538, NM.052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902,
  • Polynucleotide sequences at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homologous with, or complements of a polynucleotide sequence shown in accession numbers NM_004036, NM 300683, NM_001651, NM_001671 , NM_012104, NM_032027, NM 01777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NMJ03641, NMJ02213, NM.002223, NMJ04631 , NM_005898, NM_003953, NM_002538, NM.052932, NM_002845, NM_006511, NM_000343, NM 03982, NM_022902, NM.000232, NM_006474, NM_003264, NMJ2020
  • the polynucleotides are vertebrate genes and, in particularly preferred embodiments, include all or a portion of a nucleotide sequence corresponding to the coding regions of polynucleotides shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM.001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NM.003641, NM.002213, NMJ02223, NM_004631 , NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_
  • the polynucleotides of the invention encode the given plasma membrane proteins from any species.
  • Preferred polynucleotide sequences encode vertebrate plasma membrane proteins of the invention. More preferred are polynucleotide sequences that encode mammalian plasma membrane proteins of the invention. Even more preferred are polynucleotide sequences that encode plasma membrane proteins of the invention, specifically of primate origins (e.g. human).
  • the invention further includes polypeptides that are at least 50% identical or similar to an amino acid sequence encoding a biologically active plasma membrane protein of the invention selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NPJ0162, NPJ01662,
  • Polypeptides which are at least about 60%, and even more preferably at least about 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical or similar to an amino acid sequence shown in accession numbers NP_004027, NP_000674, NP_00162, NPJ01662, NP_036236, NPJ04416, NPJ01768, NP_006254, NP_002987, NP_015568, NP.004404, NPJ01238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NPJ03973, NP_075053, NP_000223, NP_006465, NP_003255, NP.064593, NP_00
  • polypeptides include such plasma membrane proteins from any species.
  • Preferred polypeptides are vertebrate plasma membrane proteins of the invention. More preferred polypeptides are mammalian plasma membrane proteins of the invention including primates e.g., human polypeptides.
  • polynucleotide sequences encoding plasma membrane proteins of the invention will further allow for the generation of probes and primers for use determining the presence and/or level of polynucleotide expression within a biological sample.
  • Preferred polynucleotide sequences for use as a probe include polynucleotide sequences comprising a nucleotide sequence having at least about 15, at least about 30, preferably at least about 50, more preferably at least about 100, and even more preferably at least about 200 consecutive nucleotides from a polynucleotide sequence encoding a plasma membrane protein of the invention shown, or a fragment thereof.
  • a portion of the polynucleotide sequence corresponds to any segment of the complete coding sequence of a gene encoding a plasma membrane protein of the invention.
  • a portion can be a specific polynucleotide sequence encoding a conserved motif or domain of a given plasma membrane protein of the invention, e.g. extracellular domain, ligand binding domain, transmembrane domain etc.
  • a portion can be a polynucleotide sequence located between polynucleotide sequences encoding conserved motifs of a given plasma membrane protein of the invention.
  • the invention further pertains to polynucleotide sequence molecules for use as probes and/or primer (i.e. non- coding polynucleotide sequence molecules), which can comprise at least about 15, 18, 20, 25, 30, 40, 50, 100, 125, 150 or 200 nucleotides or base pairs.
  • polynucleotide sequences comprise at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or at least about 600 nucleotides of a polynucleotide sequence encoding a plasma membrane protein of the invention.
  • the polynucleotide sequences of the invention correspond to the 5' portion of a polynucleotide sequence encoding a encoding a plasma membrane protein of the invention.
  • polynucleotide sequences that are capable of hybridising to a polynucleotide sequence encoding a plasma membrane protein of the invention, and fragments thereof, under various conditions of stringency.
  • Conditions that promote hybridisation of polynucleotide sequences are known to those skilled in the art (see Current Protocols in Molecular Biology (1989, John Wiley & Sons, N.Y. 6.3.1- 6.3.6; Jowett, T. (2001) Methods 23, 345-358).
  • the present invention also provides a probe and/or primer comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region of a polynucleotide sequence that hybridises under stringent conditions to at least about 15, preferably about 30, more preferably about 50, 100 or 200 consecutive nucleotides of sense or antisense sequence of a polynucleotide sequence encoding a plasma membrane protein of the invention, or naturally occurring mutants thereof.
  • primers based on a given polynucleotide sequence of the invention can be used in PCR reactions to clone a given homolog. Such primers are preferably selected in a region that does not share significant homology to other genes.
  • probes based on a given polynucleotide sequence that encodes a plasma membrane protein of the invention can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto and which can be detected, e.g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
  • probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which over- express a given plasma membrane protein of the invention, such as by measuring the level of its corresponding polynucleotide sequence in a sample of cells from a patient; e.g. detecting mRNA levels.
  • the use of probes directed to the polynucleotide sequences of the invention, or to the genomic polynucleotide sequences encoding a plasma membrane protein of the invention can be used for both predictive and therapeutic evaluation of altered gene expression levels which might be manifested in, for example, unwanted cell growth or abnormal differentiation of tissue.
  • kits for determining whether a subject is at risk of developing a non-steroid dependent cancer resulting from the over-expression of a polynucleotide sequence encoding a plasma membrane protein of the invention may include probes/primers specific for a given polynucleotide sequence of the invention, reaction solutions, and instructions of how to use the kit.
  • polynucleotide sequences which are used as templates for probes and/or primers and/or for complementary sequences are selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651 , NM.001671, NM_012104, NM_032027, NM.001777,
  • template polynucleotide sequences are selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NMJ301671,
  • template polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM.004036, NM_000683, NM_001651 , NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NMJ01247, NM_000569, NM.004004, NM_003641, NMJ02213, NMJ02223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NM_005765, NMJ312463, NM_002250,
  • template polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM.001777, NM_032865, NM_002996, NM.007329, NM_004413, NM_001247, NM.000569, NM_004004, NM_003641, NM_002213, NM_002223, NM.004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NMJ03264, NM.020208, NM_005765, NM.012463, NM_002250,
  • template polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651 , NM.001671, NM.012104, NM_032027, NM_001777, NM_032865, NM_002996, NM.007329, NM.004413, NM_001247, NM_000569, NMJ04004, NM_003641, NM.002213, NMJ02223, NMJ04631, NM_005898, NM_003953, NM_002538, NM.052932, NM_002845, NM_006511 , NM_000343, NMJ03982, NM_022902, NM_000232, NMJ306474, NMJ03264, NM_020208, NM_005765, NM_012463, NMJ02250, NM_002856,
  • expression vectors used for screening a library of molecules and/compounds for therapeutic agents that modulate the expression of a polynucleotide sequence encoding a plasma membrane protein of the invention contain a polynucleotide sequence encoding a reporter protein operably linked to a regulatory sequence specific of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53,
  • Reporters proteins used according to the methods of invention include, but are not limited to, ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), or neomycin.
  • expression vectors used for screening a library of molecules and/compounds for therapeutic agents that modulate the biological activity of a plasma membrane protein of the invention contain a polynucleotide sequence encoding a plasma membrane protein of the invention selected from the polynucleotide sequences shown in NM_004036, NM_000683, NM_001651, NMJ01671, NM_012104, NMJ32027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM.001247, NM_000569, NMJ04004, NM.003641 , NMJ02213, NM_002223, NM_004631 , NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NMJ06511, NM_000343, NM_003982, NM_022902, NM_000232
  • Expression vectors containing a polynucleotide sequence encoding an antagonistic polypeptide, peptide or antisense polynucleotide sequence to a given polynucleotide sequence of the invention, and being operably linked to at least one transcriptional regulatory sequence may be used for practicing the present invention.
  • Regulatory sequences are recognized by those skilled in the art, and are specifically selected to inhibit the expression and/or biologically activity of an endogenous ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10
  • the expression vector includes a polynucleotide sequence encoding an antisense polynucleotide sequence for the pu ⁇ ose of inhibiting the expression of a given plasma membrane protein of the invention.
  • the expression vector contains a polynucleotide sequence encoding a polypeptide or peptide which can be used to inhibit the biological activity of a given plasma membrane protein of the invention.
  • Such expression vectors can be used as a part of a gene therapy protocol.
  • another aspect of the invention features expression vectors for in vivo, in vitro, or ex vivo transfection and expression of an antagonistic polypeptide, peptide or an antisense polynucleotide sequence to abrogate the biological activity of a given plasma membrane protein of the invention (e.g. differentiation of epithelial or cancer cells). This could be desirable, for example, when the naturally occurring form of the protein is over-expressed.
  • transcriptional regulatory sequences are recognised by those skilled in the art and include, but are not limited to, the cytomegalovirus (CMV), SV40 and he ⁇ es simplex virus (HSV) thymidine kinase (TK) promoters, as well as T7, T3 or SP6 promoter sequences.
  • CMV cytomegalovirus
  • HSV SV40
  • HSV he ⁇ es simplex virus
  • TK thymidine kinase
  • Example 1 RNA isolation from colon and rectal tissue and cDNA labelling methods.
  • tissue samples were collected from 16 colon and 9 rectal cancer patients at varying stages of the disease, together with healthy colon and rectal tissue taken from a distant site (sample set).
  • Tissue samples were collected from both male and female patients of varying ages at hospitals in Cottbus (Caii- Thiem-Klinikum Cottbus, Chirurgische réelle, 03048 Cottbus, Germany), Magdeburg (Otto-von-Guericke- réelle, fürer Strasse 44, 39120 Magdeburg, Germany) and Eriangen (Friedrich-Alexander-Universitat, Chirurgischegraphy, 91023 Eriangen, Germany).
  • Epithelial cells were isolated according to the methods described in the patent WO98/43091 'Diagnosis of Epithelial Cell Abnormalities'.
  • the tissue samples were initially prepared by incubating with RNAIaterTM (Ambion, UK) for 15 min. on ice. Following incubation, the tissue samples were reduced in size (ca. 1 mm) using a scalpel and mechanically separated using a steel mesh of 300 ⁇ M.
  • the cell suspension was mixed with ⁇ O ⁇ l anti-BerEP4 Dynabeads® (Deutsche Dynal GmbH, Germany) and incubated for 30 min at 2-8°C in wash buffer.
  • the epithelial cell-bound Dynabeads® were collected using a magnet (Deutsche Dynal GmbH, Germany), aliquoted on ice and stored at -80°C.
  • each sample set was analysed using oligo microarrays containing more than 19000 spotted 60-mer oligonucleotides representing more than 16000 different human genes (human-1A oligo microarray, Agilent Technologies, USA). Cy-3 labelled aRNAs from epithelial cells isolated from non-tumour tissue, were mixed with Cy-5 labelled aRNAs from tumour tissue and allowed to hybridise with the oligo microarray at 65°C overnight (according to the manufacturers instructions for the humanlA oligo microarrays, Agilent Technologies, USA). All hybridisations were performed with flour reversal pairs.
  • RNAs that hybridised to their complementary sequences using a dual laser microarray scanner from Agilent Technologies.
  • Feature extraction software (Version A.7.1.1) from Agilent Technologies was used to correlate the location of the various chip features (spots) with the annotated coding sequences on the oligo microarray.
  • the software was also employed to calculate the signal intensity of a single chip feature in order to provide information regarding the expression level of its respective coding sequence in tumour and healthy tissue (figure 1).
  • data analysis was performed using the Rosseta Resolver system (Rosetta Inpharmatics, Kirkland, USA).
  • genes CTEN, CX3CL1, PROM1 , PVRL3, SCARB1, SLC30A5, TNFRS12A are represented as FLY4950, SCYD1 , PROML1, DKFZP566B0846, CD36L1, ZTL1 , FN14 on the heat-map provided ( Figure 1), respectively.
  • oligo microarrays only contain short 16-mer nucleotide sequences, it is possible that the up- regulation of gene expression for a given plasma membrane protein may be an artefact of the experimental system (non-specific hybridisation).
  • a polynucleotide sequence encoding a plasma membrane protein of the invention is genuinely up-regulated in tumour tissue, its expression pattern can be alternatively analyzed by real-time, quantitative RT (reverse transcription)-PCR (polymerase chain reaction).
  • RNA from a minimum of 20 tumour and non-tumour pairs (isolated epithelial cells) of the tissue of interest and 3-4 different cell lines (controls for intra-assay variability) is reverse transcribed in triplicate using random hexamers and then amplified in parallel by real-time quantitative PCR in the presence of SYBR Green I using a pair of primers specific for the sequence of interest (in each case, the mRNA of a ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2,
  • primers are designed using Applied Biosystems' Primer Express 2.0 software; they should span an intron to avoid the detection of contaminating DNA, and should not be complementary to any other sequence of the human genome as detected when doing a BLAST search.
  • a similar cDNA pool can used to amplify several polynucleotide sequences consecutively, as well as an endogenous reference (28S rRNA) for the normalisation of the data (correction of sample to sample pipetting variations, etc.).
  • Amplification and real-time detection and analysis of the fluorescent signals produced when SYBR Green intercalates into the double strand of the newly amplified sequences is routinely performed using the ABI Prism 7000 Sequence Detection System and associated software (v. 1.0) from Applied Biosystems.
  • Ct threshold cycle
  • Extrapolation of each Ct value from an appropriate standard curve (plot of Ct values vs. quantity obtained by amplification of the target and reference genes from a series of known dilutions of a total RNA sample) will provide the relative amounts of a given gene and reference genes in a particular replicate sample, while the ratio [mean ⁇ SD]t ar get gene/[mean ⁇ SD]reference gene will normalise the amount of a given target within that particular sample, allowing comparison between samples.
  • the amount of a given polynucleotide sequence in each tumour will be compared to the amount of the same polynucleotide sequence in the corresponding non- tumour sample to determine if it is over- or under expressed in a particular sample, or if its expression does not change.
  • Example 4 Determination of protein expression levels for plasma membrane proteins of the invention.
  • the confirmation of over-expression of plasma membrane proteins of the invention at the protein level is performed by western blot and immunohistochemistry (IHC) analyses.
  • IHC immunohistochemistry
  • protein expression by either technique is analyzed using commercially available antibodies. However, for many proteins this is not possible and therefore, new antibodies will need to be produced.
  • the production of polyclonal antibodies against the protein(s) of interest is out-sourced, and is normally based on the immunization of 2 rabbits with 2 different peptides (administrated mixed or separately) of our own design, supported by the design of the company producing the antibodies.
  • Protein sequences are selected from the public domain (i.e. SwissProt, NCBI).
  • Peptide design can also be performed using publicly available bioinformatics tools such as Prosite, surface probability, flexibility and antigenicity profiles, etc., or commercially available ones, for example, Protean from DNASTAR or Metalife's Epitope Validator. Whenever possible peptides are designed in the extracellular regions of the protein; transmembrane regions are to be avoided. Furthermore, predicted phosphorylation, glycosylation, myristylation as well as other post-translational modification sites should also be avoided. The immune sera and/or purified antibodies provided by the company are first tested by western blotting using protein extracts (isolated with strong detergent conditions, e.g.
  • the protein is cloned fused to a tag (e.g. FLAG tag) to control the over-expression (e.g. with an anti-FLAG tag monoclonal antibody).
  • a tag e.g. FLAG tag
  • the immune sera and/or purified antibodies should recognize only the over-expressed protein (also the endogenous one if the cells used express it), while the pre-immune sera should not.
  • immunoprecipitation is performed to confirm recognition of the protein by the sera and/or purified antibodies.
  • the expression of the plasma membrane proteins of the invention are analyzed by both western blotting and IHC using standard protocols known to those skilled in the art.
  • Western blotting and IHC is performed on a minimum of ten or more non-tumour and tumour pairs of tissue to confirm over-expression of a given protein of the invention in the tumour tissue.
  • additional normal and tumour tissues are also analyzed to determine if the over-expression of a given protein of the invention occurs in all tumourigenic tissues or if it is restricted to one or a few tumour types.
  • results of protein expression are correlated with clinical data. Altogether, the pattern and frequency of over-expression and the correlation to clinical parameters help to prioritize the targets for further study.
  • RNAi RNA interference
  • siRNA synthetic double stranded RNA duplexes
  • Synthetic siRNAs will be ordered from Dharmacon or MWG biotech. Two different human tumour cell lines are transfected in triplicate (using Lipofectamine 2000, Invitrogen) with 50nM of each siRNA for three consecutive days and will be analyzed on day 4-post first transfection for both gene knockdown and proliferation. Analysis of the gene knockdowns is performed either by quantitative RT-PCR or - if an antibody against a given protein is available - by Western blot.
  • This example describes a simple assay for isolating therapeutic agents that alters the biological activity of a plasma membrane protein of the invention. Based at least in part on the results described in the previous examples, such therapeutic agents will inhibit the biological activity of a ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8,
  • a therapeutic agent of a given plasma membrane protein of the invention is identified by using an in vitro assay, in which the interaction between a plasma membrane protein of the invention and a molecule known to bind and/interaction with said protein is determined in the presence and in the absence of a test compound.
  • a soluble binding fragment of a plasma membrane protein of the invention is prepared by expressing its corresponding polynucleotide sequence as selected from the polynucleotide sequences listed in Table 1 (pp. 64-66), or a fragment thereof, in £ coli according to methods known to those skilled in the art.
  • a molecule known to bind/interact with a plasma membrane protein of the invention can be produced recombinantly (i.e. if the molecule is of amino acid nature), chemically (i.e. nucleic acids, chemical compounds), or biologically synthesised as in the case of antibodies.
  • Test molecules or compounds are then tested to determine whether they inhibit the interaction between a plasma membrane protein of the invention, and a protein known to bind and/or interact with a given protein by using an ELISA type assay.
  • the molecules or compounds to be tested are labelled using appropriate methods known to those skilled in the art (i.e. tagging polypeptides with an epitope that is recognised by an antibody, or tagging a nucleic acid with a fluorescent label).
  • Unoccupied sites on the plate may be blocked with a BSA solution.
  • various amounts of the test compounds and/or molecules are added, together with a molecule that is known to bind and/or interact with a given protein of the invention, to the wells of the microtiter plate in an appropriate buffer suitable for a specific interaction between a given protein and the added molecules.
  • the wells are washed to removed any unbound molecules and the amount of test molecule bound to said protein can be determined by determining the optical density using an ELISA reader. A higher optical density indicates that the test molecule inhibits the interaction of a given protein of the invention and a molecule known to bind and/or interact with said protein and is indicative of its ability to inhibit the said protein's function.
  • Therapeutic agents specific for plasma membrane proteins of the invention can also be identified using a reporter assay in which the level of expression of a reporter construct under the control of a ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF
  • Gene promoters specific for polynucleotide sequences encoding plasma membrane proteins of the invention can be isolated by screening a genomic library with a cDNA encoding a given protein of the invention; preferably the 5' end of the cDNA. A portion of the promoter, typically from about 50 to 500 base pairs long in then cloned upstream of a reporter gene such as, but not limited to a ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), neomycin gene, in an expression plasmid.
  • a reporter gene such as, but not limited to a ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein
  • This reporter construct is then transfected into cells, e.g., epithelial cells. Transfected cells are then distributed into wells of a multi-well plate and various concentrations of the test molecule or compound are added to the wells. After several hours of incubation, the level of expression of the reporter construct is determined according to methods know to those skilled in the art. The level of expression of the reporter construct in transfected cells incubated with the test compound is compared to the level of expression of the reporter construct in transfected cells incubated without the test compound.
  • test molecule or compound is capable of regulating the expression of a polynucleotide sequence encoding a given plasma membrane protein of the invention, or regulating the biological activity of a given plasma membrane protein of the invention, thereby indicating a specific therapeutic activity.
  • Table 1 List of up-regulated genes encoding membrane proteins in tumourigenic tissue.

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Abstract

The invention relates to agents and methods for the diagnosis, prognosis and treatment of non-steroid dependent cancer. Specifically, the invention relates to the use of nucleic and amino acid sequences encoding plasma membrane proteins of the invention that are differentially expressed in tumour tissues for the diagnosis of both early and late stage non-steroid specific cancers, cancer prognosis, as well as screening for therapeutic agents that regulate the gene expression and/or biological activity of such proteins. This invention further relates to the biological technologies designed to inhibit the gene expression and/or biological activity of the proteins plasma membrane proteins of the invention, including using agents identified in screening assays described herein, as well as molecules that are complementary to polynucleotide sequences encoding plasmas membrane proteins of the invention and antibody targeting of said proteins. In specific embodiments, the proteins are of human origin.

Description

Membrane Markers for use in Cancer Diagnosis and Therapy
The invention relates to agents and methods for the diagnosis, prognosis and treatment of non-steroid dependent cancer. Specifically, the invention relates to the use of nucleic and amino acid sequences encoding plasma membrane proteins of the invention that are differentially expressed in tumour tissues for the diagnosis of both early and late stage non-steroid specific cancers, cancer prognosis, as well as screening for therapeutic agents that regulate the gene expression and/or biological activity of such proteins. This invention further relates to the biological technologies designed to inhibit the gene expression and/or biological activity of the proteins plasma membrane proteins of the invention, including using agents identified in screening assays described herein, as well as molecules that are complementary to polynucleotide sequences encoding plasmas membrane proteins of the invention and antibody targeting of said proteins. In specific embodiments, the proteins are of human origin.
BACKGROUND TO THE INVENTION Despite improved therapies for certain forms of non-steroid dependent cancers, cancer still remains the leading cause of the death worldwide. Early detection of the disease often greatly improves the chances of complete remission, thereby making it the choice focus area of research regarding development of appropriate diagnostic and therapeutic tools. Diagnostic companies around the world invest a great deal of their allocated research funds in developing such early detection tools, with a primary focus on the detection of cancer prior to the development of a substantial tumour. Furthermore, these companies are also developing detection tools for post-operative analysis, in order to provide a method of monitoring any trace of cancer (e.g. either in the form of residual tissue from the primary tumour or of secondary tumours caused by metastasis) in the patient following surgical treatment of a tumour. The latter allows the physician to prescribe an appropriate treatment such as chemotherapy, to supplement the surgical therapy.
In order to detect cancer in a patient, large quantities of non-steroid cancer cells must be present within the cancer site in order for the physician to make an effective diagnosis. In practice, this is not the case. Too small quantities of detectable non-steroid dependent cancer cells limit detection during a physical examination of the cancer site. Furthermore, the cancer site may not be susceptible to direct visual observation leaving the physician with no means of making a clear diagnosis. And in the case of potential secondary tumour development, it is not possible to predict where the tumours are likely to occur, thereby making the detection of secondary tumours difficult by visual observation. In order to overcome these problems, sensitive diagnostic tests based on the detection of cancer-associated proteins, in a patient who has, or is about to develop, cancer cells have been developed and are currently commercially available. Examples of such diagnostic tests include alpha-fetoprotein and teratocarcinoma for the identification of primary liver cancer in humans (IZOTOP, Hungary) detection of gastrointestinal cancers using carcinoembryoπic antigen (Abbot/Roche, Switzerland), chorionic gonadotropin for the detection of trophoblasts and germ cell cancers (IZOTOP, Hungary; BioCheck Inc., CA), and prostatic acid phosphatase or prostate specific antigens for prostate carcinomas. Although these markers are being used to detect various cancers, the disadvantage of these markers lies in their ability to detect advanced rather than early stage cancers.
Furthermore, many of the commercially available tests are only applicable to a very narrow range of non-steroid dependent cancer types, meaning that such tests often fail to detect other forms of cancer. Furthermore, the narrow applicability of these tests means that it may be necessary to perform multiple tests on a single patient for such diagnostic purposes. Multiple testing is expense, and the risk that one of the many tests may produce a false-positive test result is high. Therefore, there is a pertinent need for a single diagnostic test that is capable of not only detecting a small number of cancer cells within a patient, but also capable of detecting these cells in a wide variety of non-steroid dependent cancers such as gastrointestinal cancers. An ideal marker would be one that is genetically expressed in a variety of transformed cells, as well as specific and universally applicable for the purpose of cancer detection.
To date, no effective pre-symptomatic clinical signs or biomarkers indicating susceptibility to non-steroid dependent cancers exist, making early detection a high priority in the medical management of the disease. Since current therapeutic strategies for early stages of cancer have a higher cure rate than those for later stage cancer, the survival rate of the patient can be increased through early detection. For this reason, the identification of a molecular marker specific for early oncogenic tissues will assist in the diagnosis as well as prognostic monitoring of a developing cancer. Furthermore, such a molecular marker could also be used to screen a library of molecules or compounds for the purpose of developing an effective therapeutic agent which, when administered at an early stage of cancer development, would provide an effective treatment against malignant disease.
The present invention addresses these issues by providing tumour-specific plasma membrane proteins for the diagnosis and prognosis of non-steroid dependent cancers. Furthermore, the plasma membrane protein encoding amino acid sequences and their corresponding polynucleotide sequences are used to screen for agents that alter their biologically activity and/or gene expression for the purpose of identifying and developing a therapeutic agent for the treatment of non-steroid dependent cancers.
SUMMARY OF THE INVENTION
Generally, the present invention relates to the use of polynucleotide sequences encoding plasma membrane proteins of the invention, the amino acid sequences encoding such proteins, derivatives or fragments thereof, for the diagnosis, prognosis and treatment of non-steroid dependent cancers. In specific embodiments, the given proteins are of human origin. Specifically, the present invention relates to the use of adenylate cyclase type III (ADCY3), alpha-2C adrenergic receptor (ADRA2C), aquaporin 5 (AQP5), asialoglycoprotein receptor 1 (ASGR1), beta-secretase (BACE), beta-amyloid peptide-binding protein (BBP), CD47, C-terminal tensin-like protein (CTEN), fractalkine (CX3CL1), deleted in malignant brain tumours (DMBT1), microsomal dipeptidase precursor (DPEP1), ectonucleoside triphosphate disphosphohydrolase 6 (ENTPD6), FCGR3A, gap junction beta-2 protein (GJB2), interferon-induced transmembrane protein 1 (IFITM1), integrin beta-5 protein (ITGB5), inositol 1 ,4,5-triphosphate receptor type 2 (ITPR2), low density lipoprotein receptor-related protein 8 (LRP8), membrane component chromosome 11 surface marker 1 protein (M11S1), myelin protein zero-like 1 (MPZL1), occludin (OCLN), pro-oncosis receptor inducing membrane injury protein (PORIMIN), receptor-type protein-tyrosine phosphatase mu precursor (PTPRM), regulatory solute carrier protein family 1 member 1 (RSC1A1), sodium-glucose cotransporter (S C5A1), Y+L amino acid transporter 1 (SL.C7A7), ZnT-like transporter 1 (SLC30A5), beta sarcoglycan (SGCB), lung type-l cell membrane-associated glycoprotein (T1A-2), toll-like receptor 2 (TLR2), X transporter protein 3 (XT3), H+ transporting ATPase lysosomal interacting protein 2 (ATP6IP2), vacuolar proton translocating ATPase 116 kDa subunit A isoform 2 (ATP6V0A2), intermediate conductance calcium-activated potassium channel protein 4 (KCNN4), poliovirus receptor-related 2 protein (PVRL2), poliovirus receptor-related 3 protein (PVRL3), transmembrane protein 4 (TMEM4), transmembrane protein 5 (TMEM5), CD163, CD164, CD48, CD58, CD53, CD81 , tetraspanin 5 (TSPAN-5), CD83, insulin-like growth factor II receptor (IGF2R), mucin 13 (muc13), scavenger receptor class B type I (SCARB1), solute carrier family 21 member 8 (SLC21A8), solute carrier family 29 member 1 (SLC29A1), claudin-1 (CLDN1), claudin-2 (CLDN2), tumour necrosis factor receptor superfamily member Fn14 (TNFRSF12A), tumour necrosis factor receptor superfamily member 10B (TNFRSF10B), sodium/potassium-transporting ATPase alpha-3 chain (ATP1A3), leukocyte immunoglobulin-like receptor 2 (LILRB2), mal2 protein (MAL2), endothelial protein C receptor (PROCR), prominin (PROM1), tyrosine-protein kinase RYK (RYK), transfem'ng receptor protein 1 (TFRC), TGF-beta receptor type II (TGFBR2), thrombomodulin (THBD), integrin alpha 5 (ITGAV), integrin beta-2 (ITGB2), HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, frizzled-7 (FZD7), protein tyrosine phosphatase receptor type A (PTPRA), and vascular cell adhesion molecule 1 (VCAMI)-encoding polynucleotide sequences selected from the polynucleotide sequences shown in Table 1 (pp. 64-66), the amino acid sequences encoding said proteins also shown in Table 1 (pp. 64-66), and derivatives or fragments thereof, for the diagnosis, prognosis and treatment of non-steroid dependent cancers. The use of said polynucleotide and amino acid sequences in methods for screening for gene and/or protein expression modulators, as well as modulators of biological activities of said proteins are included within the scope of this invention. In specific embodiments, the given proteins are of human origin.
The present invention relates to a method for screening a library of test molecules or compounds to identify at least one therapeutic molecule or compound which specifically modulates the expression of a plasma membrane protein-encoding gene of the invention comprising, contacting a reporter construct under the control of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3C 1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3| TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, ILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or a VCAM1 promoter with a test molecule or compound, or a library of test molecules or compounds, under conditions that allow for specific binding and/or interaction, and detecting the level of expression of the reporter construct. A decrease in the level of expression relative to a control indicates a potential therapeutic activity.
The present invention also relates to a method for screening a library of test molecules or compounds to identify at least one therapeutic molecule or compound which specifically binds and/or interacts with a plasma membrane protein of the invention, a derivative or fragment thereof with a test molecule or compound, or a library of test molecules or compounds, under conditions that allow for specific binding and/or interaction, and detecting the level of specific binding and/or interaction. A decrease in the level of binding and/or interaction relative to a control indicates a potential therapeutic activity.
The library of molecules or compounds is selected from the group consisting of DNA molecules, peptides, polypeptides, agonists, antagonists, polyclonal antibodies, monoclonal antibodies, immunoglobulins, pharmaceutical agents, and naturally occurring or synthetic molecules or compounds. Furthermore, the library of molecules or compounds may also be composed of molecules or compounds previously known to modulate the gene expression and/or the biological activity of an unrelated protein, or play a therapeutic role in a non-cancer related disease and/or disorder.
According to the invention, the test molecules or compounds identified in the methods of the invention demonstrate the ability to decrease, suppress or inhibit the expression of a given plasma membrane protein-encoding gene of the invention, and/or the biological activity of a given plasma membrane protein of the invention.
Methods are also provided for treating a non-steroid dependent cancer resulting from the aberrant biological activity of a given plasma membrane protein, and/or the aberrant expression of a gene encoding a plasma membrane protein of the invention in a mammalian, preferably a human subject: comprising providing a composition that comprises a therapeutic agent identified to bind and/or interact with a given plasma membrane protein, or modulate the level of expression of a polynucleotide encoding a given plasma , membrane, and administering to a mammalian subject a therapeutically effective amount of said composition. The invention provides an alternative method for treating a non-steroid dependent cancer resulting from the aberrant expression of a polynucleotide sequence encoding a given plasma membrane protein of the invention comprising: administering a therapeutically effective amount of a polynucleotide sequence complementary to an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or a VCAM1 -encoding polynucleotide sequence selected from the polynucleotide sequences shown in Table 1 (pp. 64-66) to a human subject.
Preferably, complementary polynucleotide molecules used according to the invention are selected from siRNA, moφholinos, PNAs, triple-helix forming oligonucleotides, double and single stranded polynucleotide sequences.
Alternatively, a method is provided by the invention for treating a non-steroid dependent cancer resulting from the aberrant biological activity of a plasma membrane of the invention comprising: administering a therapeutically effective amount of an antibody directed agaisnt an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein or fragment thereof, to a human subject.
According to the alternative methods of the invention, complementary polynucleotide molecules and antibodies specific for a given plasma membrane protein of the invention decrease, suppress or inhibit gene expression or the biological activity of a given plasma membrane protein of the invention, respectively.
A further aspect of the invention is a method of modulating proliferation, differentiation and/or cell migration of target cells comprising administering a test molecule or compound identified in the screening methods of the invention to said target cells. Preferably, said target cells are neoplastic, epithelial or cancer cells.
One embodiment of the invention provides a method for determining whether a subject is at risk of developing or has a non-steroid dependent cancer caused by the aberrant expression and/or biological activity of a plasma membrane protein of the invention, comprising the means for measuring the level of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or a VCAM1 -encoding polynucleotide sequence selected from the polynucleotide sequences shown in Table 1 (pp. 64-66), or an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein shown in Table 1 (pp. 64-66), in a sample of cells of the subject. In another embodiment, the invention provides a kit with instructions to use the kit based on the above-mentioned method, whereby the kit comprises an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or a VCAM1 -encoding polynucleotide sequence selected from the polynucleotide sequences shown in Table 1 (pp. 64-66), or an ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein shown in Table 1 (pp. 64-66).
Genes encoding plasma membrane proteins of the invention are shown to be up-regulated in tumour versus normal tissue, as provided in the accompanying examples.
DESCRIPTION TO THE FIGURES
Figure 1. Heat map of genes found to be up-regulated in colon and rectal cancer. Whereas rows represent individual nucleotide sequences spotted on the oiigo microarray employed, columns correspond to the individual patient samples analysed. Each square in the matrix represents the expression level of a single nucleotide sequence. Individual genes can be represented on the oligo microarray by more than one sequence. The genes shown are more than 1.5 fold up-regulated, with a frequency of at least 30% within the 25 experiments described herein. Squares that are represented by the colour red indicate an up-regulation of a given gene, whereas green squares signify gene down-regulation. Furthermore, black squares denote either no gene regulation or no data for the mentioned sequence in the indicated experiment. Patient samples are given along the X-axis, whereas genes encoding the plasma membrane proteins of the invention are given along the Y-axis. Samples derived from cancer patients are labelled with an internal coding system: samples from rectal cancer patients begin with the letters RNC, whereas those samples commencing with the letters CNC signify samples collected from colon cancer patients.
DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that the present invention is not limited to the particular materials and methods described or equipment, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.
It should also be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plethora of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and derivatives thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any materials and methods, or equipment comparable to those specifically described herein can be used to practice or test the present invention, the preferred equipment, materials and methods are described below. All publications mentioned herein are cited for the puφose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to precede such disclosure by virtue of prior invention.
1. Definitions
The phrase 'a plasma membrane protein of the invention' refers to a protein of the invention listed in Table 1 (pp. 64-66) that contains one or more membrane-spanning α-helices, wherein the α-helices allow the protein to extend across the lipid bilayer of a plasma membrane. When spanning the plasma membrane of a cell, the amino acid sequences of the protein can be typically divided into sequences that are localized within the cell cytosol, those spanning the lipid bilayer, or those extending into the extracellular space, depending on the number of membrane-spanning α-helices. This definition also includes proteins that are attached to the non-cytoplasmic monolayer (extracellular) of a plasma membrane via a covalent linkage (via a specific oligosaccharide) to phosphatidylinositol, also referred to as a glycosylphosphatidylinositol (GPI) anchor. These proteins generally lack one or more membrane spanning α-helices. Membrane-spanning and associated proteins of the invention include adenylate cyclase type III (ADCY3), alpha-2C adrenergic receptor (ADRA2C), aquaporin 5 (AQP5), asialoglycoprotein receptor 1 (ASGR1), beta-secretase (BACE), beta- amyloid peptide-binding protein (BBP), CD47, C-terminal tensin-like protein (CTEN), fractalkine (CX3CL1), deleted in malignant brain tumours (DMBT1), microsomal dipeptidase precursor (DPEP1), ectonucleoside triphosphate disphosphohydrolase 6 (ENTPD6), FCGR3A, gap junction beta-2 protein (GJB2), interferon-induced transmembrane protein 1 (IFITM1), integrin beta-5 protein (ITGB5), inositol 1 ,4,5-triphosphate receptor type 2 (ITPR2), low density lipoprotein receptor-related protein 8 (LRP8), membrane component chromosome 11 surface marker 1 protein (M11S1), myelin protein zero-like 1 (MPZL1), occludin (OCLN), pro-oncosis receptor inducing membrane injury protein (PORIMIN), receptor-type protein-tyrosine phosphatase mu precursor (PTPRM), regulatory solute carrier protein family 1 member 1 (RSC1A1), sodium-glucose cotransporter (SLC5A1), Y+L amino acid transporter 1 (SLC7A7), ZnT-like transporter 1 (SLC30A5), beta sarcoglycan (SGCB), lung type-l cell membrane-associated glycoprotein (T1A-2), toll-like receptor 2 (TLR2), X transporter protein 3 (XT3), H+ transporting ATPase lysosomal interacting protein 2 (ATP6IP2), vacuolar proton translocating ATPase 116 kDa subunit A isoform 2 (ATP6V0A2), intermediate conductance calcium-activated potassium channel protein 4 (KCNN4), poliovirus receptor-related .2 protein (PVRL2), poliovirus receptor-related 3 protein (PVRL3), transmembrane protein 4 (TMEM4), transmembrane protein 5 (TMEM5), CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, insulin-like growth factor II receptor (IGF2R), mucin 13 (mud 3), scavenger receptor class B type I (SCARB1), solute carrier family 21 member 8 (SLC21A8), solute carrier family 29 member 1 (SLC29A1), claudin-1 (CLDN1), claudin-2 (CLDN2), tumour necrosis factor receptor superfamily member Fn14 (TNFRSF12A), tumour necrosis factor receptor superfamily member 10B (TNFRSF10B), sodium/potassium-transporting ATPase alpha-3 chain (ATP1A3), leukocyte immunoglobulin-like receptor 2 (LILRB2), mal2 protein (MAL2), endothelial protein C receptor (PROCR), prominin (PROM1), tyrosine-protein kinase RYK (RYK), transferring receptor protein 1 (TFRC), TGF-beta receptor type II (TGFBR2), thrombomodulin (THBD), integrin alpha 5 (ITGAV), integrin beta-2 (ITGB2), HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, frizzled-7 (FZD7), protein tyrosine phosphatase receptor type A (PTPRA), and vascular cell adhesion molecule 1 (VCAM1). The corresponding accession numbers for the polynucleotide and amino acid sequences encoding these proteins are outlined in Table 1 (pp. 64-66). Membrane-spanning α-helices are also commonly referred to as transmembrane (TM) domains.
The term 'derivative' refers to a modification of a polypeptide sequence, polynucleotide sequence, or a complementary polynucleotide sequence. Derivatives of the proteins of the invention include polypeptides that have been modified chemically, and retain a biological activity of a plasma membrane protein listed in Table 1 (pp. 64-66). Derivatives of a given plasma membrane protein of the invention also include polypeptides that have an altered length and/or amino acid sequence as compared to the wild-type amino acid sequence. Such derivatives are at least 60% identical or similar to an amino acid sequence encoding a plasma membrane protein of the invention selected from the amino acid sequences shown in Table 1 (pp. 64-66) Preferred derivatives are at least about 65%, 70%, and even more preferably at least 80%, 85%, 90%, 95%, or 98% identical or similar to an amino acid sequence encoding a plasma membrane protein of the invention selected from the amino acid sequences shown in Table 1.
In a particularly preferred embodiment derivatives having an overall amino acid sequence homology, similarity or identity of at least 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% with an amino acid sequence encoding a plasma membrane protein of the invention selected from the amino acid sequences listed in Table 1 (pp. 64-66), are used according to the present invention.
Also included under the definition of 'derivative' are modified polynucleotide sequences that encode a biologically active plasma membrane protein of the invention, and are selected from the polynucleotide sequences shown in Table 1 (pp. 64-66). Modified nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from a polynucleotide sequence encoding a given polypeptide of the invention, due to the degeneracy of the genetic code. Furthermore, chemical modifications of polynucleotide sequences include, but are not limited to, the replacement of hydrogen by an alkyl, acyl, or amino group.
The term 'fragment' refers to a portion of a polypeptide sequence that comprises at least 5 consecutive amino acid residues and retains the biological activity of a plasma membrane protein of the invention, or to those polynucleotide sequences which, when translated, produce a polypeptide retaining some functional characteristic of a plasma membrane protein of the invention, e.g. antigenicity, or structural domain characteristics.
The term 'biological activity' may be used interchangeably with the terms 'biologically active', 'bioactivity' or 'activity' and, for the purposes herein, means an effector or antigenic function that is directly or indirectly performed by a plasma membrane protein of the invention (whether in its native or denatured conformation), derivative or fragment thereof. Effector functions include phosphorylation (kinase activity) or activation of other molecules, induction of differentiation, mitogenic or growth promoting activity, signal transduction, immune modulation, DNA regulatory functions and the like, whether presently known or inherent. Antigenic functions include possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured, plasma membrane protein of the invention, derivative or fragment thereof. Accordingly, a biological activity of such a protein can be that it functions as regulator of a signalling pathway of a target cell. Such a signalling pathway can, for example, modulate cell differentiation, proliferation and/or migration of such a cell, as well as tissue invasion, tumour development and/or metastasis. A target cell according to the invention can be an epithelial, neoplastic or cancer cell.
The terms 'nucleotide sequence', 'oligonucleotide sequence' and 'polynucleotide sequence' refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to a peptide polynucleotide sequence (PNA), or to any DNA-like or RNA-like material.
The terms 'protein' and 'polypeptide' are used interchangeably and refer to a linear polymer of amino acids linked together by peptide bonds in a specific sequence. Such polymers are generally flexible and may take on a variety of shapes, however based on the backbones and side chains of the amino acids associated with one another, these molecules tend to fold into their correct confirmation.
As is well known, genes for a particular protein may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitution, additions or deletions, all of which still code for polypeptides having substantially the same biological activity. The phrase 'a polynucleotide sequence encoding a biologically active plasma membrane protein of the invention' may thus refer to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences- may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in the amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
The term 'a non-steroid dependent cancer1 refers to a cancer that arises from epithelial cell origin and may include, but is not limited to, breast, lung, gastrointestinal, prostate, ovarian, cervical, endometrial cancers, bladder and/or other cancers. Within the context of the invention epithelial cancers may be of different stages, for example precancerous, early and/or late stage cancers. Cancers may also be of varying degrees in grading, wherein grading refers to the extent of histological differentiation the cancer has progressed. Guidelines to the staging and grading of cancer are known to those skilled in the art, and are described in the 'Cancer Staging Handbook' from the American Joint Committee on Cancer. Also included in the context of the invention, an epithelial cancer may be referred to as a neoplasm of epithelial origin.
The term 'gastrointestinal cancer1 refers to a cancer state associated with the gastrointestinal tract of a given subject. In the context of the invention gastrointestinal cancers include, but are not limited to oesophageal, stomach, small intestine, colon, rectal, pancreatic, liver, gallbladder, and biliary tract cancers. Within the context of the invention gastrointestinal cancers may be at different stages, as well as varying degrees of grading (see the 'Cancer Staging Handbook' from the American Joint Committee on Cancer).
The term 'neoplasm' refers to any new and abnormal growth, specifically a new growth of tissue in which the growth is uncontrolled and progressive.
The terms 'biological sample' and 'test sample' refer to all biological fluids, excretions, tissues and cells isolated from any given subject. In the context of the invention such samples include, but are not limited to, blood, blood serum, plasma, nipple aspirate, urine, semen, seminal fluid, seminal plasma, prostatic fluid, excreta, tears, saliva, sweat, biopsy, ascites, cerebrospinal fluid, milk, lymph, or tissue extract samples.
The phrase 'library of test molecules or compounds' includes, but is not limited to, libraries containing DNA molecules, peptides, antagonists, monoclonal antibodies, polyclonal antibodies, immunoglobulins and/or pharmaceutical agents. These may include new or already known molecules or compounds. Furthermore, natural or chemically synthesised molecules or compounds are also included within the scope of the invention. Furthermore, the terms 'monoclonal antibodies' and 'immunoglobulins' used herein include fragments or derivatives thereof.
The term "biologically active surface" refers to any two- or three-dimensional extension of a material that biomolecules can bind to, or interact with, due to the specific biochemical properties of this material and those of the biomolecules. Such biochemical properties include, but are not limited to, ionic character (charge), hydrophobicity, or hydrophilicity.
The term "adsorbent" refers to any material that is capable of accumulating (binding) a biomolecule. The adsorbent typically coats a biologically active surface and is composed of a single material or a plurality of different materials that are capable of binding a biomolecule. Such materials include, but are not limited to, anion exchange materials, cation exchange materials, metal chelators, polynucleotides, oiigonucleotides, peptides, antibodies, metal chelators etc.
The term 'modulate' refers to an alteration in the gene expression and/or biological activity of a plasma protein of the invention as compared to wild-type gene expression and/or biological activity. Such alterations include an up-regulation, i.e. stimulation or increase, or a down-regulation, i.e. suppression, inhibition or decrease of said responses.
The terms 'Cells', 'host cells', 'target cells' and 'recombinant host cells' can be used interchangeably and refer to a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence. It will be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in succeeding generations as a result of mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within this definition.
The phrase 'delivery complex' refers to a targeting means (e.g., a molecule that results in higher affinity binding of an antisense polynucleotide sequence, protein, polypeptide, or peptide to a target cell surface and/or increased cellular uptake by a target cell). Examples of such targeting means include, but are not limited to sterols (e.g. cholesterol), lipids (e.g. a cationic lipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associated virus, or retrovirus). Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to intemalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell such that the polynucleotide, protein, polypeptide, or peptide is released in a functional form.
The term 'reporter construct' refers to a target polynucleotide sequence linked in-frame to another polynucleotide sequence to provide a coding unit whose product is easily assayed. Examples of such reporter genes include, but are not limited to, β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein
(GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), or neomycin etc.
Within the context of this invention, the phrase 'a complementary polynucleotide sequence' can be used interchangeably with the term "antisense polynucleotide sequence", and refers to a polynucleotide or anti- gene agent that is comprised of at least about 10 nucleotides and is capable of hybridising with its complementary nucleotide sequence in vivo and in vitro. In certain embodiments, a complementary polynucleotide sequence is comprised of at least 15, 18, 20, 25, 30, 35, 40, or 50 nucleotides. Such polynucleotide sequences are typically complementary to the 5' untranslated (UTR) region of the mRNA (up to and including the AUG translation initiation codon), the 3'UTR, or a non-coding region of a given polynucleotide. Specific binding of such sequences to a polynucleotide sequence encoding a given protein of the invention is effective in altering transcription or translation of the corresponding mRNA in a host cell expressing said mRNA. Within the context of the invention, such polynucleotide sequences may be linear, circular, or triple helix forming and are complementary to a plasma membrane protein-encoding polynucleotide sequence of the invention selected from the polynucleotide sequences shown in Table 1 (pp. 64-66). Furthermore, such polynucleotide sequences may be a single or double stranded nucleic acid (e.g., Moφholinos, PNAs or siRNA) and comprise: a) at least one modified base moiety which is selected from the group of 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino- methyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine; and/or at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose, or lacking a pentose sugar moiety and/or at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, a formacetal or analogue thereof, as well as α-DNA, 2'-0-methyl RNA, a moφholine backbone, repeating N-(2-aminoethyl)-glycine units linked by peptide bonds (peptide nucleic acids; PNAs) or locked nucleic acids (LNAs). As an alternative, the use of RNA interference (RNAi) to inhibit expression of a given protein of known function is also included in the invention. Small interfering RNAs (siRNAs) generated by ribonuclease III cleavage of longer dsRNAs are used to block translation, and are preferably between 20-25 nucleotides in length. Furthermore, the invention also includes the use of RNA Lassos to inhibit the translation of a given protein of known function.
The term 'antagonist' refers to a molecule or compound which, when bound to or interacts with a plasma membrane protein of the invention, decreases the biological activity of such a protein. Such an antagonist may interact with the ligand-binding domain of a given plasma membrane protein of the invention, thereby preventing the ligand-induced activation of such a protein. An antagonist may also inhibit the interaction between a given plasma membrane protein of the invention and another molecule by stencally hindering a critical protein-protein interaction site e.g. a dimerisation domain. Furthermore, an antagonist may decrease, suppress or inhibit the expression of a plasma membrane protein-encoding polynucleotide sequence of the invention selected from the polynucleotide sequences shown in Table 1 (pp. 64-66) by interacting with the regulatory region (promoter region) of the given polynucleotide sequence. Alternatively, an antagonist can be a molecule or compound which inhibits or decreases the expression or biological activity of a protein which is located downstream of a plasma membrane protein of the invention, or which interacts with a given protein of the invention. An antagonist can also be a molecule or compound which decreases the amount or the duration of the effect of the biological or immunological activity of a plasma membrane protein of the invention. Antagonists may include proteins, polynucleotide sequences, carbohydrates, antibodies or fragments thereof, or any other molecule that exerts such effects.
The terms 'neoplastic cell' and 'neoplastic tissue' refer to a cell or tissue, respectively, that has undergone significant cellular changes (transformation). Such cellular changes are manifested by an escape from specific control mechanisms, increased growth potential, alteration in the cell surface, karyotypic abnormalities, morphological and biochemical deviations from the norm, and other attributes conferring the ability to invade, metastasise and kill. The term 'antibody' refers to a polypeptide substantially encoded by an immunoglobulin gene or fragments thereof, which bind and recognise a specific antigen. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterised fragments produced by peptidase digestion. Included within the context of the invention are antibody fragments that are produced either by modifying whole antibodies or synthesised using recombinant DNA methodologies. Within a heterogeneous population of proteins and other biologically active molecules an antibody will, under appropriate binding conditions, interact with its specific antigen or fragment thereof. In this context, the antibodies used within the scope of the invention bind preferentially to a plasma membrane protein of the invention selected from the group of membrane proteins shown in Table 1 (pp. 64-66).
2. Screening for therapeutics
The invention provides methods for screening for therapeutic agents for the treatment of a non-steroid dependent cancer resulting from the aberrant expression and/or biological activity of a plasma membrane protein of the invention. The methods identify candidates, test molecules or compounds, or agents (e.g. peptides, peptidomimetics, small molecules or other drugs) which decrease and/or inhibit the biological activity of a plasma membrane protein of the invention, derivative or fragment thereof, or have an inhibitory effect on, for example, the expression of a polynucleotide sequence encoding a plasma membrane protein of the invention. Plasma membrane proteins of the invention include ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1 , CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or VCAM1 proteins. Preferred proteins of the invention include ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, FZD7, PTPRA, and/or VCAM1 proteins. More preferred are ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2 proteins. Even more preferred are ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, muc13, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, and/or TNFRSF10B plasma membrane proteins. The most preferred plasma membrane proteins of the invention are ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, and/or TMEM5.
Proteins or peptides capable of interacting directly or indirectly with a given plasma membrane protein of the invention, derivative or fragment thereof, can be identified by various methods. For example, such molecules can be identified using methods based on various binding assays (see references on: yeast-2-hybrid Bemis et al. (1995) Methods Cell Biol. 46, 139-151, Fields and Stemglanz (1994) Trends Genet. 10, 286-292, Topcu and Borden (2000) Pharm. Res. 17, 1049-1055; yeast 3 hybrid: Zhang et al. (1999) Methods Enzymol. 306, 93-113; GST pull-downs as in Palmer et al. (1998) EMBO J. 17, 5037-5047; and phage display as in Scott and Smith (1990) Science 249] 386-390).
In preferred embodiments, the biological activity of a given plasma membrane protein of the invention is to modulate cell differentiation, proliferation, transformation, migration of specific target cells, e.g. epithelial or cancer cells, as well as tissue invasion, tumour development and/or metastasis. It is also understood that the biological activity of a protein is dependent upon several molecular aspects. including, but not limited to, the gene expression of a given polynucleotide sequence encoding a given protein; the translation of the corresponding polynucleotide sequence, post-translational modification, and/or protein folding, as well as interactions with other molecules. An alteration in gene expression, translation, post-translation modification, or protein folding may affect the biological activity of a given protein of the invention such that cellular differentiation, proliferation, transformation, as well as tissue invasion, tumour development and/or metastasis will be affected. Assays for determining whether a protein has at least one biological activity of a plasma membrane protein of the invention are described herein. Furthermore, reporter assays are described herein for the determination of gene expression of polynucleotide sequence encoding a plasma membrane protein of the invention.
In one embodiment, the invention provides assays for screening test molecules or compounds that bind to, interact with, or modulate the biologically active form of a plasma membrane protein of the invention, derivative or fragment thereof. In yet another embodiment of the invention, assays for screening test molecules or compounds that modulate the expression of a polynucleotide sequence encoding a plasma membrane of the invention are also provided. The test compounds according to the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries, aptially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the One-bead-one-compound' library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Bindseil et al. (2001) Drug Discov. Today 6, 840-847; Grabley et al. (2000) Ernst Schering Res. Found. Workshop, pp. 217-252; Houghten et al. (2000) Drug Discov. Today 5, 276-285; Rader, C. (2001) Drug Discov. Today 6, 36-43).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6909-6913; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91, 11422- 11426; Gallop et al. (1994) J. Med. Chem. 37, 1233-1251 ; Gordon et al. (1994) J. Med. Chem. 37, 1385-1401.
Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13, 412-421), or on beads (Lam et al. (1991) Nature 354, 82-84), chips (Fodor et al. (1993) Nature 364, 555-556), bacteria (U.S. Patent No. 5,223,409, published June 1993), spores [U.S. Patent Nos. 5,571,698 (published in November 1996); 5,403,484 (published in April 1995); and 5,223,409 (published in June 1993)], plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1865-1869) or phages (Scott and Smith (1990) Science. 249, 386-390; Devlin et al. (1990) Science. 249, 404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382; Felici et al. (1991) J. Mol. Biol. 222, 301-310).
In one embodiment, the assay is a cell-based assay in which a cell expresses a biologically active plasma membrane protein of the invention, derivative or fragment thereof. The expressed protein is contacted with a test molecule or compound and the ability of the test molecule or compound to bind to or interact with the protein is determined. The cell can, for example, be a eukaryotic cell such as, but not limited to a yeast cell, an invertebrate cell (e.g. C. elegans), an insect cell, a teleost cell, an amphibian cell, or a cell of mammalian origin. Determining the ability of the test molecule or compound to bind to or interact with a given protein can be accomplished, for example, by coupling the test molecule or compound with a radioisotope (e.g. 125l, 35S, 14C, or 3H) or enzymatic (e.g. horseradish peroxidase, alkaline phosphatase, or luciferase) label such that binding or interaction of the test molecule or compound to the biologically active polypeptide, derivative or fragment thereof, can be determined by detecting the labelled molecule or compound in the complex. Methods of labelling and detecting interactions of test molecules or compounds with plasma membrane proteins are known to those skilled in the art. In a preferred embodiment, the assay comprises contacting a cell, which expresses a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a known molecule or compound which binds or interacts with the given protein to form an assay mixture, contacting the assay mixture with a test molecule or compound, and determining the ability of the test molecule or compound to bind to or interact with the given protein, wherein determining the ability of the test molecule or compound to bind or interact with a given plasma membrane protein of the invention is compared to a control. The determination of the ability of the test molecule or compound to bind to or interact with a given protein is based on competitive binding/inhibition kinetics of the test molecule or compound and known target molecules or compounds for a given protein of the invention. Methods of detecting competitive binding, or the interaction of two molecules for the same target, wherein the target is a plasma membrane protein of the invention, derivative or fragment thereof, are known to those skilled in the art.
In another embodiment, the assay is a cell-based assay comprising contacting a cell expressing a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a test molecule or compound and determining the ability of the test molecule or compound to inhibit the biological activity of the given polypeptide. This can be accomplished, for example, by determining whether a given protein of the invention continues to bind to or interact with a known target molecule, or whether a specific cellular function has been abrogated. For example, a target molecule can be an extracellular molecule that activates a signal transduction pathway within a cell, a second intercellular protein that has a catalytic activity, a protein that regulates transcription of specific genes, or a protein that initiates protein translation. Determining the ability of a biologically active plasma membrane protein of the invention, derivative or fragment thereof, to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target [e.g., intracellular Ca2+, diacylglycerol and inositol triphosphate IP3)], detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction (via a regulatory element that may be responsive to a given polypeptide) of a reporter gene operably linked to a polynucleotide encoding a detectable marker, e.g., β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), neomycin etc, or detecting a cellular response, for example, cellular differentiation, proliferation or migration.
In yet another embodiment, the assay of the present invention is a cell-free assay comprising contacting a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a test molecule or compound, and determining the ability of the test molecule or compound to bind to, or interact with a given plasma membrane protein of the invention. Binding or interaction of the test molecule or compound to a given protein of the invention can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting a plasma membrane protein of the invention with a known target molecule or compound, which binds, or interacts with a given plasma membrane protein of the invention to form an assay mixture. The assay mixture is contacted with a test molecule or compound, and the determination of the ability of the test molecule or compound to interact with a given plasma membrane protein of the invention is based on competitive binding/inhibition kinetics of the test molecule or compound and known molecules or compounds for a given plasma membrane protein of the invention. Methods of detecting competitive binding, or interaction, of two molecules for the same target, wherein the target is a plasma membrane protein of the invention, derivative or fragment thereof, are known to those skilled in the art.
In another embodiment, the assay is a cell-free assay comprising contacting a biologically active plasma membrane protein of the invention, derivative or fragment thereof, with a test molecule or compound, and determining the ability of the test molecule or compound to inhibit the activity of the plasma membrane protein. Determining the ability of the test molecule or compound to inhibit the activity of a given plasma membrane protein of the invention can be accomplished, for example, by determining the ability of a given plasma membrane protein of the invention to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test molecule or compound to modulate the biological activity of a given plasma membrane protein of the invention can be accomplished by determining the ability of the plasma membrane protein to further modulate a target molecule.
In embodiments of the above assay methods of the present invention, it may be desirable to immobilize either a plasma membrane protein of the invention, derivative or fragment thereof, or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test molecule or compound to a plasma membrane protein of the invention with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test molecule or compound and either the non- adsorbed target protein or a biologically active plasma membrane protein of the invention, derivative or fragment thereof. The mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of said polypeptide can be determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, a biologically active plasma membrane protein of the invention, derivative or fragment thereof, or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. In another embodiment, inhibitors of the expression of a plasma membrane protein of the invention are identified in a method in which cells are contacted with a candidate molecule or compound and the expression of the selected mRNA or protein [i.e., the mRNA or protein corresponding to a polynucleotide or a biologically active polypeptide] in the cell is determined. In a preferred embodiment, the cell is an animal cell. Even more preferred, the cell can be derived from an insect, fish, amphibian, mouse, rat, or human. The level of expression of the selected mRNA or protein in the presence of the candidate molecule or compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate molecule or compound. The candidate molecule or compound can then be identified as a inhibitor of expression of a given plasma membrane protein of the invention based on this comparison. For example, when expression of the selected mRNA or protein is less (statistically significantly less) in the presence of the candidate molecule or compound than in its absence, the candidate molecule or compound is identified as an inhibitor of the selected mRNA or protein expression. The level of the selected mRNA or protein expression in the cells can be determined by methods described herein.
In yet another embodiment, a therapeutic agent specific for a plasma membrane protein of the invention can also be identified by using a reporter assay, in which the level of expression of a reporter construct, under the control of a gene promoter specific for a given protein of the invention, is measured in the presence or absence of a test molecule or compound or a library of test molecules are compounds. Such a promoter can be isolated by screening a genomic library with a cDNA encoding a plasma membrane protein of the invention; preferably containing the 5' end of the cDNA. A portion of said promoter, typically from 20 to about 500 base pairs long is then cloned upstream of a reporter gene, e.g., a β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), neomycin gene, in a plasmid. This reporter construct is then transfected into cells, e.g., mammalian cells. The transfected cells are distributed into wells of a multi-well plate and various concentrations of test molecules or compounds are added to the wells. After several hours of incubation, the level of expression of the reporter construct is determined according to methods known in the art. A difference in the level of expression of the reporter construct in transfected cells incubated with the test molecule or compound relative to transfected cells incubated without the test molecule or compound will indicate that the test molecule or compound is capable of modulating the gene expression of a given plasma membrane protein of the invention and is thus considered a therapeutic agent specific for a plasma membrane protein of the invention. Due to the cellular nature of this assay, it is considered according to the invention as a cell-based assay.
According to the invention, polynucleotide sequences used in screening for molecules or compounds that modulate the expression of a gene encoding a plasma membrane protein of the invention, derivative or fragment thereof, are selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671 , NMJ12104, NM.032027, NM_001777, NM_032865, NM 02996, NM_007329, NMJ04413, NM_001247, NM.000569, NM.004004, NM_003641, NM_002213, NMJ02223, NM_004631, NM_005898, NMJ03953, NM.002538, NM_052932, NM_002845, NM_006511 , NM 00343, NM_003982, NM_022902, NMJ00232, NMJ06474, NM_003264, NM_020208, NM_005765, NMJ12463, NM_002250, NM_002856, NM_015480, NMJ14255, NM_014254, NM_004244, NM_006016, NMJ01778, NM_001779, NMJ00560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NMJ04955, NM_021101 , NM_020384, NM_016639, NMJ03842, NM_152296, NM_005874, NM_052886, NMJ06404, NMJ306O17, NM_002958, NM_003234, NM_003242, NM_000361, NMJ302210, NM_000211, NMJ05514, NM_002117, NM_019111, NM.022555, NM_005516, NMJ02127, NM_003507, NM_002836, and NM_001078. Preferably, polynucleotide sequences used in screening for molecules or compounds that bind to, interact with, or modulate the biologically active form of a plasma membrane protein of the invention are selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NMJ01671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NMJ07329, NMJ04413, NMJ01247, NM_000569, NM 04004, NM_003641, NM_002213, NM_002223, NMJ04631, NM.005898, NM.003953, NM_002538, NM.052932, NM_002845, NM_006511, NM_000343, NMJ03982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_002856, NM_015480, NM_014255, NM.014254, NMJ04244, NM_006016, NM.001778, NMJ01779, NM_000560, NM_004356, NM_004233, NM 33049, NM.005505, NM_019844, NM_004955, NMJ21101, NM_020384, NM.016639, NM_003842, NM_152296, NM_005874, NM_052886, NM_006404, NMJ06017, NM_002958, NM_003234, NM_003242, NMJ00361, NM_002210, NM_000211, NM_003507, NMJ02836, and NM_001078. More preferred are polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM 04036, NM_000683, NM_001651, NM_001671 , NMJ12104, NM_032027, NMJ01777, NM_032865, NMJ02996, NM_007329, NM.004413, NM_001247, NMJ00569, NM_004004, NM.003641, NM_002213, NMJ02223, NM_004631, NM_005898, NM_003953, NMJ02538, NM_052932, NM_002845, NM_006511, NM.000343, NM_003982, NM.022902, NM_000232, NMJ06474, NM.003264, NM_020208, NM_005765, NM.012463, NM_002250, NMJ302856, NM_015480, NM.014255, NM_014254, NM_004244, NM_006016, NMJ01778, NMJ01779, NM_000560, NM_004356, NMJ04233, NM_033049, NM_005505, NM_019844, NM.004955, NM_021101, NM_020384, NM_016639, NMJ03842, NM_152296, NM_005874, NMJ52886, NM.006404, NM_006017, NM_002958, NM_003234, NMJ03242, NM_000361, NMJ02210, and NM_000211. Even more preferred are polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NMJ32027, NMJ301777, NM_032865, NM_002996, NMJ07329, NM.004413, NMJ01247, NM_000569, NMJ04004, NM_003641, NM_002213, NM_002223, NMJ04631, NM_005898, NM_003953, NMJ02538, NM_052932, NMJ02845, NM_006511 , NM_000343, NMJ03982, NM.022902, NM_000232, NMJ06474, NMJ03264, NMJ320208, NMJ05765, NMJ12463, NMJ02250, NMJ02856, NM_015480, NMJ14255, NMJ14254, NMJ04244, NM_006016, NM_001778, NMJ01779, NM_000560, NM_004356, NMJ04233, NMJ33049, NM_005505, NM_019844, NM_004955, NMJ21101, NM_020384, NM_016639 and NM_003842. Most preferred are polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NM.032865, NM_002996, NM_007329, NMJ304413, NM_001247, NM_000569, NMJ04004, NM_003641, NM_002213, NM.002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NMJ00343, NM_003982, NM.022902, NM.000232, NM_006474, NM_003264, NM.020208, NMJ05765, NM_012463, NM_002250, NM.002856, NM_015480, NM_014255, and NM_014254.
Furthermore, amino acid sequences used in screening for molecules or compounds that bind to, interact with, or modulate the biologically active form a plasma membrane protein of the invention, derivative or fragment thereof, are selected from the amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NPJ01662, NP_036236, NPJ304416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NPJ01238, NP_000566, NP.003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP.000334, NP_003973, NP_075053, NPJ00223, NP_006465, NP_003255, NPJ64593, NP_005756, NP.036595, NPJ02241, NP_002847, NP_056295, NPJ55070, NP_055069, NP_004235, NP_006007, NP_001769, NP.001770, NP_000551, NP_004347, NP_005714, NP_004224, NPJ49038, NP_005496, NP_062818, NP_004946, NP.066924, NPJ65117, NP_057723, NP_003833, NP_689509, NP_005865, NP_443118, NP_006395, NP_006008, NP_002949, NP_003225, NP_003233, NP_000352, NP_002201, NP.000202, NP_005505, NP_002108, NP.061984, NP_072049, NP.005507, NP_002118, NP_003498, NP_002827, and NP_001069. Preferably, amino acid sequences used in screening for molecules or compounds that bind to, interact with, or modulate the biologically active form of a plasma membrane protein of the invention are selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP_001662, NP_036236, NP_004416, NP_001768, NP_006254, NPJ02987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NP_002204, NP.002214, NP.004622, NP_005889, NP.003944, NP.002529, NP_443164, NP_002836, NP_006502, NP_000334, NP.003973, NP_075053, NP_000223, NP_006465, NP_003255, NP.064593, NP_005756, NP_036595, NP_002241, NP_002847, NP_056295, NP_055070, NP_055069, NPJ04235, NP_006007, NP_001769, NPJ301770, NP_000551 , NP_004347, NP_005714, NP_004224, NPJ49038, NP_005496, NP_062818, NP_004946, NP.066924, NPJ365117, NP_057723, NP_003833, NP.689509, NP_005865, NP_443118, NP.006395, NP_006008, NP_002949, NPJ03225, NPJ03233, NP_000352, NP_002201 , NP_000202, NP_003498, NP_002827, and NP_001069. More preferred are amino acids sequences selected from the group of amino acid sequences shown in accession numbers NP.004027, NP_000674, NP_00162, NPJ01662, NP_036236, NP_004416, NP_001768, NPJ06254, NP_002987, NPJ15568, NP_004404, NPJ01238, NP_000566, NP_003995, NP_003632, NP.002204, NP_002214, NP_004622, NPJ05889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP_005756, NP_036595, NPJ02241, NP.002847, NP_056295, NP_055070, NP_055069, NP_004235, NP_006007, NPJ301769, NP_001770, NP_000551, NP_004347, NP 05714, NP_004224, NP_149038, NP_005496, NP_062818, NP_004946, NPJ66924, NP_065117, NP.057723, NP_003833, NP_689509, NP_005865, NP_443118, NP_006395, NP_006008, NP_002949, NPJ03225, NP_003233, NP_000352, NP_002201 , and NP_000202. Even more preferred are amino acid sequence selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP.001662, NP_036236, NP_004416, NP_001768, NPJ06254, NP.002987, NPJ15568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP 303944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP.006465, NP_003255, NP_064593, NP_005756, NP_036595, NP_002241, NP_002847, NP.056295, NP_055070, NP_055069, NP_004235, NP_006007, NP_001769, NPJ01770, NP_000551, NP_004347, NP_005714, NP_004224, NP_149038, NP_005496, NP_062818, NPJ04946, NP_066924, NP_065117, NP_057723, and NPJ303833. Most preferred are amino acid sequences selected from the group of NPJ04027, NP_000674, NPJ30162, NP_001662, NP_036236, NP_004416, NP_001768, NP_006254, NP_002987, NP_015568, NP.004404, NP_001238, NPJ300566, NP_003995, NPJ03632, NP_002204, NP_002214, NP.004622, NP_005889, NP_003944, NP_002529, NP_443164, NPJ02836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP_005756, NP_036595, NPJ02241 , NP_002847, NP.056295, NP_055070, and NP_055069.
Those test molecules or compounds identified in the above-described assays are considered within the context of the invention as specific plasma membrane protein therapeutic agents.
More specifically, the test molecules or compounds considered to be therapeutic agents specific for a plasma membrane protein of the invention, are those molecules or compounds specific for ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or VCAM1 proteins. Preferably, therapeutic agents of the invention are specific for ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN- 5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, FZD7, PTPRA, and/or VCAM1 proteins. More preferred are those therapeutic agents specific for ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, 1FITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2. Even more preferred are therapeutic agents specific for ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP61P2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B proteins. Most preferred therapeutic agents of the invention are those that are specific for ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5 proteins.
In one embodiment of the invention, a therapeutic agent specific for a plasma membrane protein of the invention can be used for treating a non-steroid dependent cancer, and may be applied to any patient in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, and primates. But most preferably, the patient in need of such therapy is a human.
Therapeutic agents of the invention used for treating non-steroid dependent cancer include those agents that are specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN- 5, CD83, mud 3, SCARB1 , SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1 3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or VCAM1 proteins. Preferably, therapeutic agents that are specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN- 5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, FZD7, PTPRA, and/or VCAM1 proteins are used for the treatment of non-steroid dependent cancer. Even more preferred are therapeutic agents that are specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of specific for an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B proteins. Most preferred are therapeutic agents specific for polypeptides and/or regulatory sequences of polynucleotides encoding the plasma membrane proteins of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5 proteins for the treatment of non-steroid dependent cancer.
This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for the treatment of a non-steroid dependent cancer as described herein. Treating a non-steroid dependent cancer
In one embodiment of the invention, a non-steroid dependent cancer resulting from aberrant gene expression and/or biological activity of a plasma membrane protein of the invention can be treated with a therapeutic agent specific for a given protein of the invention. The aberrant gene expression of a polynucleotide sequence encoding a given plasma membrane protein of the invention may result in the altered level of its corresponding polypeptide. According to the invention, increased polypeptide levels of a given plasma membrane protein, as a consequence of aberrant.gene expression can result in -abnormal cell proliferation, cell differentiation, cell migration, tumour development or metastasis within a given subject. Subjects identified as having a non-steroid dependent cancer or abnormal cell proliferation, cell differentiation, cell migration, tumour development or metastasis can be treated by administering a therapeutic agent specific for a given plasma membrane protein of the invention which has been shown to decrease the level of expression of its respective gene, or has been shown to inhibit the biological activity of the given protein.
Among the approaches which may be used to treat a non-steroid dependent cancer resulting from the aberrant expression of a polynucleotide sequence encoding a plasma membrane protein of the invention are, for example, using complementary polynucleotide sequences, siRNA, and/or triple helix molecules to inhibit gene expression at the polynucleotide level, or using therapeutic agents identified in the various screening methods provided by the invention. Furthermore, according to the invention antibodies specific for a given polypeptide may also be used to treat a non-steroid dependent cancer. Such antibodies are able to inhibit the biological activity of a plasma membrane protein of the invention. Therapeutic agents identified as decreasing, suppressing, or inhibiting the gene expression or biological activity of a plasma membrane protein of the invention can be administered to a subject at a therapeutically effective dose to treat a non-steroid dependent cancer.
The invention also provides methods for preventing the formation and/or development of tumours. For example, the development of a tumour can be preceded by the presence of a specific lesion, such as a pre- neoplastic lesion, e.g., hypeφlasia, metaplasia, and dysplasia. In the context of the invention such lesions can be found in epithelial tissue. Therefore, the invention provides a method for inhibiting the development of such a lesion into a neoplastic lesion, comprising administering to a subject having a pre-neoplastic lesion, a therapeutic amount of an agent specific for a given a plasma membrane protein of the invention. In the context of the invention, a therapeutic amount of such an agent will effectively decrease, suppress or inhibit the development of a pre-neoplastic lesion into a neoplastic lesion with minimal side effects.
In a preferred embodiment, the invention provides a method for decreasing, suppressing or inhibiting epithelial cell proliferation, differentiation or migration, comprising contacting a tissue in which epithelial cells display an abnormally high proliferative rate, abnormal differentiation and/or migration, with a therapeutic agent specific for a given plasma membrane protein of the invention. The inhibition of the development of a non-steroid dependent cancer is anticipated by the anti-proliferative, anti-differentiating and/or anti-migrative effects of the given therapeutic agent, identified within the assays of the invention.
Within-the context .of the invention, the abnormally proliferating, differentiating or migrating cells are epithelial cells that are present in breast, lung, oesophageal, stomach, small intestinal, colonic, rectal, pancreatic, liver, gallbladder, biliary, prostatic, ovarian, cervical, and endometrial tissues.
Diagnostics
Another aspect of the present invention relates to diagnostic assays for determining the expression of a biologically active plasma membrane protein of the invention, or a polynucleotide sequence encoding a biologically active a plasma membrane protein of the invention in the context of a biological sample (e.g., blood, serum, cells, tissue) to determine whether an individual is afflicted with a non-steroid dependent • cancer, or is at risk of developing a non-steroid dependent cancer resulting from an aberrant expression or biological activity of a plasma membrane protein of the invention. For example, the level of gene expression can be assayed in a biological sample to determine if a given protein may be present in a biological sample at raised levels as compared to a standard. Such assays can be used for prognostic or predictive puφoses for the prophylactic treatment of an individual prior to the development of a non-steroid dependent cancer.
A method for detecting the presence or absence of a plasma membrane protein of the invention or a polynucleotide sequence encoding a plasma membrane protein of the invention, in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or agent capable of detecting a given polypeptide or polynucleotide (e.g., mRNA, genomic DNA) such that the presence of a given polypeptide or polynucleotide is directly labelled. Examples include detection of a primary antibody using a fluorescently labelled secondary antibody and end labelling of a DNA probe with biotin such that it can be detected with fluorescently labelled streptavidin. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for the detection of mRNA are known to those skilled in the art and include, but are not limited to, Northern hybridizations, in situ hybridizations, reverse transcription-polymerase chain reaction (RT-PCR), microarrays (chips) coated with a polynucleotide active surface (e.g. cDNA or oligonucleotide sequences). In vitro techniques for the detection of a plasma membrane protein of the invention include, but are not limited to, various techniques known to those skilled in the art such as enzyme linked immunosorbent assays (ELISAs), Western blots, immuno-precipitations, immunofluorescence, tissue-array analysis using antibodies specific for a plasma membrane protein of the invention, mass spectroscopy analysis of microarrays (chips) coated with a biologically active surface (e.g. cationic, anionic, hydrophobic, metal ion or antibody surface etc.), or fragment thereof. In vitro techniques for detection of genomic DNA include Southern hybridizations.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample contains mRNA molecules from the test subject or genomic DNA molecules from the test subject. Preferred biological samples include, but are not limited to, blood, blood serum, plasma, nipple aspirate, urine, semen, seminal fluid, seminal plasma, prostatic fluid, excreta, tears, saliva, sweat, biopsy, ascites, cerebrospinal fluid, milk, lymph, or tissue extract samples isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting said polypeptide of the invention, or mRNA, or genomic DNA encoding a given plasma protein of the invention, such that the presence of the given protein, or mRNA, or genomic DNA encoding the given polypeptide is detected in the biological sample, and comparing the presence of the given polypeptide of the invention, or mRNA or genomic DNA encoding a given protein of the invention in the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the given protein of the invention in the test sample.
In yet another embodiment, the polynucleotide sequence encoding a plasma membrane protein of the invention is used as a template for the generation of probes and/or primers designed for use in determining the level of expression of a polynucleotide sequence encoding a plasma membrane protein of the invention In a preferred embodiment of the present invention, a probe and/or primer comprising a substantially purified oligonucleotide is used in which the oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 15, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of sense or antisense sequences encoding a plasma membrane protein of the invention. For instance, primers based on a polynucleotide sequence encoding a given plasma membrane protein of the invention, can be used in PCR reactions to clone its corresponding homolog. Such primers are preferably selected in a region that does not share significant homology to other genes. Likewise, probes based on a polynucleotide sequence of the invention can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto and which can be detected, e.g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
Furthermore, such probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which over-express a plasma membrane protein of the invention, such as by measuring the level of a polynucleotide sequence encoding a given plasma membrane protein of the invention in a sample of cells from a patient; e.g. detecting the mRNA level of a given gene encoding a plasma membrane protein of the invention. Briefly, specific nucleotide probes can be generated from a polynucleotide sequence encoding a plasma membrane protein of the invention that facilitates histological screening of intact tissue and tissue samples for the presence of a specific polynucleotide sequence that encodes a given plasma membrane protein of the invention. The use of such probes can be for both predictive and therapeutic evaluation, wherein the subsequent expression or presence of a.given plasma membrane protein of the invention manifests, for example, unwanted cell growth, abnormal differentiation or the activation of cell migration within a tissue. Used in conjunction with immunoassays, the oligonucleotide probes can help facilitate the determination of the molecular basis for cancer associated with the expression of a plasma membrane protein of the invention. Also within the scope of the invention are kits for determining whether a subject is at risk of developing a non-steroid dependent cancer caused by or contributed by an aberrant gene expression and/or biological activity of a plasma membrane protein of the invention.
In another embodiment of the invention, antibodies directed against an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, ULRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or VCAM1 protein may be used to detect abnormalities in the level of protein expression of said proteins. Proteins from a biological sample can be isolated and/or detected using techniques known to those skilled in the art, including but not limited to western blot analysis, immunohistochemistry, or mass spectrometry, wherein mass spectrometry includes, but is not limited to matrix-assisted laser desoφtion ionisation/time of flight (MALDI-TOF), surface-enhanced laser desorption ionisation/time of flight (SELDI-TOF), MS-MS, or ESI-MS .
For example, antibodies or fragments thereof, may be utilised for the detection of a given plasma membrane protein of the invention in a biological sample comprising applying a labelled antibody directed against to a given plasma membrane protein of the invention to said sample under conditions that favour an interaction between the labelled antibody and its corresponding protein. Depending on the nature of the biological sample, it is possible to determine not only the presence of a plasma membrane protein of the invention, but also its cellular distribution. For example, in a blood serum sample, only the serum levels of a given protein can be detected, whereas its level of expression and cellular localisation can be detected in histological samples. It will be obvious to those skilled in the art, that a wide variety of methods can be modified in order to achieve such detection.
Methods of labelling of antibodies used in the invention are known to those skilled in the art. Such labelling methods include, but not limited to, linking the antibody to an enzyme as used in an enzyme linked immunosorbent assay (ELISA), tagging the antibody with a radioactive isotope as used in a radioimmunoassay (RIA), linking the antibody to a fluorescent compound, attaching fluorescence emitting metals such as .sup.152 Eu, or others of the lanthanide series via metal chelating groups such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) to the antibody, or coupling the antibody to a chemi- or bioluminescent compound. It is also understood by those skilled in the art that detection of complexes formed between a plasma membrane protein of the invention and a labelled antibody vary depending on the type of label used.
For example, an antibody coupled to an enzyme is detected using a chromogenic substrate that is recognised and cleaved by the enzyme to produce a chemical moiety, which is readily detected using spectrometric, fluorimetric or visual means. Enzymes used to for labelling include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection may also be accomplished by visual comparison of the extent of the enzymatic reaction of a substrate with that of similarly prepared standards. Alternatively, radiolabelled antibodies can be detected using a gamma or a scintillation counter, or they can be detected using autoradiography. In another example, fluorescently labelled antibodies are detected based on the level at which the attached compound fluoresces following exposure to a given wavelength. Fluorescent compounds typically used in antibody labelling include, but are not limited to, fluorescein isothiocynate, rhodamine, phycoerthyrin, phycocyanin, allophycocyani, o-phthaldehyde and fluorescamine. In yet another example, antibodies coupled to a chemi- or bioluminescent compound can be detected by determining the presence of luminescence. Such compounds include, but are not limited to, luminal, isoluminal, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, luciferin, luciferase and aequorin.
Furthermore, in vivo techniques for the detection of a plasma membrane protein of the invention include introducing into a subject a labelled antibody directed against a given polypeptide or fragment thereof.
An alternative method for the detection of a given plasma membrane protein of the invention within a biological sample comprises contacting said sample with an adsorbent present on a biologically active surface under specific binding conditions such that a given protein within the sample is able to bind to the said adsorbent, and detecting the presence of bound protein using mass spectrometry. In the context of the invention, the adsorbent present on a biologically active surface is composed of a single material or a plurality of different materials that are capable of binding a given plasma membrane protein of the invention. Such materials include, but are not limited to, polynucleotides, oligonucleotides, peptides, antibodies directed against a given protein of the invention, known binding molecules and/or compounds of a given protein of the invention, as well as the therapeutic agents identified within specific embodiments of the invention.
Furthermore, such material may also include anion exchange materials, cation exchange materials, and metal chelators. Alternatively, the above method may also be used to detect a polynucleotide sequence encoding a plasma membrane protein of the invention within a biological sample.
Mass spectrometric methods used to detect bound protein may include, but are not limited to, matrix-assisted laser desoφtion ionisation/time-of-flight (MALDI-TOF), surface-enhanced laser desorption ionisation/time-of-flight (SELDI-TOF), liquid chromatography coupled with MS, MSS, or ESI-MS. Typically, following binding of protein to the biologically active surface under specific conditions, the presence of bound protein can be analysed by introducing the biologically active surface into a mass spectrometer, ionising said biomolecules to generate ions that are collected and analysed by methods known to those skilled in the art.
The methods used to detect plasma membrane proteins of the invention and/or the polynucleotide sequences encoding them can also be used for determining whether a subject is at risk of developing a non-steroid dependent cancer or has developed a non-steroid dependent cancer. Such methods may also be employed in the form of a diagnostic kit comprising at least one probe and/or primer, a plasma membrane protein specific antibody or a biologically active surface described herein, which may be conveniently used, for example, in clinical settings to diagnose patients exhibiting symptoms or a family history of a non-steroid dependent cancer. Such diagnostic kits also include solutions and materials necessary for the detection of a polynucleotide or polypeptide encoding a plasma membrane protein of the invention selected from the group of ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or VCAM1 protein and instructions to use the kit based on the above-mentioned methods.
According to the methods of the invention, the polynucleotide sequences detected in a biological sample are selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511 , NM_000343, NM_003982, NM_022902, NMJ00232, NM_006474, NM_003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NMJ301778, NM_001779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101, NM_020384, NMJ16639, NM.003842, NM_152296, NM_005874, NM_052886, NM_006404, NM_006017, NM_002958, NM_003234, NM_003242, NM 00361, NM 02210, NM_000211, NM_005514, NM_002117, NM_019111, NM_022555, NMJD05516, NMJ02127, NMJ03507, NM.002836, and NM_001078. Preferably detected within a biological sample are polynucleotide sequences selected from the polynucleotide sequences shown in accession numbers NM.004036, NM_000683, NM_001651, NMJ301671, NM_012104, NMJ32027, NM_001777, NM_032865, NMJ02996, NMJ07329, NM_004413, NM_001247, NM_000569, NM_004004, NM_003641 , NM_002213, NM_002223, NM_004631, NM_005898, NM.003953, NM_002538, NMJ52932, NM_002845, NM_006511, NM.000343, NM_003982, NM_022902, NM_000232, NM.006474, NM.003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NM_001778, NM_001779, NM_000560, NM_004356, NM_004233, NMJ33049, NM_005505, NM_019844, NMJ04955, NM_021101, NM_020384, NM_016639, NM_003842, NM_152296, NMJ05874, NM_052886, NM_006404, NMJ06017, NM_002958, NMJ03234, NM_003242, NMJ00361, NM_002210, NM_000211, NM_003507, NM_002836, and NM_001078.. More preferred are polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NMJ04036, NM_000683, NM_001651, NMJ301671, NMJ12104, NM_032027, NMJ01777, NM_032865, NM_002996, NM.007329, NM_004413, NMJ01247, NMJ00569, NM_004004, NMJ03641 , NM_002213, NMJ02223, NM.004631 , NM_005898, NMJ03953, NMJ02538, NM_052932, NM_002845, NM_006511, NMJ00343, NM.003982, NM_022902, NMJ00232, NMJ06474, NM_003264, NM_020208, NM.005765, NM 12463, NMJ02250, NM_002856, NMJ15480, NMJ14255, NM_014254, NM_004244, NM_006016, NMJ01778, NMJ01779, NM_000560, NM_004356, NMJ04233, NM_033049, NM_005505, NM_019844, NMJ04955, NM_021101 , NM_020384, NMJ16639, NMJ03842, NMJ52296, NM_005874, NM_052886, NMJ06404, NM.006017, NM_002958, NMJ03234, NM_003242, NMJ00361, NM_002210, and NM_000211. Even more preferred are polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NMJ300569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511 , NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NM_001778, NM_001779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NMJ04955, NM_021101, NM_020384, NM_016639 and NM_003842. Most preferred are polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NMJ32865, NM_002996, NM_007329, NM.004413, NM_001247, NM_000569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NMJ352932, NM_002845, NM_006511 , NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NMJ320208, NM_005765, NM.012463, NM_002250, NM_002856, NMJ315480, NM_014255, and NM_014254.
Furthermore, the polypeptide sequences detected within a biological sample according to the methods of the invention are selected from the amino acid sequences shown in accession numbers, NP_004027, NP_000674, NP_00162, NP_001662, NP_036236, NP_004416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP.003632, NP.002204, NP.002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP_005756, NP_036595, NP_002241, NP_002847, NP_056295, NP_055070, NPJ55069, NP_004235, NP_006007, NP_001769, NP_001770, NP_000551, NP_004347, NP_005714, NP_004224, NPJ49038, NP_005496, NP_062818, NP_004946, NP_066924, NP_065117, NP_057723, NP_003833, NP.689509, NP_005865, NP_443118, NP_006395, NP_006008, NP_002949, NP_003225, NP_003233, NP_000352, NP_002201 , NP_000202, NP_005505, NP_002108, NPJ61984, NP_072049, NP_005507, NP_002118, NP_003498, NP_002827, and NP_001069. Preferably, polypeptide sequences detected within a biological sample are selected from the amino acid sequences shown in accession numbers NP_004027, NP_000674, NP 0162, NP_001662, NP_036236, NP_004416, NP.001768, NP.006254, NP_002987, NP_015568, NP.004404, NP.001238, NP_000566, NP_003995, NP.003632, NP.002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NPJ02836, NP_006502, NP_000334, NPJ03973, NP_075053, NP_000223, NPJ06465, NP_003255, NP_064593, NP.005756, NP_036595, NP_002241 , NP_002847, NP_056295, NP_055070, NP_055069, NP.004235, NP_006007, NP_001769, NP_001770, NP_000551, NP_004347, NP_005714, NP_004224, NPJ49038, NP_005496, NPJ62818, NPJ04946, NP_066924, NP_065117, NP_057723, NP_003833, NP_689509, NP_005865, NP_443118, NP_006395, NP_006008, NP_002949, NPJ03225, NP_003233, NP_000352, NP_002201, NP_000202, NP_003498, NPJ02827, and NP_001069. More preferred are amino acids sequences selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP.001662, NP_036236, NP.004416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP.000334, NP_003973, NP_075053, NP_000223, NPJ306465, NPJ03255, NP_064593, NP_005756, NP_036595, NP_002241, NP_002847, NP_056295, NP_055070, NP_055069, NP_004235, NP_006007, NP_001769, NP_001770, NP_000551 , NP_004347, NP_005714, NPJ04224, NP_149038, NP_005496, NP_062818, NP_004946, NPJ66924, NP.065117, NP_057723, NP.003833, NP_689509, NP_005865, NP.443118, NP_006395, NP_006008, NP_002949, NP.003225, NP.003233, NP_000352, NP_002201, and NP_000202. Even more preferred are amino acid sequence selected from the group of amino acid sequences shown in accession numbers NPJ304027, NPJ300674, NP_00162, NP_001662, NP_036236, NPJ04416, NP_001768, NP_006254, NP_002987, NP_015568, NPJ04404, NP.001238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP.443164, NP_002836, NP_006502, NP_000334, NP.003973, NP_075053, NP_000223, NP_006465, NP_003255, NP_064593, NP.005756, NP_036595, NP_002241, NP_002847, NP_056295, NP_055070, NPJ55069, NP_004235, NP_006007, NP_001769, NPJ301770, NP_000551, NP_004347, NP_005714, NP_004224, NPJ49038, NP_005496, NP_062818, NP_004946, NPJ66924, NP_065117, NP_057723, and NP_003833. Most preferred are amino acid sequences selected from the group of NPJ04027, NP_000674, NP_00162, NP_001662, NP_036236, NP_004416, NP_001768, NP.006254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NPJ02204, NP_002214, NP_004622, NP_005889, NP.003944, NPJ02529, NP_443164, NP_002836, NP_006502, NPJ300334, NP_003973, NP.075053, NP_000223, NP_006465, NPJD03255, NP_064593, NP_005756, NP_036595, NP_002241 , NP_002847, NP_056295, NP_055070, and NP_055069.
Complementary polynucleotide sequences
In one embodiment of the invention, a non-steroid dependent cancer resulting from aberrant gene expression of a polynucleotide sequence encoding a plasma membrane protein of the invention can be treated by the administration and/or in situ generation of complementary polynucleotide sequences or their derivatives which specifically hybridise (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a given plasma membrane protein of the invention, so as to inhibit the expression of that protein, e.g. by inhibiting transcription and/or translation. The binding may be by classical base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, this form of treatment is know to those skilled in the art as 'antisense' therapy, and refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding of the complementary polynucleotide sequence to its target polynucleotide sequences. Complementary polynucleotide sequences used in selected embodiments of the invention are capable of specifically hybridising (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a given plasma membrane protein selected from the polynucleotide sequences shown in accession numbers NMJ304036, NM_000683, NM_001651 , NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM 04413, NM.001247, NM_000569, NM_004004, NMJ03641, NM_002213, NMJ02223, NM_004631 , NMJ05898, NMJ303953, NM.002538, NM_052932, NM_002845, NM_006511, NMJ00343, NM_003982, NMJ22902, NM_000232, NM_006474, NM_003264, NM_020208, NM 05765, NM_012463, NM_002250, NM.002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NMJ01778, NM_001779, NMJ00560, NM_004356, NM_004233, NM_033049, NM_005505, NMJ19844, NMJ04955, NM_021101 , NMJ20384, NM_016639, NM_003842, NMJ52296, NM_005874, NM.052886, NM.006404, NM_006017, NM_002958, NM_003234, NM_003242, NM_000361, NM.002210, NM_000211, NMJ05514, NM_002117, NM_019111, NM_022555, NM_005516, NM_002127, NM_003507, NM_002836, and NM_001078. Preferably, polynucleotide sequences used in selected embodiments of the invention are capable of specifically hybridising (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a given plasma membrane protein selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NMJ01777, NM_032865, NM.002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NMJ03641, NMJ02213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NMJ02845, NM_006511 , NM.000343, NM 03982, NM_022902, NM_000232, NM_006474, NM_003264, NMJ20208, NM_005765, NMJ12463, NM_002250, NM_002856, NM_015480, NMJ14255, NMJ)14254, NMJ)04244, NM_006016, NM_001778, NM 01779, NMJ00560, NM_004356, NMJ04233, NM_033049, NM_005505, NM_019844, NM.004955, NM_021101 , NM_020384, NM_016639, NMJ03842, NM_152296, NM.005874, NM_052886, NMJ06404, NM_006017, NM_002958, NM_003234, NMJ03242, NM_000361, NMJ02210, NM_000211, NM.003507, NMJ02836, and NMJ01078. More preferred are polynucleotide sequences possessing complementarity to a polynucleotide sequence selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM 01671, NM.012104, NMJ32027, NM_001777, NM.032865, NM.002996, NM_007329, NMJ04413, NM.001247, NMJ00569, NM_004004, NMJ03641, NM_002213, NM_002223, NM_004631, NMJ05898, NM_003953, NMJ02538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NMJ22902, NM.000232, NMJ06474, NMJ03264, NM_020208, NMJ05765, NM_012463, NMJ02250, NMJ02856, NM_015480, NMJ14255, NM_014254, NMJ04244, NM_006016, NM_001778, NM_001779, NMJ00560, NM_004356, NM.004233, NM_033049, NM_005505, NMJ19844, NM_004955, NM_021101, NMJ20384, NM_016639, NM.003842, NM_152296, NMJ05874, NM_052886, NM_006404, NM_006017, NMJ02958, NM_003234, NM_003242, NM_000361, NM_002210, and NM_000211. Even more preferred are polynucleotide sequences capable of specifically hybridising (e.g. bind) under cellular conditions, a polynucleotide sequence encoding a given protein of the invention selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NMJ01671, NMJ12104, NM_032027, NM.001777, NM.032865, NM_002996, NM 07329, NM_004413, NM.001247, NM_000569, NM_004004, NMJ03641, NMJ02213, NM_002223, NMJ04631, NM_005898, NMJ03953, NM_002538, NM_052932, NM 02845, NM_006511, NM_000343, NM.003982, NM_022902, NMJ00232, NM_006474, NM_003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NM_001778, NM_001779, NM_000560, NM.004356, NMJ04233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101 , NMJ20384, NM_016639 and NM_003842. Most preferred are polynucleotide sequences that possess complementarity to a polynucleotide sequence encoding a given plasma membrane protein selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651 , NM_001671, NMJ12104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM.004413, NM_001247, NMJ300569, NM.004004, NM_003641, NM_002213, NMJ02223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NMJ22902, NM_000232, NM.006474, NM_003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_002856, NM.015480, NM_014255, and NM_014254.
Typically, such complementary polynucleotide sequences are delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA encoding a plasma membrane protein of the invention. Alternatively, the complementary polynucleotide sequence is a nucleic acid molecule that is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridising with the mRNA and/or genomic sequences of a gene encoding a plasma membrane protein of the invention. Such polynucleotide sequences are preferably modified such that they are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo. Molecules for use as complementary polynucleotide sequences are selected from the group of, but not limited to phosphodiamidate, phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA (Froehler et al. (1988) Nucleic Acids Res. 16, 4831-4839; Hyrup and Nielsen (1996) Bioorg. Med. Chem. 4, 5-23; Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451; Stein et al. (1988) Nucleic Acids Res. 16, 3209-3221 ; Summerton J. (1999) Biochim. Biophys. Acta 1489, 141-158; Summerton and Weller (1997) Antisense polynucleotide sequences Drug Dev. 7, 187-195; Orum and Wengel (2001) Curr. Opin. Mol. Then 3, 239-243; Wahlestedt et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5633-5638). Additionally, general approaches to constructing antisense polynucleotide sequences useful for 'antisense' therapy have been reviewed in Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6553-6556; Summerton and Weller (1997) Antisense polynucleotide sequences Drug Dev. 7, 187-195; Froehler et al. (1988) Nucleic Acids Res. 76, 4831-4839.
Such approaches involve the design of oligonucleotides (either DNA or RNA, or derivatives thereof) that are complementary to the mRNA encoding a given plasma membrane protein of the invention. The complementary polynucleotide sequence will bind to said mRNA transcript and prevent translation. Absolute complementarity, although preferred, is not required. A sequence 'complementary' to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridise with the RNA, forming a stable duplex; in the case of double-stranded antisense polynucleotide sequences, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridise will depend on both the degree of complementarity and the length of the complementary polynucleotide sequence. Generally, the longer the hybridising polynucleotide sequence, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridised complex.
Such polynucleotide sequences are complementary to the 5' end of the mRNA, e.g., the 5' UTR up to and including the AUG translation initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' UTR of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well (Wagner, R. (1994) Nature 372, 333-335). Therefore, polynucleotide sequences complementary to either the 5' UTR, 3' UTR, or non-coding regions of a gene encoding a given plasma membrane protein of the invention could be used in an antisense approach to inhibit the translation of the endogenous mRNA of a given plasma membrane protein of the invention. Polynucleotide sequences that are complementary to the 5' UTR of the mRNA should include the complement of the AUG start codon (e.g. -20 to +20 nucleotides). Polynucleotide sequences complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridise to the 5', 3' or non-coding region of an mRNA encoding a plasma membrane protein of the invention, complementary polynucleotide sequences should be at least 10 nucleotides in length, and are preferably polynucleotide sequences ranging from 15 to about 50 nucleotides in length. In certain embodiments, the antisense polynucleotide sequence is at least 15 nucleotides, at least 18 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, or at least 50 nucleotides in length.
Regardless of the choice of the target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the complementary polynucleotide sequence to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between specific gene inhibition via a given complementary polynucleotide sequence, and non-specific biological effects of such polynucleotide sequences. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using complementary polynucleotide sequences are compared with those obtained using control sequences. It is preferred that the control oligonucleotide sequence is of approximately the same length as the test complementary sequence and should have a similar nucleotide composition, molecular weight and melting temperature. However, these parameters should differ no more than necessary to prevent specific hybridisation to the target sequence. Such sequences may be of sense, inverse or of scrambled nature.
The complementary polynucleotide sequences can be DNA or RNA, or chimeric mixtures, or derivatives thereof, single-stranded or double-stranded, or triple helix-forming oligonucleotides. Such polynucleotide sequences can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridisation, etc., or may include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84, 648-652; PCT Publication No. WO 88/09810, published in Dec. 1988) or the blood-brain baπier (see PCT Publication No. WO 89/10134, published in Nov. 1989), hybridisation-triggered cleavage agents (van der Krol et al. (1988) Biotechniques 6, 958-976) or intercalating agents (Zon, G. (1988) Pharm. Res. 5/539-549). To this end, the complementary polynucleotide sequences may be conjugated to another molecule, e.g., a peptide, hybridisation triggered cross-linking agent, transport agent, triggered cleavage agent, etc. For example, a special delivery system for facilitated moφholino transport using ethoxylated polyethylenimine (EPEI) is described in Morcos et al. U.S. Patent No. 6,228,392 published in May 2001 and Morcos, P.A. (2001) Genesis 30, 94-102.
The complementary polynucleotide sequences may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyiuracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6- diaminopurine.
The complementary polynucleotide sequence may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. Furthermore, complementary polynucleotide sequences lacking a pentose sugar moiety are also within the scope of the invention. In yet another embodiment, the complementary polynucleotide sequence comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate (Stein and Cohen (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.97-117, J. Cohen, ed. (Boca Raton, FL: CRC Press, Inc.), a phosphorothioate, a phosphoramidothioate, a phosphoramidate (Froehler et al. (1988) Nucleic Acids Res. 16, 4831-4839), a phosphordiamidate, a methylphosphonate (Miller, P. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.85-92), an alkyl phosphotriester (Miller, P. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.82-85), a formacetal or analogue thereof, as well as α-DNA (Rayner et al. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression. pp.119-136, J. Cohen, ed. (Boca Raton, FL: CRC Press, Inc.)), 2'-0-methyl RNA (Shibahara et al. (1989) Nucleic Acids Res. 17, 239-252), a moφholine backbone (Summerton and Weller (1997) Antisense polynucleotide sequences Drug Dev. 7, 187-195), repeating N-(2-aminoethyl)-glycine units linked by peptide bonds (peptide nucleic acids; PNAs) (Hanvey et al. (1992) Science 258, 1481-1485; Nielsen et al. (1993) Anticaπcer Drug Des. 8, 53-63; Nielsen, P.E. (2000) Curr. Opin. Mol. Ther. 2, 282-287), or locked nucleic acids (LNAs) (Kumar et al. (1998) Bioorg. Med. Chem. Lett. 8, 2219-2222; Petersen et al. (2000) J. Mol. Recognit. 13, 44-53).
Complementary polynucleotides of the invention may be synthesized by standard methods known in the art. As examples, phosphorothioate oligomers may be synthesized by the method of Stein et al. (1988) Nucleic Acids Res. 16, 3209-3021) and methylphosphonate oligomers can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451). Moφholino oligomers may be synthesized by the method of Summerton and Weller, U.S. Patent Nos. 5,217,866 (published in June 1993) and 5,185,444 (February 1993), etc.
Furthermore, the use of RNA interference (RNAi) to alter post-transcriptional expression of a gene encoding a plasma membrane protein of the invention is also within the scope of the invention (Boutla et al. (2001) Curr. Bi . 11, 1776-1780; Moss, E.G. (2001) Curr. Biol. 7,R772-R775; Bernstein et al. (2001) RNA 7, 1509-1521). RNAi is the process of sequence-specific, post-transcriptional gene silencing, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Generally, the length of siRNAs is between 20-25 nucleotides (Elbashir et al. (2001) Nature 411, 494-498).
Triple helix-forming oligonucleotides to modify expression of a gene encoding a plasma membrane protein of the invention are also within the scope of the invention. Triple helix formation is used to inhibit the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
Furthermore, the invention also includes the use of circular RNA (i.e. RNA Lassos) to inhibit the translation of a specific protein of known function. Nucleic Acids Res. 16, 4831-4839), a phosphordiamidate, a methylphosphonate (Miller, P. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.85-92), an alkyl phosphotriester (Miller, P. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression, pp.82-85), a formacetal or analogue thereof, as well as α-DNA (Rayner et al. (1989) In: Oligonucleotides: Antisense Inhibitors of Gene Expression. pp.119-136, J. Cohen, ed. (Boca Raton, FL CRC Press, Inc.)), 2'-0-methyl RNA (Shibahara et al. (1989) Nucleic Acids Res. 17, 239-252), a moφholine backbone (Summerton and Weller (1997) Antisense polynucleotide sequences Drug Dev. 7, 187-195), repeating N-(2-aminoethyl)-glycine units linked by peptide bonds (peptide nucleic acids; PNAs) (Hanvey et al. (1992) Science 258, 1481-1485; Nielsen et al. (1993) Anticancer Drug Des. 8, 53-63; Nielsen, P.E. (2000) Curr. Opin. Mol. Ther. 2, 282-287), or locked nucleic acids (LNAs) (Kumar et al. (1998) Bioorg. Med. Chem. Lett. 8, 2219-2222; Petersen et al. (2000) J. Mol. Recognit. 13, 44-53).
Complementary polynucleotides of the invention may be synthesized by standard methods known in the art. As examples, phosphorothioate oligomers may be synthesized by the method of Stein et al. (1988) Nucleic Acids Res. 16, 3209-3021) and methylphosphonate oligomers can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451). Moφholino oligomers may be synthesized by the method of Summerton and Weller, U.S. Patent Nos. 5,217,866 (published in June 1993) and 5,185,444 (February 1993), etc.
Furthermore, the use of RNA interference (RNAi) to alter post-transcriptional expression of a gene encoding a plasma membrane protein of the invention is also within the scope of the invention (Boutla et al. (2001) Curr.
Bid. 11, 1776-1780; Moss, E.G. (2001) Curr. Biol. 7.-R772-R775; Bernstein et al. (2001) RNA 7, 1509-1521).
RNAi is the process of sequence-specific, post-transcriptional gene silencing, initiated by double-stranded
RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Generally, the length of siRNAs is between 20-25 nucleotides (Elbashir et al. (2001)
Nature 411, 494-498).
Triple helix-forming oligonucleotides to modify expression of a gene encoding a plasma membrane protein of the invention are also within the scope of the invention. Triple helix formation is used to inhibit the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
Furthermore, the invention also includes the use of circular RNA (i.e. RNA Lassos) to inhibit the translation of a specific protein of known function. Preferred complementary polynucleotides sequences of the above embodiments of the present invention include silencing (RNAi) antisense polynucleotides, morpholino oligomers (Summerton and Weller (1999) Antisense polynucleotide sequences Drug Dev. 7, 187-195), peptide nucleic acids (PNAs) (Egholm et al. (1992) J. Am. Chem. Soc. 114, 1895-1897) and/or locked nucleic acids (LNAs) (Kumar et al. (1998) Bioorg. Med. Chem. Lett. 8, 2219-2222; Petersen et al. (2000) J. Mol. Recognit. 13, 44-53).
In specific embodiments, complementary polynucleotide sequences are delivered to cells that express a plasma membrane protein of the invention in vivo. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense polynucleotide sequences, designed to target the desired cells e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface can be administered systematically.
Polynucleotides and Polypeptides of the Invention
In preferred embodiments of the invention, polynucleotide sequences used in the screening for therapeutic agents, the design of probes and/or primers for the detection of polynucleotide levels, and as target sequences for complementary polynucleotide sequences for the treatment of a non-dependent steroid cancer are selected from the group of nucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM 01671, NM_012104, NM_032027, NM_001777, NMJ332865, NM_002996, NM_007329, NM_004413, NM 01247, NM.000569, NM_004004, NM 303641, NM.002213, NMJ02223, NMJ304631, NMJD05898, NMJ03953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NMJ03982, NM.022902, NM.000232, NM.006474, NM_003264, NM_020208, NM_005765, NM.012463, NM 02250, NM_002856, NMJ15480, NM_014255, NM_014254, NMJ304244, NM_006016, NMJ01778, NM 01779, NM_000560, NMJ04356, NM_004233, NM.033049, NM_005505, NM_019844, NMJ04955, NMJ21101, NM_020384, NM_016639, NM_003842, NMJ52296, NM_005874, NM_052886, NMJ06404, NMJ06017, NMJ02958, NM_003234, NM_003242, NM_000361, NM_002210, NM_000211, NM.005514, NMJ02117, NM_019111, NM_022555, NMJ05516, NM_002127, NM_003507, NM_002836, and NM_001078. These polynucleotide sequences encode polypeptides comprising of amino acid sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP_001662, NP.036236, NP_004416, NP_001768, NPJ06254, NPJ02987, NPJ515568, NP_004404, NP_001238, NP_000566, NPJ03995, NP_003632, NPJ302204, NP_002214, NP_004622, NP_005889, NP.003944, NP_002529, NP.443164, NP_002836, NP.006502, NPJ00334, NP.003973, NP_075053, NP_000223, NP_006465, NP.003255, NP 64593, NP.005756, NP_036595, NP_002241, NP_002847, NP.056295, NP_055070, NP_055069, NPJ04235, NP.006007, NP_001769, NP_001770, NP_000551, NP_004347, NP_005714, NPJ04224, NP_149038, NP.005496, NP_062818, NP.004946, NP_066924, NP_065117, NP_057723, NPJ03833, NP_689509, NPJ05865, NP_443118, NP_006395, NP_006008, NPJ02949, NP_003225, NPJ03233, NP_000352, NP_002201, NP_000202, NP_005505, NP_002108, NP_061984, NP_072049, NP_005507, NP_002118, NP_003498, NP.002827, and NP_001069, respectively.
The invention also includes polynucleotide variants having at least 50% polynucleotide sequence identity to a polynucleotide shown in accession numbers NM J04036, NM_000683, NM_001651, NM_001671 , NM_012104, NM_032027, NMJ01777, NM_032865, NM_002996, NM_007329, NM_004413, NMJ01247, NM_000569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631 , NM_005898, NMJ03953, NM_002538, NM_052932, NMJ02845, NM_006511, NMJ00343, NM.003982, NM_022902, NM.000232, NM_006474, NM_003264, NM.020208, NM_005765, NMJ12463, NMJ302250, NM_002856, NM_015480, NM_014255, NM_014254, NMJ04244, NM_006016, NM_001778, NM_001779, NM_000560, NMJ04356, NM_004233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101, -NM_020384, NMJ16639, NM_003842, NM_152296, NM_005874, NM_052886, NM_006404, NM_006017, NM_002958, NM_003234, NM_003242, NM.000361, NM_002210, NM_000211, NM_005514, NM_002117, NM_019111, NM_022555, NM_005516, NM_002127, NM_003507, NM_002836, and NM 01078. A polynucleotide sequence sharing nucleotide sequence identity with a polynucleotide sequence shown in accession numbers NM_004036, NM_000683, NMJXJ1651, NM_001671 , NM_012104, NM_032027, NM_001777, NM_032865, NM 302996, NM_007329, NM.004413, NM_001247, NM_000569, NM_004004, NM_003641, NM_002213, NMJ02223, NM_004631, NM_005898, NMJ03953, NM_002538, NM_052932, NM_002845, NM_006511, NMJ00343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NM_005765, NM 12463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NM_001778, NM_001779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101, NMJ320384, NM_016639, NM_003842, NM_152296, NM_005874, NM_052886, NMJ06404, NM_006017, NM.002958, NM_003234, NM_003242, NM_000361, NM.002210, NM_000211, NMJ05514, NMJ02117, NM_019111 , NM_022555, NM_005516, NM_002127, NM_003507, NM 02836, and NM_001078, can be non-coding (e.g. probes, primers, antisense molecules), or can encode a biologically active polypeptide.
Preferred polynucleotides have a sequence at least about 50% homologous, more preferably at least about 60%, 65%, 70%, 75%, or 80%, and even more preferably at least about 85% homologous with, or a complement of a polynucleotide sequence shown in accession numbers NM_004036, NM_000683, NMJ01651, NM_001671 , NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NMJ07329, NM_004413, NM_001247, NM_000569, NM_004004, NM.003641, NM_002213, NM_002223, NMJ04631, NM_005898, NM_003953, NM_002538, NM.052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NM_005765, NM_012463, NMJ02250, NM_002856, NM_015480, NMJ14255, NM.014254, NM_004244, NM_006016, NMJ01778, NMJ01779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101, NM_020384, NM_016639, NM_003842, NM_152296, NM_005874, NM_052886, NM_006404, NM 06017, NM_002958, NM_003234, NMJ03242, NM_000361, NM_002210, NM_000211, NM_005514, NM_002117, NM_019111, NMJ22555, NMJ05516, NM_002127, NM X33507, NM_002836, and NM_001078. Polynucleotide sequences at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homologous with, or complements of a polynucleotide sequence shown in accession numbers NM_004036, NM 300683, NM_001651, NM_001671 , NM_012104, NM_032027, NM 01777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NMJ03641, NMJ02213, NM.002223, NMJ04631 , NM_005898, NM_003953, NM_002538, NM.052932, NM_002845, NM_006511, NM_000343, NM 03982, NM_022902, NM.000232, NM_006474, NM_003264, NMJ20208, NM_005765, NM_012463, NM_002250, NM_002856, NM_015480, NM.014255, NM.014254, NMJ04244, NM_006016, NM_001778, NM_001779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101 , NM_020384, NM_016639, NM_003842, NM 52296, NM_005874, NM_052886, NM_006404, NM_006017, NM_002958, NM.003234, NM_003242, NM_000361, NM.002210, NM_000211, NM_005514, NM_002117, NM_019111, NM_022555, NM_005516, NMJ02127, NM_003507, NM_002836, and NM_001078, are of course also within the scope of the invention. In preferred embodiments, the polynucleotides are vertebrate genes and, in particularly preferred embodiments, include all or a portion of a nucleotide sequence corresponding to the coding regions of polynucleotides shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM.001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NM.003641, NM.002213, NMJ02223, NM_004631 , NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NMJ05765, NM_012463, NM_002250, NM_002856, NM_015480, NM_014255, NMJ14254, NM_004244, NM_006016, NM.001778, NM_001779, NMJ00560, NM_004356, NM_004233, NM_033049, NM_005505, NM.019844, NM_004955, NM_021101, NM_020384, NM.016639, NM_003842, NM_152296, NM_005874, NM_052886, NM_006404, NM_006017, NM_002958, NM_003234, NM_003242, NM_000361, NM_002210, NMJ00211, NM.005514, NM_002117, NM_019111 , NM_022555, NM_005516, NM_002127, NM_003507, NM_002836, and NM_001078.
The polynucleotides of the invention encode the given plasma membrane proteins from any species. Preferred polynucleotide sequences encode vertebrate plasma membrane proteins of the invention. More preferred are polynucleotide sequences that encode mammalian plasma membrane proteins of the invention. Even more preferred are polynucleotide sequences that encode plasma membrane proteins of the invention, specifically of primate origins (e.g. human).
The invention further includes polypeptides that are at least 50% identical or similar to an amino acid sequence encoding a biologically active plasma membrane protein of the invention selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NPJ0162, NPJ01662,
NP_036236, NP_004416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NPJ01238, NPJ300566, NP_003995, NP_003632, NPJ02204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NPJ306502, NP_000334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NP.064593, NPJ05756, NP_036595, NP_002241, NPJ02847, NP_056295, NP_055070, NP_055069, NP.004235, NP_006007, NP_001769, NP_001770, NP_000551 , NP_004347, NP_005714, NP_004224, NP_149038, NPJ05496, NP_062818, NP_004946, NP_066924, NP_065117, NP_057723, NP_003833, NP_689509, NP_005865, NP_443118, NP_006395, NP_006008, NP_002949, NP_003225, NP_003233, NP_000352, NP_002201, NP_000202, NP_005505, NPJ02108, NP_061984, NP_072049, NP_005507, NP_002118, NP_003498, NP_002827, and NP_001069. Polypeptides which are at least about 60%, and even more preferably at least about 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical or similar to an amino acid sequence shown in accession numbers NP_004027, NP_000674, NP_00162, NPJ01662, NP_036236, NPJ04416, NPJ01768, NP_006254, NP_002987, NP_015568, NP.004404, NPJ01238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NPJ03973, NP_075053, NP_000223, NP_006465, NP_003255, NP.064593, NP_005756, -NP_036595, NP_002241, NP_002847, NP_056295, NP_055070, NP_055069, NP_004235, NP_006007, NP_001769, NP_001770, NP_000551, NP_004347, NP_005714, NP_004224, NP_149038, NP_005496, NP_062818, NP_004946, NP_066924, NP_065117, NP_057723, NP_003833, NP_689509, NP_005865, NP_443118, NP_006395, NP_006008, NP_002949, NP_003225, NP.003233, NPJ00352, NP_002201, NP_000202, NPJ05505, NP_002108, NP_061984, NP.072049, NP_005507, NP_002118, NP.003498, NP_002827, and NPJ01069, are also within the scope of the invention.
In a particularly preferred embodiment, polypeptides containing an overall amino acid sequence homology or identity of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% with an amino acid sequence encoding a biologically active polypeptide shown in accession numbers NP_004027, NP_000674, NP_00162, NP_001662, NP_036236, NP_004416, NPJ01768, NPJ06254, NPJ02987, NP.015568, NP_004404, NP_001238, NP_000566, NP_003995, NP.003632, NPJ02204, NP 02214, NP.004622, NP_005889, NP_003944, NP_002529, NP_443164, NP_002836, NP_006502, NP_000334, NP.003973, NP_075053, NPJ00223, NP_006465, NP_003255, NP_064593, NPJ05756, NP_036595, NP_002241, NP_002847, NPJ56295, NP.055070, NPJ55069, NP_004235, NP_006007, NPJ01769, NP.001770, NP_000551, NPJ04347, NPJ05714, NP_004224, NPJ49038, NPJ05496, NP_062818, NP.004946, NP_066924, NP_065117, NP 57723, NP.003833, NP_689509, NPJ05865, NP_443118, NP_006395, NP_006008, NPJ02949, NPJ03225, NP_003233, NP_000352, NP 02201, NP_000202, NP_005505, NP_002108, NPJ61984, NPJ72049, NPJ05507, NP_002118, NP_003498, NP_002827, and NP_001069, are within the scope of the present invention. Said polypeptides include such plasma membrane proteins from any species. Preferred polypeptides are vertebrate plasma membrane proteins of the invention. More preferred polypeptides are mammalian plasma membrane proteins of the invention including primates e.g., human polypeptides.
Probes and Primers Within the context of the invention, polynucleotide sequences encoding plasma membrane proteins of the invention will further allow for the generation of probes and primers for use determining the presence and/or level of polynucleotide expression within a biological sample.
Preferred polynucleotide sequences for use as a probe, according to the methods of the invention, include polynucleotide sequences comprising a nucleotide sequence having at least about 15, at least about 30, preferably at least about 50, more preferably at least about 100, and even more preferably at least about 200 consecutive nucleotides from a polynucleotide sequence encoding a plasma membrane protein of the invention shown, or a fragment thereof. In a preferred embodiment, a portion of the polynucleotide sequence corresponds to any segment of the complete coding sequence of a gene encoding a plasma membrane protein of the invention. Alternatively, a portion can be a specific polynucleotide sequence encoding a conserved motif or domain of a given plasma membrane protein of the invention, e.g. extracellular domain, ligand binding domain, transmembrane domain etc. Alternatively, a portion can be a polynucleotide sequence located between polynucleotide sequences encoding conserved motifs of a given plasma membrane protein of the invention.
The invention further pertains to polynucleotide sequence molecules for use as probes and/or primer (i.e. non- coding polynucleotide sequence molecules), which can comprise at least about 15, 18, 20, 25, 30, 40, 50, 100, 125, 150 or 200 nucleotides or base pairs. Yet other preferred polynucleotide sequences comprise at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, or at least about 600 nucleotides of a polynucleotide sequence encoding a plasma membrane protein of the invention In some embodiments, the polynucleotide sequences of the invention correspond to the 5' portion of a polynucleotide sequence encoding a encoding a plasma membrane protein of the invention.
Also used in the invention are polynucleotide sequences that are capable of hybridising to a polynucleotide sequence encoding a plasma membrane protein of the invention, and fragments thereof, under various conditions of stringency. Conditions that promote hybridisation of polynucleotide sequences are known to those skilled in the art (see Current Protocols in Molecular Biology (1989, John Wiley & Sons, N.Y. 6.3.1- 6.3.6; Jowett, T. (2001) Methods 23, 345-358).
In a preferred embodiment, the present invention also provides a probe and/or primer comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region of a polynucleotide sequence that hybridises under stringent conditions to at least about 15, preferably about 30, more preferably about 50, 100 or 200 consecutive nucleotides of sense or antisense sequence of a polynucleotide sequence encoding a plasma membrane protein of the invention, or naturally occurring mutants thereof. For instance, primers based on a given polynucleotide sequence of the invention can be used in PCR reactions to clone a given homolog. Such primers are preferably selected in a region that does not share significant homology to other genes. Likewise, probes based on a given polynucleotide sequence that encodes a plasma membrane protein of the invention, can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto and which can be detected, e.g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
Such probes can also be used as a part of a diagnostic test kit for identifying cells or tissue which over- express a given plasma membrane protein of the invention, such as by measuring the level of its corresponding polynucleotide sequence in a sample of cells from a patient; e.g. detecting mRNA levels. The use of probes directed to the polynucleotide sequences of the invention, or to the genomic polynucleotide sequences encoding a plasma membrane protein of the invention, can be used for both predictive and therapeutic evaluation of altered gene expression levels which might be manifested in, for example, unwanted cell growth or abnormal differentiation of tissue. Also within the scope of the invention are kits for determining whether a subject is at risk of developing a non-steroid dependent cancer resulting from the over-expression of a polynucleotide sequence encoding a plasma membrane protein of the invention. The kits may include probes/primers specific for a given polynucleotide sequence of the invention, reaction solutions, and instructions of how to use the kit.
According to the invention, polynucleotide sequences which are used as templates for probes and/or primers and/or for complementary sequences are selected from the polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651 , NM.001671, NM_012104, NM_032027, NM.001777,
NM.032865, NM_002996, NMJ07329, NMJ04413, NM_001247, NM_000569, NM_004004, NMJ03641,
NMJ02213, NM_002223, NMJ04631, NM_005898, NMJ303953, NM.002538, NM_052932, NMJ02845,
NM_006511, NM_000343, NM.003982, NMJ322902, NM_000232, NMJ06474, NM_003264, NM 20208, NM_005765, NM_012463, NMJ02250, NM.002856, NM_015480, NMJ14255, NM.014254, NMJ04244,
NMJ06016, NM_001778, NMJ01779, NM_000560, NM_004356, NMJ04233, NM.033049, NM_005505,
NMJ19844, NM_004955, NMJ21101, NM_020384, NM_016639, NMJ03842, NMJ52296, NM.005874,
NM_052886, NM_006404, NMJ06017, NM.002958, NM_003234, NM_003242, NM_000361, NM_002210,
NM_000211, NM_005514, NMJ02117, NM_019111, NM_022555, NM_005516, NM_002127, NM_003507, NM_002836, and NM_001078. Preferably, template polynucleotide sequences are selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NMJ301671,
NM_012104, NM_032027, NMJ01777, NMJ32865, NM_002996, NMJ07329, NMJ04413, NMJ01247, NM_000569, NMJ04004, NMJJ03641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM.002538, NM.052932, NMJ02845, NM_006511, NM_000343, NM_003982, NM_022902, NM.000232, NM_006474, NM_003264, NM_020208, NM_005765, NM_012463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM.004244, NM_006016, NM_001778, NM_001779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NM.004955, NM_021101, NM.020384, NM_016639, NM_003842, NM_152296, NM.005874, NM_052886, NM_006404, NM_006017, NMJ02958, NM_003234, NM_003242, NM.000361, NM_002210, NM_000211, NM_003507, NMJ02836, and NM_001078. More preferred are template polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM.004036, NM_000683, NM_001651 , NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NMJ01247, NM_000569, NM.004004, NM_003641, NMJ02213, NMJ02223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NM_020208, NM_005765, NMJ312463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NM_001778, NM.001779, NM_000560, NM_004356, NMJ04233, NM_033049, NM_005505, NM.019844, NMJ04955, NM_021101, NM_020384, NM_016639, NM_003842, NM_152296, NM_005874, NM_052886, NM_006404, NM_006017, NM_002958, NM_003234, NM_003242, NM_000361, NM_002210, and NM_000211. Even more preferred are template polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM.001777, NM_032865, NM_002996, NM.007329, NM_004413, NM_001247, NM.000569, NM_004004, NM_003641, NM_002213, NM_002223, NM.004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NMJ03264, NM.020208, NM_005765, NM.012463, NM_002250, NM_002856, NM_015480, NM.014255, NMJ314254, NM_004244, NM_006016, NM_001778, NM_001779, NM_000560, NM_004356, NM_004233, NMJ33049, NM_005505, NM.019844, NM_004955, NM_021101, NM_020384, NM_016639 and NM_003842. Most preferred are template polynucleotide sequences selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651 , NM.001671, NM.012104, NM_032027, NM_001777, NM_032865, NM_002996, NM.007329, NM.004413, NM_001247, NM_000569, NMJ04004, NM_003641, NM.002213, NMJ02223, NMJ04631, NM_005898, NM_003953, NM_002538, NM.052932, NM_002845, NM_006511 , NM_000343, NMJ03982, NM_022902, NM_000232, NMJ306474, NMJ03264, NM_020208, NM_005765, NM_012463, NMJ02250, NM_002856, NM_015480, NM_014255, and NM_014254.
Vectors
In several embodiments of the invention, expression vectors used for screening a library of molecules and/compounds for therapeutic agents that modulate the expression of a polynucleotide sequence encoding a plasma membrane protein of the invention contain a polynucleotide sequence encoding a reporter protein operably linked to a regulatory sequence specific of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1 , SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1 , CLDN1 , CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 gene. Reporters proteins used according to the methods of invention include, but are not limited to, β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), or neomycin.
In alternative embodiments of the invention, expression vectors used for screening a library of molecules and/compounds for therapeutic agents that modulate the biological activity of a plasma membrane protein of the invention contain a polynucleotide sequence encoding a plasma membrane protein of the invention selected from the polynucleotide sequences shown in NM_004036, NM_000683, NM_001651, NMJ01671, NM_012104, NMJ32027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM.001247, NM_000569, NMJ04004, NM.003641 , NMJ02213, NM_002223, NM_004631 , NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NMJ06511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NMJ03264, NM_020208, NMJ05765, NM.012463, NM_002250, NM_002856, NM_015480, NM_014255, NM_014254, NM_004244, NM_006016, NM_001778, NM_001779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101, NM_020384, NM_016639, NM.003842, NMJ52296, NM_005874, NM.052886, NM.006404, NM_006017, NM_002958, NM.003234, NM_003242, NM_000361 , NM_002210, NM_000211, NM_005514, NM_002117, NM_019111, NM_022555, NM_005516, NM 02127, NM_003507, NMJ02836, and NM_001078, operably linked to at least one transcriptional regulatory sequence.
Expression vectors containing a polynucleotide sequence encoding an antagonistic polypeptide, peptide or antisense polynucleotide sequence to a given polynucleotide sequence of the invention, and being operably linked to at least one transcriptional regulatory sequence may be used for practicing the present invention. Regulatory sequences are recognized by those skilled in the art, and are specifically selected to inhibit the expression and/or biologically activity of an endogenous ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein. In one embodiment, the expression vector includes a polynucleotide sequence encoding an antisense polynucleotide sequence for the puφose of inhibiting the expression of a given plasma membrane protein of the invention. In another embodiment, the expression vector contains a polynucleotide sequence encoding a polypeptide or peptide which can be used to inhibit the biological activity of a given plasma membrane protein of the invention. Such expression vectors can be used as a part of a gene therapy protocol. Thus, another aspect of the invention features expression vectors for in vivo, in vitro, or ex vivo transfection and expression of an antagonistic polypeptide, peptide or an antisense polynucleotide sequence to abrogate the biological activity of a given plasma membrane protein of the invention (e.g. differentiation of epithelial or cancer cells). This could be desirable, for example, when the naturally occurring form of the protein is over-expressed.
Such transcriptional regulatory sequences are recognised by those skilled in the art and include, but are not limited to, the cytomegalovirus (CMV), SV40 and heφes simplex virus (HSV) thymidine kinase (TK) promoters, as well as T7, T3 or SP6 promoter sequences.
The present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications), as cited throughout this application, are hereby expressly incoφorated by reference. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are known to those skilled in the art. Such techniques are explained fully in the literature.
Example 1. RNA isolation from colon and rectal tissue and cDNA labelling methods.
For the diagnosis and treatment of various forms of non-steroid dependent cancers, particularly those cancers stemming from the neoplastic transformation of epithelial cells, the identification of disease-specific molecular markers is of utmost importance. A prerequisite for the successful identification of molecular-based differences between tumour tissues and those isolated from healthy individuals lies in the comparative transcriptomic analysis of differentially regulated genes in isolated epithelial cells from both healthy and tumourigenic tissues.
For this puφose, tissue samples were collected from 16 colon and 9 rectal cancer patients at varying stages of the disease, together with healthy colon and rectal tissue taken from a distant site (sample set). Tissue samples were collected from both male and female patients of varying ages at hospitals in Cottbus (Caii- Thiem-Klinikum Cottbus, Chirurgische Klinik, 03048 Cottbus, Germany), Magdeburg (Otto-von-Guericke- Klinik, Leipziger Strasse 44, 39120 Magdeburg, Germany) and Eriangen (Friedrich-Alexander-Universitat, Chirurgische Klinik, 91023 Eriangen, Germany). Epithelial cells were isolated according to the methods described in the patent WO98/43091 'Diagnosis of Epithelial Cell Abnormalities'. The tissue samples were initially prepared by incubating with RNAIater™ (Ambion, UK) for 15 min. on ice. Following incubation, the tissue samples were reduced in size (ca. 1 mm) using a scalpel and mechanically separated using a steel mesh of 300μM. Following separation, cells were washed with 50ml of 1X PBS, 50ml RNAIater™ (Ambion, UK), and 0.8 mM Benzamidine, 3mM EDTA, 5mg/100ml Leupeptin® and 2mM Pefabloc® (wash buffer), centrifuged for 10 minutes at 4°C at 300g and suspended in 4 ml of wash buffer. In order to isolate the epithelial cells from surrounding cells, the cell suspension was contacted with an antibody specific for epithelial cells (anti-BerEP4) covalently linked to magnetic beads. More specifically, the cell suspension was mixed with δOμl anti-BerEP4 Dynabeads® (Deutsche Dynal GmbH, Germany) and incubated for 30 min at 2-8°C in wash buffer. The epithelial cell-bound Dynabeads® were collected using a magnet (Deutsche Dynal GmbH, Germany), aliquoted on ice and stored at -80°C.
Total RNA was isolated using RNAeasy (Qiagen, Germany) according to the manufacturers instructions. RNA quality was confirmed using RNA 6000 Nano Lab Chips (Agilent, Palo Alto) and Agilent's 2100 Bioanalyzer system. Using Agilents Low RNA Input Fluorescence Linear Amplification Kit, 2μg of total RNA from each tissue sample was used to generate cyanine-3 (Cy-3) and cyanine-5 (Cy-5) labelled amplified RNA (aRNA). The samples were prepared for use on Agilents 22k oligo-arrays according to the manufacturers instructions.
In order to identify which genes are differentially expressed in tumour versus healthy tissue samples, each sample set was analysed using oligo microarrays containing more than 19000 spotted 60-mer oligonucleotides representing more than 16000 different human genes (human-1A oligo microarray, Agilent Technologies, USA). Cy-3 labelled aRNAs from epithelial cells isolated from non-tumour tissue, were mixed with Cy-5 labelled aRNAs from tumour tissue and allowed to hybridise with the oligo microarray at 65°C overnight (according to the manufacturers instructions for the humanlA oligo microarrays, Agilent Technologies, USA). All hybridisations were performed with flour reversal pairs. Following hybridisation, the oligo microarrays were washed to remove any unbound molecules, and subsequently scanned for aRNAs that hybridised to their complementary sequences using a dual laser microarray scanner from Agilent Technologies. Feature extraction software (Version A.7.1.1) from Agilent Technologies was used to correlate the location of the various chip features (spots) with the annotated coding sequences on the oligo microarray. The software was also employed to calculate the signal intensity of a single chip feature in order to provide information regarding the expression level of its respective coding sequence in tumour and healthy tissue (figure 1). Following feature extraction, data analysis was performed using the Rosseta Resolver system (Rosetta Inpharmatics, Kirkland, USA).
Example 2. Data analysis using Rosetta Resolver (Rosetta Inpharmatics. Kirkland, WA)
The expression of polynucleotide sequences encoding plasma membrane or plasma membrane-associated proteins was investigated using the Rosetta Resolver platform from Rosetta Inpharmatics (Kirkland, WA). All genes known to encode membrane proteins were extracted from the commercially available Human PSD database (Proteome Inc. Beverly, MA). At the time of the study, 1325 genes coding for membrane proteins were documented with supporting experimental evidence. Of the 1325 genes, 1299 were represented as probes on the Human 1A oligo catalogue microarray (Agilent Technologies Inc. Palo-Alto, CA). These 1299 genes were combined in a bioset within Rosetta Resolver (Rosetta Inpharmatics. Kirkland, WA) and their expression in 16 colon and 9 rectum samples (tumour and healthy) was analysed. Genes encoding plasma membrane proteins were identified based on observed differential expression, wherein the difference in gene expression (fold change) in normal versus tumour tissue was at least 1.5. The degree of confidence was measured using a specific error model (performed by the Rosetta Resolver) that estimates the total error for a given measurement. This specific error model accounts for random errors as well as any unaccounted systematic errors, and calculates a P-value, a measure of confidence, for a given null (statistical) hypothesis. Plasma membrane or plasma membrane-associated genes that were expressed at a fold increase of >1.5 in tumour tissue vs. normal, had a P-value ≤ 0.01), and whose differential expression was found in 30% or more of the investigated patient samples, were selected (Table 1 ; pp. 64-66). It should be noted that genes CTEN, CX3CL1, PROM1 , PVRL3, SCARB1, SLC30A5, TNFRS12A are represented as FLY4950, SCYD1 , PROML1, DKFZP566B0846, CD36L1, ZTL1 , FN14 on the heat-map provided (Figure 1), respectively.
Example 3. Confirmation of differential gene expression patterns.
Since the oligo microarrays only contain short 16-mer nucleotide sequences, it is possible that the up- regulation of gene expression for a given plasma membrane protein may be an artefact of the experimental system (non-specific hybridisation). In order to determine whether a polynucleotide sequence encoding a plasma membrane protein of the invention is genuinely up-regulated in tumour tissue, its expression pattern can be alternatively analyzed by real-time, quantitative RT (reverse transcription)-PCR (polymerase chain reaction). Briefly, the total RNA from a minimum of 20 tumour and non-tumour pairs (isolated epithelial cells) of the tissue of interest and 3-4 different cell lines (controls for intra-assay variability) is reverse transcribed in triplicate using random hexamers and then amplified in parallel by real-time quantitative PCR in the presence of SYBR Green I using a pair of primers specific for the sequence of interest (in each case, the mRNA of a ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein). These primers are designed using Applied Biosystems' Primer Express 2.0 software; they should span an intron to avoid the detection of contaminating DNA, and should not be complementary to any other sequence of the human genome as detected when doing a BLAST search. A similar cDNA pool can used to amplify several polynucleotide sequences consecutively, as well as an endogenous reference (28S rRNA) for the normalisation of the data (correction of sample to sample pipetting variations, etc.). Amplification and real-time detection and analysis of the fluorescent signals produced when SYBR Green intercalates into the double strand of the newly amplified sequences is routinely performed using the ABI Prism 7000 Sequence Detection System and associated software (v. 1.0) from Applied Biosystems. Values for every sample are normally expressed as Ct (threshold cycle) values, i.e. the cycle at which, once the background signal fixed, the fluorescent signal is first detected over a certain threshold. Extrapolation of each Ct value from an appropriate standard curve (plot of Ct values vs. quantity obtained by amplification of the target and reference genes from a series of known dilutions of a total RNA sample) will provide the relative amounts of a given gene and reference genes in a particular replicate sample, while the ratio [mean±SD]target gene/[mean±SD]reference gene will normalise the amount of a given target within that particular sample, allowing comparison between samples. Thus, once normalised, the amount of a given polynucleotide sequence in each tumour will be compared to the amount of the same polynucleotide sequence in the corresponding non- tumour sample to determine if it is over- or under expressed in a particular sample, or if its expression does not change. Finally, analysis of the expression of a given polynucleotide sequence of the invention (ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1 , SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAMl-encoding polynucleotide sequence) in all samples will provide information as to its global or partial (and at what frequency) over- (or under-) expressed in a certain tumour type.
Example 4. Determination of protein expression levels for plasma membrane proteins of the invention. The confirmation of over-expression of plasma membrane proteins of the invention at the protein level is performed by western blot and immunohistochemistry (IHC) analyses. Whenever possible, protein expression by either technique is analyzed using commercially available antibodies. However, for many proteins this is not possible and therefore, new antibodies will need to be produced. The production of polyclonal antibodies against the protein(s) of interest is out-sourced, and is normally based on the immunization of 2 rabbits with 2 different peptides (administrated mixed or separately) of our own design, supported by the design of the company producing the antibodies. Protein sequences are selected from the public domain (i.e. SwissProt, NCBI). Peptide design can also be performed using publicly available bioinformatics tools such as Prosite, surface probability, flexibility and antigenicity profiles, etc., or commercially available ones, for example, Protean from DNASTAR or Metalife's Epitope Validator. Whenever possible peptides are designed in the extracellular regions of the protein; transmembrane regions are to be avoided. Furthermore, predicted phosphorylation, glycosylation, myristylation as well as other post-translational modification sites should also be avoided. The immune sera and/or purified antibodies provided by the company are first tested by western blotting using protein extracts (isolated with strong detergent conditions, e.g. 4% SDS) from eukaryotic and/or prokaryotic cells over-expressing and not over-expressing the cloned protein, the corresponding pre-immune sera serving as negative controls. Usually the protein is cloned fused to a tag (e.g. FLAG tag) to control the over-expression (e.g. with an anti-FLAG tag monoclonal antibody). Ideally, the immune sera and/or purified antibodies should recognize only the over-expressed protein (also the endogenous one if the cells used express it), while the pre-immune sera should not. When necessary, immunoprecipitation is performed to confirm recognition of the protein by the sera and/or purified antibodies.
Once the antibodies are available, the expression of the plasma membrane proteins of the invention are analyzed by both western blotting and IHC using standard protocols known to those skilled in the art. Western blotting and IHC is performed on a minimum of ten or more non-tumour and tumour pairs of tissue to confirm over-expression of a given protein of the invention in the tumour tissue. Eventually, additional normal and tumour tissues are also analyzed to determine if the over-expression of a given protein of the invention occurs in all tumourigenic tissues or if it is restricted to one or a few tumour types. Whenever possible, results of protein expression are correlated with clinical data. Altogether, the pattern and frequency of over-expression and the correlation to clinical parameters help to prioritize the targets for further study.
Example 5. Antisense suppression of protein expression
To address the essential question of whether a given plasma membrane protein of the invention is important for tumour cell growth or cell proliferation in general, the gene expression of a given protein of the invention can be specifically down-regulated in two or three different human tumour- cell -lines using the RNAi (RNA interference) technology (reference). This recently developed and widely applied technology uses short (21 bp) synthetic double stranded RNA duplexes (silencer RNA, siRNA) that are complementary to the coding sequence of the target gene mRNA and trigger specific mRNA degradation. Many mammalian genes have already been "knocked down" using transfection of siRNA into tumour cell lines and resulting phenotypes have been described.
Briefly, for each of following genes, ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1 , CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 , 3 to 5 siRNAs, at different positions on their corresponding mRNAs, are designed using the Dharmacon "siDESIGN Center" Software as well as an additional BlastN2 in Human Unigene to minimize off-target homologies. Synthetic siRNAs will be ordered from Dharmacon or MWG biotech. Two different human tumour cell lines are transfected in triplicate (using Lipofectamine 2000, Invitrogen) with 50nM of each siRNA for three consecutive days and will be analyzed on day 4-post first transfection for both gene knockdown and proliferation. Analysis of the gene knockdowns is performed either by quantitative RT-PCR or - if an antibody against a given protein is available - by Western blot. In those cases where efficient gene knock-downs correspond to reduced cell proliferation, further phenotype analyses will be performed, namely, staining for apoptosis (Annexin V), cell cycle analysis (using RNase treatment and propidium iodide staining) and a metabolic activity assay (WST-1, Roche). Alternatively, to exclude non-transfected cells from the assays, these analyses will be performed with the corresponding siRNA expression plasmids (pSilencer from Ambion or pRNA from GenScript) under G418 (Neomycin) selection. An inhibition of expression of a gene encoding a given plasma membrane protein, is expected to increased apoptosis, cell cycle arrest and/or reduced metabolic activity.
Example 6. Identification of therapeutic agents.
This example describes a simple assay for isolating therapeutic agents that alters the biological activity of a plasma membrane protein of the invention. Based at least in part on the results described in the previous examples, such therapeutic agents will inhibit the biological activity of a ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, - HLA-B, HLA-Gr- HLA-DRA, HLA-DRB3, HLA-E..HLA-G, FZD7, PTPRA, or VCAMI protein and can therefore be used for treating non-steroid dependent cancers.
Accordingly, a therapeutic agent of a given plasma membrane protein of the invention is identified by using an in vitro assay, in which the interaction between a plasma membrane protein of the invention and a molecule known to bind and/interaction with said protein is determined in the presence and in the absence of a test compound. A soluble binding fragment of a plasma membrane protein of the invention is prepared by expressing its corresponding polynucleotide sequence as selected from the polynucleotide sequences listed in Table 1 (pp. 64-66), or a fragment thereof, in £ coli according to methods known to those skilled in the art. Similarly, a molecule known to bind/interact with a plasma membrane protein of the invention can be produced recombinantly (i.e. if the molecule is of amino acid nature), chemically (i.e. nucleic acids, chemical compounds), or biologically synthesised as in the case of antibodies.
Test molecules or compounds are then tested to determine whether they inhibit the interaction between a plasma membrane protein of the invention, and a protein known to bind and/or interact with a given protein by using an ELISA type assay. The molecules or compounds to be tested are labelled using appropriate methods known to those skilled in the art (i.e. tagging polypeptides with an epitope that is recognised by an antibody, or tagging a nucleic acid with a fluorescent label). For example, an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein of the invention can be linked to the wells of a microtiter (96-well) plate by overnight incubation of said protein in an appropriate buffer. Unoccupied sites on the plate may be blocked with a BSA solution. Subsequently, various amounts of the test compounds and/or molecules are added, together with a molecule that is known to bind and/or interact with a given protein of the invention, to the wells of the microtiter plate in an appropriate buffer suitable for a specific interaction between a given protein and the added molecules. Following incubation for a period of time necessary for binding saturation, the wells are washed to removed any unbound molecules and the amount of test molecule bound to said protein can be determined by determining the optical density using an ELISA reader. A higher optical density indicates that the test molecule inhibits the interaction of a given protein of the invention and a molecule known to bind and/or interact with said protein and is indicative of its ability to inhibit the said protein's function.
Therapeutic agents specific for plasma membrane proteins of the invention can also be identified using a reporter assay in which the level of expression of a reporter construct under the control of a ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 gene promoter is measured in the presence or absence of a test compound. Gene promoters specific for polynucleotide sequences encoding plasma membrane proteins of the invention can be isolated by screening a genomic library with a cDNA encoding a given protein of the invention; preferably the 5' end of the cDNA. A portion of the promoter, typically from about 50 to 500 base pairs long in then cloned upstream of a reporter gene such as, but not limited to a β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Ds-Red fluorescent protein, far-red fluorescent protein (He-red), secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), neomycin gene, in an expression plasmid. This reporter construct is then transfected into cells, e.g., epithelial cells. Transfected cells are then distributed into wells of a multi-well plate and various concentrations of the test molecule or compound are added to the wells. After several hours of incubation, the level of expression of the reporter construct is determined according to methods know to those skilled in the art. The level of expression of the reporter construct in transfected cells incubated with the test compound is compared to the level of expression of the reporter construct in transfected cells incubated without the test compound. The difference in the level of expression and/or biological activity indicates that the test molecule or compound is capable of regulating the expression of a polynucleotide sequence encoding a given plasma membrane protein of the invention, or regulating the biological activity of a given plasma membrane protein of the invention, thereby indicating a specific therapeutic activity.
Table 1. List of up-regulated genes encoding membrane proteins in tumourigenic tissue.
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001

Claims

We claim:
1. A method of screening for a therapeutic agent for the treatment of a non-steroid dependent cancer resulting from the aberrant expression of a polynucleotide sequence encoding a biologically active plasma membrane protein, comprising: a. contacting a reporter construct under the control of regulatory sequence specific for a polynucleotide sequence encoding a plasma membrane protein with a test molecule or compound, or a library of test molecules or compounds, under conditions to allow specific binding and/or interaction; and b. detecting the level of expression of the reporter construct, wherein an alteration in the level of expression to a control indicates a potential therapeutic activity.
2. The method of claim 1, wherein the polynucleotide sequence encoding a biologically active plasma membrane protein is selected from the group of polynucleotide sequences shown in accession numbers NMJ04036, NM_000683, NM_001651, NM 01671, NM.012104, NMJ332027,
NM_001777, NMJ32865, NM_002996, NM.007329, NM.004413, NMJ301247, NM_000569,
NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NMJ303953,
NM_002538, NM.052932, NMJ02845, NM_006511, NM_000343, NM_003982, NMJ22902, NM_000232, NM.006474, NM_003264, NM_020208, NM_005765, NM_012463, NM.002250,
NM_002856, NMJ15480, NM_014255, NM.014254, NM_004244, NM_006016, NM_001778,
NM_001779, NM_000560, NM_004356, NM.004233, NM_033049, NM_005505, NM_019844,
NM_004955, NM_021101, NM_020384, NM_016639, NM.003842, NMJ52296, NM_005874,
NM_052886, NM.006404, NM_006017, NM_002958, NM_003234, NM_003242, NM_000361, NM.002210, NM_000211, NM_005514, NM_002117, NM.019111, NM_022555, NMJ05516,
NM_00212, NM_003507, NM.002836, and NM_001078.
3. A method of screening for a therapeutic agent for the treatment of a non-steroid dependent cancer resulting from an altered biological activity of a ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1,
MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3,
ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53,
CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A,
TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, and/or VCAM1 protein comprising: a. contacting an amino acid sequence selected from the sequences shown in NP_004027,
NP_000674, NP_00162, NP 01662, NPJ36236, NP_004416, NP_001768, NPJ06254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NPJ03995, NP_003632, NP_002204, NPJ02214, NP_004622, NP_005889, NP.003944, NP_002529, NP_443164, NP_002836, NP_006502, NPJ00334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NPJ64593, NP_005756, NP_036595, NP_002241, NPJ02847, NPJ56295, NP_055070, NP_055069, NP 04235, NP_006007, NP_001769, NP_001770, NPJ00551,
NP_004347, NPJ05714, NP_004224, NP_149038, NP_005496, NP_062818, NPJ04946, NP_066924, NP_065117, NPJ57723, NP.003833, NP_689509, NPJ05865, NP_443118, NP_006395, NPJ06008, NP_002949, NP_003225, NPJ03233, NP_000352, NP_002201, NP_000202, NP.005505, NP_002108, NP_061984, NP_072049, NPJ05507, NP_002118, NP_003498, NPJ02827, or NPJ01069, derivative or fragments thereof with a test molecule or compound, or a library of test molecules or compounds, under conditions to allow specific binding and/or interaction; and b. detecting the level of specific binding and/or interaction, wherein an alteration in the level of interaction relative to a control indicates a potential therapeutic activity.
4. The method of claim 1, wherein the therapeutic agent suppresses and/or inhibits the expression of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3,
TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein-encoding polynucleotide sequence.
5. The method of claim 2, wherein the therapeutic agent suppresses and/or inhibits the biological activity of an
ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1,
SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein.
6. The methods of claims 1-5, wherein the library of test molecules or compounds is selected from the group consisting of DNA molecules, peptides, antagonists, monoclonal antibodies, immunoglobulins, small molecule drugs and pharmaceutical agents.
7. A method for the suppression and/or inhibition of a polynucleotide sequence encoding an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4,
TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud3, SCARB1, SLC21A8, SLC29A1, CLDN1 , CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAMI protein for the puφose of modulating proliferation and/or differentiation or cell death of target cells comprising: a. providing a composition that comprises a therapeutic agent identified in any one of claims 1- 6, and b. contacting a therapeutically effective amount of the composition from (a) with said target cells.
8. A method for the suppression and/or inhibition of the biologically activity of an ADCY3, ADRA2C, AQP5,
ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1,
CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein for the purpose of modulating proliferation and/or differentiation or cell death of target cells comprising: c. providing a composition that comprises a therapeutic agent identified in any one of claims 1-
6, and d. contacting a therapeutically effective amount of the composition from (a) with said target cells.
9. The methods of claims 7-8, wherein said target cells are epithelial cells, neoplastic cells, or cancer cells.
10. A method for treating a non-steroid dependent cancer resulting from the aberrant expression of a polynucleotide encoding a ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2,
ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1 , CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein, comprising administering an effective amount of a therapeutic agent from any of claims 1-7 to a subject.
11. A method for treating a non-steroid dependent cancer resulting from an altered biological activity of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR,
PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein, comprising administering an effective amount of a therapeutic agent from any of claims 1-7 to a subject.
12. A method for the suppression and/or inhibition of a polynucleotide sequence encoding an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1 , MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK,
"TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7,
PTPRA, or VCAM1 protein for the purpose of modulating proliferation and/or differentiation or cell death of target cells comprising administering a therapeutically effective amount of a polynucleotide sequence complementary to a polynucleotide sequence selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027,
NMJ301777, NM_032865, NM_002996, NMJ07329, NM_004413, NM_001247, NM_000569, NM.004004, NM_003641, NMJ02213, NMJ02223, NM.004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NMJ00343, NM_003982, NM_022902, NM_000232, NM_006474, NMJ03264, NM_020208, NM.005765, NM_012463, NM_002250, NM_002856, NM_015480, NMJ14255, NM_014254, NMJ04244, NM_006016, NM_001778,
NM_001779, NM_000560, NM 04356, NMJ04233, NM_033049, NM_005505, NM_019844, NM_004955, NM_021101, NM_020384, NM.016639, NMJ03842, NMJ52296, NM.005874, NM_052886, NM_006404, NMJ06017, NM.002958, NM_003234, NM_003242, NM_000361, NM_002210, NM_000211, NMJ05514, NM_002117, NM.019111, NM_022555, NM.005516, NM_00212, NM_003507, NMJ02836, and NMJ301078 to said target cells.
13. The method of claim 12, wherein said target cells are neoplastic cells, epithelial cells or cancer cells.
14. A method for treating a non-steroid dependent cancer resulting from the aberrant expression of a polynucleotide sequence encoding an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1 , DPEP1 , ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2,
ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein comprising administering an effective amount of a polynucleotide sequence complementary to a polynucleotide sequence selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671 , NM_012104, NM_032027, NMJJ01777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NMJ300569, NM_004004, NM_003641 , NM_002213, NM_002223, NM_004631, NM_005898, NM.003953, NM_002538, NM_052932, NM.002845, NM_006511, NMJ300343, NM_003982, NMJ22902, NM_000232, NM_006474, NM_003264,
NM_020208, NM.005765, NM_012463, NM_002250, NM_002856, NM_015480, NMJ14255, NM_014254, NM 04244, NM_006016, NM_001778, NM_001779, NM_000560, NMJ04356, NM_004233, NMJ33049, NM_005505, NM_019844, NMJ04955, NM_021101 , NM.020384, NM_016639, NM_003842, NMJ52296, NMJ05874, NM_052886, NM_006404, NMJ06017, NM_002958, NMJ03234, NM_003242, NMJ00361, NM_002210, NM_000211, NM_005514,
NM_002117, NM_019111, NM_022555, NM.005516, NM_00212, NM.003507, NM_002836, and NM_001078.
15. The methods of claims 12-14, wherein binding of the complementary polynucleotide sequence is effective in suppressing and/or inhibiting transcription or translation of an mRNA encoding an ADCY3, ADRA2C,
AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC,
TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein in a host cell expressing said mRNA.
16. The methods of claims 12-15, wherein the complementary polynucleotide sequence is expressed from a viral vector.
17. The method of claim 16, wherein the viral vector is a vaccinia virus, a retrovirus, an adenovirus, or a combination thereof.
18. The method of claim 17, wherein the polynucleotide sequence is in a delivery complex.
19. The method of claim 18, wherein the delivery complex is a sterol, lipid, virus.
20. A method for the treatment of a non-steroid dependent cancer resulting from an altered biological activity of an ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM,
RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1, CLDN1 , CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein comprising administering an effective amount of a therapeutic antibody specific for a given plasma membrane protein encoded by an amino acid sequence selected from the group of amino acids sequences shown in accession numbers NP_004027, NP_000674, NP_00162, NP_001662, NP_036236, NPJ04416, NP_001768, NP_006254, NP_002987, NP_015568, NP_004404, NP_001238, NP_000566, NP_003995, NP_003632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP_002529, NP_443164,
NPJ02836, NP_006502, NP_000334, NP_003973, NP_075053, NPJ00223, NP_006465, NPJ03255, NP_064593, NP_005756, NPJ36595, NP_002241, NP_002847, NP_056295, NP_055070, NP_055069, NP_004235, NP.006007, NPJ01769, NP_001770, NP_000551, NPJ04347, NP_005714, NP_004224, NPJ49038, NP_005496, NP.062818, NP_004946, NP_066924, NP_065117, NP_057723, NPJ03833, NP_689509, NP_005865, NP_443118,
NPJ06395, NP_006008, NP_002949, NP 03225, NPJ03233, NP_000352, NPJ02201, NPJ00202, NP_005505, NP_002108, NP.061984, NP.072049, NP_005507, NP_002118, NP 03498, NP_002827, or NP_001069 .
21. A method for determining whether a subject is at risk of developing or has a non-steroid dependent cancer caused by the aberrant expression of a polynucleotide sequence encoding an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1,
CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein comprising determining the level of a polynucleotide sequence selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NMJ32027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NM_000569, NM_004004, NM_003641, NM_002213, NM_002223, NM_004631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845,
NM_006511, NM_000343, NM_003982, NM_022902, NM_000232, NM_006474, NM_003264, NMJ20208, NM.005765, NM.012463, NMJ02250, NM_002856, NMJ15480, NM_014255, NMJ14254, NM_004244, NM.006016, NMJ01778, NM_001779, NM_000560, NM_004356, NMJ04233, NM_033049, NM_005505, NM_019844, NM.004955, NM_021101 „ NM_020384, NMJ16639, NM_003842, NMJ52296, NMJ05874, NM_052886, NM.006404, NM_006017,
NMJ02958, NM_003234, NMJ03242, NMJ00361, NM_002210, NM_000211 , NMJ05514, NMJ02117, NM_019111, NM_022555, NM.005516, NM_00212, NMJ03507, NM_002836, or NM_001078 in a sample of cells of said subject, and comparing said level with a control level that is representative of a level in a normal, cancer-free subject.
22. A method for determining whether a subject is at risk of developing or has a non-steroid dependent cancer resulting from an altered biological activity of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1, DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53,
CD81, TSPAN-5, CD83, muc13, SCARB1 , SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein comprising determining the level of a polypeptide encoded by an amino acid sequence selected from the group of amino acid sequences shown in accession numbers NP_004027, NP_000674, NPJ0162, NP_001662,
NP_036236, NP_004416, NP_001768, NPJ06254, NP_002987, NP_015568, NP.004404, NP 301238, NP_000566, NP_003995, NPJ03632, NP_002204, NP_002214, NP_004622, NP_005889, NP_003944, NP.002529, NP 43164, NP_002836, NP.006502, NP_000334, NPJ03973, NP_075053, NP.000223, NPJ06465, NP_003255, NP.064593, NP_005756, NPJ36595, NP_002241, NPJ02847, NPJ56295, NP_055070, NP_055069, NPJ04235,
NPJ06007, NP_001769, NP_001770, NP_000551, NP_004347, NP.005714, NP_004224, NPJ49038, NP_005496, NP_062818, NP.004946, NP.066924, NP.065117, NP_057723, NPJ03833, NP_689509, NP_005865, NP_443118, NP_006395, NP_006008, NP 02949, NPJ03225, NP_003233, NP 00352, NP 02201, NP_000202, NP_005505, NP 02108, NPJ61984, NP_072049, NP_005507, NP_002118, NP_003498, NP_002827, or NP_001069 in a sample of cells of said subject, and comparing said protein levels with a control level that is representative of a level in a normal subject.
23. A kit for identifying a subject at risk of developing or has a non-steroid dependent cancer caused by the aberrant expression of a polynucleotide sequence encoding an ADCY3, ADRA2C, AQP5, ASGR1 , BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1 , OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1 , SLC7A7, SLC30A5,
SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1, RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein comprising, the means for measuring the level of a polynucleotide sequence selected from the group of polynucleotide sequences shown in accession numbers NM_004036, NM_000683, NM_001651, NM_001671, NM_012104, NM_032027, NM_001777, NM_032865, NM_002996, NM_007329, NM_004413, NM_001247, NMJ00569, NM_004004, NM_003641, NM.002213, NMJ02223, NMJ04631, NM_005898, NM_003953, NM_002538, NM_052932, NM_002845, NM_006511, NMJ00343, NM.003982, NM.022902, NMJ00232, NM_006474, NM_003264,
NM_020208, NM.005765, NM_012463, NMJ02250, NMJ02856, NM_015480, NM_014255, NMJ14254, NM_004244, NM_006016, NMJ01778, NM_001779, NM_000560, NM_004356, NM_004233, NM_033049, NM_005505, NM.019844, NM_004955, NM.021101, NM_020384, NM_016639, NM_003842, NM_152296, NM_005874, NM_052886, NM_006404, NM.006017, NM.002958, NMJ03234, NM.003242, NM_000361, NM_002210, NM_000211, NM_005514,
NM_002117, NM_019111, NMJ22555, NM.005516, NM_00212, NM_003507, NMJ02836, or NM_001078 in a sample of cells of said subject, and instructions to use the kit.
24. A kit for identifying a subject at risk of developing or has a non-steroid dependent cancer caused resulting from an altered biological activity of an ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47,
CTEN, CX3CL1 , DMBT1 , DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1 , ITGB5, ITPR2, LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81, TSPAN-5, CD83, muc13, SCARB1, SLC21A8, SLC29A1 , CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2,
HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 protein comprising, the means for measuring the level of a polypeptide encoded by an amino acid sequence selected from the group of amino acid sequences shown in accession numbers NP_004027, NPJ300674, NP_00162, NPJ01662, NP_036236, NPJ304416, NPJ01768, NP.006254, NP_002987, NPJ15568, NPJ04404, NP_001238, NP_000566, NPJ03995, NP_003632, NP_002204, NPJ02214,
NPJ04622, NP_005889, NP_003944, NPJ02529, NP_443164, NP_002836, NP_006502, NPJ00334, NP_003973, NP_075053, NP_000223, NP_006465, NP_003255, NPJ64593, NP_005756, NP_036595, NPJ02241, NP_002847, NP.056295, NP_055070, NPJ55069,
NP_004235, NP_006007, NPJ01769, NP_001770, NP.000551, NP_004347, NPJ05714,
NP_004224, NP_149038, NP.005496, NP_062818, NP_004946, NP_066924, NP_065117,
NP_057723, NPJ03833, NP_689509, NP_005865, NP.443118, NP_006395, NP_006008, NP_002949, NP_003225, NPJ03233, NP.000352, NP.002201, NP_000202, NP_005505,
NP_002108, NP_061984, NPJ72049, NP_005507, NP_002118, NP_003498, NP_002827, or NP_001069 in a sample of cells of said subject, and instructions to use the kit.
25. The methods and kits, of any of claims 1-24, wherein said ADCY3, ADRA2C, AQP5, ASGR1, BACE, BBP, CD47, CTEN, CX3CL1 , DMBT1, DPEP1, ENTPD6, FCGR3A, GJB2, IFITM1, ITGB5, ITPR2,
LRP8, M11S1, MPZL1, OCLN, PORIMIN, PTPRM, RSC1A1, SLC5A1, SLC7A7, SLC30A5, SGCB, T1A-2, TLR2, XT3, ATP6IP2, ATP6V0A2, KCNN4, PVRL2, PVRL3, TMEM4, TMEM5, CD163, CD164, CD48, CD58, CD53, CD81 , TSPAN-5, CD83, mud 3, SCARB1, SLC21A8, SLC29A1, CLDN1, CLDN2, TNFRSF12A, TNFRSF10B, ATP1A3, LILRB2, MAL2, PROCR, PROM1 , RYK, TFRC, TGFBR2, THBD, ITGAV, ITGB2, HLA-B, HLA-C, HLA-DRA, HLA-DRB3, HLA-E, HLA-G, FZD7, PTPRA, or VCAM1 proteins are of human origin.
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