EP1214414A2 - Rip-3-ähnliche totverbundene kinase - Google Patents

Rip-3-ähnliche totverbundene kinase

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Publication number
EP1214414A2
EP1214414A2 EP00973364A EP00973364A EP1214414A2 EP 1214414 A2 EP1214414 A2 EP 1214414A2 EP 00973364 A EP00973364 A EP 00973364A EP 00973364 A EP00973364 A EP 00973364A EP 1214414 A2 EP1214414 A2 EP 1214414A2
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EP
European Patent Office
Prior art keywords
polypeptide
r3dak
polypeptides
amino acid
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00973364A
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English (en)
French (fr)
Inventor
G. Duke Virca
Timothy A. Bird
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Immunex Corp
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Immunex Corp
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Publication date
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Publication of EP1214414A2 publication Critical patent/EP1214414A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is directed to purified and isolated novel RIP-3-L ⁇ ke Death-Associated Kinase
  • R3DAK polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.
  • apoptosis is termed programmed cell death
  • the morphological changes that occur during apoptosis are characterized by DNA degradation by endonucleases, cytoplasmic and nuclear condensation, and the formation of membrane "blebs" or apoptotic bodies (T G Cotter et al , Anticancer Res, Sept- Oct: 10(5A): 1153-9, 1990) Neighboring cells then move in to engulf the remaining cellular debris.
  • Apoptosis is an important process during development and adult life: misregulation of apoptotic processes can result in inflammatory, malignant, autoimmune, and neurodegenerative conditions; and infectious agents, including viruses, can use cellular apoptosis in the host to evade the immune system
  • infectious agents including viruses
  • the balance between apoptosis and cell proliferation is also believed to play a role in vascular function, with an imbalance in these processes leading to vascular diseases such as abnormal vascular remodeling, ischemic and non-ischemic heart failure, myocardial infarction, and arrhythmias.
  • the biochemical mechanism driving apoptosis can begin with a ligand/receptor induced signal that activates (in part through phosphorylation or desphosyphorylation) other proteins, such as kinases, along the signal transduction pathway and ultimately concludes with the activation of the cell death program
  • ligand/receptor pairs that can induce apoptosis are, for example, TNF/TNF-RI, TNF TNF-R2, CD95 hgand/CD95, TRAIIVTRAIL-Rl, and TRAIUTRAIL-R2.
  • DD death domain
  • eukaryotic protein kinases e.g., cell death related kinases, cell proliferation related kinases, etc.
  • cell death related kinases e.g., cell death related kinases, cell proliferation related kinases, etc.
  • cell proliferation related kinases e.g., cell proliferation related kinases, etc.
  • protein kinase genes have been identified from a wide selection of invertebrates and lower eukaryotes, including Drosophila, Caenorhabditis elegans, Aplysia, Hydra, Dictyostelium, and budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe). Homologous genes have also been identified in higher plants. Protein kinases, however, are not limited to the eukaryotes.
  • RIP Receptor- Interacting Proteins
  • the third member, RIP3 does not possess a death domain or a CARD motif at its C-terminus but instead the C-terminal segment binds RIP and through this interaction engages the NF- ⁇ B pathway. (Sun et al., 1999, J. Biol. Chem. 274: 16871-16875.)
  • kinases Given the important functions of kinases, there is a need in the art for additional members of the kinase family. In particular, there is a need in the art for additional members of the RIP kinase family. There is also a need in the art for the identity and function of proteins having kinase activities. Moreover, given the important roles kinases may play in apoptosis, there is an unmet need for therapeutic compounds that interfere with apoptosis.
  • R3DAK RIP-3-Like Death-Associated Kinase
  • Particular embodiments of the invention are directed to an isolated R3DAK nucleic acid molecule comprising the DNA sequence of SEQ ID NO: l and an isolated R3DAK nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2, as well as nucleic acid molecules complementary to these sequences.
  • the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
  • an amino acid sequence having kinase activity comprising at least 20 amino acids, and sharing amino acid identity with the amino acid sequences of any of (a)-(k), wherein the percent amino acid identity is selected from the group consisting of: at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%; and
  • polypeptides comprising the above amino acid sequences and having a molecular weight of approximately 52 kD as determined by SDS-PAGE, and polypeptides comprising the above amino acid sequences in non-glycosylated form.
  • isolated nucleic acids encoding polypeptides of the invention and isolated nucleic acids, preferably having a length of at least 17 nucleotides, that hybridize under conditions of moderate stringency to the nucleic acids encoding polypeptides of the invention.
  • nucleic acids encode a polypeptide having kinase activity, or comprise a nucleotide sequence that shares nucleotide sequence identity with the nucleotide sequences of the nucleic acids of the invention, wherein the percent nucleotide sequence identity is selected from the group consisting of: at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99%, and at least 99.5%.
  • isolated polypeptides and nucleic acids consisting of amino acid sequences and nucleotide sequences, respectively, disclosed herein.
  • expression vectors and recombinant host cells comprising at least one nucleic acid of the invention, and preferred recombinant host cells wherein said nucleic acid is integrated into the host cell genome.
  • the expression of these polypeptides in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
  • a preferred process provided by the invention further comprises purifying said polypeptide.
  • the polypeptide produced by said process is provided.
  • inventions are isolated antibodies that bind to the polypeptides of the invention, preferably monoclonal antibodies, also preferably humanized antibodies or humanized antibodies.
  • the invention additionally provides a method of designing an inhibitor of the polypeptides of the invention, the method comprising the steps of determining the three-dimensional structure of any such polypeptide, analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predicted reactive site, and determining the poiypeptide-inhibiting activity of the molecule
  • a method for identifying compounds that alter R3DAK kinase activity comprising
  • the invention encompasses methods of using the nucleic acids noted above to identify nucleic acids encoding proteins having kinase activity or proteins involved in apoptosis signal transduction
  • the polypeptides of the invention can be used to study cellular processes such as immune regulation, cell death, cell migration, cell-to-cell interaction, and inflammatory responses These polypeptides can also be used to identify proteins associated with R3DAK kinases
  • the invention also provides a method for increasing kinase activity, comprising providing at least one compound, wherein the compound is selected from the group consisting of a polypeptide of the invention and an agonist of the kinase activity of said polypeptide, with a preferred embodiment of the method further comprising increasing said activities in a patient by administering at least one polypeptide of the invention or an agonist of said polypeptide
  • a method for decreasing kinase activity comprising providing at least one antagonist of the
  • Also provided by the invention is a method for use of a compound, wherein the compound is selected from the group consisting of a polypeptide of the invention and an agonist of the kinase activity of said polypeptide, in the manufacture of a medicament for treating apoptosis-related conditions promoted by lower levels of R3DAK kinase activity
  • the invention also provides a method for the use of a compound that inhibits the kinase activity of the polypeptide of the invention in the manufacture of a medicament for treating apoptosis-related conditions promoted by higher levels of R3DAK kinase activity DETAILED DESCRIPTION OF THE INVENTION
  • R3DAK RIP-3-L ⁇ ke Death- Associated Kinase
  • R3DAK polypeptides such as R3DAK have kinase activity and bind proteinaceous substrates
  • preferred R3DAK polypeptides include those having at least one kinase catalytic domain and exhibiting at least one such substrate-bmding activity
  • Preferred R3DAK polypeptides further include o gomers or fusion polypeptides comprising at least one kinase domain of one or more R3DAK polypeptides, and fragments of any of these polypeptides that have kinase and/or substrate-binding activity
  • the binding activity or activities of R3DAK polypeptides may be determined, for example, in a yeast two-hybrid assay, or in an in vitro assay that measures binding between an R3DAK polypeptide and one of its substrates or binding partners, where either the R3DAK polypeptide or its binding partner is labeled with a radioactive, fluorescent, or biolum
  • R3DAK activity includes any one or more of the following kinase activity, substrate-binding activity, and binding activities via the R3DAK C-terminal domain, as well as the ex vivo and in vivo activities of R3DAK polypeptides
  • binding partner includes without limitation ligands, receptors, native cognates, counter-structures, substrates, antibodies, other RIP- 3-L ⁇ ke polypeptides, the same R3DAK polypeptide (in the case of homotypic interactions), and any other molecule that interacts with a R3DAK polypeptide through contact or proximity between particular portions of the binding partner and the R3DAK polypeptide
  • the degree to which R3DAK polypeptides and fragments and other derivatives of these polypeptides exhibit these biological activities and partner-binding properties may be assayed by standard methods and by those representative assays described herein Those of skill in the art will appreciate that other, similar types of assays can be used
  • the following conditions involving apoptosis are those that are known or are likely to involve the biological activities of R3DAK polypeptides: cell hyperproliferation conditions such as tumor growth or metastasis, autoimmune disorders, and vascular cell hyper-proliferation; and conditions involving excess apoptosis such as inflammation, neurodegenerative disorders, infection, abnormal vascular remodeling, ischemia, and myocardial infarction.
  • cell hyperproliferation conditions such as tumor growth or metastasis, autoimmune disorders, and vascular cell hyper-proliferation
  • conditions involving excess apoptosis such as inflammation, neurodegenerative disorders, infection, abnormal vascular remodeling, ischemia, and myocardial infarction.
  • Blocking or inhibiting the interactions between R3DAK. polypeptides and their substrates, binding partners, and or other interacting polypeptides is an aspect of the invention and provides methods for treating or ameliorating these diseases and conditions through the use of inhibitors of R3DAK activity. Examples of such inhibitors or antagonists are described in
  • methods of treating or ameliorating these conditions comprise increasing the amount or activity of R3DAK polypeptides by providing isolated R3DAK polypeptides or active fragments or fusion polypeptides thereof, or by providing compounds (agonists) that activate endogenous or exogenous R3DAK polypeptides. Additional uses for R3DAK polypeptides and agonists and antagonists thereof include their use in studies of signal transduction, and in regulating cellular processes associated with transduction of biological signals. R3DAK polypeptide fragments also may be employed as immunogens, in generating antibodies.
  • nucleic acids of the invention can be used as probes to identify nucleic acid encoding proteins having kinase activity. Because homologs of SEQ ID NO: 1 from other mammalian species are contemplated herein, probes based on the rattus DNA sequence of SEQ ID NO: l may be used to screen cDNA libraries derived from other mammalian species, using conventional cross-species hybridization techniques.
  • R3DAK Polypeptides A R3DAK polypeptide is a polypeptide that shares a sufficient degree of amino acid identity or similarity to the R3DAK amino acid sequence shown as SEQ ID NO:2 to (A) be identified by those of skill in the art as a polypeptide likely to share particular structural domains and/or (B) have biological activities in common with R3DAK and/or (C) bind to antibodies that also specifically bind to other R3DAK polypeptides.
  • R3DAK polypeptides may be isolated from naturally occurring sources, or have the same structure as naturally occurring R3DAK polypeptides, or may be produced to have structures that differ from naturally occurring R3DAK polypeptides.
  • Polypeptides derived from any R3DAK polypeptide by any type of alteration are also R3DAK polypeptides. Therefore, the polypeptides provided by the invention include polypeptides characterized by amino acid sequences similar to those of the R3DAK polypeptides described herein, but into which modifications are naturally provided or deliberately engineered.
  • a polypeptide that shares biological activities in common with R3DAK polypeptides is a polypeptide having R3DAK activity.
  • polypeptides of the invention include full length proteins (amino acids 1 to 478 of SEQ ID NO 2) encoded by the nucleic acid sequences set forth above
  • Preferred polypeptides comprise the amino acid sequence of SEQ ID NO 2, with particularly preferred fragments comprising ammo acids 1 to 300, 22 to 338, 22 to 291 , 103 to 216, 139 to 165, 139 to 194, and 184 to 216 of SEQ ID NO 2
  • Additional preferred embodiments of the invention include a truncated version of R3DAK containing, for example, only the kinase catalytic domain or a catalytically inactive mutant thereof
  • Catalyticallv inactivated variants include variants in which, for example, invariant lysine residue (amino acid 51 of SEQ ID NO 2) is substituted for an arginine or alanine residue or in which any non-glycine residue is substituted for certain glycine residues (ammo acids 29, 31, and/or 34 of S
  • a purified R3DAK polypeptide of the invention (SEQ ID NO 2) has a calculated molecular weight of approximately 52,218 Daltons Fragmentation of the polypeptide of SEQ ID NO 2 with cyanogen bromide generates a unique set of fragmented peptide molecular weight markers with molecular weights as shown in Table 1 The distribution of methionine residues determines the number of amino acids in each peptide and the unique amino acid composition of each peptide determines its molecular weight
  • Full- length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated
  • the amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame ("ORF") of a cDNA molecule
  • ORF complete open reading frame
  • full-length polypeptides may be encoded by a single genetic locus if multiple mRNA forms are produced from that locus by alternative splicing or by the use of multiple translation initiation sites
  • the "mature form" of a polypeptide refers to a polypeptide that has undergone post-translational processing steps such as cleavage of the signal sequence or proteolytic cleavage to remove a prodomain.
  • Multiple mature forms of a particular full-length polypeptide may be produced, for example by cleavage of the signal sequence at multiple sites, or by differential regulation of proteases that cleave the polypeptide.
  • the mature form(s) of such polypeptide may be obtained by expression, in a suitable mammalian cell or other host cell, of a nucleic acid molecule that encodes the full-length polypeptide.
  • the sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites.
  • the R3DAK polypeptides of the invention also include those that result from post-transcriptional or post-translational processing events such as alternate mRNA processing which can yield a truncated but biologically active polypeptide, for example, a naturally occurring soluble form of the polypeptide. Also encompassed within the invention are variations attributable to proteolysis such as differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptide (generally from 1 to 5 terminal amino acids).
  • the invention further includes R3DAK polypeptides with or without associated native-pattern glycosyiation.
  • Polypeptides expressed in yeast or mammalian expression systems e.g., COS-1 or CHO cells
  • yeast or mammalian expression systems e.g., COS-1 or CHO cells
  • Expression of polypeptides of the invention in bacterial expression systems, such as E. coll provides non-glycosylated molecules.
  • a given preparation can include multiple differentially glycosylated species of the polypeptide. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).
  • Species homologues of R3DAK polypeptides and of nucleic acids encoding them are also provided by the present invention.
  • a "species homologue” is a polypeptide or nucleic acid with a different species of origin from that of a given polypeptide or nucleic acid, but with significant sequence similarity to the given polypeptide or nucleic acid, as determined by those of skill in the art.
  • Species homologues may be isolated and identified by making suitable probes or primers from polynucleotides encoding the amino acid sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of R3DAK polypeptides and nucleic acids encoding them; that is, naturally-occurring alternative forms of such polypeptides and nucleic acids in which differences in amino acid or nucleotide sequence are attributable to genetic polymorphism (allelic variation among individuals within a population).
  • R3DAK polypeptides of the present invention are encompassed by the present invention and may be in linear form or cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 1 14 9245-9253 (1992), both of which are incorporated by reference herein.
  • Polypeptides and polypeptide fragments of the present invention, and nucleic acids encoding them include polypeptides and nucleic acids with amino acid or nucleotide sequence lengths that are at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of a R3DAK polypeptide and have at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97 5%, or at least 99%, and most preferably at least 99 5%) with that R3DAK polypeptide or encoding nucleic acid, where sequence identity is determined by comparing the amino acid sequences of the polypeptides when aligned so as to maximize overlap and identity while minimizing sequence gaps
  • polypeptides and polypeptide fragments, and nucleic acids encoding them that contain or encode a segment preferably comprising at least 8, or at least 10, or
  • polypeptides of the present invention and nucleic acids encoding them include those comprising or encoding two or more copies of a domain such as the kinase domain, two or more copies of a domain such as the C-terminal domain, or at least one copy of each domain, and these domains may be presented in any order within such polypeptides
  • Modifications of interest in the polypeptide sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid A given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics Examples of such conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, substitutions of one polar residue for another, such as between Lys
  • the D As of the invention include variants that differ from a native DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide
  • one or more of the cyste e residues may be deleted or replaced with another amino acid to alter the conformation of the molecule, an alteration which may involve preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e g , U S Pat No 4,518,584)
  • N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosyiation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems N-glycosylation sites in eukaryotic polypeptide
  • oligomers or fusion polypeptides that contain a R3DAK polypeptide, one or more fragments of R3DAK polypeptides, or any of the derivative or variant forms of R3DAK polypeptides as disclosed herein
  • the oligomers comprise soluble R3DAK polypeptides
  • Oligomers can be in the form of covalently linked or non-covalently-linked multimers, including dimers, t ⁇ mers, or higher oligomers
  • the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, t ⁇ valent, etc , binding sites
  • the invention is directed to oligomers comprising multiple R3DAK polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides, such peptides having the property of promoting oligomerization Leucme zippers and certain polypeptides derived from antibodies are among the peptide
  • the polypeptides of the invention or fragments thereof may be fused to molecules such as immunoglobulins for many purposes, including increasing the valency of polypeptide bindmg sites
  • fragments of a R3DAK polypeptide may be fused directly or through linker sequences to the Fc portion of an lmmunoglobulin
  • a bivalent form of the polypeptide such a fusion could be to the Fc portion of an IgG molecule
  • Other lmmunoglobulin isotypes may also be used to generate such fusions
  • a polypeptide-IgM fusion would generate a decavalent form of the polypeptide of the invention
  • the term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising any or all of the CH domains of the Fc region Truncated forms of such polypeptides containing the hinge region that promotes dime ⁇ zation are also
  • the oligomer is a fusion polypeptide comprising multiple R3DAK polypeptides, with or without peptide linkers (spacer peptides)
  • suitable peptide linkers are those described in U S Patents 4,751,180 and 4,935,233, which are hereby incorporated by reference
  • a DNA sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as, the DNA sequences of the invention, using any suitable conventional technique
  • a chemically synthesized o gonucleotide encoding the linker can be ligated between the sequences
  • a fusion polypeptide comprises from two to four soluble R3DAK polypeptides, separated by peptide linkers
  • Suitable peptide linkers, their combination with other polypeptides, and their use are well known by those skilled in the art
  • Leucme-Zippers Another method for preparing the oligomers of the invention involves use of a leucine zipper
  • Leucme zipper domains are peptides that promote oligomerization of the polypeptides in which they are found.
  • Leucine zippers were originally identified in several DNA-binding polypeptides (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different polypeptides.
  • the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • the zipper domain (also referred to herein as an oligomerizing, or oligomer- forming, domain) comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids.
  • leucine zippers and preparation of oligomers using leucine zippers are well known in the art.
  • nucleic acids encoding R3DAK polypeptides can be identified in several ways, including isolation of genomic or cDNA molecules from a suitable source. Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for the isolation of nucleic acids or as query sequences for database searches, can be obtained by "back-translation" from the amino acid sequences, or by identification of regions of amino acid identity with polypeptides for which the coding DNA sequence has been identified.
  • PCR polymerase chain reaction
  • Nucleic acid molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences.
  • DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • the nucleic acid molecules of the invention include full-length genes or cDNA molecules as well as a combination of fragments thereof.
  • the nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.
  • isolated nucleic acid is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from naturally-occurring sources.
  • nucleic acids synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or oligonucleotides for example it is understood that the nucleic acids resulting from such processes are isolated nucleic acids.
  • An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct.
  • the invention relates to certain isolated nucleic acids that are substantially free from contaminating endogenous material.
  • the nucleic acid molecule has preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al.. Molecular Cloning: A Laboratory Manual. 2nd sed.. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).
  • sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5' or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.
  • the present invention also includes nucleic acids that hybridize under moderately stringent conditions, and more preferably highly stringent conditions, to nucleic acids encoding R3DAK polypeptides described herein.
  • the basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis ( 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. N.Y., chapters 9 and 1 1 ; and Current Protocols in Molecular Biology, 1995, F. M.
  • One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6 x SSC, and a hybridization temperature of about 55 degrees C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 degrees C), and washing conditions of about 60 degrees C, in 0.5 x SSC, 0.1% SDS.
  • highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68 degrees C, 0.2 x SSC, 0.1% SDS.
  • SSPE (lxSSPE is 0.15M NaCl, 10 M NaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (lxSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete.
  • wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al., 1989).
  • the hybrid length is assumed to be that of the hybridizing nucleic acid.
  • the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity.
  • each such hybridizing nucleic acid has a length that is at least 15, 18, 20, 25, 30, 40, or more preferably 50 nucleotides, or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with the nucleic acid of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned so as to maximize overlap and identity while minimizing sequence gaps as described in more detail above.
  • the present invention also provides genes corresponding to the nucleic acid sequences disclosed herein.
  • “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA nucleic acid sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements.
  • the corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.
  • Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
  • An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
  • R3DAK polypeptides are described below. Expression, isolation, and purification of the polypeptides and fragments of the invention can be accomplished by any suitable technique, including but not limited to the following methods.
  • the isolated nucleic acid of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991 ); and Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985), in order to produce the polypeptide recombinantly. Many suitable expression control sequences are known in the art.
  • operably linked means that the nucleic acid of the invention and an expression control sequence are situated within a construct, vector, or cell in such a way that the polypeptide encoded by the nucleic acid is expressed when appropriate molecules (such as polymerases) are present.
  • At least one expression control sequence is operably linked to the nucleic acid of the invention in a recombinant host cell or progeny thereof, the nucleic acid and/or expression control sequence having been introduced into the host cell by transformation or transfection, for example, or by any other suitable method.
  • at least one expression control sequence is integrated into the genome of a recombinant host cell such that it is operably linked to a nucleic acid sequence encoding a polypeptide of the invention.
  • at least one expression control sequence is operably linked to a nucleic acid of the invention through the action of a trans-acting factor such as a transcription factor, either in vitro or in a recombinant host cell
  • a sequence encoding an appropriate signal peptide can be incorporated into expression vectors
  • the choice of signal peptide or leader can depend on factors such as the type of host ceils in which the recombinant polypeptide is to be produced
  • examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for ⁇ nterleukm-7 (IL-7) described in United States Patent 4,965,195, the signal sequence for ⁇ nterleuk ⁇ n-2 receptor described in Cosman et al , Nature 312 768 (1984), the ⁇ nterleuk ⁇ n-4 receptor signal peptide described in EP 367,566, the type I interleukin- 1 receptor signal peptide described in U S Patent 4,968,607, and the type II interleukin- 1 receptor signal peptide described in EP 460,846
  • a DNA sequence for a signal peptide (secretory leader) can be fused in frame to the nucleic acid sequence of the invention so that
  • a number of types of cells may act as suitable host cells for expression of the polypeptide Mammalian host cells include, for example, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al , Cell 23 175, 1981), L cells.
  • C 127 cells 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line C V 1 (ATCC CCL 70) as described by McMahan et al (EMBO J 10 2821, 1991), human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells Alternatively, it may be possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides Potentially suitable bacterial strain
  • the polypeptide of the invention may be prepared by cultu ⁇ ng transformed host cells under culture conditions suitable to express the recombinant polypeptide
  • the resulting expressed polypeptide may then be purified from such culture (l e , from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography
  • the purification of the polypeptide may also include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanava n A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®, one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether, or lmmunoaffinity chromatography
  • the polypeptide of the invention may also be expressed in a form which will facilitate purification For example, it may be expressed as a fusion polypeptide, such as those of maltose
  • an affinity column comprising a polypeptide-bmding polypeptide of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides
  • polypeptides can be removed from an affinity column using conventional techniques, e g , in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention
  • polypeptide-binding polypeptides such as the anti-polypeptide antibodies of the invention or other polypeptides that can interact with the polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface Adherence of polypeptide-binding polypeptides
  • the desired degree of purity depends on the intended use of the polypeptide.
  • a relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example.
  • the polypeptides are purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel eiectrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide can be visualized by SDS-PAGE, due to differential glycosyiation, differential post-translational processing, and the like.
  • the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.
  • any method which neutralizes R3DAK polypeptides or inhibits expression of the R3DAK genes can be used to reduce the biological activities of R3DAK polypeptides.
  • antagonists inhibit the binding of at least one R3DAK polypeptide to binding partners expressed on cells, thereby inhibiting biological activities induced by the binding of those R3DAK polypeptides to the cells.
  • antagonists can be designed to reduce the level of endogenous R3DAK gene expression, e.g., using well-known antisense or ribozyme approaches to inhibit or prevent translation of R3DAK mRNA transcripts; triple helix approaches to inhibit transcription of R3DAK genes; or targeted homologous recombination to inactivate or "knock out" the R3DAK genes or their endogenous promoters or enhancer elements.
  • antisense, ribozyme, and triple helix antagonists may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant R3DAK gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
  • Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing polypeptide translation.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to a R3DAK 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 "complementary" to a portion of a nucleic acid as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the nucleic acid, forming a stable duplex (or triplex, as appropriate).
  • a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
  • Oligonucleotides that are complementary to the 5' end of the message e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation.
  • oligonucleotides complementary to either the 5'- or 3'- non- translated, non-coding regions of the R3DAK gene transcript could be used in an antisense approach to inhibit translation of endogenous R3DAK mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon
  • Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length In specific aspects the o gonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides
  • the oligonucleotides can be DNA or RNA or chime ⁇ c mixtures or derivatives or modified versions thereof, single-stranded or double-stranded
  • the ohgonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc
  • the ohgonucleotide may include other appended groups such as peptides (e g , for targeting host cell
  • Ribozyme molecules designed to catalytically cleave R3DAK mRNA transcripts can also be used to prevent translation of R3DAK mRNA and expression of R3DAK polypeptides (See, e g , PCT International Publication WO90/1 1364, published Oct 4, 1990, US Patent No 5,824,519)
  • the ⁇ bozymes that can be used in the present invention include hammerhead ⁇ bozymes (Haseloff and Gerlach, 1988, Nature, 334 585-591), RNA endo ⁇ bonuc leases (hereinafter "Cech-type ⁇ bozymes") such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (International Patent Application No WO 88/04300, Been and Cech, 1986, Cell, 47 207-216)
  • the ⁇ bozymes can be composed of modified
  • endogenous R3DAK gene expression can be reduced by targeting deoxy ⁇ bonucleotide sequences complementary to the regulatory region of the target gene (1 e , the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target
  • R3DAK gene See generally, Helene, 1991, Anticancer Drug Des , 6(6), 569-584. Helene, et al , 1992,
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules These include techniques for chemically synthesizing oligodeoxy ⁇ bonucleotides and oligo ⁇ bonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis Oligonucleotides can be synthesized by standard methods known in the art, e g by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc ) As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al , 1988, Nucl Acids Res 16 3209 Methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sa ⁇ n et al , 1988, Proc
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule
  • DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines
  • 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 , 1985, Nature 317, 230-234, Thomas and Capecchi, 1987, Cell 51, 503-512, Thompson, et al , 1989, Cell 5, 313- 321 , each of which is incorporated by reference herein in its entirety)
  • a mutant, non- functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous 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 Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene
  • Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to
  • R3DAK in in vitro or in vivo procedures Encompassed within the invention are kinase or C-terminal domains of R3DAK polypeptides that act as "dominant negative" inhibitors of native R3DAK polypeptide function when expressed as fragments or as components of fusion polypeptides
  • a purified C- terminal domain of the present invention can be used to inhibit binding of R3DAK polypeptides to endogenous binding partners Such use effectively would block R3DAK polypeptide interactions and inhibit R3DAK polypeptide activities
  • antibodies which bind to R3DAK polypeptides or binding partners may inhibit R3DAK activity and act as antagonists
  • antibodies that specifically recognize one or more epitopes of R3DAK binding partners, R3DAK polypeptides, or epitopes of conserved variants of R3DAK polypeptides, or peptide fragments of the R3DAK polypeptide can be used in the invention to inhibit R3DAK activity
  • Such antibodies include but are not limited
  • specifically binding antibodies are those that will specifically recognize and bind with R3DAK polypeptides, homologues, and variants, but not with other molecules
  • the antibodies are specific for the polypeptides of the present invention and do not cross-react with other polypeptides
  • the R3DAK polypeptides, fragments, variants, fusion polypeptides, etc as set forth above can be employed as "immunogens" in producing antibodies immunoreactive therewith
  • the polypeptides, fragment, variants, fusion polypeptides, etc contain antigenic determinants or epitopes that elicit the formation of antibodies
  • These antigenic determinants or epitopes can be either linear or conformational (discontinuous) Linear epitopes are composed of a single section of ammo acids of the polypeptide, while conformational or discontinuous epi
  • both polyclonal and monoclonal antibodies can be prepared by conventional techniques See, for example, Monoclonal Antibodies, Hvbridomas A New Dimension in Biological Analyses, Kennet et al (eds ), Plenum Press, New York ( 1980), and Antibodies A Laboratory Manual, Harlow and Land (eds ), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Kohler and Milstein, (U S Pat No 4,376,1 10), the human B-cell hyb ⁇ doma technique (Kosbor et al , 1983, Immunology Today 4 72, Cole et al , 1983, Proc Natl Acad Sci USA 80 2026-2030), and the EBV-hybndoma technique (Cole et al , 1985, Monoclonal Antibodies And Cancer Therapy, Alan
  • chime ⁇ c antibodies are a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a porcine mAb and a human lmmunoglobulin constant region
  • the monoclonal antibodies of the present invention also include humanized versions of mu ⁇ ne monoclonal antibodies Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans
  • a humanized monoclonal antibody comprises the variable region of a mu ⁇ ne antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody
  • a humanized antibody fragment can comprise the antigen bmding site of a murine monoclon
  • Antibodies can be screened for agonistic (/ e , gand- mimicking) properties
  • Such antibodies upon binding to a R3DAK binding partner, induce biological effects (e g , transduction of biological signals) similar to the biological effects induced when the R3DAK binding partner binds to R3DAK
  • Agonistic antibodies can be used to induce R3DAK-med ⁇ ated stimulatory pathways
  • conjugates comprising a detectable (e g , diagnostic) or therapeutic agent, attached to the antibody Examples of such agents are presented herein
  • the conjugates find use in in vitro or in vivo procedures
  • the antibodies of the invention can also be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo
  • the antibodies also can be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, e g , inhibitors, agonists, antagonists, etc Any of these examples can be used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo (Hodgson J (1991) Biotechnology 9: 19-21, incorporated herein by reference).
  • the three-dimensional structure of a polypeptide of interest, or of a polypeptide-inhibitor complex is determined by x-ray crystallography, by nuclear magnetic resonance, or by computer homology modeling or, most typically, by a combination of these approaches.
  • Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous polypeptides. In both cases, relevant structural information is used to design analogous serpin-like molecules, to identify efficient inhibitors, or to identify small molecules that may bind serpins.
  • Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton S and Wells JA ( 1992 Biochemistry 31 :7796-7801) or which act as inhibitors, agonists, or antagonists of native peptides as shown by Athauda SB et al (1993 J Biochem 1 13:742-746), incorporated herein by reference.
  • the use of R3DAK polypeptide structural information in molecular modeling software systems to assist in inhibitor design and inhibitor-R3DAK polypeptide interaction is also encompassed by the invention.
  • a particular method of the invention comprises analyzing the three dimensional structure of R3DAK polypeptides for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described further herein.
  • a target-specific antibody selected by functional assay, as described further herein, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass polypeptide crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • the purified R3DAK polypeptides of the invention are useful in a variety of assays.
  • the R3DAK molecules of the present invention can be used to identify binding partners of R3DAK polypeptides, which can also be used to modulate intracellular communication or cell activity.
  • they can be used to identify non-binding-partner molecules or substances that modulate intracellular communication or cell activity.
  • Polypeptides of the R3DAK and fragments thereof can be used to identify binding partners. For example, they can be tested for the ability to bind a candidate binding partner in any suitable assay, such as a conventional binding assay.
  • the R3DAK polypeptide can be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like).
  • a detectable reagent e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like.
  • the labeled polypeptide is contacted with cells expressing the candidate binding partner.
  • the cells then are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.
  • a binding assay procedure is as follows.
  • a recombinant expression vector containing the candidate binding partner cDNA is constructed.
  • CVl-EBNA-1 cells in 10 cm 2 dishes are transfected with this recombinant expression vector.
  • CV-l/EB A-1 cells constitutive ly express EBV nuclear antigen- 1 driven from the CMV Immediate-early enhancer/promoter.
  • CV l-EBNA- 1 was derived from the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan et al., (EMBO J. 10:2821, 1991).
  • the transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate.
  • the transfected cells (about 4 x 10 4 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added.
  • the cells then are incubated for 1 hour at 37°C with various concentrations of, for example, a soluble polypeptide/Fc fusion polypeptide made as set forth above. Cells then are washed and incubated with a constant saturating concentration of a 125 I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37°C.
  • the mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA.
  • the antibody is radioiodinated using the standard chloramine-T method.
  • the antibody will bind to the Fc portion of any polypeptide/Fc polypeptide that has bound to the ceils.
  • non-specific binding of l25 I-antibody is assayed in the absence of the Fc fusion polypeptide/Fc, as well as in the presence of the Fc fusion polypeptide and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody.
  • Binding can also be detected using methods that are well suited for high-throughput screening procedures, such as scintillation proximity assays (Udenfriend S, Gerber LD, Brink L, Spector S, 1985, Proc Natl Acad Sci U S A 82: 8672-8676), homogeneous time-resolved fluorescence methods (Park YW, Cummings RT, Wu L, Zheng S, Cameron PM, Woods A, Zaller DM, Marcy Al, Hermes JD, 1999, Anal Biochem 269: 94- 104), fluorescence resonance energy transfer (FRET) methods (Clegg RM, 1995, Curr Opin Biotechnol 6: 103-1 10), or methods that measure any changes in surface plasmon resonance when a bound polypeptide is exposed to a potential binding partner, such methods using for example a biosensor such as that supplied by Biacore AB (Uppsala, Sweden).
  • a biosensor such as that supplied by Biacore AB (
  • the nucleic acid encoding the R3DAK polypeptide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify nucleic acids encoding the other polypeptide with which binding occurs or to identify inhibitors of the binding interaction.
  • interaction trap assays such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)
  • Polypeptides involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Suitable binding assays Another type of suitable binding assay is a competitive binding assay
  • biological activity of a variant can be determined by assaying for the variant's ability to compete with the native polypeptide for binding to the candidate binding partner
  • Competitive binding assays can be performed by conventional methodology
  • Reagents that can be employed in competitive binding assays include radiolabeled R3DAK
  • a radiolabeled R3DAK fragment can be used to compete with a R3DAK variant for binding to a R3DAK binding partner
  • a soluble binding partner/Fc fusion polypeptide bound to a solid phase through the interaction of Polypeptide A or Polypeptide G (on the solid phase) with the Fc moiety could be utilized in such an assay Chromatography columns that contain Polypeptide A and Polypeptide G include those available from Pharmacia Biotech, Inc . Piscataway, NJ Assays to Identify Modulators of Intracellular Communication or Cell Activity The influence of
  • R3DAK polypeptides on intracellular communication or cell activity can be manipulated to control these activities in target cells
  • the disclosed R3DAK polypeptides, nucleic acids encoding the disclosed R3DAK polypeptides, or agonists or antagonists of such polypeptides can be administered to a cell or group of cells to induce, enhance, suppress, or arrest intracellular signaling or R3DAK kinase activity in the target cells
  • Identification of R3DAK polypeptides, agonists or antagonists that can be used in this manner can be carried out via a variety of assays known to those skilled in the art Included in such assays are those that evaluate the ability of an R3DAK polypeptide to influence intracellular communication or cell activity
  • Such an assay would involve, for example, the analysis of intracellular signaling in the presence of an R3DAK polypeptide In such an assay, one would determine a rate of signaling or cell stimulation or cell death or survival in the presence of the R3DAK polypeptide and then determine
  • Kinase assays are typically carried out by combining R3DAK, or an active kinase domain, with radiolabeled ATP ( ⁇ 32 P-ATP) and a peptide or protein substrate in a buffer solution
  • the peptide substrates generally range from 8 to 30 amino acids in length or the substrate may also be a protein known to be phosphorylated readily by R3DAK
  • Many such general kinase substrates are known, e g , ⁇ or ⁇ casein, histone HI, myelin basic protein, etc
  • the R3DAK mediated transfer of radioactive phosphate from ATP to the substrate protein or substrate peptide can be determined by methods well known in the art, such as, for example, spotting the radioactive products onto phosphocellulose paper, followed by washing and liquid scintillation counting, gel eletrophoresis followed by autoradiography, and scintillation proximity assay
  • the purpose of such an assay is to identify substances
  • R3DAK like other kinases, could play a central role in apoptosis which involves cellular signal transduction pathways As such, alterations in the expression and/or activation of R3DAK can have profound effects on the plethora of cellular processes Expression of cloned R3DAK, functionally inactive mutants of R3DAK, or the kinase domain can be used to identify the role a particular protein plays in mediating specific signaling events
  • TUNEL terminal transferase UTP nick end labeling
  • polypeptides of the present invention may also be used in a screening assay to identify compounds and small molecules which inhibit (antagonize) or enhance (agonize) activation of the polypeptides of the instant invention
  • polypeptides of the invention may be used to identify antagonists and agonists from cells, cell-free preparations, chemical libraries, and natural product mixtures
  • the antagonists and agonists may be natural or modified substrates, gands, enzymes, receptors, etc of the polypeptides of the instant invention, or may be structural or functional mimetics of the polypeptides
  • Potential antagonists of the polypeptides of the instant invention may include small molecules, peptides, and antibodies that bind to and occupy a binding site of the polypeptides, causing them to be unavailable to bind to their hgands and therefore preventing normal biological activity
  • Other potential antagonists are antisense molecules which may hybridize to mRNA in vivo and block translation of the mRNA into the polypeptides of the instant invention
  • Antibodies which include intact molecules as well as fragments such as Fab and F(ab')2 fragments, may be used to bind to and inhibit the polypeptides of the instant invention by blocking the commencement of a signaling cascade It is preferable that the antibodies are humanized, and more preferable that the antibodies are human
  • the antibodies of the present invention may be prepared by any of a variety of well-known methods
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, cell based assays, etc These assay formats are well known in the art
  • the screening assays of the present invention are amenable to screening of chemical libraries and are suitable for the identification of small molecule drug candidates, antibodies, peptides and other antagonists and agonists
  • One embodiment of a method for identifying molecules which antagonize or inhibit the polypeptides involves adding a candidate molecule to a medium which contains cells that express the polypeptides of the instant invention, changing the conditions of said medium so that, but for the presence of the candidate molecule, the polypeptides would be bound to their hgands, and observing the binding and stimulation or inhibition of a functional response
  • the activity of the cells which were contacted with the candidate molecule may then be compared with the identical cells which were not contacted and agonists and antagonists of the polypeptides of the instant invention may be identified
  • the measurement of biological activity may be performed by a number of well-known methods such as measuring the amount of protein present (e g an ELISA) or of the protein's activity A decrease in biological stimulation or activation would indicate an antagonist
  • An increase would indicate an agonist
  • one embodiment of the instant invention includes agonists and antagonists of R3DAK
  • Screening assays can further be designed to find molecules that mimic the biological activity of the polypeptides of the instant invention Molecules which mimic the biological activity of a polypeptide may be useful for enhancing the biological activity of the polypeptide
  • Molecules which mimic the biological activity of a polypeptide may be useful for enhancing the biological activity of the polypeptide
  • a binding candidate molecule is then added to a biological assay to determine its biological effects The biological effects of the candidate molecule are then compared to those of the polypeptide
  • the nucleic acids encoding the R3DAK polypeptides provided by the present invention can be used for numerous diagnostic or other useful purposes
  • the nucleic acids of the invention can be used to express recombinant polypeptide for analysis, characterization or therapeutic use, as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states), as molecular weight markers on Southern gels, as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions, to compare with endogenous DNA sequences m patients to identify potential genetic disorders, as probes to hybridize and thus discover novel, related DNA sequences, as a source of information to derive PCR primers for genetic fingerprinting, as a probe to "subtract-out" known sequences in the process of discovering other novel nucleic acids, for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns, to raise anti-
  • Probes and Primers Among the uses of the disclosed R3DAK nucleic acids, and combinations of fragments thereof, is the use of fragments as probes or primers Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence In other embodiments, a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence
  • a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence
  • sets of degenerate oligonucleotides can be prepared Such oligonucleotides are useful as primers, e g , in polymerase cha reactions (PCR), whereby DNA fragments are isolated and amplified
  • degenerate primers can be used as probes for non- human genetic libraries Such libraries would include but are not limited to cDNA libraries, genomic libraries, and even electronic EST (ex
  • nucleic acids encoding R3DAK polypeptides can be used by those skilled in the art using well-known techniques to identify the human chromosome to which these nucleic acids map
  • Useful techniques include, but are not limited to, using the sequence or portions, including oligonucleotides, as a probe in various well-known techniques such as radiation hybrid mapping (high resolution), in situ hybridization to chromosome spreads (moderate resolution), and Southern blot hybridization to hybrid cell lines containing individual human chromosomes (low resolution)
  • radiation hybrid mapping high resolution
  • in situ hybridization to chromosome spreads moderate resolution
  • Southern blot hybridization to hybrid cell lines containing individual human chromosomes
  • chromosomes can be mapped by radiation hybridization
  • PCR is performed using the Whitehead Institute/MIT Center for Genome Research Geneb ⁇ dge4 panel of 93 radiation hybrids http //www-genome wi mit edu ftp/d ⁇ str ⁇ but ⁇ on/human_STS_
  • nucleic acids encoding R3DAK polypeptides, and the disclosed fragments and combinations of these nucleic acids can be used by one skilled in the art using well-known techniques to analyze abnormalities associated with the genes corresponding to these polypeptides This enables one to distinguish conditions in which this marker is rearranged or deleted
  • nucleic acids of the invention or a fragment thereof can be used as a positional marker to map other genes of unknown location
  • the DNA can be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, the genes corresponding to the nucleic acids of the invention Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement thereof with normal genes Defective genes can be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in this gene
  • the R3DAK polypeptides of the invention each can be used as reagents in methods to screen for or identify binding partners
  • the R3DAK polypeptides can be attached to a solid support material and may bind to their binding partners in a manner similar to affinity chromatography
  • a polypeptide is attached to a solid support by conventional procedures
  • chromatography columns containing functional groups that will react with functional groups on ammo acid side chains of polypeptides are available (Pharmacia Biotech, Inc , Piscataway, NJ)
  • a polypeptide/Fc polypeptide (as discussed above) is attached to Polypeptide A- or Polypeptide G-containing chromatography columns through interaction with the Fc moiety
  • the R3DAK polypeptides also find use in identifymg cells that express a binding partner on the cell surface Polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate For example, magnetic microporous aluminum silicates, a polypeptides are
  • Measuring Biological Activity Polypeptides also find use in measuring the biological activity of R3DAK-b ⁇ nd ⁇ ng polypeptides in terms of their binding affinity
  • the polypeptides thus can be employed by those conducting "quality assurance" studies, e g , to monitor shelf life and stability of polypeptide under different conditions
  • the polypeptides can be employed in a binding affinity study to measure the biological activity of a binding partner polypeptide that has been stored at different temperatures, or produced in different cell types
  • the polypeptides also can be used to determine whether biological activity is retained after modification of a binding partner polypeptide (e g , chemical modification, truncation, mutation, etc )
  • the binding affinity of the modified polypeptide is compared to that of an unmodified binding polypeptide to detect any adverse impact of the modifications on biological activity of the binding polypeptide
  • the biological activity of a binding polypeptide thus can be ascertained before it is used m a research study, for example
  • the polypeptides also find use as carriers for delivering agents attached thereto within cells in which they are expressed
  • the polypeptides thus can be used to deliver diagnostic or therapeutic agents within such cells in ex vivo, in vitro, or in vivo procedures
  • Detectable (diagnostic) and therapeutic agents that can be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuc des, chromophores, enzymes that catalyze a colo ⁇ met ⁇ c or fluoromet ⁇ c reaction, and the like, with the particular agent being chosen according to the intended application
  • the toxins are ⁇ cin, ab ⁇ n, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ⁇ bosomal inactivating polypeptides, mycotoxins such as t ⁇ chothecenes, and derivatives and fragments (e g , single chains) thereof
  • Radionuchdes suitable for diagnostic use include, but are
  • R3DAK polypeptides, fragments, variants, antagonists, agonists, antibodies, and binding partners of the invention will be useful for treating medical conditions and diseases including, but not limited to, conditions involving kinase activity or apoptosis as described further herein.
  • the therapeutic molecule or molecules to be used will depend on the etiology of the condition to be treated and the biological pathways involved, and variants, fragments, and binding partners of R3DAK polypeptides may have effects similar to or different from R3DAK polypeptides.
  • R3DAK polypeptides or antagonists refers to all R3DAK polypeptides, fragments, variants, antagonists, agonists, antibodies, and binding partners etc. of the invention, and it is understood that a specific molecule or molecules can be selected from those provided as embodiments of the invention by individuals of skill in the art, according to the biological and therapeutic considerations described herein.
  • R3DAK polypeptides or antagonists, compositions and combination therapies described herein are useful in medicines for treating bacterial, viral or protozoal infections, and complications resulting therefrom.
  • One such disease is Mycoplasma pneumonia.
  • R3DAK polypeptides or antagonists to treat AIDS and related conditions, such as AIDS dementia complex, AIDS associated wasting, lipidistrophy due to antiretroviral therapy; and Kaposi's sarcoma.
  • R3DAK polypeptides or antagonists for treating protozoal diseases, including malaria and schistosomiasis.
  • R3DAK polypeptides or antagonists to treat erythema nodosum leprosum; bacterial or viral meningitis; tuberculosis, including pulmonary tuberculosis; and pneumonitis secondary to a bacterial or viral infection.
  • R3DAK polypeptides or antagonists to prepare medicaments for treating louse-bome relapsing fevers, such as that caused by Borrelia recurrentis.
  • the R3DAK polypeptides or antagonists of the invention can also be used to prepare a medicament for treating conditions caused by Herpes viruses, such as herpetic stromal keratitis, comeal lesions, and virus-induced corneal disorders.
  • R3DAK polypeptides or antagonists can be used in treating human papillomavirus infections.
  • the R3DAK polypeptides or antagonists of the invention are used also to prepare medicaments to treat influenza.
  • Cardiovascular disorders are treatable with the disclosed R3DAK polypeptides or antagonists, pharmaceutical compositions or combination therapies, including aortic aneurisms; arteritis; vascular occlusion, including cerebral artery occlusion; complications of coronary by-pass surgery; ischemia/reperfusion injury; heart disease, including atherosclerotic heart disease, myocarditis, including chronic autoimmune myocarditis and viral myocarditis; heart failure, including chronic heart failure (CHF), cachexia of heart failure; myocardial infarction; restenosis after heart surgery; silent myocardial ischemia; post-implantation complications of left ventricular assist devices; Raynaud's phenomena; thrombophlebitis; vasculitis, including Kawasaki's vasculitis; giant cell arteritis, Wegener's granulomatosis; and Schoenlein- Henoch purpura.
  • aortic aneurisms including cerebral artery occlusion
  • a combination of at least one R3DAK polypeptide or antagonist and one or more other anti- angiogenesis factors may be used to treat solid tumors, thereby reducing the vascularization that nourishes the tumor tissue.
  • Suitable anti-angiogenic factors for such combination therapies include IL-8 inhibitors, angiostatin, endostatin, kringle 5, inhibitors of vascular endothelial growth factor (such as antibodies against vascular endothelial growth factor), angiopoietin-2 or other antagonists of angiopoietin- 1 , antagonists of platelet-activating factor and antagonists of basic fibroblast growth factor
  • R3DAK polypeptides or antagonists, compositions and combination therapies are used to treat chronic pain conditions, such as chronic pelvic pain, including chronic prostatitis/pelvic pain syndrome.
  • R3DAK polypeptides or antagonists and the compositions and combination therapies of the invention are used to treat post-herpetic pain.
  • R3DAK polypeptides or antagonists are used to treat various disorders of the endocrine system.
  • the R3DAK polypeptides or antagonists are used to treat juvenile onset diabetes (includes autoimmune and insulin- dependent types of diabetes) and also to treat maturity onset diabetes (includes non-insulin dependent and obesity-mediated diabetes).
  • the subject compounds, compositions and combination therapies are used to treat secondary conditions associated with diabetes, such as diabetic retinopathy, kidney transplant rejection in diabetic patients, obesity-mediated insulin resistance, and renal failure, which itself may be associated with proteinurea and hypertension.
  • endocrine disorders also are treatable with these compounds, compositions or combination therapies, including polycystic ovarian disease, X-Iinked adrenoleukodystrophy, hypothyroidism and thyroiditis, including Hashimoto's thyroiditis (i.e., autoimmune thyroiditis).
  • Conditions of the gastrointestinal system also are treatable with R3DAK polypeptides or antagonists, compositions or combination therapies, including coeliac disease.
  • the compounds, compositions and combination therapies of the invention are used to treat Crohn's disease; ulcerative colitis; idiopathic gastroparesis; pancreatitis, including chronic pancreatitis and lung injury associated with acute pancreatitis; and ulcers, including gastric and duodenal ulcers.
  • R3DAK polypeptides or antagonists, compositions or combination therapies for treating disorders of the genitourinary system, such as glomerulonephritis, including autoimmune glomerulonephritis, glomerulonephritis due to exposure to toxins or glomerulonephritis secondary to infections with haemolytic streptococci or other infectious agents.
  • glomerulonephritis including autoimmune glomerulonephritis, glomerulonephritis due to exposure to toxins or glomerulonephritis secondary to infections with haemolytic streptococci or other infectious agents.
  • uremic syndrome and its clinical complications for example, renal failure, anemia, and hypertrophic cardiomyopathy
  • Further conditions treatable with the compounds, compositions and combination therapies of the invention are complications of hemodialysis; prostate conditions, including benign prostatic hypertrophy, nonbacterial prostatitis and chronic prostatitis; and complications of hemodialysis.
  • R3DAK polypeptides or antagonists are used to treat various forms of cancer, including acute myelogenous leukemia, Epstein-Barr virus-positive nasopharyngeal carcinoma, g oma, colon, stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer (SCLC and NSCLC), including cancer-associated cachexia, fatigue, asthenia, paraneoplastic syndrome of cachexia and hypercalcemia
  • R3DAK polypeptides or antagonists, compositions or combination therapies are solid tumors, including sarcoma, osteosarcoma, and carcinoma, such as adenocarcinoma (for example, breast cancer) and squamous cell carcinoma
  • the subject compounds, compositions or combination therapies are useful for treating leukemia, including acute myelogenous leukemia, chronic or acute
  • lymphoproliferative disorders also are treatable with the disclosed R3DAK polypeptides or antagonists, compositions or combination therapies
  • autoimmune lymphoproliferative syndrome ALPS
  • chronic lymphoblastic leukemia hairy cell leukemia
  • chronic lymphatic leukemia peripheral T-cell lymphoma
  • small lymphocytic lymphoma mantle cell lymphoma
  • follicular lymphoma Burkitt's lymphoma
  • Epstein-Barr virus-positive T cell lymphoma histiocytic lymphoma
  • Hodgk 's disease diffuse aggressive lymphoma, acute lymphatic leukemias, T gamma lymphoproliferative disease, cutaneous B cell lymphoma, cutaneous T cell lymphoma (I e , mycosis fungoides) and Sezary syndrome
  • R3DAK polypeptides or antagonists, compositions and combination therapies are used to treat hereditary conditions such as Gaucher's disease, Huntington's disease, linear IgA disease, and muscular dystrophy
  • R3DAK polypeptides or antagonists, compositions and combination therapies include those resulting from injuries to the head or spmal cord, and including subdural hematoma due to trauma to the head
  • the disclosed R3DAK polypeptides or antagonists, compositions and combination therapies are further used to treat conditions of the liver such as hepatitis, including acute alcoholic hepatitis, acute drug- induced or viral hepatitis, hepatitis A, B and C, scleros g cholangitis and inflammation of the liver due to unknown causes
  • the disclosed R3DAK polypeptides or antagonists, compositions and combination therapies are used to treat various disorders that involve hearing loss and that are associated with abnormal TNF ⁇ expression
  • One of these is inner ear or cochlear nerve-associated hearing loss that is thought to result from an autoimmune process, l e , autoimmune hearing loss
  • This condition currently is treated with steroids, methotrexate and/or cyclophosphamide, which may be administered concurrently with the R3DAK polypeptides or antagonists
  • cholesteatoma a middle ear disorder often associated with hearing loss
  • the subject invention provides R3DAK polypeptides or antagonists, compositions and combination therapies for the treatment of non-arthritic medical conditions of the bones and joints
  • osteoclast disorders that lead to bone loss, such as but not limited to osteoporosis, including post-menopausal osteoporosis, pe ⁇ odontitis resulting in tooth loosening or loss, and prosthesis loosening after joint replacement (generally associated with an inflammatory response to wear debris)
  • This latter condition also is called “orthopedic implant osteolysis
  • Another condition treatable by administering R3DAK polypeptides or antagonists, is temporal mandibular joint dysfunction (TMJ)
  • TMJ temporal mandibular joint dysfunction
  • a number of pulmonary disorders also can be treated with the disclosed R3DAK polypeptides or antagonists, compositions and combination therapies
  • One such condition is adult respiratory distress syndrome (ARDS), which is associated with elevated TNF ⁇ , and may be triggered by a variety of causes, including exposure to toxic chemicals, pancreatiti
  • Cystic fibrosis is an inherited condition characterized primarily by the accumulation of thick mucus, predisposing the patient to chronic lung infections and obstruction of the pancreas, which results in maiabsorption of nutrients and malnutrition
  • R3DAK polypeptides or antagonists may be administered to treat cystic fibrosis
  • treatment with R3DAK polypeptides or antagonists may be administered concurrently with corticosteroids, mucus-thinning agents such as inhaled recombinant deoxy ⁇ bonuclease I (such as PULMOZYME ® , Genentech, Inc ) or inhaled tobramycin (TOBI ® , Pathogenesis, Inc )
  • the R3DAK polypeptides or antagonists of the invention also may be administered concurrently with corrective gene therapy, drugs that stimulate cystic fibrosis cells to secrete chloride or other yet-to-be-discovered treatments Sufficiency of treatment may be assessed, for example, by observmg a decrease in the
  • the R3DAK polypeptides or antagonists of the invention may be used for treating cystic fibrosis or fibrotic lung diseases, such as ldiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis and bleomycin-induced pulmonary fibrosis
  • cystic fibrosis or fibrotic lung diseases such as ldiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis and bleomycin-induced pulmonary fibrosis
  • this combination is useful for treating other diseases characterized by organ fibrosis, including systemic sclerosis (also called "scleroderma"), which often involves fibrosis of the liver
  • R3DAK polypeptides or antagonists and IFN ⁇ -lb may be combined with PULMOZYME ® or TOBI ® or other treatments for cystic fibrosis
  • the R3DAK polypeptides or antagonists of the invention alone or in combination with IFN ⁇ -lb may be administered together with other treatments presently used for treating fibrotic lung disease
  • additional treatments include glucocorticoids, azathiop ⁇ ne cyclophosphamide, penicillamine, colchisicine, supplemental oxygen and so forth
  • Patients with fibrotic lung disease, such as IPF often present with nonproductive cough, progressive dyspnea and show a restrictive ventilatory pattern in pulmonary function tests Chest radiographs reveal fibrotic accumulations in the patient's lungs
  • sufficiency of treatment may be detected by observing a decrease in the patient's coughing (when cough is present), or by using standard lung function tests to detect improvements in total lung capacity, vital capacity, residual lung volume or by administering a arterial blood gas determination measuring desaturation under exercising conditions, and showing that the patient's lung function has improved according to one or more of these measures
  • patient improvement may be determined through chest
  • R3DAK polypeptides or antagonists are useful for treating organ fibrosis when administered in combination with relaxin, a hormone that down-regulates collagen production thus inhibiting fibrosis, or when given in combination with agents that block the fibrogenic activity of TGF- ⁇
  • Combination therapies using R3DAK polypeptides or antagonists and recombinant human relaxin are useful, for example, for treating systemic sclerosis or fibrotic lung diseases, including cystic fibrosis, ldiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis and bleomycin-induced pulmonary fibrosis
  • R3DAK polypeptides or antagonists, compositions or combination therapies provide methods for using the disclosed R3DAK polypeptides or antagonists, compositions or combination therapies to treat a variety of rheumatic disorders These include adult and juvenile rheumatoid arthritis, systemic lupus erythematosus, gout, osteoarth ⁇ tis, polymyalgia rheumatica, seronegative spondylarthropathies, including ankylosing spondy tis, and Reiter's disease
  • the subject R3DAK polypeptides or antagonists, compositions and combination therapies are used also to treat pso ⁇ atic arthritis and chronic Lyme arthritis Also treatable with these compounds, compositions and combination therapies are Still's disease and uveitis associated with rheumatoid arthritis
  • the compounds, compositions and combination therapies of the invention are used in treating disorders resulting in inflammation of the voluntary muscle, including dermatomyositis and polymyositis
  • the R3DAK polypeptides or antagonists, compositions and combination therapies of the invention may be used to inhibit hypertrophic scarring, a phenomenon believed to result in part from excessive TNF ⁇ secretion
  • the R3DAK polypeptides or antagonists of the invention may be administered alone or concurrently with other agents that inhibit hypertrophic scarring, such as inhibitors of TGF- ⁇
  • Cervicogenic headache is a common form of headache arising from dysfunction in the neck area, and which is associated with elevated levels of TNF ⁇ , which are believed to mediate an inflammatory condition that contributes to the patient's discomfort (Martelletti, Chn Exp Rheumatol 18(2 Suppl 19) S33-8
  • Cervicogenic headache may be treated by administering R3DAK polypeptides or antagonists as disclosed herein, thereby reducing the inflammatory response and associated headache pam
  • R3DAK polypeptides or antagonists, compositions and combination therapies of the invention are useful for treating primary amyloidosis
  • the secondary amyloidosis that is characteristic of various conditions also are treatable with R3DAK polypeptides or antagonists such as R3DAK polypeptides or antagonists, and the compositions and combination therapies described herein
  • Such conditions include Alzheimer's disease, secondary reactive amyloidosis, Down's syndrome, and dialysis-associated amyloidosis
  • Also treatable with the compounds, compositions and combination therapies of the invention are inherited periodic fever syndromes, including familial Mediterranean fever, hype ⁇ mmunoglobu n D and periodic fever syndrome and TNF-receptor associated periodic syndromes (TRAPS)
  • R3DAK polypeptides or antagonists such as graft-versus-host disease, and complications resulting from solid organ transplantation, including transplantion of heart, liver, lung, skin, kidney or other organs
  • R3DAK polypeptides or antagonists may be administered, for example, to prevent or inhibit the development of bronchiolitis ob terans after lung transplantation
  • Ocular disorders also are treatable with the disclosed R3DAK polypeptides or antagonists, compositions or combination therapies, including rhegmatogenous retinal detachment, and inflammatory eye disease, and inflammatory eye disease associated with smoking and macular degeneration
  • R3DAK polypeptides or antagonists of the invention and the disclosed compositions and combination therapies also are useful for treating disorders that affect the female reproductive system Examples include, but are not limited to, multiple implant failure/infertility, fetal loss syndrome or IV embryo loss (spontaneous abortion), preeclamptic pregnancies or eclampsia, and endomet ⁇ osis
  • the disclosed R3DAK polypeptides or antagonists, compositions and combination therapies are useful for treating obesity, including treatment to bring about a decrease in leptin formation
  • the compounds, compositions and combination therapies of the invention are used to treat sciatica, symptoms of aging, severe drug reactions (for example, 11-2 toxicity or bleomycm-induced pneumopathy and fibrosis), or to suppress the inflammatory response prior, during or after the transfusion of allogeneic red blood cells in cardiac or other surgery, or in treating a traumatic injury to a limb or joint, such as traumatic knee injury
  • Various other medical disorders treatable with the disclosed R3DAK polypeptides or antagonists, compositions and combination therapies include multiple sclerosis, Behcet's syndrome, Sjogren's syndrome, autoimmune hemolytic anemia, beta thalassemia, amyotrophic lateral sclerosis (Lou Geh ⁇ g's Disease), Parkinson's disease, and tenosynovitis of unknown cause, as well as various autoimmune disorders or
  • disorders involving the skin or mucous membranes also are treatable using the disclosed R3DAK polypeptides or antagonists, compositions or combination therapies
  • Such disorders include acantholytic diseases, including Da ⁇ er's disease, keratosis fol cularis and pemphigus vulga ⁇ s
  • acantholytic diseases including Da ⁇ er's disease, keratosis fol cularis and pemphigus vulga ⁇ s
  • Also treatable with the subject R3DAK polypeptides or antagonists, compositions and combination therapies are acne, acne rosacea, alopecia areata, aphthous stomatitis, bullous pemphigoid, burns, eczema, erythema, including erythema multiforme and erythema multiforme bullosum (Stevens-Johnson syndrome), inflammatory skin disease, lichen planus, linear IgA bullous disease (chronic bullous dermato
  • the R3DAK polypeptides or antagonists of the invention may also exhibit one or more of the following additional activities or effects inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites, effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape), effecting biorhythms or ca ⁇ cadic cycles or rhythms, effecting the fertility of male or female subjects, effecting the metabolism, catabohsm, anabo sm, processing, utilization, storage or elimination of dietary fat, lipid, polypeptide, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s), effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors, providing
  • This invention provides compounds, compositions, and methods for treating a patient, preferably a mammalian patient, and most preferably a human patient, who is suffering from a medical disorder, and in particular a R3DAK-med ⁇ ated disorder
  • R3DAK-med ⁇ ated disorders include conditions caused (directly or indirectly) or exacerbated by bindmg between R3DAK and a binding partner
  • the terms "illness,” “disease,” “medical condition,” “abnormal condition” and the like are used interchangeably with the term “medical disorder "
  • the terms “treat”, “treating”, and “treatment” used herein includes curative, preventative (e g , prophylactic) and palliative or ameliorative treatment
  • R3DAK polypeptides and fragments, R3DAK nucleic acids encoding the R3DAK polypeptides, and/or agonists or antagonists of the R3DAK polypeptide such as antibodies can be administered to the patient in need through well-known
  • Suitable dosages will vary, depending upon such factors as the nature and severity of the disorder to be treated, the patient's body weight, age, general condition, and prior illnesses and/or treatments, and the route of administration
  • Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices such as standard dosing trials
  • the therapeutically effective dose can be estimated initially from cell culture assays The dosage will depend on the specific activity of the compound and can be readily determined by routine experimentation
  • a dose may be formulated animal models to achieve a circulating plasma concentration range that includes the IC50 (/ e , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture, while minimizing toxicities
  • the attending physician will decide the amount of polypeptide of the present invention with which to treat each individual patient Initially, the attending physician will administer low doses of polypeptide of the present invention
  • compositions comprising an effective amount of a R3DAK polypeptide of the present invention (from whatever source derived, including without limitation from recombinant and non- recombinant sources), in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active mgred ⁇ ent(s)
  • Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents
  • the polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically
  • R3DAK polypeptide of the present invention may be active in multimers (e g , heterodimers or homodimers) or complexes with itself or other polypeptides
  • pharmaceutical compositions of the invention may comprise a polypeptide of the invention in such multime ⁇ c or complexed form
  • the pharmaceutical composition of the invention may be in the form of a complex of the polypept ⁇ de(s) of present invention along with polypeptide or peptide antigens
  • the invention further includes the administration of R3DAK polypeptides or antagonists concurrently with one or more other drugs that are administered to the same patient in combination with the R3DAK polypeptides or antagonists, each drug being administered according to a regimen suitable for that medicament "Concurrent administration" encompasses simultaneous or sequential treatment with the components of the combination, as well as regimens in which the drugs are alternated, or wherein one component is administered long-term and the other(s) are administered intermittently Components may be administered in the same or in separate compositions, and
  • R3DAK polypeptides or antagonists thereof include those compositions comprising nucleic acids
  • Parenteral administration includes injection, for example, via intra-articular, intravenous, intramuscular, mtralesional, lntrape ⁇ toneal or subcutaneous routes by bolus injection or by continuous infusion , and also includes localized administration, e g , at a site of disease or injury
  • Other suitable means of administration include sustained release from implants, aerosol inhalation and/or insufflation , eyedrops, vaginal or rectal suppositories, buccal preparations, oral preparations, including pills, syrups, lozenges or chewing gum; and topical preparations such as lotions, gels, sprays, ointments or other suitable techniques
  • polypeptideaceous R3DAK polypeptides or antagonists may be administered by implanting cultured cells that express the polypeptide, for example, by implanting cells that express R3DAK polypeptide, for example, by implanting cells that express R3DA
  • polypeptide of the present invention When administered orally, polypeptide of the present invention will be in the form of a tablet, capsule, powder, solution or elixir
  • the pharmaceutical composition of the invention may additionally contain a solid ca ⁇ ier such as a gelatin or an adjuvant
  • the tablet, capsule, and powder contain from about 5 to 95% polypeptide of the present invention, and preferably from about 25 to 90% polypeptide of the present invention
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccha ⁇ de solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol
  • the pharmaceutical composition contains from about 0 5 to 90% by weight of polypeptide of the present invention, and preferably from about 1 to 50% polypeptide of
  • polypeptide of the present invention When a therapeutically effective amount of polypeptide of the present invention is administered by intravenous, cutaneous or subcutaneous injection, polypeptide of the present invention will be the form of a pyrogen-free, parenterally acceptable aqueous solution
  • polypeptide of the present invention will be the form of a pyrogen-free, parenterally acceptable aqueous solution
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to polypeptide of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than a polypeptide of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering the polypeptide-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • a matrix capable of delivering the polypeptide-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure polypeptides or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate- phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethyl-cellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorbtion of the polypeptide from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the polypeptide the opportunity to assist the osteogenic activity of the progenitor cells
  • polypeptides of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- alpha and TGF- beta ), and insulin-like growth factor (IGF)
  • EGF epidermal growth factor
  • the original cDNA clone from which the sequence is derived was obtained from a rat dermal papilla cell library that is maintained by Genesis Research and Development Co ⁇ . Ltd. (Aukland, New Zealand).
  • a partial sequence from this clone was identified as a putative kinase, and subsequent double- strand sequencing extended the sequence of the clone and confirmed its identity as a kinase (see Table 2).
  • R3DAK amino acid sequence (SEQ ID NO:2) are predicted to be more likely to alter or disrupt R3DAK polypeptide activities if they result in changes to the residues of SEQ ID NO:2 indicated by a vertical line as shown in Table 2, and particularly if those changes do not substitute a residue present in other kinase polypeptides at that conserved position for the R3DAK residue
  • R3DAK amino acid sequence resulting in substitution of one or more conserved kinase sequence residues for the R3DAK residue at that corresponding position, it is less likely that such an alteration will affect R3DAK polypeptide function
  • Embodiments of the invention include R3DAK polypeptides and fragments of R3DAK polypeptides comprising altered ammo acid sequences Altered R3DAK polypeptide sequences share at least 30%, or more preferably at least 40% or more preferably at least
  • a substrate mix [100 1 per well of a 1 1 premix of the TMB Peroxidase Substrate and Peroxidase Solution B (Kirkegaard Perry Laboratories, Gaithersburg, Maryland)] is added to the wells After sufficient color reaction, the enzymatic reaction is terminated by addition of 2N H 2 S0 4 (50 1 per well) Color intensity (indicating R3DAK-b ⁇ ndmg activity) is determined by measuring extinction at 450 nm on a V Max plate reader (Molecular Devices, Sunnyvale, CA)
  • This example illustrates a method for preparing monoclonal antibodies that bind R3DAK
  • Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified R3DAK polypeptide or an lmmunogenic fragment thereof such as the extracellular domain, or fusion proteins containing R3DAK (e g , a soluble R3DAK Fc fusion protein)
  • R3DAK can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in U S Patent 4,411,993 Briefly, mice are immunized with R3DAK immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 micrograms subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional R3DAK immunogen emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule.
  • Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for R3DAK antibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay), inhibition of R3DAK-binding partner binding or inhibition of kinase catalytic activity.
  • dot blot assay ELISA (Enzyme-Linked Immunosorbent Assay)
  • inhibition of R3DAK-binding partner binding or inhibition of kinase catalytic activity.
  • spleen cells are fused to a murine myeloma cell line, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hypoxanthine, aminopterin and thymidine
  • hybridoma cells are screened by ELISA for reactivity against purified R3DAK by adaptations of the techniques disclosed in Engvall et al., Immunochem. 8:871 , 1971 and in U.S. Patent 4,703,004.
  • a preferred screening technique is the antibody capture technique described in Beckmann et al., (J. Immunol. 144:4212, 1990).
  • Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti- R3DAK monoclonal antibodies.
  • hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques.
  • Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography.
  • affinity chromatography based upon binding of antibody to Protein A or Protein G can also be used, as can affinity chromatography based upon binding to R3DAK.
  • the tissue distribution of R3DAK mRNA was investigated by Northern blot analysis, as follows. An aliquot of a radiolabeled probe was added to two different human multiple tissue Northern blots
  • Hybridization was conducted as recommended by the manufacturer and using Clontech's ExpressHyb hybridization solution. The post-hybridization wash protocol was also as described by the manufacturer.
  • Nucleic acids encoding R3DAK polypeptides may be isolated by various techniques known to those skilled in the art. As described below, ohgonucleotide primers may be designed on the basis of conserved amino acid sequences present in rat R3DAK and other kinases. An example of conserved R3DAK kinase amino acid sequences are indicated in Table 2 above.
  • a pool of 'degenerate' ohgonucleotide primers could be designed to include every codon at each position that would encode the conserved stretch of amino acids from amino acid 205 to amino acid 216 of SEQ ID NO:2, and all of these 'degenerate' oligonucleotides may then be synthesized simultaneously on an automated DNA synthesizer by optionally adding a mixture of nucleotides at any given step in the synthesis.
  • such oligonucleotides also include at their 5' ends the recognition sequence for a restriction endonuclease in order to facilitate the manipulation of a specifically amplified nucleic acid sequence.
  • the R3DAK amino acid sequence is used to design sets of ohgonucleotide primers which will specifically amplify a portion of the R3DAK-encoding sequence located in the region between the primers utilized to perform the specific amplification reaction. It is contemplated that such R3DAK-derived primers would allow one to specifically amplify corresponding R3DAK-encoding sequences from mRNA, cDNA, or genomic DNA templates obtained from any species, preferably Homo sapiens, Mus musculus, or another mammalian or vertebrate species.
  • Ohgonucleotide primers designed on the basis of the R3DAK sequences are predicted to allow the specific amplification of human R3DAK-encoding nucleic acid sequences from pre-established human cDNA libraries which are commercially available from companies such as Stratagene (La Jolla, California) or Clontech Laboratories, Inc. (Palo Alto, California).
  • a cDNA library may be constructed: mRNA is selected by oligo (dT) cellulose chromatography and cDNA is synthesized and cloned using established techniques into lambda gtlO or other lambda bacteriophage vectors known to those skilled in the art, for example, lambda ZAP. It is also possible to perform the ohgonucleotide primer directed amplification reaction, described above, directly on a pre-established human cDNA or genomic library which has been cloned into a lambda bacteriophage vector.
  • a library which yields a specifically amplified nucleic acid product encoding a portion of the human R3DAK polypeptide could be screened directly, utilizing the fragment of amplified R3DAK-encoding nucleic acid as a probe. Analogous procedures can be used to isolate R3DAK-encoding nucleic acids from any species.
  • Genomic DNA from any species can be used as a template to perform specific amplification reactions which would result in the identification of R3DAK-encoding nucleic acids.
  • Genomic DNA (such as human genomic DNA) is sheared by repeated passage through a 25 gauge needle, denatured at 100 degrees C for 5 minutes and then chilled on ice before adding to a reaction mixture containing 200 microM each deoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP), 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.001% gelatin, 1.25 units Taq DNA polymerase, and 100 pM of each ohgonucleotide primer, where each primer has a predicted hybridization temperature of 55 degrees C.
  • This reaction mixture is incubated at 94 degrees C for two minutes and then subjected to thermal cycling in the following manner: 1 minute at 94 degrees C, 1 minute at 40 degrees C, 1 minute at 72 degrees C for three cycles; then 1 minute at 94 degrees C, 1 minute at 55 degrees C, 1 minute at 72 degrees C for thirty-seven cycles; followed by a 10 minute incubation at 72 degrees C.
  • the DNA which is specifically amplified by this reaction is ethanol precipitated, digested with selected restriction endonucleases and subjected to agarose gel electrophoresis.
  • the digested DNA product is diluted in 10 mM Tris-HCl pH 8.0, 1 mM EDTA followed by centrifugation through a CentriconTM 30 microconcentrator (W. R. Grace & Co., Beverly, Md.; Product #4209), cloned into a plasmid vector, and these clones are then sequenced to confirm the cloned inserts as R3DAK- encoding fragments.
  • the gene corresponding to the R3DAK coding sequence disclosed herein is mapped using PCR- based mapping strategies.
  • Initial human chromosomal assignments are made using R3DAK-specific PCR primers and a BIOS Somatic Cell Hybrid PCRable DNA kit from BIOS Laboratories (New Haven, CT), following the manufacturer's instructions. More detailed mapping is performed using a Genebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville, AL; described in Walter, MA et al., Nature Genetics 7:22-28, 1994).
  • Isolated R3DAK polypeptides or fusion proteins containing the isolated protein kinase domain of R3DAK can be used in an assay of protein kinase activity. Typically this would be carried out by combining R3DAK with radiolabeled ATP( ⁇ 32 P-ATP) and a magnesium salt in buffer solution containing a peptide or protein substrate.
  • the peptides substrates are generally from 8-30 amino acids in length and may terminate at the N- or C-terminus with three or more lysine or arginine residues to facilitate binding of the peptide to phosphocellulose paper.
  • the substrates may also be a protein known to phosphorylated readily by R3DAK.
  • kinase substrates such as ⁇ or ⁇ casein, histone HI, myelin basic protein, etc.
  • the transfer of radioactive phosphate from ATP to the substrate protein or substrate peptide may be monitored, by spotting of the reaction mixture onto phosphocellulose paper, and subsequent washing of the paper with a dilute solution of phosphoric acid, in the case of a peptide substrate, or by application of the reaction products to a gel electrophoresis system followed by autoradiographic detection in the case of proteins.
  • Other methods are available to conveniently measure the R3DAK-meidated transfer of phosphaste to substrate proteins, such as the scintillation proximity assay. These methods are well known to those practiced in the art.

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