WO2002006454A2 - Regulation d'une enzyme humaine de type carboxylesterase - Google Patents

Regulation d'une enzyme humaine de type carboxylesterase Download PDF

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WO2002006454A2
WO2002006454A2 PCT/EP2001/007919 EP0107919W WO0206454A2 WO 2002006454 A2 WO2002006454 A2 WO 2002006454A2 EP 0107919 W EP0107919 W EP 0107919W WO 0206454 A2 WO0206454 A2 WO 0206454A2
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carboxylesterase
enzyme
polypeptide
polynucleotide
seq
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PCT/EP2001/007919
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WO2002006454A3 (fr
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Yonghong Xiao
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Bayer Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the area of regulation of human carboxylesterase-like enzyme to provide therapeutic effects.
  • organophosphoms compounds in pesticides, as well as their application in chemical weapons, has resulted in increased exposure to these toxic compounds. Exposure to organophosphoms compounds typically occurs tlirough inhalation of such compounds that have been dispersed in aerosols. Organophosphoms compounds primarily exert their toxicity through the inhibition of acetylcholinesterase, which in turn causes an excess of acetylcholine at nerve cholinergic receptors, thereby inhibiting synaptic nerve transmission (Wallace et ah, Am. J. Respir. Cell Mol. Biol. 20, 1201-08, 1999). The toxic effects of organophosphoms compounds have been evidenced in brain, plasma, lung and liver, and if untreated, culminate in respiratory failure.
  • organophosphoms compounds The detoxification of organophosphoms compounds occurs in large part through the irreversible binding of such compounds by carboxylesterases (Wallace et ah, supra). By binding organophosphoms compounds, carboxylesterases reduce or eliminate the amount of such compounds that are available to inhibit acetylcholinesterase. Carboxylesterases can therefore potentially be administered after exposure to organophosphoms compounds as a therapeutic, or prior to anticipated exposure as a prophylactic. Compounds that increase the ability of carboxylesterases to act as scavengers of organophosphoms compounds may prove useful as detoxifying agents. Similarly, compounds that effectively increase the levels of carboxylesterases, such as by increasing its expression, may prove useful as detoxifying agents.
  • Such compounds need not only be used as therapeutic agents following exposure to organophosphoms compounds, but also have the potential to serve as prophylatics in the instances where there is a likelihood of exposure to organophosphoms compounds.
  • compounds or agents that can effectively increase the ability of carboxylesterases to detoxify organophosphoms compounds will prove useful in many applications.
  • One embodiment of the invention is a carboxylesterase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO: 5.
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a carboxylesterase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO: 5.
  • a test compound which binds to the carboxylesterase-like enzyme polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the activity of the carboxylesterase-like enzyme.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a carboxylesterase-like enzyme polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; and the nucleotide sequence shown in SEQ ID NO: 4.
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the carboxylesterase-like enzyme through interacting with the carboxylesterase-like enzyme mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a carboxylesterase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO: 5.
  • a carboxylesterase-like enzyme activity of the polypeptide is detected.
  • a test compound which increases carboxylesterase-like enzyme activity of the polypeptide relative to carboxylesterase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases carboxylesterase-like enzyme activity of the polypeptide relative to carboxylesterase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a carboxylesterase-like enzyme product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; and the nucleotide sequence shown in SEQ ID NO: 4.
  • Binding of the test compound to the carboxylesterase-like enzyme product is detected.
  • a test compound which binds to the carboxylesterase-like enzyme product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a carboxylesterase-like enzyme polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; and the nucleotide sequence shown in SEQ ID NO: 4.
  • Carboxylesterase-like enzyme activity in the cell is thereby decreased.
  • the invention thus provides a human carboxylesterase-like enzyme which can be used to identify test compounds which may act, for example, as agonists or antagonists at the enzyme's active site.
  • Human carboxylesterase-like enzyme and fragments thereof also are useful in raising specific antibodies which can block the enzyme and effectively reduce its activity.
  • Fig. 1 shows the DNA-sequence encoding a carboxylesterase-like enzyme polypeptide (SEQ ID NO:l).
  • Fig. 2 shows the DNA-sequence encoding a carboxylesterase-like enzyme polypeptide (SEQ ID NO:2).
  • Fig. 3 shows the DNA-sequence encoding a carboxylesterase-like enzyme polypeptide (SEQ ID NO:3).
  • Fig. 4 shows the DNA-sequence encoding a carboxylesterase-like enzyme polypeptide (SEQ ID NO:4).
  • Fig. 5 shows the amino acid sequence deduced from the DNA-sequence ofFig. 4 (SEQ ID NO:5).
  • Fig. 5 shows the amino acid sequence deduced from the DNA-sequence ofFig. 4 (SEQ ID NO:5).
  • FIG. 6 shows the DNA-sequence encoding a carboxylesterase-like enzyme polypeptide (SEQ ID NO:6).
  • Fig. 7 shows the DNA-sequence encoding a carboxylesterase-like enzyme polypeptide (SEQ ID NO:7).
  • Fig. 8 shows the amino acid sequence of the protein identified with
  • Fig. 9 shows the BLASTX alignment of carboxylesterase (SEQ ID NO:5) with the protein identified with EMBL Accession No. AB010632 (SEQ ID NO: 8).
  • Fig. lO shows the GENSCANW output for SEQ ID NO:5.
  • Fig. 11 shows the BLASTP alignment of SEQ ID NO:5 with SEQ ID NO:8.
  • Fig. 12 shows the BLOCKS search results.
  • the invention relates to an isolated polynucleotide encoding a heparanase-like enzyme polypeptide and being selected from the group consisting of: a. a polynucleotide encoding a heparanase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO: 5.
  • Human carboxylesterase-like enzyme comprises the amino acid sequence shown in SEQ ID NO:4, as encoded by the coding sequence shown in SEQ ID NO: 5.
  • a number of ESTs (SEQ ID NOS:l-3) are contained within the coding sequence of human carboxylesterase-like enzyme, indicating that SEQ ID NO:5 is expressed.
  • a BLASTX alignment shows that human carboxylesterase-like enzyme is 43% identical over an 81 amino acid overlap and 41% identical over a 46 amino acid overlap to the rat protein identified by EMBL Accession No. ABO 10632 (SEQ ID NO: 8) and annotated as carboxylesterase precursor (FIG. 1).
  • BLASTP alignment of human carboxylesterase-like enzyme with the rat protein shows that the two proteins are 43% identical over 293 amino acids (FIG. 3).
  • human carboxylesterase-like enzyme of the invention is expected to be useful for the same purposes as previously identified carboxylesterases.
  • human carboxylesterase-like enzyme can be used in therapeutic methods to treat disorders such as organophosphoms intoxication, cancer, and osteoporosis.
  • Human carboxylesterase-like enzyme also can be used to screen for human carboxylesterase-like enzyme agonists and antagonists.
  • Carboxylesterase-like enzyme polypeptides according to the invention comprise at least 11, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 5 or a biologically active variant thereof, as defined below.
  • a carboxylesterase-like enzyme polypeptide of the invention therefore can be a portion of a carboxylesterase-like enzyme protein, a full-length carboxylesterase-like enzyme protein, or a fusion protein comprising all or a portion of a carboxylesterase-like enzyme protein.
  • Carboxylesterase-like enzyme polypeptide variants which are biologically active, i.e., retain an carboxylesterase-like activity also are carboxylesterase-like enzyme polypeptides.
  • naturally or non-naturally occurring carboxylesterase-like enzyme polypeptide variants have amino acid sequences which are at least about 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, or 98% identical to the amino acid sequence shown in SEQ ID NO: 5 or a fragment thereof. Percent identity between a putative carboxylesterase-like enzyme polypeptide variant and an amino acid sequence of SEQ ID NO: 5 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar stmctural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a carboxylesterase-like enzyme polypeptide can be found using computer programs well known in the art, such as D ⁇ ASTAR software. Whether an amino acid change results in a biologically active carboxylesterase-like enzyme polypeptide can readily be determined by assaying for carboxylesterase-like activity, as described for example, in the specific Examples, below.
  • Fusion proteins are useful for generating antibodies against carboxylesterase-like enzyme polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a carboxylesterase-like enzyme polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drag screens.
  • a carboxylesterase-like enzyme polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises at least 11, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 contiguous amino acids of SEQ ID ⁇ O:5 or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full-length carboxylesterase-like enzyme protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include -galactosidase, -glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constmctions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, NSN-G tags, and thioredoxin (Trx) tags.
  • fusion constmctions can include maltose binding protein (MBP), S-tag, Lex a D ⁇ A binding domain (DBD) fusions, GAL4 D ⁇ A binding domain fusions, and herpes simplex vims (HSN) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 D ⁇ A binding domain fusions GAL4 D ⁇ A binding domain fusions
  • HSN herpes simplex vims
  • a fusion protein also can be engineered to contain a cleavage site located between the carboxylesterase-like enzyme polypeptide-encoding sequence and the heterologous protein sequence, so that the carboxylesterase-like enzyme polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant D ⁇ A methods can be used to prepare fusion proteins, for example, by making a D ⁇ A construct which comprises coding sequences selected from SEQ ID ⁇ O:4 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • kits for constmcting fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cmz Biotechnology (Santa Cmz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS). Identification of Species Homologs
  • Species homologs of human carboxylesterase-like enzyme polypeptide can be obtained using carboxylesterase-like enzyme polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of carboxylesterase-like enzyme polypeptide, and expressing the cDNAs as is known in the art.
  • a carboxylesterase-like enzyme polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a carboxylesterase-like enzyme polypeptide.
  • a coding sequence for human carboxylesterase-like enzyme is shown in SEQ ID NO:4.
  • nucleotide sequences encoding human carboxylesterase-like enzyme polypeptides as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence shown in SEQ ID NO:4 also are carboxylesterase-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • cDNA Complementary DNA molecules, species homologs, and variants of carboxylesterase-like enzyme polynucleotides which encode biologically active carboxylesterase-like enzyme polypeptides also are carboxylesterase-like enzyme polynucleotides.
  • Variants and homologs of the carboxylesterase-like enzyme polynucleotides described above also are carboxylesterase-like enzyme polynucleotides.
  • homologous carboxylesterase-like enzyme polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known carboxylesterase-like enzyme polynucleotides under stringent conditions, as is known in the art.
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5- 15% basepair mismatches.
  • Species homologs of the carboxylesterase-like enzyme polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of carboxylesterase-like enzyme polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et ah, J. Mol. Biol. 81, 123 (1973).
  • Variants of human carboxylesterase-like enzyme polynucleotides or carboxylesterase-like enzyme polynucleotides of other species can therefore be identified by hybridizing a putative homologous carboxylesterase-like enzyme polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:4 or the complement thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to carboxylesterase-like enzyme polynucleotides or their complements following stringent hybridization and/or wash conditions also are carboxylesterase-like enzyme polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et ah, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m of a hybrid between a carboxylesterase-like enzyme polynucleotide having a nucleotide sequence shown in SEQ ID NO:4 or the complement thereof and a polynucleotide sequence which is at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C.
  • Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.
  • a carboxylesterase-like enzyme polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated carboxylesterase-like enzyme polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises carboxylesterase-like enzyme nucleotide sequences.
  • Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • Carboxylesterase-like enzyme cDNA molecules can be made with standard molecular biology techniques, using carboxylesterase-like enzyme mRNA as a template. Carboxylesterase-like enzyme cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et a (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • restriction site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et ah, Nucleic Acids Res. 16, 8186, 1988).
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 2230 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 °C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et ah, PCR Methods Applic. 1, 111119, 1991).
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries can be useful for extension of sequence into 5' nontranscribed regulatory regions. Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • Carboxylesterase-like enzyme polypeptides can be obtained, for example, by purification from human cells, by expression of carboxylesterase-like enzyme polynucleotides, or by direct chemical synthesis.
  • Carboxylesterase-like enzyme polypeptides can be purified from any cell which expresses the enzyme, including host cells which have been transfected with carboxylesterase-like enzyme expression constmcts. Fetal lung and testis provide useful sources of carboxylesterase-like enzyme polypeptides.
  • a purified carboxylesterase-like enzyme polypeptide is separated from other compounds which normally associate with the carboxylesterase-like enzyme polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a preparation of purified carboxylesterase-like enzyme polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding carboxylesterase-like enzyme polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a carboxylesterase-like enzyme polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with vims expression vectors (e.g., baculovims), plant cell systems transformed with vims expression vectors (e.g., cauliflower mosaic vims, CaMV; tobacco mosaic vims, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with vims expression vectors (e.g., baculovims)
  • control elements or regulatory sequences are those nontranslated regions of the vector enhancers, promoters, 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJoUa, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovims polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant vimses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian vimses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a carboxylesterase-like enzyme polypeptide, vectors based on SN40 or EBN can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for the carboxylesterase-like enzyme polypeptide. For example, when a large quantity of a carboxylesterase-like enzyme polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • a sequence encoding the carboxylesterase-like enzyme polypeptide can be ligated into the vector in frame with sequences for the amino terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced.
  • pI ⁇ vectors Na Heeke & Schuster, J. Biol. Chem. 264, 55035509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • sequences encoding carboxylesterase-like enzyme polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et ah, EMBO J. 3, 16711680, 1984; Broglie et ah, Science 224, 838843, 1984; Winter et ah, Results Probl. Cell Differ. 17, 85105, 1991).
  • constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • pathogen-mediated transfection Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191196, 1992).
  • An insect system also can be used to express a carboxylesterase-like enzyme polypeptide.
  • a carboxylesterase-like enzyme polypeptide for example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in T ⁇ choplusia larvae. Sequences encoding carboxylesterase-like enzyme polypeptides can be cloned into a nonessential region of the vims, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of carboxylesterase-like enzyme polypeptides will render the polyhedrin gene inactive and produce recombinant vims lacking coat protein.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the recombinant vimses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which carboxylesterase-like enzyme polypeptides can be expressed (Engelhard et ah, Proc. Nat. Acad. Sci. 91, 32243227, 1994).
  • a number of viral-based expression systems can be used to express carboxylesterase-like enzyme polypeptides in mammalian host cells.
  • sequences encoding carboxylesterase-like enzyme polypeptides can be ligated into an adenovims transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a carboxylesterase-like enzyme polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 36553659, 1984).
  • transcription enhancers such as the Rous sarcoma vims (RS V) enhancer, can be used to increase expression in mammalian host cells.
  • RS V Rous sarcoma vims
  • HACs Human artificial chromosomes
  • 6M to 10M are constracted and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding carboxylesterase-like enzyme polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a carboxylesterase-like enzyme polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et ah, Results Probl. Cell Differ. 20, 125162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed carboxylesterase-like enzyme polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Posttranslational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express carboxylesterase-like enzyme polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 12 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced carboxylesterase-like enzyme sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986. Any number of selection systems can be used to recover transformed cell lines.
  • herpes simplex virus thymidine kinase (Wigler et ah, Cell 11, 22332, 1977) and adenine phosphoribosyltransferase (Lowy et ah, Cell 22, 81723, 1980) genes which can be employed in tk " or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methofrexate (Wigler et ah, Proc. Natl. Acad. Sci.
  • npt confers resistance to the aminoglycosides, neomycin and G418 (Colbere-Garapin et ah, J. Mol. Biol. 150, 114, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 804751, 1988).
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et ah, Methods Mol. Biol. 55, 121131, 1995).
  • marker gene expression suggests that the carboxylesterase-like enzyme polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a carboxylesterase-like enzyme polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode a carboxylesterase-like enzyme polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a carboxylesterase-like enzyme polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the carboxylesterase-like enzyme polynucleotide.
  • host cells which contain a carboxylesterase-like enzyme polynucleotide and which express a carboxylesterase-like enzyme polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding a carboxylesterase-like enzyme polypeptide can be detected by DNA-DNA or DNA- RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a carboxylesterase-like enzyme polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a carboxylesterase-like enzyme polypeptide to detect transformants which contain a carboxylesterase-like enzyme polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a carboxylesterase-like enzyme polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a carboxylesterase-like enzyme polypeptide can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding carboxylesterase-like enzyme polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a carboxylesterase-like enzyme polypeptide can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a carboxylesterase-like polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode carboxylesterase-like enzyme polypeptides can be designed to contain signal sequences which direct secretion of soluble carboxylesterase-like enzyme polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound carboxylesterase-like enzyme polypeptide.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Imrnunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the carboxylesterase-like enzyme polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a carboxylesterase-like enzyme polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et ah, Prot. Exp.
  • enterokinase cleavage site provides a means for purifying the carboxylesterase-like enzyme polypeptide from the fusion protein.
  • Vectors which contain fusion proteins are disclosed in Kroll et ah, DNA Cell Biol. 12, 441453, 1993.
  • Sequences encoding a carboxylesterase-like enzyme polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Carathers et ah, Nucl. Acids Res. Symp. Ser. 215223, 1980; Horn et a Nucl. Acids Res. Symp. Ser. 225232, 1980).
  • a carboxylesterase-like enzyme polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc. 85, 21492154, 1963; Roberge et ah, Science 269, 202204, 1995).
  • Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of carboxylesterase-like enzyme polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983).
  • composition of a synthetic carboxylesterase-like enzyme polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the carboxylesterase-like enzyme polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter carboxylesterase-like enzyme polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a carboxylesterase-like enzyme polypeptide.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope.
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a carnitine palmitoyltransferase I-like enzyme polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
  • an antibody which specifically binds to a carboxylesterase-like enzyme polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to carboxylesterase-like enzyme polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a carboxylesterase-like enzyme polypeptide from solution.
  • Carboxylesterase-like enzyme polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • a carboxylesterase-like enzyme polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to a carboxylesterase-like enzyme polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the EBV HYBRIDOMA technique (Kohler et ah, Nature 256, 495497, 1985; Kozbor et ah, J. Immunol. Methods 81, 3142, 1985; Cote et ah, Proc. Natl. Acad. Sci. 80, 20262030, 1983; Cole et ah, Mol. Cell Biol. 62, 109120, 1984).
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et ah, Proc. Natl. Acad. Sci. 81, 68516855, 1984; Neuberger et ah, Nature 312, 604608, 1984; Takeda et ah, Nature 314, 452454, 1985).
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues.
  • rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
  • Antibodies which specifically bind to a carboxylesterase-like enzyme polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Patent 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to carboxylesterase-like enzyme polypeptides.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 1112023, 1991).
  • Single-chain antibodies also can be constracted using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et ah, 1996, Eur. J. Cancer Prev. 5, 507-11).
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Constmction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J Biol. Chem. 269, 199-206.
  • a nucleotide sequence encoding a single-chain antibody can be constracted using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et ah, 1995, Int. J. Cancer 61, 497-501; Nicholls et ah, 1993, J Immunol. Meth. 165, 81-91).
  • Antibodies which specifically bind to carboxylesterase-like enzyme polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et ah, Proc. Natl. Acad. Sci. 86, 38333837, 1989; Winter et ah, Nature 349, 293299, 1991).
  • chimeric antibodies can be constracted as disclosed in WO 93/03151.
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a carboxylesterase-like enzyme polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of carboxylesterase-like enzyme gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester intemucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioat.es, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 18, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et ah, Chem. Rev. 90, 543583, 1990.
  • Modifications of carboxylesterase-like enzyme gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the carboxylesterase-like enzyme gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions 10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a carboxylesterase-like enzyme polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent carboxylesterase-like enzyme nucleotides, can provide sufficient targeting specificity for carboxylesterase-like enzyme mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular carboxylesterase-like enzyme polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a carboxylesterase-like enzyme polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5' substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et ah, Trends Biotechnol. 10, 152158, 1992; Uhlmann et ah, Chem. Rev. 90, 543584, 1990; Uhlmann et ah, Tetrahedron. Lett. 215, 35393542, 1987.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 15321539; 1987; Cech, Ann. Rev. Biochem. 59, 543568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et ah, U.S. Patent 5,641,673).
  • ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a carboxylesterase-like enzyme polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the carboxylesterase-like enzyme polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et ah Nature 334, 585591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et ah, EP 321,201).
  • Specific ribozyme cleavage sites within a carboxylesterase-like enzyme RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate carboxylesterase-like enzyme RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease carboxylesterase-like enzyme expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA constmct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destmction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • the invention provides assays for screening test compounds which bind to or modulate the activity of a v enzyme polypeptide or a carboxylesterase-like enzyme polynucleotide.
  • a test compound preferably binds to a carboxylesterase-like enzyme polypeptide or polynucleotide. More preferably, a test compound decreases or increases carboxylesterase-like activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to bind to carboxylesterase-like enzyme polypeptides or polynucleotides or to affect carboxylesterase-like enzyme activity or carboxylesterase-like enzyme gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instmments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et ah, Proc. Natl. Acad. Sci. U.S.A. 19, 161418 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 710, 1995).
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV LIGHT. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule which binds to and occupies, for example, the active site of the carboxylesterase-like enzyme polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the carboxylesterase-like enzyme polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound which is bound to the carboxylesterase-like enzyme polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a carboxylesterase-like enzyme polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a carboxylesterase-like enzyme polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a carboxylesterase-like enzyme polypeptide (McConnell et ah, Science 257, 19061912, 1992).
  • Determining the ability of a test compound to bind to a carboxylesterase-like enzyme polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 23382345, 1991, and Szabo et ah, Curr. Opin. Struct. Biol. 5, 699705, 1995).
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • a carboxylesterase-like enzyme polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et ah, Cell 72, 223232, 1993; Madura et ah, J. Biol. Chem.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA BINDING and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a carboxylesterase-like enzyme polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA BINDING and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the carboxylesterase-like enzyme polypeptide.
  • a reporter gene e.g., LacZ
  • either the carboxylesterase-like enzyme polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • any method known in the art can be used to attach the carboxylesterase-like enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a carboxylesterase-like enzyme polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the carboxylesterase-like enzyme polypeptide is a fusion protein comprising a domain that allows the carboxylesterase-like enzyme polypeptide to be bound to a solid support.
  • glutathione S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the nonadsorbed carboxylesterase-like enzyme polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • binding of the interactants can be determined either directly or indirectly, as described above.
  • the complexes can be dissociated from the solid support before binding is determined.
  • Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a carboxylesterase-like enzyme polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated carboxylesterase-like enzyme polypeptides (or polynucleotides) or test compounds can be prepared from biotinNHS(Nhydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, 111.
  • antibodies which specifically bind to a carboxylesterase-like enzyme polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the carboxylesterase-like enzyme polypeptide can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the carboxylesterase-like enzyme polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the carboxylesterase-like enzyme polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Any cell which comprises a carboxylesterase-like enzyme polypeptide or polynucleotide can be used in a cell-based assay system.
  • a carboxylesterase-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a carboxylesterase-like enzyme polypeptide or polynucleotide is determined as described above. Enzyme Assays
  • Test compounds can be tested for the ability to increase or decrease the carboxylesterase-like activity of a human carboxylesterase-like enzyme polypeptide.
  • carboxylesterase-like activity can be measured, for example, as described in Wallace et ah, 1999 (see Example 2).
  • Enzyme assays can be carried out after contacting either a purified carboxylesterase-like enzyme polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound which decreases a carboxylesterase-like activity of a carboxylesterase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing carboxylesterase-like enzyme activity.
  • a test compound which increases a carboxylesterase-like activity of a human carboxylesterase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human carboxylesterase-like enzyme activity.
  • test compounds which increase or decrease carboxylesterase-like enzyme gene expression are identified.
  • a carboxylesterase-like enzyme polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the carboxylesterase-like enzyme polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of carboxylesterase-like enzyme mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of a carboxylesterase-like enzyme polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a carboxylesterase-like enzyme polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses a carboxylesterase-like enzyme polynucleotide can be used in a cell-based assay system.
  • the carboxylesterase-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the invention can comprise, for example, a carboxylesterase-like enzyme polypeptide, carboxylesterase-like enzyme polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a carboxylesterase-like enzyme polypeptide, or mimetics, agonists, antagonists, or inhibitors of a carboxylesterase-like enzyme polypeptide activity.
  • compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • agent such as stabilizing compound
  • the compositions can be administered to a patient alone, or in combination with other agents, drags or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 150 mM histidine, 0.1 %2% sucrose, and 27% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • Human carboxylesterase-like enzyme can be regulated to treat or prevent toxicity due to exposure to organophosphoras compounds.
  • Compounds that increase the ability of human carboxylesterase-like enzyme to bind to organophosphoms compounds are useful as detoxifying agents.
  • compounds that effectively increase the levels of human carboxylesterase-like enzyme, such as by increasing its expression also can be used as detoxifying agents.
  • Such compounds need not only be used as therapeutic agents following exposure to organophosphoras compounds, but also have the potential to serve as prophylatics in the instances where there is a likelihood of exposure to organophosphoras compounds.
  • compounds or agents that can effectively increase the ability of human carboxylesterase-like enzyme to detoxify organophosphoras compounds will prove useful in many applications.
  • human carboxylesterase-like enzyme itself can be administered as a detoxifying agent.
  • Human carboxylesterase-like enzyme also can be regulated to treat cancer.
  • Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression.
  • Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.
  • Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Agonists and/or antagonists of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drag development and subsequent testing in humans.
  • Osteoporosis is a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. It is the most common human metabolic bone disorder. Established osteoporosis includes the presence of fractures. Bone turnover occurs by the action of two major effector cell types within bone: the osteoclast, which is responsible for bone resorption, and the osteoblast, which synthesizes and mineralizes bone matrix. The actions of osteoclasts and osteoblasts are highly co-ordinated. Osteoclast precursors are recruited to the site of turnover; they differentiate and fuse to form mature osteoclasts which then resorb bone.
  • osteoclasts Attached to the bone surface, osteoclasts produce an acidic microenvironment in a tightly defined junction between the specialized osteoclast border membrane and the bone matrix, thus allowing the localized solubilization of bone matrix. This in turn facilitate the proteolysis of demineralized bone collagen. Matrix degradation is thought to release matrix-associated growth factor and cytokines, which recmit osteoblasts in a temporally and spatially controlled fashion. Osteoblasts synthesise and secrete new bone matrix proteins, and subsequently mineralise this new matrix. In the normal skeleton this is a physiological process which does not result in a net change in bone mass. In pathological states, such as osteoporosis, the balance between resorption and formation is altered such that bone loss occurs.
  • osteoclast itself is the direct or indirect target of all currently available osteoporosis agents with the possible exception of fluoride.
  • Antireso ⁇ tive therapy prevents further bone loss in treated individuals.
  • Osteoblasts are derived from multipotent stem cells which reside in bone marrow and also gives rise to adipocytes, chondrocytes, fibroblasts and muscle cells. Selective enhancement of osteoblast activity is a highly desirable goal for osteoporosis therapy since it would result in an increase in bone mass, rather than a prevention of further bone loss. An effective anabolic therapy would be expected to lead to a significantly greater reduction in fracture risk than currently available treatments.
  • the agonists or antagonists to the newly discovered polypeptides may act as antiresorptive by directly altering the osteoclast differentiation, osteoclast adhesion to the bone matrix or osteoclast function of degrading the bone matrix.
  • the agonists or antagonists could indirectly alter the osteoclast function by interfering in the synthesis and/or modification of effector molecules of osteoclast differentiation or function such as cytokines, peptide or steroid hormones, proteases, etc.
  • the agonists or antagonists to the newly discovered polypeptides may act as anabolics by directly enhancing the osteoblast differentiation and/or its bone matrix forming function.
  • the agonists or antagonists could also indirectly alter the osteoblast function by enhancing the synthesis of growth factors, peptide or steroid hormones or decreasing the synthesis of inhibitory molecules.
  • the agonists and antagonists may be used to mimic, augment or inhibit the action of the newly discovered polypeptides which may be useful to treat osteoporosis, Paget's disease, degradation of bone implants particularly dental implants.
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a carboxylesterase-like enzyme polypeptide binding molecule
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above described screening assays for treatments as described herein.
  • a reagent which affects carboxylesterase-like enzyme activity can be administered to a human cell, either in vitro or in vivo, to reduce carboxylesterase-like enzyme activity.
  • the reagent preferably binds to an expression product of a human carboxylesterase-like enzyme gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours.
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • from about 0.1 ⁇ g to about 10 ⁇ g of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et a Trends in Biotechnol. 11, 202-05 (1993); Chiou et ah, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et ah, J. Biol. Chem. 269, 542-46 (1994); Zenke et ah, Proc. Natl. Acad. Sci. USA. 87, 3655-59 (1990); Wu et ah, J. Biol. Chem. 266, 338-42 (1991). Determination of a Therapeutically Effective Dose
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases carboxylesterase-like enzyme activity relative to the carboxylesterase-like enzyme activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 5 Q.
  • compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drag combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a carboxylesterase-like enzyme gene or the activity of a carboxylesterase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of a carboxylesterase-like enzyme gene or the activity of a carboxylesterase-like enzyme polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to carboxylesterase-like enzyme-specific mRNA, quantitative RT-PCR, immunologic detection of a carboxylesterase-like enzyme polypeptide, or measurement of carboxylesterase-like enzyme activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Diagnostic Methods
  • Human carboxylesterase-like enzyme also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding carboxylesterase-like enzyme in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g. , Myers et ah, Science 230, 1242, 1985).
  • Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et ah, Proc. Natl. Acad. Sci. USA 85, 43974401, 1985).
  • nuclease protection assays such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et ah, Proc. Natl. Acad. Sci. USA 85, 43974401, 1985).
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • mutations can also be detected by in situ analysis.
  • Altered levels of a carboxylesterase-like enzyme also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
  • the polynucleotide of SEQ LD NO: 4 is inserted into the expression vector pCEV4 and the expression vector pCEN4- carboxylesterase-like enzyme polypeptide obtained is transfected into human embryonic kidney 293 cells.
  • the carboxylesterase-like enzyme activity of these cells is measured by the release of p-nitrophenol from p-nitrophenol acetate as described by Wallace et al, 1999, supra.
  • the cells are incubated for 48 hours and harvested in buffer that contains 250 mM sodium phosphate, pH 7.4, and 250 mM sucrose.
  • the cell suspension is sonicated, and cell debris is removed by centrifugation at 10,000 x g for 30 minutes at 4 °C.
  • the post-mitochondrial supernatant is used as the enzyme preparation. Protein is measured with Pierce BCA reagent (Pierce, Rockford, IL) using bovine seram albumin as a standard. To measure enzyme activity toward p-nitrophenyl acetate (Sigma Chemical Co., St. Louis, MO), an aliquot of the supernatant is diluted to 1 ml with 200 mM phosphate buffer, 5 mM beta-mercaptoethanol, and 80 mM potassium chloride. The reaction is started by the addition of p-nitrophenol acetate to a final concentration of 0.2 mM and incubated at 37 °C for 5 minutes. The release of p-nitrophenol is monitored at 400 nm.
  • an enzyme preparation (as described above) containing between 35 and 45 micrograms protein is incubated with 0.1, 1.0, or 10.0 micromolar paraoxon for 15 or 30 minutes at 37 °C. The enzyme activity is then assayed as above. It is shown that SEQ ID NO: 5 has carboxylesterase-like enzyme activity.
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human carboxylesterase-like polypeptides in yeast.
  • the carboxylesterase-like enzyme-encoding DNA sequence is derived from SEQ LD NO:4.
  • the DNA sequence is modified by well known methods in such a way that it contains at its 5' end an initiation codon and at its 3' end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (NiNTAResin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human carboxylesterase-like enzyme polypeptide is obtained.
  • Carboxylesterase-like activity can be assayed by testing the activity of carboxylesterase-like enzymes expressed in a eukaryotic expression system, such as transfected COS-7 cells.
  • Test compounds from a small molecule library can be assayed for their ability to regulate the binding of organophosphoras compounds by a human carboxylesterase-like activity by contacting enzyme preparations with the test compounds. Control preparations, in the absence of a test compound, are also assayed.
  • Carboxylesterase-like activity can be measured by the release of p-nitrophenol from p-nitrophenol acetate as described by Wallace et ah, 1999, supra.
  • Enzyme preparations are made from COS-7 cells that are transfected with a constmct encoding a human carboxylesterase-like enzyme.
  • the COS-7 cells are transfected with lipofectamine (GLBCO BRL) according to the manufacturer's instructions. After 48 hours of incubation, the cells are harvested in buffer that contains 250 mM sodium phosphate, pH 7.4, and 250 mM sucrose. The cell suspension is sonicated, and cell debris is removed by centrifugation at 10,000 x g for 30 minutes at 4 °C.
  • the post-mitochondrial supernatant is used, as the enzyme preparation. Protein is measured with Pierce BCA reagent (Pierce, Rockford, IL) using bovine serum albumin as a standard. To measure enzyme activity toward p-nitrophenyl acetate (Sigma Chemical Co., St. Louis, MO), an aliquot of the supernatant is diluted to 1 ml with 200 mM phosphate buffer, 5 mM beta-mercaptoethanol, and 80 mM potassium chloride. The reaction is started by the addition of p-nitrophenol acetate to a final concentration of 0.2 mM and incubated at 37 °C for 5 minutes. The release of p-nitrophenol is monitored at 400 nm.
  • an enzyme preparation (as described above) containing between 35 and 45 micrograms protein is incubated with 0.1, 1.0, or 10.0 micromolar paraoxon for 15 or 30 minutes at 37 °C. The enzyme activity is then assayed as above.
  • a test compound which increases the inhibitory effect of paraoxon on carboxylesterase-like activity by at least 20% is identified as a compound that increases the organophosphoras compound binding by human carboxylesterase-like enzyme.
  • Purified carboxylesterase-like enzyme polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Carboxylesterase-like enzyme polypeptides comprise the amino acid sequence shown in SEQ ID NO: 5.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a carboxylesterase-like enzyme polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a carboxylesterase-like enzyme polypeptide.
  • a test compound is administered to a culture of human cells transfected with a carboxylesterase-like enzyme expression construct and incubated at 37 °C for 10 to 45 minutes.
  • a culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et ah, Biochem. 18, 5294-99, 1979).
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled carboxylesterase-like enzyme-specific probe at 65 ° C in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:4.
  • a test compound which increases the carboxylesterase-like enzyme-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of carboxylesterase-like enzyme gene expression.
  • a liver biopsy is performed to obtain hepatocytes.
  • the hepatocytes are genetically engineered to contain an expression construct which expresses human carboxylesterase-like enzyme and reintroduced into the patient. See U.S. Patent 6,063,630.
  • the patient is now capable of producing higher levels of human carboxylesterase-like enzyme.
  • the level of carboxylesterase-like enzyme mRNA or polypeptide expression in the patient's cells is determined by performing a second liver biopsy and detecting human carboxylesterase-like mRNA by Northern blotting. Higher levels of human carboxylesterase-like enzyme mRNA are produced due to the presence of the expression construct.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des réactifs qui régulent une enzyme humaine de type carboxylestérase et des réactifs qui se lient à des produits géniques d'enzyme humaine de type carboxylestérase. Ces réactifs peuvent jouer un rôle dans la prévention, l'amélioration ou la correction de disfonctionnements ou de maladies, notamment, mais pas exclusivement, de l'intoxication due à l'organo-phosphore, du cancer et de l'ostéoporose.
PCT/EP2001/007919 2000-07-17 2001-07-10 Regulation d'une enzyme humaine de type carboxylesterase WO2002006454A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001281969A AU2001281969A1 (en) 2000-07-17 2001-07-10 Regulation of human carboxylesterase-like enzyme

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21856400P 2000-07-17 2000-07-17
US60/218,564 2000-07-17

Publications (2)

Publication Number Publication Date
WO2002006454A2 true WO2002006454A2 (fr) 2002-01-24
WO2002006454A3 WO2002006454A3 (fr) 2002-07-11

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AU (1) AU2001281969A1 (fr)
WO (1) WO2002006454A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002050256A2 (fr) * 2000-12-18 2002-06-27 Millennium Pharmaceuticals, Inc. 53010, nouveau membre de la famille des carboxylesterases humaines et utilisations
WO2009113758A1 (fr) * 2008-03-14 2009-09-17 Industry-Academic Cooperation Foundation, Yonsei University Outil biomarqueur de plasma pour le diagnostic d'un cancer du foie comportant de la carboxylestérase hépatique 1 et procédé de criblage d'un cancer du foie

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2001057272A2 (fr) * 2000-02-04 2001-08-09 Aeomica, Inc. Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans le placenta humain

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2001057272A2 (fr) * 2000-02-04 2001-08-09 Aeomica, Inc. Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans le placenta humain

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] 30 April 2001 (2001-04-30) HASHIMOTO, K. ET AL.: "Macaca fascicularis brain cDNA clone:QtrA-12316, full insert sequence." Database accession no. AB060873 XP002197703 *
DATABASE EMBL [Online] 8 July 1999 (1999-07-08) STRAUSBERG, R.: "wf67d05.x1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone IMAGE:2360649 3' similar to TR:Q16859 Q16859 CARBOXYLESTERASE ;, mRNA sequence." Database accession no. AI808985 XP002197702 *
SCHWER H ET AL: "MOLECULAR CLONING AND CHARACTERIZATION OF A NOVEL PUTATIVE CARBOXYLESTERASE, PRESENT IN HUMAN INTESTINE AND LIVER" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 233, no. 1, 1997, pages 117-120, XP000891817 ISSN: 0006-291X *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002050256A2 (fr) * 2000-12-18 2002-06-27 Millennium Pharmaceuticals, Inc. 53010, nouveau membre de la famille des carboxylesterases humaines et utilisations
WO2002050256A3 (fr) * 2000-12-18 2003-07-24 Millennium Pharm Inc 53010, nouveau membre de la famille des carboxylesterases humaines et utilisations
US6664091B2 (en) 2000-12-18 2003-12-16 Millennium Pharmaceuticals, Inc. 53010, a human carboxylesterase family member and uses thereof
US7094589B2 (en) 2000-12-18 2006-08-22 Millennium Pharmaceuticals, Inc. 53010, A novel human carboxylesterase family member and uses thereof
WO2009113758A1 (fr) * 2008-03-14 2009-09-17 Industry-Academic Cooperation Foundation, Yonsei University Outil biomarqueur de plasma pour le diagnostic d'un cancer du foie comportant de la carboxylestérase hépatique 1 et procédé de criblage d'un cancer du foie
US8198038B2 (en) 2008-03-14 2012-06-12 Industry-Academic Cooperation Foundation, Yonsei University Plasma biomarker tool for the diagnosis of liver cancer comprising liver carboxylesterase 1 and liver cancer screening method
USRE46572E1 (en) 2008-03-14 2017-10-17 Industry-Academic Cooperation Foundation, Yonsei University Plasma biomarker tool for the diagnosis of liver cancer comprising liver carboxylesterase 1 and liver cancer screening method

Also Published As

Publication number Publication date
WO2002006454A3 (fr) 2002-07-11
AU2001281969A1 (en) 2002-01-30

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