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WO2002036070A2 - Nouvelle chimiokine de poisson et procedes d'utilisation de celle-ci - Google Patents

Nouvelle chimiokine de poisson et procedes d'utilisation de celle-ci Download PDF

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Publication number
WO2002036070A2
WO2002036070A2 PCT/US2001/045366 US0145366W WO0236070A2 WO 2002036070 A2 WO2002036070 A2 WO 2002036070A2 US 0145366 W US0145366 W US 0145366W WO 0236070 A2 WO0236070 A2 WO 0236070A2
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Prior art keywords
polypeptide
sequence
amino acid
nucleic acid
seq
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PCT/US2001/045366
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English (en)
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WO2002036070A3 (fr
Inventor
Roy S. Sundick
Lei Liu
Brian Dixon
Kazuhiro Fujiki
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Wayne State University
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Priority to AU2002220047A priority Critical patent/AU2002220047A8/en
Priority to AU2002220047A priority patent/AU2002220047A1/en
Publication of WO2002036070A2 publication Critical patent/WO2002036070A2/fr
Publication of WO2002036070A3 publication Critical patent/WO2002036070A3/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a novel chemokine found in fish, such as rainbow trout, and referred to herein as chemokine-2 or CK-2.
  • the invention also relates to isolated nucleic acids which encode CK-2 and to antibodies that specifically recognize and bind to CK-2.
  • the novel chemokines of the invention are useful, e.g., as adjuvants in vaccines.
  • the invention also relates to methods for using the CK-2 chemokine of the invention in vaccines, e.g., as an adjuvant.
  • the invention also relates to novel adjuvants, for use in vaccines, which comprise a CK-2 chemokine of the invention and, additionally, to vaccines which contain these adjuvants.
  • vaccines under development include both conventional vaccines, which contain disease-inducing infectious agents that have been killed or attenuated, as well as genetically engineered vaccines which contain recombinant proteins derived from pathogens (see, e.g., Christie, supra), and naked DNA vaccines which encode proteins derived from pathogens. See also, Traxler et al, Dis. Aquat Organ. 1999 38:183-190; Gomez-Chiarri & Chiaverini, Genet. Anal 1999, 15:121-124; Lorenzen et /., Virus Res. 1999, 63:19-25; Kanellos et al, Immunology 1999, 96:307-313; Anderson et al, Mol. Mar. Biol.
  • Chemokines are small (typically about 7-12 kDa in molecular weight) polypeptide molecules, produced and secreted by cells, which are chemotactic for leukocyte cells, such as lymphocytes, monocytes and polymorphonuclear leukocytes. Secretion of chemokines at a site of infection or trauma attracts these blood-borne cells to the site. Deliberate release of chemokines at a vaccination site has been shown to similarly draw mobile cells to the site and thereby augment immune responses to vaccine antigens. For example, Biragyn et al.
  • chemokines have been identified in humans, and homologous chemokines have been identified in other species as well, including other mammalian species (e.g., mice) and some non-mammalian species, including avian species such as chickens (see, for example, Kaiser et al, Immunogenetics 1999, 49:673-684).
  • mammalian species e.g., mice
  • avian species such as chickens
  • Fujiki et al. Immunogenetics 1999, 49:909-914 describe the cloning and characterization of a chemokine from carp (i.e., from Cyprinus carpi ⁇ ) referred to as CC chemokine- 1 or "CC- 1".
  • the present invention overcomes the above-discussed and other problems in the prior art by providing a novel chemokine, referred to herein as chemokine-2 or CK-2, that is expressed in fish such as rainbow trout (Oncorhynchus mykiss).
  • chemokine-2 referred to herein as chemokine-2 or CK-2
  • the invention provides novel polypeptide sequences, such as the polypeptide sequence set forth in FIG. 2 (SEQ ID NO:3), for CK-2 chemokines, including amino acid sequences for individual domains of CK-2.
  • the invention also provides fusion proteins and fusion polypeptides which comprise a CK-2 polypeptide sequence and a non-CK-2 polypeptide sequence.
  • the invention provides fusion polypeptides that comprise an amino acid sequence of a CK-2 polypeptide and an amino acid sequence of an antigen or immunogenic polypeptide.
  • Nucleic acids which encode the CK-2 polypeptides and fusion polypeptides of the invention are also provided, as well as antibodies that specifically bind to the CK-2 polypeptides and fusion polypeptides of the invention. Such nucleic acids and antibodies are useful, e.g., for expressing and detecting the CK-2 and fusion polypeptides of the invention.
  • the CK-2 polypeptides and nucleic acids of the invention are particularly useful for enhancing an immune response to an immunogen. Accordingly, the invention also provides methods for enhancing an immune response in an organism (preferably a fish, such as rainbow trout) to an immunogen by administering a CK-2 polypeptide and/or nucleic acid of the invention to the organism in combination with the immunogen.
  • the invention also provides pharmaceutical compositions which comprise an immunogen and an amount of a CK-2 polypeptide or nucleic acid effective for enhancing an immune response to the immunogen. Such compounds are useful, e.g., as vaccines, to enhance an immune response in an organism such as a fish.
  • the invention also provides novel screening methods for identifying nucleic acids that have a controlled half-life in a cell, or for identifying fragments of nucleic acids that have a controlled half-life in a cell.
  • the screening methods comprise identifying, from a plurality of nucleic acids, nucleic acid molecules having a sequence motif associated with a reduced half-life, such as the ATTTA sequence motif.
  • the screening methods are particularly useful to identify nucleic acids that encode gene products such as cytokines, chemokines and oncogene products (i.e., gene products encoded by oncogenes).
  • FIGS. 1 shows the cDNA sequence (SEQ ID NO:l) of the rainbow trout chemokine-2 (CK-2).
  • FIG. 2 sets forth the protein coding sequence of rainbow trout CK-2 cDNA (SEQ ID NO:2) and the amino acid sequence of the predicted CK-2 gene product (SEQ ID NO:3) it encodes.
  • 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, i.e., components of the cells in which the material is found or produced, hi the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
  • 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.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in 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.
  • 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.
  • sample refers to a biological material which can be tested, e.g., for the presence of CK-2 polypeptides or CK-2 nucleic acids, e.g., to identify cells that specifically express the CK-2 gene and its gene product.
  • samples can be obtained from any source, including tissue, blood and blood cells, including circulating hematopoietic stem cells (for possible detection of protein or nucleic acids), plural effusions, cerebrospinal fluid (CSF), ascites fluid, and cell culture.
  • samples are obtained from bone marrow.
  • 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.
  • non-human animals also include fish, including rainbow trout (Oncorhynchus mykiss), salmon (e.g., Salmo sola and Oncoryhynchus choica), carp (Cyprinus carpio) and catfish, to name a few.
  • the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • molecule means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes, for example, polypeptides and polynucleotides.
  • a vaccine generally comprises a therapeutically effective dose of an immunogen (e.g. , an antigen of an infectious agent) and, preferably, an adjuvant and/or a pharmaceutically acceptable carrier.
  • a vaccine may be admimstered to an organism, e.g., by inhalation or insufflation (either through the mouth or the nose), or by oral, buccal, rectal or parenteral administration (e.g., by subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal or intravenous injection and the like) or by submersion (e.g., in the case offish and other aquatic organisms).
  • a vaccine may also be administered by particle-mediated transfer (e.g., using a "particle gun"). See for example, Gainer et al, J. Neurooncol 2000, 47:23-30; Koide et al., Jpn J.
  • a vaccine may comprise, for example, a suspension of an attenuated or killed infectious agent (e.g., a microorganism such as a bacterium or a virus, a parasite or other pathogen, etc.) that causes an infectious disease.
  • a vaccine of the invention may be a polypeptide vaccines or a DNA vaccine.
  • the term "polypeptide vaccine” refers to a vaccine comprising an immunogenic polypeptide, for example a polypeptide derived from an infectious agent which may be an antigen, and therefore activates an immune response in an organism.
  • the term "DNA vaccine” is an informal term of art, and is used herein to refer to vaccines delivered by means of a recombinant vector.
  • vector vaccine since some potential vectors, for example retroviruses and lentiviruses, are RNA viruses and since in some instances non- viral RNA instead of DNA may be delivered to cells).
  • a therapeutically effective dose refers to that amount of a compound or compositions that is sufficient to result in a desired activity.
  • a therapeutically effective dose refers to the amount of a compound or compositions (e.g., an antigen) that is sufficient to produce an effective immune response.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve, e.g. , as a tissue depot that slowly releases the antigen, and also as a lymphoid system activator that enhances the immune response (see, Hood et al, Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, California, p. 384).
  • Exemplary adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels (for example, aluminum hydroxide), surface active substances (for example, lysolecithin), pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions.
  • Exemplary adjuvants also include potentially useful human adjuvants such as BCG (bacille Calmette-Gueri ⁇ ) and Corynebacterium parvum.
  • immunostimulatory proteins such as chemokines, may be provided as an adjuvant to increase the immune response to a vaccine.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction (for example, gastric upset, dizziness and the like) when administered to an individual.
  • pharmaceutically acceptable may mean approved by a regulatory agency (for example, the U.S. Food and Drug Agency) or listed in a generally recognized pharmacopeia for use in animals (for example, the U.S. Pharmacopeia).
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered.
  • Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • Exemplary suitable pharmaceutical carriers are described in "Reminington's Pharmaceutical Sciences” by E. . Martin.
  • polymer means any substance or compound that is composed of two or more building blocks ('mers') that are repetitively linked together.
  • a "dimer” is a compound in which two building blocks have been joined togther; a “trimer” is a compound in which three building blocks have been joined together; etc.
  • polynucleotide or "nucleic acid molecule” as used herein refers to a polymeric molecule having a backbone that supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer backbone presents the bases in a manner to permit such hydrogen bonding in a specific fashion between the polymeric molecule and a typical polynucleotide (e.g., single-stranded DNA).
  • bases are typically inosine, adenosine, guanosine, cytosine, uracil and thymidine.
  • Polymeric molecules include "double stranded” and “single stranded” DNA and RNA, as well as backbone modifications thereof (for example, methylphosphonate linkages).
  • a "polynucleotide” or “nucleic acid” sequence is a series of nucleotide bases (also called “nucleotides”), generally in DNA and RNA, and means any chain of two or more nucleotides.
  • a nucleotide sequence frequently carries genetic information, including the information used by cellular machinery to make proteins and enzymes. The terms include genomic DNA, cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides.
  • PNA protein nucleic acids
  • the polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, 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 of the 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 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 to name a few.
  • the polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidite 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.
  • Other non-limiting examples of modification which may be made are provided, below, in the description of the present invention.
  • a “polypeptide” is a chain of chemical building blocks called amino acids that are linked together by chemical bonds called “peptide bonds”.
  • the term “protein” refers to polypeptides that contain the amino acid residues encoded by a gene or by a nucleic acid molecule (e.g., an mRNA or a cDNA) transcribed from that gene either directly or indirectly.
  • a protein may lack certain amino acid residues that are encoded by a gene or by an mRNA.
  • a gene or mRNA molecule may encode a sequence of amino acid residues on the N-terminus of a protein (i. e. , a signal sequence) that is cleaved from, and therefore may not be part of, the final protein.
  • a protein or polypeptide, including an enzyme may be a "native” or “wild-type”, meaning that it occurs in nature; or it may be a “mutant”, “variant” or “modified”, meaning that it has been made, altered, derived, or is in some way different or changed from a native protein or from another mutant.
  • the term "cytokine”, as used herein, is a general term for any of a wide variety of proteins and polypeptides that are secreted by cells and have specific effects on cell-cell interactions, communication and/or behavior of other cells. Typically, cytokines are secreted by cells of the immune system of an organism (e.g., lymphocytes) and regulate immune responses in the organism.
  • Cyclones are a particular subclass of cytokines which are chemotactic for leukocyte cells such as lymphocytes, monocytes and leukocytes.
  • the term “chemotactic” refers to the movement of a cell or organism along a chemical concentration gradient either towards or away from a chemical stimulant.
  • a compound such as a chemokine, is chemotactic for a cell if the compound causes the cell to move towards or away from that compound.
  • Routine assays are known in the art for determining whether a compound is chemotactic for cells, including the micropore filter method (see, Ward & Maderazo, in Manual of Clinical Immunology, 2nd Ed., American Society for Microbiology, Washington DC, 1980) and the agarose method (see, Nelson et al, J. Immunol. 1975, 115:1650-1655).
  • Chemokines are small (typically about 7-12 kDa molecular weight) polypeptides. Chemokines may be subdivided into four groups, referred to as CC, CXC, CXXXC and C chemokines, which are characterized by the arrangement of the first two cysteines in the amino acid sequence of the mature, secreted protein. Specifically, CC chemokines comprise a pair of conserved, adjacent cysteine residues. CXC and CXXXC chemokines each comprise a pair of conserved cysteine residues that are separated by one (CXC) or three (CXXXC) nonconserved intervening amino acid residues.
  • C chemokines are characterized by having only a single conserved cysteine residues (i.e., no adjacent cysteine residue) in a consensus sequence. All chemokines comprise a globular tertiary protein structure having an ⁇ -helix and both ⁇ - and a ⁇ -sheets. For a review see Miller & Krangel, Crit. Rev. Immunol 1992, 12:17-46.
  • a “ligand” is, broadly speaking, any molecule that binds to another molecule.
  • the ligand is either a soluble molecule or the smaller of the two molecule or both.
  • the other molecule is referred to as a "receptor".
  • both a ligand and its receptor are molecules (preferably proteins or polypeptides) produced by cells.
  • a ligand is a soluble molecule and the receptor is an integral membrane protein (i.e., a protem expressed on the surface of a cell).
  • a ligand is CK-2 or another chemokine and the receptor is a chemokine receptor, expressed on the surface of a leukocyte, that specifically binds to the chemokine.
  • the binding of a ligand to its receptor is frequently a step of signal transduction within a cell.
  • chemokines such as CK-2
  • chemokine receptors on a leukocyte generates a signal in the leukocyte cells which, in turn, stimulates the leukocyte to move towards the source of the chemokines (i.e., towards the higher concentration of chemokines).
  • Other exemplary ligand- receptor interactions include, but are not limited to, binding of a hormone to a hormone receptor (for example, the binding of estrogen to the estrogen receptor) and the binding of a neurotransmitter to a receptor on the surface of a neuron.
  • Amplification of a polynucleotide denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences.
  • PCR polymerase chain reaction
  • “Chemical sequencing” of DNA denotes methods such as that of Maxam and Gilbert (Maxam-Gilbert sequencing; see Maxam & Gilbert, Proc. Natl. Acad. Sci. U.S.A. 1977, 74:560), in which DNA is cleaved using individual base-specific reactions.
  • Enzymatic sequencing of DNA denotes methods such as that of Sanger (Sanger et al, Proc. Natl. Acad. Sci. U.S.A. 1977, 74:5463) and variations thereof well known in the art, in a single-stranded DNA is copied and randomly terminated using DNA polymerase.
  • a “gene” is a sequence of nucleotides which code for a functional "gene product”.
  • a gene product is a functional protein.
  • a gene product can also be another type of molecule in a cell, such as an RNA (e.g. , a tRNA or a rRNA).
  • a gene product also refers to an mRNA sequence which may be found in a cell.
  • measuring gene expression levels according to the invention may correspond to measuring mRNA levels.
  • a gene may also comprise regulatory (i.e., non-coding) sequences as well as coding sequences. Exemplary regulatory sequences include promoter sequences, which determine, for example, the conditions under which the gene is expressed.
  • the transcribed region of the gene may also include untranslated regions including introns, a 5'-untranslated region (5'-UTR) and a 3 '-untranslated region (3'-UTR).
  • 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” that RNA or it encodes the amino acid sequence for that polypeptide, protein or enzyme.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently found, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control of or is “operatively associated with” transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, which is then trans-RNA spliced (if it contains introns) and, if the sequence encodes a protein, is translated into that protein.
  • RNA such as rRNA or mRNA
  • a DNA sequence is expressed by a cell to form an "expression product” such as an RNA (e.g. , a mRNA or a rRNA) or a protein.
  • the expression product itself e.g. , the resulting RNA or protein, may also said to be “expressed” by the cell.
  • 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 into a host cell so that the host cell will express the introduced gene or sequence to produce a desired substance, in this invention typically an RNA coded by the introduced gene or sequence, but also a protein or an 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 (e.g., 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 of the 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 may include plasmids, phages, viruses, etc. and are discussed in greater detail below.
  • 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 of the cassette in the proper reading frame.
  • foreign DNA is inserted at one or more restriction sites of the 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.
  • 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.
  • a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme.
  • Host cells can further be used for screening or other assays that are described infra.
  • Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g. , a transgenic animal or a transiently transfected animal).
  • 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 such as Sf9, Hi5 or S2 cells and Baculovirus vectors, Drosophila cells (Schneider cells) and expression systems, fish cells and expression systems (including, for example, RTH-149 cells from rainbow trout, which are available from the American Type Culture Collection and have been assigned the accession no. CRL-1710) and mammalian host cells and vectors.
  • heterologous refers to a combination of elements not naturally occurring.
  • the present invention includes chimeric RNA molecules that comprise an rRNA sequence and a heterologous RNA sequence which is not part of the rRNA sequence.
  • the heterologous RNA sequence refers to an RNA sequence that is not naturally located within the ribosomal RNA sequence.
  • the heterologous RNA sequence may be naturally located within the ribosomal RNA sequence, but is found at a location in the rRNA sequence where it does not naturally occur.
  • heterologous DNA refers to DNA that is not naturally located in the cell, or in a chromosomal site of the cell.
  • heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is a regulatory element operatively associated with a different gene that the one it is operatively associated with in nature.
  • 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. , RNA, 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, RNA, enzyme, cell, etc. ; i. e. , any kind of mutant.
  • the present invention relates to altered or "chimeric" RNA molecules that comprise an rRNA sequence that is altered by inserting a heterologous RNA sequence that is not naturally part of that sequence or is not naturally located at the position of that rRNA sequence.
  • chimeric RNA sequences as well as DNA and genes that encode them, are also referred to herein as "mutant" sequences.
  • Sequence-conservative variants of a polynucleotide 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” of a polypeptide or polynucleotide are those in which a given amino acid residue in the polypeptide, or the amino acid residue encoded by a codon of the polynucleotide, has been changed or altered without altering the overall conformation and function of the polypeptide.
  • function-conservative variants may include, but are not limited to, replacement of an amino acid with one having similar properties (for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic and the like). Amino acid residues with similar properties are well known in the art.
  • amino acid residues arginine, histidine and lysine are hydrophilic, basic amino acid residues and may therefore be interchangeable.
  • amino acid residue isoleucine which is a hydrophobic amino acid residue, 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 of the polypeptide.
  • Amino acid residues other than those indicated as conserved may also differ in a protein or enzyme so that the percent protein or amino acid sequence similarity (e.g., percent identity or homology) 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 the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
  • "Function-conservative variants" of a given polypeptide also include polypeptides that have at least 60% amino acid sequence identity to the given polypeptide as determined, e.g., by the BLAST or FASTA algorithms.
  • function-conservative variants of a given polypeptide have at least 75%, more preferably at least 85% and still more preferably at least 90% amino acid sequence identity to the given polypeptide and, preferably, also have the same or substantially similar properties (e.g., of molecular weight and/or isoelectric point) Or functions (e.g., biological functions or activities) as the native or parent polypeptide to which it is compared.
  • function-conservative variants may not only have between at least 75% and at least 90% amino acid sequence identity to a given chemokine, but preferably also have similar properties, such as conserved domains (e.g., as in CC, CXC and CXXC chemokines, described supra) and/or similar biological function or activities, such as chemotaxis of cells (particularly leukocytes).
  • homologous in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a "common evolutionary origin", including proteins from superfamilies (e.g., the immunoglobulin superfamily) in the same species of organism, as well as homologous proteins from different species of organism (for example, myosin light chain polypeptide, etc.; see, Reeck et al, Cell 1987, 50:667).
  • proteins and their encoding nucleic acids
  • sequence homology as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions.
  • homologous proteins are chemokines
  • homologous chemokines in closely related species of organisms such as between mammals (e.g., between mice and humans) or between closely related species offish (e.g., between salmon and trout) typically share greater than 50% sequence identity, and more preferably share at least about 60 to 65% sequence identity.
  • homologous chemokines between closely related species of organisms may also be cross reactive (e.g., chemotactic) in both species of organisms.
  • chemokines between more divergent species of organisms such as between humans and fish (e.g., between humans and carp, trout or salmon) or between divergent species offish (e.g., between trout and carp, or between salmon and carp) share less sequence identity and generally are not cross reactive in both species.
  • homologous chemokines between divergent species of organisms typically share less than 50% sequence identity, and may share only 25% sequence identity.
  • homologous chemokines between divergent species preferably share a higher level of sequence identity, such as between about 35% to 45% sequence identity.
  • sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences 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 nucleic acid sequences are "substantially homologous" or “substantially similar” when at least about 80%, and more preferably at least about 90% or at least about 95% of the nucleotides match over a defined length of the nucleic acid sequences, as determined by a sequence comparison algorithm known such as BLAST, FASTA, DNA Strider, CLUSTAL, etc.
  • a sequence comparison algorithm known such as BLAST, FASTA, DNA Strider, CLUSTAL, etc.
  • An example of such a sequence is an allelic or species variant of the specific genes of the present invention.
  • Sequences that are substantially homologous may also be identified by hybridization, e.g., in a Southern hybridization experiment under, e.g., stringent conditions as defined for that particular system.
  • two amino acid sequences are "substantially homologous" or “substantially similar” when greater than 80% of the amino acid residues are identical, or when greater than about 90% of the amino acid residues are similar (i.e., are functionally identical).
  • the similar or homologous polypeptide sequences are identified by aligmnent using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison Wisconsin) pileup program, or using any of the programs and algorithms described above (e.g., BLAST, FASTA, CLUSTAL, etc.).
  • 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 or a fluorescent dye (for example, Cy3 or Cy5) 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 ck-2, or to detect the presence of nucleic acids encoding CK-2.
  • an oligonucleotide of the invention can form a triple helix with a CK-2 DNA molecule.
  • oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer.
  • 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 a CK-2 gene or its gene product.
  • 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.
  • antisense broadly includes RNA-RNA interactions, RNA-DNA interactions, triple helix 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).
  • Other specific examples of antisense nucleic acid molecules of the invention are provided infra.
  • oligonucleotides envisioned for this invention include, in addition to the nucleic acid moieties described above, 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. Pat. 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 of the polyamide backbone (Nielsen et al, Science 254:1497, 1991).
  • oligonucleotides may contain substituted sugar moieties comprising one of the 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; to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-; S-, or N- alkyl; O-, S-, orN-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 oligonu
  • 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.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the 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 T m (melting temperature) of 55 °C, can be used, e.g.
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • SCC is a 0.15M NaCl, 0.015M Na-citrate.
  • 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 T m for hybrids of nucleic acids having those sequences.
  • the relative stability corresponding to higher T- of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al, supra, 9.50-9.51).
  • 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 T m of 55 °C, and utilizes conditions as set forth above.
  • the T m is 60 °C; in a more preferred embodiment, the T m 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.
  • Suitable hybridization conditions for oligonucleotides are typically somewhat different than for full-length nucleic acids (e.g. , full-length cDNA), because of the oligonucleotides' lower melting temperature. Because the melting temperature of oligonucleotides will depend on the length of the oligonucleotide sequences involved, suitable hybridization temperatures will vary depending upon the oligoncucleotide molecules used.
  • Exemplary temperatures may be 37 °C (for 14-base oligonucleotides), 48 °C (for 17-base oligoncucleotides), 55 °C (for 20-base oligonucleotides) and 60 °C (for 23 -base oligonucleotides).
  • Exemplary suitable hybridization conditions for oligonucleotides include washing in 6x SSC/0.05% sodium pyrophosphate, or other conditions that afford equivalent levels of hybridization.
  • the present invention relates to a novel chemokine, referred to herein as chemokine-2 or CK-2, which is expressed in fish such as rainbow trout (i. e. , Oncorhynchus mykiss).
  • a CK-2 chemokine is, in general, a polypeptide that is encoded by a gene which hybridizes to the complement of a CK-2 nucleic acid sequence as described in Section 5.3, infra.
  • a full-length non-glycosylated CK-2 polypeptide of the invention has an apparent molecular weight of about 20 kDa.
  • a gfycosylated form of a full length, wild-type CK-2 polypeptide will have an apparent molecular weight between about 25 and 50 kDa.
  • CK-2 chemokines and polypeptides of the present invention may also be characterized by their specific bioactivity.
  • a CK-2 chemokine or polypeptide of the invention may also modulate the immune system of an organism (for example, a fish such as a trout or a salmon) expressing a CK-2 polypeptide or a CK-2 gene.
  • a CK-2 chemokine or polypeptide of the invention may also modulate the immune system of an organism (for example, a fish such as a trout or a salmon) when administered to the organism, e.g., as an adjuvant in a vaccine.
  • a CK-2 chemokine or polypeptide of the invention is chemotactic for leukocytes (including T lymphocytes such as CD8 and CD4 cells, monocytes, and B lymphocytes) as well as for other cells (for example, brain microglial cells). Accordingly, introduction of a CK-2 chemokine (e.g., by injection or secretion) at the site of an infection or trauma in an organism may attract leukocytes to the site and augment an immune response.
  • the CK-2 polypeptide is further characterized as comprising at least two domains: a globular domain and a "mucin-like stalk" domain.
  • the globular domain preferably shares amino acid sequence homology and/or amino acid sequence identity with other CC chemokines, such as the carp (Cyprinus carpi ⁇ ) CC chemokine (Fujiki et al. , Immunogenetics 1999, 49:909-914) and the human chemokine MCP1 (Yoshimura et al, FEBS Lett. 1989, 244:487-493).
  • a CK-2 polypeptide of the invention is derived from a rainbow trout or has an amino acid sequence of a polypeptide derived from a rainbow trout cell.
  • a rainbow trout CK-2 polypeptide of the invention may comprise the amino acid sequence set forth in FIG. 2 (SEQ ID NO:3). This sequence comprises amino acid sequences corresponding to at least three distinct domains: a signal peptide sequence (comprising amino acid residues 1-28 of SEQ ID NO:3), a "globular” domain (comprising amino acid residues 29-100 of SEQ ID NO:3) and a "mucin-like” stalk domain (comprising amino acid residues 101-191 of SEQ ID NO:3).
  • the "mucin-like stalk domain” is also referred to herein as the "stalk domain”.
  • amino acid residue numbers used to delineate these different domains are preferably approximate.
  • the globular domain of SEQ ID NO:3 may merely comprise amino acid residues 29-90 of SEQ ID NO:3 or may, alternatively, comprise amino acid residues 29-119 of SEQ ID NO:3.
  • the stalk domain of SEQ ID NO:3 may, in certain embodiments, merely comprise amino acid residues 120-191.
  • a mature CK-2 polypeptide lacks a signal peptide sequence.
  • a CK-2 polypeptide of the invention comprises an amino acid sequence corresponding to amino acid residues 29- 191 of the sequence set forth in FIG. 2 (SEQ ID NO:3).
  • a CK-2 polypeptide of the invention may also lack a stalk domain. Accordingly, in still other specific aspects a CK-2 polypeptide of the invention comprises an amino acid sequence corresponding to amino acid residues 29-90 of the sequence set forth in FIG. 2 (SEQ ID NO:3). Alternatively, the CK-2 polypeptides of the invention also include polypeptides which comprise a stalk domain; e.g., an amino acid sequence corresponding to amino acid residues 101-191 of the sequence set forth in FIG. 2 (SEQ ID NO:3). In still other embodiments, the CK-2 polypeptides of the invention include polypeptides having amino acid sequences corresponding to any combination of the individual, above-described domains.
  • the CK-2 polypeptides of the present invention may include polypeptides that comprise amino acid sequences corresponding to any combination of the above-described domains; i.e., polypeptides comprising at least two amino acid sequences selected from the group consisting of amino acid residues 1-28, amino acid residues 29-90 and amino acid residues 101-191 of the sequence set forth in FIG. 2 (SEQ ID NO :3).
  • CK-2 polypeptides of the invention also include polypeptides comprising an amino acid sequence of an epitope of a full length CK-2 polypeptide, such as epitopes of the full length CK-2 polypeptide set forth in FIG. 2 (SEQ ID NO:3).
  • An epitope of a CK-2 polypeptide represents a site on the polypeptide against which an antibody may be produced and to which the antibody binds. Therefore, polypeptides comprising the amino acid sequence of a CK-2 epitope are useful for making antibodies to a CK-2 polypeptide of chemokine.
  • an epitope comprises a sequence of at least 5, more preferably at least 10, 15, 20, 25 or 50 amino acid residues in length.
  • CK-2 polypeptides of the invention that comprise epitopes of a CK-2 chemokine preferably contain an amino acid sequence corresponding to at least 5, at least 10, at least 15, at least 20, at least 25 or at least 50 amino acid residues of the CK-2 chemokine's polypeptide sequence.
  • the epitope is an epitope of the full length CK-2 polypeptide set forth in FIG. 2 (SEQ ID NO:3)
  • a CK-2 polypeptide of the invention preferably comprising an amino acid sequence corresponding to at least 5, at least 10, at least 15, at least 20, at least 25 or at least 50 amino acid residues of the sequence set forth in FIG. 2 (SEQ ID NO:3).
  • the CK-2 polypeptides of the invention also include analogs and derivatives of the full length CK-2 polypeptides (e.g., of SEQ ID NO:3). Analogs and derivatives of the CK-2 polypeptides of the invention have the same or homologous characteristics of CK-2 polypeptides set forth above.
  • truncated forms of a CK-2 polypeptide can be provided.
  • Such truncated forms may include a CK-2 polypeptide with a specific deletion.
  • amino acid residues corresponding to one or more domains of a full length CK-2 polypeptide e.g., a signal sequence domain, a globular domain or a stalk domain
  • a truncated CK-2 polypeptide of the invention is one wherein a signal sequence domain has been deleted or otherwise removed; i. e. , a CK-2 polypeptide which does not comprise a signal sequence domain.
  • a truncated CK-2 polypeptide of the invention is one wherein a stalk domain has been deleted or otherwise removed so that the truncated CK-2 polypeptide is one that does not comprise a stalk domain.
  • a CK-2 polypeptide derivative is functionally active; i.e., it is capable of exhibiting one or more functional activities associated with a full-length, wild-type CK-2 polypeptide of the invention.
  • a CK-2 polypeptide derivative may be chemotactic for certain cells, such as for leukocytes (including T lymphocytes such as CD8 and CD4 cells, monocytes, and B lymphocytes), or for other cells (for example, brain microglial cells).
  • a CK-2 polypeptide derivative may, when secreted or introduced into an organism (e.g., at the site of an infection or trauma), may modulate or enhance an immune response in the organism.
  • a CK-2 chimeric or fusion polypeptide may also be prepared in which the CK- 2 portion of the fusion polypeptide has one or more characteristics of the CK-2 polypeptide.
  • Such fusion polypeptides therefore represent embodiments of the CK-2 polypeptides of this invention.
  • Exemplary CK-2 fusion polypeptides include ones which comprise a full length, derivative or truncated CK-2 amino acid sequence, as well as fusions which comprise a fragment of a CK-2 polypeptide sequence (e.g., a fragment corresponding to an epitope or to one or more domains).
  • Such fusion polypeptides may also comprise the amino acid sequence of a marker polypeptide; for example FLAG, a histidine tag, glutathione S-transferase (GST), or Fc portion of an IgG.
  • a CK-2 polypeptide may be expressed with (e.g., fused to) a bacterial protein such as ⁇ -galactosidase.
  • CK-2 fusion polypeptides may comprise amino acid sequences that increase solubility of the polypeptide, such as a thioreductase amino acid sequence or the sequence of one or more immunoglobulin proteins (e.g., IgGl or IgG2).
  • a fusion CK-2 polypeptide of the invention is prepared by combining a CK-2 polypeptide sequence (or a portion thereof) with an antigen.
  • an exemplary DNA vaccine comprising a plasmid which encodes a truncated CK-2 polypeptide fused to a ⁇ -galactosidase polypeptide as an antigen.
  • fusion proteins of chemokines are already known in the art (see, e.g., Biragyn et al, Nature Biotechnology 1999, 17:253-258), and have been shown to induce a stronger immune response to the antigen than either administration of the antigen alone or co-administration of the antigen and chemokine as separate polypeptides.
  • CK-2 analogs or variants can also be made by altering encoding nucleic acid molecules, for example by substitutions, additions or deletions.
  • Such altered nucleic acid molecules encode functionally similar molecules ( . e. , molecules that perfo ⁇ n one or more CK-2 functions or have one or more CK-2 bioactivities).
  • an analog of a CK-2 polypeptide is a function-conservative variant.
  • Amino acid residues may differ among variants of a protein or polypeptide. Accordingly, the percentage of protein or amino acid sequence similarity between any two CK-2 polypeptides of similar function may vary. Typically, the percentage of protein or amino acid sequence similarity between different CK-2 polypeptide variants may be from 70% to 99%, as determined according to an alignment scheme such as the Cluster Method and/or the MEGALIGN algorithm.
  • “Function-conservative variants” also include polypeptides that have greater than or at least 20%, at greater than or at least 25%, preferably greater than or at least 45%), more preferably greater than or at least 50%, still more preferably at least 75%, even more preferably at least 85%, and more preferably at least 90% or at least 95% sequence identity to a CK-2 polypeptide of the invention (e.g., the polypeptide set forth in FIG. 2 and in SEQ ID NO:3) or to one or more fragments or domains thereof.
  • function- conservative variants also have the same or similar properties, functions or bioactivities as the native polypeptide to which they are compared.
  • function-conservative variants of the present invention include, not only variants of the full length CK-2 polypeptides of the invention (e.g., variants of a polypeptide comprising the sequence set forth in FIG. 2 and in SEQ ID NO: 3), but also include function-conservative variants of modified CK-2 polypeptides (e.g. , truncations and deletions) and of fragments (e.g. , corresponding to domains or epitopes) of full length CK-2 polypeptides.
  • an analog of a CK-2 polypeptide is an allelic variant or mutant of a CK-2 polypeptide.
  • allelic variant and mutant when used herein to describe a polypeptide, refers to a polypeptide encoded by an allelic variant or mutant gene.
  • allelic variant and mutant CK-2 polypeptides of the invention are polypeptides encoded by allelic variants or mutants of the CK-2 nucleic acid molecules of the present invention.
  • an analog of a CK-2 polypeptide is a substantially homologous polypeptide from the same species (e.g., allelic variants) or from another species (e.g., an orthologous polypeptide); preferably from another species of fish such as from another species of trout, salmon, carp, catfish, goldfish, etc.
  • CK-2 homologs of the invention may, however, be from any species of animal, including mammals (e.g., mouse, rat, rabbit, hamster, guinea pig, dog, cat, sheep, goat, pig, horse, cow and human) to name a few.
  • the Example presented in Section 6.3 infra describes at least four different, exemplary allelic variations that may be found in a CK-2 polypeptide.
  • the specific allelic variants described in the Examples below include ones having the polypeptide sequence set forth in FIG. 2 (SEQ ID NO:3), or a fragment or domain thereof, but having a deletion at amino acid residue 26 (i. e. , wherein the amino acid residue at position 26 of SEQ ID NO:3 is deleted).
  • Other, exemplary CK-2 allelic variants of the invention include ones having the polypeptide sequence set forth in FIG.
  • CK-2 polypeptides of the invention therefore include (but are not limited to) polypeptides having any one or more, any two or more, any three or more, or all four of the amino acid sequence substitutions and/or deletions described here.
  • variant CK-2 polypeptides of the invention include other CK-2 polypeptides having equivalent amino acid substitutions, deletions or insertions.
  • the variant CK-2 polypeptides of the invention also include fragments of the full length CK-2 polypeptide set forth in FIG.2 (SEQ ID NO:3) that have one or more of the amino acid substitutions, deletions or insertions described above for the full length CK-2 polypeptide.
  • CK-2 polypeptide sequences include allelic or species variants of the specific CK-2 polypeptide sequence set forth in FIG. 2 (SEQ ID NO:3). Sequences that are substantially homologous can be readily identified by comparing the sequences using standard software packages available in sequence data banks, including the BLAST algorithms (e.g., BLASTP, BLASTN, BLASTX), FASTA, DNA Strider, the GCG pileup program, CLUSTAL, and other such programs which are known in the art and/or described herein.
  • BLAST algorithms e.g., BLASTP, BLASTN, BLASTX
  • FASTA FASTA
  • DNA Strider the GCG pileup program
  • CLUSTAL and other such programs which are known in the art and/or described herein.
  • variants of a CK-2 polypeptide are polypeptides encoded by nucleic acid molecules that hybridize to the complement of a nucleic acid molecule encoding a CK-2 polypeptide (e.g., in a Southern hybridization experiment under defined conditions).
  • analogs and/or homologs of a CK-2 polypeptide comprise amino acid sequences encoded by nucleic acid molecules that hybridize to a complement of a CK-2 nucleic acid sequence, such as any of the coding sequences set forth in FIG. 1 (SEQ ID NO:l) or in FIG.
  • the analogs and/or homologs of a CK-2 polypeptide may comprise amino acid sequences encoded by nucleic acid molecules that hybridize to a complement of a CK-2 nucleic acid sequence (e.g., the coding sequences set forth in FIGS 1 and 2 and in SEQ ID NOS: 1-2, respectively) under moderately stringent hybridization conditions (i.e., 40% formamide with 5x 6x SSC) or under low stringency conditions (e.g., in 5x SSC, 0.1% SDS, 0.25% milk, no formamide, 30% formamide, 5x SSC, or 0.5% SDS).
  • moderately stringent hybridization conditions i.e., 40% formamide with 5x 6x SSC
  • low stringency conditions e.g., in 5x SSC, 0.1% SDS, 0.25% milk, no formamide, 30% formamide, 5x SSC, or 0.5% SDS.
  • variants (including analogs, homologs and orthologs) of a CK-2 polypeptide can also be identified by isolating variant CK-2 genes, e.g. , by PCR using degenerate oligonucleotide primers designed on the basis of amino acid sequences of the CK-2 polypeptide and as described below).
  • Derivatives of the CK-2 polypeptides of the invention further include, but are by no means limited to, phosphorylated CK-2, myristylated CK-2, methylated CK-2 and other CK-2 polypeptides that are chemically modified.
  • CK-2 polypeptides of the invention also include labeled variants; for example, radio-labeled with iodine or phosphorous (see, e.g., EP 372707B) or other detectable molecule such as, but by no means limited to, biotin, a fluorescent dye (e.g., Cy5 or Cy3), a chelating group complexed with a metal ion, a chromophore or fluorophore, a gold colloid, a particle such as a latex bead, or attached to a water soluble polymer.
  • labeled variants for example, radio-labeled with iodine or phosphorous (see, e.g., EP 372707B) or other detectable molecule such as, but by no means limited to, biotin, a fluorescent dye (e.g., Cy5 or Cy3), a chelating group complexed with a metal ion, a chromophore or fluoro
  • CK-2 nucleic acids or polypeptides may provide additional advantages under certain circumstances. See, for example, U.S. Patent No. 4,179,337 issued December 18, 1970 to Davis et al. Also, for a review see Abuchowski et al. , in Enzymes as Drugs (J.S. Holcerberg and J. Roberts, eds. 1981), pp. 367-383. A review article describing protein modification and fusion proteins is also found in Fracis, Focus on Growth Factors 1992, 3:4-10, Mediscript: Mountview Court, Friern Barnet Lane, London N20, OLD, UK.
  • a CK-2 nucleic acid molecule of the present invention comprises a nucleic acid sequence that encodes a CK-2 polypeptide as defined, supra, in Section 5.2, the complement of a nucleic acid sequence that encodes a CK-2 polypeptide, and fragments thereof.
  • the CK-2 nucleic acid molecules of the invention comprise a nucleotide sequence that encodes the amino acid sequence set forth in FIG. 2 (SEQ ID NO:3), such as the particular CK-2 nucleic acid sequences set forth in FIG. 1 (SEQ ID NO: 1) and in FIG. 2 (SEQ ID NO:2).
  • the CK-2 nucleic acid molecules of the invention comprise nucleic acid sequences that encode one or more domains of a CK-2 polypeptide (e.g., a signal sequence domain, a "globular” or a "stalk” domain), or nucleic acid sequences that encode any combination of domains of a CK-2 polypeptide.
  • the CK-2 nucleic acid molecules of the invention also include nucleic acids which comprise a sequence encoding one or more fragments of a CK-2 polypeptide.
  • Such fragments include, for example, polynucleotides encoding an epitope of a CK-2 polypeptide; e.g., nucleic acids that encode a sequence of at least 5, more preferably at least, 10, 15, 20, 25 or 50 amino acid residues of a CK-2 polypeptide sequence (e.g., of the polypeptide sequence set forth in FIG. 2 and in SEQ ID NO:3).
  • the CK-2 nucleic acid molecules of the invention also include nucleic acid molecules that comprise coding sequences for modified CK-2 polypeptides (e.g., having amino acid substitutions, deletions or truncations) and for variants (including analogs and homologs from the same or different species) of CK-2 polypeptides.
  • such nucleic acid molecules have at least 50%, preferably at least 75% and more preferably at least 90% sequence identity to a CK-2 coding sequence (e.g., the coding sequence set forth in FIG. 1 or in FIG. 2, and in SEQ ID NOS: 1-2, respectively).
  • variants CK-2 nucleic acid molecules of this invention include that allelic variants described in Section 6.3 infra.
  • the variant CK-2 nucleic acids of the invention include, but are not limited to nucleic acids encoding a polypeptide sequence as set forth in FIG. 2 (SEQ ID NO:2) wherein: (a) the amino acid residue at position 26 of that sequence is deleted; (b) the amino acid residue at position 37 of that sequence is an alanine; (c) the amino acid residue at position 39 of the sequence is a proline; and/or (d) the amino acid residue at position 176 of the sequence is an arginine.
  • the variant CK-2 nucleic acids of the invention may encode polypeptides having any one or more, any two or more, any three or more, or all four of the amino acid substitutions and/or deletion recited above.
  • nucleic acids that encode such variant CK-2 polypeptides include CK-2 nucleic acid sequences, such as SEQ ID NO:l, wherein: (a) nucleic acid residues 140-142 of the sequence are deleted; (b) nucleic acid residue 172 of the sequence is a guanine; (c) nucleic acid residue 178 of the sequence is a cytosine; or (d) nucleic acid residue 589 of the sequence is a cytosine.
  • the variant CK-2 nucleic acids of the invention also include CK-2 nucleic acids comprising one or more "silent" mutations or polymorphisms that do not affect the sequence of the CK-2 polypeptide encoded by the variant nucleic acid.
  • Examplary silent CK-2 polymorphisms include those described in Section 6.2, infra; including CK-2 nucleic acid sequences such as SEQ ID NO: 1, wherein: (a) nucleic acid residue 528 is a cytosine; or (b) nucleic acid residue 667 is a cytosine.
  • the variant CK-2 nucleic acids of the invention therefore include (but are not limited to) nucleic acids having any one or more, any two or more, any three of more, any four or more, any five or more, or all six of the exemplary nucleic acid substitutions, deletions and/or insertions described hereabove.
  • variant CK-2 nucleic acids of the invention include other CK-2 nucleic acids having equivalent nucleotide substitutions, deletions or insertions.
  • the variant CK-2 nucleic acids of the invention also include fragments of the full length CK-2 cDNA sequence set forth in FIG. 1 (SEQ ID NO:l) that have one or more of the nucleotide substitutions, deletions or insertions described above for the full length CK-2 cDNA sequence.
  • a specific embodiment of such equivalent CK-2 nucleotide substitutions, insertions or deletions include corresponding nucleic acid substitutions, deletions and insertions in the CK-2 coding sequence set forth in FIG. 2 (SEQ ID NO:2).
  • the variant CK-2 nucleic acids of the invention include nucleic acids having any one or more, any two or more, or all three of these nucleic acid substitutions and/or deletions.
  • CK-2 nucleic acid molecules of the invention may also be ones that hybridize to a CK-2 nucleic acid molecules, e.g., in a Southern blot assay under defined conditions.
  • a CK-2 nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to a complement of a CK-2 nucleic acid sequence, such as any of the coding sequences set forth in FIGS. 1 and 2 (SEQ ID NOS:l-2, respectively) under highly stringent hybridization conditions that comprise 50% formamide and 5x or 6x SSC.
  • the nucleic acid molecules hybridize to a complement of a CK-2 nucleic acid sequence (e.g., to any of the coding sequences set forth in FIGS. 1 and 2, and in SEQ ID NOS: 1-2, respectively) under moderately stringent hybridization conditions (e.g., 40% formamide with 5x or 6x SSC), or under low stringency conditions (e.g., in 5x SSC, 0.1% SDS, 0.25% mile, no formamide, 30% formamide, 5x SSC or 0.5% SDS).
  • a nucleic acid molecule of the invention may hybridize, under the same defined hybridization conditions, to the complement of a fragment of a nucleotide sequence encoding a full length CK-2 polypeptide.
  • S9, S96 and S5 SEQ ID NOS:6-8, respectively
  • S9, S96 and S5 SEQ ID NOS:6-8, respectively
  • the Examples further demonstrate hybridization of such probes to clones in a cDNA library under defined, exemplary hybridization conditions (i.e., hybridization to nylon filters at 42 °C followed by washing in 2x SSC, 0.1% SDS at room temperature, followed by washes in 0.5x SSC, 0.1% SDS at 68 °C and in 0.2x SSC, 0.1% SDS at 68 °C) and isolation of a full length CK-2 cDNA clone.
  • the nucleic acid molecules of the invention comprise fragments of a full length CK-2 nucleic acid sequence.
  • Such fragments include, but are not limited to, the fragment CK-2 nucleic acid sequences contained in the cDNA inserts of clones S9, S96 and S5 (SEQ ID NOS: 6-8, respectively) described in the Examples, supra.
  • such CK-2 nucleic acid fragments comprise a nucleotide sequence that corresponds to a sequence of at least 10 nucleotides, preferably at least 15 nucleotides and more preferably at least 20 nucleotides of a full length coding CK-2 nucleotide sequence.
  • the fragments correspond to a portion (e.g., of at least 10, 15 or 20 nucleotides) of the CK-2 coding sequences set forth in FIG.
  • the CK-2 nucleic acid fragments comprise sequences of at least 10, preferably at least 15, and more preferably at least 20 nucleotides that are complementary and/or hybridize to a full length coding CK-2 nucleic acid sequence (e.g., in the sequences set forth in FIGS. 1 and 2 and in SEQ ID NOS: 1-2, respectively) or to a fragment thereof.
  • Suitable hybridization conditions for such oligonucleotides are described supra, and include washing in 6x SSC/0.05% sodium pyrophosphate.
  • suitable hybridization temperatures will vary depending upon the oligonucleotide molecules used. Exemplary temperatures will be 37 °C (e.g., for 14-base oligonucleotides), 48 °C (e.g., for 17-base oligonucleotides), 55 °C (e.g., for 20-base oligonucleotides) and 60 °C (e.g., for 23 -base oligonucleotides).
  • Nucleic acid molecules comprising such fragments are useful, for example, as oligonucleotide probes and primer (e.g. , PCR primers) to detect and amplify other nucleic acid molecules encoding a CK-2 polypeptide, including genes the encode variant CK-2 polypeptides such as CK-2 analogs and homologs.
  • Oligonucleotide fragments of the invention may also be used, e.g., as antisense nucleic acids, triple helix forming oligonucleotides or as ribozymes; e.g., to modulate levels of CK-2 gene expression or transcription in cells.
  • the nucleic acid molecules of the invention also include "chimeric" CK-2 nucleic acid molecules.
  • Such chimeric nucleic acid molecules are polynucleotides which comprise at least one CK-2 nucleic acid sequence (which may be any of the full length or partial CK-2 nucleic acid sequences described above), and also at least one non-CK-2 nucleic acid sequence.
  • the non-CK-2 nucleic acid sequence may be a heterologous regulatory sequence (for example a promoter sequence) that is derived from another, non-CK- 2 gene and is not normally associated with a naturally occurring CK-2 gene.
  • the non-CK-2 nucleic acid sequence may also be a coding sequence of another, non-CK-2 polypeptide such as FLAG, a histidine tag, glutathione S-transferase (GST), hemaglutinin, ⁇ -galactosidase, thioreductase or an immunoglobulin domain or domains (for examples, an Fc region).
  • a chimeric nucleic acid molecule of the invention encodes a CK-2 fusion polypeptide of the invention.
  • a chimeric nucleic acid molecules of the invention comprises a CK-2 nucleic acid sequence and also a sequence encoding an antigen.
  • the Examples, infra describe the administration of an exemplary DNA vaccine comprising a plasmid having the nucleic acid sequence for a truncated CK-2 polypeptide fused to a ⁇ -galactosidase polypeptide antigen.
  • constructs encoding similar fusion proteins of chemokines are already known in the art (see, e.g., Biragyn et al, Nature Biotechnology 1999, 17:253-258) and induce a stronger immune response to the antigen than either administration of a plasmid encoding the antigen alone, or administration of separate plasmids encoding an antigen and a chemokine (e.g., a CK-2 polypeptide), respectively.
  • a chemokine e.g., a CK-2 polypeptide
  • CK-2 nucleic acid molecules of the invention can be isolated from any source including, for example, fish (e.g., rainbow trout, salmon or carp) cDNA or genomic libraries. Methods for obtaining CK-2 genes 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 (for example, from a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein (e.g., from hemopoietic cells or from tissue, such as the kidney, containing a large number of such cells).
  • the DNA is obtained from a "subtraction" library, as described in the Examples, infra, to enrich the library for cDNAs of genes specifically expressed by a particular cell type or under certain conditions.
  • a a subtractive library may be prepared using activated T-lymphocytes (e.g., by treating cells with PHAP-5) as a source of "activated” mRNA and untreated cells as a source of "resting” mRNA, so that a substantial fraction of cDNA clones corresponding to genes that are expressed by both activated and resting T-lymphocytes are removed.
  • activated T-lymphocytes e.g., by treating cells with PHAP-5
  • untreated cells as a source of "resting” mRNA
  • a library may be prepared 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. Vols. I and II).
  • a cDNA library may be screened for a CK-2 nucleic acid by identifying cDNA inserts that encode a polypeptide which is homologous or substantially similar to a CK-2 polypeptide, such as the polypeptide set forth in FIG. 2 (SEQ ID NO: 3) or a fragment thereof.
  • a cDNA library may be screened for a CK-2 nucleic acid by identifying cDNA inserts having a nucleic acid sequence that is homologous or substantially similar to a CK-2 nucleic acid sequence, such as the nucleic acid sequence set forth in FIG. 1 or in FIG.
  • a cDNA library may be screened as described in the Examples, infra, to identify cDNA inserts that encode a polypeptide which is homologous or substantially similar to a known chemokine or cytokine, such as the carp CC-1 chemokine or fractalkine.
  • the known chemokine or cytokine may, however, be any cytokine or chemokine that is known in the art, including, for example, a chemokine or cytokine from another species of fish (e.g. , from trout, carp, catfish, Tilapia or sea bream to name a few), or from another vertebrate such as a human or other primate, a mouse, a rat, a chicken or other species of bird, etc.
  • a chemokine or cytokine from another species of fish (e.g. , from trout, carp, catfish, Tilapia or sea bream to name a few), or from another vertebrate such as a human or other primate, a mouse, a rat, a chicken or other species of bird, etc.
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions. Clones derived from cDNA generally will not contain intron sequences. Whatever the source, the gene is preferably molecularly cloned into a suitable vector for propagation of the gene. Identification of the specific DNA fragment containing the desired CK-2 gene may be accomplished in a number of ways. For example, a portion of a CK-2 gene exemplified infra can be purified and labeled to prepare a labeled probe (Benton & Davis, Science 1977, 196:180; Grunstein & 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 CK-2 gene.
  • CK-2 gene product 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 a CK-2 polypeptide.
  • the presence of the gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.
  • the selection methods of the present invention may comprise screening a cDNA or other nucleic acid library for a particular sequence or motif associated with a property of interest, or by screening a genomic library for the complement of such a sequence motif.
  • nucleic acid molecules in particular, mRNA molecules
  • polypeptides such as chemokines and cytokines typically have controlled, short half-lives in cells.
  • nucleic acids that encode the gene products of many oncogenes for example, the RAS oncogene
  • RAS oncogene also frequently have controlled, short half- lives in cells.
  • the half-life of such nucleic acids is believed to be regulated in cells by one or more endonuclease enzymes that recognize particular sequence motifs in the nucleic acid, for example an AUUUA motif in mRNA which may be reverse transcribed into the sequence ATTTA as described, infra, in the Examples.
  • the recognized sequence or motif is in an untranslated region of the nucleic acid, for example the 3 '-untranslated region or the 5'- untranslated region.
  • the present invention also provides methods of screening for nucleic acids having a controlled half-life in cells (e.g., a short half-life) by identifying and/or selecting nucleic acid molecules which comprise a particular sequence (for example, the ATTTA sequence), or complement thereof, associated with a controlled half-life in cells.
  • Such methods may be used to identify, not only novel chemokines such as CK-2, but may also be used to identify novel cytokines (for example, novel homologs of cytokines such as IL-2, IL-4 etc.) and/or novel oncogenes (for example, novel homologs of the RAS and other oncogenes).
  • Such screening methods are particularly useful, and are therefore preferably used, for selecting and/or isolating nucleic acids encoding polypeptides for which no close homologs or orthologs are known in the art.
  • chemokine, cytokine and oncogene nucleic acids have been isolated from mammalian species such as humans, mice, rats, etc.
  • homologs of these nucleic acids have been identified in other, more distantly related species such as fish (e.g., salmon, trout, carp, catfish, sea bream, Tilapia and the like) and birds (for instance, chickens, turkey, pheasants, etc.) to name a few.
  • nucleic acid homologs do typically share a certain level of conserved sequence motifs and sequence homology, even across distantly related species of organisms, such levels of homology or sequence identity are typically much lower across distantly related species. As a result, it may be difficult to identify novel nucleic acid in organisms, such as fish and birds, by selecting sequences that have have a certain level of sequence identity or sequence similarity (either at the nucleotide or amino acid level) to homologs in a distantly related species (for example, a mammalian species).
  • the Example set forth in Section 6.2, infra describes the screening of a cDNA library prepared from rainbow trout hemopoetic cells using probes that contain either a nucleotide sequence corresponding to a particular sequence motif (ATTTA), or the complement of the nucleotide sequence corresponding to this particular sequence motif (i.e., TAAAT).
  • ATTTA a nucleotide sequence corresponding to a particular sequence motif
  • TAAAT the complement of the nucleotide sequence corresponding to this particular sequence motif
  • This sequence motif is one commonly found in nucleic acids encoding gene products, such as cytokines, chemokines and oncogene products (i.e., polypeptides and other gene products encoded by oncogenes), whose mRNAs have short half-lives in cells.
  • the Example particularly describes the use of a specific cDNA insert containing the ATTTA sequence, referred to as S 5 (SEQ ID NO: 8), as a probe to screen a library full length, rainbow trout hemopoetic cell cDNAs.
  • S 5 a specific cDNA insert containing the ATTTA sequence
  • the S5 probe hybridizes to, and is thereby used to isolate, a novel CK-2 nucleic acid of the present invention (specifically, the CK-2 cDNA sequence set forth in FIG. 1 and in SEQ ID NO:l).
  • the S5 clone comprises a nucleotide sequence (SEQ ID NO: 8) that encodes only the last 39 amino acid residues of the CK-2 polypeptide sequence shown in FIG.2 (SEQ ID NO:3).
  • amino acid residues correspond to a mucin-like stalk domain and, in particular, do not share any of the conserved amino acid residue sequence homologies or motifs commonly associated with chemokines.
  • traditional methods for selecting nucleic acid probes e.g., by identifying nucleic acids having common sequence homologies or motifs commonly associated with chemokines, would not have identified S5 as a probe which would isolated a novel chemokine nucleic acid (e.g., the CK-2 cDNA sequence shown in FIG. 2 and in SEQ ID NO:l).
  • nucleic acid sequences and/or probes may be used, e.g., for novel cytokine and/or chemokine nucleic acids (for example, nucleic acid molecules that encode novel cytokine and/or chemokine homologs such as novel homologs of IL-2, IL-4, IL-8, IL- l ⁇ , interferons such as interferon type I and interferon type II, GMCSF, etc.) or for novel oncogene nucleic acids (for example, nucleic acid molecules that are novel homologs of oncogenes such as the RAS oncogene). Accordingly, the methods of the present invention are intended to comprise these applications, as well as other similar applications that will be appreciated by those skilled in the art.
  • DNA sequences which encode substantially the same amino acid sequence as a CK-2 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.
  • the nucleic acid sequences of the invention include both "function-conservative variants" and "sequence-conservative variants”. Nucleic acid substitutions may be made for example, to alter the amino acid residue encoded by a particular codon, and thereby substitute an amino acid in a CK-2 polypeptide for one with a particularly preferable property.
  • a Cysteine amino acid residue may be introduced at a potential site for disulfide bridges with another Cysteine amino acid residue.
  • an amino acid residue for example a serine amino acid residue
  • a cystein amino acid residue in CK-2 may be substituted for a cystein amino acid residue in CK-2.
  • substitutions may be useful, for example, to facilitate solubilization of a recombinant CK-2 polypeptide.
  • the genes encoding CK-2 derivatives and analogs of the 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 CK-2 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 CK-2-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 also be made to introduce restriction sites and facilitate cloning the CK-2 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.
  • 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.
  • 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 pBR322 derivatives or pUC plasmid derivatives, e.g., pG ⁇ X vectors, pmal-c, pFLAG, pKK plasmids (Clonetech), p ⁇ T plasmids (Novagen, Inc., Madison, WI), pRS ⁇ T or pR ⁇ P plasmids, pcDNA (Invitrogen, Carlsbad, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), etc.
  • pG ⁇ X vectors pmal-c
  • pFLAG pFLAG
  • pKK plasmids Clonetech
  • p ⁇ T plasmids Novagen, Inc., Madison, WI
  • pRS ⁇ T or pR ⁇ P plasmids pcDNA (Invitrogen, Carlsbad, CA), or pMAL
  • 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 enzyrnatically 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.
  • 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 S ⁇ cch ⁇ romyces cerevisi ⁇ e by linking sequences from an E. coli plasmid with sequences form the yeast 2m plasmid.
  • a nucleotide sequence coding for CK-2, for an antigenic fragment, derivative or analog of CK-2, or for a functionally active derivative of CK-2 (including a chimeric protein) may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • a nucleic acid encoding a CK-2 polypeptide of the invention can be operationally associated with a promoter in an expression vector of the 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 CK-2 polypeptides.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector.
  • Potential host- vector systems include but are not limited to mammalian or other vertebrate (e.g., fish) 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., baculo virus); 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.
  • CK-2 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 CK-2 gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent ⁇ os. 5,385,839 and 5,168,062), the SN40 early promoter region (Benoist and Chambon, Nature 1981, 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell 1980, 22:787-797), the herpes thymidine kinase promoter (Wagner et al, Proc.
  • CMV cytomegalovirus
  • U.S. Patent ⁇ os. 5,385,839 and 5,168,062 the SN40 early promoter region
  • the promoter contained in the 3' long terminal repeat of Rous sarcoma virus Y
  • 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 hematopoietic tissue specificity, in particular: beta-globin gene control region which is active in myeloid cells (Mogram et al, Nature 1985, 315:338-340; Kollias et al, Cell 1986, 46:89-94), hematopoietic stem cell differentiation factor promoters, erythropoietin receptor promoter (Maouche et al, Blood 1991, 15:2557), etc.
  • 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 hem
  • any type of plasmid, cosmid, YAC or viral vector may be used to prepare a recombinant nucleic acid construct which can be introduced to a cell, or to tissue, where expression of a CK-2 gene product is desired.
  • viral vectors that selectively infect the desired cell type or tissue type can be used.
  • the invention provides methods for expressing CK-2 polypeptides by using a non-endogenous promoter to control expression of an endogenous CK-2 gene within a cell.
  • An endogenous CK-2 gene within a cell is a CK-2 gene of the present invention which is ordinarily (i.e., naturally) found in the genome of tht cell.
  • a non- endogenous promoter is a promoter or other nucleotide sequence that may be used to control expression of a gene but is not ordinarily or naturally associated with the endogenous CK-2 gene.
  • methods of homologous recombination may be employed (preferably using non-protein encoding CK-2 nucleic acid sequences of the invention) to insert an amplifiable gene or other regulatory sequence in the proximity of an endogenous CK-2 gene.
  • the inserted sequence may then be used, e.g. , to provide for higher levels of CK-2 gene expression than normally occurs in that cell, or to overcome one or more mutations in the endogenous CK-2 regulatory sequences winch prevent normal levels of CK- 2 gene expression.
  • Such methods of homologous recombination are well known in the art. See, for example, International Patent Publication No. WO 91/06666, published May 16, 1991 by Skoultchi; International Patent Publication No.
  • Soluble forms of the protein can be obtained by collecting culture fluid, or solubilizing inclusion bodies, e.g. , by treatment with detergent, and if desired sonication or other mechanical processes, as described above.
  • the solubilized or soluble 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.
  • 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 SN40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al, Gene 1988, 67:31-40), pCR2.1 and pcD ⁇ A 3.1+ (Invitrogen, Carlsbad, California), pMB9 and their derivatives, plasmids such as RP4; phage D ⁇ As, e.g., the numerous derivatives of phage 1, e.g., ⁇ M989, and other phage DNA, e.g.
  • yeast plasmids such as the 2m 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.
  • Preferred vectors are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism.
  • viral vectors such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular tropism.
  • a gene encoding a functional or mutant CK-2 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 Public
  • 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 of the 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), 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.
  • 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 (HS V), papillomavirus, Epstein Barr virus (EB V), 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.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al, Molec. Cell Neurosci.
  • 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.
  • 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. 1987, 84:7413-7417; Feigner and Ringold, Science 1989, 337:387-388; Mackey et al, Proc. Natl. Acad. Sci. U.S.A. 1988, 85:8027-8031; Ulmer et al, Science 1993, 259:1745-1748).
  • 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, Proc. Natl. Acad. Sci. U.S.A. 1988, 85:8027-8031).
  • 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 WO 95/21931).
  • 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 WO 95/21931
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinj ection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu etal., J. Biol. Chem. 1992, 267:963-967; Wu and Wu, J Biol. Chem. 1988, 263 : 14621- 14624; Hartmut et al. , Canadian Patent Application No. 2,012,311 , filed March 15, 1990; Williams et al, Proc.
  • an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g. , adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells.
  • the viral vector e.g. , adenovirus vector
  • immunosuppressive cytokines such as interleukin-12 (IL-12), interferon-g (IFN- ⁇ ), or anti- CD4 antibody
  • IL-12 interleukin-12
  • IFN- ⁇ interferon-g
  • anti- CD4 antibody can be administered to block humoral or cellular immune responses to the viral vectors (see, e.g. , Wilson, Nat. Med. 1995, 1:887-889).
  • a viral vector that is engineered to express a minimal number of antigens.
  • Antibodies to CK-2 are useful, ter alia, for diagnostics and intracellular regulation of CK-2 activity, as set forth below.
  • CK-2 polypeptides produced e.g. , recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize the CK-2 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 preferably specific for (i.e., specifically binds to) a rainbow trout CK-2 polypeptide of the present invention.
  • the antibody may, alternatively, be specific for a CK-2 ortholog from some other species of organism, preferably another species offish such as salmon, carp or catfish.
  • the antibody may recognize a mutant form of CK-2, or wild-type CK-2, or both.
  • CK-2 polypeptide or derivative or analog thereof various procedures known in the art may be used for the production of polyclonal antibodies to CK-2 polypeptide or derivative or analog thereof.
  • various host animals can be immunized by injection with the CK-2 polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • the CK-2 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 (b ⁇ cille C ⁇ lmette-Guerin) and Coryneb ⁇ cterium p ⁇ rvum.
  • BCG b ⁇ cille C ⁇ lmette-Guerin
  • Coryneb ⁇ cterium p ⁇ rvum for preparation of monoclonal antibodies directed toward the CK-2 polypeptide, or fragment, analog, or derivative thereof.
  • monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690).
  • techniques developed for the production of "chimeric antibodies” may also be used.
  • such techniques comprise splicing the genes from an antibody molecule from a first species of organism (e.g., a mouse) that is specific for a CK-2 polypeptide together with genes from an antibody molecule of appropriate biological activity derived from a second species of organism (e.g., from a human).
  • a first species of organism e.g., a mouse
  • an antibody molecule of appropriate biological activity derived from a second species of organism (e.g., from a human).
  • Antibody fragments which contain the idiotype of the 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 of the antibody molecule; the Fab' fragments which can be generated by reducing the disulf ⁇ de bridges of the 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. , radioimmunoassay, 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.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays,
  • 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 a CK-2 polypeptide, one may assay generated hybridomas for a product which binds to a CK-2 polypeptide fragment containing such epitope. For selection of an antibody specific to a CK-2 polypeptide from a particular species of animal, one can select on the basis of positive binding with CK-2 polypeptide expressed by or isolated from cells of that species of animal.
  • antibodies can be used in methods known in the art relating to the localization and activity of the CK-2 polypeptide, e.g., for Western blotting, imaging CK- 2 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 US 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.
  • anti-CK-2 antibodies may also be used to isolate cells which express a CK-2 polypeptide by panning or related immunoadsorption techniques.
  • antibodies that agonize or antagonize the activity of a CK-2 polypeptide can be generated.
  • intracellular single chain Fv antibodies can be used to regulate (inhibit) CK-2 activity (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.
  • CK-2 chemokines including applications and uses for CK-2 nucleic acids of the invention (defined in Section 5.3, supra), for CK-2 polypeptides of the invention (defined, supra, in Section 5.2) and for antibodies directed against such CK-2 polypeptides and nucleic acids (described in Section 5.5, supra).
  • the CK-2 chemokines of the present invention may induce or enhance and immune response in an organism.
  • the chemokines of the invention are chemotactic for leukocyte cells (including T lymphocytes, such as CD8 and CD4 cells, activated B cells and monocytes).
  • leukocyte cells including T lymphocytes, such as CD8 and CD4 cells, activated B cells and monocytes.
  • the CK-2 nucleic acids, polypeptides and antibodies of the present invention may be used in prognostic and diagnostic applications to identify or detect an immune response stimulated by CK-2.
  • the CK-2 nucleic acids, polypeptides and antibodies of the invention may also be used in screening assays, e.g., to identify compounds, such as a chemokine receptor that specifically bind to a CK-2 chemokine of the invention or, alternatively, to identify compounds that modulate binding of a CK-2 chemokine to a chemokine receptor or other molecule. Such compounds may be useful, e.g., as immune enhancing or suppressing drugs.
  • the CK-2 nucleic acids and polypeptides of the invention may also be used in pharmaceutical preparations.
  • a CK-2 polypeptide or CK-2 nucleic acid of the invention is used in a vaccine, e.g., as an adjuvant to enhance an immune response to the vaccine antigen.
  • a variety of methods can be employed for diagnostic and prognostic methods using reagents such as the CK-2 nucleic acids and polypeptides described supra (including fragments, chimeras and fusions thereof), as well as antibodies directed against these polypeptides. For example, by using the methods described here to detect a CK-2 nucleic acid or a CK-2 polypeptide in an individual it is possible to detect, in the individual, the presence or absence of an immune response associated with a CK-2 chemokine.
  • the prognostic and diagnostic methods described here may be used in the prognostic or diagnostic evaluation of a disease or disorder, e.g., an immune disorder, which is associated with a CK-2 chemokine (for example, a disease or disorder associated with abnormal levels of a CK-2 chemokine, or a disease or disorder associated with a mutant or variant CK-2 chemokine).
  • a disease or disorder e.g., an immune disorder, which is associated with a CK-2 chemokine
  • a CK-2 chemokine for example, a disease or disorder associated with abnormal levels of a CK-2 chemokine, or a disease or disorder associated with a mutant or variant CK-2 chemokine.
  • CK-2 gene product e.g., a CK-2 mRNA
  • an unaffected state i.e., in a subject not having or predisposed to having a bone growth associated disorder
  • detection of either and over- or an under-abundance of a CK-2 gene product relative to abundance in an unaffected state i.e., in a subject not having or predisposed to having a bone growth associated disorder
  • kits may comprise at least one specific CK-2 nucleic acid or a CK-2 specific antibody reagent of the invention.
  • the kit and any reagent(s) contained therein can be used, for example, in a clinical setting, to diagnose patients exhibiting abnormalities, such as an immune disorder.
  • a sample comprising a nucleated cell (of any cell type) from an individual may be used in such diagnostic and prognostic methods as a starting source for genomic nucleic acid and to detect mutations of a CK-2 gene.
  • a sample comprising a cell of any cell type or tissue of any tissue type in which a CK-2 gene is expressed may also be used in such diagnostic methods, e.g., for detection of CK-2 gene expression or of CK-2 gene products (such as CK-2 proteins), as well as for identifying cells, particularly hemopoietic cells, that express a CK-2 gene or a CK-2 gene product.
  • CK-2 nucleic acids For the detection of CK-2 mutations or to assay levels of CK-2 nucleic acid sequences in a sample, a variety of methods may be employed. For example, mutations within a CK-2 gene may be detected by utilizing a number of techniques known in the art and with nucleic acid derived from any nucleated cell. The nucleic acid may be isolated according to standard nucleic acid preparation procedures that are already well known to those of skill in the art.
  • CK-2 nucleic acid sequences may be used in hybridization or amplification assays of such biological samples to detect abnormalities involving CK-2 gene structure.
  • Exemplary abnormalities that can be detected in such methods include point mutations, single nucleotide polymorphisms (SNPs), insertions, deletions, inversions, translocations and chromosomal rearrangements.
  • Exemplary assays that can be used to detect these abnormalities include Southern analyses, fluorescence in situ hybridization (FISH), single- stranded conformational polymorphism analyses (SSCP) and polymerase chain reaction (PCR) analyses.
  • FISH fluorescence in situ hybridization
  • SSCP single- stranded conformational polymorphism analyses
  • PCR polymerase chain reaction
  • diagnostic methods for the detection of CK-2 gene-specific mutations can involve contacting and incubating nucleic acids (including recombinant DNA molecules, clones genes or degenerate variants thereof) obtained from a sample with one or more labeled nucleic acid reagents, such as recombinant CK-2 DNA molecules, cloned genes or degenerate variants thereof, under conditions favorable for specifically annealing or hybridizing these reagents to their complementary sequences in the sample nucleic acids.
  • the lengths of these nucleic acid reagents are at least 15 to 30 nucleotides.
  • CK-2 gene sequences to which the nucleic acid reagents have annealed may be compared to the annealing pattern expected from a normal (i.e., a wild-type) CK-2 gene sequence in order to dete ⁇ nine whether a CK-2 gene mutation is present.
  • the nucleic acid from the cell type or tissue of interest may be immobilized, for example, to a solid support such as a membrane or a plastic surface (for example, on a nylon membrane, a microtiter plate or on polystyrene beads).
  • a solid support such as a membrane or a plastic surface (for example, on a nylon membrane, a microtiter plate or on polystyrene beads).
  • non-annealed, labeled CK-2 nucleic acid reagents may be easily removed and detection of the remaining, annealed, labeled CK-2 nucleic acid reagents may be accomplished using standard techniques that are well-known in the art.
  • CK-2 gene specific nucleic acids in patient samples or in other cell sources may involve their amplification, e.g., by PCR (see, for example, the experimental embodiment taught in U.S. Patent No. 4,683,202) followed by detection of the amplified molecules using techniques that are well known to those of skill in the art.
  • the resulting amplified sequences may be compared to those that would be expected if the nucleic acid being amplified contained only normal copies of a CK- 2 gene in order to determine whether a CK-2 mutation is present in the samples
  • genotyping techniques may also be used to identify individuals carrying CK-2 mutations. Such techniques include, for example, the use of restriction fragment length polymorphisms (RFLPs). Other methods which may be used to identify CK-2 mutations capitalize on the presence of variable numbers of short tandemly repeated DNA sequences between the restriction enzyme sites.
  • RFLPs restriction fragment length polymorphisms
  • U.S. Patent No. 5,075,217 describes a DNA marker based on length polymorphisms in blocks of short tandem repeats. The average separation of such blocks is estimated to be 30 to 70 kb.
  • Markers that are so closely spaced exhibit a high frequency of co-inheritance and are extremely useful in the identification of genetic mutations, including for example mutations within the CK-2 gene, as well as for the diagnosis of diseases and disorders related to genetic mutations, e.g., within a CK-2 gene.
  • the diagnostic and prognostic methods of the invention also include methods for assaying the level of CK-2 gene expression.
  • RNA from a cell type or tissue such as hemopoietic cells, that is known or suspected to express the CK-2 gene may be isolated and tested utilizing hybridization or PCR techniques such as those described supra.
  • the isolated cells may be, for example, cells derived from a cell culture or from an individual.
  • the analysis of cells taken from a cell culture may be useful, e.g., to test the effect of compounds on the expression of a CK-2 gene, or alternatively, to verify that the cells are ones of a particular cell type that expresses a CK-2 gene.
  • a cDNA molecule is synthesized from an RNA molecule of interest (e.g., by reverse transcription). A sequence within the cDNA may then be used as a template for a nucleic acid amplification reaction such as PCR. Nucleic acid reagents used as synthesis intitation reagents (e.g., primers) in the reverse transcription and amplification steps of such an assay are preferably chosen from the CK-2 nucleic acid sequences described herein or are fragments thereof. Preferably, the nucleic acid reagents are at least about 9 to 30 nucleotides in length.
  • the amplification may be performed using, e.g., radioactively labeled or fluorescently labeled nucleotides, for detection.
  • enough amplified product may be made such that the product can be visualized by standard ethidium bromide or other staining methods.
  • CK-2 gene expression assays of the invention may also be performed in situ (i. e. , directly upon tissue sections of patient tissue, which may be fixed and/or frozen), thereby eliminating the need of nucleic acid purification.
  • CK-2 nucleic acid reagents may be used as probes or as primers for such in situ procedures (see, for example, Nuovo, PCR In Situ Hybridization: Protocols And Application, 1992, Raven Press, New York).
  • Standard Northern analysis can be performed to determine the level of CK-2 gene express by detecting levels of CK-2 mRNA.
  • the diagnostic and prognostic methods of the invention also include ones that comprise detecting levels of a CK-2 protein or other CK- 2 polypeptide and including functionally conserved variants and fragments thereof.
  • antibodies directed against unimpaired, wild-type or mutant CK-2 gene products or against functionally conserved variants or peptide fragments of a CK-2 gene product may be used as diagnostic and prognostic reagents for immune disorders or, alternatively, to detect and immune response associated with a CK-2 chemokine.
  • Such reagents may be used, for example, to detect abnormalities in the level of CK-2 gene product synthesis or expression, or to detect abnormalities in the structure, temporal expression or physical location of a CK-2 gene product.
  • Antibodies and immunoassay methods such as those described hereinbelow also have important in vitro applications for assessing the efficacy of treatments, e.g., for immune disorders.
  • Such antibodies and immunoassays also have important applications for assessing efficacy of vaccines, e.g., by assaying a vaccine's ability to stimulate an immune response.
  • antibodies, or fragments of antibodies can be used in screens of potentially therapeutic compounds in vitro to ascertain a compound's effects on CK-2 gene expression and CK-2 polypeptide production.
  • Compounds that may have beneficial effects on a CK-2 associated disorder can be identified and a therapeutically effective dose for such compounds may be determined using such assays.
  • In vitro immunoassays can also be used to assess the efficacy of cell-based gene therapy for a CK-2 associated disorder.
  • antibodies directed against CK-2 polypeptides may be used in vitro to determine the level of CK-2 gene or polypeptide expression achieved in cells genetically engineered to produce a CK-2 polypeptide.
  • Such methods may be used to detect intracellular CK-2 gene products, preferably using cell lysates or extracts, to detect expression of CK-2 gene products of cell surfaces, or to detect CK-2 gene products secreted into the cell culture media.
  • Such an assessment can be used to determine the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.
  • tissue or cell types analyzed using such methods will include ones, such as hemopoietic cells, that are known to express a CK-2 gene product.
  • Protein isolation methods such as those described by Harlow & Lane (Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) may be employed.
  • the isolated cells may be cells derived from cell culture or from an individual (e.g., a patient suspected of having a CK-2 associated disorder or suspected of having a propensity for a CK-2 associated disorder).
  • antibodies or fragments of antibodies may be used to detect the presence of a CK-2 gene product, a variant of a CK-2 gene product or fragments thereof, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric or fluorimetric detection methods.
  • Antibodies or fragments thereof may also be employed bistologically, for example in immunofluorescence or immunoelectron microscopy techniques, for in situ detection of a CK-2 gene product.
  • In situ detection may be accomplished by removing a histological specimen (e.g., a tissue sample) from a patient and applying thereto a labeled antibody of the present invention or a fragment of such an antibody.
  • the antibody or antibody fragment is preferably applied by overlaying the labeled antibody or antibody fragment onto a biological sample.
  • Immunoassays for CK-2 gene products will typically comprise incubating a biological sample (for example, a biological fluid, a tissue extract, freshly harvested cells or cell lysates) in the presence of a detectably labeled antibody that is capable of specifically binding a CK-2 gene product (including, for example, a functionally conserved variant or a peptide fragment thereof). The bound antibody may then be detected by any of a number of techniques well known in the art.
  • a biological sample for example, a biological fluid, a tissue extract, freshly harvested cells or cell lysates
  • a detectably labeled antibody that is capable of specifically binding a CK-2 gene product (including, for example, a functionally conserved variant or a peptide fragment thereof).
  • the bound antibody may then be detected by any of a number of techniques well known in the art.
  • Screening Assays such as those described hereinbelow, it is also possible to identify compounds that bind to or otherwise interact with a CK-2 gene product, including intracellular compounds (for example, proteins or portions of proteins) that interact with a CK-2 gene product, natural and synthetic ligands (or receptors) for a CK-2 gene product, compounds that interfere with the interaction of a CK-2 gene product (for example, compounds that interfere with specific binding of a CK-2 gene product to a receptor or intracellular compound), and compounds that modulate the activity of a CK-2 gene (for example, by modulating the level of CK-2 gene expression) or the activity (for example, the bioactivity) of a CK-2 polypeptide or other CK-2 gene products.
  • intracellular compounds for example, proteins or portions of proteins
  • natural and synthetic ligands (or receptors) for a CK-2 gene product for example, compounds that interfere with specific binding of a CK-2 gene product to a receptor or intracellular compound
  • the CK-2 chemokines of the invention are believed to specifically bind to a CK-2 specific chemokine receptor (referred to herein as a "CK-2 chemokine receptor" or "CK-2 receptor") expressed by certain types of cells and, particularly, by leukocyte cells (including T lymphocytes, such as CD8 and CD4 cells, activated B cells and monocytes). Such binding is believed the activated those cells expressing a CK-2 specific receptor and attract or draw those cells to the site where a CK-2 gene product is present or secreted ( . e. , to the site where concentration of a CK-2 gene product is highest), thereby stimulating an immune response.
  • CK-2 chemokine receptor referred to herein as a "CK-2 chemokine receptor” or "CK-2 receptor”
  • the screening methods of the present invention may be used to identify receptors and cells expressing receptors, that specifically bind to and are therby activated by a CK-2 gene product of the invention.
  • the screening methods may also be used to identify compounds that inhibit or modulate such a binding interaction and are therefore useful as agonist or antagonist for CK-2 binding to a CK-2 specific receptor.
  • such compounds may be used, e.g., to enhance or suppress a CK-2 associated immune response.
  • the screening assays described here may be used to identify compounds that bind to a promoter or other regulatory sequence of a CK-2 gene, and so may modulate the level of CK-2 gene expression (see, for example, Platt, J Biol. Chem. 1994, 269:28558-28562).
  • Classes of compounds that may be identified by such screening assays include, but are not limited to, small molecules (e.g., organic or inorganic molecules which are less than about 2 kDa in molecular weight, are more preferably less than about 1 kDa in molecular weight, and/or are able to cross the blood-brain barrier or gain entry into an appropriate cell and affect expression of either a CK-2 gene or of some gene involved in a CK-2 regulatory pathway) as well as macromolecules (e.g., molecules greater than about 2 kDa in molecular weight).
  • Compounds identified by these screening assays may also include peptides and polypeptides.
  • Examples of such compounds include but are not limited to: soluble peptides; fusion peptide members of combinatorial libraries (such as ones described by Lam et al, Nature 1991, 354:82-84; and by Houghten et al, Nature 1991, 354:84-86); members of libraries derived by combinatorial chemistry, such as molecular libraries of D- and/or L-configuration amino acids; phosphopeptides, such as members of random or partially degenerate, directed phosphopeptide libraries (see, e.g., Songyang et al, Cell 1993, 72:767-778); antibodies, including but not limited to polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies; antibody fragments, including but not limited to Fab, F(ab') 2 , Fab expression library fragments, and epitope-binding fragments thereof.
  • Assays for binding compounds In vitro systems can be readily designed to identify compounds capable of binding the CK-2 gene products of the present invention. Such compounds can be useful, for example, in modulating the activity of a wild-type CK-2 gene product or, alternatively, to modulate the activity of a mutant or other variant CK-2 gene product.
  • screening assays involve preparation of a reactive mixture comprising a CK-2 gene product and a test compound under conditions and for a time sufficient to allow the two compounds to interact (e.g. , bind), thereby forming a complex that may be detect.
  • the assays may be conducted in any of a variety of different ways. For example, one embodiment comprises anchoring a CK-2 polypeptide or a test compound onto a solid phase and detecting complexes of the CK-2 polypeptide and the test compound that are on the solid phase at the end of the reaction and after removing (e.g. , by washing) unbound compounds.
  • a CK-2 gene product may be anchored onto a solid surface and a labeled compound (e.g., labeled according to any of the methods described supra) is contacted to the surface.
  • a labeled compound e.g., labeled according to any of the methods described supra
  • unbound molecules of the test compound are removed from the surface (e.g., by washing) and labeled molecules which remain are detected.
  • molecules of one or more different test compounds are attached to the solid phase and molecules of a labeled CK-2 polypeptide may be contacted thereto.
  • the molecules of different test compounds are preferably attached to the solid phase at a particular location on the solid phase so that test compounds that bind to a CK-2 polypeptide may be identified by determining the location of bound CK-2 polypeptides on the solid phase or surface.
  • Assays for compounds that interact with CK-2 Any of a variety of known methods for detecting protein-protein interactions may also be used to detect and/or identify proteins that interact with a CK-2 gene product. For example, co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns as well as other techniques known in the art may be employed. Proteins which may be identified using such assays include, but are not limited to, extracellular proteins, such as CK-2 specific receptors, as well as intracellular proteins such as signal transducing proteins. As an example, and not by way of limitation, an expression cloning assay may be used to identify CK-2 specific receptors and other proteins that specifically interact with a CK-2 gene product.
  • a cDNA expression library may be generated from any cell line that expresses a CK-2 specific receptor (for example, leukocyte cells, such as monocytes, B lymphocytes and T lymphocytes, including CD8 and CD4 cells).
  • a CK-2 specific receptor for example, leukocyte cells, such as monocytes, B lymphocytes and T lymphocytes, including CD8 and CD4 cells.
  • Clones from such an expression library may then be transfected or infected into cells, such as a B cell lymphoma line (e.g., CHI 2 cells, A20.25 cells or LBB1 cells) that do not normally express a CK-2 specific receptor.
  • a B cell lymphoma line e.g., CHI 2 cells, A20.25 cells or LBB1 cells
  • Cells that are, transfected with a clone that encodes a CK-2 specific receptor may then express this gene product, and can be identified and isolated using standard techniques such as FACS or using magnetic beads that have a CK-2 polypeptide (for example, a CK-2-Fc fusion polypeptide) attached thereto.
  • CK-2 specific receptor may be isolated from a cell line, including any of the CK-2 receptor expressing cell lines recited above, using immunoprecipitation techniques that are well known in the art.
  • CK-2 specific receptors may also be isolated using any of the screening assays discussed, supra for identifying CK-2 binding compounds.
  • a CK-2-Fc fusion polypeptide may be bound or otherwise attached to a solid surface, and a labeled compound (e.g., a candidate CK-2 receptor) may be contacted to the surface for a sufficient time and under conditions that permit formation of a complex between the CK-2-Fc fusion polypeptide and the test compound. Unbound molecules of the test compound can then be removed from the surface (e.g., by washing), and labeled compounds that remain bound can be detected.
  • standard techniques may be used to identify any protein detected in such assays.
  • amino acid sequence of a protein that interacts with the CK-2 gene product can be ascertained using techniques well known in the art, such as the Edman degradation technique (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman&Co., New York, pages 34-49). Once such proteins have been identified, their amino acid sequence may be used as a guide for the generation of oligonucleotide mixtures to screen for gene sequences encoding such proteins; e.g., using standard hybridization or PCR techniques described supra.
  • plasmids may be constructed which encode two hybrid proteins: one of which preferably comprises of the DNA-binding domain of a transcription activator protein fused to a CK-2 gene product.
  • the other hybrid protein preferably comprises an activation domain of the transcription activator protein used in the first hybrid, fused to an unknown protein that is encoded by a cDNA recombined into the plasmid library as part of a cDNA library.
  • Both the DNA-binding domain fusion plasmid and the cDNA library may be co-transformed into a strain of S ⁇ cch ⁇ romyces cerevisi ⁇ e or other suitable organism which contains a reporter gene (for example, HBS, lacZ, HIS 3 or GFP).
  • the regulatory region of this reporter gene comprises a binding site for the transcription activator moiety of the two hybrid proteins. In such a two-hybrid system, the presence of either of the two hybrid proteins alone cannot activate transcription of the reporter gene.
  • the DNA-binding domain hybrid protein cannot activate transcription because it cannot localize to the necessary activation function.
  • the activation domain hybrid protein cannot activate transcription because it cannot localize to the DNA binding site on the reporter gene.
  • interaction between the two hybrid proteins reconstitutes that functional transcription activator protein and results in expression of the reporter gene.
  • an interaction between a CK-2 polypeptide i.e., the CK-2 polypeptide fused to the transcription activator's DNA binding domain
  • a test polypeptide i.e., a protein fused to the transcription activator's DNA binding domain
  • cDNA libraries for screening in such two-hybrid and other assays may be made according to any suitable technique known in the art.
  • cDNA fragments may be inserted into a vector so that they are translationally fused to the transcriptional activation domain of GAL4, and co-transformed along with a "bait" CK- 2-GAL4 fusion plasmid into a strain of Saccharomyces cerevisiae or other suitable organism that contains a HIS3 gene driven by a promoter that contains a GAL4 activation sequence.
  • a protein from this cDNA library, fused to the GAL4 transcriptional activation domain, which interacts with the CK-2 polypeptide moiety of the CK-2-GAL4 fusion will reconstitute and active GAL4 protein, and can thereby drive expression of the HIS3 gene.
  • Colonies that express the HIS3 gene may be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA may then be purified from these strains, sequenced and used to identify the encoded protein which interacts with the CK-2 polypeptide.
  • the screening methods described in these methods may also be used to identify other compounds (e.g., small molecules, peptides and proteins) which bind to these binding compounds.
  • Such compounds may also be useful to modulating CK-2-related bioactivities, for example by binding to a natural CK-2 receptor or binding partner, and presenting its interaction with a CK-2 gene product. For instance, these compounds could be tested for their ability to inhibit the binding of CK-2-Fc to cell lines which express CK-2 specific receptor. Assays for compounds that interfere with a CK-2-receptor interaction.
  • a CK-2 gene product of the invention may interact with one or more molecules (i.e., receptors) in vivo or in vitro.
  • molecules i.e., receptors
  • Compounds that disrupt or otherwise interfere with this binding interaction are therefore useful in modulating activity of a CK-2 gene product, and, in particular, may serve to enhance or suppress an immune response associated with a CK-2 gene or gene product of the invention.
  • Such compounds include, but are not limit to, compounds identified according to the screening assays described supra, for identifying compounds that bind to a CK-2 gene product, including any of the numerous exemplary classes of compounds described therein.
  • assays for identifying compounds that interfere with the interaction between a CK-2 gene product and a binding partner involve preparing a test reaction mixture that contains the CK-2 gene product and its binding partner under conditions and for a time sufficient for the CK-2 gene product and its binding partner to bind and form a complex.
  • the test compound preferably is also present in the test reaction mixture.
  • the test compound may be initially included in the test reaction mixture with the CK-2 gene product and its binding partner. Alternatively, however, the test compound may be added to the test reaction mixture at a later time, subsequent to the addition of the CK-2 gene product and its binding partner.
  • one or more control reaction mixtures which do not contain the test compound, may also be prepared. Typically, a control reaction mixture will contain the same CK-2 gene product and binding partner that are in the test reaction mixture, but will not contain a test compound. A control reaction mixture may also contain a placebo, not present in the test reaction mixture, in place of the test compound. The formation of a complex between the CK-2 gene product and the binding partner may then be detected in the reaction mixture.
  • test compound is one which interferes with or modulates the interaction of a CK-2 polypeptide and a binding partner.
  • assays for compounds that modulate the interaction of a CK-2 gene product and a binding partner may be conducted in a heterogenous format or, alternatively, in a homogeneous format.
  • Heterogeneous assays typically involve anchoring either a CK-2 gene product or a binding partner onto a solid phase and detecting compounds anchored to the solid phase at the end of the reaction.
  • such assays are similar to the solid phase assays described supra for detecting and/or identifying CK-2 nucleic acids and gene products and for detecting or identifying CK-2 binding partners. Indeed, those skilled in the art will recognize that many of the principles and techniques described above for those assays may be modified and applied without undue experimentation in the solid phase assays described here, for identifying compounds that modulate interaction(s) between and CK-2 gene product and a binding partner. Regardless of the particular assay used, the order to which reactants are added to a reaction mixture may be varied; for example, to identify compounds that interfere with the interaction of a CK-2 gene product with a binding partner by competition, or to identify compounds that disrupt a preformed binding complex.
  • Test compounds that interfere with the interaction of a CK-2 gene product with a binding partner by competition may be identified by conducting the reaction in the presence of a test compound. Specifically, in such assays a test compound may be added to the reaction mixture prior to or simultaneously with the CK-2 gene product and the binding partner. Test compounds that disrupt preformed complexes of a CK-2 gene product and a binding partner may be tested by adding the test compound to a reaction mixture after complexes have been formed.
  • the screening assays described herein may also be practiced using peptides or polypeptides that correspond to portions of a full length CK-2 polypeptide or protein, or with fusion proteins comprising such peptide or polypeptide sequences.
  • screening assays for identifying compounds the modulate interactions of a CK-2 polypeptide with a binding partner may be practiced using peptides or polypeptides corresponding to particular regions or domains of a full length CK-2 polypeptide that bind to a binding partner (e.g. , receptor "binding sites").
  • screening assays may be carried out using polypeptides (or fusions thereof) that comprise an amino acid sequence corresponding to a mature, full length CK-2 polypeptide (e.g., comprising the sequence of amino acid residues 29-191 of the CK-2 polypeptide set forth in FIG. 2 and in SEQ ID NO:3).
  • screening assays may be carried out using a truncated CK-2 polypeptide which, preferably, retains the biological activity of a full length CK-2 chemokine.
  • screening assays are carried out using a polypeptide comprising a CK-2 globular domain, such as a polypeptide comprising the sequence of amino acid residues 29-90 in the polypeptide set forth in FIG. 2 (SEQ ID NO:3).
  • binding sites may be identified by mutating a CK-2 gene and screening for disruptions of binding as described above.
  • a gene encoding the binding partner may also be mutated in such assays to identify mutations that compensate for disruptions from the mutation to the CK-2 gene. Sequence analysis of these mutations can then reveal mutations that correspond to the binding region of the two proteins.
  • a protein e.g., a CK-2 protein or a protein binding partner to a CK-2 protein
  • Another labeled protein which binds to the protein anchored to the solid surface may be treated with a proteolytic enzyme, and its fragments may be allowed to interact with the protein attached to the solid surface, according to the methods of the binding assays described supra. After washing, short, labeled peptide fragments of the treated protein may remain associated with the anchored protein. These peptides can be isolated and the region of the full length protein from which they are derived may be identified by the amino acid sequence.
  • compounds that interfere with a CK-2-receptor interaction may also be identified by screening for compounds that modulate binding of a CK-2 polypeptide (for example, a CK-2-Fc fusion polypeptide) to cells that express a CK-2 specific receptor, such as leukocyte cells (including monocytes, B lymphocytes and T lymphocytes, including CD8 and CD4 cells).
  • a CK-2 polypeptide for example, a CK-2-Fc fusion polypeptide
  • leukocyte cells including monocytes, B lymphocytes and T lymphocytes, including CD8 and CD4 cells.
  • CK-2 nucleic acid molecules, polypeptides and antibodies of the present invention may also be used in therapeutic methods and compositions, e.g., to modulate or enhance an immune response.
  • CK-2 chemokines of the present invention are believed to enhance or stimulate an immune response by activating leukocytes (including, for example, monocytes, B lymphocytes and T lymphocytes such as CD8 and CD4 cells) and drawing such cells to a site where the CK-2 chemokine is introduced or released.
  • leukocytes including, for example, monocytes, B lymphocytes and T lymphocytes such as CD8 and CD4 cells
  • the introduction of a chemokine, such as CK-2 to a site of infection or trauma may draw mobile cells (e.g., leukocytes) to that site and thereby augment an immune response to the infection or traume.
  • the therapeutic methods of this invention include methods for enhanceing an immune response in an organism by administering a CK-2 nucleic acid or polypeptide.
  • the CK-2 nucleic acids and polypeptides of the invention of the invention are administered either before, after or substantially simultaneously with an antigen or immunogen (e.g. , in a vaccine) so that an immune response to the antigen or immunogen is enhanced in the organism.
  • compounds that bind to a CK-2 nucleic acid or polypeptide of the invention may also be useful, e.g., in methods for enhancing or suppressing an immune response by enhancing or suppressing CK- • 2 activity.
  • the CK-2 nucleic acids and polypeptides of the present invention may be used to enhance or stimulate an immune response in an organism and, in particular, to enhance or stimulate an immune response to an immunogen. Accordingly, the invention provides methods and compositions for enhancing or stimulating an immune response in an organism.
  • the organism is a vertebrate.
  • the CK-2 chemokines of the invention include CK-2 chemokines that are derived from fish, including CK-2 chemokines derived from rainbow trout (Onchorhychus mykiss) and other species of trout, as well as ones from salmon (e.g., Salmo sola and Oncoryhynchus choica) and carp (e.g., Cyprinus carpio).
  • the CK-2 chemokines of the invention may be used to enhance or stimulate an immune response in a fish, including but not limited to any of the particular species offish identified in this specification.
  • the methods of the invention comprise administering a CK-2 polypeptide or nucleic acid of the invention to the organism (e.g., fish) in an amount effective to enhance or stimulate an immune response to the immunogen.
  • the CK-2 polypeptide or nucleic acid may, alternatively, be administered before or after the immunogen is administered.
  • the CK-2 nucleic acid or polypeptide is preferably administered either immediately (e.g., within a few minutes) after or prior to administration of the immunogen.
  • the CK-2 polypeptide or nucleic acid may be administered some time (e.g., within a few days or even a few weeks) before or after administration of the immunogen.
  • an immunogen may first be administered to the organism, and a CK-2 nucleic acid or polypeptide may be administered some time later (e.g., between 1-4 weeks later) as a booster.
  • the immunogen may be any composition which is capable of eliciting or inducing an immune response in the organism, including but not limited to a protein, a lipoprotein, a polysaccharide or a nucleic acid.
  • the immunogen is one that is used as part of a vaccine.
  • the immunogen may comprise a suspension of live (preferably attenuated) or killed infectious agent (for example a microorganism such as a bacterium or virus, a parasite, or other pathogen, etc.) that causes an infectious disease.
  • the immunogen may comprise an immunogenic polypeptide, for example a polypeptide derived from an infectious agent which may be an antigen and which therefore activates an immune response in an organism.
  • the immunogen may be a nucleic acid, such as a recombinant vector (including DNA vectors or plasmids, retroviral vectors and lentivirus vectors) that encodes an antigen and may be administered, e.g., as part of a DNA vaccine (for a review, see Heppell & Davis, Adv. DrugDeliv. Rev. 2000, 43:29-43).
  • a CK-2 polypeptide or nucleic acid of the present invention may be used to enhance or stimulate an immune response by expressing the CK-2 polypeptide or nucleic acid in an infectious agent which may then be administered to an organism, e.g., in a vaccine.
  • the CK-2 polypeptide or nucleic acid may be expressed in a microorganism, for example in a virus or bacterium, according to any of the methods described in Section 5.4, supra.
  • the CK-2 polypeptide or nucleic acid may be recombinantly expressed in a microorganism that co-expresses an antigen.
  • Vaccines that comprise administering an infectious agent or other microorganism co-expressing an antigen and either a cytokine or chemokine have previously been described in the art and are known to effectively enhance an immune response to an infectious agent. See, for example, Gherardi et al, J. Immunol.
  • the antigen may also be an antigen (e.g., a protein or protein fragment) that is recombinantly expressed in the microorganism.
  • the antigen may be an antigen from another infectious agent, which is different type or species of infectious agent than that administered to the organism and which preferably causes or is associated with an infectious disease in the organism.
  • the microorganism that co-expresses the antigen and CK-2 may be used, e.g., in a vaccine, to enhance an immune response against the infectious agent which causes or is associated with the infectious disease.
  • the antigen may be an antigen (e.g.
  • a protein or a protein fragment that is normally expressed in an infectious agent (e.g., microorganism) which is administered to an organism.
  • an infectious agent e.g., microorganism
  • a CK-2 polypeptide or nucleic acid may be expressed in a live (preferably attenuated) infectious agent that causes or is associated with an infectious disease, and the infectious agent may be administered, e.g., in a vaccine against that disease.
  • the recombinantly expressed CK-2 polypeptide may then function as an adjuvant, e.g., to enhnace an immune response against that infectious agent or to enhance an immune response against an agent of that infectious agent.
  • infectious pancreatic necrosis virus examples include infectious pancreatic necrosis virus and infectious hematopoietic necrosis virus (which infect salmon), viral hemorrhagic septicaemia virus (which infects trout) and infectious pancreatic necrosis virus (which infects salmon and trout), to name a few (see, Christie, Dev. Biol. Stand. 1997, 90:191-199).
  • the CK-2 nucleic acids and polypeptides which may be administered in these methods include any of the CK-2 polypeptides and nucleic acids of the present invention (see, e.g., Sections 5.2-5.3, supra).
  • the CK-2 polypeptide administered is a functional (i.e., biologically active) CK-2 polypeptide.
  • the CK-2 polypeptide administered comprises the amino acid sequence of the mature CK-2 polypeptide set forth in FIG. 2 and in SEQ ID NO: 3 (i.e., amino acid residues 29-191 of the polypeptide sequence set forth in FIG. 2 and in SEQ ID NO: 3).
  • the CK-2 polypeptide administered may be a truncated CK-2 polypeptide, e.g. , comprising one or more functional domains.
  • the CK-2 polypeptide administered comprises the amino acid sequence of the globular domain of a CK- 2 polypeptide such as the globular domain of the CK-2 polypeptide set forth in FIG. 2 and in SEQ ID NO: 3 (i.e., amino acid residues 29-90 of the polypeptide set forth in FIG. 2 and in SEQ ID NO.-3).
  • the CK-2 nucleic acid preferably encodes a functional (i.e., biologically active) CK-2 polypeptide.
  • the CK-2 polypeptide administered encodes a polypeptide having the amino acid sequence of the mature CK-2 polypeptide set forth in FIG. 2 and in SEQ ID NO: 3 (i.e., amino acid residues 29-191 of the polypeptide sequence set forth in FIG. 2 and in SEQ ID NO: 3).
  • such a nucleic acid may be one having the amino acid sequence set forth in FIG. 2 (SEQ ID NO:2) or in FIG.
  • the CK-2 nucleic acid administered may be one that encodes a truncated CK-2 polypeptide, e.g., comprising one or more functional domains.
  • the CK-2 nucleic acids administered may be one that encodes a polypeptide having the amino acid sequence of a CK-2 globular domain, such as the globular domain of the CK-2 polypeptide set forth in FIG. 2 and in SEQ ID NO: 3 (i.e., amino acid residues 29-90 of the polypeptide set forth in FIG. 2 and in SEQ ID NO:3).
  • the CK- 2 chemokines of the invention are believed to stimulate or enhance an immune response by attracting leukocytes (including, for example, T lymphocytes, such as CD8 and CD4 cells, activated B cells and monocytes) to the site of an infection or trauma in an organism.
  • leukocytes including, for example, T lymphocytes, such as CD8 and CD4 cells, activated B cells and monocytes
  • the CK-2 polypeptides administered to an organisms are preferably ones that are biologically active in the organism and, in particular, are chemotactic for leukocytes of that particular organism.
  • the nucleic acid preferably encodes a CK-2 polypeptide that is biologically active in the organism and, in particular, encodes a CK-2 polypeptide that is chemotactic for leukocytes of that particular organism.
  • the CK-2 nucleic acid or polypeptide is one derived from the same species of organism as that species to which it is administered.
  • the CK-2 polypeptides or nucleic acids administered comprise a polypeptide or nucleic acid sequence set forth in FIG.
  • the CK-2 nucleic acid or polypeptide is preferably administered to a rainbow trout.
  • the CK-2 chemokines of the invention may be cross functional across different species of organisms, particularly across species that share a high level of CK-2 sequence homology or identity.
  • mouse and human fractalkine are approximately 65% identical at the amino acid level and are biologically active across both species (i.e., human fractalkine is biologically active in mice).
  • a CK-2 nucleic acid or polypeptide of the invention which is derived from a first species (e.g.
  • the CK-2 polypeptide set forth in FIG. 2 (SEQ ID NO:3), a portion thereof or a nucleic acid encoding such a polypeptide may be administered, e.g., to another species of trout, including any fish of the family Salmonidae (including fish of the Salmo, Salvelinus and allied genera such as brook trout, speckeled trout, red-spotted trout, river or brown trout, spotted trout, lake trout, mountain trout and river trout, to name a few).
  • Salmonidae including fish of the Salmo, Salvelinus and allied genera such as brook trout, speckeled trout, red-spotted trout, river or brown trout, spotted trout, lake trout, mountain trout and river trout, to name a few.
  • SEQ ID NOS: 1-3 may also be administered to salmon (e.g., Salmo sola and Oncoryhynchus choica and varieties thereof) and other fish such as perch, carp catfish, sea bream (e.g., fish of the Pagellus and allied genera), and Tilapia to name a few.
  • salmon e.g., Salmo sola and Oncoryhynchus choica and varieties thereof
  • other fish such as perch, carp catfish, sea bream (e.g., fish of the Pagellus and allied genera), and Tilapia to name a few.
  • the CK-2 polypeptide should be biologically active in the two species of organism (e.g., chemotactic to leukocytes of both species).
  • CK-2 polypeptides and nucleic acids derived from the two species of organisms preferably share a high level of sequence identity and, in particular, are preferably at least 65% identical, are more preferably at least 75% identical, and still more preferably are at least 85%, 90%, 95% or 99% identical.
  • the CK-2 chemokine (i.e., the CK-2 polypeptide or nucleic acid) and the immunogen may be administered as separate compositions or molecules or, alternatively, as a single molecules (e.g., a fusion or chimeric molecule).
  • the methods of the invention may comprise administering a first polypeptide, which is an antigen, and a second polypeptide, which is a CK-2 polypeptide, to the organism.
  • fusion polypeptides of a chemokine and an antigen are also known in the art (see, e.g., Biragyn et al, Nature Biotechnology 1999, 17:253-258) and, moreover, administration of such fusion proteins induces a stronger immune response to an antigen than administration of either the antigen alone, or the antigen and chemokine as separate polypeptides.
  • the methods of the invention also provide a preferred embodiment wherein a fusion protein is also administered to the organism.
  • Such fusion proteins which are described supra in Section 5.2, may comprise a polypeptide sequence for an antigen fused to a CK-2 polypeptide sequence of the invention.
  • the invention also provides methods for administering CK-2 as part of a DNA or vector vaccine.
  • the methods of the invention may comprise administering a first nucleic acid molecule, which encodes an antigen, and a second nucleic acid molecule which encodes a CK-2 polypeptide of the invention.
  • the methods of the invention also include techniques that comprise administering a nucleic acid that encodes both an antigen and a CK-2 polypeptide.
  • such methods may include ones where a single nucleic acid vector is administered which comprises coding sequences for two separate polypeptides: a first polypeptide, which is an antigen; and a second polypeptide, which is a CK-2 polypeptide of the invention.
  • the nucleic acid vector comprises a coding sequence for a single, fusion polypeptide of the invention.
  • the nucleic acid may encode a single fusion polypeptide having the sequence for an antigen fused to a CK-2 polypeptide of the invention.
  • the nucleic acid preferably comprises an expression vector (for example, any of the expression vectors described in Section 5.4, supra) that contains nucleic acid sequences encoding either an antigen, a CK-2 polypeptide or both.
  • an expression vector for example, any of the expression vectors described in Section 5.4, supra
  • the methods described here may also include ones where both nucleic acids and polypeptides are administered to enhance or stimulate an immune response.
  • the methods may comprise administering a polypeptide, which is an antigen, and a nucleic acid which encodes a CK-2 polypeptide to an organism.
  • the methods of the invention may also comprise administering to an organism a nucleic acid which encodes and antigen, and a CK-2 polypeptide.
  • the amount of the CK-2 nucleic acid or polypeptide administered in these methods is an "effective amount"; i.e., an amount which is sufficient to enhance or stimulate an immune response to the immunogen.
  • an effective amount i.e., an amount which is sufficient to enhance or stimulate an immune response to the immunogen.
  • a variety of methods are known in the art to determine whether an immune response has been enhanced. For example, the experiments described in the Examples presented in Section 6.4, infra include ones wherein levels of antibodies to the immunogen are determined, e.g., by an ELISA assay. Alternatively, lymphocytes may also be tested to T cell mediated responses, e.g., by measuring their proliferative responses to the immunogen in vitro.
  • Such measurements are preferably made in organisms vaccinated according to the above-described methods and compared to similar measurements in an organism vaccinated with the immunogen alone.
  • An increase in either the level of antibodies to the immunogen, T cell proliferative responses to the immunogen, or both may be indicative of an enhanced or stimulated immune response to the immunogen.
  • the amount of a CK-2 nucleic acid or polypeptide which is an effective amount for enhancing or stimulating an immune response in a particular organism will depend on the total weight of that organism.
  • exemplary effective amounts of a peptide or polypeptide antigen will be, for each 100 grams offish, between about 20-200 ⁇ g.
  • typical, exemplary effective amounts of a CK-2 polypeptide administered as an adjuvant in such a vaccines may be between about 1-20 ⁇ g for each 100 grams offish.
  • exemplary effective amounts of a nucleic acid e.g.
  • a plasmid) encoding an antigen will be from about 1-200 ⁇ g per 100 grams offish, and exemplary effective amounts of a nucleic acid (e.g., a plasmid) encoding a CK-2 polypeptide will be from about 1-20 ⁇ g per 100 grams of fish.
  • Both the immunogen and the CK-2 nucleic acids or polypeptides may be administered to an organism, e.g. , by inhalation, immersion or insufflation (either through the mouth or through the nose), or by oral, bucal, rectal or parenteral administration (e.g., by subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal or intravenous injection and the like).
  • a vaccine may also be administered by particle-mediated transfer (e.g., using a "particle gun"). Particle transfer methods of administration are particularly preferred for DNA vaccines. See, for example, Gainer et al, J. Neurooncol 2000, 47:23-30; Koide et al, Jpn J. Pharmacol 2000, 83:167-174; Kuriyama et al, Gene Ther. 2000, 7:1 132-1136; and Yamauchi et al, J. Exp. Zool. 2000, 287:285-293.
  • the present invention also contemplates the transmucosal (e.g., oral) administration of immunogen and CK-2, e.g., as part of a transmucosal vaccine.
  • transmucosal e.g., oral
  • CK-2 e.g., as part of a transmucosal vaccine.
  • Various strategies have been employed to deliver immunogenic proteins to the mucosa. These strategies includes ones for the delivery of DNA vaccines as well, e.g., by using the specific mucosal targeting proteins as vector targeting proteins, or by delivering the vaccine vector in an admixture with the mucosal targeting protein.
  • the methods may be readily adapted, without undue experimentation, to administer CK-2 nucleic acids and/or polypeptides as well as antigens.
  • the polypeptide or vector vaccine (comprising both the immunogen and CK-2) may be administered in an admixture with, or as a conjugate or chimeric fusion protein with, cholera toxin, such as cholera toxin B or cholera toxin A/B chimera (Hajishengallis et al, J. Immunol 1995, 154:4322-4332; Jobling and Homes, Infect. Immun. 1992, 60:4915-4924).
  • Mucosal vaccines based on use of the cholera toxin B subunit have been previously described (see, Lebens & Holmgren, Dev. Biol. Stand. 1994, 82:215-227).
  • an admixture with heat labile enterotoxin (LT) may be prepared for mucosal vaccination.
  • mucosal immunization strategies include encapsulating the immunogen and CK-2 in icrocapsules (U.S. Patent Nos. 5,075, 109; 5,820,883; and 5,853,763) and using an immunopotentiating membranous carrier (International Patent Publication WO 98/0558). Immunogenicity of orally administered immunogens may be further enhanced by using red blood cells (RBC) or RBC ghost (U.S. Patent No. 5,643,577) or by using blue tongue antigen (U.S. Patent No. 5,690,938). System administration of a target immunogen (with CK-2) may also produce mucosal immunization (see, U.S. Patent No. 5,518,725).
  • Various strategies may also be used to deliver genes for expression in mucosal tissues (e.g., as part of transmucosally administered DNA vaccines), including use of chimeric rhinoviruses (U.S. Patent No. 5,714,374), adenoviruses, or specific targeting of nucleic acid (International Patent Publication No. WO 97/05267).
  • the invention also contemplates use of CK-2 nucleic acids and/or polypeptides to stimulate or enhance an immune response in a passive immunization strategy.
  • Passive immunization methods are ones which comprise administering, instead of or in addition to an immunogen, an antibody that is reactive with, and is preferably generated against, the immunogen.
  • Passive immunization strategies are particularly effective for an incipient or established infection before the host's immune system can respond.
  • the methods of the invention also include ones for enhancing or stimulating an immune response to an immunogen by administering an antibody reactive with (preferably generated against) the immunogen, and a CK-2 polypeptide or nucleic acid of the invention.
  • Antibodies for use in a passive immunization therapy may be obtained, e.g., from the serum of affected animals which are preferably of the same species as the infected host.
  • antibodies from fish blood serum e.g., from rainbow trout
  • antibodies from antibodies from fish blood serum can be isolated, preferably by affinity purification against an epitope of the immunogen, and used to passively immunize newly infected fish of the same species (e.g., newly infected rainbow trout).
  • antibodies may be generated against the immunogen or an antigen derived therefrom.
  • the above-described vaccination strategy may also be used to generate antibodies for passive immunization.
  • the invention also provides pharmaceutical compositions that may be used, e.g., as vaccines, in the above-described methods to enhance or stimulate an immune response to an immunogen.
  • These pharmaceutical compositions comprise a CK-2 polypeptide or a CK-nucleic acid of the present invention in an amount effective to stimulate an immune response and in combination with one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical compositions further comprise an immunogen.
  • the pharmaceutical compositions may also comprise an immunogenic polypeptide (i.e., an antigen), or they may comprise a nucleic acid (e.g., a nucleic acid vector or plasmid) that encodes an immunogenic polypeptide.
  • Pharmaceutical compositions of the invention are further discussed below in this subsection (see “Pharmaceutical Preparations", infra).
  • the present invention provides methods and compositions for modulating (e.g., enhancing or suppressing) an immune response by modulating (e.g., increasing or decreasing) the expression or activity of a CK-2 gene or its gene product.
  • Such methods may simply comprise administering one or more compounds that modulate expression of a CK-2 gene, synthesis of a CK-2 gene product or CK-2 gene product activity so the immune response is modulated (e.g., enhanced or suppressed).
  • these one or more compounds are administered until the immune response is modulated as desired.
  • CK-2 nucleic acid Among the compounds that may exhibit an ability to modulate the activity, expression or synthesis of a CK-2 nucleic acid are antisense, ribozyme and triple-helix molecules. Such molecules may be designed to reduce or inhibit wild-type CK-2 nucleic acids and polypeptides or, alternatively, may target mutant CK-2 nucleic acids or polypeptides.
  • Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to target mRNA molecules and preventing protein translation.
  • Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence that is "complementary" to a portion of a nucleic acid refers to a sequence having sufficient complementarity to be able to hybridize with the nucleic acid and form a stable duplex.
  • the ability of nucleic acids to hybridize will depend both on the degree of sequence complementarity and the length of the antisense nucleic acid. Generally, however, the longer the hybridizing nucleic acid, the more base mismatches it may contain and still form a stable duplex (or triplex in triple helix methods).
  • a tolerable degree of mismatch can be readily ascertained, e.g., by using standard procedures to determine the melting temperature of a hybridized complex.
  • oligonucleotides complementary to non-coding regions of a CK-2 gene may be used in an antisense approach to inhibit translation of endogenous CK-2 mRNA molecules.
  • Antisense nucleic acids are preferably at least six nucleotides in length, and more preferably range from between about six to about 50 nucleotides in length.
  • the oligonucleotides may be at least 10, at least 15, at least 20, at least 25 or at least 50 nucleotides in length.
  • in vitro studies are first performed to quantitate the ability of an antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • antisense nucleotides complementary to the target gene coding region sequence could be used, those complementary to the transcribed, untranslated region are most preferred.
  • Antisense molecules are preferably delivered to cells, such as hemopoietic cells, that express the target gene in vivo.
  • cells such as hemopoietic cells
  • a number of methods have been developed for delivering antisense DNA or RNA to cells.
  • antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • Preferred embodiments achieve intracellular concentrations of antisense nucleic acid molecules which are sufficient to suppress translation of endogenous mRNAs.
  • one preferred approach uses a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.
  • the use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA.
  • a vector as set forth above, can be introduced e.g., such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in the particular cell type (for example in a hemopoietic cell).
  • any of the promoters discussed supra in connection with the expression of recombinant CK-2 nucleic acids can also be used to express a CK-2 antisense nucleic acid.
  • Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and, therefore, expression of target gene product (see, e.g., International Publication No. WO 90/11364; Sarver, et al, Science 1990, 247:1222-1225). Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (for a review, see Rossi, Current Biology 1994, 4:469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event.
  • composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Pat. No. 5,093,246.
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs
  • the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially Figure. 4, page 833) and in Haseloff and Gerlach, Nature 1988, 334:585-591.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al., Science 1984, 224:574-578; Zaug and Cech, Science 1986, 231 :470-475; Zaug et al, Nature 1986, 324:429-433; International Patent Publication No. WO 88/04300; Been and Cech, Cell 1986, 47:207-216).
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those
  • Cech-type ribozymes which target eight base-pair active site sequences that are present in the target gene.
  • the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficacy.
  • Such constructs can be introduced to cells using any of the vectors described supra.
  • Endogenous target gene expression can also be reduced by inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination (e.g., see Smithies, et al., Nature 1985, 317:230-234; Thomas and Capecchi, Cell 1987, 51:503-512; and Thompson et al, Cell 1989, 5:313-321).
  • a mutant, non-functional target gene or a completely unrelated DNA sequence flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo.
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas and Capecchi, 1987 and Thompson, 1989, supra).
  • ES embryonic stem
  • this approach can be adapted for use in other species of organism (for example, in fish, such as trout and salmon, and in mammals, including humans) provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
  • endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the target gene (t. e.
  • the target gene promoter and/or enhancers to form triple helical structures that prevent transcription of the target gene in target cells in the body, (see generally, Helene, Anticancer Drug Des. 1991, 6:569-584; Helene, et al, Ann. N.Y. Acad. Sci. 1992, 660:27-36; and Maher, Bioassays 1992, 14:807-815).
  • Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides.
  • the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the antisense, ribozyme, and/or triple helix molecules described herein are utilized to inhibit mutant gene expression, it is possible that the technique may so efficiently reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles that the possibility may arise wherein the concentration of normal target gene product present may be lower than is necessary for a normal phenotype.
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity may, be introduced into cells via gene therapy methods such as those described, below, that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized.
  • target gene encodes an extracellular protein
  • CK-2 nucleic acids and/or polypeptides of the invention may also be administered, preferably in combination with an immunogen, in an amount effective to enhance or stimulate an immune response in the organism.
  • the invention also provides pharmaceutical preparations for use, e.g., as vaccines or as therapeutic compounds, to modulate, stimulate or enhance an immune response in an organism.
  • therapeutically effective dose and “effective amount” refer to the amount of the compound that is sufficient to result in such a modulated, enhanced or stimulated immune responses.
  • Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures, for example in cell culture assays or using experimental animals to determine the LD 50 and the ED 50 .
  • the parameters LD 50 and ED 50 are well known in the art, and refer to the doses of a compound that are lethal to 50% of a population and therapeutically effective in 50% of a population, respectively.
  • the dose ratio between toxic and therapeutic effects is referred to as the therapeutic index and may be expressed as the ratio: LD 50 /ED 50 .
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used. However, in such instances it is particularly preferable to use delivery systems that specifically target such compounds to the site of affected tissue so as to minimize potential damage to other cells, tissues or organs and to reduce side effects.
  • the dosage of compounds used in therapeutic methods of the present invention preferably lie within a range of circulating concentrations that includes the ED 50 concentration but with little or no toxicity (e.g., below the LD 50 concentration).
  • the particular dosage used in any application may vary within this range, depending upon factors such as the particular dosage form employed, the route of administration utilized, the conditions of the individual (e.g., patient), and so forth.
  • a therapeutically effective dose may be initially estimated from cell culture assays and formulated in animal models to achieve a circulating concentration range that includes the IC 50 .
  • the IC 50 concentration of a compound is the concentration that achieves a half-maximal inhibition of symptoms (e.g., as determined from the cell culture assays). Appropriate dosages for use in a particular individual, for example in human patients, may then be more accurately determined using such information.
  • Measures of compounds in plasma may be routinely measured in an individual such as a patient by techniques such as high performance liquid chromatography (HPLC) or gas chromatography.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. , magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g. , magnesium stearate, talc or silica
  • disintegrants e.g., potato
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. , containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the culture conditions comprised 10 7 white blood cells per milliliter Isclove's medium, bovine serum albumin (2 mg/ml), penicillin and streptomycin.
  • the aliquots of cells were maintained in these culture conditions at 17 °C, 5% CO 2 for 5.5 hours, after which time the cells were rinsed with medium and harvested for RNA in RNAlater (Ambion Inc., Austin, Texas) by manual scraping.
  • the other half of cells extracted from this fish served as "resting" cells for subtractive hybridization, as described below.
  • a second rainbow trout fish was also exsanguinated, and its head kidney cells were activated, in vitro by incubation with PHA-P as described above. However, these cells were incubated for 19 hours under the conditions described above.
  • Two mRNA preparations were then made from the isolated cells using a QuickPrep mRNA Purification kit (Amersham Pharmacia Biotech UK, Ltd., Buckinghamshire, United Kingdom) and following the manufacturer's recommended protocol.
  • the first mRNA preparation was made from a pool of the PHA-P activated cells (5.5 hour and 9 hour), whereas the second mRNA preparation was made from the nonactivated (i.e., the "resting") cells isolated from the first fish.
  • mRNA from each of these preparations was then used to synthesize cDNA with a cDNA synthesis kit (Amersham- Pharmacia), following the manufacturer's recommended protocol.
  • nucleic acids in each preparation were PCR amplified with a forward primer (5'-ACCACTGGTATCGTGATGGAC-3'; SEQ ID NO:4) and reverse primer (5'-AGTGTTGGCATACAGGTCCTT-3'; SEQ ID NO:5) specific for ⁇ -actin nucleic acid sequences, under PCR conditions which comprised incubation at 95 °C for five minutes and 25 amplification cycles, each consisting of 95 °C for 30 seconds, 55 °C for 30 seconds and 72 °C for one minute. A sharp band of 390 bp in size was observed upon gel electrophoresis of the PCR amplified aliquots from both cDNA preparations, thus confirming successful cDNA synthesis from the mRNA preparations.
  • a forward primer 5'-ACCACTGGTATCGTGATGGAC-3'; SEQ ID NO:4
  • reverse primer 5'-AGTGTTGGCATACAGGTCCTT-3'; SEQ ID NO:5'-AGTGTTGGCATACAGGTCCTT
  • Suppression subtractive hybridization was employed as reported elsewhere (see, in particular, Fujiki etal, Immunogenetics 1999, 49:909-914) using a PCR-select cDNA Subtraction Kit (Clontech, Palo Alto, California). Briefly, both cDNA preparations were digested with Rsal. The Rsal digested, activated cDNA preparation was then divided into two separate aliquots, and ligated to two different adaptors. Efficiency of ligation was verified by PCR amplification using carp 3 OS ribosomal protein primers compared to amplification using one primer that annealed with the adaptors and one of the ribosomal protein primers. Equally strong bands of DNA were recovered from both PCR products, indicating a high ligation efficiency.
  • the ligated cDNA molecules from the aliquots of the activated cDNA preparation were denatured and hybridized with excess amounts of the resting cDNA preparation to enrich for differentially expressed sequences.
  • the hybridized mixtures were then rehybridized, without denaturation, in the presence of additional denatured resting cDNA.
  • double stranded, differentially expressed cDNAs were obtained having one adaptor (i.e., the adaptor ligated to cDNA molecules in the first aliquot of the "activated" preparation) at one end and a second adaptor (i. e. , the adaptor ligated to cDNA molecules in the second aliquot of the "activated” preparation) at the other end.
  • sequences were then preferentially amplified by PCR with primers specific for both adaptors, resulting in amplification of only the differentially expressed sequences.
  • a second nested PCR was performed using primers specific for sites within the two adaptors. The final PCR product was then electrophoresed on an agarose gel, and a
  • Transformed E. coli cells were plated on agar. Approximately 160 of the plated colonies were grown in LB broth and prepared, using a plasmid miniprep 8 Turbo kit (Qiagen, Valencia, California) to yield plasmid preparations. These plasmid preparations were digested with ⁇ coRl to determine the size of their cDNA inserts. All plasmids with inserts over 200 bp in length were sequenced on an ABI Model 377 automated sequencer using the T7 primer from the vector. The sequences were then entered into BLASTX and BLASTN searches (see, Altschul, J Mol. Biol. 1991, 219:555-565; Gish & States, Nat. Genet.
  • S9 S ⁇ Q ID NO:6
  • S96 S ⁇ Q ID NO:7
  • ATTTA motif is typically found in untranslated regions (e.g., the 3 '-untranslated region) of nucleic acids, such as mRNA or cDNA derived therefrom, that have short half-lives in cells.
  • the AUUUA motif in mRNA is believed to be recognized by at least one endonuclease enzyme that degrades mRNA molecules having such a motif.
  • Nucleic acid molecules that have short- half lives in cells may therefore be expected to contain at least one ATTTA motif, including, for example, ones that encode chemokines, cytokines and oncogenes.
  • mRNA was then prepared from the extracted RNA using a Fast Track Kit 2.0 (Invitrogen, Carsbad, California) according to the manufacturer's recommended protocol. This mRNA was converted to cDNA with a cDNA Copy Kit (Invitrogen) and using oligo-dT(Not 1) as a primer to generate unidirection inserts with Notl restriction sites at the polyT end of the cDNA. The resulting double stranded blunt ended cDNA was ligated to BSTX1 adaptors (Invitrogen) using routine conditions known in the art (see, e.g., Sambrook et al, eds., Molecular Cloning: A Laboratory Manual, 2nd Ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, New York). The cDNA was then restricted with Notl, leaving a BSTX1 site at the 5'-end of the cDNA molecules, and a Notl site at the 3 '-end.
  • cDNA molecules were extracted with phenol-chloroform, ethanol precipitated and run on a 1% agarose gel. cDNA molecules greater than about 500 bp in apparent length were extracted from the gel and ligated to the plasmid pcDNA3.1+ (Invitrogen).
  • One fourth of this reaction mixture was used to transform ultracompetent DH5alpha E. coli cells (Gibco BRL), which were plated on LB agar plates with ampicillin (100 ⁇ g/ml) to yield 120,000 colonies. 8 out of 10 of these colonies were determined to have cDNA inserts greater than 450 bp in length.
  • cDNA Library Screening Those cDNA fragments described in Section 6.1, supra, that contain at least one ATTTA motif or its complement (i. e. , a TAAAT motif) were used as probes to screen the full length cDNA library.
  • screening for cDNA sequences containing a motif, such as the ATTTA motif (or its complement) that controls or regulates the half-life of an mRNA molecule in a cell is expected to be generally useful to screen for nucleic acids corresponding to such mRNA sequences.
  • the method is generally useful to screen for nucleic acids that encode proteins, including cytokines and oncogenes, as well as chemokines, whose mRNA molecules are known or believed to have controlled (e.g., short) half-lives in cells.
  • chemokines whose mRNA molecules are known or believed to have controlled (e.g., short) half-lives in cells.
  • sequences of chemokines from other species such as chemokines from another species of fish or from another vertebrate such as a mammal
  • these methods are particularly useful in instances where no homologous sequences are known or where the known homologous sequences are expected to have low levels of homology and so may not hybridize to a corresponding cDNA clone from another, distantly related species.
  • the PHA-activated rainbow trout cDNA library prepared as described above, was plated at 50,000 colonies per 15 cm plate. 10 plates were incubated overnight at 37 °C and transferred to MagnaGraph Nylon filters (Micron Separations Inc., Massachusetts). The methods described in The Dig System User's Guide for Filter Hybridization (Mannheim Boehringer Biochemicals, Indianapolis, Indiana) were used to screen the cDNA library, as follows. Briefly, the filters were subjected to alkaline treatment to lyse the colonies, to UV crosslinking to immobilize denatured DNA, and to proteinase K treatment to digest interfering proteins.
  • Nylon filters were then prehybridized at 42 °C for one hour in DIG easy Hyb (Mannheim Boehringer Biochemicals, Indianapolis, Indiana).
  • each cDNA insert (which ranged in size from 126 to 600 nucleic acids in length) was cut from its plasmid using EcoRl, separated on an agarose gel and purified using a GeneClean kit (Qbiogene, Carlsbad, California). The cDNA fragments were then pooled, extracted once with a phenol:chloroform:isoamylalcohol (25:24:1) mixture from Gibco BRL (Rockville, Maryland) and precipitated with 4 M lithium and ethanol.
  • the probes were labeled with a Dig-label using a DIG High Prime Labeling kit (Mannheim Boehringer Biochemicals, Indianapolis, Indiana) according to the manufacturer's instructions and hybridized to the prehybridized nylon filters at 42 °C in a Hybridization Incubator Model 2710 (VWR Scientific Products, Plainfield, New Jersey). Filters were washed three times for ten minutes in 2x SSC, 0.1% SDS at room temperature with gentle agitation, followed by two additional washes for 15 minutes each in 0.5x SSC, 0.1% SDS at 68 °C, and finally washed once for 15 minutes in 0.2x SSC, 0.1% SDS at 68 °C.
  • colorimetric detection of positive clones was carried out using a DIG high prime labeling and detection starter kit I (Mannheim Boehringer Biochemicals, Indianapolis, Indiana), according to the manufacturer's recommended protocol but with the following minor modification. Briefly, the filters were equilibrated in maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) for five minutes and blocked in lx Block Solution (Mannheim Boehringer Biochemicals, Indianapolis, Indiana) with gentle agitation for 60 minutes at room temperature.
  • DIG high prime labeling and detection starter kit I Mannheim Boehringer Biochemicals, Indianapolis, Indiana
  • the filters were then incubated in blocking buffer with anti-digoxigenin-AP 1 :5000 for sixty minutes, followed by two washes, of 15 minutes each, in washing buffer (maleic acid buffer plus 30% TWEEN). After equilibration in detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, 50 mM MgCl 2 , pH 9.5), the filters were incubated in freshly prepared color solution for 16 hours in the dark.
  • the CK-2 cDNA sequence set forth in FIG. 1 (SEQ ID NO:l) encodes a polypeptide having significant (44%) sequence identity to the carp CC-1 chemokine.
  • the cDNA insert contained in one of the clones comprises the nucleotide sequence set forth in SEQ ID NO: 8 and in Table 2, below.
  • This cDNA sequence aligns with nucleotides 520-849 of the CK-2 cDNA sequence set forth in FIG. 1 (SEQ ID NO:l) which comprise most of the 3 '-untranslated region and includes at least three ATTTA motifs (underlined in Table 2).
  • the S5 cDNA insert (SEQ ID NO:8) also comprises nucleotides encoding the last 39 amino acid residues of the CK-2 polypeptide depicted in FIG. 2 and in SEQ ID NO:3.
  • nucleic acid probes represent an improvement over screening methods that are normally used in the art.
  • nucleic acid probes may be traditionally selected according to sequences that encode (or are predicted to encode) conserved or homologous polypeptide sequences (e.g., the sequence of a known fish chemokine, such as the carp CC-1 chemokine discussed supra).
  • nucleic acid probes are selected, in the instant invention, according to the presence (or absence) of particular consensus sequences that control a shared function of the corresponding mRNA molecule (for example, its half-life in a cell).
  • Such screening methods are preferable and may produce superior results when used to screen for genes encoding proteins (for example, chemokines, cytokines or oncogenes) in species (for example, in species offish or birds) for which no homologous proteins are known or, alternatively, where the only homologous proteins and polypeptides are from distantly related species (for example, mammalian species) and have low or insignificant levels of sequence homology.
  • proteins for example, chemokines, cytokines or oncogenes
  • species for example, in species offish or birds
  • distantly related species for example, mammalian species
  • the nucleotide sequence of the cDNA insert isolated as described in Section 6.2, supra, is set forth here in FIG. 1 and in SEQ ID NO:l.
  • This cDNA was isolated by specifically hybridizing clones from a rainbow trout cDNA library to rainbow trout probes comprising a sequence motif (i. e. , the "ATTTA" motif and/or its complement) characteristic of mRNAs, such as chemokine and cytokine encoding mRNA molecules, that have short half-lives in cells.
  • the chemokine identity of the polypeptide encoded by this cDNA i.e., the polypeptide sequence set forth in FIG. 2 and in SEQ ID NO:3 was confirmed by its sequence identity (44%) to a known fish chemokine referred to as carp chemokine CC-1 (Fujiki et al, Immunogenetics 1999, 49:909-914).
  • the CK-2 cDNA sequence shown in FIG. 1 (SEQ ID NO: 1) comprises a sequence of 971 nucleotides followed by a polyA tail.
  • the open reading frame (ORF) of this sequence begins at the "START” (i.e., ATG) codon located at nucleotide 64, and ends at the "STOP” or TAG codon terminating at nucleotide 639. These START and STOP codons are indicated in FIG. 1 by bold-faced type.
  • the untranslated regions of the CK-2 cDNA comprise several "ATTTA" repeats. These repeats, which are underlined in the sequence set forth in FIG. 1, are indicative of mRNA molecules that have short half-lives in cells, and are typically found, e.g., in mRNA sequences encoding cytokines, chemokines and oncogenes.
  • the CK-2 open reading frame sequence (SEQ ID NO:2) is set forth separately in FIG. 2 along with the predicted amino acid sequence (SEQ ID NO:3) of the CK-2 polypeptide it encodes.
  • BLAST searches were executed (see, Altschul, J Mol. Biol. 1991, 219:555-565; Altschul et al. , Nat. Genet. 1994, 6:119-129; Altschul & Gish, Methods Enzymol. 1996, 266:460-480; Altschul et al, Nucl Acids Res. 1997, 25:3389-3402; and Henikoff, Curr. Op. in Struc. Biol.
  • the CK-2 polypeptide contains a domain, comprising approximately amino acid residues 1-100 of the CK-2 polypeptide sequence set forth in FIG. 2 and in SEQ ID NO:3, which corresponds to a typical globular domain of a CC class of chemokine.
  • This domain of CK-2 is 44% identical to the carp CC chemokine previously described (Fujiki et al, Immunogenetics 1999, 49:909-914).
  • the first 119 amino acid residues of trout CK-2 are about 20% identical to the trout chemokine known in the art as CK-1 (Dixon et al, Imm. Reviews. 1998, 166:345-352), and are about 19% identical to the human CC chemokine known in the art as MCP1 (Yoshimura et al, FEBS Lett. 1989, 244:487-493).
  • the CK-2 polypeptide further contains a second domain comprising approximately amino acid residues 101-191 of the CK-2 polypeptide sequence set forth in FIG. 2 and in SEQ ID NO:3.
  • This second domain comprises an unusually high number of serine, proline and leucine amino acid residues.
  • the domain does not, therefore, correspond to a globular domain of the type normally observed in chemokine polypeptides. Rather, this second domain corresponds to a "mucin-like stalk” domain.
  • the BLAST searches revealed that this mucin-like stalk domain has significant sequence homology with a number of stalklike protein produced by plants, and with the proteophosphoglycan of Leishmania major (Hg et al, J. Biol. Chem. 1999, 274:31410-31420).
  • a mucin-like domain has previously been reported in only one other chemokine; the human chemokine fractalkine (see, Bazan et al, Nature 1997, 385:640-644).
  • the present invention represents, therefore, the discovery of only the second chemokine having a mucin-like stalk domain, and the first discovery of a non-human chemokine having such a domain.
  • a BLAST sequence alignment of the rainbow trout CK-2 and human fractalkine amino acid sequence reveals that the two sequences are 25% identical.
  • the mucin-like stalk domain in CK-2 is only about half the length of the mucin-like stalk domain found in human fractalkine. Analysis with the TMpred program (Persson & Argos, J Mol.
  • the CK-2 polypeptide was also analyzed using the SignallP program (Nielsen et al, Protein Engineering 1997, 10:1-6; Nielsen et al, Protein Engineering 1999, 12:3-9).
  • the CK-2 polypeptide shown in FIG. 2 does contain a signal sequence domain comprising approximately amino acid residues 1-28 of SEQ ID NO:3.
  • This signal sequence domain is cleaved in the mature (i. e. , processed) CK-2 chemokine when it is secreted by cells.
  • the mature CK-2 chemokine of the present invention may comprise the sequence of amino acid residues 29-191 of the polypeptide sequence set forth in FIG. 2 (SEQ ID NO:3).
  • the mature trout CK-2 polypeptide has several features common to CC chemokines in general.
  • CK-2 has two adjacent cysteine residues at positions homologous to conserved cysteine positions in other CC chemokines (specifically, at positions 34 and 35 in SEQ ID NO:3).
  • CK-2 contains cysteines (at positions 59 and 73 of SEQ ID NO:3) which are locations homologous to conserved cysteine positions in other CC chemokines.
  • the polypeptide sequence of CK-2 further contains a glutamic acid residue at a position five amino acid residues upstream of the first cysteine in the mature CK-2 polypeptide (i.e., at amino acid residue 29 in SEQ ID NO:3).
  • chemokines including other CC chemokines, also comprise either a glutamic acid or an aspartic acid residue at a position between 1-8 amino acid residues upstream of the first cysteine in the processed polypeptide, and mutations of the residue typically reduce chemotactic activity of the chemokine.
  • variant CK-2 Nucleic Acids and Polypeptides.
  • the partial cDNA sequence S96 (SEQ ID NO:7), described in Section 6.1 supra, aligns with nucleic acids 33-242 of the CK-2 cDNA sequence shown in FIG. 1 (SEQ ID NO:l) and described above.
  • the nucleotide sequence of S96 does not contain nucleotides corresponding to nucleic acids 140- 142 of this this CK-2 cDNA.
  • the S96 sequence therefore corresponds to a fragment of a first exemplary CK-2 allelic variant.
  • this allelic variant has a cDNA sequence identical to the nucleotide sequence set forth in FIG.
  • This CK-2 allelic variant encodes a variant CK-2 polypeptide having a single amino acid residue deletion.
  • the CK-2 allelic variant encodes a polypeptide having an amino acid sequence as set forth in FIG. 2 (SEQ ID NO:2) but with the single deletion of amino acid residue 26 (a valine amino acid residue) in SEQ ID NO:2.
  • nucleotide sequence of the probe S96 (SEQ ID NO:7), when aligned with the CK-2 cDNA sequence set forth in FIG. 1 (SEQ ID NO:l) contains at least two nucleotide mismatches.
  • nucleic acid residue 137 of the S96 nucleotide sequence set forth in SEQ ID NO:7 is a guanine (G).
  • this residue aligns with nucleotide 172 of SEQ ID NO:l, which is an adenine (A).
  • nucleotide 143 of the S96 nucleotide sequence is a cytosine
  • the aligning residue (nucleotide 178) in SEQ ID NO:l is a thymine.
  • Each of these allelic variants alters a codon in the coding region of the CK-2 cDNA and encodes a variant CK-2 polypeptide having at least one amino acid residue substitution.
  • the substitution of guanine for adenine at nucleic acid 172 in SEQ ID NO: 1 changes the codon for amino acid residue 37 in SEQ ID NO:2 (FIG.
  • SEQ ID NO:l The partial cDNA sequence S5 (SEQ ID NO:8), described in Section 6.2, supra, also contains nucleotide variants of the full length cDNA sequence set forth in FIG. 1 (SEQ ID NO:l).
  • S5 aligns with nucleotides 520-849 of the CK-2 cDNA sequence set forth in FIG. 1 (SEQ ID NO:l).
  • the alignment contains at least three nucleotide mismatches.
  • nucleic acid residue 9 of the S5 sequence is a cytosine (C).
  • the residue aligns with nucleotide 528 in SEQ ID NO:l, which is a thymine. This single nucleotide polymorphism changes the codon "CCT" (nucleic acid residues 526-528 in SEQ ID NO:l) to "CCC".
  • nucleic acid residue 148 of S5 is a cytosine
  • the aligning residue in SEQ ID NO:l is a thymine.
  • this single nucleotide polymorphism occurs in a non- coding region of the CK-2 cDNA sequence, and therefore is not expected to alter the CK-2 polypeptide sequence.
  • the S5 sequence does contain, however, at least one polymorphism that does encode a variant CK-2 polypeptide. Specifically, although nucleic acid residue 70 of S5
  • SEQ ID NO:8 is a cytosine
  • this nucleotide aligns with nucleic acid residue 589 in SEQ ID NO:l, which is a thymine.
  • This polymorphism changes the codon at this position (i.e., at nucleic acid residues 589-591 of SEQ ID NO:l) from "TGG”, which encodes a tryptophan amino acid residues (Trp or W), to "CGG”, which encodes an arginine amino acid residue (Arg or R).
  • the polymorphism encodes a single amino acid substitution in the CK-2 polypeptide set forth in FIG. 2 (SEQ ID NO: 3), wherein amino acid residue 176 of that sequence is arginine amino acid residue rather than a tryptophan amino acid residue.
  • the nucleic acids corresponding to S9 were derived from different individual fish than the full length CK-2 cDNA set forth in FIG. 1 (SEQ ID NO: 1).
  • S9, S96 and S5 were derived from a cDNA substraction library produced from the cells of two individual fish.
  • the CK-2 cDNA sequence shown in FIG. 2 was obtained from a full length cDNA library derived from a different individual fish.
  • the allelic variants described in this example are examples of naturally occurring allelic variants of CK-2.
  • the CK-2 allelic variants (i.e., the variant CK-2 nucleic acids and polypeptides) described above comprise substantially the same structural features and domains as do the wild-type CK-2 nucleic acids and polypeptides set forth in SEQ ID NOS:l and 2, respectively.
  • the CK-2 allelic variant encoded by a cDNA corresponding to the S96 probe i.e., a cDNA comprising the nucleotide sequence set forth in SEQ ID NO:7 was analyzed using the SignallP program, as described above for the wild-type CK-2 polypeptide.
  • this variant CK-2 polypeptide also contains a cleavage site and, moreover, that the cleavage site in the variant CK-2 polypeptide is identical to the cleavage site identified above in the wild-type CK-2 polypeptide.
  • This example provides specific, exemplary protocols by which one skilled in the art may readily evaluate in vivo properties of a CK-2 chemokine.
  • the example describes protocols for three experiments by which a skilled artisan may readily evaluate the efficacy of CK-2 polypeptides and gene products when administered to an organism (e.g. , a fish such as rainbow trout) as part of a vaccine.
  • an organism e.g. , a fish such as rainbow trout
  • CK-2 e.g., a fish such as rainbow trout
  • the experiments and protocols described here are merely exemplary of ones which may be used to evaluate a chemokine such as CK-2. It is understood that other protocols, which are known and routine to those skilled in the art, may also be used to prepare and use appropriate vaccines from a chemokine such as CK-2.
  • CK-2 is preferably used in vaccines for fish, such as rainbow trout (i.e., for Oncorhynchus mykiss)
  • the exemplary experiments described here are described, specifically, as using rainbow trout. It will be readily appreciated, however, that the procedures described here may be adapted, without undue experimentation, for any species of organism; including, but not limited to, other species of fish such as salmon (e.g., Salmo sala and Oncoryhynchus choica), carp (e.g., Cyprinus carpio) and other species of trout,.
  • salmon e.g., Salmo sala and Oncoryhynchus choica
  • carp e.g., Cyprinus carpio
  • adult fish between about 7 to 9 inches in length are preferred since fish of this size may be readily raised, e.g., in a laboratory environment and under controlled conditions.
  • CK-2 Nucleic Acids in a DNA Vaccine This experiment may be used by those skilled in the art to evaluate the efficacy of CK-2 as an adjuvant when co-administered in a DNA vaccine with a separate plasmid encoding an antigen.
  • three groups of fish are used in this experiment, with each group preferably having the same number of individual fish.
  • a group may have any number of fish, preferably each group has between about 5 and 50 fish, and more preferably between about 5 and 20 fish. In particularly preferred embodiments, each group has about 10 individual fish.
  • the first group offish may be injected with a plasmid (e.g., about 200 ⁇ g) encoding a protein antigen.
  • the plasmid is one, such as pcDNA3.1 (InvitroGen Corp., Carlsbad, California) that is known in the art and commonly used in DNA vaccines.
  • the plasmid may contain nucleic acid sequences for any antigen. For example, where one skilled in the art is developing a vaccine for a particular infectious agent, such artisan will preferably use a plasmid encoding for a particular antigen of that infectious agent.
  • a model antigen for example, ⁇ -galactosidase
  • the plasmid may be administered to the fish in any carrier or excipient commonly used in the art.
  • the plasmid will typically be administered in 0.1 ml saline.
  • the second group offish may be injected with the same plasmid, encoding the same antigen, admixed with a second plasmid encoding a CK-2 chemokine.
  • the plasmid may encode for a mature, full length CK-2 polypeptide (e.g. , a polypeptide comprising amino acid sequence set forth in FIG. 2 and in SEQ ID NO: 3) or, alternatively, may encode a truncated CK-2 polypeptide.
  • the truncated CK-2 polypeptide should be one which retains the biological activity of CK-2, for example a polypeptide comprising a CK-2 globular domain (e.g., amino acid residues 29-90, or 1-90 of the polypeptide set forth in FIG. 1 and in SEQ ID NO:3).
  • the plasmid encoding a CK-2 chemokine is the same plasmid (e.g., pcDNA3.1) as that encoding the antigen.
  • the amount of each plasmid administered to the second group offish is identical to the amounts administered to the first group (e.g., 200 ⁇ g of plasmid encoding ⁇ -galactosidase admixed with 200 ⁇ g of plasmid encoding CK-2).
  • the third group offish may be injected with the plasmid encoding the antigen plus a control plasmid which does not encode a protein.
  • the control plasmid is the same plasmid (e.g., pcDNA3.1) as that encoding the antigen.
  • the amount of each plasmid administered to the third group of fish is identical to the amounts administered to the first and second groups (e.g., 200 ⁇ g of plasmid encoding ⁇ -galactosidase admixed with 200 ⁇ g of control plasmid).
  • the fish are raised, preferably under controlled conditions (e.g., in a research tank at a laboratory) for a period of time (typically about one month or 28 days) at which point the fish may be sacrificed.
  • Head kidney lymphoid cells and blood serum may be collected from individual fish to determine the immune response of each group to their respective vaccines.
  • blood serum may be tested, e.g. , in an ELISA assay, for antibodies to the antigen (e.g. , for anti- ⁇ - galactosidase antibodies).
  • Lymphocytes may also be tested for T cell mediated responses, e.g. , by measuring their proliferative responses to the antigen in vitro.
  • CK-2 is then readily assessed by comparing the magnitude of blood serum antibody levels and/or T cell mediated responses between fish from the first and/or third groups (i.e., fish vaccinated solely with the antigen-encoding plasmid or with antigen-encoding plasmid and a control plasmid) to fish from the second group (i.e., fish co-injected with antigen and CK-2 encoding plasmids).
  • fish from group two which are co-injected with plasmids encoding the antigen and CK-2, respectively, will have a stronger immune response (indicated by higher levels of antibody and T cell proliferative response) to the antigen.
  • This experiment may be used by those skilled in the art to evaluate the efficacy with which a vaccine comprising CK-2 may stimulate a secondary or recall immune response to an antigen.
  • the exemplary DNA vaccines described, above, in the previous experiment may be administered to three additional groups offish. Preferably, these groups offish are identical to the groups used in the first experiment, above.
  • the fish are raised, preferably under control conditions (e.g., in a research tank at a laboratory) for a period of time (again, typically one month or 28 days), at which time a "booster" of the protein antigen (e.g., 200 ⁇ g of ⁇ -galactosidase) is administered to fish from each of the three groups.
  • a "booster" of the protein antigen e.g., 200 ⁇ g of ⁇ -galactosidase
  • the fish may be returned to their respective tanks, and some period of time later (typically about 7-14 days) may be sacrificed, e.g., as described for the first example, above.
  • head kidney lymphoid cells and blood serum may be collected from individual fish to determine the immune response of each group to their respective vaccines.
  • blood serum may be tested, e.g., in an ELISA assay, for antibodies to the antigen (e.g., for anti- ⁇ -galactosidase antibodies).
  • Lymphocytes may also be tested for T cell mediated responses, e.g., by measuring their proliferative responses to the antigen in vitro.
  • CK-2 is then readily assessed by comparing the magnitude of blood serum antibody levels and/or T cell mediated responses between fish from the first and/or third groups (i.e., fish vaccinated solely with the antigen-encoding plasmid or with antigen-encoding plasmid and a control plasmid) to fish from the second group ( . e. , fish co- injected with antigen and CK-2 encoding plasmids).
  • fish vaccinated with plasmids encoding both an antigen and CK-2 will have a stronger and quicker secondary antibody and T cell response to the booster injection of antigen protein.
  • fusion proteins of chemokines and an antigen are known in the art (see, e.g. , Biragyn et al, Nature Biotechnology 1999, 17:253-258). Further, such fusion proteins have been shown to induce a stronger immune response to the antigen than administration of the antigen alone. The fusion proteins also induce a stronger immune response than co-administration of the antigen and chemokine as separate proteins.
  • the present invention therefore provides vaccines which contain a fusion polypeptide comprising an amino acid sequence for an antigen fused to a CK-2 polypeptide sequence.
  • the invention further provides vaccines which contain a nucleic acid (e.g. , a DNA plasmid) that encodes such a fusion polypeptide.
  • CK-2 polypeptide may be a full length CK-2 polypeptide (e.g., comprising amino acid residues 1-191 of the sequence set forth in FIG.
  • the CK-2 polypeptide is a truncated polypeptide that retains the biological activity of CK-2.
  • the truncated CK-2 polypeptide comprises a CK-2 globular domain (e.g., amino acid residues 29-90 of the sequence set forth in FIG. 2 and in SEQ ID NO:3).
  • the fish may be immunized and sacrificed in this experiment as described, e.g. , in the first experiment for this Example. Head kidney lymphoid cells and blood serum may be collected from individual fish to determine the immune response of each group to their respective vaccines.
  • blood serum may be tested, e.g., in an ELISA assay, for antibodies to the antigen (e.g. , for anti- ⁇ -galactosidase antibodies).
  • Lymphocytes may also be tested for T cell mediated responses, e.g. , by measuring their proliferative responses to the antigen in vitro.
  • the efficacy of CK-2 is then readily assessed by comparing the magnitude of blood serum antibody levels and/or T cell mediated responses between fish from the first and/or third groups (i. e. , fish vaccinated solely with the antigen-encoding plasmid or with antigen-encoding plasmid and a control plasmid) to fish from the second group (i. e.
  • fish co-injected with antigen and CK-2 encoding plasmids fish co-injected with antigen and CK-2 encoding plasmids.
  • fish injected with a plasmid encoding the antigen-CK2 fusion polypeptide will have a stronger immune response (indicated by higher levels of antibody and T cell proliferative response) to the antigen than either those fish immunized with plasmid encoding the antigen alone or those fish co-immunized with DNA encoding the antigen and CK-2 on separate plasmids.

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Abstract

L'invention concerne une nouvelle chimiokine, dorénavant chimiokine-2 ou CK-2, et notamment des modes de réalisation particuliers dérivés de poissons tels que la truite arc-en-ciel (Oncorhynchus mykiss). L'invention concerne de nouveaux polypeptides correspondant aux chimiokines CK-2, ainsi que des acides nucléiques codant pour une chimiokine CK-2 et des anticorps qui se lient spécifiquement à une chimiokine CK-2. L'invention concerne également des méthodes thérapeutiques pour utiliser les nouvelles chimiokines CK-2. Plus spécifiquement, l'invention concerne des compositions pharmaceutiques, telles que des vaccins, qui contiennent une chimiokine CK-2 et un immunogène. Ces compositions pharmaceutiques peuvent être administrées à un organisme pour augmenter ou activer une réponse immunitaire à cet immunogène. Dans un autre mode de réalisation, l'invention concerne également un procédé de criblage pour identifier et/ou isoler des gènes dont l'ARNm est rapidement dégradé dans une cellule, par identification et/ou isolation d'acides nucléiques (par exemple, à partir d'une bibliothèque d'ADNc) possédant au moins un motif de séquence reconnu par une endonucléase. Ces procédés de criblage sont particulièrement utiles pour identifier et/ou isoler des gènes codant pour de nouvelles chimiokines, cytokines ou oncogènes.
PCT/US2001/045366 2000-10-30 2001-10-30 Nouvelle chimiokine de poisson et procedes d'utilisation de celle-ci WO2002036070A2 (fr)

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