EP1226150A1 - Tadg-15: an extracellular serine protease overexpressed in carcinomas - Google Patents

Tadg-15: an extracellular serine protease overexpressed in carcinomas

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Publication number
EP1226150A1
EP1226150A1 EP00973739A EP00973739A EP1226150A1 EP 1226150 A1 EP1226150 A1 EP 1226150A1 EP 00973739 A EP00973739 A EP 00973739A EP 00973739 A EP00973739 A EP 00973739A EP 1226150 A1 EP1226150 A1 EP 1226150A1
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EP
European Patent Office
Prior art keywords
tadg
protein
dna
individual
fragment
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00973739A
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German (de)
French (fr)
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EP1226150A4 (en
Inventor
Timothy J. O'brien
Hirotoshi Tanimoto
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University of Arkansas
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University of Arkansas
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Priority claimed from US09/421,213 external-priority patent/US7022821B1/en
Application filed by University of Arkansas filed Critical University of Arkansas
Priority to EP07118615A priority Critical patent/EP1887082A3/en
Publication of EP1226150A1 publication Critical patent/EP1226150A1/en
Publication of EP1226150A4 publication Critical patent/EP1226150A4/en
Withdrawn legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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

  • TADG-15 AN EXTRACELLULAR SERINE PROTEASE OVEREXPRESSED IN CARCINOMAS
  • the present invention relates generally to the fields o f cellular biology and the diagnosis of neoplastic disease. More specifically, the present invention relates to an extracellular serine protease, termed tumor antigen-derived gene 15 (TADG- 15), which is overexpressed in carcinomas.
  • TADG- 15 tumor antigen-derived gene 15
  • Extracellular proteases have been directly associated with tumor growth, shedding of tumor cells and invasion of target organs. Individual classes of proteases are involved in, but n o t limited to, (a) digestion of stroma surrounding the initial tumor area, (b) digestion of the cellular adhesion molecules to allow dissociation of tumor cells; and (c) invasion of the basement membrane for metastatic growth and activation of both tu mor growth factors and angiogenic factors.
  • proteases mediate specific proteolysis and contribute to th e removal of extracellular matrix components surrounding tu mor cells, the digestion of intercellular adhesion molecules to allow dissociation of malignant cells and the activation of many growth and angiogenic factors . 1 3
  • proteases are classified into four families: serine proteases, metalloproteases, aspartic proteases and cysteine proteases . 3
  • the metalloproteases have been well studied in relation to tumor growth and progression, and they are known to be capable o f degrading the extracellular matrix, thereby enhancing th e invasive potential of malignant cells.
  • the prior art is deficient in the lack of effective means of screening to identify proteases overexpressed in carcinoma.
  • the present invention fulfills this longstanding need and desire in the art.
  • the present invention discloses a screening program to identify proteases overexpressed in carcinoma by examining PCR products amplified using differential display in early stage tumors and metastatic tumors compared to that of normal tissues.
  • the approach herein to identify candidate genes overexpressed in tumor cells has been to utilize the well conserved domains surrounding the triad of amino acids (His- Asp-Ser) prototypical of the catalytic domain of serine proteases .
  • His- Asp-Ser His- Asp-Ser
  • a PCR product pre sent only in carcinoma was subcloned and sequenced, and was found to have a catalytic domain which was consistent with the serine protease family. Reported herein is the complete cloning and sequencing of this transcript and evidence for its expression in ovarian tumor cells.
  • a DNA encoding a tumor antigen-derived gene (TADG- 15) protein selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to the isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) a n isolated DNA differing from the isolated DNAs of (a) and ( b ) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein.
  • the embodiment further includes a vector comprising the TADG-15 DNA and regulatory elements necessary for expression of the DNA in a cell.
  • an isolated and purified TADG-15 protein coded for by DNA selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to isolated DNA o f (a) above and which encodes a TADG-15 protein; and (c) a n isolated DNA differing from the isolated DNAs of (a) and ( b ) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein.
  • a method for detecting TADG-15 mRNA in a sample comprising the steps of: (a) contacting a sample with a probe which is specific for TADG-15; and (b) detecting binding o f the probe to TADG-15 mRNA in the sample.
  • a kit for detecting TADG-15 mRNA comprising: an oligonucleotide probe specific for TADG-15. A label for detection is further embodied in the kit.
  • the present invention additionally embodies a method of detecting TADG-15 protein in a sample, comprising the steps of: (a) contacting a sample with an antibody which is specific for TADG-15 or a fragment thereof; and (b) detecting binding of th e antibody to TADG-15 protein in the sample.
  • th e present invention also embodies a kit for detecting TADG-15 protein, comprising: an antibody specific for TADG-15 protein o r a fragment thereof. Means for detection of the antibody is further embodied in the kit.
  • the present invention provides an antibody specific for the TADG-15 protein or a fragment thereof.
  • the present invention provides a method of screening for compounds that inhibit TADG-15, comprising the steps of: (a) contacting a sample comprising TADG-15 protein with a compound; and (b) assaying for TADG-15 protease activity.
  • a decrease in the TADG- 15 protease activity in the presence of the compound relative t o TADG-15 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-15.
  • a method of inhibiting expression o f TADG-15 in a cell comprising the step of: (a) introducing a vector into a cell, whereupon expression of the vector produces TADG-15 antisense mRNA in the cell which hybridizes t o endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell.
  • a method of inhibiting a TADG-15 protein in a cell comprising the step of: (a) introducing an antibody specific for a TADG-15 protein or a fragment thereof into a cell, whereupon binding of the antibody to the TADG-15 protein inhibits the TADG-15 protein.
  • a method of targeted therapy to an individual comprising the step of: (a) administering a compound containing a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-15.
  • a method of diagnosing cancer in an individual comprising the steps of: (a) obtaining a biological sample from a n individual; and (b) detecting TADG-15 in the sample, wherein the presence of TADG-15 in the sample is indicative of the presence of carcinoma in the individual and the absence of TADG-15 in th e sample is indicative of the absence of carcinoma in the individual.
  • a method of vaccinating an individual against TADG-15 comprising the steps of: (a) inoculating an individual with a TADG-15 protein or fragment thereof that lacks TADG- 15 protease activity, wherein the inoculation with the TADG- 15 protein or fragment thereof elicits an immune response in th e individual, thereby vaccinating the individual against TADG-15.
  • a method of producing immune-activated cells directed toward TADG-15 comprising the steps of: exposing dendritic cells to a TADG-15 protein or fragment thereof that lacks TADG- 15 protease activity, wherein the exposure to said TADG-15 protein or fragment thereof activates the dendritic cells, thereby producing immune-activated cells directed toward TADG-15.
  • an immunogenic composition comprising a n immunogenic fragment of a TADG-15 protein and an appropriate adjuvant.
  • Figure 1 shows a comparison of the serine protease catalytic domain of TADG-15 with Hepsin (Heps, SEQ ID No. 3 ) , SCCE (SEQ ID No. 4), Trypsin (Try, SEQ ID No. 5), Chymotrypsin (Chymb, SEQ ID No. 6), Factor 7 (Fac7, SEQ ID No. 7) and Tissue plasminogen activator (Tpa, SEQ ID No. 8).
  • the asterisks indicate conserved amino acids of catalytic triad.
  • Figure 2 shows the nucleotide sequence of the TADG- 15 cDNA and the derived amino acid sequence of the TADG-15 protein.
  • the putative start codon is located at nucleotides 23 -25.
  • the potential transmembrane sequence is underlined. Possible N- linked glycosylation sites are indicated by a broken line.
  • the asterisks indicate conserved cysteine residues of CUB domain .
  • the SDE-motifs of the LDL receptor ligand binding repeat-like domain are boxed.
  • the arrow shows the arginine-valine b o nd cleaved upon activation.
  • the conserved amino acids of the catalytic triad; histidine, aspartic acid and serine residues are circled.
  • Figure 3 shows the amino acid sequence of the TADG- 15 protease, including functional sites and domains.
  • Figure 4 shows a diagram of the TADG-15 protein. 1 ; cytoplasmic domain, (aa #1-54), 2; transmembrane domain ( aa #55-57), 3; extracellular domain (aa #78-213), 4-5; CUB repeat (aa #214-447), 6-9; LDL receptor ligand binding repeat (class A motif) like domain (aa #453-602), 10; serine protease (aa #615 - 855 ) .
  • Figure 5 shows Northern blot analysis of TADG-15 mRNA expression in normal ovary, ovarian carcinomas, carcinoma cell lines, normal fetal tissues and normal adult tissues.
  • a single intense transcript of the TADG-15 was observed in every sub-type of carcinoma and the two ovarian carcinoma cell lines, SW626 and CAOV3, whereas no visible band was detected in normal ovary or the two breast cancer cell lines.
  • normal fetal tissues fetal kidney showed increased transcript and faint expression was detected in fetal lung.
  • the TADG-15 was detected in colon with low expression in small intestine and prostate.
  • Figure 6 A shows quantitative PCR analysis of TADG- 15 expression.
  • Expression levels of TADG-15 relative to ⁇ -tubulin are significantly elevated in all LMP tumors and carcinomas compared to that of normal ovaries, m; mucinous, s; serous .
  • Figure 6B shows the ratio of TADG-15 expression to expression of ⁇ -tubulin in normal ovary, LMP tumor and ovarian carcinoma.
  • TADG-15 mRNA expression levels were significantly elevated in both LMP tumor (*; p ⁇ 0.001) and carcinoma (**; p ⁇ 0.0001 ) compared to that in normal ovary. All 10 samples of normal ovary showed a low level of TADG-15 expression.
  • Figure 7 shows the TADG-15 expression in tumor cell lines derived from both ovarian and breast carcinoma tissues.
  • Figure 8 shows the overexpression of TADG-15 in other tumor tissues.
  • Figure 9 shows SW626 and CAOV3 cell lysates that were separated by SDS-PAGE and immunoblotted. Lanes 1 and 2 were probed with rabbit pre-immune serum as a negative control . Lanes 3 and 4 were probed with polyclonal rabbit antibody generated to a carboxy terminal peptide from TADG-15 protein sequence .
  • Figure 10 shows that immunohistochemical staining of normal ovarian epithelium ( Figure 10 A ) with a polyclonal antibody to a TADG-15 protease peptide shows no staining of th e stroma or epithelium.
  • antibody staining of carcinomas confirms the presence of TADG-15 expression in the cytoplasm of a serous low malignant potential tumor ( Figure 10B ) ; a mucinous low malignant potential tumor ( Figure 10C) ; a serous carcinoma ( Figure 10D); and its presence in both the cytoplasm and cell surface of an endometrioid carcinoma (Figure 10E).
  • Figure 11 shows an alignment of the human TADG 15 protein sequence with that of mouse epithin which demonstrates that the proteins are 84% similar and 81 % identical over 843 amino acids. Residues that are identical between the two proteins are indicated by a "-", while the "*" symbol represents th e
  • TADG15 translation termination The most significant difference between these two proteins lies in the carboxy-termini, which for epithin, includes 47 amino acids that are not present in TADG15.
  • Figure 12 shows a nucleotide sequence comparison between TADG-15 and human SNC-19 (GeneBank Accession No.
  • TADG-15 tumor antigen-derived gene 15
  • the TADG-15 cDNA is 3147 base pairs long (SEQ ID No. 1) encoding for a 855 amino acid protein (SEQ ID No. 2).
  • SEQ ID No. 2 The availability of the TADG-15 gene provides numerous utilities.
  • the TADG-15 gene can be used as a diagnostic o r therapeutic target in ovarian and other carcinomas, including breast, prostate, lung and colon.
  • the present invention is directed to DNA encoding a tumor antigen-derived gene (TADG-15) protein, selected from th e following: (a) an isolated DNA which encodes a TADG-15 protein ; (b) an isolated DNA which hybridizes under high stringency conditions to the isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from th e isolated DNAs of (a) and (b) above in codon sequence due to th e degeneracy of the genetic code, and which encodes a TADG-15 protein. It is preferred that the DNA has the sequence shown in SEQ LD No. 1 and the TADG-15 protein has the amino acid sequence shown in SEQ ID No. 2.
  • the present invention is directed toward a vector comprising the TADG-15 DNA and regulatory elements necessary for expression of the DNA in a cell, or a vector in which the TADG-15 DNA is positioned in reverse orientation relative to the regulatory elements such that TADG-15 antisense mRNA is produced.
  • the DNA encodes a TADG-15 protein having the amino acid sequence shown in SEQ ID No. 2.
  • the invention is also directed toward host cells transfected with either of the above-described vector(s).
  • Representative host cells are bacterial cells, mammalian cells, plant cells and insect cells.
  • the bacterial cell is E. coli.
  • the present invention is directed toward an isolated and purified TADG-15 protein coded for by DNA selected from the following: (a) an isolated DNA which encodes a TADG- 15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein.
  • the protein has the amino acid sequence shown in SEQ ID No. 2.
  • the present invention is directed toward a method for detecting TADG-15 mRNA in a sample, comprising the steps of: (a) contacting a sample with a probe which is specific for TADG- 15; and (b) detecting binding of the probe to TADG-15 mRNA in the sample.
  • the present invention is also directed toward a method of detecting TADG-15 protein in a sample, comprising the steps of: (a) contacting a sample with an antibody which is specific for TADG-15 or a fragment thereof; and (b) detecting binding of the antibody to TADG-15 protein in the sample.
  • the sample is a biological sample; preferably, the biological sample is from an individual; and typically, the individual is suspected of having cancer.
  • the present invention is directed toward a kit for detecting TADG-15 mRNA, comprising: an oligonucleotide probe , wherein the probe is specific for TADG-15.
  • the kit may further comprise: a label with which to label the probe; and means for detecting the label.
  • the present invention is additionally directed toward a kit for detecting TADG-15 protein, comprising: a n antibody which is specific for TADG-15 protein or a fragment thereof.
  • the kit may further comprise: means to detect the antibody.
  • the present invention is directed toward a antibody which is specific for TADG-15 protein or a fragment thereof.
  • the present invention is directed toward a method o f screening for compounds that inhibit TADG-15, comprising the steps of: (a) contacting a sample containing TADG-15 protein with a compound; and (b) assaying for TADG-15 protease activity.
  • a decrease in the TADG-15 protease activity in the presence of the compound relative to TADG-15 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-15.
  • the present invention is directed toward a method o f inhibiting expression of TADG-15 in a cell, comprising the step of: (a) introducing a vector expressing TADG-15 antisense mRNA into a cell, which hybridizes to endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell.
  • a vector expressing TADG-15 antisense mRNA into a cell, which hybridizes to endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell.
  • th e inhibition of TADG-15 expression is for treating cancer.
  • the present invention is directed toward a method o f inhibiting a TADG-15 protein in a cell, comprising the step of: ( a ) introducing an antibody specific for a TADG-15 protein or a fragment thereof into a cell, which inhibits the TADG-15 protein.
  • the inhibition of the TADG-15 protein is for treating cancer.
  • the present invention is directed toward a method o f targeted therapy to an individual, comprising the step of: ( a ) administering a compound having a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-15.
  • Representative targeting moiety are a n antibody specific for TADG-15 and a ligand or ligand binding domain (e.g., CUB, LDLR, protease and extracellular) that binds TADG-15.
  • a representative therapeutic moiety is a radioisotope, a toxin, a chemotherapeutic agent and immune stimulants.
  • the above-described method is useful when the individual suffers from ovarian cancer, breast cancer o r cancers of the prostate, lung, colon and cervix.
  • the present invention is directed toward a method of diagnosing cancer in an individual, comprising the steps of: ( a ) obtaining a biological sample from an individual; and ( b ) detecting TADG-15 in the sample.
  • the presence o f TADG-15 in the sample is indicative of the presence of carcinoma in the individual, and the absence of TADG-15 in the sample is indicative of the absence of carcinoma in the individual.
  • the biological sample is blood, ascites fluid, urine, tears, saliva or interstitial fluid.
  • Typical means of detecting TADG-15 are by Northern blot, Western blot, PCR, dot blot, ELIZA, radioimmunoassay, DNA chips or tumor cell labeling. This method may be useful in diagnosing cancers such as ovarian, breast and other cancers in which TADG-15 is overexpressed, such as lung, prostate and colon cancers.
  • the present invention is also directed to an antisense oligonucleotide having the nucleotide sequence complementary to a TADG-15 mRNA sequence.
  • the present invention is also directed to a composition comprising such an antisense oligonucleotide according and a physiologically acceptable carrier therefore.
  • the present invention is also directed to a method of treating a neoplastic state in an individual syndrome in an individual in need of such treatment, comprising the step of administering to said individual an effective dose of an antisense oligonucleotide of.
  • the neoplastic state is selected from the group consisting of from ovarian cancer, breast cancer, lung cancer, prostate cancer, colon cancer and other cancers in which TADG-15 is overexpressed.
  • th e oligonucleotides alone or in combination with other anti- neoplastic agents can be formulated for a variety of modes of administration, including systemic, topical or localized administration.
  • oligonucleotide active ingredient is generally combined with a pharamceutically accceptable carrier such as a diluent or excipient which can include fillers, extenders, binders , wetting agents, disintergrants, surface active agents or lubricants, depending on the nature of the mode of administration an d dosage forms.
  • a pharamceutically accceptable carrier such as a diluent or excipient which can include fillers, extenders, binders , wetting agents, disintergrants, surface active agents or lubricants, depending on the nature of the mode of administration an d dosage forms.
  • Typical dosage forms include tablets, powders , liquid preparations including suspensions, emulsions, and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
  • oligonucleotides of th e invention are formulated in liquid solutions, preferably in physiologically compatible buffers.
  • th e oligonucleotides can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also incldued. Dosages that can be used for systemic administration preferably range from about 0.01 mg/kg to 5 0 mg/kg administered once or twice per day.
  • the present invention is directed toward a method o f vaccinating an individual against TADG-15 overexpression, comprising the steps of: (a) inoculating an individual with a TADG-15 protein or fragment thereof which lacks TADG-15 protease activity.
  • the inoculation with the TADG-15 protein o r fragment thereof elicits an immune response in the individual, thereby vaccinating the individual against TADG-15.
  • the vaccination with TADG-15 described herein is intended for a n individual who has cancer, is suspected of having cancer or is a t risk of getting cancer.
  • the TADG-15 fragment useful for vaccinating an individual are 9-residue fragments up to 20- residue fragments, with preferred 9-residue fragments shown in SEQ ID Nos. 2, 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83 , 84, 89 and 90.
  • the present invention is directed toward a method o f producing immune-activated cells directed toward TADG-15, comprising the steps of: exposing dendritic cells to a TADG- 15 protein or fragment thereof that lacks TADG-15 protease activity, wherein exposure to the TADG-15 protein or fragment thereof activates the dendritic cells, thereby producing immune- activated cells directed toward TADG-15.
  • Representative immune-activated cells are B-cells, T-cells and dendrites.
  • the TADG- 15 fragment is a 9-residue fragment up to a 20-residue fragment, with preferable 9-residue fragments shown in SEQ ID Nos. 2, 1 9 , 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.
  • the dendritic cells are isolated from an individual prior to exposure, and the activated dendritic cells reintroduced into the individual subsequent to exposure.
  • the individual for which this method may apply has cancer, is suspected of having cancer or is at risk of getting cancer.
  • the present invention is directed toward a n immunogenic composition, comprising an immunogenic fragment of a TADG-15 protein and an appropriate adjuvant.
  • the fragment is a 9-residue fragment up to a 20-residue fragment, with preferred 9-residue fragments shown in SEQ ID Nos. 2, 1 9 , 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.
  • cDNA shall refer to th e DNA copy of the mRNA transcript of a gene.
  • derived amino acid sequence shall mean the amino acid sequence determined by reading the triplet sequence of nucleotide bases in the cDNA.
  • screening a library shall refer to the process of using a labeled probe to check whether, under the appropriate conditions, there is a sequence complementary to the probe present in a particular DNA library.
  • screening a library could be performed by PCR.
  • PCR refers to th e polymerase chain reaction that is the subject of U.S. Patent Nos. 4,683, 195 and 4,683,202 to Mullis, as well as o ther improvements now known in the art.
  • amino acid described herein are preferred to b e in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long a s the desired functional property of immunoglobulin-binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers t o the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of ami no - terminus to carboxy-terminus. Furthermore, it should be no ted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of o ne or more amino-acid residues.
  • a "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i. e., capable of replication under its own control.
  • a “vector” is a replicon, such as plasmid, phage o r cosmid, to which another DNA segment may be attached so as t o bring about the replication of the attached segment.
  • a “vector” may further be defined as a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid.
  • a “DNA molecule” refers to the polymeric form o f deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single-stranded form or as a double-stranded helix.
  • This term refers only to the primary and secondary structure o f the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., viruses, plasmids, and chromosomes.
  • the structure is discussed herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i. e., the strand having a sequence homologous to the mRNA).
  • An expression vector is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of effecting expression of the polypeptide in a cell.
  • control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Methods which are well known to those skilled in the art can be used to construct expression vectors containing appropriate transcriptional and translational control signals. See, for example, techniques described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, N.Y.
  • a gene and its transcription control sequences are defined as being "operably linked” if the transcription control sequences effectively control transcription of the gene.
  • Vectors of th e invention include, but are not limited to, plasmid vectors and viral vectors.
  • Preferred viral vectors of the invention are tho se derived from retro viruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses.
  • expression vectors contain promoter sequences which facilitate the efficient transcription of the inserted DNA fragment and are used in connection with the host.
  • the expression vector typically contains an origin of replication, promoter(s), terminator(s), a s well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • the transformed hosts can b e fermented and cultured according to means known in the art t o achieve optimal cell growth.
  • An "origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal an d transcription termination sequence will usually be located 3' t o the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers , polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence .
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site a s well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters typically contain Shine-Dalgarno ribosome-binding sequences in addition to the -10 and - 35 consensus sequences.
  • An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation o f another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by th e coding sequence.
  • a "signal sequence” can be included near the coding sequence. This sequence encodes a signal peptide, N-terminal t o the polypeptide, that communicates to the host cell to direct th e polypeptide to the cell surface or secrete the polypeptide into th e media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes .
  • the terms “restriction endonucleases” and “restriction enzymes” refer to enzymes, each of which cu t double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous o r heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is o ne in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells throu gh chromosome replication.
  • This stability is demonstrated by th e ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and mo st preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • a "heterologous" region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature .
  • the gene will usually be flanked by DNA that does not flank th e mammalian genomic DNA in the genome of the source organism.
  • coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also b e labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3H, 1 C, 3 2 P, ⁇ S, 36Q, 51 C r, 57 Co , 58 Co , 59 ⁇ 90 Y> 125 131T, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized.
  • a particular assay system developed and utilized in the art is known as a receptor assay.
  • a receptor assay the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantitiy of both the label after which binding studies are conducted to determine th e extent to which the labeled material binds to the cell receptors . In this way, differences in affinity between materials can b e ascertained.
  • An assay useful in the art is known as a " cis/trans " assay.
  • this assay employs two genetic constructs, one o f which is typically a plasmid that continually expresses a particular receptor of interest when transfected into a n appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex.
  • a reporter such as luciferase
  • the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene.
  • the resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands.
  • the term "host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells.
  • a recombinant DNA molecule or gene which encodes a human TADG-15 protein of the present invention c an be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art.
  • a vector containing coding sequences for the gene which encodes a human TADG-15 protein of the present invention for purposes of prokaryote transformation .
  • Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis.
  • Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells.
  • the invention includes a substantially pure DNA encoding a TADG-15 protein, a DNA strand which will hybridize at high stringency to a probe containing a sequence of at least 1 5 consecutive nucleotides of (SEQ ID No. 1).
  • the protein encoded by the DNA of this invention may share at least 80% sequence identity (preferably 85%, more preferably 90%, and mo s t preferably 95%) with the amino acids listed in Figures 3 and 4 (SEQ ID No. 2). More preferably, the DNA includes the coding sequence of the nucleotides of Figure 2 (SEQ ID No. 1), or a degenerate variant of such a sequence.
  • This invention also includes a substantially pure DNA containing a sequence of a t least 15 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of the region from nucleotides 1 to 3147 of the nucleotides shown in Figure 2 (SEQ ID No. 1).
  • substantially pure DNA DNA that is no t part of a milieu in which the DNA naturally occurs, by virtue o f separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences.
  • PCR polymerase chain reaction
  • a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein. Also included is a recombinant DNA which includes a portion o f the nucleotides listed in Figure 2 (SEQ ID No. 1) and which encodes an alternative splice variant of TADG-15.
  • a substantially pure protein is meant a protein which has been separated from at least some of tho se components which naturally accompany it. Typically, the protein is substantially pure when it is at least 60% (by weight) free from the proteins and other naturally-occurring organic molecules with which it is naturally associated in vivo.
  • the purity of the preparation is at least 75%, more preferably a t least 90%, and most preferably at least 99%.
  • a substantially p ure TADG-15 protein may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a TADG-15 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, such a s immunoaffinity chromatography using an antibody specific for TADG-15, poly aery lamide gel electrophoresis, or HPLC analysis.
  • a protein is substantially free of naturally associated components when it is separated from at least some of those contaminants which accompany it in its natural state.
  • a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be, by definition, substantially free from its naturally associated components. Accordingly, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, o r any other organism in which they do not naturally occur.
  • oligonucleotide as used herein, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors, which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally (as in a purified restriction digest) or produced synthetically, and which is capable of initiating synthesis of a strand complementary to a nucleic acid when placed under appropriate conditions, i. e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must b e sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, sequence and/or homology of primer and th e method used.
  • the oligonucleotide primer typically contains 15-25 or m ore nucleotides, depending upon the complexity of the target sequence, although it may contain fewer nucleotides.
  • the primers herein are selected to be "substanti" complementary to particular target DNA sequences. This me ans that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment (i. e., containing a restriction site) may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary t o the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that th e primer sequence has sufficient complementary with the sequence to hybridize therewith and form the template for synthesis of the extension product.
  • a non-complementary nucleotide fragment i. e., containing a restriction site
  • non-complementary bases or longer sequences can be interspersed into the primer, provided that th e primer sequence has sufficient complementary with the sequence to hybridize therewith and form the template for synthesis of the extension product.
  • the probe to which the DNA of the invention hybridizes preferably consists of a sequence of at least 2 0 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 1 00 nucleotides or more (up to 100%) of the coding sequence of th e nucleotides listed in Figure 2 (SEQ ID No. 1) or the complement thereof.
  • Such a probe is useful for detecting expression of TADG- 15 in a cell by a method including the steps of (a) contacting mRNA obtained from the cell with a labeled TADG- 15 hybridization probe; and (b) detecting hybridization of the probe with the mRNA.
  • high stringency DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65°C at a salt concentration of approximately 0.1X SSC, or the functional equivalent thereof.
  • high stringency conditions may include hybridization at about 42°C in the presence of about 50 % formamide; a first wash at about 65°C with about 2X SSC containing 1 % SDS; followed by a second wash at about 65°C with about 0.1X SSC.
  • the DNA may have at least about 70% sequence identity to the coding sequence of the nucleotides listed in Figure 2 (SEQ ID No. 1), preferably at least 75% (e.g., at least 80%); and most preferably at least 90%.
  • the identity between two sequences is a direct function of the number of matching o r identical positions.
  • a position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then they are identical at that position.
  • the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides.
  • Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group (GCG), University o f Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705).
  • the present invention comprises a vector comprising a DNA sequence which encodes a human TADG-15 protein, wherein said vector is capable of replication in a host, and comprises, in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding for said TADG-15 protein.
  • the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No. 1.
  • Vectors may be used to amplify and/or express nucleic acid encoding a TADG-15 protein or fragment thereof.
  • the invention also includes fragments (e.g., antigenic fragments) o f the TADG-15 protein (SEQ ID No. 2).
  • fragment as applied to a polypeptide, will ordinarily be at least 6 residues , more typically at least 9-12 residues, and preferably at least 1 3 - 20 residues in length, but less than the entire, intact sequence .
  • a fragment may be an individual domain of 20- 120 residues from SEQ ID No. 2.
  • Fragments of the TADG-15 protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring o r recombinant TADG-15 protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment o f TADG-15, or by chemical synthesis.
  • TADG-15 The ability of a candidate fragment to exhibit a characteristic of TADG-15 (e.g., binding t o an antibody specific for TADG-15) can be assessed by methods described herein.
  • Purified TADG-15 or antigenic fragments o f TADG-15 can be used to generate new antibodies or to te s t existing antibodies (e.g., as positive controls in a diagnostic assay) by employing standard protocols known to those skilled in the art. Included in this invention is polyclonal antisera generated by using TADG-15 or a fragment of TADG-15 as the immunogen in, e.g., rabbits. Standard protocols for monoclonal and polyclonal antibody production known to those skilled in this art are employed. The monoclonal antibodies generated by this procedure can be screened for the ability to identify recombinant TADG-15 cDNA clones, and to distinguish them from other cDNA clones .
  • TADG- 15 proteins which are encoded, at least in part, by portions of SEQ ID No. 2, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of TADG- 15 sequence has been deleted.
  • the fragment, or the intact TADG-15 polypeptide may be covalently linked to another polypeptide, e.g., one which acts as a label, a ligand or a means to increase antigenicity.
  • the invention also includes a polyclonal o r monoclonal antibody which specifically binds to TADG-15.
  • the invention encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab) 2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and th e remaining portions of another antibody, e.g., of human origin.
  • the antibody, or a fragment thereof may be linked to a toxin or to a detectable label, e.g., a radioactive label, non-radioactive isotopic label, fluorescent label, chemiluminescent label, paramagnetic label, enzyme label, or colorimetric label.
  • a detectable label e.g., a radioactive label, non-radioactive isotopic label, fluorescent label, chemiluminescent label, paramagnetic label, enzyme label, or colorimetric label.
  • suitable toxins include diphtheria toxin, Pseudomonas exotoxin A, ricin, and cholera toxin.
  • suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc.
  • suitable radioisotopic labels include 3 H, 12S I, 131 I, 32 P, 5 S, 14 C, etc.
  • Paramagnetic isotopes for purposes of in vivo diagnosis can also be used according to the methods of this invention.
  • elements that are useful in magnetic resonance imaging.
  • fluorescent labels include a fluorescein label, an isothiocyalate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an ophthaldehyde label, a fluorescamine label, etc .
  • chemiluminescent labels include a luminal label, a n isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.
  • TADG-15 protein in a biological sample which includes the steps of contacting the sample with the labeled antibody, e.g., radioactively tagged antibody specific for TADG-15, and determining whether the antibody binds to a component of the sample.
  • Antibodies to the TADG-15 protein can be used in a n immunoassay to detect increased levels of TADG-15 protein expression in tissues suspected of neoplastic transformation . These same uses can be achieved with Northern blot assays and analyses .
  • the invention provides a number of diagnostic advantages and uses.
  • the TADG-15 protein is useful in diagnosing cancer in different tissues since this protein is highly overexpressed in tumor cells.
  • Antibodies (or antigen-binding fragments thereof) which bind to an epitope specific for TADG-15, are useful in a method of detecting TADG- 15 protein in a biological sample for diagnosis of cancerous o r neoplastic transformation.
  • This method includes the steps o f obtaining a biological sample (e.g., cells, blood, plasma, tissue, etc.) from a patient suspected of having cancer, contacting the sample with a labeled antibody (e.g., radioactively tagged antibody) specific for TADG-15, and detecting the TADG-15 protein using standard immunoassay techniques such as an ELISA.
  • a biological sample e.g., cells, blood, plasma, tissue, etc.
  • a labeled antibody e.g., radioactively tagged antibody
  • Antibody binding to the biological sample indicates that th e sample contains a component which specifically binds to a n epitope within TADG-15.
  • a standard Northern blot assay can be used to ascertain the relative amounts of TADG-15 mRNA in a cell o r tissue obtained from a patient suspected of having cancer, in accordance with conventional Northern hybridization techniques known to those of ordinary skill in the art.
  • This Northern assay uses a hybridization probe, e.g., radiolabelled TADG-15 cDNA, either containing the full-length, single stranded DNA having a sequence complementary to SEQ ID No. 1 ( Figure 2), or a fragment of that DNA sequence at least 20 (preferably at least 30 , more preferably at least 50, and most preferably at least 1 00 consecutive nucleotides in length).
  • the DNA hybridization probe can be labeled by any of the many different methods known t o those skilled in this art.
  • the specimen Upon patient hysterectomy, bilateral salpingo- oophorectomy, or surgical removal of neoplastic tissue, the specimen is retrieved and placed on ice. The specimen was then taken to the resident pathologist for isolation and identification of specific tissue samples. Finally, the sample was frozen in liquid nitrogen, logged into the laboratory record and stored at -80°C. Additional specimens were frequently obtained from the Cooperative Human Tissue Network (CHTN). These samples were prepared by the CHTN and shipped on dry ice. Upon arrival, these specimens (e.g., blood (serum), urine, saliva, tears and insterstitial fluid) were logged into the laboratory record and stored at -80°C.
  • CHTN Cooperative Human Tissue Network
  • CHTN Cooperative Human Tissue Network
  • ovarian tumors (10 low malignant potential tumors and 31 carcinomas) and 10 normal ovaries were obtained from surgical specimens and frozen in liquid nitrogen.
  • Messenger RNA (mRNA) isolation was performed according to the manufacturer's instructions using the Mini RiboSepTM Ultra mRNA Isolation Kit purchased from Becton Dickinson. In this procedure, polyA + mRNA was isolated directly from the tissue lysate using the affinity chromatography media oligo(dT) cellulose. The amount of mRNA recovered was quantitated by UV spectrophotometry.
  • cDNA First-strand complementary DNA
  • Promega T-vector plasmid and the ligation products used t o transform JM109 competent cells according to th e manufacturer's instructions (Promega). Positive colonies were cultured for amplification, the plasmid DNA isolated using the WizardTM Minipreps DNA purification system (Promega), and the plasmids were digested with Apal and Sacl restriction enzymes t o determine the size of the insert. Plasmids with inserts of the size(s) visualized by the previously described PCR product gel electrophoresis were sequenced. Individual colonies were cultured and plasmid DNA was isolated using the Wizard Miniprep DNA purification system (Promega). Applied Biosystems Model 373A DNA sequencing system was used for direct cDNA sequence determination .
  • sequencing reactions were carried out using PRISMTM Ready Reaction Dye DeoxyTM terminators (Applied Biosystems) according to the manufacturer's instructions. Residual dye terminators were removed from the completed sequencing reaction using a Centri-sepTM spin column (Princeton Separation) . Based upon the determined sequence, primers that specifically amplify the gene of interest were designed and synthesized.
  • the original TADG-15 subclone (436bp) was randomly labeled and used as a probe to screen an ovarian tumor cDNA library by standard hybridization techniques . 13
  • the library was constructed in 8ZAP using mRNA isolated from the tumor cells of a stage Ill/grade III ovarian adenocarcinoma patient. Three overlapping clones were obtained which spanned 3 147 nucleotides .
  • RNAs 10 ⁇ g mRNAs were size separated by electrophoresis through a 1% formaldehyde-agarose gel in 0.02 M MOPS, 0.05 M sodium acetate (pH 7.0), and 0.001 M EDTA. The mRNAs were then blotted to Hybond-N + nylon membrane (Amersham) by capillary action in 20x SSPE. The RNAs are fixed to the membrane by baking for 2 hours at 80°C. 32 P-labeled cDNA probes were made by Prime-a-Gene Labeling System (Promega). The PCR products amplified by the same primers described above were used for probes.
  • the blots were prehybridized for 30 min and hybridized for 60 min at 68°C with 32 P-labeled cDNA probe in ExpressHyb Hybridization Solution (CLONTECH). Control hybridization to determine relative gel loading was performed with a ⁇ -tubulin probe. Normal human tissues; spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocyte, and normal human fetal tissues; brain, lung, liver and kidney (Human Multiple Tissue Northern Blot; CLONTECH) were also examined by the same hybridization procedure. Additional multiple tissue northern (MTN) blots from CLONTECH include the Human MTN blot, the Human MTN II blot, the Human Fetal MTN II blot, and the Human Brain MTN III blot.
  • MTN Multiple tissue northern
  • Polyclonal rabbit antibody was generated by immunization with a poly-lysine linked multiple Ag peptide derived from the TADG-15 protein sequence 'LFRDWIKENTGV' (SEQ ID No. 13). Approximately 20 ⁇ g of cell lysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDF a t 100 V for 40 min at 4°C. The proteins were fixed to th e membrane by incubation in 50% MeOH for 10 min. The membrane was blocked overnight in TBS (pH 7.8) containing 0.2% non-fat milk. Primary antibody was added to the membrane at a dilution of 1 : 100 in 0.2% milk/TBS and incubated for 2 h a t room temperature.
  • the blot was washed and incubated with a 1 :3000 dilution of alkaline-phosphatase conjugated goat anti- rabbit IgG (BioRad) for 1 h at room temperature.
  • the blot was washed and incubated with a chemiluminescent substrate before a 10 sec exposure to X-ray film for visualization.
  • TADG-15 The mRNA overexpression of TADG-15 was determined using a quantitative PCR. Quantitative PCR was performed. 11 12 Oligonucleotide primers were used for TADG-15: forward 5'-ATGACAGAGGATTCAGGTAC-3' (SEQ ID No. 1 4 ) an d reverse 5'-GAAGGTGAAGTCATTGAAGA-3' (SEQ ID No. 15 ) ; and and for ⁇ -tubulin: forward 5'-CGCATCAACGTGTACTACAA-3' (SEQ ID No. 1 6 ) and reverse 5'-TACGAGCTGGTGGACTGAGA-3' (SEQ ID No. 17). ⁇ -tubulin was utilized as an internal control.
  • the PCR reaction mixture consists of cDNA derived from 50 ng of mRNA, 5 pmol of sense and antisense primers for both the TADG-15 gene and the ⁇ -tubulin gene, 200 ⁇ mol o f dNTPs, 5 ⁇ Ci of ⁇ - 32 PdCTP and 0.25 units of Taq DNA polymerase with reaction buffer (Promega) in a final volume of 25 ⁇ l.
  • the target sequences were amplified in parallel with the ⁇ -tubulin gene. Thirty cycles of PCR were carried out in a Thermal Cycler (Perkin Elmer Gene Amp 2400; Perkin-Elmer Cetus).
  • Each cycle of PCR included 30 sec of denaturation at 94°C, 30 sec of annealing at 60°C and 30 sec of extension at 72°C.
  • the annealing temperature varies according to the primers that are used in the PCR reaction. For the reactions involving degenerate primers, a n annealing temperature of 48°C was used. The appropriate annealing temperature for the TADG-15- and ⁇ -tubulin-specific primers is 62°C.
  • Immunohistochemical staining was performed using a Vectastain Elite ABC Kit (Vector). Formalin-fixed and paraffin- embedded specimens were routinely deparaffinized and processed using microwave heat treatment in 0.01 M sodium citrate buffer (pH 6.0). The specimens were incubated with normal goat serum in a moist chamber for 30 min. After incubation with biotinylated anti-rabbit IgG for 30 min, th e sections were then incubated with ABC reagent (Vector) for 3 0 min. The final products were visualized using the AEC substrate system (DAKO) and sections were counterstained with hematoxylin before mounting. Negative controls were performed using normal serum instead of the primary antibody.
  • DAKO AEC substrate system
  • TADG-15 is cloned and expressed in the opposite orientation such that an antisense RNA molecule (SEQ ID No. 1 8 ) is produced.
  • the antisense RNA is used to hybridize to the complementary RNA in the cell and thereby inhibit translation of TADG-15 RNA into protein.
  • a screening strategy to identify proteases which are overexpressed in human cancer has been developed in which RT- PCR products amplified specifically in tumors, as compared t o normal tissue, are examined. 9 During this effort, candidate genes were identified using redundant sense primers to the conserved amino acid histidine domain at the NH 3 end of the catalytic domain and antisense primers to the downstream conserved amino acid serine domain. Subcloning and sequencing th e appropriate 480 base pair band(s) amplified in such a PCR reaction provides the basis for identifying the gene(s) encoding proteases(s). Among these amplified catalytic domains, a new serine protease gene named TADG-15 (tumor antigen-derived gene 15) was identified. The catalytic domain of the newly identified TADG-15 protein is similar to other serine proteases and specifically contains conserved amino acids appropriate for the catalytic domain of the trypsin-like serine protease family.
  • the deduced open reading frame encoded by th e TADG-15 nucleotide sequence contains several distinct domains as follows: an amino terminal cytoplasmic tail (amino acids (aa) #1-54), a potential transmembrane domain (aa #55-77), an extracellular membrane domain (aa #78-213), two complement subcomponents Clr/Cls, Uegf, and bone morphogenetic protein 1 (CUB) repeats (aa #214- 447), four ligand binding repeats of the low density lipoprotein (LDL) receptor-like domain (aa #453-602) and a serine protease domain (aa #615-855).
  • amino acids amino acids
  • aa #55-77 potential transmembrane domain
  • aa #78-213 extracellular membrane domain
  • CRB bone morphogenetic protein 1
  • the TADG-15 protein also contains two potential N-linked glycosylation sites (aa #109 and 302) and a potential proteolytic cleavage site upstream from the protease domain (aa #614) which could release and/or activate the protease at the carboxy end of this protein.
  • TADG-15 contains an RGD motif (aa #249-251 ) which is commonly found in proteins involved in cell-cell adhesion.
  • transcript size for TADG-15 was determined to be approximately 3.2 kb and a single intense transcript appeared to be present in all of the carcinomas examined, whereas no visible band was detected in normal ovary ( Figure 5). This transcript size is also in good agreement with the sequence data predicting a transcript size of 3.15 kb.
  • the ovarian tumor cell lines, SW626 and CAOV3, also showed a n abundance of transcript, however little or no transcript was detectable in the breast carcinoma cell lines MDA-MB-231 and MDA-MB-4355.
  • TADG-15 was detected in colon with low levels o f expression in small intestine and prostate ( Figure 5).
  • TADG-15 transcripts were noted in all ovarian carcinomas, but were not present at detectable levels in any of the following tissues: a) normal ovary, b) fetal liver and brain, c) adult spleen, thymus, testes, ovary and peripheral blood lymphocytes, d) skeletal muscle, liver, brain o r heart. This evaluation was extended to a standard panel of about 40 tumors. Using TADG-15-specific primers, the expression was also examined in tumor cell lines derived from both ovarian and breast carcinoma tissues as shown in Figure 7 and in other tumor tissues as shown in Figure 8. Expression of TADG-15 was also observed in carcinomas of the breast, colon, prostate and lung.
  • Polyclonal antibodies developed to a synthetic peptide (a 12-mer) at the carboxy terminus of the protease domain were used to examine TADG-15 expression in cell lines by Western blot and by immunolocalization in normal ovary and ovarian tumors .
  • Western blots of cell extracts from SW626 and CAOV3 cells were probed with both antibody and preimmune sera ( Figure 9 ) .
  • Several bands were detected with the antibody, including bands of approximately 100,000 daltons, approximately 60,000 daltons and 32,000 daltons.
  • the anticipated molecular size of th e complete TADG-15 molecule is estimated to be approximately 100,000 daltons, and the protease domain which may be released by proteolytic cleavage at aa #614 is estimated to b e approximately 32,000 daltons. Some intermediate proteolytic product may be represented by the 60,000 dalton band.
  • Antibody staining of tumor cells confirms the presence of the TADG-15 protease in the cytoplasm of a serous LMP tumor, mucinous LMP tumor and serous carcinoma ( Figure 10B, C & D, respectively). This diffuse staining pattern may b e due to detection of TADG 15 within the cell as it is being packaged and transported to the cell surface.
  • Carcinoma 31 31 (100%) 0.771 ⁇ 0.380 serous 18 18 (100%) 0.779 ⁇ 0.332 mucinous 7 7 (100%) 0.907 ⁇ 0.584 endometrioid 3 3 (100%) 0.502 + 0.083 clear cell 3 3 ⁇ OO% 0.672 + 0.077
  • H-6201 clear cell carcinoma ++ H-5640 clear cell carcinoma ⁇
  • Epithin is a 902 amino acid protein which contains a similar structure to TADG-15 in that it has a cytoplasmic domain, transmembrane domain, two CUB domains, four LDLR-like domains and a carboxy terminal serine protease domain.
  • TADG-15 and epithin are 84% similar over 843 amino acids, suggesting that the proteins may b e orthologous ( Figure 11).
  • the precise role of epithin remains t o be elucidated.
  • a search of GeneBank for similar previously identified sequences yielded one such sequence with relatively high homology to a portion of the TADG-15 gene.
  • TADG-15 has a n open reading frame of 855 amino acids whereas the longest open reading frame of SNC-19 is 173 amino acids.
  • SNC- 19 does not include a proper start site for the initiation o f translation, nor does it include the amino terminal portion of th e protein encoded by TADG-15.
  • SNC-19 does n o t include an open reading frame for a functional serine protease because the His, Asp and Ser residues of the catalytic triad that are necessary for function are encoded in different reading frames .
  • the overall structure of the TADG-15 protein is relatively similar to the members of the tolloid/BMP-1 family and the complement subcomponents, Clr/Cls. These proteins contain both CUB and protease domains, and complex formation through the ligand binding domain is essential for their function. Activation of the serine protease domains of Clr and Cls requires proteolytic cleavage of Arg-Gly and Arg-Ile bonds, respectively. 1 5 Similarly, it might be expected that the TADG-15 protein is synthesized as a zymogen, which is activated by cleavage between Arg 614 and Val 615 and analogous to the activation mechanism of other serine protease zymogens.
  • CUB domains were first found in complement subcomponents C l r/C l s 16 18 and are known to be a widespread module in developmentally regulated proteins, such as the b one morphogenetic protein-1 (BMP-1) and the tolloid gene product. 18- 20 The role of these repeats remains largely unknown. However, some models suggest that the CUB domain may be involved in protein-protein interactions.
  • the CUB domain of Clr and Cls participates in the assembly of the Cls-Clr-Clr-Cls tetrameric complex in the activation of the classical pathway of complement by providing protein-protein interaction domains . 15
  • BMP-1 b one morphogenetic protein-1
  • Drosophila decapentaplegic (DPP) protein is essential for dorsal- ventral specification of the embryo, and the Drosophila tolloid (TLD) forms a complex with DPP to regulate its activity.
  • DPP Drosophila decapentaplegic
  • TLD Drosophila tolloid
  • the TADG-15 protein contains two tandem repeats o f CUB-like domains between amino acid residues 214 and 447. Each of these is approximately 110 amino acids long and each has four conserved cysteine residues characteristic of other CUBs (amino acids 214, 244, 268, 294, 340, 366, 397, 410).
  • the CUB repeats of the TADG-15 protein may form a n interactive domain capable of promoting multimeric complex formation and regulating the activity of the target protein o r TADG- 15 itself.
  • the TADG-15 protein also contains the LDL receptor ligand binding repeat (class A motif) -like domain, which consists of four contiguous cysteine-rich repeats (amino acid residues 453 to 602). Each cysteine-rich repeat is approximately 40 amino acids long and contains a conserved, negatively-charged sequence (Ser-Asp-Glu) with six cysteine residues. In the LDL receptor protein, this repeat is thought to function as a protein-binding domain which interacts with the lysine and arginine residues present in lipoproteins. 21 ,22 In addition, the first repeat of the LDL receptor appears to bind Ca 2+ and not the lipoproteins .
  • LDL receptor ligand binding repeat class A motif
  • LDL receptor-like repeat in TADG- 15 may act in a similar fashion, interacting with positively charged regions of other proteins and/or as a Ca 2+ binding site.
  • LDL receptor is internalized via clathrin-coated pits .
  • TADG-15 does not contain this motif in its cytosolic region, and furthermore, no similarities with other known protein sequences were found in the cytoplasmic domain of the TADG-15. This finding suggests th at TADG-15 functions in a different manner from the endocytic receptors (such as the LDL receptor), although TADG-15 possesses similar ligand-binding repeats in the extracellular matrix .
  • TADG-15 Although the precise role of TADG-15 is unknown, this gene is clearly overexpressed in ovarian tumors.
  • a variety o f proteases such as type IV collagenase and plasminogen activator, appear to be involved in the process of tumor invasion and are constituents of a protease cascade in malignant progression.
  • TADG-15 may constitute such an activity and directly digest extracellular matrix components surrounding a tumor, or activate other proteases by cleavage of inactive precursors, indirectly enhancing tumor growth and invasion. It is also possible that TADG-15 may function like a member of the tolloid/BMP-1 family by forming complexes with other growth factors or signal transduction proteins to modulate their activities.

Abstract

The present invention provides DNA encoding a TADG-15 protein as well as a TADG-15 protein. Also provided is a vector capable of expressing the DNA of the present invention adapted for expression in a recombinant cell and regulatory elements necessary for expression of the DNA in the cell. The present invention further provides for methods of inhibiting TADG-15 expression and/or protease activity, methods of detecting TADG-15 mRNA and/or protein and methods of screening for TADG-15 inhibitors. Additionally, the present invention provides for cell-specific targeting via TADG-15 and methods of vaccinating an individual against TADG-15. The methods described are useful in the diagnosis, treatment and prevention of cancer, particularly breast and ovarian cancer.

Description

TADG-15: AN EXTRACELLULAR SERINE PROTEASE OVEREXPRESSED IN CARCINOMAS
BACKGROUND OF THE INVENTION
Cross-Reference to Related Application
This application in a continuation-in-part of USSN 09/027,337, filed February 20, 1998 and thereby claims the benefit of priority under 35 USC § 120.
Field of the Invention
The present invention relates generally to the fields o f cellular biology and the diagnosis of neoplastic disease. More specifically, the present invention relates to an extracellular serine protease, termed tumor antigen-derived gene 15 (TADG- 15), which is overexpressed in carcinomas.
Description of the Related Art Extracellular proteases have been directly associated with tumor growth, shedding of tumor cells and invasion of target organs. Individual classes of proteases are involved in, but n o t limited to, (a) digestion of stroma surrounding the initial tumor area, (b) digestion of the cellular adhesion molecules to allow dissociation of tumor cells; and (c) invasion of the basement membrane for metastatic growth and activation of both tu mor growth factors and angiogenic factors.
In the process of cancer progression and invasion, proteases mediate specific proteolysis and contribute to th e removal of extracellular matrix components surrounding tu mor cells, the digestion of intercellular adhesion molecules to allow dissociation of malignant cells and the activation of many growth and angiogenic factors .1 3 Depending on the nature of their catalytic domain, proteases are classified into four families: serine proteases, metalloproteases, aspartic proteases and cysteine proteases .3 Among these proteases, the metalloproteases have been well studied in relation to tumor growth and progression, and they are known to be capable o f degrading the extracellular matrix, thereby enhancing th e invasive potential of malignant cells. -5 For serine proteases , previous studies have demonstrated an increased production o f plasminogen activator in tumor cells and a positive correlation between plasminogen activator activity and aggressiveness o f cancer.6 7 Prostate specific antigen (a serine protease) has also been widely used as an indicator of abnormal prostate growth.8 More recently, several other serine proteases have been reported, viz. hepsin and the stratum corneum chymotryptic enzyme (SCCE), which are overexpressed in ovarian cancer and which may contribute to malignant progression by increasing the extracellular lytic activity of these tumor cells.9
The prior art is deficient in the lack of effective means of screening to identify proteases overexpressed in carcinoma. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The present invention discloses a screening program to identify proteases overexpressed in carcinoma by examining PCR products amplified using differential display in early stage tumors and metastatic tumors compared to that of normal tissues. The approach herein to identify candidate genes overexpressed in tumor cells has been to utilize the well conserved domains surrounding the triad of amino acids (His- Asp-Ser) prototypical of the catalytic domain of serine proteases . Herein, evidence is presented for a unique form of serine protease not previously described in the literature which is highly expressed in ovarian carcinomas. Through the screening approach using differential PCR amplification of normal, low malignant potential and overt carcinomas, a PCR product pre sent only in carcinoma was subcloned and sequenced, and was found to have a catalytic domain which was consistent with the serine protease family. Reported herein is the complete cloning and sequencing of this transcript and evidence for its expression in ovarian tumor cells. In one embodiment of the present invention, there is provided a DNA encoding a tumor antigen-derived gene (TADG- 15) protein, selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to the isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) a n isolated DNA differing from the isolated DNAs of (a) and ( b ) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein. The embodiment further includes a vector comprising the TADG-15 DNA and regulatory elements necessary for expression of the DNA in a cell. Additionally embodied is a vector in which the TADG-15 DNA is positioned in reverse orientation relative to the regulatory elements such that TADG-15 antisense mRNA is produced. In another embodiment of the present invention, there is provided an isolated and purified TADG-15 protein coded for by DNA selected from the following: (a) an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to isolated DNA o f (a) above and which encodes a TADG-15 protein; and (c) a n isolated DNA differing from the isolated DNAs of (a) and ( b ) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein.
In yet another embodiment of the present invention, there is provided a method for detecting TADG-15 mRNA in a sample, comprising the steps of: (a) contacting a sample with a probe which is specific for TADG-15; and (b) detecting binding o f the probe to TADG-15 mRNA in the sample. In still yet another embodiment of the present invention, there is provided a kit for detecting TADG-15 mRNA, comprising: an oligonucleotide probe specific for TADG-15. A label for detection is further embodied in the kit.
The present invention additionally embodies a method of detecting TADG-15 protein in a sample, comprising the steps of: (a) contacting a sample with an antibody which is specific for TADG-15 or a fragment thereof; and (b) detecting binding of th e antibody to TADG-15 protein in the sample. Similarly, th e present invention also embodies a kit for detecting TADG-15 protein, comprising: an antibody specific for TADG-15 protein o r a fragment thereof. Means for detection of the antibody is further embodied in the kit.
In another embodiment, the present invention provides an antibody specific for the TADG-15 protein or a fragment thereof.
In yet another embodiment, the present invention provides a method of screening for compounds that inhibit TADG-15, comprising the steps of: (a) contacting a sample comprising TADG-15 protein with a compound; and (b) assaying for TADG-15 protease activity. Typically, a decrease in the TADG- 15 protease activity in the presence of the compound relative t o TADG-15 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-15.
In still yet another embodiment of the pre sent invention, there is provided a method of inhibiting expression o f TADG-15 in a cell, comprising the step of: (a) introducing a vector into a cell, whereupon expression of the vector produces TADG-15 antisense mRNA in the cell which hybridizes t o endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell.
Further embodied by the present invention, there is provided a method of inhibiting a TADG-15 protein in a cell, comprising the step of: (a) introducing an antibody specific for a TADG-15 protein or a fragment thereof into a cell, whereupon binding of the antibody to the TADG-15 protein inhibits the TADG-15 protein.
In an embodiment of the present invention, there is provided a method of targeted therapy to an individual, comprising the step of: (a) administering a compound containing a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-15.
In an embodiment of the present invention, there is provided a method of diagnosing cancer in an individual, comprising the steps of: (a) obtaining a biological sample from a n individual; and (b) detecting TADG-15 in the sample, wherein the presence of TADG-15 in the sample is indicative of the presence of carcinoma in the individual and the absence of TADG-15 in th e sample is indicative of the absence of carcinoma in the individual. In another embodiment of the present invention, there is provided a method of vaccinating an individual against TADG-15, comprising the steps of: (a) inoculating an individual with a TADG-15 protein or fragment thereof that lacks TADG- 15 protease activity, wherein the inoculation with the TADG- 15 protein or fragment thereof elicits an immune response in th e individual, thereby vaccinating the individual against TADG-15.
In an embodiment of the present invention, there is provided a method of producing immune-activated cells directed toward TADG-15, comprising the steps of: exposing dendritic cells to a TADG-15 protein or fragment thereof that lacks TADG- 15 protease activity, wherein the exposure to said TADG-15 protein or fragment thereof activates the dendritic cells, thereby producing immune-activated cells directed toward TADG-15. In another embodiment of the present invention, there is provided an immunogenic composition, comprising a n immunogenic fragment of a TADG-15 protein and an appropriate adjuvant.
Other and further aspects, features, and advantages o f the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features, advantages and objects of the invention, as well a s others which will become clear, are attained and can b e understood in detail, more particular descriptions of th e invention briefly summarized above may be had by reference t o certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of th e specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
Figure 1 shows a comparison of the serine protease catalytic domain of TADG-15 with Hepsin (Heps, SEQ ID No. 3 ) , SCCE (SEQ ID No. 4), Trypsin (Try, SEQ ID No. 5), Chymotrypsin (Chymb, SEQ ID No. 6), Factor 7 (Fac7, SEQ ID No. 7) and Tissue plasminogen activator (Tpa, SEQ ID No. 8). The asterisks indicate conserved amino acids of catalytic triad. Figure 2 shows the nucleotide sequence of the TADG- 15 cDNA and the derived amino acid sequence of the TADG-15 protein. The putative start codon is located at nucleotides 23 -25. The potential transmembrane sequence is underlined. Possible N- linked glycosylation sites are indicated by a broken line. The asterisks indicate conserved cysteine residues of CUB domain . The SDE-motifs of the LDL receptor ligand binding repeat-like domain are boxed. The arrow shows the arginine-valine b o nd cleaved upon activation. The conserved amino acids of the catalytic triad; histidine, aspartic acid and serine residues are circled.
Figure 3 shows the amino acid sequence of the TADG- 15 protease, including functional sites and domains.
Figure 4 shows a diagram of the TADG-15 protein. 1 ; cytoplasmic domain, (aa #1-54), 2; transmembrane domain ( aa #55-57), 3; extracellular domain (aa #78-213), 4-5; CUB repeat (aa #214-447), 6-9; LDL receptor ligand binding repeat (class A motif) like domain (aa #453-602), 10; serine protease (aa #615 - 855 ) . Figure 5 shows Northern blot analysis of TADG-15 mRNA expression in normal ovary, ovarian carcinomas, carcinoma cell lines, normal fetal tissues and normal adult tissues. A single intense transcript of the TADG-15 was observed in every sub-type of carcinoma and the two ovarian carcinoma cell lines, SW626 and CAOV3, whereas no visible band was detected in normal ovary or the two breast cancer cell lines. In normal fetal tissues, fetal kidney showed increased transcript and faint expression was detected in fetal lung. In normal adult tissues, the TADG-15 was detected in colon with low expression in small intestine and prostate.
Figure 6 A shows quantitative PCR analysis of TADG- 15 expression. Expression levels of TADG-15 relative to β-tubulin are significantly elevated in all LMP tumors and carcinomas compared to that of normal ovaries, m; mucinous, s; serous . Figure 6B shows the ratio of TADG-15 expression to expression of β-tubulin in normal ovary, LMP tumor and ovarian carcinoma. TADG-15 mRNA expression levels were significantly elevated in both LMP tumor (*; p<0.001) and carcinoma (**; p<0.0001 ) compared to that in normal ovary. All 10 samples of normal ovary showed a low level of TADG-15 expression.
Figure 7 shows the TADG-15 expression in tumor cell lines derived from both ovarian and breast carcinoma tissues. Figure 8 shows the overexpression of TADG-15 in other tumor tissues.
Figure 9 shows SW626 and CAOV3 cell lysates that were separated by SDS-PAGE and immunoblotted. Lanes 1 and 2 were probed with rabbit pre-immune serum as a negative control . Lanes 3 and 4 were probed with polyclonal rabbit antibody generated to a carboxy terminal peptide from TADG-15 protein sequence .
Figure 10 shows that immunohistochemical staining of normal ovarian epithelium (Figure 10 A ) with a polyclonal antibody to a TADG-15 protease peptide shows no staining of th e stroma or epithelium. However, antibody staining of carcinomas confirms the presence of TADG-15 expression in the cytoplasm of a serous low malignant potential tumor (Figure 10B ) ; a mucinous low malignant potential tumor (Figure 10C) ; a serous carcinoma (Figure 10D); and its presence in both the cytoplasm and cell surface of an endometrioid carcinoma (Figure 10E).
Figure 11 shows an alignment of the human TADG 15 protein sequence with that of mouse epithin which demonstrates that the proteins are 84% similar and 81 % identical over 843 amino acids. Residues that are identical between the two proteins are indicated by a "-", while the "*" symbol represents th e
TADG15 translation termination. The most significant difference between these two proteins lies in the carboxy-termini, which for epithin, includes 47 amino acids that are not present in TADG15.
Figure 12 shows a nucleotide sequence comparison between TADG-15 and human SNC-19 (GeneBank Accession No.
#U20428 ) .
DETAILED DESCRIPTION OF THE INVENTION
Proteases have been implicated in the extracellular modulation required for tumor growth and invasion. In an effort to categorize those proteases contributing to ovarian carcinoma progression, redundant primers directed towards conserved amino acid domains surrounding the catalytic triad of His, Asp and Ser were utilized to amplify serine proteases differentially expressed in carcinomas. Using this method, a serine protease named TADG-15 (tumor antigen-derived gene 15) has been identified that is overexpressed in ovarian tumors. TADG- 15 appears to be a transmembrane multidomain serine protease . TADG-15 is highly overexpressed in ovarian tumors based on PCR, Northern blot and immunolocalization. The TADG-15 cDNA is 3147 base pairs long (SEQ ID No. 1) encoding for a 855 amino acid protein (SEQ ID No. 2). The availability of the TADG-15 gene provides numerous utilities. For example, the TADG-15 gene can be used as a diagnostic o r therapeutic target in ovarian and other carcinomas, including breast, prostate, lung and colon.
The present invention is directed to DNA encoding a tumor antigen-derived gene (TADG-15) protein, selected from th e following: (a) an isolated DNA which encodes a TADG-15 protein ; (b) an isolated DNA which hybridizes under high stringency conditions to the isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from th e isolated DNAs of (a) and (b) above in codon sequence due to th e degeneracy of the genetic code, and which encodes a TADG-15 protein. It is preferred that the DNA has the sequence shown in SEQ LD No. 1 and the TADG-15 protein has the amino acid sequence shown in SEQ ID No. 2.
The present invention is directed toward a vector comprising the TADG-15 DNA and regulatory elements necessary for expression of the DNA in a cell, or a vector in which the TADG-15 DNA is positioned in reverse orientation relative to the regulatory elements such that TADG-15 antisense mRNA is produced. Generally, the DNA encodes a TADG-15 protein having the amino acid sequence shown in SEQ ID No. 2. The invention is also directed toward host cells transfected with either of the above-described vector(s). Representative host cells are bacterial cells, mammalian cells, plant cells and insect cells. Preferably, the bacterial cell is E. coli. The present invention is directed toward an isolated and purified TADG-15 protein coded for by DNA selected from the following: (a) an isolated DNA which encodes a TADG- 15 protein; (b) an isolated DNA which hybridizes under high stringency conditions to isolated DNA of (a) above and which encodes a TADG-15 protein; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-15 protein. Preferably, the protein has the amino acid sequence shown in SEQ ID No. 2.
The present invention is directed toward a method for detecting TADG-15 mRNA in a sample, comprising the steps of: (a) contacting a sample with a probe which is specific for TADG- 15; and (b) detecting binding of the probe to TADG-15 mRNA in the sample. The present invention is also directed toward a method of detecting TADG-15 protein in a sample, comprising the steps of: (a) contacting a sample with an antibody which is specific for TADG-15 or a fragment thereof; and (b) detecting binding of the antibody to TADG-15 protein in the sample. Generally, the sample is a biological sample; preferably, the biological sample is from an individual; and typically, the individual is suspected of having cancer.
The present invention is directed toward a kit for detecting TADG-15 mRNA, comprising: an oligonucleotide probe , wherein the probe is specific for TADG-15. The kit may further comprise: a label with which to label the probe; and means for detecting the label. The present invention is additionally directed toward a kit for detecting TADG-15 protein, comprising: a n antibody which is specific for TADG-15 protein or a fragment thereof. The kit may further comprise: means to detect the antibody.
The present invention is directed toward a antibody which is specific for TADG-15 protein or a fragment thereof. The present invention is directed toward a method o f screening for compounds that inhibit TADG-15, comprising the steps of: (a) contacting a sample containing TADG-15 protein with a compound; and (b) assaying for TADG-15 protease activity. Typically, a decrease in the TADG-15 protease activity in the presence of the compound relative to TADG-15 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-15.
The present invention is directed toward a method o f inhibiting expression of TADG-15 in a cell, comprising the step of: (a) introducing a vector expressing TADG-15 antisense mRNA into a cell, which hybridizes to endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in the cell. Generally, th e inhibition of TADG-15 expression is for treating cancer.
The present invention is directed toward a method o f inhibiting a TADG-15 protein in a cell, comprising the step of: ( a ) introducing an antibody specific for a TADG-15 protein or a fragment thereof into a cell, which inhibits the TADG-15 protein. Generally, the inhibition of the TADG-15 protein is for treating cancer. The present invention is directed toward a method o f targeted therapy to an individual, comprising the step of: ( a ) administering a compound having a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-15. Representative targeting moiety are a n antibody specific for TADG-15 and a ligand or ligand binding domain (e.g., CUB, LDLR, protease and extracellular) that binds TADG-15. Likewise, a representative therapeutic moiety is a radioisotope, a toxin, a chemotherapeutic agent and immune stimulants. Typically, the above-described method is useful when the individual suffers from ovarian cancer, breast cancer o r cancers of the prostate, lung, colon and cervix.
The present invention is directed toward a method of diagnosing cancer in an individual, comprising the steps of: ( a ) obtaining a biological sample from an individual; and ( b ) detecting TADG-15 in the sample. Generally, the presence o f TADG-15 in the sample is indicative of the presence of carcinoma in the individual, and the absence of TADG-15 in the sample is indicative of the absence of carcinoma in the individual. Generally, the biological sample is blood, ascites fluid, urine, tears, saliva or interstitial fluid. Typical means of detecting TADG-15 are by Northern blot, Western blot, PCR, dot blot, ELIZA, radioimmunoassay, DNA chips or tumor cell labeling. This method may be useful in diagnosing cancers such as ovarian, breast and other cancers in which TADG-15 is overexpressed, such as lung, prostate and colon cancers.
The present invention is also directed to an antisense oligonucleotide having the nucleotide sequence complementary to a TADG-15 mRNA sequence. The present invention is also directed to a composition comprising such an antisense oligonucleotide according and a physiologically acceptable carrier therefore.
The present invention is also directed to a method of treating a neoplastic state in an individual syndrome in an individual in need of such treatment, comprising the step of administering to said individual an effective dose of an antisense oligonucleotide of. Preferably, the neoplastic state is selected from the group consisting of from ovarian cancer, breast cancer, lung cancer, prostate cancer, colon cancer and other cancers in which TADG-15 is overexpressed. For such therapy, th e oligonucleotides alone or in combination with other anti- neoplastic agents can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations generally can b e found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. The oligonucleotide active ingredient is generally combined with a pharamceutically accceptable carrier such as a diluent or excipient which can include fillers, extenders, binders , wetting agents, disintergrants, surface active agents or lubricants, depending on the nature of the mode of administration an d dosage forms. Typical dosage forms include tablets, powders , liquid preparations including suspensions, emulsions, and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous. For injection, the oligonucleotides of th e invention are formulated in liquid solutions, preferably in physiologically compatible buffers. In addition, th e oligonucleotides can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also incldued. Dosages that can be used for systemic administration preferably range from about 0.01 mg/kg to 5 0 mg/kg administered once or twice per day. However, different dosing schedules can be utilized depending on (1) the potency o f an individual oligonucleotide at inhibiting the activity of its target DNA, (2) the severity or extent of the pathological disease state, or (3) the pharmacokinetic behavior of a given oligonucleotide.
The present invention is directed toward a method o f vaccinating an individual against TADG-15 overexpression, comprising the steps of: (a) inoculating an individual with a TADG-15 protein or fragment thereof which lacks TADG-15 protease activity. The inoculation with the TADG-15 protein o r fragment thereof elicits an immune response in the individual, thereby vaccinating the individual against TADG-15. The vaccination with TADG-15 described herein is intended for a n individual who has cancer, is suspected of having cancer or is a t risk of getting cancer. Generally, the TADG-15 fragment useful for vaccinating an individual are 9-residue fragments up to 20- residue fragments, with preferred 9-residue fragments shown in SEQ ID Nos. 2, 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83 , 84, 89 and 90. The present invention is directed toward a method o f producing immune-activated cells directed toward TADG-15, comprising the steps of: exposing dendritic cells to a TADG- 15 protein or fragment thereof that lacks TADG-15 protease activity, wherein exposure to the TADG-15 protein or fragment thereof activates the dendritic cells, thereby producing immune- activated cells directed toward TADG-15. Representative immune-activated cells are B-cells, T-cells and dendrites. Generally, the TADG- 15 fragment is a 9-residue fragment up to a 20-residue fragment, with preferable 9-residue fragments shown in SEQ ID Nos. 2, 1 9 , 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90. Preferably, the dendritic cells are isolated from an individual prior to exposure, and the activated dendritic cells reintroduced into the individual subsequent to exposure. Typically, the individual for which this method may apply has cancer, is suspected of having cancer or is at risk of getting cancer.
The present invention is directed toward a n immunogenic composition, comprising an immunogenic fragment of a TADG-15 protein and an appropriate adjuvant. Generally, the fragment is a 9-residue fragment up to a 20-residue fragment, with preferred 9-residue fragments shown in SEQ ID Nos. 2, 1 9 , 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.
In accordance with the present invention there may b e employed conventional molecular biology, microbiology, an d recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and π (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed . 1984); "Nucleic Acid Hybridization" (B.D. Hames & S.J. Higgins eds. 1985); "Transcription and Translation" (B.D. Hames & S.J. Higgins eds. 1984); "Animal Cell Culture" (R.I. Freshney, ed . 1986); "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.
As used herein, the term "cDNA" shall refer to th e DNA copy of the mRNA transcript of a gene. As used herein, the term "derived amino acid sequence" shall mean the amino acid sequence determined by reading the triplet sequence of nucleotide bases in the cDNA.
As used herein the term "screening a library" shall refer to the process of using a labeled probe to check whether, under the appropriate conditions, there is a sequence complementary to the probe present in a particular DNA library.
In addition, "screening a library" could be performed by PCR.
As used herein, the term "PCR" refers to th e polymerase chain reaction that is the subject of U.S. Patent Nos. 4,683, 195 and 4,683,202 to Mullis, as well as o ther improvements now known in the art.
The amino acid described herein are preferred to b e in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long a s the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers t o the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J Biol. Chem., 243:3552-59 ( 1969), abbreviations for amino acid residues are used as in customary in the art.
It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of ami no - terminus to carboxy-terminus. Furthermore, it should be no ted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of o ne or more amino-acid residues. A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i. e., capable of replication under its own control. A "vector" is a replicon, such as plasmid, phage o r cosmid, to which another DNA segment may be attached so as t o bring about the replication of the attached segment. A "vector" may further be defined as a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid. A "DNA molecule" refers to the polymeric form o f deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single-stranded form or as a double-stranded helix. This term refers only to the primary and secondary structure o f the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. The structure is discussed herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i. e., the strand having a sequence homologous to the mRNA).
An expression vector is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of effecting expression of the polypeptide in a cell. The need for such control sequences will vary depending upon the cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Methods which are well known to those skilled in the art can be used to construct expression vectors containing appropriate transcriptional and translational control signals. See, for example, techniques described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, N.Y. A gene and its transcription control sequences are defined as being "operably linked" if the transcription control sequences effectively control transcription of the gene. Vectors of th e invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the invention are tho se derived from retro viruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses. In general, expression vectors contain promoter sequences which facilitate the efficient transcription of the inserted DNA fragment and are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), a s well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can b e fermented and cultured according to means known in the art t o achieve optimal cell growth.
An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.
A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal an d transcription termination sequence will usually be located 3' t o the coding sequence.
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers , polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence . For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within th e promoter sequence will be found a transcription initiation site, a s well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters typically contain Shine-Dalgarno ribosome-binding sequences in addition to the -10 and - 35 consensus sequences.
An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation o f another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by th e coding sequence.
A "signal sequence" can be included near the coding sequence. This sequence encodes a signal peptide, N-terminal t o the polypeptide, that communicates to the host cell to direct th e polypeptide to the cell surface or secrete the polypeptide into th e media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes .
As used herein, the terms "restriction endonucleases " and "restriction enzymes" refer to enzymes, each of which cu t double-stranded DNA at or near a specific nucleotide sequence. A cell has been "transformed" by exogenous o r heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes , yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is o ne in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells throu gh chromosome replication. This stability is demonstrated by th e ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
Two DNA sequences are "substantially homologous " when at least about 75% (preferably at least about 80%, and mo st preferably at least about 90% or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
A "heterologous" region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature . Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank th e mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also b e labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3H, 1 C, 32P, ^S, 36Q, 51Cr, 57Co, 58Co, 59^ 90Y> 125 131T, and 186Re. Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
A particular assay system developed and utilized in the art is known as a receptor assay. In a receptor assay, the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantitiy of both the label after which binding studies are conducted to determine th e extent to which the labeled material binds to the cell receptors . In this way, differences in affinity between materials can b e ascertained. An assay useful in the art is known as a " cis/trans " assay. Briefly, this assay employs two genetic constructs, one o f which is typically a plasmid that continually expresses a particular receptor of interest when transfected into a n appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex. Thus, for example, if it is desired t o evaluate a compound as a ligand for a particular receptor, one o f the plasmids would be a construct that results in expression o f the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted. If the compound under test is an agonist for the receptor, the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands. The foregoing protocol is described in detail in U.S. Patent No. 4,981,784. As used herein, the term "host" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. A recombinant DNA molecule or gene which encodes a human TADG-15 protein of the present invention c an be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a vector containing coding sequences for the gene which encodes a human TADG-15 protein of the present invention for purposes of prokaryote transformation . Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells.
The invention includes a substantially pure DNA encoding a TADG-15 protein, a DNA strand which will hybridize at high stringency to a probe containing a sequence of at least 1 5 consecutive nucleotides of (SEQ ID No. 1). The protein encoded by the DNA of this invention may share at least 80% sequence identity (preferably 85%, more preferably 90%, and mo s t preferably 95%) with the amino acids listed in Figures 3 and 4 (SEQ ID No. 2). More preferably, the DNA includes the coding sequence of the nucleotides of Figure 2 (SEQ ID No. 1), or a degenerate variant of such a sequence. This invention also includes a substantially pure DNA containing a sequence of a t least 15 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of the region from nucleotides 1 to 3147 of the nucleotides shown in Figure 2 (SEQ ID No. 1).
By "substantially pure DNA" is meant DNA that is no t part of a milieu in which the DNA naturally occurs, by virtue o f separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein. Also included is a recombinant DNA which includes a portion o f the nucleotides listed in Figure 2 (SEQ ID No. 1) and which encodes an alternative splice variant of TADG-15. By a "substantially pure protein" is meant a protein which has been separated from at least some of tho se components which naturally accompany it. Typically, the protein is substantially pure when it is at least 60% (by weight) free from the proteins and other naturally-occurring organic molecules with which it is naturally associated in vivo. Preferably, the purity of the preparation (by weight) is at least 75%, more preferably a t least 90%, and most preferably at least 99%. A substantially p ure TADG-15 protein may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a TADG-15 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, such a s immunoaffinity chromatography using an antibody specific for TADG-15, poly aery lamide gel electrophoresis, or HPLC analysis. A protein is substantially free of naturally associated components when it is separated from at least some of those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be, by definition, substantially free from its naturally associated components. Accordingly, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, o r any other organism in which they do not naturally occur. The term "oligonucleotide", as used herein, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors, which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term "primer", as used herein, refers to an oligonucleotide, whether occurring naturally (as in a purified restriction digest) or produced synthetically, and which is capable of initiating synthesis of a strand complementary to a nucleic acid when placed under appropriate conditions, i. e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must b e sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, sequence and/or homology of primer and th e method used. For example, in diagnostic applications, the oligonucleotide primer typically contains 15-25 or m ore nucleotides, depending upon the complexity of the target sequence, although it may contain fewer nucleotides.
The primers herein are selected to be " substantially" complementary to particular target DNA sequences. This me ans that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment (i. e., containing a restriction site) may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary t o the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that th e primer sequence has sufficient complementary with the sequence to hybridize therewith and form the template for synthesis of the extension product. The probe to which the DNA of the invention hybridizes preferably consists of a sequence of at least 2 0 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 1 00 nucleotides or more (up to 100%) of the coding sequence of th e nucleotides listed in Figure 2 (SEQ ID No. 1) or the complement thereof. Such a probe is useful for detecting expression of TADG- 15 in a cell by a method including the steps of (a) contacting mRNA obtained from the cell with a labeled TADG- 15 hybridization probe; and (b) detecting hybridization of the probe with the mRNA.
By "high stringency" is meant DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65°C at a salt concentration of approximately 0.1X SSC, or the functional equivalent thereof. For example, high stringency conditions may include hybridization at about 42°C in the presence of about 50 % formamide; a first wash at about 65°C with about 2X SSC containing 1 % SDS; followed by a second wash at about 65°C with about 0.1X SSC. The DNA may have at least about 70% sequence identity to the coding sequence of the nucleotides listed in Figure 2 (SEQ ID No. 1), preferably at least 75% (e.g., at least 80%); and most preferably at least 90%. The identity between two sequences is a direct function of the number of matching o r identical positions. When a position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then they are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group (GCG), University o f Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705).
The present invention comprises a vector comprising a DNA sequence which encodes a human TADG-15 protein, wherein said vector is capable of replication in a host, and comprises, in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding for said TADG-15 protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No. 1. Vectors may be used to amplify and/or express nucleic acid encoding a TADG-15 protein or fragment thereof. In addition to substantially full-length proteins, the invention also includes fragments (e.g., antigenic fragments) o f the TADG-15 protein (SEQ ID No. 2). As used herein, "fragment, " as applied to a polypeptide, will ordinarily be at least 6 residues , more typically at least 9-12 residues, and preferably at least 1 3 - 20 residues in length, but less than the entire, intact sequence . Alternatively, a fragment may be an individual domain of 20- 120 residues from SEQ ID No. 2. Fragments of the TADG-15 protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring o r recombinant TADG-15 protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment o f TADG-15, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of TADG-15 (e.g., binding t o an antibody specific for TADG-15) can be assessed by methods described herein. Purified TADG-15 or antigenic fragments o f TADG-15 can be used to generate new antibodies or to te s t existing antibodies (e.g., as positive controls in a diagnostic assay) by employing standard protocols known to those skilled in the art. Included in this invention is polyclonal antisera generated by using TADG-15 or a fragment of TADG-15 as the immunogen in, e.g., rabbits. Standard protocols for monoclonal and polyclonal antibody production known to those skilled in this art are employed. The monoclonal antibodies generated by this procedure can be screened for the ability to identify recombinant TADG-15 cDNA clones, and to distinguish them from other cDNA clones .
Further included in this invention are TADG- 15 proteins which are encoded, at least in part, by portions of SEQ ID No. 2, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of TADG- 15 sequence has been deleted. The fragment, or the intact TADG-15 polypeptide, may be covalently linked to another polypeptide, e.g., one which acts as a label, a ligand or a means to increase antigenicity.
The invention also includes a polyclonal o r monoclonal antibody which specifically binds to TADG-15. The invention encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab)2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and th e remaining portions of another antibody, e.g., of human origin.
In one embodiment, the antibody, or a fragment thereof, may be linked to a toxin or to a detectable label, e.g., a radioactive label, non-radioactive isotopic label, fluorescent label, chemiluminescent label, paramagnetic label, enzyme label, or colorimetric label. Examples of suitable toxins include diphtheria toxin, Pseudomonas exotoxin A, ricin, and cholera toxin. Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc. Examples of suitable radioisotopic labels include 3H, 12SI, 131I, 32P, 5S, 14C, etc. Paramagnetic isotopes for purposes of in vivo diagnosis can also be used according to the methods of this invention. There are numerous examples of elements that are useful in magnetic resonance imaging. For discussions on in vivo nuclear magnetic resonance imaging, see, for example, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986) Magn. Reson. Med. 3, 336-340; Wolf, G. L., (1984) Physiol. Chem. Phys. Med. NMR Xβ, 93-95; Wesbey et al., ( 1984) Physiol. Chem. Phys. Med. NMR 16, 145-155 ; Runge et al., (1984) Invest. Radiol. 19 , 408-415. Examples of suitable fluorescent labels include a fluorescein label, an isothiocyalate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an ophthaldehyde label, a fluorescamine label, etc . Examples of chemiluminescent labels include a luminal label, a n isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.
Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies o r fragments thereof can be accomplished using standard techniques commonly known and used by those of ordinary skill in the art. Typical techniques are described by Kennedy et al. , ( 1976) Clin. Chim. Ada 70, 1-31 ; and Schurs et al., (1977) Clin. Chim. Ada 81, 1-40. Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, th e dimaleimide method, the m-maleimidobenzyl-N-hydroxy- succinimide ester method. All of these methods are incorporated by reference herein. Also within the invention is a method of detecting
TADG-15 protein in a biological sample, which includes the steps of contacting the sample with the labeled antibody, e.g., radioactively tagged antibody specific for TADG-15, and determining whether the antibody binds to a component of the sample. Antibodies to the TADG-15 protein can be used in a n immunoassay to detect increased levels of TADG-15 protein expression in tissues suspected of neoplastic transformation . These same uses can be achieved with Northern blot assays and analyses .
As described herein, the invention provides a number of diagnostic advantages and uses. For example, the TADG-15 protein is useful in diagnosing cancer in different tissues since this protein is highly overexpressed in tumor cells. Antibodies (or antigen-binding fragments thereof) which bind to an epitope specific for TADG-15, are useful in a method of detecting TADG- 15 protein in a biological sample for diagnosis of cancerous o r neoplastic transformation. This method includes the steps o f obtaining a biological sample (e.g., cells, blood, plasma, tissue, etc.) from a patient suspected of having cancer, contacting the sample with a labeled antibody (e.g., radioactively tagged antibody) specific for TADG-15, and detecting the TADG-15 protein using standard immunoassay techniques such as an ELISA. Antibody binding to the biological sample indicates that th e sample contains a component which specifically binds to a n epitope within TADG-15.
Likewise, a standard Northern blot assay can be used to ascertain the relative amounts of TADG-15 mRNA in a cell o r tissue obtained from a patient suspected of having cancer, in accordance with conventional Northern hybridization techniques known to those of ordinary skill in the art. This Northern assay uses a hybridization probe, e.g., radiolabelled TADG-15 cDNA, either containing the full-length, single stranded DNA having a sequence complementary to SEQ ID No. 1 (Figure 2), or a fragment of that DNA sequence at least 20 (preferably at least 30 , more preferably at least 50, and most preferably at least 1 00 consecutive nucleotides in length). The DNA hybridization probe can be labeled by any of the many different methods known t o those skilled in this art.
The following examples are given for the purpose o f illustrating various embodiments of the invention and are no t meant to limit the present invention in any fashion.
EXAMPLE 1
Tissu collection and storage
Upon patient hysterectomy, bilateral salpingo- oophorectomy, or surgical removal of neoplastic tissue, the specimen is retrieved and placed on ice. The specimen was then taken to the resident pathologist for isolation and identification of specific tissue samples. Finally, the sample was frozen in liquid nitrogen, logged into the laboratory record and stored at -80°C. Additional specimens were frequently obtained from the Cooperative Human Tissue Network (CHTN). These samples were prepared by the CHTN and shipped on dry ice. Upon arrival, these specimens (e.g., blood (serum), urine, saliva, tears and insterstitial fluid) were logged into the laboratory record and stored at -80°C. Participation of the following divisions of th e Cooperative Human Tissue Network (CHTN) in providing tumor tissues is acknowledged: Western Division, Case Western Reserve University, (Cleveland, OH); Midwestern Division, Ohio state University, (Columbus, OH); Eastern Division, NDRI, (Philadelphia, PA); Pediatric Division, Children's Hospital, (Columbus, OH) ; Southern Division, University of Alabama at Birmingham, (Birmingham, AL).
EXAMPLE 2 mRNA isolation and cDNA synthesis
Forty-one ovarian tumors (10 low malignant potential tumors and 31 carcinomas) and 10 normal ovaries were obtained from surgical specimens and frozen in liquid nitrogen. The human ovarian carcinoma cell lines SW626 and CAOV3, and th e human breast carcinoma cell lines MDA-MB-231 and MDA-MB- 435S, were purchased from the American Type Culture Collection (Rockville, MD). Cells were cultured to sub-confluency in Dulbecco' s modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum and antibiotics. Messenger RNA (mRNA) isolation was performed according to the manufacturer's instructions using the Mini RiboSep™ Ultra mRNA Isolation Kit purchased from Becton Dickinson. In this procedure, polyA+ mRNA was isolated directly from the tissue lysate using the affinity chromatography media oligo(dT) cellulose. The amount of mRNA recovered was quantitated by UV spectrophotometry.
First-strand complementary DNA (cDNA) was synthesized using 5.0 μg of mRNA and either random hexamer o r oligo(dT) primers according to the manufacturer's protocol utilizing a first strand synthesis kit obtained from CLONTECH (Palo Alto, CA). The purity of the cDNA was evaluated by PCR using primers specific for the p53 gene. These primers span a n intron such that pure cDNA can be distinguished from cDNA that is contaminated with genomic DNA. EXAMPLE 3
PCR with redundant primers, cloning of TADG-15 r.DNA, T-ve.clor ligation and transformations and DNA sequencing Redundant primers, forward 5'-TGGGTIGTIACIGCIGCICA(C/T)TG-3' (SEQ ID No.
11) and reverse 5'-A(A/G)IGGICCICCI(C/G)(T/A)(A/G)TCICC-3' (SEQ ID No. 12), corresponding to the amino acids surrounding the catalytic triad for serine proteases, were used to compare the PCR products from normal and carcinoma cDNAs. The purified PCR products were ligated into th e
Promega T-vector plasmid and the ligation products used t o transform JM109 competent cells according to th e manufacturer's instructions (Promega). Positive colonies were cultured for amplification, the plasmid DNA isolated using the Wizard™ Minipreps DNA purification system (Promega), and the plasmids were digested with Apal and Sacl restriction enzymes t o determine the size of the insert. Plasmids with inserts of the size(s) visualized by the previously described PCR product gel electrophoresis were sequenced. Individual colonies were cultured and plasmid DNA was isolated using the Wizard Miniprep DNA purification system (Promega). Applied Biosystems Model 373A DNA sequencing system was used for direct cDNA sequence determination . Utilizing a plasmid-specific primer near the cloning site, sequencing reactions were carried out using PRISM™ Ready Reaction Dye Deoxy™ terminators (Applied Biosystems) according to the manufacturer's instructions. Residual dye terminators were removed from the completed sequencing reaction using a Centri-sep™ spin column (Princeton Separation) . Based upon the determined sequence, primers that specifically amplify the gene of interest were designed and synthesized.
The original TADG-15 subclone (436bp) was randomly labeled and used as a probe to screen an ovarian tumor cDNA library by standard hybridization techniques .13 The library was constructed in 8ZAP using mRNA isolated from the tumor cells of a stage Ill/grade III ovarian adenocarcinoma patient. Three overlapping clones were obtained which spanned 3 147 nucleotides . EXAMPLE 4
Northern blot analysis
10 μg mRNAs were size separated by electrophoresis through a 1% formaldehyde-agarose gel in 0.02 M MOPS, 0.05 M sodium acetate (pH 7.0), and 0.001 M EDTA. The mRNAs were then blotted to Hybond-N+ nylon membrane (Amersham) by capillary action in 20x SSPE. The RNAs are fixed to the membrane by baking for 2 hours at 80°C. 32P-labeled cDNA probes were made by Prime-a-Gene Labeling System (Promega). The PCR products amplified by the same primers described above were used for probes. The blots were prehybridized for 30 min and hybridized for 60 min at 68°C with 32P-labeled cDNA probe in ExpressHyb Hybridization Solution (CLONTECH). Control hybridization to determine relative gel loading was performed with a β-tubulin probe. Normal human tissues; spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocyte, and normal human fetal tissues; brain, lung, liver and kidney (Human Multiple Tissue Northern Blot; CLONTECH) were also examined by the same hybridization procedure. Additional multiple tissue northern (MTN) blots from CLONTECH include the Human MTN blot, the Human MTN II blot, the Human Fetal MTN II blot, and the Human Brain MTN III blot.
EXAMPLE S
Western blot analysis
Polyclonal rabbit antibody was generated by immunization with a poly-lysine linked multiple Ag peptide derived from the TADG-15 protein sequence 'LFRDWIKENTGV' (SEQ ID No. 13). Approximately 20 μg of cell lysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDF a t 100 V for 40 min at 4°C. The proteins were fixed to th e membrane by incubation in 50% MeOH for 10 min. The membrane was blocked overnight in TBS (pH 7.8) containing 0.2% non-fat milk. Primary antibody was added to the membrane at a dilution of 1 : 100 in 0.2% milk/TBS and incubated for 2 h a t room temperature. The blot was washed and incubated with a 1 :3000 dilution of alkaline-phosphatase conjugated goat anti- rabbit IgG (BioRad) for 1 h at room temperature. The blot was washed and incubated with a chemiluminescent substrate before a 10 sec exposure to X-ray film for visualization.
EXAMPLE 6
Quantitative PCR The mRNA overexpression of TADG-15 was determined using a quantitative PCR. Quantitative PCR was performed.11 12 Oligonucleotide primers were used for TADG-15: forward 5'-ATGACAGAGGATTCAGGTAC-3' (SEQ ID No. 1 4 ) an d reverse 5'-GAAGGTGAAGTCATTGAAGA-3' (SEQ ID No. 15 ) ; and and for β-tubulin: forward 5'-CGCATCAACGTGTACTACAA-3' (SEQ ID No. 1 6 ) and reverse 5'-TACGAGCTGGTGGACTGAGA-3' (SEQ ID No. 17). β-tubulin was utilized as an internal control.
The PCR reaction mixture consists of cDNA derived from 50 ng of mRNA, 5 pmol of sense and antisense primers for both the TADG-15 gene and the β-tubulin gene, 200 μmol o f dNTPs, 5 μCi of α-32PdCTP and 0.25 units of Taq DNA polymerase with reaction buffer (Promega) in a final volume of 25 μl. The target sequences were amplified in parallel with the β-tubulin gene. Thirty cycles of PCR were carried out in a Thermal Cycler (Perkin Elmer Gene Amp 2400; Perkin-Elmer Cetus). Each cycle of PCR included 30 sec of denaturation at 94°C, 30 sec of annealing at 60°C and 30 sec of extension at 72°C. The annealing temperature varies according to the primers that are used in the PCR reaction. For the reactions involving degenerate primers, a n annealing temperature of 48°C was used. The appropriate annealing temperature for the TADG-15- and β-tubulin-specific primers is 62°C.
A portion of the PCR products were separated on 2 % agarose gels and the radioactivity of each PCR product was determined by using a Phospholmager (Molecular Dynamics). In the present study, the expression ratio (TADG- 15/β-tubulin) was used to evaluate gene expression and defined the value at mean ± 2SD of normal ovary as the cut-off value to determine overexpression. The student's t test was used for comparison o f the mean values of normal ovary and tumors. EXAMPLE 7
Immunohi stoch mi try
Immunohistochemical staining was performed using a Vectastain Elite ABC Kit (Vector). Formalin-fixed and paraffin- embedded specimens were routinely deparaffinized and processed using microwave heat treatment in 0.01 M sodium citrate buffer (pH 6.0). The specimens were incubated with normal goat serum in a moist chamber for 30 min. After incubation with biotinylated anti-rabbit IgG for 30 min, th e sections were then incubated with ABC reagent (Vector) for 3 0 min. The final products were visualized using the AEC substrate system (DAKO) and sections were counterstained with hematoxylin before mounting. Negative controls were performed using normal serum instead of the primary antibody.
EXAMPLE S Antisense TADG-15
TADG-15 is cloned and expressed in the opposite orientation such that an antisense RNA molecule (SEQ ID No. 1 8 ) is produced. For example, the antisense RNA is used to hybridize to the complementary RNA in the cell and thereby inhibit translation of TADG-15 RNA into protein.
EXAMPLE 9 Peptide ranking
For vaccine or immune stimulation, individual 9-mers to 11-mers were examined to rank the binding of individual peptides to the top 8 haplotypes in the general population (Parker et al., (1994)). The computer program used for this analyses c an be found at <http://www-bimas .dcrt.nih.gov/molbio/hla_bind/>. Table 1 shows the peptide ranking based upon the predicted half- life of each peptide's binding to a particular HLA allele. A larger half-life indicates a stronger association with that peptide and the particular HLA molecule. The TADG-15 peptides that strongly bind to an HLA allele are putative immunogens, and are used t o innoculate an individual against TADG-15.
TABLE 1
c 3
HLA Type Predicted SEQ
&. Ranking Start Peptide Dissociation^ TD No
HLA A0201
1 68 VLLGIGFLV 2537.396 19
2 126 LLYSGVPFL 1470.075 20
3 644 SLLSPNWLV 521.640 21
4 379 KVSFKFFYL 396.525 22
5 386 YLLEPGVPA 346.677 23
6 257 SLTFRSFDL 123.902 24
7 762 ILQKGEIRV 118.238 25
8 841 RLPLFRDWI 106.842 26
9 64 GLLLVLLGI 88,783 27
10 57 VLAAVLIGL 83.527 28
HLA A0205
1 67 LVLLGIGFL 142.800 29
2 379 KVSFKFFYL 100.800 30
3 126 LLYSGVPFL 71.400 31
4 88 KVFNGYMRI 36.000 32
5 670 TQWTAFLGL33.600 33
6 119 KVKDALKLL 25.200 34
7 60 AVLIGLLLV 24.000 35
8 62 UGLLLVLL 23.800 36
9 57 VLAAVLIGL 23.800 37
10 61 VUGLLLVL 23.800 38
HLAA1
1 146 FSEGSVTAY 337.500 39
2 658 YIDDRGFRY 125.000 40
3 449 SSDPCPGQF 75.000 41 4 401 YVFNGEKY 45.000 42
5 387 TIFPGVPAG 18.000 43
6 553 GSDEASCPK 15.000 44
7 97 TNENFVDAY 11.250 45
8 110 STEFVSLAS 11.250 46
9 811 SVEADGRΓF 9.000 47
10 666 YSDPTQWTA 7.500 48
HLAA24
1 709 DYDIATIFJ, 220.000 49
2 408 KYCGERSQF 200.000 50
3 754 QYGGTGALI 50.000 51
4 153 AYYWSEFSI 50.000 52
5 722 EYSSMVRPI 50.000 53
6 326 GFEATFFQL 36.000 54
7 304 TFHSSQNVL 24.000 55
8 707 TFDYDIALL 20.000 56
9 21 KYNSRHEKV 16.500 57
10 665 RYSDPTQWT 14.400 58
HLAB7
1 686 APGVQERRL 240.000 59
2 12 GPKDFGAGL 80.000 60
3 668 DPTQWTAFL80.000 61
4 461 TGRCJRKEL 60.000 62
5 59 AAVLIGLLL 36.000 63
6 379 KVSFKFFYL 20.000 64
7 119 KVKDALKLL 20.000 65
8 780 LPQQΠPRM 20.000 66
9 67 LVLLGIGFL 20.000 67
10 283 SPMEPHALV 18.000 68
HLAB8
1 12 GPKDFGAGL 24.000 69
2 257 SLTFRSFDL 8.000 70 3 1 80 MLPPRARSL 8.000 71
4 2 17 GLHARGVEL 8.000 72
5 173 MAEERWML4.800 73
6 267 SCDERGSDL 4.800 74
7 567 CTKHTYRCL 4.000 75
8 724 SSMVRPICL 4.000 76
9 409 YCGERSQFV 3.600 77
1 0 495 TCKNKFCKP 3.200 78
HLA B2702
1 427 VRFHSDQSY 1000.000 79
2 695 KRΠSHPFF 600.000 80
3 664 FRYSDPTQW 500.000 81
4 220 ARGVELMRF 200.000 82
5 492 HQFTCKNKF 100.000 83
6 5 3 GRWVVLA A V 100.000 84
7 248 LRGDADSVL 60.000 85
8 572 YRCLNGLCL 60.000 86
9 692 RRLKRΠSH 6O.000 87
1 0 24 SRHEKVNGL 60.000 88
HLA B4403
1 147 SEGSVTAYY 360.000 89
2 7 15 LELEKPAEY 360.000 90
3 105 YENSNSTEF 60.000 91
4 1 4 KDFGAGLKY 50.625 92
5 129 SGVPFLGPY 36.000 93
6 436 TDTGFLAEY 33.750 94
7 766 GEIRVINQT 30.000 95
8 402 VFJNGEKYC 30.000 96
9 482 DELNCSCDA 24.000 97
1 0 82 RDVRVQKVF22.500 98 EXAMPLE 10
TADG-15 cDNA
A screening strategy to identify proteases which are overexpressed in human cancer has been developed in which RT- PCR products amplified specifically in tumors, as compared t o normal tissue, are examined.9 During this effort, candidate genes were identified using redundant sense primers to the conserved amino acid histidine domain at the NH3 end of the catalytic domain and antisense primers to the downstream conserved amino acid serine domain. Subcloning and sequencing th e appropriate 480 base pair band(s) amplified in such a PCR reaction provides the basis for identifying the gene(s) encoding proteases(s). Among these amplified catalytic domains, a new serine protease gene named TADG-15 (tumor antigen-derived gene 15) was identified. The catalytic domain of the newly identified TADG-15 protein is similar to other serine proteases and specifically contains conserved amino acids appropriate for the catalytic domain of the trypsin-like serine protease family.
A computerized search of GenEMBL databases using the FASTA program (Wisconsin Package Version 9.1, GCG, Madison, Wisconsin) for amino acid sequences homologous t o the TADG-15 protease domain revealed that homologies with other known human proteases never exceeds 55%. Figure 1 shows the alignment of the protease domain of TADG-15 compared with other human serine proteases. Using the BESTFTT program available through GCG, the similarities between TADG-15 and trypsin, chymotrypsin, and tissue-type plasminogen activator are 51 %, 46% and 52%, respectively. From the sequence derived from the TADG- 15 catalytic domain, specific primers were synthesized to amplify a TADG-15-specific probe for library screening. After screening a n ovarian carcinoma library, one 1785 bp clone was obtained which included the 3' end of the TADG-15 transcript. Upon further screening using the 5' end of the newly detected clone, two additional clones were identified which provided another 1362 b p of the cDNA, including the 5' end of the TADG-15 transcript. The total length of the sequenced cDNA was approximately 3.15 kb . The total nucleotide sequence obtained includes a Kozak' s consensus sequence preceding a single open reading frame encoding a predicted protein of 855 amino acids (Figure 2).
The deduced open reading frame encoded by th e TADG-15 nucleotide sequence (Figures 2, 3 and 4) contains several distinct domains as follows: an amino terminal cytoplasmic tail (amino acids (aa) #1-54), a potential transmembrane domain (aa #55-77), an extracellular membrane domain (aa #78-213), two complement subcomponents Clr/Cls, Uegf, and bone morphogenetic protein 1 (CUB) repeats (aa #214- 447), four ligand binding repeats of the low density lipoprotein (LDL) receptor-like domain (aa #453-602) and a serine protease domain (aa #615-855). The TADG-15 protein also contains two potential N-linked glycosylation sites (aa #109 and 302) and a potential proteolytic cleavage site upstream from the protease domain (aa #614) which could release and/or activate the protease at the carboxy end of this protein. In addition, TADG-15 contains an RGD motif (aa #249-251 ) which is commonly found in proteins involved in cell-cell adhesion. EXAMPLE 11
TADG-15 expression
To examine the size of the transcript for TADG-15 and its pattern of expression in various tissues, Northern blot hybridization was performed for representative histological types of carcinoma and in a series of cell lines, fetal tissues and normal adult tissues (Figure 5). The transcript size for the TADG-15 message was determined to be approximately 3.2 kb and a single intense transcript appeared to be present in all of the carcinomas examined, whereas no visible band was detected in normal ovary (Figure 5). This transcript size is also in good agreement with the sequence data predicting a transcript size of 3.15 kb. The ovarian tumor cell lines, SW626 and CAOV3, also showed a n abundance of transcript, however little or no transcript was detectable in the breast carcinoma cell lines MDA-MB-231 and MDA-MB-4355. Among normal human fetal tissues, fetal kidney showed an abundance of the TADG-15 transcript and low expression was also detected in fetal lung. In normal adult tissues, TADG-15 was detected in colon with low levels o f expression in small intestine and prostate (Figure 5).
To evaluate mRNA transcript expression of TADG- 15 in ovarian tumors and normal ovary, semi-quantitative PCR (Figure 6) was performed. In a preliminary study, the linearity o f this assay1 1 12 was confirmed and its efficacy correlated with b o th Northern blots and immunohistochemistry. The data was quantified using a phosphoimager and compared as a ratio o f expression (TADG- 15/β-tubulin). Results herein indicate that TADG-15 transcript expression is elevated above the cut-off value (mean for normal ovary ± 2 SD) in all of the tumor cases examined and is either not detected or detected at extremely low levels in normal ovaries (Figure 6A and B). Analysis of ovarian carcinoma subtypes, including early stage and late stage disease, confirms overexpression of TADG-15 in all carcinomas examined (Table 2). All of the carcinomas studied, which included 5 stage I and 3 stage II carcinomas, showed overexpression of the TADG-15 gene.
These data can also be examined with regard to tumor stage and histological sub-type, and results indicated that every carcinoma of every stage and histological sub-type overexpressed the TADG-15 gene. The expression ratio (mean value ± SD) for normal ovary group was determined as 0.182 ± 0.024, for LMP tumor group as 0.847 ± 0.419 and for carcinoma group as 0.771 ± 0.380 (Table 2). A comparison between the normal ovary group and tumor groups showed that overexpression of th e TADG-15 gene is statistically significant in both the LMP tumor group and the carcinoma group (LMP tumor: p<0.001 , carcinoma: p<0.0001 ) .
As shown in Figure 6, TADG-15 transcripts were noted in all ovarian carcinomas, but were not present at detectable levels in any of the following tissues: a) normal ovary, b) fetal liver and brain, c) adult spleen, thymus, testes, ovary and peripheral blood lymphocytes, d) skeletal muscle, liver, brain o r heart. This evaluation was extended to a standard panel of about 40 tumors. Using TADG-15-specific primers, the expression was also examined in tumor cell lines derived from both ovarian and breast carcinoma tissues as shown in Figure 7 and in other tumor tissues as shown in Figure 8. Expression of TADG-15 was also observed in carcinomas of the breast, colon, prostate and lung. Polyclonal antibodies developed to a synthetic peptide (a 12-mer) at the carboxy terminus of the protease domain were used to examine TADG-15 expression in cell lines by Western blot and by immunolocalization in normal ovary and ovarian tumors . Western blots of cell extracts from SW626 and CAOV3 cells were probed with both antibody and preimmune sera (Figure 9 ) . Several bands were detected with the antibody, including bands of approximately 100,000 daltons, approximately 60,000 daltons and 32,000 daltons. The anticipated molecular size of th e complete TADG-15 molecule is estimated to be approximately 100,000 daltons, and the protease domain which may be released by proteolytic cleavage at aa #614 is estimated to b e approximately 32,000 daltons. Some intermediate proteolytic product may be represented by the 60,000 dalton band. Antibody staining of tumor cells confirms the presence of the TADG-15 protease in the cytoplasm of a serous LMP tumor, mucinous LMP tumor and serous carcinoma (Figure 10B, C & D, respectively). This diffuse staining pattern may b e due to detection of TADG 15 within the cell as it is being packaged and transported to the cell surface. In endometrioid carcinoma, the antigen is clearly detectable on the surface of tumor cells (Figure 10E). No staining was detected in normal ovarian epithelium or stromal cells (Figure 10A). Immunohistochemical staining of a series of 27 tumors indicates the presence of the TADG-15 protein in all the carcinoma subtypes examined, including the low malignant potential group. Strong staining was noted in 7 of 9 low malignant potential tumors and 13 of 1 8 carcinomas (Table 3). TABLE 2
Number of cases with overexpression of TADG-15 in normal ovaries and ovarian tumors
N of TADG-15
Normal 10 0 (0%) 0.182 ± 0.024
LMP 10 10 (100%) 0.847 ± 0.419 serous 6 6 (100%) 0.862 ± 0.419 mucinous 4 4 (100%) 0.825 ± 0.483
Carcinoma 31 31 (100%) 0.771 ± 0.380 serous 18 18 (100%) 0.779 ± 0.332 mucinous 7 7 (100%) 0.907 ± 0.584 endometrioid 3 3 (100%) 0.502 + 0.083 clear cell 3 3 ΠOO% 0.672 + 0.077
"The ratio of expression level of TADG-15 to β-tubulin (mean ± SD)
TABLE 3
Tmmnnohistochemical staining using TADG-15
Lah No. Histology TADG-1 Surface epithelium of the ovary -
H-3194 serous (LMP) ++
H-162 serous (LMP) ++
H-1182 serous (LMP) ++
H-4818 serous (LMP) ++ H-4881 serous (LMP) ++
H-675 mucinous (LMP) +
H-2446 mucinous (LMP) +
H-0707 mucinous (LMP) ++
H-2042 mucinous (LMP) ++ H-2555 serous carcinoma ++
H-1858 serous carcinoma ++
H-5266 serous carcinoma ++
H-5316 serous carcinoma +
H-2597 serous carcinoma + H-4931 mucinous carcinoma ++
H-1867 mucinous carcinoma ++
H-5998 mucinous carcinoma ++
H-2679 endometrioid adenocarcinoma +
H-5718 endometrioid adenocarcinoma ++ H-3993 endometrioid adenocarcinoma +
H-2991 endometrioid adenocarcinoma ++
H-2489 endometrioid adenocarcinoma ++
H-5994 clear cell carcinoma ++
H-6718 clear cell carcinoma ++ H- 1661 clear cell carcinoma ++
H-6201 clear cell carcinoma ++ H-5640 clear cell carcinoma ±
- Negative; + Weak Positive; ++ Strong Positive (more than 50 % of cell staining)
EXAMPLE 12 TADG-1 5 homology
Recently, a mouse protein named epithin (GenBank Accession No. AF042822) has been described.14 Epithin is a 902 amino acid protein which contains a similar structure to TADG-15 in that it has a cytoplasmic domain, transmembrane domain, two CUB domains, four LDLR-like domains and a carboxy terminal serine protease domain. TADG-15 and epithin are 84% similar over 843 amino acids, suggesting that the proteins may b e orthologous (Figure 11). The precise role of epithin remains t o be elucidated. A search of GeneBank for similar previously identified sequences yielded one such sequence with relatively high homology to a portion of the TADG-15 gene. The similarity between the portion of TADG-15 from nucleotide #182 to 3 1 39 and SNC-19 GeneBank Accession No. #U20428) is approximately 97% (Figure 12). There are however significant differences between SNC-19 and TADG-15. For example, TADG-15 has a n open reading frame of 855 amino acids whereas the longest open reading frame of SNC-19 is 173 amino acids. Additionally, SNC- 19 does not include a proper start site for the initiation o f translation, nor does it include the amino terminal portion of th e protein encoded by TADG-15. Moreover, SNC-19 does n o t include an open reading frame for a functional serine protease because the His, Asp and Ser residues of the catalytic triad that are necessary for function are encoded in different reading frames .
Impl i cati on s
The overall structure of the TADG-15 protein is relatively similar to the members of the tolloid/BMP-1 family and the complement subcomponents, Clr/Cls. These proteins contain both CUB and protease domains, and complex formation through the ligand binding domain is essential for their function. Activation of the serine protease domains of Clr and Cls requires proteolytic cleavage of Arg-Gly and Arg-Ile bonds, respectively.1 5 Similarly, it might be expected that the TADG-15 protein is synthesized as a zymogen, which is activated by cleavage between Arg614 and Val615 and analogous to the activation mechanism of other serine protease zymogens. Western blot analysis o f cultured cell lysates confirmed both a 100 kDa and 32 kDa peptide, which correspond to the putative zymogen (whole molecule) and a cleaved protease product of TADG-15 (Figure 9 ) . These data support a model for proteolytic release an d/o r activation of TADG-15 as occurs for similar type II serine proteases .
CUB domains were first found in complement subcomponents C l r/C l s 16 18 and are known to be a widespread module in developmentally regulated proteins, such as the b one morphogenetic protein-1 (BMP-1) and the tolloid gene product.18- 20 The role of these repeats remains largely unknown. However, some models suggest that the CUB domain may be involved in protein-protein interactions. The CUB domain of Clr and Cls participates in the assembly of the Cls-Clr-Clr-Cls tetrameric complex in the activation of the classical pathway of complement by providing protein-protein interaction domains .15 The
Drosophila decapentaplegic (DPP) protein is essential for dorsal- ventral specification of the embryo, and the Drosophila tolloid (TLD) forms a complex with DPP to regulate its activity.19 20 Missense mutations in the CUB domain of the tolloid protein results in a phenotype that does not allow a protein interaction with the DPP complex.19
The TADG-15 protein contains two tandem repeats o f CUB-like domains between amino acid residues 214 and 447. Each of these is approximately 110 amino acids long and each has four conserved cysteine residues characteristic of other CUBs (amino acids 214, 244, 268, 294, 340, 366, 397, 410). By analogy, the CUB repeats of the TADG-15 protein may form a n interactive domain capable of promoting multimeric complex formation and regulating the activity of the target protein o r TADG- 15 itself.
The TADG-15 protein also contains the LDL receptor ligand binding repeat (class A motif) -like domain, which consists of four contiguous cysteine-rich repeats (amino acid residues 453 to 602). Each cysteine-rich repeat is approximately 40 amino acids long and contains a conserved, negatively-charged sequence (Ser-Asp-Glu) with six cysteine residues. In the LDL receptor protein, this repeat is thought to function as a protein-binding domain which interacts with the lysine and arginine residues present in lipoproteins.21 ,22 In addition, the first repeat of the LDL receptor appears to bind Ca2+ and not the lipoproteins .23 By analogy, it is possible that the LDL receptor-like repeat in TADG- 15 may act in a similar fashion, interacting with positively charged regions of other proteins and/or as a Ca2+ binding site. As a result of ligand binding and the formation of receptor-ligand complex, LDL receptor is internalized via clathrin-coated pits .24 These types of plasma membrane receptors contain a characteristic amino acid sequence in their cytoplasmic domain for binding to clathrin-coated pits.24 TADG-15 does not contain this motif in its cytosolic region, and furthermore, no similarities with other known protein sequences were found in the cytoplasmic domain of the TADG-15. This finding suggests th at TADG-15 functions in a different manner from the endocytic receptors (such as the LDL receptor), although TADG-15 possesses similar ligand-binding repeats in the extracellular matrix .
Although the precise role of TADG-15 is unknown, this gene is clearly overexpressed in ovarian tumors. A variety o f proteases, such as type IV collagenase and plasminogen activator, appear to be involved in the process of tumor invasion and are constituents of a protease cascade in malignant progression. TADG-15 may constitute such an activity and directly digest extracellular matrix components surrounding a tumor, or activate other proteases by cleavage of inactive precursors, indirectly enhancing tumor growth and invasion. It is also possible that TADG-15 may function like a member of the tolloid/BMP-1 family by forming complexes with other growth factors or signal transduction proteins to modulate their activities. These data raise the possibility that the TADG-15 gene and its translated protein will be a useful marker for the early detection of ovarian carcinoma through release of the protease domain into the extracellular matrix and ultimately the circulation. These data also suggest the possibility of using TADG-15 as a target for therapeutic intervention through delivery systems directed at the CUB/LDLR ligand binding domains.
The following references were cited herein:
I . Liotta, L.A., et al. Cell, 64: 327-336, 1991. 2 . Duffy, M.J. Clin. Exp. Metastasis, 10: 145-155, 1992.
3 . Tryggvason, K, et al. Biochem. Biophys. Acta., 907: 19 1 - 217, 1987.
4. Levy, A.T., et al. Cancer Res., 57 : 439-444, 1991.
5 . Monsky, W.L. et al. Cancer Res., 53 : 3159-3164, 1993. 6 . Duffy, M.J. et al. Cancer, 62 : 531-533, 1988.
7 . Hackel, C, et al. Cancer, 79: 53-58, 1997.
8 . Watt, et al. Proc.Natl. Acad. Sci.U.S.A., 83:3166-3170, 1986.
9. Tanimoto, H. et al. Cancer Res., 57: 2884-2887, 1997.
I I . Shigemasa, K. et al. J. Soc. Gynecol. Invest., 4 : 95 - 102, 1997.
1 2. Tanimoto, H. et al. Gynecol. Oncol., 66: 308-312, 1997.
1 3 . Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular Cloning, p. 309-361 Cold Spring Harbor Laboratory, New York, 1982.
14. Kim, M.G., et al. Immunogenetics, 49(5): 420-428, 1999. 15 . Arlaud et al. Method in Enzymology, 223 : 61-82, 1993.
1 6. Journet, A. & Tosi, M. Biochem. J., 240: 783-787, 1986.
1 7. Mackinnon, et al. Eur. J. Biochem., 169: 547-553, 1987.
1 8. Bork, P. & Beckmann, G. J. Mol. Biol., 231 : 539-545, 1993.
19. Childs, & O'Connor, M.B. Dev. Biol., 162: 209-220, 1994. 20. Blader, PL, et al. Science, 278: 1937-1940, 1997.
2 1 . Yamamoto, T. et al. Cell, 39: 27-38, 1984.
22. Daly, N.L., et al. Proc. Natl. Acad. Sci., 92 : 6334-6338, 1995.
23. van Driel, I.R., et al. J. Biol. Chem., 262 : 17443 - 17449, 1987.
24. Lodish, H. et al. Sorting of membrane proteins internalized from the cell surface. In: Molecular Cell Biology, 3rd ed., p .722- 733 Scientific American Books, Inc., New York, 1995.
25 . Parker, KC et al. J. Immunol. 152: 163, 1994. Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods , procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompas sed within the spirit of the invention as defined by the scope of the claims.

Claims

WHAT IS CLAIMED IS:
1 . DNA encoding a tumor antigen-derived gene (TADG-15) protein, selected from the group consisting of: ( a ) isolated DNA which encodes a TADG-15 protein;
( b ) isolated DNA which hybridizes under high stringency conditions to the isolated DNA of (a) above and which encodes a TADG- 15 protein; and
( c ) isolated DNA differing from the isolated DNAs o f (a) and (b) above in codon sequence due to the degeneracy o f the genetic code, and which encodes a TADG-15 protein.
2. The DNA of claim 1, wherein said DNA has the sequence shown in SEQ ID No. 1.
3 . The DNA of claim 1, wherein said TADG-15 protein has the amino acid sequence shown in SEQ ID No. 2.
4. A vector comprising the DNA of claim 1 and regulatory elements necessary for expression of said DNA in a cell.
5 . The vector of claim 4, wherein said DNA encodes a TADG-15 protein having the amino acid sequence shown in SEQ ID No. 2.
6. The vector of claim 4, wherein said DNA is positioned in reverse orientation relative to said regulatory elements such that TADG-15 antisense mRNA is produced.
7 . A host cell transfected with the vector of claim 4, said vector expressing a TADG-15 protein.
8 . The host cell of claim 7, wherein said cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells and insect cells.
9 . The host cell of claim 8, wherein said bacterial cell is E. coli.
1 0. Isolated and purified TADG-15 protein coded for by DNA selected from the group consisting of:
( a ) isolated DNA which encodes a TADG-15 protein;
( b ) isolated DNA which hybridizes under high stringency conditions to isolated DNA of (a) above and which encodes a TADG-15 protein; and
( c ) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy o f the genetic code, and which encodes a TADG-15 protein.
1 1 . The TADG-15 protein of claim 10, wherein said protein has the amino acid sequence shown in SEQ ID No. 2.
1 2. A method for detecting TADG-15 mRNA in a sample, comprising the steps of:
( a ) contacting a sample with a probe, wherein said probe is specific for TADG-15; and
( b ) detecting binding of said probe to TADG-15 mRNA in said sample.
1 3 . The method of claim 12, wherein said sample is a biological sample.
1 4. The method of claim 13, wherein said biological sample is from an individual.
15. The method of claim 14, wherein said individual is suspected of having cancer.
1 6. A kit for detecting TADG-15 mRNA, comprising: an oligonucleotide probe, wherein said probe is specific for TADG-15.
17. The kit of claim 16, further comprising: a label with which to label said probe; and means for detecting said label.
1 8. A method of detecting TADG-15 protein in a sample, comprising the steps of:
( a ) contacting a sample with an antibody, wherein said antibody is specific for TADG-15 or a fragment thereof; and
( b ) detecting binding of said antibody to TADG- 15 protein in said sample.
1 9. The method of claim 18, wherein said sample is a biological sample.
20. The method of claim 19, wherein said biological sample is from an individual.
2 1 . The method of claim 20, wherein said individual is suspected of having cancer.
22. A kit for detecting TADG-15 protein, comprising: an antibody, wherein said antibody is specific for TADG-15 protein or a fragment thereof.
23. The kit of claim 22, further comprising: means to detect said antibody.
24. An antibody, wherein said antibody is specific for TADG-15 protein or a fragment thereof.
25. A method of screening for compounds th at inhibit TADG-15, comprising the steps of: ( a ) contacting a sample with a compound, wherein said sample comprises TADG-15 protein; and
( b ) assaying for TADG-15 protease activity, wherein a decrease in said TADG-15 protease activity in the presence of said compound relative to TADG-15 protease activity in th e absence of said compound is indicative of a compound that inhibits TADG-15.
26. A method of inhibiting expression of TADG-15 in a cell, comprising the step of introducing the vector of claim 6 into a cell, wherein expression of said vector produces TADG- 15 antisense mRNA in said cell, wherein said TADG-15 antisense mRNA hybridizes to endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 in said cell.
27. A method of inhibiting a TADG-15 protein in a cell, comprising the step of introducing an antibody into a cell, wherein said antibody is specific for a TADG-15 protein or a fragment thereof, wherein binding of said antibody to said TADG- 15 protein inhibits said TADG-15 protein.
28. A method of targeted therapy to an individual, comprising the step of:
( a ) administering a compound to an individual, wherein said compound has a targeting moiety and a therapeutic moiety, wherein said targeting moiety is specific for TADG-15.
29. The method of claim 28, wherein said targeting moiety is selected from the group consisting of an antibody specific for TADG-15 and a ligand or ligand binding domain that binds TADG-15.
30. The method of claim 28, wherein said therapeutic moiety is selected from the group consisting of a radioisotope, a toxin, a chemotherapeutic agent, an immune stimulant and a cytotoxic agent.
3 1 . The method of claim 28, wherein said individual suffers from ovarian cancer, lung cancer, prostate cancer, colon cancer and other cancers in which TADG-15 is overexpressed.
32. A method of diagnosing cancer in an individual, comprising the steps of:
( a ) obtaining a biological sample from an individual;
( b ) detecting TADG-15 in said sample, wherein the presence of TADG-15 in said sample is indicative of the presence of carcinoma in said individual, wherein the absence of TADG-15 in said sample is indicative of the absence of carcinoma in said individual.
33 . The method of claim 32, wherein said biological sample is selected from the group consisting of blood, urine, saliva, tears, interstitial fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells.
34. The method of claim 32, wherein said detection of said TADG-15 is by means selected from the group consisting of Northern blot, Western blot, PCR, dot blot, ELIZA sandwich assay, radioimmunoassay, DNA array chips and flow cytometry.
35 . The method of claim 32, wherein said carcinoma is selected from the group consisting of ovarian, breast, lung, colon, prostate and others in which TADG-15 is overexpressed.
36. A method of vaccinating an individual against TADG-15, comprising the steps of: inoculating an individual with a TADG-15 protein o r fragment thereof, wherein said TADG-15 protein or fragment thereof lacks TADG-15 protease activity, wherein said inoculation with said TADG-15 protein or fragment thereof elicits an immune response in said individual, thereby vaccinating said individual against TADG-15.
37. The method of claim 36, wherein said individual has cancer, is suspected of having cancer or is at risk of getting cancer.
38. The method of claim 36, wherein said TADG-15 fragment is selected from the group consisting of a 9-residue fragment up to a 20-residue fragment.
39. The method of claim 38, wherein said 9-residue fragment is selected from the group consisting of SEQ ID Nos. 2 , 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.
40. A method of producing immune-activated cells directed toward TADG-15, comprising the steps of: exposing dendritic cells to a TADG-15 protein o r fragment thereof, wherein said TADG-15 protein or fragment thereof lacks TADG-15 protease activity, wherein said exposure to said TADG-15 protein or fragment thereof activates said dendritic cells, thereby producing immune-activated cells directed toward TADG-15.
41 . The method of claim 40, wherein said immune- activated cells are selected from the group consisting of B-cells, T-cells and dendrites.
42. The method of claim 40, wherein said TADG-15 fragment is selected from the group consisting of a 9-residue fragment up to a 20-residue fragment.
43 . The method of claim 42, wherein said 9-residue fragment is selected from the group consisting of SEQ ID Nos. 2 , 19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.
44. The method of claim 40, wherein said dendritic cells are isolated from an individual prior to said exposure, wherein said activated dendritic cells are reintroduced into said individual subsequent to said exposure.
45 . The method of claim 44, wherein said individual has cancer, is suspected of having cancer or is at risk of getting cancer.
46. An immunogenic composition, comprising a n immunogenic fragment of a TADG-15 protein and an appropriate adjuvant.
47. The immunogenic composition of claim 46 , wherein said fragment is selected from the group consisting of a 9-residue fragment up to a 20-residue fragment.
48. The immunogenic composition of claim 47 , wherein said 9-residue fragment is selected from the group consisting of SEQ ID Nos. 2, 19, 20, 21, 29, 39, 49, 50, 59, 79, 80 , 81 , 82, 83, 84, 89 and 90.
49. An oligonucleotide having the nucleotide sequence complementary to a sequence of claim 1.
50. A composition comprising the oligonucleotide according to claim 49 and a physiologically acceptable carrier therefore .
5 1 . A method of treating a neoplastic state in a n individual syndrome in an individual in need of such treatment, comprising the step of administering to said individual a n effective dose of the oligonucleotide of claim 49.
52. The method of claim 51 , wherein said neoplastic state is selected from the group consisting of ovarian cancer, breast cancer, lung cancer, colon cancer, prostate cancer and other cancers in which TADG-15 is overexpressed.
EP00973739A 1999-10-20 2000-10-20 Tadg-15: an extracellular serine protease overexpressed in carcinomas Withdrawn EP1226150A4 (en)

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