WO2001085786A2 - Human histamine h4 receptor - Google Patents

Human histamine h4 receptor Download PDF

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Publication number
WO2001085786A2
WO2001085786A2 PCT/US2001/014527 US0114527W WO0185786A2 WO 2001085786 A2 WO2001085786 A2 WO 2001085786A2 US 0114527 W US0114527 W US 0114527W WO 0185786 A2 WO0185786 A2 WO 0185786A2
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Prior art keywords
receptor
sequence
nucleic acid
receptor protein
protein
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PCT/US2001/014527
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French (fr)
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WO2001085786A9 (en
WO2001085786A3 (en
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Philip G. Jones
Maria Blatcher
Shujian Wu
Mark H. Pausch
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Wyeth
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Priority to AU2001259513A priority Critical patent/AU2001259513A1/en
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Publication of WO2001085786A3 publication Critical patent/WO2001085786A3/en
Publication of WO2001085786A9 publication Critical patent/WO2001085786A9/en

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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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention discloses the identification of a novel histamine receptor, termed H 4 .
  • H H 2 histamine produces numerous physiological effects in the body through interaction with one of three different cell surface receptors, classified as H H 2 , and H 3 . These receptors belong to the guanine nucleotide binding protein coupled receptor (G-protein coupled receptors, GPCR) class.
  • G-protein coupled receptors GPCR
  • Stimulation of histamine H- receptors produces symptoms that are typically associated with physiological responses to allergic stimuli (Ash and Schild, Br. J. Pharmacol.
  • H- antagonists such as, for example, diphenhydramine.
  • H* antagonists are generally defined as "classical antihistamines". Classical antihistamines are the active ingredient in most over-the-counter allergy medications. Pharmacological studies indicate that agonist activation of these receptors stimulates the inositol phosphate pathway, and thus stimulates formation of inositol triphosphate (IP 3 ) and diacylglycerol (DAG).
  • IP 3 inositol triphosphate
  • DAG diacylglycerol
  • Histamine H 2 receptors have been shown to play a role in gastric acid secretions (Black et al, Nature 1972, 236:385).
  • Histamine H 2 receptor antagonists such as, for example, cimetidine and ranitidine, are often the active ingredient in over-the-counter and prescription drugs that are used to treat duodenal ulcers, gastric ulcers, heartburn, indigestion, and other disorders of the gastrointestinal tract.
  • Activation of histamine H 2 stimulates adenylyl cyclase activity and stimulates formation of c AMP.
  • Histamine H 3 receptors are a relatively new member of the histamine receptor class. These receptors were originally described as histamine autoreceptors.
  • H 3 receptors were shown to be located on presynaptic histamine nerve terminals and to control the rate of histamine synthesis and release in the brain (Arrang etal, Nature 1983, 302:832). Recent studies indicate that H 3 receptors also are present on non-histamine presynaptic nerve terminals. H 3 receptors may modulate the synthesis and release of other neurotransmitters such as dopamine, serotonin, acetylcholine, and norepinephrine. H 3 receptors also have been found in peripheral tissues. Histamine activation of H 3 receptor inhibits adenylyl cyclase activity and formation of cAMP.
  • H 3 receptor subtypes Leurs et al JPET 1996, 276:1009-1015; Gumming and Gjede, Brain Res. 1994, 641:203-207; Calpham and Kilpatrick, Br. J. Pharmacol 1996, 107:919-923; Schworer et al Naunyn-Schmiedeberg's Arch. Pharmacol. 1994, 350:375-379; Schkicker et al, Naunyn- Schmiedeberg's Arch. Pharmacol. 1996, 353:482-488).
  • the present invention contemplates an isolated histamine H 4 receptor protein having an amino acid sequence at least 51 % identical or comprising at least 10 contiguous amino acids from the sequence depicted in SEQ ID NO:2.
  • the receptor protein upon binding histamine or a histamine agonist the receptor protein inhibits second messenger formation.
  • the second messenger is cAMP.
  • the present application also discloses an isolated H 4 receptor protein having an amino acid sequence with at least 95% sequence identity to human H 4 receptor protein having an amino acid sequence as depicted in SEQ ID NO: 2.
  • the protein is a human H 4 receptor protein, e.g. , having an amino acid sequence as depicted in SEQ ID NO:2 or an allelic variant of that sequence.
  • nucleic acids e.g., cDNAs encoding the H 4 receptor proteins as discussed above.
  • an isolated nucleic acid encoding an H 4 receptor protein which nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence of at least 20 nucleotides identical to a corresponding nucleotide sequence ofthe same number of bases in SEQ ID NO:l or its complement.
  • the isolated nucleic acids encoding H 4 receptors can be part of vectors, e.g., for cloning, expression, and/or expansion.
  • An expression vector comprises the nucleic acid encoding the H 4 receptor protein operably associated with an expression control sequence.
  • the invention further provides host cells and non-human transgenic animals containing such an expressible vector, and methods for producing an H 4 receptor polypeptide using such host cells.
  • the invention provides an isolated nucleic acid, such as a primer or probe, of at least 10 bases having a nucleotide sequence identical to a corresponding nucleotide sequence ofthe same number of bases in SEQ ID NO:l, or its complement.
  • the invention also provides an antibody that specifically binds an H 4 receptor.
  • the present invention further contemplates a method for detecting expression of H 4 receptor, which method comprises detecting mRNA encoding H 4 receptor in a sample from a cell suspected of expressing H 4 receptor or detecting the H 4 receptor protein with an antibody ofthe invention.
  • the present invention also contemplates an assay system for identifying H 4 receptor ligands.
  • the assay system comprises a- sufficient number of transformed host cells to be able to detect an alteration in second messenger accumulation.
  • the second messenger is cAMP.
  • the present invention also contemplates a method for identifying a test compound that antagonizes or agonizes histamine H 4 receptors.
  • the method comprises detecting an alteration in the level of a second messenger in the assay system contacted with the test compound.
  • an increase in the level ofthe second messenger indicates that the test compound antagonizes the H 4 receptor.
  • a decrease in the level of the second messenger indicates that the test compound agonizes the H 4 receptor.
  • the present invention also discloses an isolated nucleic acid that specifically hybridizes under highly stringent conditions to the complement ofthe sequence depicted in SEQ ID NO:l, where the nucleic acid encodes a histamine H 4 receptor protein.
  • FIGS. 2A, 2B, and 2C (A) Agonist assay, Square: histamine; triangle: R- ⁇ - methylhistamine; inverted triangle: clobenpropit; diamond: thioperamide. (B) Antagonist assay.
  • Figure 4 H 4 expression in from 6 human T cell clones derived from a single human donor. Three of these clones were CD4+ cell clones (RG4.3B, RG4.3A, and RG4.3), two were CD8+ cell clones (RG8.1C and RG8.1A), and one was an NKT cell clone (RG1).
  • FIG. H 4 expression in bulk populations of CD4+ and CD8+ T cells isolated from three different individuals.
  • B-CD8 and B-CD4 were CD8+ and CD4+ cells, respectively, obtained from individual 1.
  • L-CD8 and L-CD4 were CD8+ and CD4+ cells, respectively, obtained from individual 2.
  • U-CD8 and U-CD4 were CD8+ and CD4+ cells, respectively, obtained from individual 3.
  • the present invention is based, in part, on discovery of a no vel histamine receptor, which has been termed H 4 .
  • the new histamine receptor was cloned from a human heart library using H 3 specific primers, but proved to be a new histamine receptor.
  • Yeast and human endothelial kidney (HEK) cells were transformed with a human H 4 expression vector.
  • Transformed cells in multi- ell plates were treated with test compounds, and regulation ofthe intracellular second messenger cyclic adenosine monophosphate (cAMP) formation was determined. Modulation of cAMP formation is ligand and concentration dependent.
  • cAMP second messenger cyclic adenosine monophosphate
  • H 4 shows homology to known G- protein coupled receptors. Specifically, the H 4 protein shows homology to biogenic-amine G- protein coupled receptors. As in other biogenic amine receptors, the present protein contains conserved aspartate residues within transmembrane (TM) domains 2 and 3 (at positions 61 and
  • a DY motif (D94 and Y95) in TM3 is found only in histamine and muscarinic receptors.
  • a DRY sequence motif that exists at the cytoplasmic interface of TM3 of all GPCRs also can be found in the present sequence (amino acids 111- 113). Also present are two conserved cysteine residues in the first and second extracellular loops (position 88 and 164), which are predicted to form a disulfide bond; two conserved tryptophan residues in TM4 and TM6 (positions 140 and
  • the putative TM domains are shown in Figure 1.
  • H 4 shares sequence identity with human H 3 receptors. Sequence comparison indicates an overall sequence identity of about 44% and overall sequence similarity of about 51%.
  • the present invention also contemplates an assay method and system for identifying selective H 4 receptor ligands. The method involves detecting binding of a test compound to isolated cell membranes containing the histamine H 4 receptor.
  • the assay system comprises transformed host cells that express H 4 receptors, where the number of cells in the assay system is sufficient to detect an alteration in second messenger accumulation.
  • the test system also includes an appropriate cell culture medium to permit cell growth and viability, and preferably tissue culture plates or arrays containing the host cells in cell culture medium.
  • the second messenger that is detected is cAMP.
  • the receptor is a human receptor.
  • the invention also discloses a method for identifying a test compound that antagonizes or agonizes histamine H 4 receptors.
  • the method comprises detecting an increase
  • the present invention advantageously provides H 4 protein, including fragments, derivatives, and analogs of H 4 ; H 4 nucleic acids, including oligonucleotide primers and probes, and H 4 regulatory sequences (especially an H 4 primer and splice sites with introns);
  • H 4 -specific antibodies and related methods of using these materials to detect the presence of H 4 proteins or nucleic acids, H 4 binding partners, and in screens for agonists and antagonists of H 4 .
  • an isolated nucleic acid means that the referenced material is removed from the environment in which it is normally found.
  • an isolated biological material can be free of cellular components, z ' .e., components of the cells in which the material is found or produced in nature.
  • an isolated nucleic acid includes a
  • an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
  • the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • a protein expressed from a vector in a cell, particularly a cell, in which the protein is normally not expressed is also a regarded as isolated.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in a cell or an organism.
  • An isolated material may be, but need not be, purified.
  • purified refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell;
  • a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure; and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • nucleic acids can be purified by precipitation, chromatography (including preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means.
  • Polypeptides and proteins can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and countercurrent distribution.
  • the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST.
  • the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
  • antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents.
  • Cells can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting [FACS]). Other purification methods are possible.
  • a purified material may contain less than about 50%), preferably less than about 75%), and most preferably less than about 90%, of the cellular components with which it was originally associated. The "substantially pure" indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art.
  • the term "about” or “approximately” means within a scientifically acceptable error range for a given value relative to the precision with which the value is or can be measured, e.g., within 20% > , preferably within 10%, and more preferably within 5%> of a given value or range.
  • the term can mean within an order of magnitude, preferably within 5 -fold and more preferably within 2-fold of a given value.
  • sample refers to a biological material which can be tested for the presence of H 4 protein or H 4 nucleic acids.
  • samples can be obtained from animal subjects, such as humans and non-human animals, and include tissue, especially muscle, biopsies, blood and blood products; plural effusions; cerebrospinal fluid (CSF); ascites fluid; and cell culture.
  • Non-human animals include, without limitation, laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animals such as dogs and cats; and, farm animals such as sheep, goats, pigs, horses, and cows.
  • ligand refers to a compound that recognizes and binds to a receptor binding site. In a specific embodiment, the ligand binds to the histamine H 4 receptors of the invention. Upon binding to the receptor, the ligand may produce agonist or antagonist functional effects.
  • agonist refers to a ligand that binds to the receptor and produces a functional effect similar to that produced by the endogenous ligand for the receptor.
  • the agonist at the histamine H 4 receptor produces an effect similar to that produced by histamine, the endogenous ligand (histamine) for the H 4 receptor.
  • examples of such agonists include, but are not limited to, R- ⁇ -methyl histamine and imetit.
  • antagonist refers to a ligand that binds to the receptor and blocks a functional effect produced by an agonist for the receptor or the endogenous ligand ofthe receptor.
  • Examples of such antagonists include, but are not limited to, thioperamide.
  • selective refers to the ability of a histamine H 4 agonist or antagonist to elicit a response from the H 4 receptor while eliciting minimal responses from another receptor.
  • a selective H 4 agonist may be a potent agonist for the H 4 receptor while agonizing another receptor, such as another G-protein coupled receptor and particularly another histamine receptor, poorly or not at all.
  • the term "ability to elicit a response” refers to the ability of a H 4 agonist or antagonist ligand to agonize or antagonize H receptor activity.
  • transformed cell refers to a modified host cell that expresses a functional H 4 receptor expressed from a vector encoding the histamine receptor.
  • a "functional histamine receptor” is a receptor that binds histamine or H 4 agonists and transduces a signal upon such binding.
  • the H 4 receptor is a human H 4 .
  • the signal that is transduced is accumulation of a second messenger, preferably cAMP.
  • Histamine H 4 receptors may be derived from a variety of sources, including mammal, e.g., human, bovine, porcine, and canine; and avian.
  • the cells of the invention are particularly suitable for an assay system for histamine H 4 receptor ligands that modulate second messenger accumulation.
  • An "assay system" for histamine H 4 receptor ligands that modulate second messenger accumulation.
  • ' is one or more collections of such cells, e.g., in a microwell plate or some other culture system.
  • the number of cells in a single assay system is sufficient to express a detectable amount of the regulated second messenger accumulation at least under conditions of maximum second messenger accumulation.
  • a "second messenger” is an intracellular molecule or ion, where formation and/or accumulation ofthe second messenger is regulated by activation of cellular membranes.
  • cellular membranes contain G-protein coupled receptor, ion channels, and tyroinse kinase receptors.
  • the cellular membrane is a G-protein coupled receptor, preferably a histamine H 4 receptor.
  • the second messenger is one or more of cAMP, cGMP, inositol phosphate, DAG, and ions such as calcium and potassium.
  • the second messenger is cAMP.
  • test compound or “candidate compound” is any molecule that can be tested for its ability to bind H 4 receptors, and preferably modulate second messenger accumulation through the H 4 receptor, as set forth herein.
  • H 4 is a "lead compound" suitable for further testing and development as an H 4 agonist or antagonist.
  • the term "provide” refers to supplying the compounds or pharmaceutical compositions of the present invention to cells or to an animal, preferably a human, in any form.
  • a prodrug form ofthe compounds may be provided the subj ect, which then is metabolized to the compound in the body.
  • H 4 receptor refers to a polypeptide having about 390 amino acids.
  • H 4 refers to orthologs and allelic variants, e.g. , a protein having greater than about 50%), preferably greater than 80%, more preferably still greater than 90%, and even more preferably greater than 95% overall sequence identity to SEQ ID NO: 2.
  • Allelic variants may differ from 1 to about 5 amino residues from SEQ ID NO:2.
  • H 4 has an amino acid sequence as shown in SEQ ID NO: 2.
  • H 4 receptors like H 2 and H 3 receptors, modulates adenylyl cyclase activity. Therefore, the receptor modulates accumulation of the intracellular messenger cAMP. Modulation of H 4 receptors may be a treatment for transplanted organ rejection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis. Additionally, CNS functions such as cognitive and memory defects may be treated.
  • H 4 fragments, derivatives, and analogs can be characterized by one or more of the characteristics of H 4 protein.
  • antibodies in order to develop the specific C-terminal and N-terminal H 4 antibodies, antibodies can be raised against extracellular or cytoplasmic portions of the H 4 protein, preferably or antigenic peptides identified using a hydrophobicity profile or other algorithms.
  • Analogs and derivatives of the H 4 receptor of the invention have the same or homologous characteristics of H 4 as set forth above.
  • a truncated form of H 4 can be provided. Such a truncated form includes H 4 with a either an N-terminal, C-terminal, or internal deletion.
  • the derivative is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type H 4 of the invention.
  • Such functions include, but are not limited to, inhibition of adenylyl cyclase activity and cAMP formation.
  • a H 4 chimeric fusion protein can be prepared in which the H 4 portion of the fusion protein has one or more characteristics of H 4 .
  • Such fusion proteins include fusions ofthe H 4 receptor with a marker polypeptide, such as FLAG, a histidine tag, a myc tag, or glutathione-S-transferase (GST).
  • the H 4 receptor can be fused with an expression-related peptide, such as yeast ⁇ -mating factor, a heterogeneous signal peptide, or a peptide that renders the protein more stable upon expression.
  • H 4 can also be fused with a unique phosphorylation site for labeling.
  • H 4 analogs can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally similar molecules, i.e., molecules that perform one or more H 4 functions.
  • an analog of H 4 is a sequence-conservative variant of H 4 .
  • an analog of H 4 is a function- conservative variant.
  • an analog of H 4 is an allelic variant or a homologous variant from another species.
  • human variants of H 4 are described.
  • H 4 derivatives include, but are by no means limited to, phosphorylated H 4 , glycosylated H 4 , methylated H 4 , and other H 4 proteins that are otherwise chemically modified.
  • H 4 derivatives also include labeled variants, e.g., radio-labeled with iodine (or, as pointed out above, phosphorous); a detectable molecule, such as but by no means limited to biotin, a chelating group complexed with a metal ion, a chromophore " br fluorophore, a gold colloid, or a particle such as a latex bead; or attached to a water soluble polymer.
  • the present invention contemplates analysis and isolation of a gene encoding a functional or mutant H 4 , including a full length, or naturally occurring form of H 4 , and any antigenic fragments thereof from any source, preferably human. It further contemplates expression of functional or mutant H 4 protein for evaluation, diagnosis, or therapy.
  • PCR polymerase chain reaction
  • sequence-specific oligonucleotides refers to related sets of oligonucleotides that can be used to detect allelic variations or mutations in the H 4 gene.
  • nucleic acid molecules herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
  • the nucleic acids may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include methylation, "caps”, substitution of one or more ofthe naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • the polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly.
  • exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, results in ' the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme.
  • a coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
  • gene also called a "structural gene” means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include introns and regulatory DNA sequences, such as promoter sequences, 5 '-untranslated region, or 3'-untranslated region which affect for example the conditions under which the gene is expressed.
  • promoter sequence is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site
  • the present invention includes the H 4 receptor gene promoter found in the genome, which can be operatively associated with a H 4 coding sequence with a heterologous coding sequence.
  • the term "host cell” means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays, as described infra.
  • a coding sequence is "under the control of or “operatively associated with” transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if it contains introns) and translated, in the case of mRNA, into the protein encoded by the coding sequence.
  • express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product” such as a protein.
  • the expression product itself e.g., the resulting protein, may also be said to be “expressed” by the cell.
  • An expression product can be characterized as intracellular, extracellular, transmembrane, or secreted.
  • intracellular means something that is inside a cell.
  • extracellular means something that is outside a cell.
  • the H 4 receptor is a seven transmembrane protein with intracellular and extracellular domains.
  • transfection means the introduction of a foreign nucleic acid into a cell.
  • transformation means the introduction of a "foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
  • the introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery.
  • the gene or sequence may include nonfunctional sequences or sequences with no known function.
  • a host cell that receives and expresses introduced DNA or RNA has been "transformed” and is a "transformant” or a “clone.”
  • the DNA or RNA introduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or cells of a different genus or species.
  • the terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors include plasmids, phages, viruses, etc.; they are discussed in greater detail below.
  • Vectors typically comprise the DNA of a transmissible agent, into which foreign
  • DNA is inserted.
  • a common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites.
  • restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites.
  • a "cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites.
  • the cassette restriction sites are designed to ensure insertion ofthe cassette in the proper reading frame.
  • foreign DNA is inserted at one or more restriction sites ofthe vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA.
  • a segment or sequence of DNA having inserted or added DNA, such as an expression vector can also be called a "DNA construct.”
  • a common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • a plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA.
  • Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme.
  • Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression ofthe coding DNA.
  • Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms.
  • a large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
  • expression system means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site ofthe cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is such an element operatively associated with a different gene than the one it is operatively associated with in nature.
  • an H 4 gene is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e.g., a HEK cell.
  • mutant and mutant mean any detectable change in genetic material, e.g., DNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence.
  • variant may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.
  • Sequence-conservative variants of apolynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.
  • “Function-conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function ofthe polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine.
  • a “function-conservative variant” also includes a polypeptide or enzyme which has at least 60 % ⁇ amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%>, and even more preferably at least 90%o, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared.
  • homologous in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al, Cell 1987, 50:667). Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs at conserved positions.
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al, supra).
  • sequence similarity when modified with an adverb such as "highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • two DNA sequences are "substantially homologous" or “substantially similar” when at least about 80%>, and most preferably at least about 90 or 95%> of the nucleotides match over the defined length of the DNA sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc.
  • sequence comparison algorithms such as BLAST, FASTA, DNA Strider, etc.
  • An example of such a sequence is an allelic or species variant of the specific H 4 gene of the invention.
  • Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.
  • two amino acid sequences are "substantially homologous” or “substantially similar” when greater than 80% ofthe amino acids are identical, or greater than about 90%) are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any ofthe programs described above (BLAST, FASTA, etc)
  • GCG Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin
  • FASTA FASTA, etc
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form ofthe nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, supra).
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a Tm (melting temperature) of 55°C
  • Tm melting temperature
  • Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40%> formamide, with 5x or 6x SSC.
  • High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5x or 6x SSC.
  • SSC is a 0.15M NaCl, 0.015M Na-citrate buffer.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency ofthe hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al.
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the. length is at least about 20 nucleotides.
  • standard hybridization conditions refers to a Tm of 55 °C, and utilizes conditions as set forth above.
  • the Tm is 60 °C; in a more preferred embodiment, the Tm is 65 °C.
  • “high stringency” refers to hybridization and/or washing conditions at 68 °C in 0.2xSSC, at 42°C in 50%) formamide, 4xSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
  • oligonucleotide refers to a nucleic acid, generally of at least 10, preferably at least 15 , and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
  • a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.
  • oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of H 4 , or to detect the presence of nucleic acids encoding H 4 .
  • an oligonucleotide of the invention can form a triple helix with a H 4 DNA molecule.
  • oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
  • the present invention provides antisense nucleic acids (including ribozymes), which may be used to inhibit expression of H 4 ofthe invention. Inhibition of H 4 expression may be desired when upregulation of H 4 receptor expression or excessive inhibition of cAMP formation induces disease states such as, transplant organ rejection; asthma; allergies; autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis; and CNS functions such as cognitive and memory defects.
  • An "antisense nucleic acid” is a single stranded nucleic acid molecule which, on hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid. As presently used, "antisense” broadly includes
  • Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent No. 5,814,500; U.S. Patent No. 5,811,234), or alternatively they can be prepared synthetically (e.g., U.S. Patent No. 5,780,607).
  • synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds.
  • oligonucleotides having morpholino backbone structures U.S. Patent No. 5,034,506.
  • the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms ofthe polyamide backbone (Nielsen et al, Science 254:1497, 1991).
  • oligonucleotides may contain substituted sugar moieties comprising one ofthe following at the 2' position: OH, SH, SCH 3 , F, OCN, O(CH 2 ) n NH 2 or O(CH 2 ) n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; O-; S-, or N-alkyl; O-, S-, or
  • N-alkenyl SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substitued silyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group.
  • Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine, such as inosine, may be used in an oligonucleotide molecule.
  • H 4 Nucleic Acids A gene encoding H 4 , whether genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining H 4 gene are well known in the art, as described above (see, e.g., Sambrook et al, 1989, supra).
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression ofthe protein, by chemical synthesis, by cDNA cloning, or by the cloning of genomic
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation ofthe gene. Identification ofthe specific DNA fragment containing the desired H 4 gene may be accomplished in a number of ways.
  • a portion of a H 4 gene exemplified infra can be purified and labeled to prepare a labeled probe, and the generated DNA may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, Science 1977, 196:180; Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 1975, 72:3961). Those DNA fragments with substantial homology to the probe, such as an allelic variant from another individual, will hybridize. In a specific embodiment, highest stringency hybridization conditions are used to identify a homologous H 4 gene.
  • the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, partial or complete amino acid sequence, antibody binding activity, or ligand binding profile of H 4 protein as disclosed herein.
  • the presence ofthe gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.
  • DNA sequences which encode substantially the same amino acid sequence as a H 4 gene may be used in the practice of the present invention. These include but are not limited to allelic variants, species variants, sequence conservative variants, and functional variants.
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • H 4 derivatives and analogs ofthe invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned H 4 gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al, 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the v-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • modifications can be made to introduce restriction sites and facilitate cloning the H 4 gene into an expression vector.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, et al, J. Biol. Chem. 253:6551, 1978; Zoller and Smith, DNA
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as Bluescript, pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • simple PCR or overlapping PCR may be used to insert a fragment into a cloning vector. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2 ⁇ plasmid.
  • Elements of the H 4 promoter can be identified by scanning the human genomic region upstream of the H 4 start site, e.g., by creating deletion mutants and checking for expression, or with the TRANSFAC algorithm. Sequences up to about 6 kilobases (kb) or more upstream from the H 4 start site can contain tissue-specific regulatory elements.
  • H 4 promoter encompasses artificial promoters. Such promoters can be prepared by deleting nonessential intervening sequences from the upstream region ofthe H 4 promoter, or by joining upstream regulatory elements from the H 4 promoter with a heterologous minimal promoter, such as the CMV immediate early promoter.
  • An H 4 promoter can be operably associated with a heterogenous coding sequence, e.g., for reporter gene (luciferase and green fluorescent proteins are examples of reporter genes) in a construct.
  • This construct will result in expression ofthe heterologous coding sequence under control the H 4 promoter, e.g., a reporter gene can be expressed, under conditions that under normal conditions cause H 4 expression.
  • This construct can be used in screening assays, described below, for H 4 agonists and antagonists.
  • the nucleotide sequence coding for H 4 , or antigenic fragment, derivative or analog thereof, or a functionally active derivative, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, /. e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • a nucleic acid encoding H 4 of the invention can be operationally associated with a promoter in an expression vector ofthe invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • Such vectors can be used to express functional or functionally inactivated H 4 polypeptides.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding H 4 and/or its flanking regions.
  • Potential host-vector systems include but are not limited to mammalian cell systems transfected with expression plasmids or infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, herpes virus, etc.); insect cell systems infected with virus (e.g, baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, adeno-associated virus, herpes virus, etc.
  • insect cell systems infected with virus e.g, baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA e.g., bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors
  • H 4 protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used to control H 4 gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell 22:787-
  • CMV cytomegalovirus
  • herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981), the regulatory sequences of the metallothionein gene (Brinster et al, Nature 296:39-42, 1982); prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa- Komaroff, et al , Proc. Natl. Acad. Sci. U.S.A. 75 :3727-3731 , 1978), or the tac promoter (DeBoer, et al, Proc. Natl. Acad. Sci. U.S.A.
  • Solubilized forms ofthe protein can be obtained by solubilizing inclusion bodies or reconstituting membrane components, e.g., by treatment with detergent, and if desired sonication or other mechanical processes, as described above.
  • the solubilized protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.
  • PAGE polyacrylamide gel electrophoresis
  • isoelectric focusing e.g., isoelectric focusing
  • 2-dimensional gel electrophoresis e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.
  • Vectors A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al, Gene 67:31-40, 1988), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • phage DNAS e.g., the numerous derivatives of phage 1, e.g., NM9
  • Viral vectors such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphavirus, and other recombinant viruses with desirable cellular tropism are also useful.
  • a gene encoding a functional or mutant H 4 protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA.
  • Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.
  • Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques 1992, 7:980- 990).
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the genome ofthe replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part) or be rendered non-functional by any technique known to a person skilled in the art.
  • DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HS V 1 ) vector (Kaplitt et al, Molec. Cell. Neurosci. 2:320-330, 1991), defective herpes virus vector lacking a glyco-protein L gene (Patent Publication RD 371005 A), or other defective herpes virus vectors (International Patent Publication No.
  • HS V 1 defective herpes virus 1
  • Patent Publication RD 371005 A defective herpes virus vector lacking a glyco-protein L gene
  • viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc.
  • Avigen, Inc. Almeda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc.
  • an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation ofthe viral vector and transfected cells.
  • the viral vector e.g., adenovirus vector
  • immunosuppressive cytokines such as interleukin-12 (IL-12), interferon- ⁇ (IFN- ⁇ ), or anti-CD4 antibody
  • IL-12 interleukin-12
  • IFN- ⁇ interferon- ⁇
  • anti-CD4 antibody can be provided to block humoral or cellular immune responses to the viral vectors (see, e.g., Wilson, Nature Medicine, 1995).
  • IL-12 interleukin-12
  • IFN- ⁇ interferon- ⁇
  • anti-CD4 antibody can be provided to block humoral or cellular immune responses to the viral vectors (see, e.g., Wilson, Nature Medicine, 1995).
  • the vector can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.).
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et al, Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417, 1987; Feigner and Ringold, Science 337:387-388, 1989; see Mackey, et al, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031, 1988; Ulmer, et al, Science 259:1745-1748, 1993).
  • lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • a nucleic acid in vivo, is also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO 96/25508), or a cationic polymer (e.g., International Patent Publication WO95/21931).
  • a cationic oligopeptide e.g., International Patent Publication WO 95/21931
  • peptides derived from DNA binding proteins e.g., International Patent Publication WO 96/25508
  • a cationic polymer e.g., International Patent Publication WO95/21931.
  • non- viral DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection; see, e.g., U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,853,663, U.S. Pat. No. 5,885,795, and U.S. Pat. No. 5,702,384 and see Sanford, TIB-TECH, 6:299-302, 1988; Fynan et al, Proc. Natl. Acad. Sci.
  • H ⁇ Ligands and Binding Partners The present invention further permits identification of physiological ligands and binding partners of H 4 .
  • One method for evaluating and identifying H 4 binding partners is the yeast two-hybrid screen.
  • the yeast two-hybrid screen is performed using an cell library with yeast that are transformed with recombinant H 4 .
  • H 4 can be used as a capture or affinity purification reagent.
  • labeled H 4 can be used as a probe for binding, e.g., by immunoprecipitation or Western analysis.
  • Expected H 4 binding partners are G-proteins.
  • binding interactions between H 4 and any of its binding partners will be strongest under conditions approximating those found in the cytoplasm, i.e., physiological conditions of ionic strength, pH and temperature. Perturbation of these conditions will tend to disrupt the stability of a binding interaction.
  • Antibodies to H 4 are useful, inter alia, for diagnostics and intracellular regulation of H 4 activity, as set forth below.
  • a H 4 polypeptide produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as immunogens to generate antibodies that recognize the
  • H 4 polypeptide Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Such an antibody is preferfably specific for human H 4 and it may recognize either a mutant form of H 4 or wild-type H 4 , or both.
  • H 4 polypeptide or derivative or analog thereof various procedures known in the art may be used for the production of polyclonal antibodies to H 4 polypeptide or derivative or analog thereof.
  • various host animals can be immunized by injection with the H 4 polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • the H 4 polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 1975,
  • monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690).
  • chimeric antibodies Infact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al. , J. Bacteriol. 1984, 159:870; Neuberger et al, Nature 1984, 312:604-608; Takeda et al, Nature 1985, 314:452-454) by splicing the genes from a mouse antibody molecule specific for an H 4 polypeptide together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.
  • Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves.
  • Antibody fragments which contain the idiotype ofthe antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab')2 fragment which can be produced by pepsin digestion ofthe antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges ofthe F(ab')2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radio immunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radio immunoassay e.g., radio immunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of an H 4 polypeptide, one may assay generated hybridomas for a product which binds to an H 4 polypeptide fragment containing such epitope. For selection of an antibody specific to an H 4 polypeptide from a particular species of animal, one can select on the basis of positive binding with H 4 polypeptide expressed by or isolated from cells of that species of animal.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity ofthe H 4 polypeptide, e.g., for Western blotting, imaging H 4 polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art.
  • Such antibodies can also be used in assays for ligand binding, e.g., as described in U.S. Patent No. 5,679,582.
  • Antibody binding generally occurs most readily under physiological conditions, e.g., pH of between about 7 and 8, and physiological ionic strength. The presence of a carrier protein in the buffer solutions stabilizes the assays. While there is some tolerance of perturbation of optimal conditions, e.g., increasing or decreasing ionic strength, temperature, or pH, or adding detergents or chaotropic salts, such perturbations will decrease binding stability.
  • antibodies that act as ligands and agonize or antagonize the activity of H 4 polypeptide can be generated.
  • intracellular single chain Fv antibodies can be used to regulate cAMP formation (Marasco et al., Proc. Natl. Acad. Sci. U.S.A. 1993, 90:7884-7893; Chen., Mol. Med. Today 1997, 3:160-167; Spitz et al., Anticancer Res.
  • nucleotide sequences encoding H 4 and the H 4 receptor structure which can be modeled from the amino acid sequence based on homology to other GPCR proteins, are useful targets to identify drugs that are effective in treating disorders associated with histamine-regulated processes.
  • Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding H 4 (e. g. , antisense or ribozyme molecules) and (ii)small molecule compounds that recognize and bind the receptor.
  • identification and isolation of H 4 provides for development of screening assays, particularly for high throughput screening of molecules that up- or down- regulate the activity of H 4 .
  • the present invention contemplates methods for identifying specific histamine receptor ligands that interact with H 4 receptors, using various screening assays known in the art.
  • Any screening technique known in the art can be used to screen for H 4 agonists or antagonists.
  • the present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and agonize or antagonize H 4 activity in vivo.
  • natural products libraries can be screened using assays ofthe invention for molecules that agonize or antagonize H 4 expression or activity.
  • synthetic libraries (Needels et al, Proc. Natl. Acad. Sci. USA 1993, 90:10700-4; Ohlmeyer et al, Proc. Natl. Acad. Sci. USA 1993, 90:10922-10926; Lam etal, PCT Publication No. WO 92/00252; Kocis etal, PCT Publication No. WO 9428028) and the like can be used to screen for ligands that regulate H 4 activity. Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co.
  • test wells containing an aliquot of a lipid bilayer membranes that contain the histamine H 4 receptor are incubated with an known concentration of a radiolabeled ligand for the receptor.
  • the lipid bilayer may be prepared by any known protocol that separates the membrane containing receptor component from the cytoplasmic components.
  • Each well also is incubated with a different concentration of a unlabeled test compound.
  • Cell membranes are then separated from the incubation mixture by any method known in the art including, but not limited to, centrifugation and vacuum filtration on a cell harvester.
  • the radioactivity of each well is then determined using any device that can detect radioactivity, such as a scintillation counter. As increasing concentrations of the test compound compete for the receptor binding site, the radioactivity detected decreases. The data then can be converted using the Cheng-Prusoff equation (Biochem Pharmacol. 1973, 22:3099-3108) to determine the affinity (K-) ofthe compound for the receptor.
  • In vivo screening methods Intact cells or whole animals expressing a gene encoding H 4 can be used in screening methods to identify candidate drugs.
  • a permanent cell line is established.
  • cells including without limitation mammalian, insect, yeast, or bacterial cells
  • Identification of candidate compounds can be achieved using any suitable assay, including without limitation
  • H 4 knockout mammals can be prepared for evaluating the molecular pathology of this defect in greater detail than is possible with human subjects. Such animals also provide excellent models for screening drug candidates.
  • a "knockout mammal” is an mammal (e.g., mouse, rabbit) that contains within its genome a specific gene that has been inactivated by the method of gene targeting (see, e.g., U.S. Patent Nos. 5,777,195 and 5,616,491).
  • a knockout mammal includes both a heterozygote knockout (i.e., one defective allele and one wild-type allele) and a homozygous mutant (i.e., two defective alleles; a heterologous construct for expression of an H 4 , such as a human H 4 , could be inserted to permit the knockout mammal to live if lack of H 4 expression was lethal).
  • Preparation of a knockout mammal requires first introducing a nucleic acid construct that will be used to suppress expression of a particular gene into an undifferentiated cell type termed an embryonic stem cell. This cell is then injected into a mammalian embryo. A mammalian embryo with an integrated cell is then implanted into a foster mother for the duration of gestation.
  • Zhou, et al. (Genes and Development 1995, 9:2623-34) describes PPCA knock-out mice.
  • knockout refers to partial or complete suppression ofthe expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell.
  • knockout construct refers to a nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell.
  • the nucleic acid sequence used as the knockout construct is typically comprised of (1) DNA from some portion ofthe gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell.
  • the knockout construct is inserted into a cell, and integrates with the genomic DNA ofthe cell in such a position so as to prevent or interrupt transcription of the native DNA sequence.
  • Such insertion usually occurs by homologous recombination (i.e., regions ofthe knockout construct that are homologous to endogenous DNA sequences hybridize to each other when the knockout construct is inserted into the cell and recombine so that the knockout construct is inco ⁇ orated into the corresponding position ofthe endogenous DNA).
  • the knockout construct nucleic acid sequence may comprise (1) a full or partial sequence of one or more exons and/or introns ofthe gene to be suppressed, (2) a full or partial promoter sequence ofthe gene to be suppressed, or (3) combinations thereof.
  • the knockout construct is inserted into an embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination. This ES cell is then injected into, and integrates with, the developing embryo.
  • ES cell embryonic stem cell
  • disruption ofthe gene and “gene disruption” refer to insertion of a nucleic acid sequence into one region ofthe native DNA sequence (usually one or more exons) and/or the promoter region of a gene so as to decrease or prevent expression of that gene in the cell as compared to the wild-type or naturally occurring sequence of the gene.
  • a nucleic acid construct can be prepared containing a DNA sequence encoding an antibiotic resistance gene which is inserted into the DNA sequence that is complementary to the DNA sequence (promoter and/or coding region) to be disrupted. When this nucleic acid construct is then transfected into a cell, the construct will integrate into the genomic DNA. Thus, many progeny ofthe cell will no longer express the gene at least in some cells, or will express it at a decreased level, as the DNA is now disrupted by the antibiotic resistance gene.
  • the DNA will be at least about 1 kb in length and preferably 3-4 kb in length, thereby providing sufficient complementary sequence for recombination when the knockout construct is introduced into the genomic DNA ofthe ES cell (discussed below).
  • a mammal in which two or more genes have been knocked out. Such mammals can be generated by repeating the procedures set forth herein for generating each knockout construct, or by breeding to mammals, each with a single gene knocked out, to each other, and screening for those with the double knockout genotype.
  • Regulated knockout animals can be prepared using various systems, such as the tet-repressor system (see U.S. Patent No.5,654,168) or the Cre-Lox system (see U.S. Patent Nos. 4,959,317 and 5,801,030).
  • transgenic animals are created in which (i) a human H 4 is stably inserted into the genome ofthe transgenic animal; and/or (ii) the endogenous H 4 genes are inactivated and replaced withhuman H 4 genes. See, e.g., Coffman, Semin. Nephrol.
  • H 4 Activation Assay Any cell assay system that allows for assessment of functional activity of H 4 agonists and antagonists is defined by the present invention.
  • the assay can be used to identify compounds that selectively interact with H 4 , which can be evaluated by assessing the effects of H 4 transformed cells contacted with a test compound, which modulates cAMP accumulation.
  • the assay system can thus be used to identify compounds that selectively produce a functional effect through histamine H 4 receptors.
  • Compounds that increase cAMP formation and accumulation may be useful as novel therapeutics in the prevention of transplanted organ rej ection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis. Additionally, CNS functions such as cognitive and memory defects may be treated.
  • each experiment is performed in triplicate at multiple different dilutions of test compound.
  • An agonist and/or antagonist screen involves detecting cAMP accumulation by the host cell when contacted with H 4 ligand. If cAMP accumulation is increased, the test compound is a candidate antagonist of H 4 receptors. If cAMP accumulation is decreased, the test compound is a candidate agonist of H 4 receptors. If there is no change in cAMP formation, the test compound is not an effective H 4 ligand.
  • the invention provides immunoassays for detecting cAMP.
  • immunoassays use either a labeled antibody or a labeled antigenic component (e.g., that competes with the antigen in the sample for binding to the antibody).
  • Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules.
  • Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labelled immunoassays, such as ELISA assays.
  • labeled antigenic component may be quantified by scintillation techniques.
  • the second messenger preferably cAMP, will be separated on a high performance liquid chromatograph and quantified by a UV detector.
  • the assay system described here also may be used in a high-throughput primary screen for agonists and antagonists, or it may be used as a secondary functional screen for candidate compounds identified by a different primary screen, e.g., a binding assay screen that identifies compounds that interact with the receptor.
  • High-Throughput Screen Agents according to the invention may be identified by screening in high- throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time. Such high- throughput screening methods are particularly preferred. The use of high-throughput screening assays to test for agents is greatly facilitated by the availability of large amounts of purified polypeptides, as provided by the invention.
  • Hetamine refers to a neurotransmitter that is produced and released from neurons. Histamine is formed from the amino acid histidine by histidine decarboxylase.
  • histamine is an imidazolethylamine.
  • histamine is comprised of an imidazole moiety and an amino group connected by an alkyl chain.
  • the pharmacologically active form of histamine is proposed to be the monocationic tautomer, where one ofthe nitrogen present in the imidazole ring is positively charged.
  • different forms of histamine may interact with histamine receptors to produce a functional effect. Histamine is produced intracellularly and stored until released in response to a physiological stimulus.
  • Hyistamine analogs refers to compounds that comprise the imidazole, alkyl chain, and a nitrogen moiety (e.g., amine (unsubstituted or substituted), piperidine, pyridine) of histamine, but may be modified at other positions. These modifications may be performed to alter affinity and/or selectivity ofthe compound for the histamine receptors.
  • Heistamine compounds refers to compounds that may bind to the histamine receptors.
  • Histamine analogs and compounds can be classified as agonists or antagonists. As discussed previously, agonists are ligands that bind to the receptor and produce a functional effect similar to that produced by the endogenous ligand (i.e., histamine) for the receptor, whereas antagonists are ligands that bind to the receptor and block a functional effect produced by an agonist for the receptor or the endogenous ligand ofthe receptor. Histamine analogs and compounds are further described in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996.
  • Agonists that may be contemplated by this invention include, but are not limited to, R-( ⁇ )-methylhistamine, imetit, and immepip. Antagonists, burimamide, impromidine, dimaprit, and thioperamide clobenpropit and iodophenpropit impentamine, GT2016 and iodoproxyfan. Other compounds include derivatives, metabolites, and precursors.
  • genetic variants of H 4 can be detected to diagnose an H 4 associated disease, such as treatment for transplanted organ rejection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis. Additionally, CNS functions such as cognitive and memory defects may be treated. The various methods for detecting such variants are described herein. Where such variants impact H 4 function, either as a result of a mutated amino acid sequence or because the mutation results in expression of a truncated protein, or no expression at all, they are expected to result in disregulation ofthe allergic response, the immune response, cognition and memory.
  • the DNA may be obtained from any cell source.
  • DNA is extracted from the cell source or body fluid using any ofthe numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature ofthe source. Generally, the minimum amount of DNA to be extracted for use in the present invention is about 25 pg (corresponding to about 5 cell equivalents of a genome size of 4 x
  • RNA is isolated from biopsy tissue using standard methods well known to those of ordinary skill in the art such as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznski et al, Anal. Biochem., 162: 156, 1987).
  • the isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a selected site.
  • RT-PCR polymerase chain reaction
  • Conditions for primer annealing are chosen to ensure specific reverse transcription and amplification; thus, the appearance of an amplification product is diagnostic ofthe presence of a particular genetic variation.
  • RNA is reverse-transcribed and amplified, after which the amplified sequences are identified by, e.g., direct sequencing.
  • cDNA obtained from the RNA can be cloned and sequenced to identify a mutation.
  • biopsy tissue is obtained from a subject.
  • Antibodies that are capable of specifically binding to H 4 are then contacted with samples of the tissue to determine the presence or absence of a H 4 polypeptide specified by the antibody.
  • the antibodies may be polyclonal or monoclonal, preferably monoclonal. Measurement of specific antibody binding to cells may be accomplished by any known method, e.g., quantitative flow cytometry, enzyme-linked or fluorescence-linked immunoassay, Western analysis, etc. Therapeutic Uses
  • stimulation of H 4 receptor activity may be used as a treatment option in patients with histamine-related disease states.
  • Stimulation of H 4 receptor activity may be by methods, such as, but not limited to, (i) providing polypeptides that stimulate receptor activity and (ii) providing compounds that stimulate receptor activity.
  • vectors comprising a sequence encoding a protein, including, but not limited to, full-length H 4 , are provided to treat or prevent a disease or disorder associated with the function of H 4 in peripheral blood leukocytes.
  • the therapeutic vector encodes a sequence that produces the protein ofthe invention.
  • the therapeutic vector comprises a nucleic acid that expresses a protein of the invention in a suitable host.
  • a vector has a promoter operationally linked to the coding sequence for the protein.
  • the promoter can be inducible or constitutive and, optionally, tissue-specific.
  • a nucleic acid molecule is used in which the protein coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the protein (Koller and Smithies, Proc. Natl. Acad. Sci.
  • Delivery ofthe vector into a patient may be either direct, in which case the patient is directly exposed to the vector or a delivery complex, or indirect, in which case, cells are first transformed with the vector in vitro then transplanted into the patient.
  • direct in which case the patient is directly exposed to the vector or a delivery complex
  • indirect in which case, cells are first transformed with the vector in vitro then transplanted into the patient.
  • the vector is directly provided in vivo, where it enters the cells ofthe organism and mediates expression ofthe protein.
  • This can be accomplished by any of numerous methods known in the art, by constructing it as part of an appropriate expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, U.S. Patent No.
  • a nucleic acid ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publication Nos. WO 92/06180, WO 92/22635, WO 92/20316 and WO 93/14188).
  • the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.
  • the form and amount of therapeutic nucleic acid envisioned for use depends on the type of disease and the severity ofthe desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Exogenous compounds may interact with extracellular and/or intracellular messenger systems, such as, but not limited to, adenosine triphosphate, nitric oxide, and guanosine triphosphate; to regulate protein synthesis.
  • exogenous compounds that stimulate or inhibit H 4 protein synthesis may be used in the prevention and/or treatment for transplanted organ rejection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis.
  • CNS functions such as cognitive and memory defects may be treated.
  • the present invention provides antisense nucleic acids (including ribozymes), which may be used to inhibit expression of H 4 ofthe invention.
  • the antisense nucleic acid upon hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule, inhibits the role ofthe RNA or DNA. Additionally, hybridization ofthe antisense nucleic acid to the DNA or RNA may inhibit transcription ofthe DNA into RNA and/or translation of the RNA into the protein. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid.
  • Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S.
  • Patent No. 5,814,500 can be prepared synthetically (e.g., U.S. Patent No. 5,780,607).
  • antibody molecules can also be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (Proc. Natl. Acad Sci. USA, 1993, 90:7889-7893).
  • Therapeutically suggested compounds may be provided to the patient in formulations that are known in the art and may include any pharmaceutically acceptable additives, such as excipents, lubricants, diluents, flavorants, colorants, and disintegrants.
  • the formulations may be produced in useful dosage units such as tablet, caplet, capsule, liquid, or injection.
  • the form and amount of therapeutic compound envisioned for use depends on the type of disease and the severity ofthe desired effect, patient state, etc., and can be determined by one skilled in the art.
  • a GPCR sequence profile was generated from a sequence alignment of members of this subfamily based on hidden Markov Models (Eddy Bioinformatics, 1998 14:755-763; Durbin et al. A tutorial introduction to hidden Markov models and other probabilistic modeling approaches in computational sequence analysis, Cambridge University Press, 1998) to look for novel members in the human genome database.
  • GPCR proteins ofthe biogenic amine subfamily were retrieved from S wiss-Prot database and a sequence alignment was generated by a multiple sequence alignment tool, named CLUSTALW (Thompson et al. Nucleic Acids Res, 1994, 22:4673-80).
  • CLUSTALW Thimpson et al. Nucleic Acids Res, 1994, 22:4673-80.
  • LLFLLTLPPW ALYYLVGGSE DWPFGSALCK LVTALDVVNM YASILLLTAI SIDRYLAIVH PLRYRRRRTS PRRAKVVILL VWVLALLLSL PPLLFSWVKT VEEGNGTLNV NVTVCLIDFP EESTAS VSTW LRS YVLLSTL VGFLLPLLVI LVC YTRJLRT LRKAAKTLLV VVVVFVLCWL PYFIVLLLDT LCLSIIMSST CELERVLPTA LLVTLWLAYV NSCLNPIIY; SEQ ID NO: 3) was developed from the biogenic amine subfamily members. The consensus sequence contained the unique 7-transmembrane sequence structure of biogenic amine GPCRs.
  • Quantitative RT-PCR was performed on an ABI 7700 "Taqman" sequence detection system to determine the tissue distribution ofthe H 4 receptor.
  • Primers spanning the exon 1-2 boundary (Forward primer: 5'-taacttggccattgacttctt-3' (SEQ ID NO:4); Reverse primer:
  • 5'-attcgaacagcatgtgagggat-3' (SEQ ID NO: 5) and a Probe: 5'-(6-carboxyfluorescein)- tacaaaggaatggagatcacacccaca- (6-carboxy-N,N,N'N'-tetramethylrhodamine)-3' (SEQ ID NO: 6)) were used to determine H 4 expression levels in a mRNA prepared from a series of human tissues, purchased from Clontech (Palo Alto). A 2 step reaction procedure was performed as per manufacturers directions. Briefly, 2mg RNA was reverse transcribed using random hexamers
  • H 4 receptor protein coding sequences were amplified using Forward oligo:
  • the yeast expression plasmid, pMP327 was introduced into MPN578i5 cells (MATa ura3 his3 tipl leu2 lys2 ade2 farl::LNS2 fusl ::FUSl-HIS3 sst2::SST2-G418R ste2::LEU2, gpal::GPAli5) (Hadcock, J.R. and Pausch, M.H. submitted) using LiOAc and selected for ura prototrophy.
  • MPN578i5 cells express a chimeric
  • the G alpha protein coupled to the mating signal transduction pathway is expressed from the GPA1 locus and is composed of Gpal sequences in which the 5'-C-terminal amino acids have been replaced with those of Gai3.
  • a multicopy FUSl-LacZ reporter gene plasmid, pMP283 (Hadcock, J.R. and Pausch, M.H. submitted), was subsequently introduced into H 4 R-containing MPY578i5 cells and selected on media lacking tip and ura. The resulting yeast strain, MPY733, was used for further analysis.
  • the ORF was modified by PCR for mammalian expression of H 4 .
  • a 5' Hindlll restriciton enzymes site and a Kozak consensus sequence were added using the primer 5'- aagcttccaccatgccagatactaatagcacaatcaatttatc-3' (SEQ ID NO:9), and a 3' Xbal site added 5'- tctagattaagaagatactgaccgactgtgttg-3 ' (SEQ ID NO : 10) .
  • the sequence was confirmed and ligated into the Hindlll and Xba I sites of the mammalian expression vector pCDNA3.1+ zeo (Invitrogen, Carlsbad CA).
  • HEK 293 cells (approximately 10 7 cells) were transfected with the pCDN A3.1 +zeo/H 4 using standard lipofectamine plus reagent (Life Technologies) .
  • Cells were maintained in DMEM containing 10% fetal calf serum and penicillin ( 100units/ml)/streptomycin (lOOmg/ml).
  • 48 hours post transfection cells were selected using 500mg/ml zeocin.
  • Zeocin resistant clones were assayed using the cAMP assay and by RT-PCR.
  • RNA was extracted from approximately 10 6 cells using One step PCR kit (Life technologies).
  • the primers used in the extraction were 5'-ggaaggatgaaggtagtgaatg-3' (SEQ ID NO: 11) and 5'-cagaatctgattgggaggaagg-3' (SEQ ID NO: 12).
  • HEK cells stably expressing the H 4 receptor were assessed functionally in cAMP assays using the cAMP scintillation proximity assay (SPA) (Amersham Pharmacia Biotech, Pistcataway, NJ). Briefly, 40 000 cells were plated into wells of a 96 well plate. 24 hours later the media was removed and replaced with lOO ⁇ l Krebs bicarbonate buffer and the cells were incubated at 37°C for 15 minutes. Following this, the cells were incubated in Krebs buffer containing 0.5mM isobutylmethylxanthine, a phosphodiesterase inhibitor, to prevent cAMP breakdown. The effect of H 4 on the forskolin induced formation was determined by incubating the cells in the presence of forskolin (lO ⁇ M) and agonist for 12 minutes. cAMP levels were determined using the cAMP SPA kit according to the manufacturers directions.
  • SPA cAMP scintillation proximity assay
  • EXAMPLE N H ⁇ EXPRESSION IN INDUCED CD4 AND CD8 CLONES
  • RNA for the standard curve was polyA+ human leukocyte RNA from Clontech.
  • H 4 expression was normalized with GAPDH.
  • HMM Hidden Markov Model
  • the predicted protein sequence contains residues that are conserved across the biogenic amine receptors. As in other biogenic amine receptors, the present protein contains conserved aspartate residues within transmembrane (TM) domains 2 and 3 (at positions 61 and 94). A DY (D94 and Y95) motif in TM3 is found only in histamine and muscarinic receptors.
  • a DRY sequence motif that exists at the cytoplasmic interface of TM3 of all GPCRs also can be found in the H 4 sequence (amino acids 111- 113). Also present are two conserved cysteine residues in the first and second extracellular loops (position 88 and 164), which are predicted to form a disulfide bond; two conserved tryptophan residues in TM4 and TM6 (positions 140 and 316); and conserved proline residues in TM5, TM6, and TM7 (positions 186, 318, and 355, respectively). Based on these results, H 4 cDNA appears to encode a biogenic amine-like receptor.
  • the predicted open reading frame is 1173 base pairs long and encodes a protein of 390 amino acids. Sequence comparison using BLASTP, under standard conditions, analysis reveals that the novel protein is most similar to the human H 3 receptor (44%> identical and 51 %> similar). Based on sequence homology it is proposed that the receptor belongs to the histamine receptor family, therefore we have termed it the H 4 receptor.
  • Receptor distribution studies indicate that the present receptor is highly expressed in peripheral blood leukocytes. Trace amounts ofthe H 4 receptor are expressed in heart, lung and placenta. It is proposed that these trace amounts of expression represent blood cell mRNA present in the samples. Functional studies further confirmed the categorization ofthe protein as a novel histamine receptor. Histamine and the H 3 selective agonist R- ⁇ -methylhistamine stimulated cAMP accumulation, suggesting these compounds are agonists at the H 4 receptor (See Figure 2A).
  • R- ⁇ -methylhistamine exhibited both lower potency and efficacy than histamine at the H 4 receptor despite being more potent at the related cloned H 3 receptor and in several tissue based assays for the H 3 receptor (Vollinga, et al, J. Med. Chem. 1995, 38:266-271; Ha ⁇ er, etal, Br. J. Pharmacol. 1999, 128:751-759).
  • the selective H 3 receptor antagonist clobenpropit was a partial agonist at the H 4 receptor (See Figure 2A).
  • H 3 antagonist thioperamide almost fully inhibited the stimulatory response produced by histamine (See Figure 2B). Comparatively, clobenpropit partially blocked histamine-induced stimulation of cAMP formation, further suggesting that clobenpropit is a partial agonist at the H 4 receptor.
  • the interaction ofthe H 4 receptor to a GPAI/ G ⁇ i3 chimeric G-protein alpha subunit also predicts its coupling specificity in mammalian cells.
  • H 4 receptor in HEK 293tsa cells confirms the coupling ofthe H 4 receptor to the inhibition of cAMP formation (See Figure 2C).
  • Forskolin stimulated 8-fold greater cAMP formation compared to basal levels.
  • H 4 expression normalized with GAPDH, was detectable in all samples at all time points ( Figure 5).
  • Anti-CD3 stimulation produced significant induction of H 4 expression in all 6 samples, most peaking at the 8 hour time point.
  • the highest level of induction was found in the 8 hour CD4+ and CD8+ samples from the same donor (designated 'U'), peaking at greater than 100 copies of H 4 RNA per copy of GAPDH. Combined, these studies indicate that the pharmacological profile of the H 4 receptor is not similar to any known histamine H 3 receptor.

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Abstract

The present invention discloses the identification of a novel histamine receptor, termed H4. Amino acid sequences, nucleic acid sequences, vectors, and host cells are also discussed. Additionally, methods of detecting agonists and antagonists for the receptor are disclosed herein.

Description

HUMAN HISTAMINE H4 RECEPTOR This application claims priority under 35 U.S.C. § 119 from provisional patent application Serial Nos. 60/202,151, filed May 5, 2000, 60/227,567, filed August 23, 2000, and 60/247,855, filed November 13, 2000; which all are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
The present invention discloses the identification of a novel histamine receptor, termed H4.
BACKGROUND OF THE INVENTION Histamine produces numerous physiological effects in the body through interaction with one of three different cell surface receptors, classified as H H2, and H3. These receptors belong to the guanine nucleotide binding protein coupled receptor (G-protein coupled receptors, GPCR) class.
Stimulation of histamine H- receptors produces symptoms that are typically associated with physiological responses to allergic stimuli (Ash and Schild, Br. J. Pharmacol.
1966, 27:427). These effects are blocked by H- antagonists such as, for example, diphenhydramine. H* antagonists are generally defined as "classical antihistamines". Classical antihistamines are the active ingredient in most over-the-counter allergy medications. Pharmacological studies indicate that agonist activation of these receptors stimulates the inositol phosphate pathway, and thus stimulates formation of inositol triphosphate (IP3) and diacylglycerol (DAG).
Histamine H2 receptors have been shown to play a role in gastric acid secretions (Black et al, Nature 1972, 236:385). Histamine H2receptor antagonists such as, for example, cimetidine and ranitidine, are often the active ingredient in over-the-counter and prescription drugs that are used to treat duodenal ulcers, gastric ulcers, heartburn, indigestion, and other disorders of the gastrointestinal tract. Activation of histamine H2 stimulates adenylyl cyclase activity and stimulates formation of c AMP. Histamine H3 receptors are a relatively new member of the histamine receptor class. These receptors were originally described as histamine autoreceptors. These receptors were shown to be located on presynaptic histamine nerve terminals and to control the rate of histamine synthesis and release in the brain (Arrang etal, Nature 1983, 302:832). Recent studies indicate that H3 receptors also are present on non-histamine presynaptic nerve terminals. H3 receptors may modulate the synthesis and release of other neurotransmitters such as dopamine, serotonin, acetylcholine, and norepinephrine. H3 receptors also have been found in peripheral tissues. Histamine activation of H3 receptor inhibits adenylyl cyclase activity and formation of cAMP. Multiple pharmacological studies have indicated the presence of H3 receptor subtypes (Leurs et al JPET 1996, 276:1009-1015; Gumming and Gjede, Brain Res. 1994, 641:203-207; Calpham and Kilpatrick, Br. J. Pharmacol 1996, 107:919-923; Schworer et al Naunyn-Schmiedeberg's Arch. Pharmacol. 1994, 350:375-379; Schkicker et al, Naunyn- Schmiedeberg's Arch. Pharmacol. 1996, 353:482-488). Additionally, pharmacological characterization of a histamine receptor on eosinophils describes the greater potency of histamine compared to R-α-methylhistamine (Raible, etal Am. J. Respir. Crit. Care.Med 1994 , 149:1506- 1511.). However, the existence of these receptor subtypes has yet to be substantiated by molecular biological techniques.
SUMMARY OF THE INVENTION
The present invention contemplates an isolated histamine H4 receptor protein having an amino acid sequence at least 51 % identical or comprising at least 10 contiguous amino acids from the sequence depicted in SEQ ID NO:2. The H4 receptor protein binds ligands comprising an imidazole and an amine, which imidazole and amine are attached by an alkyl chain, where the rank order of efficacy of modulation of second messenger formation of the ligands at the H4 receptor protein is 5>6=10>8=4, where the number represents the number of carbons in the alkyl chain. In one embodiment, upon binding histamine or a histamine agonist the receptor protein inhibits second messenger formation. Preferably the second messenger is cAMP. The present application also discloses an isolated H4 receptor protein having an amino acid sequence with at least 95% sequence identity to human H4 receptor protein having an amino acid sequence as depicted in SEQ ID NO: 2. In a specific embodiment, the protein is a human H4 receptor protein, e.g. , having an amino acid sequence as depicted in SEQ ID NO:2 or an allelic variant of that sequence.
Also provided are nucleic acids, e.g., cDNAs encoding the H4 receptor proteins as discussed above. In another embodiment, an isolated nucleic acid encoding an H4 receptor protein, which nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence of at least 20 nucleotides identical to a corresponding nucleotide sequence ofthe same number of bases in SEQ ID NO:l or its complement.
The isolated nucleic acids encoding H4 receptors can be part of vectors, e.g., for cloning, expression, and/or expansion. An expression vector comprises the nucleic acid encoding the H4 receptor protein operably associated with an expression control sequence. The invention further provides host cells and non-human transgenic animals containing such an expressible vector, and methods for producing an H4 receptor polypeptide using such host cells.
In addition, the invention provides an isolated nucleic acid, such as a primer or probe, of at least 10 bases having a nucleotide sequence identical to a corresponding nucleotide sequence ofthe same number of bases in SEQ ID NO:l, or its complement. The invention also provides an antibody that specifically binds an H4 receptor.
The present invention further contemplates a method for detecting expression of H4 receptor, which method comprises detecting mRNA encoding H4 receptor in a sample from a cell suspected of expressing H4 receptor or detecting the H4 receptor protein with an antibody ofthe invention.
The present invention also contemplates an assay system for identifying H4 receptor ligands. The assay system comprises a- sufficient number of transformed host cells to be able to detect an alteration in second messenger accumulation. Preferably, the second messenger is cAMP.
The present invention also contemplates a method for identifying a test compound that antagonizes or agonizes histamine H4 receptors. The method comprises detecting an alteration in the level of a second messenger in the assay system contacted with the test compound. In the method, an increase in the level ofthe second messenger indicates that the test compound antagonizes the H4 receptor. A decrease in the level of the second messenger indicates that the test compound agonizes the H4 receptor. The present invention also discloses an isolated nucleic acid that specifically hybridizes under highly stringent conditions to the complement ofthe sequence depicted in SEQ ID NO:l, where the nucleic acid encodes a histamine H4 receptor protein.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Sequence of the human H4 cDNA (SEQ ID NO: 1) and the deduced amino acid sequence ofthe protein it encodes (SEQ ID NO:2). The TM domains are denoted by lines.
Figures 2A, 2B, and 2C. (A) Agonist assay, Square: histamine; triangle: R-α- methylhistamine; inverted triangle: clobenpropit; diamond: thioperamide. (B) Antagonist assay.
Assay were conducted in duplicate and presented as the average ± SEM. (C) Activity in mammalian cells.
Figure 3. Antagonist activity of a series of histamine analogs. Square: clobenpropit; triangle: thioperamide. Assay were conducted in duplicate and presented as the average ± SEM."
Figure 4. H4 expression in from 6 human T cell clones derived from a single human donor. Three of these clones were CD4+ cell clones (RG4.3B, RG4.3A, and RG4.3), two were CD8+ cell clones (RG8.1C and RG8.1A), and one was an NKT cell clone (RG1).
Figure 5. H4 expression in bulk populations of CD4+ and CD8+ T cells isolated from three different individuals. B-CD8 and B-CD4 were CD8+ and CD4+ cells, respectively, obtained from individual 1. L-CD8 and L-CD4 were CD8+ and CD4+ cells, respectively, obtained from individual 2. U-CD8 and U-CD4 were CD8+ and CD4+ cells, respectively, obtained from individual 3.
DETAILED DESCRIPTION
The present invention is based, in part, on discovery of a no vel histamine receptor, which has been termed H4. The new histamine receptor was cloned from a human heart library using H3 specific primers, but proved to be a new histamine receptor. Yeast and human endothelial kidney (HEK) cells were transformed with a human H4 expression vector. Transformed cells in multi- ell plates were treated with test compounds, and regulation ofthe intracellular second messenger cyclic adenosine monophosphate (cAMP) formation was determined. Modulation of cAMP formation is ligand and concentration dependent.
The nucleic acid and protein sequences of H4 shows homology to known G- protein coupled receptors. Specifically, the H4 protein shows homology to biogenic-amine G- protein coupled receptors. As in other biogenic amine receptors, the present protein contains conserved aspartate residues within transmembrane (TM) domains 2 and 3 (at positions 61 and
94). A DY motif (D94 and Y95) in TM3 is found only in histamine and muscarinic receptors. A DRY sequence motif that exists at the cytoplasmic interface of TM3 of all GPCRs also can be found in the present sequence (amino acids 111- 113). Also present are two conserved cysteine residues in the first and second extracellular loops (position 88 and 164), which are predicted to form a disulfide bond; two conserved tryptophan residues in TM4 and TM6 (positions 140 and
316); and conserved proline residues in TM5, TM6, and TM7 (positions 186, 318, and 355, respectively). The putative TM domains are shown in Figure 1. H4 shares sequence identity with human H3 receptors. Sequence comparison indicates an overall sequence identity of about 44% and overall sequence similarity of about 51%. The present invention also contemplates an assay method and system for identifying selective H4 receptor ligands. The method involves detecting binding of a test compound to isolated cell membranes containing the histamine H4 receptor. The assay system comprises transformed host cells that express H4 receptors, where the number of cells in the assay system is sufficient to detect an alteration in second messenger accumulation. The test system also includes an appropriate cell culture medium to permit cell growth and viability, and preferably tissue culture plates or arrays containing the host cells in cell culture medium. In a specific embodiment, the second messenger that is detected is cAMP. In a further embodiment, the receptor is a human receptor.
The invention also discloses a method for identifying a test compound that antagonizes or agonizes histamine H4 receptors. The method comprises detecting an increase
(antagonist) or decrease (agonist) in the level of a second messenger in the assay system when contacted with the test compound.
Thus, the present invention advantageously provides H4 protein, including fragments, derivatives, and analogs of H4; H4 nucleic acids, including oligonucleotide primers and probes, and H4 regulatory sequences (especially an H4 primer and splice sites with introns);
H4-specific antibodies; and related methods of using these materials to detect the presence of H4 proteins or nucleic acids, H4 binding partners, and in screens for agonists and antagonists of H4.
The following sections ofthe application, which are delineated by headings (in bold) and sub-headings (in bold italics), which cover these three aspects of the invention, are provided for clarity, and not by way of limitation.
General Definitions As used herein, the term "isolated" means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, z'.e., components of the cells in which the material is found or produced in nature. In the case of nucleic acid molecules, an isolated nucleic acid includes a
PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. A protein expressed from a vector in a cell, particularly a cell, in which the protein is normally not expressed is also a regarded as isolated. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in a cell or an organism. An isolated material may be, but need not be, purified.
The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material.
Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure; and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
Methods for purification are well-known in the art. For example, nucleic acids can be purified by precipitation, chromatography (including preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means. Polypeptides and proteins can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents. Cells can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting [FACS]). Other purification methods are possible. A purified material may contain less than about 50%), preferably less than about 75%), and most preferably less than about 90%, of the cellular components with which it was originally associated. The "substantially pure" indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art. In a specific embodiment, the term "about" or "approximately" means within a scientifically acceptable error range for a given value relative to the precision with which the value is or can be measured, e.g., within 20%>, preferably within 10%, and more preferably within 5%> of a given value or range. Alternatively, particularly with biological systems, the term can mean within an order of magnitude, preferably within 5 -fold and more preferably within 2-fold of a given value.
A "sample" as used herein refers to a biological material which can be tested for the presence of H4 protein or H4 nucleic acids. Such samples can be obtained from animal subjects, such as humans and non-human animals, and include tissue, especially muscle, biopsies, blood and blood products; plural effusions; cerebrospinal fluid (CSF); ascites fluid; and cell culture. Non-human animals include, without limitation, laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animals such as dogs and cats; and, farm animals such as sheep, goats, pigs, horses, and cows.
The use of italics indicates a nucleic acid molecule; normal text indicates the polypeptide or protein. The term "ligand" refers to a compound that recognizes and binds to a receptor binding site. In a specific embodiment, the ligand binds to the histamine H4 receptors of the invention. Upon binding to the receptor, the ligand may produce agonist or antagonist functional effects.
The term "agonist" refers to a ligand that binds to the receptor and produces a functional effect similar to that produced by the endogenous ligand for the receptor. In a specific embodiment, the agonist at the histamine H4 receptor produces an effect similar to that produced by histamine, the endogenous ligand (histamine) for the H4 receptor. Examples of such agonists include, but are not limited to, R-α-methyl histamine and imetit.
The term "antagonist" refers to a ligand that binds to the receptor and blocks a functional effect produced by an agonist for the receptor or the endogenous ligand ofthe receptor.
Examples of such antagonists include, but are not limited to, thioperamide.
The term "selective" refers to the ability of a histamine H4 agonist or antagonist to elicit a response from the H4 receptor while eliciting minimal responses from another receptor.
Stated differently, a selective H4 agonist may be a potent agonist for the H4 receptor while agonizing another receptor, such as another G-protein coupled receptor and particularly another histamine receptor, poorly or not at all.
The term "ability to elicit a response" refers to the ability of a H4 agonist or antagonist ligand to agonize or antagonize H receptor activity.
As used herein the term "transformed cell" refers to a modified host cell that expresses a functional H4 receptor expressed from a vector encoding the histamine receptor.
Any cell can be used, preferably a mammalian cell, and more preferably a HEK cell. A "functional histamine receptor" is a receptor that binds histamine or H4 agonists and transduces a signal upon such binding. Preferably the H4 receptor is a human H4. Preferably, the signal that is transduced is accumulation of a second messenger, preferably cAMP.
Histamine H4 receptors may be derived from a variety of sources, including mammal, e.g., human, bovine, porcine, and canine; and avian.
The cells of the invention are particularly suitable for an assay system for histamine H4 receptor ligands that modulate second messenger accumulation. An "assay system"
' is one or more collections of such cells, e.g., in a microwell plate or some other culture system.
To permit evaluation ofthe effects of a test compound on the cells, the number of cells in a single assay system is sufficient to express a detectable amount of the regulated second messenger accumulation at least under conditions of maximum second messenger accumulation.
A "second messenger" is an intracellular molecule or ion, where formation and/or accumulation ofthe second messenger is regulated by activation of cellular membranes. In one embodiment, cellular membranes contain G-protein coupled receptor, ion channels, and tyroinse kinase receptors. In the context of this invention, the cellular membrane is a G-protein coupled receptor, preferably a histamine H4 receptor. In a specific embodiment, the second messenger is one or more of cAMP, cGMP, inositol phosphate, DAG, and ions such as calcium and potassium. Preferably, the second messenger is cAMP.
A "test compound" or "candidate compound" is any molecule that can be tested for its ability to bind H4 receptors, and preferably modulate second messenger accumulation through the H4 receptor, as set forth herein. A compound that binds, and preferably modulates
H4 is a "lead compound" suitable for further testing and development as an H4 agonist or antagonist.
As used herein, the term "provide" refers to supplying the compounds or pharmaceutical compositions of the present invention to cells or to an animal, preferably a human, in any form. For example, a prodrug form ofthe compounds may be provided the subj ect, which then is metabolized to the compound in the body. H4 Receptor
H4 receptor, as defined herein, refers to a polypeptide having about 390 amino acids.
The protein is transcribed from a nucleic acid sequence that is about 1173 base pairs in length. The H4 protein has significant homology to the H3 receptor. Thus, H4 refers to orthologs and allelic variants, e.g. , a protein having greater than about 50%), preferably greater than 80%, more preferably still greater than 90%, and even more preferably greater than 95% overall sequence identity to SEQ ID NO: 2. Allelic variants may differ from 1 to about 5 amino residues from SEQ ID NO:2. In a specific embodiment, H4 has an amino acid sequence as shown in SEQ ID NO: 2.
Sequence comparison studies between human H4 protein and human H3 protein, indicates sequence identity of about 44%>. The predicted protein sequence contains residues which are characteristic of the class of biogenic amine receptors. As in other biogenic amine receptors, the present protein contains conserved aspartate residues within TM2 and TM3 (at positions 61 and 94). A DY motif, found only in histamine and muscarinic receptors, is found at positions 94-95. A DRY sequence motif that exists at the cytoplasmic interface of TM3 of all GPCRs also can be found in amino acids 111-113. Also present are two conserved cysteine residues in the first and second extracellular loops (position 88 and 164), two conserved tryptophan residues in TM4 and TM6 (positions 140 and 316); and conserved proline residues in TM5, TM6, and TM7 (positions 186, 318, and 355, respectively).
H4 receptors, like H2 and H3 receptors, modulates adenylyl cyclase activity. Therefore, the receptor modulates accumulation of the intracellular messenger cAMP. Modulation of H4 receptors may be a treatment for transplanted organ rejection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis. Additionally, CNS functions such as cognitive and memory defects may be treated.
H4 fragments, derivatives, and analogs can be characterized by one or more of the characteristics of H4 protein. In a specific embodiment, in order to develop the specific C-terminal and N-terminal H4 antibodies, antibodies can be raised against extracellular or cytoplasmic portions of the H4 protein, preferably or antigenic peptides identified using a hydrophobicity profile or other algorithms. Analogs and derivatives of the H4 receptor of the invention have the same or homologous characteristics of H4 as set forth above. For example, a truncated form of H4 can be provided. Such a truncated form includes H4 with a either an N-terminal, C-terminal, or internal deletion. In a specific embodiment, the derivative is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type H4 of the invention. Such functions include, but are not limited to, inhibition of adenylyl cyclase activity and cAMP formation. Alternatively, a H4 chimeric fusion protein can be prepared in which the H4 portion of the fusion protein has one or more characteristics of H4. Such fusion proteins include fusions ofthe H4 receptor with a marker polypeptide, such as FLAG, a histidine tag, a myc tag, or glutathione-S-transferase (GST). Alternatively, the H4 receptor can be fused with an expression-related peptide, such as yeast α-mating factor, a heterogeneous signal peptide, or a peptide that renders the protein more stable upon expression. H4 can also be fused with a unique phosphorylation site for labeling.
H4 analogs can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally similar molecules, i.e., molecules that perform one or more H4 functions. In a specific embodiment, an analog of H4 is a sequence-conservative variant of H4. In another embodiment, an analog of H4 is a function- conservative variant. In yet another embodiment, an analog of H4 is an allelic variant or a homologous variant from another species. In a specific embodiment, human variants of H4 are described.
H4 derivatives include, but are by no means limited to, phosphorylated H4, glycosylated H4, methylated H4, and other H4 proteins that are otherwise chemically modified. H4 derivatives also include labeled variants, e.g., radio-labeled with iodine (or, as pointed out above, phosphorous); a detectable molecule, such as but by no means limited to biotin, a chelating group complexed with a metal ion, a chromophore"br fluorophore, a gold colloid, or a particle such as a latex bead; or attached to a water soluble polymer.
Cloning and Expression of H^
The present invention contemplates analysis and isolation of a gene encoding a functional or mutant H4, including a full length, or naturally occurring form of H4, and any antigenic fragments thereof from any source, preferably human. It further contemplates expression of functional or mutant H4 protein for evaluation, diagnosis, or therapy.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill ofthe art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)];
B.Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
Molecular Biology - Definitions "Amplification" of DNA as used herein denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of
DNA sequences. For a description of PCR see Saiki et al, Science, 239:487, 1988.
As used herein, "sequence-specific oligonucleotides" refers to related sets of oligonucleotides that can be used to detect allelic variations or mutations in the H4 gene.
The nucleic acid molecules (polynucleotides) herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more ofthe naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like. A "coding sequence" or a sequence "encoding" an expression product, such as a
RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in' the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon. The term "gene", also called a "structural gene" means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include introns and regulatory DNA sequences, such as promoter sequences, 5 '-untranslated region, or 3'-untranslated region which affect for example the conditions under which the gene is expressed. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site
(conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The present invention includes the H4 receptor gene promoter found in the genome, which can be operatively associated with a H4 coding sequence with a heterologous coding sequence. The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays, as described infra.
A coding sequence is "under the control of or "operatively associated with" transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if it contains introns) and translated, in the case of mRNA, into the protein encoded by the coding sequence.
The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself , e.g., the resulting protein, may also be said to be "expressed" by the cell. An expression product can be characterized as intracellular, extracellular, transmembrane, or secreted. The term "intracellular" means something that is inside a cell. The term "extracellular" means something that is outside a cell. The H4 receptor is a seven transmembrane protein with intracellular and extracellular domains. A substance is
"secreted" by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
The term "transfection" means the introduction of a foreign nucleic acid into a cell. The term "transformation" means the introduction of a "foreign" (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone." The DNA or RNA introduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or cells of a different genus or species. The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.; they are discussed in greater detail below. Vectors typically comprise the DNA of a transmissible agent, into which foreign
DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A "cassette" refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion ofthe cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites ofthe vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct." A common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression ofthe coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England
Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
The term "heterologous" refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site ofthe cell. Preferably, the heterologous DNA includes a gene foreign to the cell. A heterologous expression regulatory element is such an element operatively associated with a different gene than the one it is operatively associated with in nature. In the context ofthe present invention, an H4 gene is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e.g., a HEK cell.
The terms "mutant" and "mutation" mean any detectable change in genetic material, e.g., DNA, or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" may also be used to indicate a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.
"Sequence-conservative variants" of apolynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.
"Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function ofthe polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point ofthe protein or polypeptide. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70%) to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide or enzyme which has at least 60 %ι amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%>, and even more preferably at least 90%o, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared.
As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al, Cell 1987, 50:667). Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs at conserved positions. Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al, supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin.
In a specific embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 80%>, and most preferably at least about 90 or 95%> of the nucleotides match over the defined length of the DNA sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc. An example of such a sequence is an allelic or species variant of the specific H4 gene of the invention.
Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.
Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80% ofthe amino acids are identical, or greater than about 90%) are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any ofthe programs described above (BLAST, FASTA, etc) A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form ofthe nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm (melting temperature) of 55°C, can be used, e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS. Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40%> formamide, with 5x or 6x SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5x or 6x SSC. SSC is a 0.15M NaCl, 0.015M Na-citrate buffer. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency ofthe hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al. , supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al, supra, 11.7-11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the. length is at least about 20 nucleotides.
In a specific embodiment, the term "standard hybridization conditions" refers to a Tm of 55 °C, and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60 °C; in a more preferred embodiment, the Tm is 65 °C. In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68 °C in 0.2xSSC, at 42°C in 50%) formamide, 4xSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15 , and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of H4, or to detect the presence of nucleic acids encoding H4. In a further embodiment, an oligonucleotide of the invention can form a triple helix with a H4 DNA molecule. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
The present invention provides antisense nucleic acids (including ribozymes), which may be used to inhibit expression of H4 ofthe invention. Inhibition of H4 expression may be desired when upregulation of H4 receptor expression or excessive inhibition of cAMP formation induces disease states such as, transplant organ rejection; asthma; allergies; autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis; and CNS functions such as cognitive and memory defects. An "antisense nucleic acid" is a single stranded nucleic acid molecule which, on hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid. As presently used, "antisense" broadly includes
RNA-RNA interactions, RNA-DNA interactions, ribozymes and RNase-H mediated arrest. Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent No. 5,814,500; U.S. Patent No. 5,811,234), or alternatively they can be prepared synthetically (e.g., U.S. Patent No. 5,780,607). Specific non-limiting examples of synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH2-NH-O-CH2, CH2-N(CH3)-O- CH2, CH2-O-N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and O-N(CH3)-CH2-CH2 backbones (where phosphodiester is O-PO2-O-CH2). U.S. Patent No. 5,677,437 describes heteroaromatic olignucleoside linkages. Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U.S. Patents No. 5,792,844 and No. 5,783,682). U.S. Patent No. 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds. Also envisioned are oligonucleotides having morpholino backbone structures (U.S. Patent No. 5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms ofthe polyamide backbone (Nielsen et al, Science 254:1497, 1991). Other synthetic oligonucleotides may contain substituted sugar moieties comprising one ofthe following at the 2' position: OH, SH, SCH3, F, OCN, O(CH2)nNH2 or O(CH2)nCH3 where n is from 1 to about 10; CI to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3; OCF3; O-; S-, or N-alkyl; O-, S-, or
N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substitued silyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group. Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine, such as inosine, may be used in an oligonucleotide molecule.
H4 Nucleic Acids A gene encoding H4, whether genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining H4 gene are well known in the art, as described above (see, e.g., Sambrook et al, 1989, supra). The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library"), and preferably is obtained from a cDNA library prepared from tissues with high level expression ofthe protein, by chemical synthesis, by cDNA cloning, or by the cloning of genomic
DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al. , 1989, supra; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II). Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation ofthe gene. Identification ofthe specific DNA fragment containing the desired H4 gene may be accomplished in a number of ways. For example, a portion of a H4 gene exemplified infra can be purified and labeled to prepare a labeled probe, and the generated DNA may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, Science 1977, 196:180; Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 1975, 72:3961). Those DNA fragments with substantial homology to the probe, such as an allelic variant from another individual, will hybridize. In a specific embodiment, highest stringency hybridization conditions are used to identify a homologous H4 gene.
Further selection can be carried out on the basis ofthe properties ofthe gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, partial or complete amino acid sequence, antibody binding activity, or ligand binding profile of H4 protein as disclosed herein. Thus, the presence ofthe gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.
Other DNA sequences which encode substantially the same amino acid sequence as a H4 gene may be used in the practice of the present invention. These include but are not limited to allelic variants, species variants, sequence conservative variants, and functional variants.
Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys.
The genes encoding H4 derivatives and analogs ofthe invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned H4 gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al, 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production ofthe gene encoding a derivative or analog of H4, care should be taken to ensure that the modified gene remains within the same translational reading frame as the H4 gene, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded. Additionally, the v-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Such modifications can be made to introduce restriction sites and facilitate cloning the H4 gene into an expression vector. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, et al, J. Biol. Chem. 253:6551, 1978; Zoller and Smith, DNA
3:479-488, 1984; Oliphant et al, Gene 44:177, 1986; Hutchinson et al, Proc. Natl. Acad. Sci. U.S.A. 83:710, 1986), use of TAB" linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).
The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as Bluescript, pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In addition, simple PCR or overlapping PCR may be used to insert a fragment into a cloning vector. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2μ plasmid.
H4 Regulatory Nucleic Acids
Elements of the H4 promoter can be identified by scanning the human genomic region upstream of the H4 start site, e.g., by creating deletion mutants and checking for expression, or with the TRANSFAC algorithm. Sequences up to about 6 kilobases (kb) or more upstream from the H4 start site can contain tissue-specific regulatory elements.
The term "H4 promoter" encompasses artificial promoters. Such promoters can be prepared by deleting nonessential intervening sequences from the upstream region ofthe H4 promoter, or by joining upstream regulatory elements from the H4 promoter with a heterologous minimal promoter, such as the CMV immediate early promoter.
An H4 promoter can be operably associated with a heterogenous coding sequence, e.g., for reporter gene (luciferase and green fluorescent proteins are examples of reporter genes) in a construct. This construct will result in expression ofthe heterologous coding sequence under control the H4 promoter, e.g., a reporter gene can be expressed, under conditions that under normal conditions cause H4 expression. This construct can be used in screening assays, described below, for H4 agonists and antagonists.
Expression ofH4 Polypeptides
The nucleotide sequence coding for H4, or antigenic fragment, derivative or analog thereof, or a functionally active derivative, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, /. e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Thus, a nucleic acid encoding H4 of the invention can be operationally associated with a promoter in an expression vector ofthe invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. Such vectors can be used to express functional or functionally inactivated H4 polypeptides.
The necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding H4 and/or its flanking regions.
Potential host-vector systems include but are not limited to mammalian cell systems transfected with expression plasmids or infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, herpes virus, etc.); insect cell systems infected with virus (e.g, baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used. Expression of H4 protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control H4 gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell 22:787-
797, 1980), the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981), the regulatory sequences of the metallothionein gene (Brinster et al, Nature 296:39-42, 1982); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff, et al , Proc. Natl. Acad. Sci. U.S.A. 75 :3727-3731 , 1978), or the tac promoter (DeBoer, et al, Proc. Natl. Acad. Sci. U.S.A. 80:21-25, 1983); see also "Useful proteins from recombinant bacteria" in Scientific American, 242:74-94, 1980; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and transcriptional control regions that exhibit tissue specificity, particularly endothelial cell-specific promoters. Solubilized forms ofthe protein can be obtained by solubilizing inclusion bodies or reconstituting membrane components, e.g., by treatment with detergent, and if desired sonication or other mechanical processes, as described above. The solubilized protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.
Vectors A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al, Gene 67:31-40, 1988), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
Viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphavirus, and other recombinant viruses with desirable cellular tropism are also useful. Thus, a gene encoding a functional or mutant H4 protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.
Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques 1992, 7:980- 990). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome ofthe replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part) or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles. DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HS V 1 ) vector (Kaplitt et al, Molec. Cell. Neurosci. 2:320-330, 1991), defective herpes virus vector lacking a glyco-protein L gene (Patent Publication RD 371005 A), or other defective herpes virus vectors (International Patent Publication No. WO 94/21807, published September 29, 1994; International Patent Publication No. WO 92/05263, published April 2, 1994); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al (J. Clin. Invest.
90:626-630, 1992; see also La Salle etal, Science 259:988-990, 1993); and a defective adeno- associated virus vector (Samulski etα/., J. Virol. 61:3096-3101, 1987; Samulski et α/., J. Virol. 63:3822-3828, 1989; Lebkowski et al. , Mol. Cell. Biol. 8:3988-3996, 1988).
Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and heφes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).
Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation ofthe viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, can be provided to block humoral or cellular immune responses to the viral vectors (see, e.g., Wilson, Nature Medicine, 1995). In that regard, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.
In another embodiment, the vector can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et al, Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417, 1987; Feigner and Ringold, Science 337:387-388, 1989; see Mackey, et al, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031, 1988; Ulmer, et al, Science 259:1745-1748, 1993). Useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127. Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al, supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO 96/25508), or a cationic polymer (e.g., International Patent Publication WO95/21931).
Alternatively, non- viral DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection; see, e.g., U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,853,663, U.S. Pat. No. 5,885,795, and U.S. Pat. No. 5,702,384 and see Sanford, TIB-TECH, 6:299-302, 1988; Fynan et al, Proc. Natl. Acad. Sci. U.S.A., 90:11478-11482, 1993; and Yang et al, Proc. Natl. Acad. Sci. U.S.A., 87:1568-9572, 1990), or use of a DNA vector transporter (see, e.g., Wu, et al, J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem. 263:14621-14624, 1988; Hartmut, et al, Canadian Patent
Application No. 2,012,311, filed March 15, 1990; Williams, et al, Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al, Hum. Gene Ther. 3:147-154, 1992; Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987). U.S. Patent Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir, et al. , C.P. Acad. Sci., 321:893, 1998; WO 99/01157; WO 99/01158; WO 99/01175).
H^ Ligands and Binding Partners The present invention further permits identification of physiological ligands and binding partners of H4. One method for evaluating and identifying H4 binding partners is the yeast two-hybrid screen. Preferably, the yeast two-hybrid screen is performed using an cell library with yeast that are transformed with recombinant H4. Alternatively, H4 can be used as a capture or affinity purification reagent. In another alternative, labeled H4 can be used as a probe for binding, e.g., by immunoprecipitation or Western analysis. Expected H4 binding partners are G-proteins.
Generally, binding interactions between H4 and any of its binding partners will be strongest under conditions approximating those found in the cytoplasm, i.e., physiological conditions of ionic strength, pH and temperature. Perturbation of these conditions will tend to disrupt the stability of a binding interaction.
Antibodies to H,,
Antibodies to H4 are useful, inter alia, for diagnostics and intracellular regulation of H4 activity, as set forth below. According to the invention, a H4 polypeptide produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as immunogens to generate antibodies that recognize the
H4 polypeptide. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Such an antibody is preferfably specific for human H4 and it may recognize either a mutant form of H4 or wild-type H4, or both.
One can use the hydropathic index of amino acids, as discussed by Kyte and Doolittle(JMolBiol. 1982, 157:105-132). See, for example, U.S. Patent 4,554,101, whichstates that the greatest local average hydrophilicity of a "protein," as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid. In using either the hydrophilicity index or hydropathic index, which assigns values to each amino acid, it is preferred to introduce substitutions of amino acids where these values are ± 2, with ± 1 being particularly preferred, and those within ± 0.5 being the most preferred substitutions.
Various procedures known in the art may be used for the production of polyclonal antibodies to H4 polypeptide or derivative or analog thereof. For the production of antibody, various host animals can be immunized by injection with the H4 polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the H4 polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
For preparation of monoclonal antibodies directed toward the H4 polypeptide, or fragment, analog, or derivative thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 1975,
256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today 1983, 4:72; Cote et al, Proc. Natl. Acad. Sci. 1983, 80:2026-2030), and the EB V-hybridoma technique to produce human monoclonal antibodies (Cole et al , in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96). In an additional embodiment ofthe invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690). Infact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al. , J. Bacteriol. 1984, 159:870; Neuberger et al, Nature 1984, 312:604-608; Takeda et al, Nature 1985, 314:452-454) by splicing the genes from a mouse antibody molecule specific for an H4 polypeptide together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves. Antibody fragments which contain the idiotype ofthe antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragment which can be produced by pepsin digestion ofthe antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges ofthe F(ab')2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
According to the invention, techniques described for the production of single chain antibodies (U.S. Patent Nos. 5,476,786, 5,132,405, and U.S. Patent 4,946,778) can be adapted to produce H4 polypeptide-specific single chain antibodies. An additional embodiment ofthe invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al. , Science 1989, 246: 1275- 1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for an H4 polypeptide, or its derivatives, or analogs.
In the production and use of antibodies, screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radio immunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of an H4 polypeptide, one may assay generated hybridomas for a product which binds to an H4 polypeptide fragment containing such epitope. For selection of an antibody specific to an H4 polypeptide from a particular species of animal, one can select on the basis of positive binding with H4 polypeptide expressed by or isolated from cells of that species of animal.
The foregoing antibodies can be used in methods known in the art relating to the localization and activity ofthe H4 polypeptide, e.g., for Western blotting, imaging H4 polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art. Such antibodies can also be used in assays for ligand binding, e.g., as described in U.S. Patent No. 5,679,582. Antibody binding generally occurs most readily under physiological conditions, e.g., pH of between about 7 and 8, and physiological ionic strength. The presence of a carrier protein in the buffer solutions stabilizes the assays. While there is some tolerance of perturbation of optimal conditions, e.g., increasing or decreasing ionic strength, temperature, or pH, or adding detergents or chaotropic salts, such perturbations will decrease binding stability.
In a specific embodiment, antibodies that act as ligands and agonize or antagonize the activity of H4 polypeptide can be generated. In addition, intracellular single chain Fv antibodies can be used to regulate cAMP formation (Marasco et al., Proc. Natl. Acad. Sci. U.S.A. 1993, 90:7884-7893; Chen., Mol. Med. Today 1997, 3:160-167; Spitz et al., Anticancer Res.
1996, 16:3415-22; Indolfi et al., Nat. Med. 1996, 2:634-635; Kijma et al., Pharmacol. Ther. 1995, 68 :247-267). Such antibodies can be tested using the assays described infra for identifying ligands.
Screening and Chemistry
According to the present invention, nucleotide sequences encoding H4 and the H4 receptor structure, which can be modeled from the amino acid sequence based on homology to other GPCR proteins, are useful targets to identify drugs that are effective in treating disorders associated with histamine-regulated processes. Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding H4 (e. g. , antisense or ribozyme molecules) and (ii)small molecule compounds that recognize and bind the receptor.
In particular, identification and isolation of H4 provides for development of screening assays, particularly for high throughput screening of molecules that up- or down- regulate the activity of H4. Accordingly, the present invention contemplates methods for identifying specific histamine receptor ligands that interact with H4 receptors, using various screening assays known in the art.
Any screening technique known in the art can be used to screen for H4 agonists or antagonists. The present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and agonize or antagonize H4 activity in vivo. For example, natural products libraries can be screened using assays ofthe invention for molecules that agonize or antagonize H4 expression or activity.
Another approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" (Scott and Smith, Science 1990, 249:386-390; Cwirla, et al, Proc. Natl. Acad. Sci., USA 1990, 87:6378-6382; Devlin etal, Science 1990, 49:404-406), very large libraries can be constructed (106-108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al, Molecular Immunology 1986, 23:709-715; Geysen etal. J. Immunologic Method 1987 102:259-274; and the method of Fodor et al. (Science 1991, 251 :767-773) are examples. Furka et al. (14th International Congress of Biochemistry, Volume #5 1988, Abstract FR:013; Furka, Int. J. Peptide Protein Res. 1991, 37:487-493), Houghton (U.S. Patent No. 4,631,211) and Rutter (U.S. Patent No. 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
In another aspect, synthetic libraries (Needels et al, Proc. Natl. Acad. Sci. USA 1993, 90:10700-4; Ohlmeyer et al, Proc. Natl. Acad. Sci. USA 1993, 90:10922-10926; Lam etal, PCT Publication No. WO 92/00252; Kocis etal, PCT Publication No. WO 9428028) and the like can be used to screen for ligands that regulate H4 activity. Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al, Tib Tech 1996, 14:60). Knowledge ofthe primary sequence of H4, and the similarity of that sequence with proteins of known function, can provide an initial clue as to the structure of agonists or antagonists of the receptor. Identification and screening of agonists antagonists is further facilitated by determining structural features ofthe receptor, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, homology studies, structure- activity relationships, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists.
One technique that may be used to assess the affinity of a test compound for the H4 receptor is a competition binding assay. In this assay, test wells containing an aliquot of a lipid bilayer membranes that contain the histamine H4 receptor are incubated with an known concentration of a radiolabeled ligand for the receptor. The lipid bilayer may be prepared by any known protocol that separates the membrane containing receptor component from the cytoplasmic components. Each well also is incubated with a different concentration of a unlabeled test compound. Cell membranes are then separated from the incubation mixture by any method known in the art including, but not limited to, centrifugation and vacuum filtration on a cell harvester. The radioactivity of each well is then determined using any device that can detect radioactivity, such as a scintillation counter. As increasing concentrations of the test compound compete for the receptor binding site, the radioactivity detected decreases. The data then can be converted using the Cheng-Prusoff equation (Biochem Pharmacol. 1973, 22:3099-3108) to determine the affinity (K-) ofthe compound for the receptor.
In vivo screening methods Intact cells or whole animals expressing a gene encoding H4 can be used in screening methods to identify candidate drugs.
In one series of embodiments, a permanent cell line is established. Alternatively, cells (including without limitation mammalian, insect, yeast, or bacterial cells) are transiently programmed to express an H4 gene by introduction of appropriate DNA or mRNA. Identification of candidate compounds can be achieved using any suitable assay, including without limitation
(i) assays that measure binding of test compounds to H4 (ii) assays that measure the ability of a test compound to modify (i. e. , inhibit or enhance) a measurable activity or function of H4 and (iii) assays that measure the ability of a compound to modify (i.e., inhibit or enhance) the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions ofthe H4 gene.
H4 knockout mammals can be prepared for evaluating the molecular pathology of this defect in greater detail than is possible with human subjects. Such animals also provide excellent models for screening drug candidates. A "knockout mammal" is an mammal (e.g., mouse, rabbit) that contains within its genome a specific gene that has been inactivated by the method of gene targeting (see, e.g., U.S. Patent Nos. 5,777,195 and 5,616,491). A knockout mammal includes both a heterozygote knockout (i.e., one defective allele and one wild-type allele) and a homozygous mutant (i.e., two defective alleles; a heterologous construct for expression of an H4, such as a human H4, could be inserted to permit the knockout mammal to live if lack of H4 expression was lethal). Preparation of a knockout mammal requires first introducing a nucleic acid construct that will be used to suppress expression of a particular gene into an undifferentiated cell type termed an embryonic stem cell. This cell is then injected into a mammalian embryo. A mammalian embryo with an integrated cell is then implanted into a foster mother for the duration of gestation. Zhou, et al. (Genes and Development 1995, 9:2623-34) describes PPCA knock-out mice.
The term "knockout" refers to partial or complete suppression ofthe expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell. The term
"knockout construct" refers to a nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell. The nucleic acid sequence used as the knockout construct is typically comprised of (1) DNA from some portion ofthe gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell. The knockout construct is inserted into a cell, and integrates with the genomic DNA ofthe cell in such a position so as to prevent or interrupt transcription of the native DNA sequence. Such insertion usually occurs by homologous recombination (i.e., regions ofthe knockout construct that are homologous to endogenous DNA sequences hybridize to each other when the knockout construct is inserted into the cell and recombine so that the knockout construct is incoφorated into the corresponding position ofthe endogenous DNA). The knockout construct nucleic acid sequence may comprise (1) a full or partial sequence of one or more exons and/or introns ofthe gene to be suppressed, (2) a full or partial promoter sequence ofthe gene to be suppressed, or (3) combinations thereof. Typically, the knockout construct is inserted into an embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination. This ES cell is then injected into, and integrates with, the developing embryo.
The phrases "disruption ofthe gene" and "gene disruption" refer to insertion of a nucleic acid sequence into one region ofthe native DNA sequence (usually one or more exons) and/or the promoter region of a gene so as to decrease or prevent expression of that gene in the cell as compared to the wild-type or naturally occurring sequence of the gene. By way of example, a nucleic acid construct can be prepared containing a DNA sequence encoding an antibiotic resistance gene which is inserted into the DNA sequence that is complementary to the DNA sequence (promoter and/or coding region) to be disrupted. When this nucleic acid construct is then transfected into a cell, the construct will integrate into the genomic DNA. Thus, many progeny ofthe cell will no longer express the gene at least in some cells, or will express it at a decreased level, as the DNA is now disrupted by the antibiotic resistance gene.
Generally, the DNA will be at least about 1 kb in length and preferably 3-4 kb in length, thereby providing sufficient complementary sequence for recombination when the knockout construct is introduced into the genomic DNA ofthe ES cell (discussed below). Included within the scope of this invention is a mammal in which two or more genes have been knocked out. Such mammals can be generated by repeating the procedures set forth herein for generating each knockout construct, or by breeding to mammals, each with a single gene knocked out, to each other, and screening for those with the double knockout genotype. Regulated knockout animals can be prepared using various systems, such as the tet-repressor system (see U.S. Patent No.5,654,168) or the Cre-Lox system (see U.S. Patent Nos. 4,959,317 and 5,801,030).
In another series of embodiments, transgenic animals are created in which (i) a human H4 is stably inserted into the genome ofthe transgenic animal; and/or (ii) the endogenous H4 genes are inactivated and replaced withhuman H4 genes. See, e.g., Coffman, Semin. Nephrol.
1997, 17:404; Esther et al, Lab. Invest. 1996, 74:953; Murakami et al., Blood Press. Suppl. 1996, 2:36.
H4 Activation Assay Any cell assay system that allows for assessment of functional activity of H4 agonists and antagonists is defined by the present invention. In a specific embodiment, exemplified infra, the assay can be used to identify compounds that selectively interact with H4, which can be evaluated by assessing the effects of H4 transformed cells contacted with a test compound, which modulates cAMP accumulation. The assay system can thus be used to identify compounds that selectively produce a functional effect through histamine H4 receptors. Compounds that increase cAMP formation and accumulation may be useful as novel therapeutics in the prevention of transplanted organ rej ection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis. Additionally, CNS functions such as cognitive and memory defects may be treated. Preferably, each experiment is performed in triplicate at multiple different dilutions of test compound. An agonist and/or antagonist screen involves detecting cAMP accumulation by the host cell when contacted with H4 ligand. If cAMP accumulation is increased, the test compound is a candidate antagonist of H4 receptors. If cAMP accumulation is decreased, the test compound is a candidate agonist of H4 receptors. If there is no change in cAMP formation, the test compound is not an effective H4 ligand.
Any convenient method permits detection of the formed product, cAMP. For example, the invention provides immunoassays for detecting cAMP. Typically, immunoassays use either a labeled antibody or a labeled antigenic component (e.g., that competes with the antigen in the sample for binding to the antibody). Suitable labels include without limitation enzyme-based, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labelled immunoassays, such as ELISA assays. Alternatively, labeled antigenic component may be quantified by scintillation techniques. In another method, the second messenger, preferably cAMP, will be separated on a high performance liquid chromatograph and quantified by a UV detector.
The assay system described here also may be used in a high-throughput primary screen for agonists and antagonists, or it may be used as a secondary functional screen for candidate compounds identified by a different primary screen, e.g., a binding assay screen that identifies compounds that interact with the receptor.
High-Throughput Screen Agents according to the invention may be identified by screening in high- throughput assays, including without limitation cell-based or cell-free assays. It will be appreciated by those skilled in the art that different types of assays can be used to detect different types of agents. Several methods of automated assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period of time. Such high- throughput screening methods are particularly preferred. The use of high-throughput screening assays to test for agents is greatly facilitated by the availability of large amounts of purified polypeptides, as provided by the invention.
Compounds
"Histamine" refers to a neurotransmitter that is produced and released from neurons. Histamine is formed from the amino acid histidine by histidine decarboxylase.
Structurally, histamine is an imidazolethylamine. In other words, histamine is comprised of an imidazole moiety and an amino group connected by an alkyl chain. The pharmacologically active form of histamine is proposed to be the monocationic tautomer, where one ofthe nitrogen present in the imidazole ring is positively charged. However, different forms of histamine may interact with histamine receptors to produce a functional effect. Histamine is produced intracellularly and stored until released in response to a physiological stimulus.
"Histamine analogs" refers to compounds that comprise the imidazole, alkyl chain, and a nitrogen moiety (e.g., amine (unsubstituted or substituted), piperidine, pyridine) of histamine, but may be modified at other positions. These modifications may be performed to alter affinity and/or selectivity ofthe compound for the histamine receptors. "Histamine compounds" refers to compounds that may bind to the histamine receptors.
Histamine analogs and compounds can be classified as agonists or antagonists. As discussed previously, agonists are ligands that bind to the receptor and produce a functional effect similar to that produced by the endogenous ligand (i.e., histamine) for the receptor, whereas antagonists are ligands that bind to the receptor and block a functional effect produced by an agonist for the receptor or the endogenous ligand ofthe receptor. Histamine analogs and compounds are further described in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996.
Agonists that may be contemplated by this invention include, but are not limited to, R-(α)-methylhistamine, imetit, and immepip. Antagonists, burimamide, impromidine, dimaprit, and thioperamide clobenpropit and iodophenpropit impentamine, GT2016 and iodoproxyfan. Other compounds include derivatives, metabolites, and precursors.
Methods of Diagnosis
According to the present invention, genetic variants of H4 can be detected to diagnose an H4 associated disease, such as treatment for transplanted organ rejection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis. Additionally, CNS functions such as cognitive and memory defects may be treated. The various methods for detecting such variants are described herein. Where such variants impact H4 function, either as a result of a mutated amino acid sequence or because the mutation results in expression of a truncated protein, or no expression at all, they are expected to result in disregulation ofthe allergic response, the immune response, cognition and memory.
Nucleic A cid Assays
The DNA may be obtained from any cell source. DNA is extracted from the cell source or body fluid using any ofthe numerous methods that are standard in the art. It will be understood that the particular method used to extract DNA will depend on the nature ofthe source. Generally, the minimum amount of DNA to be extracted for use in the present invention is about 25 pg (corresponding to about 5 cell equivalents of a genome size of 4 x
109 base pairs).
In another alternate embodiment, RNA is isolated from biopsy tissue using standard methods well known to those of ordinary skill in the art such as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznski et al, Anal. Biochem., 162: 156, 1987). The isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a selected site. Conditions for primer annealing are chosen to ensure specific reverse transcription and amplification; thus, the appearance of an amplification product is diagnostic ofthe presence of a particular genetic variation. In another embodiment, RNA is reverse-transcribed and amplified, after which the amplified sequences are identified by, e.g., direct sequencing. In still another embodiment, cDNA obtained from the RNA can be cloned and sequenced to identify a mutation.
Protein Assays In an alternate embodiment, biopsy tissue is obtained from a subject. Antibodies that are capable of specifically binding to H4 are then contacted with samples of the tissue to determine the presence or absence of a H4 polypeptide specified by the antibody. The antibodies may be polyclonal or monoclonal, preferably monoclonal. Measurement of specific antibody binding to cells may be accomplished by any known method, e.g., quantitative flow cytometry, enzyme-linked or fluorescence-linked immunoassay, Western analysis, etc. Therapeutic Uses
According to the present invention, stimulation of H4 receptor activity may be used as a treatment option in patients with histamine-related disease states. Stimulation of H4 receptor activity may be by methods, such as, but not limited to, (i) providing polypeptides that stimulate receptor activity and (ii) providing compounds that stimulate receptor activity.
Gene Therapy
In a specific embodiment, vectors comprising a sequence encoding a protein, including, but not limited to, full-length H4, are provided to treat or prevent a disease or disorder associated with the function of H4 in peripheral blood leukocytes. In this embodiment of the invention, the therapeutic vector encodes a sequence that produces the protein ofthe invention.
Any ofthe methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews ofthe methods of gene therapy, see, Goldspiel etal. , Clinical Pharmacy, 1993, 12:488-505; Wu and Wu, Biotherapy, 1991, 3:87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 1993, 32:573-596; Mulligan, Science, 1993, 260:926-932; andMorganand
Anderson, Ann. Rev. Biochem., 1993, 62: 191-217; May, TIBTECH, 1993, 11:155-215. Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al, (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13 , Dracopoli et al., (eds.), 1994, Current Protocols in Human Genetics, John
Wiley & Sons, NY. Vectors suitable for gene therapy are described above.
In one aspect, the therapeutic vector comprises a nucleic acid that expresses a protein of the invention in a suitable host. In particular, such a vector has a promoter operationally linked to the coding sequence for the protein. The promoter can be inducible or constitutive and, optionally, tissue-specific. In another embodiment, a nucleic acid molecule is used in which the protein coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the protein (Koller and Smithies, Proc. Natl. Acad. Sci. U.S.A, 1989, 86:8932-8935; Zijlstra et α/., Nature, 1989, 342:435-438). Delivery ofthe vector into a patient may be either direct, in which case the patient is directly exposed to the vector or a delivery complex, or indirect, in which case, cells are first transformed with the vector in vitro then transplanted into the patient. These two approaches are known, respectively, as in vivo and ex vivo gene therapy.
In a specific embodiment, the vector is directly provided in vivo, where it enters the cells ofthe organism and mediates expression ofthe protein. This can be accomplished by any of numerous methods known in the art, by constructing it as part of an appropriate expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in biopolymers (e.g., poly-S-l-64-N-acetylglucosamine polysaccharide; see, U.S. Patent No.
5,635,493), encapsulation in liposomes, microparticles, or microcapsules; by administering it in linkage to a peptide or other ligand known to enter the nucleus; or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 1987, 62:4429-4432), etc. In another embodiment, a nucleic acid ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publication Nos. WO 92/06180, WO 92/22635, WO 92/20316 and WO 93/14188). Alternatively, the nucleic acid can be introduced intracellularly and incoφorated within host cell DNA for expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.
USA, 1989, 86:8932-8935; Zijlstra, et al, Nature, 1989, 342:435-438). These methods are in addition to those discussed above in conjunction with "Viral and Non- viral Vectors".
The form and amount of therapeutic nucleic acid envisioned for use depends on the type of disease and the severity ofthe desired effect, patient state, etc., and can be determined by one skilled in the art.
Inhibition or stimulation of protein synthesis Gene transcription and protein translation may be inhibited or stimulated by administration of exogenous compounds. Exogenous compounds may interact with extracellular and/or intracellular messenger systems, such as, but not limited to, adenosine triphosphate, nitric oxide, and guanosine triphosphate; to regulate protein synthesis. In this embodiment, exogenous compounds that stimulate or inhibit H4 protein synthesis may be used in the prevention and/or treatment for transplanted organ rejection, asthma, allergies, and autoimmune pathologies, such as, but not limited to, multiple sclerosis, type I diabetes, and rheumatoid arthritis. Additionally, CNS functions such as cognitive and memory defects may be treated. The present invention provides antisense nucleic acids (including ribozymes), which may be used to inhibit expression of H4 ofthe invention. The antisense nucleic acid, upon hybridizing under cytoplasmic conditions with complementary bases in an RNA or DNA molecule, inhibits the role ofthe RNA or DNA. Additionally, hybridization ofthe antisense nucleic acid to the DNA or RNA may inhibit transcription ofthe DNA into RNA and/or translation of the RNA into the protein. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a countertranscript or mRNA-interfering complementary nucleic acid. Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent No. 5,814,500; U.S. Patent No. 5,811,234) or can be prepared synthetically (e.g., U.S. Patent No. 5,780,607). Alternatively, antibody molecules can also be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (Proc. Natl. Acad Sci. USA, 1993, 90:7889-7893).
Therapeutically suggested compounds may be provided to the patient in formulations that are known in the art and may include any pharmaceutically acceptable additives, such as excipents, lubricants, diluents, flavorants, colorants, and disintegrants. The formulations may be produced in useful dosage units such as tablet, caplet, capsule, liquid, or injection.
The form and amount of therapeutic compound envisioned for use depends on the type of disease and the severity ofthe desired effect, patient state, etc., and can be determined by one skilled in the art.
EXAMPLES
The present invention will be better understood by reference to the following Examples, which are provided as exemplary ofthe invention, and not by way of limitation. EXAMPLE I: CHARACTERIZATION OF THE EL, RECEPTOR
A GPCR sequence profile was generated from a sequence alignment of members of this subfamily based on hidden Markov Models (Eddy Bioinformatics, 1998 14:755-763; Durbin et al. A tutorial introduction to hidden Markov models and other probabilistic modeling approaches in computational sequence analysis, Cambridge University Press, 1998) to look for novel members in the human genome database. In detail, GPCR proteins ofthe biogenic amine subfamily were retrieved from S wiss-Prot database and a sequence alignment was generated by a multiple sequence alignment tool, named CLUSTALW (Thompson et al. Nucleic Acids Res, 1994, 22:4673-80). Using the HMMER program (Eddy, HMMER User's Guide and Program, Version 2.1, 1998), a consensus sequence (GNLLVILVIL RTKKLRTPTN IFILNLAVAD
LLFLLTLPPW ALYYLVGGSE DWPFGSALCK LVTALDVVNM YASILLLTAI SIDRYLAIVH PLRYRRRRTS PRRAKVVILL VWVLALLLSL PPLLFSWVKT VEEGNGTLNV NVTVCLIDFP EESTAS VSTW LRS YVLLSTL VGFLLPLLVI LVC YTRJLRT LRKAAKTLLV VVVVFVLCWL PYFIVLLLDT LCLSIIMSST CELERVLPTA LLVTLWLAYV NSCLNPIIY; SEQ ID NO: 3) was developed from the biogenic amine subfamily members. The consensus sequence contained the unique 7-transmembrane sequence structure of biogenic amine GPCRs.
A weekly update of nucleotide sequence from GenBank database is maintained in-house. An auto-search script using TBLASTN program was written and the biogenic amine GPCR consensus sequence was used to search this database weekly. Every TBLASTN search result was carefully examined and potential open reading frame (ORF) fragments were extracted from nucleotide sequence. Each fragment was further verified to determine its novelty.
Four peptide fragments translated from a recently released human genomic sequence (Accession number: AC007922) of chromosome 18 clone RP11-178F10, from the Whitehead Institute/MIT Center for Genome Research, were shown to have modest homology to different regions of this GPCR consensus sequence. These four fragments located to different regions of sequence AC009722 in both plus and minus strands. Interestingly, the highest scoring hit from searching the protein database with these peptides by BLASP was in all cases, the human histamine receptor 3 (H3). It is very likely that these four fragments are exons of a GPCR gene and their appearance in both strands of this genomic sequence may result from incorrect genomic contig assembly. To clone the full length ORF, multiple primer sets were designed against the predicted sequence. Using multiple primer pairs, 3 overlapping pieces spanning the entire ORF were obtained from a human heart marathon cDNA library (Clontech, Palo Alto, CA). Using the marathon adapter primer API, and a reverse primer corresponding to ORF bases 644-615, a fragment ofthe H4 cDNA was obtained which corresponds to bases 276-644 of the H4 coding region (exon sequence) and 39 bases of intronic sequence at the 5' end. Primers comprising nucleotides 461-482 and 1173-1146 were used to PCR a 712bp band. The 5' sequence was obtained using primers comprising nucleotides 1-32 and 339-309. The outer primers (1-32, 1173-1146) were used to piece the 3 fragments together. The sequence was ligated into the mammalian expression vector pCDNA3.1 + zeo (Invitrogen, Carlsbad, CA).
EXAMPLE II: TISSUE EXPRESSION OF THE EL, RECEPTOR
Quantitative RT-PCR was performed on an ABI 7700 "Taqman" sequence detection system to determine the tissue distribution ofthe H4 receptor. Primers spanning the exon 1-2 boundary (Forward primer: 5'-taacttggccattgacttctt-3' (SEQ ID NO:4); Reverse primer:
5'-attcgaacagcatgtgagggat-3' (SEQ ID NO: 5) and a Probe: 5'-(6-carboxyfluorescein)- tacaaaggaatggagatcacacccaca- (6-carboxy-N,N,N'N'-tetramethylrhodamine)-3' (SEQ ID NO: 6)) were used to determine H4 expression levels in a mRNA prepared from a series of human tissues, purchased from Clontech (Palo Alto). A 2 step reaction procedure was performed as per manufacturers directions. Briefly, 2mg RNA was reverse transcribed using random hexamers
(2.5mM in a final volume of 20ml. 14ul of this was used in the PCR reaction. The cyling conditions were as follows, 95 °C for 10 minutes, followed by 40 cycles of (a) 95 °C for 15 seconds and (b) 60 °C for 1 minute.
EXAMPLE III: PHARMACOLOGICAL PROFILE OF THE H, RECEPTOR IN YEAST
CELLS
The H4 receptor protein coding sequences were amplified using Forward oligo:
5'-aaggatccaaaatgccagatactaatagc-3' (SEQ ID NO:7) and Reverse oligo: 5'- aagtcgacttaagaagatactgaccgac-3' (SEQ ID NO: 8) that add BamHI and yeast consensus translational initiation sites to the 5' end and a Sail site to the 3' end. The fragment was cloned into corresponding sites in the multicopy yeast expression vector, p426GPD, thus placing receptor expression under control of the strong constitutive GPDl promoter. The yeast expression plasmid, pMP327, was introduced into MPN578i5 cells (MATa ura3 his3 tipl leu2 lys2 ade2 farl::LNS2 fusl ::FUSl-HIS3 sst2::SST2-G418R ste2::LEU2, gpal::GPAli5) (Hadcock, J.R. and Pausch, M.H. submitted) using LiOAc and selected for ura prototrophy. In order to facilitate coupling ofthe receptor to the G protein, MPN578i5 cells express a chimeric
G alpha protein coupled to the mating signal transduction pathway. The chimeric construct is expressed from the GPA1 locus and is composed of Gpal sequences in which the 5'-C-terminal amino acids have been replaced with those of Gai3. A multicopy FUSl-LacZ reporter gene plasmid, pMP283 (Hadcock, J.R. and Pausch, M.H. submitted), was subsequently introduced into H4R-containing MPY578i5 cells and selected on media lacking tip and ura. The resulting yeast strain, MPY733, was used for further analysis.
Samples (250 ng) of compounds present in the LOP AC panel (Sigma RBI, Νatick MA), were dispensed to 96 well microtiter dishes. MPY733 cells (5 x lOVml, 200 ml/well) in assay medium (SCD-ura-frp, pH 6.8, 25 mM PIPES, O.lmg/ml Chlorophenylred bglactopyranoside(CPRG), 2 mM 3-AT) were added and cultured overnight at 30 °C. The presence of active compounds was detected the next day by measurement of absorbance at 570 nm using a Wallac Nictor II. The LOP AC panel was screened in duplicate and in parallel with another yeast strain containing a different oφhan GPCR. Only compounds that produced significantly elevated absorbance in both receptor containing plates and not in the other GPCR containing plates were deemed active.
EXAMPLE IN: PHARMACOLOGICAL PROFILE OF THE EL, RECEPTOR IN MAMMALIAN CELLS
The ORF was modified by PCR for mammalian expression of H4. A 5' Hindlll restriciton enzymes site and a Kozak consensus sequence were added using the primer 5'- aagcttccaccatgccagatactaatagcacaatcaatttatc-3' (SEQ ID NO:9), and a 3' Xbal site added 5'- tctagattaagaagatactgaccgactgtgttg-3 ' (SEQ ID NO : 10) . The sequence was confirmed and ligated into the Hindlll and Xba I sites of the mammalian expression vector pCDNA3.1+ zeo (Invitrogen, Carlsbad CA). HEK 293 cells (approximately 107 cells) were transfected with the pCDN A3.1 +zeo/H4 using standard lipofectamine plus reagent (Life Technologies) . Cells were maintained in DMEM containing 10% fetal calf serum and penicillin ( 100units/ml)/streptomycin (lOOmg/ml). 48 hours post transfection cells were selected using 500mg/ml zeocin. Zeocin resistant clones were assayed using the cAMP assay and by RT-PCR. RNA was extracted from approximately 106 cells using One step PCR kit (Life technologies). The primers used in the extraction were 5'-ggaaggatgaaggtagtgaatg-3' (SEQ ID NO: 11) and 5'-cagaatctgattgggaggaagg-3' (SEQ ID NO: 12).
HEK cells stably expressing the H4 receptor were assessed functionally in cAMP assays using the cAMP scintillation proximity assay (SPA) (Amersham Pharmacia Biotech, Pistcataway, NJ). Briefly, 40 000 cells were plated into wells of a 96 well plate. 24 hours later the media was removed and replaced with lOOμl Krebs bicarbonate buffer and the cells were incubated at 37°C for 15 minutes. Following this, the cells were incubated in Krebs buffer containing 0.5mM isobutylmethylxanthine, a phosphodiesterase inhibitor, to prevent cAMP breakdown. The effect of H4 on the forskolin induced formation was determined by incubating the cells in the presence of forskolin (lOμM) and agonist for 12 minutes. cAMP levels were determined using the cAMP SPA kit according to the manufacturers directions.
EXAMPLE N: H^ EXPRESSION IN INDUCED CD4 AND CD8 CLONES
This examination was undertaken using the TaqMan EZ RT PCR kit and the following oligonucleotides (provided by Philip Jones at Wyeth Neuroscience) (i) H4 EX IF (forward oligo): 5'-taacttggccatctttgac-3' (SEQ ID NO: 13), (ii) H4 EX 1R (reverse primer): 5'- attcgaacagcgtgtgag-3' (SEQ ID NO.T4), and H4 EX 1 Probe: 5'-(6-carboxyfluorescein)- tacaaaggaatggagatca-3' (SEQ ID NO: 15). RNA for the standard curve was polyA+ human leukocyte RNA from Clontech.
50 nanograms of total RNA was assayed in duplicate from 6 human T cell clones derived from a single human donor. Three of these clones were CD4+, two were CD8+, and one was an NK T cell clone. These T cell clones were stimulated with anti-CD3 and RNA was isolated at 0, 2, 4, 8, 24 and 48 hours after stimulation. Expression was normalized with GAPDH.
In order to determine whether the pattern of H4 expression was specific to clonal populations of human lymphocytes, the same TaqMan quantitative PCR assay on 10 nanograms of RNA from bulk populations of CD4+ and CD 8+ T cells isolated from three different individuals. These lymphocytes were stimulated with anti-CD3 and harvested at the same time points listed above. H4 expression was normalized with GAPDH.
RESULTS
Using a GPCR sequence profile generated from a Hidden Markov Model (HMM) of the biogenic amine subfamily, several sequences with homology to the human H3 receptor were identified from the human genomic sequence of chromosome 18 (clone RP11- 178F10 Accession number: AC007922 from the Whitehead Institute/MIT Center for Genome Research) (Eddy Bioinformatics, 1998, 14:755-763). A contig ofthe predicted exons forms a sequence encoding aputative GPCR whose nucleotide sequence and translated peptide sequence are shown in Figure 1 and SEQ ID NOS:l and 2. The sequence was deposited with Genbank (Accession
Number AF307973).
The predicted protein sequence contains residues that are conserved across the biogenic amine receptors. As in other biogenic amine receptors, the present protein contains conserved aspartate residues within transmembrane (TM) domains 2 and 3 (at positions 61 and 94). A DY (D94 and Y95) motif in TM3 is found only in histamine and muscarinic receptors.
A DRY sequence motif that exists at the cytoplasmic interface of TM3 of all GPCRs also can be found in the H4 sequence (amino acids 111- 113). Also present are two conserved cysteine residues in the first and second extracellular loops (position 88 and 164), which are predicted to form a disulfide bond; two conserved tryptophan residues in TM4 and TM6 (positions 140 and 316); and conserved proline residues in TM5, TM6, and TM7 (positions 186, 318, and 355, respectively). Based on these results, H4 cDNA appears to encode a biogenic amine-like receptor.
The predicted open reading frame is 1173 base pairs long and encodes a protein of 390 amino acids. Sequence comparison using BLASTP, under standard conditions, analysis reveals that the novel protein is most similar to the human H3 receptor (44%> identical and 51 %> similar). Based on sequence homology it is proposed that the receptor belongs to the histamine receptor family, therefore we have termed it the H4 receptor.
Receptor distribution studies indicate that the present receptor is highly expressed in peripheral blood leukocytes. Trace amounts ofthe H4 receptor are expressed in heart, lung and placenta. It is proposed that these trace amounts of expression represent blood cell mRNA present in the samples. Functional studies further confirmed the categorization ofthe protein as a novel histamine receptor. Histamine and the H3 selective agonist R-α-methylhistamine stimulated cAMP accumulation, suggesting these compounds are agonists at the H4 receptor (See Figure 2A). R-α-methylhistamine exhibited both lower potency and efficacy than histamine at the H4 receptor despite being more potent at the related cloned H3 receptor and in several tissue based assays for the H3 receptor (Vollinga, et al, J. Med. Chem. 1995, 38:266-271; Haφer, etal, Br. J. Pharmacol. 1999, 128:751-759). The selective H3 receptor antagonist clobenpropit was a partial agonist at the H4 receptor (See Figure 2A).
The H3 antagonist thioperamide almost fully inhibited the stimulatory response produced by histamine (See Figure 2B). Comparatively, clobenpropit partially blocked histamine-induced stimulation of cAMP formation, further suggesting that clobenpropit is a partial agonist at the H4 receptor. The interaction ofthe H4 receptor to a GPAI/ Gαi3 chimeric G-protein alpha subunit also predicts its coupling specificity in mammalian cells.
Expression ofthe H4 receptor in HEK 293tsa cells confirms the coupling ofthe H4 receptor to the inhibition of cAMP formation (See Figure 2C). In these cells, forskolin stimulated 8-fold greater cAMP formation compared to basal levels. Addition of histamine (1 μM) inhibited forskolin-induced stimulation of cAMP accumulation by about 40%>. These studies suggest that the H4 receptor couples an inhibitory G-protein to inhibit adenylyl cyclase activity and cAMP accumulation. Structure-activity relationship studies were conducted with several histamine antagonists to further define the pharmacological profile of this receptor. Studies were conducted to correlate the effect ofthe alkyl chain to antagonist activity at the histamine H4 receptor (See Figure 3). The rank order of efficacy obtained for the human H4 was (number represents the length ofthe alkyl chain) 5>6= 1 0>8=4. This rank order of efficacy contrasts with the human H3 receptor where the rank order is 5>4>6>8>10.
Prior studies have indicated the expression of at least two subtypes of the H3 receptor. These two potential H3 subtypes, localized in the rat brain and guinea pig j ej enum, have been shown to have rank order of efficiencies of 4=5>3>6>8 and 5>6=4>8>3, respectively. These assays were different (rat was a radioligand binding assay and guinea pig was an organ bath experiment using isolated guinea pig ileum). However, the relative potencies ofthe series can be compared, so its possible to say that as the rank order differs then the receptors are likely to be different (Leurs et al J. Pharm. Exp. Ther.,'l996, 276:1009-1015.)
The present studies also indicate that thioperamide is more efficacious and potent than impentamine at the H4 receptor. Comparatively, impentamine has greater affinity for cloned histamine H3 receptors (4-fold) than thioperamide (Ki=50.8nM and 193nM respectively). H4 expression was consistently detected in all three CD4+ clones at most time points (Figure 4). There was no consistent temporal pattern to anti-CD3 induction of expression between these three clones. One CD8+ clone showed a very low but detectable level of expression at most time points, while the other as well as the NKT clone had no detectable H4 expression despite good GAPDH amplification. H4 expression, normalized with GAPDH, was detectable in all samples at all time points (Figure 5). Anti-CD3 stimulation produced significant induction of H4 expression in all 6 samples, most peaking at the 8 hour time point. The highest level of induction was found in the 8 hour CD4+ and CD8+ samples from the same donor (designated 'U'), peaking at greater than 100 copies of H4 RNA per copy of GAPDH. Combined, these studies indicate that the pharmacological profile of the H4 receptor is not similar to any known histamine H3 receptor.
H= * *
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope ofthe appended claims.
It is further to be understood that values are approximate, and are provided for description.
Patents, patent applications, publications, procedures, and the like are cited throughout this application, the disclosures of which are incoφorated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. An isolated H4 receptor protein having an amino acid sequence at least 51% identical or comprising at least 10 contiguous amino acids from the sequence depicted in SEQ ID NO: 2 which H4 receptor protein binds ligands comprising an imidazole and an amine, which imidazole and amine are attached by an alkyl chain, wherein the rank order of efficacy of modulation second messenger formation ofthe ligands at the H4 receptor protein is 5>6= 10>8=4, where the number represents the number of carbons in the alkyl chain.
2. The H4 receptor protein of claim 1 , wherein upon binding histamine or a histamine agonist the receptor protein inhibits second messenger formation.
3. The H4 receptor protein of claim 2, wherein the second messenger is cAMP.
4. The H4 receptor protein of claim 1 which is a human H4 receptor protein.
5. The H4 receptor protein of claim 4 which has an amino acid sequence as depicted in SEQ ID NO: 2.
6. The H4 receptor protein of claim 4 which is encoded by a nucleic acid having a sequence as depicted in SEQ ID NO: 1.
7. An isolated H4 receptor protein having an amino acid sequence with at least 95%) sequence identity to human H4 receptor protein having an amino acid sequence as depicted in SEQ ID NO : 2.
8. An isolated nucleic acid encoding the H4 receptor protein of claim 1 or 7.
9. The nucleic acid of claim 8 which is a cDNA.
10. The nucleic acid of claim 8, wherein the H4 receptor protein is a human H4 receptor protein.
11. An isolated nucleic acid encoding an H4 receptor protein, which nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence of at least 20 nucleotides identical to a corresponding nucleotide sequence ofthe same number of bases in SEQ ID NO: 1 or its complement.
12. The nucleic acid of claim 8, which encodes an H4 receptor protein having an amino acid sequence as depicted in SEQ ID NO:2.
13. The nucleic acid of claim 12, which comprises a nucleotide sequence as depicted in SEQ ID NO: 1.
14. A vector comprising the nucleic acid of claim 8 operably associated with an expression control sequence.
15. A host cell transfected with the vector of claim 14.
16. A non-human animal transformed with the vector of claim 14, wherein the animal expresses a H4 receptor protein at a detectable level, whereby the cells expressing the H4 receptor protein suppress cAMP formation when contacted with an H4 receptor agonist.
17. A method for producing a H4 receptor protein, which method comprises culturing host cells of claim 15 under conditions that provide for expression ofthe H4 receptor protein by the vector.
18. An isolated nucleic acid of at least ten bases having a nucleotide sequence identical to a corresponding nucleotide sequence ofthe same number of bases in SEQ ID NO: 1 or its complement.
19. The nucleic acid of claim 18 which is detectably labeled.
20. An antibody that specifically binds to the H4 receptor protein of claim 1 or 7.
21. A method for detecting an H4 receptor protein, which method comprises detecting binding of the antibody of claim 20 to a protein in a sample suspected of containing a H4 receptor protein, wherein the antibody is contacted with the sample under conditions that permit specific binding with any H4 receptor protein present in the sample.
22. A method for detecting expression of H4 receptor, which method comprises detecting mRNA encoding H4 receptor in a sample from a cell suspected of expressing H4 receptor.
23. The method according to claim 22 wherein mRNA encoding H4 receptor is detected by hybridization to a H4 receptor-specific nucleic acid.
24. The method according to claim 23 wherein the H4 receptor-specific nucleic acid is at least 10 nucleotides in length and has a sequence identical to a sequence ofthe same number of bases in SEQ ID NO: 1, or the complementary sequence thereof.
25. An assay system for identifying H4 receptor ligands, comprising a sufficient number of cells of claim 15 to detect an alteration in second messenger accumulation.
26. The assay system of claim 25, wherein the second messenger is cAMP.
27. The assay system of claim 25, wherein the receptor is a human receptor.
28. A method for identifying a test compound that antagonizes histamine H4 receptors, which method comprises detecting an increase in the level of a second messenger in an assay system of claim 25 contacted with the test compound, wherein an increase in the level ofthe second messenger indicates that the test compound antagonizes the H4 receptor.
29. A method for identifying a test compound that agonizes histamine H4 receptors, which method comprises detecting a decrease in the level of a second messenger in an assay system of claim 25 contacted with the test compound, wherein the decrease in the level ofthe second messenger indicates that the test compound agonizes the H4 receptor.
30. A method for identifying a compound that binds an H4 receptor, which method comprises detecting binding of a test compound to the H4 receptor protein of claim 1.
31. The method according to claim 29, wherein binding ofthe test compound is detecting by inhibiting binding of a labeled H4 ligand.
32. The method according to claim 29, wherein the H4 receptor protein is in a lipid bilayer membrane.
33. An isolated nucleic acid that specifically hybridizes under highly stringent conditions to the complement ofthe sequence depicted in SEQ ID NO:l, wherein said nucleic acid encodes a histamine H4 receptor protein.
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WO2004021999A2 (en) * 2002-09-06 2004-03-18 Janssen Pharmaceutica, N.V. Use of histamine h4 receptor modulators for the treatment of allergy and asthma
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US7115395B2 (en) 2000-05-31 2006-10-03 Ortho-Mcneil Pharmaceutical, Inc. DNAs encoding guinea pig histamine receptors of the H4 subtype
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WO2003057919A3 (en) * 2001-12-21 2003-10-30 Ortho Mcneil Pharm Inc The use of histamine h4 receptor antagonist for the treatment of inflammatory response
WO2004021999A2 (en) * 2002-09-06 2004-03-18 Janssen Pharmaceutica, N.V. Use of histamine h4 receptor modulators for the treatment of allergy and asthma
WO2004021999A3 (en) * 2002-09-06 2004-10-07 Janssen Pharmaceutica Nv Use of histamine h4 receptor modulators for the treatment of allergy and asthma

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