EP0840787A2 - Menschlicher edg-2 rezeptorhomolog - Google Patents

Menschlicher edg-2 rezeptorhomolog

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Publication number
EP0840787A2
EP0840787A2 EP96945463A EP96945463A EP0840787A2 EP 0840787 A2 EP0840787 A2 EP 0840787A2 EP 96945463 A EP96945463 A EP 96945463A EP 96945463 A EP96945463 A EP 96945463A EP 0840787 A2 EP0840787 A2 EP 0840787A2
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EP
European Patent Office
Prior art keywords
hedg
polypeptide
leu
expression
ser
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EP96945463A
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English (en)
French (fr)
Inventor
Jeffrey J. Seilhamer
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Incyte Corp
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Incyte Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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 is in the field of molecular biology; more particularly, the present invention describes a nucleic acid sequence and an amino acid sequence for a novel human EDG-2 receptor homolog.
  • the EDG-2 receptor is a putative G-protein coupled seven transmembrane receptor (T7G) which was initially cloned from sheep mRNA (GenBank U 18405; asana Ml et ai (1994) unpublished).
  • Human edg-1 is commonly grouped with orphan receptors because its endogenous ligand is not known (Hla T and Maciag T (1990) J Biol Chem 265:9308-13).
  • T7G receptors have been classified as orphan receptors; they include LCR-1 from brain, the mas oncogene associated with epidermoid carcinoma, RDC-1 known from several major organs, and R334 from rat brain and testis.
  • the orphan receptors vary in number of amino acids, in molecular weight, in glycosylation sites, and presence and number of disulfide bonds (Watson S and Arkinstall S (1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego CA).
  • T7Gs transmembrane segments
  • TMS transmembrane segments
  • the activated receptor interacts with an intracellular G-protein complex which mediates further intracellular signalling activities generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate or ion channel proteins.
  • cAMP cyclic AMP
  • phospholipase C phospholipase C
  • inositol triphosphate ion channel proteins
  • T7G receptors are expressed and activated during numerous developmental and disease processes. Identification of a novel T7G receptor provides the opportunity to diagnose or intervene in such processes, and the receptor can be used in screening assays to identify physiological or pharmaceutical molecules which trigger, prolong or inhibit its activity.
  • the subject invention provides a unique nucleotide sequence which encodes a novel human EDG-2 receptor homolog (HEDG).
  • HEDG human EDG-2 receptor homolog
  • the cDNA herein designated hedg, was identified and cloned using Incyte Clone No. 80853 from a rheumat synovium cDNA library.
  • the invention relates to the use of nucleic acid and amino acid sequences of HEDG, or its variants, in the diagnosis or treatment of activated, inflamed or diseased cells and/or tissues associated with its expression.
  • aspects of the invention include the antisense DNA of hedg; cloning or expression vectors containing hedg; host cells or organisms transformed with expression vectors containing hedg; a method for the production and recovery of purified HEDG from host cells; and purified protein, HEDG, which can be used to identify inhibitors for the downregulation of signal transduction involving HEDG.
  • Figures 1A and 1 B shows the alignment of the nucleic acid sequence (coding region of SEQ ID NO: 1 ) and amino acid sequence (SEQ ID NO:2) for HEDG.
  • the alignment of the sequences was produced using MacDNAsis software (Hitachi Software Engineering Co Ltd)
  • Figure 2 displays the alignment of HEDG with sheep EDG-2 (U18405; SEQ ID NO:3) and human EDG-1 (Gl 119130; SEQ ID NO:4) receptors. Note the conserved Arg 36 and Ser 37 cleavage site characteristic of these T7G molecules. Sequences for Fig. 2 were aligned using the multisequence alignment program of DNAStar software (DNAStar Inc, Madison Wl).
  • HEDG refers to an EDG2 receptor homolog in either naturally occurring or synthetic form and active fragments thereof which have the amino acid sequence of SEQ ID NO:2.
  • the polypeptide HEDG is encoded by mRNAs transcribed from the cDNA, as designated by the Iower case abbreviation, hedg, of SEQ ID NO:1.
  • the novel human edg-2 receptor homolog, HEDG which is the subject of this patent application, was discovered among the partial cDNA sequences (Incyte Clone 80853) expressed in a rheumatoid synovium library. It is more distantly homologous to human edg-1 which was cloned from human vascular endothelial cells and expressed in epithelioid cells, fibroblasts, melanocytes, and vascular smooth muscle cells.
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double- stranded nucleic acids or be chemically synthesized. They are useful in detecting the presence of identical or similar sequences.
  • a "portion” or “fragment” of a polynucleotide or nucleic acid comprises all or any part of the nucleotide sequence having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb which can be used as a probe.
  • Such probes may be labelled with reporter molecules using, nick translation, Klenow fill-in reaction, PCR or other methods well known in the art.
  • nucleic acid probes may be used in Southern, northern or in sjtjj hybridizations to determine whether DNA or RNA encoding HEDG is present in a cell type, tissue, or organ.
  • Reporter molecules are those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with, establish the presence of, and may allow quantification of a particular nucleotide or amino acid sequence.
  • Recombinant nucleotide variants encoding HEDG may be synthesized by making use of the "redundancy" in the genetic code.
  • Various codon substitutions such as the silent changes whichvproduce specific restriction sites or codon usage-specific mutations, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic host system, respectively.
  • Chimeric molecules may be constructed by introducing ail or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one (or more than one) of the following HEDG characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signalling, etc.
  • Active refers to those forms, fragments, or domains of any HEDG polypeptide which retain the biologic and/or antigenic activities of any naturally occurring HEDG.
  • HEDG “Naturally occurring HEDG” refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation. carboxylation, glycosylation, phosphorylation, iipidation and acylation.
  • Derivative refers to those polypeptides which have been chemically modified by such techniques as ubiquitination, labelling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Recombinant polypeptide variant refers to any polypeptide which differs from naturally occurring HEDG by amino acid insertions, deletions and/or substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest may be found by comparing the sequence of HEDG with that of related polypeptides and minimizing the number of amino acid sequence changes made in highly conserved regions.
  • substitutions are conservative in nature when they result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the hedg sequence using recombinant DNA techniques.
  • a "signal or leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. It may be functionally equivalent to and the same length as (or considerably shorter than) a "fragment,” “portion,” or “segment” of a polypeptide. Such sequences comprise a stretch of amino acid residues of at least about 5 amino acids and often about 17 or more amino acids, typically at least about 9 to 13 amino acids, and of sufficient length to display biologic and/or antigenic activity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • ⁇ Animal as used herein may be defined to include human, domestic (cats dogs, etc.), agricultural (cows, horses, sheep, etc) or test species (mouse, rat, rabbit, etc).
  • the present invention provides a nucleotide sequence uniquely identifying a novel seven transmembrane receptor, human EDG-2 or HEDG. Because HEDG is specifically expressed in inflamed rheumatoid synovium, the nucleic acids (hedg), polypeptides (HEDG) and antibodies to
  • HEDG are useful in diagnostic assays which survey for increased receptor production. Excessive expression of HEDG is likely to be associated with the activation of T lymphocytes and other cells which respond to inflammation and can result in the production of abundant proteases and other molecules which can lead to tissue damage or destruction. Therefore, a diagnostic test for excessive expression of HEDG can accelerate diagnosis and proper treatment of abnormal conditions caused by viral, bacterial or fungal infections; allergic responses; mechanical injury associated with trauma; hereditary diseases; lymphoma or carcinoma; or other conditions which activate the genes of lymphoid tissues.
  • nucleotide sequences encoding HEDG have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of HEDG, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding HEDG disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • 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, e.g., the triplet genetic code, specific base pair interactions, etc.
  • HEDG-encoding nucleotide sequences may be produced. Some of these will only bear minimal homology to the nucleotide sequence of the known and naturally occurring HEDG.
  • the invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring hedg, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences which encode HEDG, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring hedg under stringent conditions, it may be advantageous to produce nucleotide sequences encoding HEDG or its derivatives possessing a substantially different codon usage. Codons can 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.
  • Nucleotide sequences encoding HEDG may be joined to a variety of other nucleotide sequences by means of well established recombinant BNA techniques (Sambrook J et al ⁇ 1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY; or Ausubel FM et al (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York City).
  • Useful nucleotide sequences for joining to hedg include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for hedg-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding HEDG. Such probes may also be used for the detection of similar T7G encoding sequences and should preferably contain at least 50% of the nucleotides from the hedg sequence.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ ID NOM of- from genomic sequences including promoter, enhancers or introns of the native gene.
  • Hybridization probes may be labeled by a variety of reporter molecules using techniques well known in the art. PCR as described US Patent Nos.
  • oligonucleotides based upon the nucleotide sequence which encodes HEDG.
  • probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of hedg in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.
  • nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are well known in the art.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • the nucleotide sequence for hedg can be used in an assay -to detect inflammation or disease associated with abnormal levels of HEDG expression.
  • the cDNA can be labeled by methods known in the*art, added to a fluid, cell or tissue sample from a patient, and incubated under hybridizing conditions. After an incubation period, the sample is washed with a compatible fluid which optionally contains a reporter molecule. After the compatible fluid is rinsed off, the reporter molecule is quantitated and compared with a standard as previously defined. If kinase expression is significantly different from standard expression, the assay indicates inflammation or disease.
  • the nucleotide sequence for hedg can be used to construct hybridization probes for mapping the native gene.
  • the gene may be mapped to a particular chromosome or to a specific region of a chromosome using well known mapping techniques. These techniques include in situ hybridization of chromosomal spreads (Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City), flow-sorted chromosomal preparations, or artificial chromosome constructions such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions or single chromosome cDNA libraries.
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers are invaluable in extending genetic maps. Examples of genetic map data can be found in the yearly genome issue of Science (eg 1994, 265:1981 f). Often locating a gene on the chromosome of another mammalian species may reveal associated markers which can be used to help identify the analogous human chromosome. New nucleotide sequences can be assigned to chromosomal subregions by physical mapping. The mapping of new genes or nucleotide sequences provide useful landmarks for investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • AT ataxia telangiectasia
  • genomic region for example, AT to 11q22-23 (Gatti et al (1988) Nature 336:577-580)
  • any sequences mapping to that area may represent or reveal genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in gene sequence between normal and carrier or affected individuals.
  • Nucleotide sequences encoding hedg may be used to produce a purified oligo- or polypeptide using well known methods of recombinant DNA technology. Goeddel (1990, Gene
  • oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
  • HEDG may be cultured under conditions suitable for the expression of T7Gs, their extracellular, transmembrane or intracellular domains and recovery of such peptides from cell culture.
  • HEDG (or any of its domains) produced by a recombinant cell may be secreted or may be contained intracellularly, depending on the particular genetic construction used. In general, it is more convenient to prepare recombinant proteins in secreted form. Purification steps vary with the production process and the particular protein produced. Often an oligopeptide can be produced from a chimeric nucleotide sequence.
  • fragments of HEDG may be produced by direct peptide synthesis using solid-phase techniques (eg Stewart et al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco CA; Merrifield J (1963) J Am Chem Soc
  • HEDG 85:2149-2154. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Foster City, CA) in accordance with the instructions provided by the manufacturer. Additionally, a particular portion of HEDG may be mutated during direct synthesis and combined with other parts of the peptide using chemical methods. jv- ⁇ EDG for antibody induction does not require biological activity; however, the protein must be antigenic. Peptides used to induce specific antibodies may have an aa sequence consisting of at least five aa, preferably at least 10 aa. They should mimic a portion of the aa sequence of the protein and may contain the entire aa sequence of a small naturally occurring molecule such as HEDG. An antigenic portion of HEDG may be fused to another protein such as keyhole limpet hemocyanin, and the chimeric molecule used for antibody production.
  • Antibodies specific for HEDG may be produced by inoculation of an appropriate animal with the polypeptide or an antigenic fragment.
  • An antibody is specific for HEDG if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein.
  • Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous processes such as the production of synthetic antibodies, the screening of recombinant immunoglobulin libraries for specific-binding molecules (eg Orlandi R et al (1989) PNAS 86:3833-3837, or Huse WD et al (1989) Science 256:1275-1281) or the in vitro stimulation of lymphocyte populations.
  • Current technology (Winter G and Milstein C (1991) Nature 349:293-299) provides for a number of highly specific binding reagents based on the principles of antibody formation. These techniques may be adapted to produce molecules which specifically bind HEDGs.
  • An additional embodiment of the subject invention is the use of HEDG specific antibodies, inhibitors, receptors or their analogs as bioactive agents to treat inflammation or disease including, but not limited to viral, bacterial or fungal infections; allergic responses; mechanical injury associated with trauma; hereditary diseases; lymphoma or carcinoma; or other conditions which activate the genes of lymphoid tissues.
  • Bioactive compositions comprising agonists, antagonists, receptors or inhibitors of HEDG may be administered in a suitable therapeutic dose determined by any of several methodologies including clinical studies on mammalian species to determine maximal tolerable dose and on normal human subjects to determine safe dose. Additionally, the bioactive agent may be complexed with a variety of well established compounds or compositions which enhance stability or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be delivered by intravenous infusion into the bloodstream or any other effective means which could be used for treating problems involving excessive lymphocyte and leukocyte trafficking.
  • HEDG Rheumatoid arthritis is currently evaluated on the basis of swelling, response to NSAIDs, x-rays, etc.
  • HEDG is most likely expressed on the surface of the fibroblasts, T and B lymphocytes, monocyte/macrophages, or mast cells which comprise the cells of the inflamed synovium.
  • an assay for the abnormal expression of HEDG is a viable diagnostic tool for assessing the extent that RA has progressed.
  • the expression of HEDG in- a sustained inflammatory response makes it a valuable therapeutic target for screening drug libraries.
  • Inhibitors of HEDG are useful for controlling signal transduction and signaling cascades in cells of the rheumatoid synovium.
  • the hedg sequence of this application was first identified in Incyte Clone No. 80853 among the sequences comprising the rheumatoid synovium library.
  • Rheumatoid synovial tissue was obtained from the hip joint removed from a 68 year old female with erosive, nodular rheumatoid arthritis. The tissue was frozen, ground to powder in a mortar and pestle, and lysed immediately in buffer containing guanidinium isothiocyanate. Lysis was followed by several phenol-chloroform extractions and ethanol precipitations.
  • Poly-A+ mRNA was isolated using biotinylated oligo d(T) and streptavidin coupled to paramagnetic particles (Poly(A) Tract Isolation System, Promega, Madison Wl). Using this Poly-A+ mRNA, a custom cDNA library was constructed by Stratagene (La Jolla CA). Synthesis of cDNA was primed with oligo d(T), and adapter oligonucleotides were ligated onto the cDNA molecules enabling them to be inserted into the Uni-ZAPTM vector system (Stratagene). Altemative unidirectional vectors might include, but are not limited to, pcDNAI (invitrogen, San Diego CA) and pSHIox-1 (Novagen, Madison Wl).
  • the phagemid forms of individual cDNA clones were obtained by the in vivo excision process, in which the host bacterial strain was co-infected with both the library phage and an f1 helper phage.
  • Polypeptides or enzymes derived from both the library-containing phage and the helper phage nicked the DNA, initiated new DNA synthesis from defined sequences on the target DNA, and created a smaller, single stranded circular phagemid DNA molecule that included all DNA sequences of the pBluescript phagemid and the cDNA insert.
  • the phagemid DNA was released from the cells, purified, and used to reinfect fresh host cells (SOLRTM, Stratagene) where double-stranded DNA was produced. DNA was purified using the QIAWELL-8 Plasmid Purification System (QIAGEN Inc,
  • the chain termination reaction products were electrophoresed on urea-acrylamide gels and detected either by autoradiography (for radionuclide-labeled precursors) or by fluorescence (for fluorescent-labeled precursors).
  • autoradiography for radionuclide-labeled precursors
  • fluorescence for fluorescent-labeled precursors
  • Peptide and protein sequence homologies were ascertained using the INHERITTM 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattem 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, is used to search for local sequence alignments (Altschul SF (1993) J Mol Evol 36:290-300; Altschul,
  • BLAST produces alignments of both nucleotide and aa 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. Whereas it is ideal for matches which do not contain gaps, it is inappropriate for performing motif-style searching.
  • the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair
  • 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.
  • Incyte 80853 was identified as a homolog of sheep EDG-2 receptc-r.
  • the partial sequence displayed 92.6% identity with nucleotide sequence of accession V U18405 in GenBank (Masana Ml et al, supra).
  • the cONA insert comprising Incyte 80853 was fully sequenced and used as the basis for cloning the full length cDNA.
  • the primers allowed the sequence to be extended "outward" generating amplicons containing new, unknown nucleotide sequence for the gene of interest.
  • the primers were designed using Oligo 4.0 (National Biosciences Inc, Plymouth MN) 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 were avoided.
  • PCR was performed using the Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the following parameters:
  • Step 1 94° C for 60 sec (initial denaturation)
  • Step 2 94° C for 15 sec
  • Step 3 65° C for 1 min
  • Step 4 68° C for 7 min
  • Step 5 Repeat step 2-4 for 15 additional cycles
  • Step 7 65° C for 1 min
  • Step 8 68° C for 7 min + 15 sec/cycle
  • Step 9 Repeat step 6-8 for 11 additional cycles
  • Step 3 68° C for (10 min + 15 sec)/cycle
  • Step 4 Repeat step 1 -3 for 9 additional cycles Step 5 72° C for 10 min
  • reaction mixture A 5-10 ⁇ l aliquot of the reaction mixture was 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. Although all extensions potentially contain a full length gene, sqme of the largest products or bands were selected and cut out of the gel. Further purification involved using a commercial gel extraction method such as QIAQuickTM (QIAGEN Inc). After recovery of the DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitated religation and cloning.
  • QIAQuickTM QIAGEN Inc
  • Step 1 94° C for 60 sec
  • Step 2 94° C for 20 sec
  • Step 5 Repeat steps 2-4 for an additional 29 cycles
  • Step 6 72° C for 180 sec Step 7 4° C (and holding)
  • the nucleotide and aa sequences for human HEDG are shown in Figure 1.
  • the coding region of hedg begins at nucleotide 309 and ends at nucleotide 1403 of SEQ ID NO:1.
  • Figure 2 shows the comparison between the amino acid sequences of HEDG, sheep EDG-2 (U18405) and human EDG-1 (Gi 119130).
  • V I Antisense analysis Knowledge of the correct, complete cDNA sequence of HEDG enables its use as a tool for antisense technology in the investigation of gene function.
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense strand of hedg are used either in vitro or in vivo to inhibit expression of the mRNA.
  • antisense molecules can be designed at various locations along the nucleotide sequences.
  • the gene of interest is effectively tumed off.
  • the function of the gene is ascertained by observing behavior at the intracellular, cellular, tissue or organismal level (eg, lethality, loss of differentiated function, changes in morphology, etc).
  • hedg is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into analogous expression hosts.
  • the cloning vector previously used for the generation of the cDNA library also provides for direct expression of hedg sequences in J . coli. Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase. Immediately following these eight residues is an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • Induction of the isolated, transfected bacterial strain with IPTG using standard methods produces a fusion protein corresponding to the first seven residues of ⁇ -galactosidase, about 15 residues of "linker", and the peptide encoded within the cDNA. Since cDNA clone inserts are generated by an essentially random process, there is one chance in three that the included cDNA will lie in the correct frame for proper translation. If the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods including in vitro mutagenesis, digestion with exonuclease III or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the hedg cDNA is shuttled into other vectors known to be useful for expression of protein in specific hosts.
  • Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR. The resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae. and bacteria such as £, £ ⁇ .
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as the rous sarcoma virus enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced HEDG are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • HEDG is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are 'hot 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 Corp, Seattle WA).
  • the inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen) between the purification domain and the HEDG sequence is useful to facilitate expression of HEDG. I X Testing of Chimeric T7Gs
  • Functional chimeric T7Gs are constructed by combining the extracellular receptive sequences of a new isoform with the transmembrane and intracellular segments of a known isoform for test purposes. This concept was demonstrated by Kobilka et al (1988, Science 240:1310-1316) who created a series of chimeric ⁇ 2- ⁇ 2 adrenergic receptors (AR) by inserting progressively greater amounts of ⁇ 2-AR transmembrane sequence into B2-AR. The binding activity of known agonists changed as the molecule shifted from having more oc2 than B2 conformation, and intermediate constructs demonstrated mixed specificity. The specificity for binding antagonists, however, correlated with the source of the domain Vll.
  • T7G domain Vll for ligand recognition was also found in chimeras utilizing two yeast ⁇ -factor receptors and is significant because the yeast receptors are classified as miscellaneous receptors. Thus, functional role of specific domains appears to be preserved throughout the T7G family regardless of category .
  • Chimeric or modified T7Gs containing substitutions in the extracellular and transmembrane regions have shown that these portions of the receptor determine ligand binding specificity.
  • two Ser residues conserved in domain V of all adrenergic and D catecholamine T7G receptors are necessary for potent agonist activity. These serines are believed to form hydrogen bonds with the catechol moiety of the agonists within the T7G binding site.
  • an Asp residue present in domain III of all T7Gs which bind biogenic amines is believed to form an ion pair with the ligand amine group in the T7G binding site.
  • T7Gs are expressed in heterologous expression systems and their biological activity assessed (eg Marullo et al (1988) Proc Natl Acad Sci 85:7551-55; King et al (1990) Science 250:121-23).
  • One heterologous system introduces genes for a mammalian cells
  • T7G and a mammalian G-protein into yeast cells.
  • the T7G is shown to have appropriate ligand specificity and affinity and trigger appropriate biological activation-growth arrest and morphological changes--of the yeast cells.
  • chimeric genes are created by combining sequences for extracellular receptive segments of any newly discovered T7G polypeptide with the nucleotides for the transmembrane and intracellular segments of the known P U molecule.
  • the P 2U system is useful for defining antagonists or inhibitors which block binding and prevent such fluorescent reactions.
  • denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 micrograms are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiodinated and used to screen potential murine B-cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate HEDG domain is analyzed to determine regions of high antigenicity.
  • Oligopeptides comprising appropriate hydrophilic regions are synthesized and used in suitable immunization protocols to raise antibodies. Analysis to select appropriate epitopes is described by Ausubel FM et al (supra).
  • the optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St Louis MO) by reaction with M-maleimidoben- zoyl-N- hydroxysuccinimide ester (MBS; Ausubel FM et al, supra). If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
  • KLH keyhole limpet hemocyanin
  • MVS M-maleimidoben- zoyl-N- hydroxysuccinimide ester
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled HEDG to identify those fusions producing the monoclonal antibody with the desired specificity.
  • wells of plates FAST; Becton-Dickinson, Palo Alto CA
  • affinity purified, specific rabbit anti-mouse (or suitable antispecies Ig) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1 % BSA, washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled HEDG at 1 mg/ml.
  • Monoclonal antibodies with affinities of at least 10 ⁇ M *1 , preferably 10 9 to 10 10 or stronger, are typically made by standard procedures as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and in Goding (1986) Monoclonal Antibodies: Principles and Practice, Academic Press, New York City, both incorporated herein by reference.
  • HEDG antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amount or distribution of HEDG or downstream products of an active signaling cascade. Since
  • HEDG was found in a human rheumatoid library, it appears to be upregulated in cell types mainly involved in immune protection or defense.
  • Diagnostic tests for HEDG include methods utilizing antibody and a label to detect HEDG in human body fluids, membranes, cells, tissues or extracts of such.
  • the polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like. Paterits teaching the use of such labels include US Patent Nos.
  • recombinant immunoglobulins may be produced as shown in US Patent No. 4,816,567, incorporated herein by reference.
  • a variety of protocols for measuring soluble or membrane-bound HEDG, 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 HEDG is preferred, but a competitive binding assay may be employed.
  • Native or recombinant HEDG is purified by immunoaffinity chromatography using antibodies specific for HEDG.
  • an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway NJ). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as
  • Such immunoaffinity columns are utilized in the purification of HEDG by preparing a fraction from cells containing HEDG in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble HEDG containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown. A soluble HEDG-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HEDG (eg, high ionic strength buffers in the presence of detergent).
  • HEDG eg, high ionic strength buffers in the presence of detergent
  • HEDG HEDG
  • a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion
  • a chaotrope such as urea or thiocyanate ion
  • HEDG HEDG binding fragments thereof in any of a variety of drug screening techniques.
  • the polypeptide or fragment employed in such a test is either free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment.
  • Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, are used for standard binding assays.
  • One measures for example, the formation of complexes between HEDG and the agent being tested.
  • the present invention provides methods of screening for drugs or any other agents which affect signal transduction. These methods, well known in the art, comprise contacting such an agent with HEDG polypeptide or a fragment thereof and assaying (i) for the presence of a complex between the agent and the HEDG polypeptide or fragment, or (ii) for the presence of a complex between the HEDG polypeptide or fragment and the cell. In such competitive binding assays, the HEDG polypeptide or fragment is typically labeled. After suitable incubation, free HEDG polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to HEDG or to interfere with the HEDG and agent complex.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the HEDG polypeptides and is described in detail in European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HEDG polypeptide and washed. Bound HEDG polypeptide is then detected by methods well known in the art. Purified HEDG are also coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies are used to capture the peptide and immobilize it on the solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding HEDG specifically compete with a test compound for binding to HEDG polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic determinants with HEDG.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (eg Hodgson J (1991 ) Bio/Technology 9:19-21 , incorporated herein by reference).
  • the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches.
  • Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design includes molecules which have improved activity or stability as shown by Braxton S and Wells JA (1992, Biochemistry 31 :7796- 7801) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda SB et al (1993 J Biochem 113:742-46), incorporated herein by reference.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • the inventive purified HEDG is a research tool for identification, characterization and purification of interacting G or other signal transduction pathway proteins. Radioactive labels are incorporated into a selected HEDG domain by various methods known in the art and used __ vitro to capture interacting molecules.
  • a preferred method involves labeling the primary amino groups in HEDG with 125 l Bolton-Hunter reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133: 529). This reagent has been used to label various molecules without concomitant loss of biological activity (Hebert CA et al (1991 ) J Biol Chem 266: 18989; McColl S et al (1993) J Immunol 150:4550-4555).
  • Labeled HEDG is useful as a reagent for the purification of molecules with which it interacts.
  • membrane-bound HEDG is covalently coupled to a chromatography column.
  • Cell-free extract derived from synovial cells or putative target cells is passed over the column, and molecules with appropriate affinity bind to HEDG.
  • HEDG-complex is recovered from the column, and the HEDG-binding ligand disassociated and subjected to N-terminal protein sequencing. This aa sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
  • antibodies are raised against HEDG, specifically monoclonal antibodies.
  • the monoclonal antibodies are screened to identify those which inhibit the binding of labeled HEDG. These monoclonal antibodies are then used therapeutically.
  • LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, half-life and antigenicity/immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are preferred as LSTs, but organic or synthetic molecules resulting from drug screens are equally effective in particular situations.
  • LSTs are delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation.
  • Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other drugs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations 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 LST. Normal dosage amounts 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.
  • abnormal signal transduction, trauma, or diseases which trigger HEDG activity are treatable with LSTs. These conditions or diseases are specifically diagnosed by the tests discussed above, and such testing should be performed in suspected cases of viral, bacterial or fungal infections; allergic responses; mechanical injury associated with trauma; hereditary diseases; lymphoma or carcinoma; or other conditions which activate the genes of lymphoid tissues.

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FR2759375B1 (fr) * 1997-02-13 1999-04-30 Inst Nat Sante Rech Med Polypeptide a activite de recepteur ob25 specifique des cellules myelinisantes chez le rat, application au criblage de medicaments et medicaments
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US5912144A (en) * 1997-04-24 1999-06-15 Incyte Pharmaceuticals, Inc. Edg-1-receptor homolog
US6020158A (en) * 1997-05-22 2000-02-01 Allelix Biopharmaceuticals, Inc. Isolated polynucleotide for novel G-protein coupled receptor
US6485922B1 (en) 1997-10-10 2002-11-26 Atairgin Technologies, Inc. Methods for detecting compounds which modulate the activity of an LPA receptor
WO1999019513A2 (en) * 1997-10-10 1999-04-22 Lxr Biotechnology, Inc. Methods for detecting compounds which modulate the activity of an lpa receptor
DE69825629T2 (de) 1997-12-30 2005-08-11 Nps Allelix Corp., Mississauga Säugertier edg-7 rezeptor homologen
AU1865499A (en) 1997-12-30 1999-07-26 Allelix Biopharmaceuticals Inc. Identification of lysolipid receptors involved in inflammatory response
EP1053323A2 (de) * 1998-02-12 2000-11-22 Incyte Pharmaceuticals, Inc. Menschliche rezeptorproteine
AU5575299A (en) * 1998-08-19 2000-03-14 Millennium Pharmaceuticals, Inc. 14274 receptor, a g-protein coupled receptor related to the edg receptor family
US6812335B1 (en) 1999-03-23 2004-11-02 The Regents Of The University Of California Human polypeptide receptors for lysophospholipids and sphingolipids and nucleic acids encoding the same
AU2002246680A1 (en) 2000-12-22 2002-07-30 University Of South Florida Sphingosine 1-phosphate receptor gene, sppr
AU2003278165A1 (en) * 2002-11-13 2004-06-03 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with human endothelial differentiation, lysophosphatidic acid g-protein-coupled receptor 2 (edg2)
US20070003928A1 (en) * 2002-12-12 2007-01-04 Shuji Hinuma Novel use of edg receptors
US20100239501A1 (en) * 2007-06-12 2010-09-23 Riken Gene sensitive to bone/joint disease and use thereof

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