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WO1998011219A1 - Proteines membranaires associees a des maladies (damp) - Google Patents

Proteines membranaires associees a des maladies (damp) Download PDF

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Publication number
WO1998011219A1
WO1998011219A1 PCT/US1997/015924 US9715924W WO9811219A1 WO 1998011219 A1 WO1998011219 A1 WO 1998011219A1 US 9715924 W US9715924 W US 9715924W WO 9811219 A1 WO9811219 A1 WO 9811219A1
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Prior art keywords
damp
sequence
seq
polypeptide
sequences
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PCT/US1997/015924
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English (en)
Inventor
Jennifer L. Hillman
Surya K. Goli
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Incyte Pharmaceuticals, Inc.
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Priority to AU42612/97A priority Critical patent/AU4261297A/en
Publication of WO1998011219A1 publication Critical patent/WO1998011219A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to nucleic acid and amino acid sequences of a novel disease associated membrane protein (DAMP) and to the use of these sequences in the diagnosis, study, prevention, and treatment of disease.
  • DAMP disease associated membrane protein
  • Proteins and lipids are all important constituents of cell membranes. Proteins, including glycoproteins, which span the lipid bilayer are called transmembrane proteins and may cross back and forth several times. While hydrophobic residues are relegated to the middle of the membrane, this crossing back and forth exposes hydrophihc residues to the internal and external milieu. (Unwin N et al (1984) Sci Am 250:78).
  • Membrane proteins participate in catalyzing reactions, transporting molecules in and out of cells, receiving and transmitting intercellular messages, anchoring cells to substratum, and providing tissue-identification tags. They are important in both paracrine and autocrine signaling and may react appropriately or inappropriately in response to growth factors. In fact, cells vary in identity, character and bioactivity largely as a result of variation in their membrane proteins.
  • PMP22 Peripheral myelin protein 22
  • PMP22 is a hydrophobic protein of 160 residues having four predicted transmembrane domains which play a role in cell proliferation (Welcher AA et ai (1991) Proc Nat Acad Sci 88:7195-9). Tissue culture experiments using NIH 3T3 cells and primary dermal fibroblasts have shown that PMP22 is upregulated under growth arrest due to serum deprivation or high cell density. This suggests a . significant role for PMP22 in cell proliferation (Manfioletti G et al (1990) Mol Cell Biol 10:2924-30).
  • EMP1 and PMP22 are co-expressed in most tissues although their relative expression levels differ.
  • EMP1 and PMP22 mRNA levels are inversely regulated in the degenerating rat sciatic nerve after injury and by growth arrest in NIH 3T3 fibroblasts (Taylor, supra; Marvin, supra).
  • the present invention discloses a novel disease associated membrane protein, hereinafter referred to as DAMP, which shares features with four transmembrane spanning proteins involved in regulating cell proliferation. Accordingly, the invention features a substantially purified DAMP, as shown in the amino acid sequence of SEQ ID NO:l .
  • polynucleotide which encode DAMP.
  • the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
  • the invention features polynucleotide sequences that hybridize under stringent conditions to SEQ ID NO:2.
  • the invention further relates to the nucleic acid sequence encoding DAMP, oligonucleotides, peptide nucleic acids, fragments, portions or antisense molecules thereof.
  • the present invention also relates, in part, to the inclusion of the nucleic acid sequence encoding DAMP in an expression vector which can be used to transform host cells or organisms.
  • the invention also provides for using similar vectors for the therapeutic transformation to prevent proliferation of cancerous cells or tissues.
  • the instant invention presents a method for producing DAMP or a fragment thereof. It contemplates the delivery of purified DAMP, alone or in a pharmaceutically acceptable excipient, to cancerous cells or tissues. It also encompasses antibodies which bind specifically to DAMP and can be used to examine prevalence of the protein in vivo.
  • SEQ ID NO:2 of the novel DAMP of the present invention produced using MacDNAsis software (Hitachi Software Engineering Co Ltd, San Bruno CA).
  • FIGS. 2A and 2B shows the northern analysis for the consensus nucleotide sequence (SEQ ID NO:2) produced electronically using the LIFESEQTM database (Incyte Pharmaceuticals, Palo Alto CA).
  • Figure 3 shows the amino acid sequence alignments among DAMP (SEQ ID NO: 1 ) and GI 1 171356, human tumor associated membrane protein homolog, and GI 951 124, mouse tumor associated membrane protein (Ben-Porath I and Benvenisty N et al 1996, in press). Sequences were aligned using the multisequence alignment program of DNAStarTM software (DNAStar Inc, Madison WI).
  • Figure 4 shows the hydrophobicity plot (generated using MacDNAsis software) for DAMP, SEQ ID NO: 1 ; the X axis reflects amino acid position, and the negative Y axis, hydrophobicity.
  • Figure 5 shows the isoelectric plot (generated using MacDNAsis software) for DAMP, SEQ ID NO: 1.
  • Nucleic acid sequence refers to an oligonucleotide, nucleotide or polynucleotide. and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • amino acid sequence refers to peptide or protein sequence.
  • Protein nucleic acid refers to a molecule which comprises an oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary (template) strand of nucleic acid (Nielsen PE et al (1993) Anticancer Drug Des 8:53-63).
  • a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • An “insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring DAMP.
  • a “substitution” results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
  • DAMP refers to the amino acid sequence of substantially purified DAMP obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic or recombinant.
  • a “variant" of DAMP is defined as an amino acid sequence differs by one or more amino acids.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, eg, replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative” changes, eg, replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNAStar software.
  • biologically active' 1 refers to DAMP having structural, regulatory or biochemical functions of a naturally occurring DAMP.
  • immunologically active defines the capability of the natural, recombinant or synthetic DAMP, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • derivative refers to the chemical modification of a nucleic acid encoding DAMP or the encoded DAMP. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative would encode a polypeptide which retains essential biological characteristics of natural DAMP.
  • substantially purified refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • hybridization shall include "any process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology. Stockton Press, New York NY). Amplification is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach CW and GS Dveksler (1995) PCR Primer, a . Laboratory Manual. Cold Spring Harbor Press, Plainview NY).
  • Stringent hybridization typically occurs in a range from about Tm-5°C (5°C below the Tm of the probe)to about 20°C to 25°C below Tm.
  • stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
  • the present invention relates to a novel human disease associated membrane protein (DAMP) identified among the cDNAS from a library constructed from synovial tissue of an individual with osteoarthritis and to the use of the nucleic acid and amino acid sequences in the study, diagnosis, prevention and treatment of disease.
  • DAMP human disease associated membrane protein
  • cDNAs encoding portions of DAMP were found in eosinophils and macrophages, and a variety of diseased tissues including rheumatoid and osteoarthritic synovia, cancerous and Crohn's colon, and tumors of the brain, heart, lung, tongue, prostate, ovary, stomach and kidney. It must be noted that naturally occurring expression of DAMP is not necessarily limited to these cells and tissues. The present invention also encompasses DAMP variants.
  • a preferred DAMP variant is one having at least 80% amino acid sequence similarity to the amino acid sequence (SEQ ID NO:l), a more preferred DAMP variant is one having at least 90% amino acid sequence similarity to SEQ ID NO:l and a most preferred DAMP variant is one having at least 95% amino acid sequence similarity to SEQ ID NO: 1.
  • Nucleic acid encoding the human DAMP of the present invention was first identified in cDNA, Incyte Clone 728086 (SEQ ID NO:2), through a computer- generated search for amino acid sequence alignments. The nucleic acid sequence, SEQ ID NO:2, encodes the 163 amino acid sequence, SEQ ID NO: 1.
  • the present invention is based, in part, on the chemical and structural homology among DAMP, GI 1171356, human tumor associated membrane protein homolog, and GI 951 124, mouse tumor associated membrane protein (Ben-Porath I and Benvenisty N et al 1996, in press).
  • DAMP residues 1-28, 62-90, 94-1 18, and 134-159 of SEQ ID NO: 1 represent four hydrophobic membrane spanning domains.
  • potential N glycosylation sites are present at residues N 4g and N 57 .
  • any nucleic acid sequence which encodes DAMP can be used to generate recombinant molecules which express DAMP.
  • a partial sequence encoding DAMP was first isolated as Incyte Clone 728086 from a osteoarthritic synovium cDNA library (SYNOOAT01).
  • nucleotide sequences which encode DAMP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring sequence under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding DAMP or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • a DNA sequence, or portions thereof, encoding DAMP or its derivative may be produced entirely by synthetic chemistry. After synthesis, the gene may be inserted into any of the many available DNA vectors and cell systems using reagents that generally available. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding DAMP or any portion thereof.
  • polynucleotide sequences that are capable of hybridizing to the nucleotide sequence of SEQ ID NO:2 under various conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques. Methods in Enzymologv. Vol 152, Academic Press, San Diego CA) inco ⁇ orated herein by reference, and on the salt concentrations under which the steps of the process are carried out .
  • Tm melting temperature
  • Altered nucleic acid sequences encoding DAMP which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent DAMP.
  • the protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent DAMP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of DAMP is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine. valine; glycine. alanine; asparagine. glutamine; serine, threonine phenylalanine, and tyrosine.
  • an "allele” or “allelic sequence” is an alternative form of the nucleic acid sequence encoding DAMP. Alleles result from a mutation,- ie, a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • Methods for DNA sequencing may be used which are well known in the art and employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Co ⁇ , Cleveland OH)), Taq polymerase (Perkin Elmer, Norwalk CT), thermostable T7 polymerase (Amersham, Chicago IL), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg MD).
  • the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the ABI 377 DNA sequencers (Perkin Elmer). Extending the Polynucleotide Sequence
  • the polynucleotide sequence encoding DAMP may be extended utilizing partial nucleotide sequence and various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • Gobinda et al (1993; PCR Methods Applic 2:318-22) use "restriction-site" polymerase chain reaction (PCR) as a direct method which uses universal primers to retrieve unknown sequence adjacent to a known locus.
  • genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are 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 can be used to amplify or extend sequences using divergent primers based on a known region (Triglia T et al (1988) Nucleic Acids Res 16:8186).
  • the primers may be designed using OLIGO® 4.06 Primer Analysis Software (1992; National Biosciences Inc, Plymouth MN), or another appropriate program, to be 22-30 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 (Lagerstrom M et al (1991) PCR Methods Applic 1 :11 1-19) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double- stranded sequence into an unknown portion of the DNA molecule before PCR.
  • PCR Parker JD et al (1991 ) Nucleic Acids Res 19:3055-60
  • PCR nested primers.
  • PromoterFinderTM Click-through libraries
  • PromoterFinder libraries can be used to walk in genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • Preferred libraries for screening for full length cDNAs are those which have been size-selected to include larger cDNAs.
  • random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
  • a randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
  • Capillary electrophoresis may be used to analyze either the size or confirm the nucleotide sequence in sequencing or PCR products.
  • Systems for rapid sequencing are available from Perkin Elmer, Beckman Instruments (Fullerton CA). and other companies.
  • Capillary sequencing may 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 devise camera.
  • Output/light intensity is converted to electrical signal using appropriate software (eg. GenotyperTM and Sequence NavigatorTM from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display is computer controlled.
  • Capillary electrophoresis is particularly suited to the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • the reproducible sequencing of up to 350 bp of Ml 3 phage DNA in 30 min has been reported (Ruiz-Martinez MC et al (1993) Anal Chem 65:2851-8).
  • Expression of the Nucleotide Sequence polynucleotide sequences which encode DAMP, fragments of the polypeptide, fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules that direct the expression of DAMP in appropriate host cells.
  • the nucleotide sequences of the present invention can be engineered in order to alter DAMP-encoding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
  • mutations may be introduced using techniques which are well known in the art, eg, site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to produce splice variants, etc. 5
  • a natural, modified or recombinant DAMP- encoding sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between DAMP and the
  • sequence encoding DAMP may be synthesized, whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al(1980) Nuc Acids Res Symp Ser
  • the protein itself may be produced using chemical methods to synthesize an amino acid sequence for DAMP, whole or in part.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the 0 manufacturer.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (eg, Creighton (1983) Proteins. Structures and Molecular Principles. WH Freeman and Co. New York NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (eg, the Edman degradation procedure; 5 Creighton, supra). Additionally the amino acid sequence of DAMP, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • the nucleotide sequence encoding DAMP 0 or its functional equivalent is inserted into an appropriate expression vector, ie, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • an appropriate expression vector ie, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems may be utilized to contain and express a sequence encoding DAMP. These include but are not limited to 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 virus expression vectors (eg, baculovirus); plant cell systems transfected with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg, Ti or pBR322 plasmid); 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 virus expression vectors (eg, baculovirus); plant cell systems transfected with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors
  • control elements or “regulatory sequences” of these systems vary in their strength and specificities and are those nontranslated regions of the vector, enhancers, promoters, and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla CA) or pSportl (Gibco BRL) and pt ⁇ -lac hybrids and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (eg, heat shock, RUBISCO; and storage protein genes) or from plant viruses (eg, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from the mammalian genes or from mammalian viruses are most appropriate. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding DAMP, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • Promoters or enhancers derived from the genomes of plant cells eg, heat shock, RUBISCO; and storage protein genes
  • plant viruses eg, viral promoters or leader sequences
  • a number of expression vectors may be selected depending upon the use intended for DAMP. For example, when large quantities of DAMP are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding DAMP may 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; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); and the like.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems are 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.
  • Saccharomvces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • the expression of a sequence encoding DAMP may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al (1984) Nature 310:51 1-514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al (1987) EMBO J 3:17-31 1 ).
  • plant promoters such as the small subunit of RUBISCO (Coruzzi et al (1984) EMBO J 3: 1671-1680; Brogue et al (1984) Science 224:838-843); or heat shock promoters (Winter J and Sinibaldi RM (1991) Results Probl Cell Differ 17:85-105) may be used. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. For reviews of such techniques, see Hobbs S or Murry LE in McGraw Hill Yearbook of Science and Technology H992 McGraw Hill New York NY, pp 191 - 196 or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology. Academic Press, New York NY, pp 421 -463.
  • An alternative expression system which may be used to express DAMP is an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequence encoding DAMP may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the sequence encoding DAMP will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat.
  • the recombinant viruses are then used to infect £. frugiperda cells or Trichoplusia larvae in which DAMP is expressed (Smith et al (1983) J Virol 46:584; Engelhard EK et al ( 1994) Proc Nat Acad Sci 91 :3224-7).
  • a number of viral-based expression systems may be utilized.
  • a sequence encoding DAMP may be ligated into an adenovirus transcription/ translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 81 :3655-59).
  • transcription enhancers such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV rous sarcoma virus
  • Specific initiation signals may also be required for efficient translation of a sequence encoding DAMP. These signals include the ATG initiation codon and adjacent sequences. In cases where the sequence encoding DAMP, its initiation codon and upstream sequences are inserted into the most appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic.
  • Enhancers appropriate to the cell system in use (Scharf D et al (1994) Results Probl Cell Differ 20: 125-62; Bittner et al (1987) Methods in Enzymol 153:516-544).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which stably express DAMP may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1 -2 days in an enriched media before they are switched to selective media.
  • the pu ⁇ ose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the he ⁇ es simplex virus thymidine kinase (Wigler M et al ( 1977) Cell 1 1 :223-32) and adenine phosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite.
  • antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin F et al (1981 ) J Mol Biol 150: 1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra).
  • marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
  • recombinant cells containing the sequence encoding DAMP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with the sequence encoding DAMP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem sequence as well.
  • host cells which contain the sequence encoding DAMP and expressing
  • DAMP may 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 hybridization and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of the nucleic acid or protein.
  • oligonucleotides or “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer.
  • a variety of protocols for detecting and measuring the expression of DAMP, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on DAMP is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al ( 1990, Serological Methods, a Laboratory Manual. APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 158:1211).
  • Means for producing labeled hybridization or PCR probes for detecting related sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the DAMP-encoding sequence, or any portion of it may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
  • Patents teaching the use of such labels include US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.
  • recombinant immunoglobulins may be produced as shown in US Patent No. 4,816,567 inco ⁇ orated herein by reference. Purification of DAMP
  • Host cells transformed with a nucleotide sequence encoding DAMP may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing sequence encoding DAMP can be designed with signal sequences which direct secretion of DAMP through a prokaryotic or eukaryotic cell membrane.
  • DAMP may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • 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 (Immunex Co ⁇ , Seattle WA).
  • the inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and DAMP is useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein comprising the sequence encoding DAMP and nucleic acid sequence encoding 6 histidine residues followed by thioredoxin and an enterokinase cleavage site.
  • the histidine residues facilitate purification while the enterokinase cleavage site provides a means for purifying DAMP from the fusion protein.
  • fragments of DAMP may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al ( 1969) Solid-Phase Peptide
  • DAMP DAMP appears to be associated with both inflammatory cells, autoimmune diseases and cancers. These diseases include rheumatoid and osteoarthritis, Crohn's diseases, melanoma and carcinomas of the brain, heart, lung, tongue, prostate, ovary, stomach and kidney.
  • DAMP has amino acid sequence homology with four other transmembrane proteins clearly associated with cell proliferation, and nucleotide homology with a growth arrest specific protein and an epithelial membrane protein. These homologies suggest that DAMP, or fragments or derivatives thereof, can be used in diagnostic methods for the detection of nucleotide sequences associated with tumors and other cancers. Furthermore, the nucleotide sequences of DAMP provide the basis for therapeutic molecules useful in the treatment of aberrant cell proliferation. Additionally, the sequences for DAMP will provide the basis for screening for agonists, antagonists or inhibitors that modulate the activity or products of DAMP
  • anti-DAMP antibodies capable of neutralizing the activity of DAMP may be used to prevent or treat diseases.
  • Neri D et al (1995, Cell Biophys 27: 47- ) and Liberatore M et al (1995, Eur J Nucl Med 22: 1326-) describe simple methods for isotope labelling of cysteine-tagged antibodies resulting in more than 50% radionuclide inco ⁇ oration and full retention of immuoreactivity.
  • DAMP-specific antibodies are also useful for the diagnosis of conditions and diseases associated with expression of the polypeptides.
  • the DAMP nucleic acid sequence of SEQ ID NO:2 can be inco ⁇ orated into effective eukaryotic expression vectors and directly administered into somatic cells for gene therapy.
  • RNA transcripts produced in vitro may be encapsulated in and administered via liposomes.
  • Such vectors and transcripts may function transiently or may be inco ⁇ orated into the host chromosomal DNA for longer term expression.
  • DAMP-specific antibodies are useful for the diagnosis and treatment of conditions and diseases associated with expression of DAMP.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain. Fab fragments and fragments produced by a Fab expression library.
  • Neutralizing antibodies, ie, those which inhibit dimer formation, are especially preferred for diagnostics and therapeutics.
  • DAMP used for antibody induction may have an amino acid sequence consisting of at least five amino acids, and preferably at least 10 amino acids. Preferably, they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of DAMP amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to DAMP.
  • various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with DAMP or any portion, fragment or oligopeptide which retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin. and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corvnebacterium parvum are potentially useful human adjuvants.
  • Monoclonal antibodies to DAMP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al ( 1985) Monoclonal Antibodies and ancer Therapy. Alan R Liss Inc, New York NY, pp 77-96).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991 ; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for DAMP may also be generated.
  • such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-1281).
  • DAMP antibodies are useful for the diagnosis of conditions or diseases characterized by expression of DAMP or in assays to monitor patients being treated with DAMP, its fragments, agonists or inhibitors. Diagnostic assays for DAMP include methods utilizing the antibody and a label to detect DAMP in human body fluids or extracts of cells or tissues.
  • the polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known, several of which were described above.
  • a variety of protocols for measuring DAMP, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on DAMP is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox,
  • normal or standard values for DAMP expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with antibody to DAMP under conditions suitable for complex formation which are well known in the art. The amount of standard complex formation may be quantified by comparing various artificial membranes containing known quantities of
  • DAMP its catalytic or immunogenic fragments or oligopeptides thereof
  • the fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between DAMP and the agent being tested, may be measured.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding DAMP specifically compete with a test compound for binding DAMP. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with DAMP.
  • a polynucleotide sequence encoding DAMP or any part thereof may be used for diagnostic and/or therapeutic pu ⁇ oses.
  • the sequence encoding DAMP of this invention may be used to detect and quantitate gene expression in biopsied tissues in which DAMP may be expressed in response to oncogenes.
  • the diagnostic assay is useful to distinguish between absence, presence, and excess expression of DAMP and to monitor regulation of DAMP levels during therapeutic intervention.
  • Included in the scope of the invention are oligonucleotide sequences, antisense RNA and DNA molecules, and peptide nucleic acids, (PNA).
  • Another aspect of the subject invention is to provide for hybridization or PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding DAMP or closely related molecules.
  • the specificity of the probe whether it is made from a highly specific region, eg, 10 unique nucleotides in the 5' regulatory region, or a less specific region, eg, especially in the 3' region, and the stringency of the hybridization or amplification (maximal, high, intermediate or low) will determine whether the probe identifies only naturally occurring DAMP, alleles or related sequences.
  • Probes may also be used for the detection of related sequences and should preferably contain at least 50% of the nucleotides from any of these sequences encoding DAMP.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence of SEQ ID NO:2 or from genomic sequence including promoter, enhancer elements and introns of the naturally occurring sequence encoding DAMP.
  • Hybridization probes may be labeled by a variety of reporter groups, including radionuclides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.
  • Polynucleotide sequences encoding DAMP may be used for the diagnosis of conditions or diseases with which the expression of DAMP is associated.
  • polynucleotide sequences encoding DAMP may be used in hybridization or PCR assays of fluids or tissues from biopsies to detect DAMP expression.
  • the form of such qualitative or quantitative methods may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin, chip and ELISA technologies. All of these techniques are well known in the art and are the basis of many commercially available diagnostic kits.
  • the DAMP-encoding nucleotide sequences disclosed herein provide the basis for assays that detect activation or induction associated with inflammation or disease.
  • the nucleotide sequence may be labeled by methods known in the art and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After an incubation period, the sample is washed with a compatible fluid which optionally contains a dye (or other label requiring a developer) if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed off, the dye is quantitated and compared with a standard.
  • the nucleotide sequence has hybridized with nucleotide sequences in the sample, and the presence of elevated levels of nucleotide sequences encoding DAMP in the sample indicates the presence of the associated inflammation and/or disease.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for DAMP expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with DAMP, or a portion thereof, under conditions suitable for hybridization or amplification.
  • Standard hybridization may be quantified by comparing the values obtained for normal subjects with a dilution series of DAMP run in the same experiment where a known amount of substantially purified DAMP is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients affected by DAMP-associated diseases. Deviation between standard and subject values establishes the presence of disease.
  • a therapeutic agent is administered and a treatment profile is generated. Such assays may be repeated on a regular basis to evaluate whether the values in the profile progress toward or return to the normal or standard pattern. Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
  • PCR may be used as described in US Patent Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the sequence encoding DAMP. Such oligomers are generally chemically synthesized, but they may be generated enzymatically or produced from a recombinant source.
  • Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'->3') and one with antisense (3' ⁇ -5'), employed under optimized conditions for identification of a specific gene or condition.
  • the same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • methods which may be used to quantitate the expression of a particular molecule include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are inte ⁇ olated. Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation. A definitive diagnosis of this type may allow health professionals to begin aggressive treatment and prevent further worsening of the condition.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known such as the triplet genetic code, specific base pair interactions, and the like.
  • Therapeutic Use Based upon its homology to EMAP-II and its expression profile, the polynucleotide encoding DAMP disclosed herein may be useful in the treatment of immune deficiency diseases.
  • Expression vectors derived from retroviruses, adenovirus, he ⁇ es or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express antisense of the sequence encoding DAMP. See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra).
  • polynucleotides comprising full length cDNA sequence and/or its regulatory elements enable researchers to use the sequence encoding DAMP as an investigative tool in sense (Youssoufian H and HF Lodish 1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60:631-652) regulation of gene function.
  • sense or antisense oligomers, or larger fragments can be designed from various locations along the coding or control regions.
  • Genes encoding DAMP can be turned off by transfecting a cell or tissue with expression vectors which express high levels of a desired DAMP fragment. Such constructs can flood cells with untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector (Mettler I, personal communication) and even longer if appropriate replication elements are part of the vector system.
  • Methodtler I personal communication
  • antisense molecules DNA, RNA or PNA, to the control regions of the sequence encoding DAMP, ie, the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, eg, between -10 and +10 regions of the leader sequence, are preferred.
  • the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA were reviewed by Gee JE et al (In: Huber BE and BI Carr (1994) Molecular and Immunologic Approaches. Futura Publishing Co, Mt Kisco NY).
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA. followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of the sequence encoding DAMP.
  • ribozyme cleavage sites within any potential RNA target are initially 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 gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding DAMP. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • vectors are introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient is presented in US Patent Nos. 5,399,493 and 5,437,994, disclosed herein by reference. Delivery by transfection and by liposome are quite well known in the art.
  • nucleotide sequences encoding DAMP disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including but not limited to such properties as the triplet genetic code and specific base pair interactions. Detection and Mapping of Related Polynucleotide Sequences
  • the nucleic acid sequence encoding DAMP can also be used to generate hybridization probes for mapping the naturally occurring genomic sequence.
  • the sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in sim hybridization to chromosomal spreads, flow-sorted chromosomal preparations, or artificial chromosome constructions such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions or single chromosome cDNA libraries as reviewed in Price CM (1993; Blood Rev 7:127-34) and Trask BJ (1991 ; Trends Genet 7:149-54).
  • telangiectasia AT
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier or affected individuals.
  • compositions for oral administration are accomplished orally or parenterally.
  • Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Maack Publishing Co, Easton PA).
  • 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.
  • Pharmaceutical preparations 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 corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also 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 may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie, 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 may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • compositions for parenteral administration include aqueous solutions of active compounds.
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may 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.
  • the suspension may also 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. Manufacture and Storage
  • compositions of the present invention may be manufactured in a manner that known in the art, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may 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 that are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder in lmM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
  • compositions comprising a compound of the invention formulated in a acceptable carrier
  • they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency and method of administration.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, eg, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors which ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eg, ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • ED50 the dose therapeutically effective in 50% of the population
  • LD50 the dose lethal to 50% of the population
  • LD50/ED50 Pharmaceutical 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 of such compounds lies preferably within a range of circulating concentrations that include the ED50 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 is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.
  • Additional factors which may be taken into account include the severity of the disease state, eg, tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may 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. See US Patent Nos. 4,657,760; 5,206,344; or 5,225,212. 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.
  • DAMP can be used to screen for therapeutic molecules which would ameliorate the adverse effects of inflammatory cells in autoimmune diseases.
  • the examples below are provided to illustrate the subject invention and are not included for the pu ⁇ ose of limiting the invention.
  • the osteoarthritic joint from a 82 year-old female used for cDNA library construction was obtained from the University of California Davis.
  • the frozen tissue was homogenized using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury NJ) and lysed in a buffer containing guanidinium isothiocyanate.
  • the lysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.
  • the RNA was extracted twice with acid phenol pH 4.0 using the reagents and extraction procedures as supplied in the
  • the cDNA was blunted with T4 polymerase, and a Sal I linker was added to the blunt ended cDNA.
  • the Sal I adapted, double stranded cDNAs were the digested with Not I and fractionated on a Sepharose CL4B column (Catalog #275105, Pharmacia). Those cDNAs exceeding 400 bp were ligated into pSport I which was subsequently transformed into DHSa -1 competent cells (Catalog #18258-012, Gibco/BRL).
  • Plasmid DNA was released from the cells and purified using the Miniprep Kit (Catalog #77468; Advanced Genetic Technologies Co ⁇ oration, Gaithersburg MD). This kit consists of a 96-well block with reagents for 960 purifications.
  • the recommended protocol was employed except for the following changes: 1) the 96 wells were each filled with only 1 ml of sterile Terrific Broth (Catalog #2271 1, LIFE TECHNOLOGIESTM) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria were cultured for 24 hours after the wells were inoculated and then lysed with 60 ml of lysis buffer; 3) a centrifugation step employing the Beckman GS-6R rotor at 2900 ⁇ m for 5 minutes was performed before the contents of the block were added to the primary filter plate; and 4) the optional step of adding isopropanol to TRIS buffer was not routinely performed. After the last step in the protocol, samples were transferred to a Beckman 96-well block for storage.
  • the cDNAs were sequenced by the method of Sanger F and AR Coulson ( 1975; J Mol Biol 94:44 If), using a Hamilton Micro Lab 2200 (Hamilton, Reno NV) in combination with four Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown MA) and Applied Biosystems 377 or 373 DNA Sequencing Systems, and the reading frame was determined.
  • Each cDNA was compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and inco ⁇ orated into the INHERIT- 670 Sequence Analysis System.
  • Pattern Specification Language TRW Inc, Los Angeles CA
  • the three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance.
  • the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith- Waterman alignments were used to display the results of the homology search.
  • Peptide and protein sequence homologies were ascertained using the INHERIT TM 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dot-matrix- homology plots were examined to distinguish regions of significant homology from chance matches.
  • BLAST which stands for Basic Local Alignment Search Tool (Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al (1990) J Mol Biol 215:403-10), was used to search for local sequence alignments.
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. BLAST is useful for matches which do not contain gaps.
  • the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).
  • An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
  • the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
  • the parameter E establishes the statistically significant threshold for reporting database sequence matches.
  • E is inte ⁇ reted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook et al. supra).
  • Analogous computer techniques using BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQTM database (Incyte, Palo Alto CA). This analysis is much faster than multiple, membrane- based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • the basis of the search is the product score which is defined as:
  • homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • the nucleic acid sequence of SEQ ID NO:2 is used to design oligo-nucleotide primers for extending a partial nucleotide sequence to full length or for obtaining 5 'sequence from genomic libraries.
  • One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF).
  • Primers allow the extension of the know sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest (US Patent Application 08/487,1 12, filed June 7, 1995, specifically inco ⁇ orated by reference).
  • the initial primers are designed from the cDNA using OLIGO ® 4.06 Primer Analysis Software (National Biosciences), or another appropriate program, to be 22-30 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. Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations is avoided.
  • the original, selected cDNA libraries, or a human genomic library are used to extend the sequence; the latter is most useful to obtain 5' upstream regions. If more extension is necessary or desired, additional sets of primers are designed to further extend the known region.
  • PCR is performed using the Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the following parameters:
  • Step 1 94° C for 1 min (initial denaturation)
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 7 Repeat step 4-6 for 15 additional cycles
  • Step 8 94° C for 15 sec
  • Step 9 65° C for 1 min
  • Step 10 68° C for 7: 15 min
  • Step 1 Repeat step 8-10 for 12 cycles
  • Step 13 4° C (and holding) A 5-10 ⁇ aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were selected and cut out of the gel. Further purification involves using a commercial gel extraction method such as
  • the whole transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra) containing 2xCarb. The following day, several colonies are randomly picked from each plate and cultured in 150 ⁇ l of liquid LB/2xCarb medium placed in an individual well of an appropriate, commercially-available, sterile 96-well microtiter plate. The following day, 5 ⁇ l of each overnight culture is transferred into a non-sterile 96-well plate and after dilution 1 : 10 with water, 5 ⁇ l of each sample is transferred into a PCR array.
  • LB Luria Bertani
  • 2xCarb 2xCarb
  • PCR amplification For PCR amplification. 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer and one or both of the gene specific primers used for the extension reaction are added to each well. Amplification is performed using the following conditions:
  • Step 1 94° C for 60 sec
  • Step 2 94° C for 20 sec
  • Step 4 72 ° C for 90 sec Step 5 Repeat steps 2-4 for an additional 29 cycles
  • Hybridization probes derived from SEQ ID NO:2 are employed to screen cDNAs, genomic DNAs or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of each oligomer and 250 mCi of [ ⁇ - 32 P] adenosine triphosphate (Amersham, Chicago IL) and T4 polynucleotide kinase (DuPont NEN ® , Boston MA).
  • the labeled oligonucleotides are substantially purified with Sephadex G-25 super fine resin column (Pharmacia). A portion containing 10 7 counts per minute of each of the sense and antisense oligonucleotides is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II. Eco RI, Pst I. Xba 1. or Pvu II; DuPont NEN ® ).
  • the sequence encoding DAMP is used to inhibit in vivo or in vitro expression of naturally occurring sequence.
  • antisense oligonucleotides comprising about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments.
  • An oligonucleotide complementary to a portion of the coding sequence of DAMP as shown in SEQ ID NO:2 is used to inhibit expression of naturally occurring sequence.
  • the complementary oligonucleotide is designed from the most unique 5' sequence and used either to inhibit transcription by preventing promoter binding to the upstream nontranslated sequence or translation of a transcript encoding DAMP by preventing the ribosome from binding.
  • an effective antisense oligonucleotide includes any 15-20 nucleotides spanning the region which translates into the signal or early coding sequence of the polypeptide as shown in Figures 1 A and IB.
  • VIII Expression of DAMP Expression of DAMP is accomplished by subcloning the cDNAs into appropriate vectors and transfecting the vectors into host cells. In this case, the cloning vector, pSport, previously used for the generation of the cDNA library is used to express DAMP in £. coli.
  • this vector Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing 5 the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
  • Induction of an isolated, transfected bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first seven residues of ⁇ -galactosidase, about 5 to 10 15 residues of linker, and the full length DAMP.
  • the signal sequence directs the secretion of DAMP into the bacterial growth media which can be used directly in the following assay for activity.
  • the chemotactic activity of DAMP is measured under agarose according to the method of 15 Nelson et al (1975; J Immunol 1 15: 1650). Mononuclear cells or granulocytes are exposed to serial dilutions of control media or media in which cells expressing recombinant DAMP were grown. After 2 hours incubation at 37°C, cells are fixed and stained. Spontaneous migration toward the control sample is compared to migration toward the test sample for various cell types. The specificity of the chemoattraction is determined by performing the agarose assay on 0 specific populations of cells.
  • Blood cells obtained from venipuncture are fractionated by density gradient centrifugation and the chemotactic activity of DAMP is tested on enriched populations of neutrophils, peripheral blood mononuclear cells, granulocytes, monocytes and lymphocytes.
  • enriched cell populations may be further fractionated using CD8 + and CD4 + specific antibodies for negative selection of CD4 + and CD8 + enriched T-cell populations, 5 respectively.
  • DAMP is substantially purified using PAGE electrophoresis (Sambrook, supra) is used to immunize rabbits and to produce antibodies using standard protocols.
  • the amino acid sequence translated from DAMP is analyzed using DNAStar software (DNAStar Inc) to determine regions 0 of high immunogenicity and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • Analysis to select appropriate epitopes, such as those near the C-terminus or in hydrophilic regions (shown in Figure 4) is described by Ausubel FM et al (supra).
  • the oligopeptides are 15 residues in length, synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-che istry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel FM et al, supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
  • Naturally occurring or recombinant DAMP is substantially purified by immunoaffinity chromatography using antibodies specific for DAMP.
  • An immunoaffinity column is constructed by covalently coupling DAMP antibody to an activated chromatographic resin such as CnBr-activated Sepharose (Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • Membrane fractions from cells expressing DAMP are prepared by methods well known in the art. Alternatively, a recombinant DAMP fragment containing an appropriate signal sequence may be secreted in useful quantity into the medium in which transfected cells are grown.
  • the DAMP-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of DAMP (eg, high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/DAMP binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and DAMP is collected.

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Abstract

L'invention porte sur un polynucléotide identifiant et codant pour une nouvelle protéine associée à certaines maladies de l'homme (DAMP), et sur la DAMP elle-même. Elle porte également sur des vecteurs d'expression et des cellules hôtes obtenus par génie génétique et comportant la séquence nucléotidique codant pour la DAMP. Elle porte de plus sur des préparations pharmaceutiques contenant des DAMP et leur utilisation pour traiter le cancer. Elle porte en outre sur des essais diagnostiques dans lesquels on utilise ledit polynucléotide pour l'hybrider à des produits de transcription codant pour la DAMP, ou pour des anticorps anti-DAMP se fixant spécifiquement à la DAMP.
PCT/US1997/015924 1996-09-11 1997-09-10 Proteines membranaires associees a des maladies (damp) WO1998011219A1 (fr)

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AU42612/97A AU4261297A (en) 1996-09-11 1997-09-10 Disease associated membrane protein (damp)

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US71402796A 1996-09-11 1996-09-11
US08/714,027 1996-09-11

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006085482A1 (fr) * 2005-02-10 2006-08-17 Riken Facteur d'autoreplication et procede d’amplification d’une cellule souche hematopoietique
EP1578980A4 (fr) * 2002-04-01 2013-01-02 Agensys Inc Acide nucleique et proteine correspondante 213p1f11 que l'on utilise dans le traitement et la detection du cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BEN-PORATH I ET AL: "Characterization of a tumor-associated gene, a member of a novel family of genes encoding membrane glycoproteins", GENE, vol. 183, no. 1, 1996, pages 69-75, XP004062729 *
BOLIN, LAUREL M. ET AL: "HNMP-1: a novel hematopoietic and neural membrane protein differentially regulated in neural development and injury", J. NEUROSCI., vol. 17, no. 14, 15 July 1997 (1997-07-15), pages 5493 - 5502, XP002048248 *
TAYLOR V ET AL: "Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family", GENE, vol. 175, no. 1, 10 October 1996 (1996-10-10), pages 115-120, XP004043302 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1578980A4 (fr) * 2002-04-01 2013-01-02 Agensys Inc Acide nucleique et proteine correspondante 213p1f11 que l'on utilise dans le traitement et la detection du cancer
WO2006085482A1 (fr) * 2005-02-10 2006-08-17 Riken Facteur d'autoreplication et procede d’amplification d’une cellule souche hematopoietique

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