WO2001019990A1

WO2001019990A1 - Rip-3-like death-associated kinase

Info

Publication number: WO2001019990A1
Application number: PCT/US2000/025201
Authority: WO
Inventors: G. Duke Virca; Timothy A. Bird
Original assignee: Immunex Corporation
Priority date: 1999-09-17
Filing date: 2000-09-15
Publication date: 2001-03-22
Also published as: WO2001019990B1; AU1188001A; EP1214414A2

Abstract

The invention is directed to purified and isolated novel R3DAK polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, agonists and antagonists of the activities of these polypeptides, and the uses of the above.

Description

RIP-3-LIKE DEATH-ASSOCIATED KINASE

BACKGROUND OF THE INVENTION Field of the Invention The invention is directed to purified and isolated novel RIP-3-Lιke 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.

Description of Related Art

Cell death occurs by one of two mechanisms, necrosis or apoptosis. Necrosis is uncontrolled cell death, which usually results from environmental stresses such as severe trauma to cells Cell death from apoptosis, however, results from specific internal signals that activate a cell's death program Therefore, 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. Programmed cell death has been observed during early development and in immune responses where cells, such as lymphocytes, are eliminated via apoptosis when they are no longer needed (C.A. Janeway, Jr. and P. Travers, Immuno Biology G.3 (Garland Publishing Inc., 2^nd ed. 1996)). 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 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 Such ligand/receptor pairs that can induce apoptosis are, for example, TNF/TNF-RI, TNF TNF-R2, CD95 hgand/CD95, TRAIIVTRAIL-Rl, and TRAIUTRAIL-R2. Many of these receptors and the lntracellular kinases that play a role in transducing the signal from the membrane to the nucleus contain a stretch of 80 am o acids that is necessary to activate cell death. This "death domain" (DD) functions by interacting with other proteins via their DD or via self-association. Over-expression of many DD-contaimng proteins results in cell death, indicating that these proteins may play a role in apoptosis (K.Schulze-Osthoff et al., Eur. J Biochem, 254:439-459, 1998)

Among the proteins involved in apoptosis are the eukaryotic protein kinases (e.g., cell death related kinases, cell proliferation related kinases, etc.) These kinases make up a large and rapidly expanding family of proteins related on the basis of homologous catalytic domains. Spurred by the development of gene cloning and sequencing methodologies, distinct 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. Enzyme activities have been well documented in prokaryotes, but the prokaryotic protein kinase genes are not obviously homologous to those of the eukaryotes. One family of kinases which has been identified as being involved in apoptosis is the Receptor- Interacting Proteins (RIP) family. This family has three identified family members, RIP, R1P2, and RIP3. The family members may be distinguished from each other by their C-terminal segment. RIP has a C- terminal death domain while RIP2 has a C-terminal caspase activation and recruitment domain (CARD). 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-κB pathway. (Sun et al., 1999, J. Biol. Chem. 274: 16871-16875.)

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.

SUMMARY OF THE INVENTION

The invention aids in fulfilling these various needs in the art by providing isolated RIP-3-Like Death-Associated Kinase (R3DAK) nucleic acids and polypeptides encoded by these nucleic acids. 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:

(a) SEQ ID NO:2; (b) amino acid 1 to amino acid 300 of SEQ ID NO:2;

(c) amino acid 22 to amino acid 338 of SEQ ID NO:2;

(d) amino acid 22 to amino acid 291 of SEQ ID NO:2;

(e) a fragment of the amino acid sequence of SEQ ID NO:2 having kinase activity;

(f) a fragment of the amino acid sequence of SEQ ID NO:2 having kinase activity, the fragment comprising the amino acid sequence of amino acid 103 to amino acid 216 of SEQ ID NO:2;

(g) a fragment of the amino acid sequence of SEQ ID NO:2 having kinase activity, the fragment comprising the amino acid sequence of amino acid 139 to amino acid 165 of SEQ ID NO:2;

(h) a fragment of the amino acid sequence of SEQ ID NO:2 having kinase activity, the fragment comprising the amino acid sequence of amino acid 139 to amino acid 194 of SEQ ID NO:2; (i) a fragment of the amino acid sequence of SEQ ID NO:2 having kinase activity, the fragment comprising the amino acid sequence of amino acid 184 to amino acid 216 of SEQ ID NO:2;

(j) fragments of the amino acid sequences of any of (a)-(i) having kinase activity and comprising at least 20 contiguous amino acids of SEQ ID NO:2; (k) fragments of the amino acid sequences of any of (a)-(i) having kinase activity and comprising at least 30 contiguous amino acids of SEQ ID NO:2;

(1) 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

(m) an amino acid sequence of (1), wherein a polypeptide comprising said amino acid sequence of (1) binds to an antibody that also binds to a polypeptide comprising an amino acid sequence of any of (a)-(k).

Additionally provided by the invention are 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.

Other aspects of the invention are 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. In preferred embodiments of the invention, such 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%. Also encompassed by the present invention are isolated polypeptides and nucleic acids consisting of amino acid sequences and nucleotide sequences, respectively, disclosed herein.

Further provided by the invention are 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.

Also provided is a process for producing a polypeptide encoded by the nucleic acids of the invention, comprising culturing a recombinant host cell under conditions promoting expression of said polypeptide, wherein the recombinant host cell comprises at least one nucleic acid of the invention. A preferred process provided by the invention further comprises purifying said polypeptide. In another aspect of the invention, the polypeptide produced by said process is provided.

Further aspects of the invention 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

In a further aspect of the invention, a method is provided for identifying compounds that alter R3DAK kinase activity comprising

(a) mixing a test compound with a polypeptide of the invention, and

(b) determining whether the test compound alters the kinase activity of said polypeptide In another aspect of the invention, a method is provided identifying compounds that inhibit the binding activity of R3DAK polypeptides comprising

(a) mixing a test compound with a polypeptide of the invention and a binding partner of said polypeptide, and

(b) determining whether the test compound inhibits the binding activity of said polypeptide In addition, 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 Further provided by the invention is a method for decreasing kinase activity, comprising providing at least one antagonist of the polypeptides of the invention, with a preferred embodiment of the method further comprising decreasing said activities in a patient by administering at least one antagonist of the polypeptides of the invention

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

Structure of R3DAK

We have identified a new member of the RIP-3 polypeptide family, RIP-3-Lιke Death- Associated Kinase (R3DAK) A cDNA clone having the nucleotide sequence of SEQ ID NO 1 and encoding R3DAK polypeptide was isolated as described in Example 1 The sequence of amino acids encoded by the DNA of SEQ ID NO 1 is shown in SEQ ID NO 2 This sequence identifies the R3DAK polynucleotide as a member of the kinase family More specifically, R3DAK is a member of the RIP3 family, which is known to be involved in apoptosis Kinases which play a role in apoptosis are characterized by an N-termmal protein seπne-threoine kinase domain and a C-terminal domain which is variable and may include a series of ankyπn repeats, a death domain, or a caspase-recruitment domain The kinase domain of R3DAK is from amino acid 22 to amino acid 291 of the R3DAK polypeptide (SEQ ID NO 2) The C-terminal domain of R3DAK, which is C-terminal to the kinase domain extending approximately from ammo acid 292 to ammo acid 478 of the R3DAK polypeptide, may bind death-domain-containing polypeptides or other proteins involved in the apoptotic regulatory cascade

Biological Activities and Functions of R3DAK

RIP-3-Lιke polypeptides such as R3DAK have kinase activity and bind proteinaceous substrates Thus, for uses requiring kinase activity and/or substrate binding activity, 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 bioluminescent protein such that binding can be detected

The term "R3DAK activity," as used herein, 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 The term "binding partner," as used herein, includes without limitation ligands, receptors, native cognates, counter-structures, substrates, antibodies, other RIP- 3-Lιke 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 to measure the biological and partner-binding activities of R3DAK polypeptides Polypeptides of the RIP-3-Like family such as R3DAK are involved in diseases or conditions that share as a common feature abnormal levels of apoptosis in their etiology. More specifically, 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. 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 more detail below. For certain conditions involving a defect in apoptosis associated with too little R3DAK activity, 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. In addition to being used to express polypeptides, the nucleic acids of the invention, including oligonucleotides, 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 (for example, but not limited to, insertions, deletions, or substitutions of amino acids; changes in the state of glycosylation of the polypeptide; refolding or isomerization to change its three- dimensional structure or self-association state; and changes to its association with other polypeptides or molecules) 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. The 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 SEQ ID NO 2) The invention also provides polypeptides and fragments of the kinase domain and polypeptides and fragments of the C-terminal domain, individually or any combination thereof, that retain a desired biological activity Particular embodiments are directed to polypeptide fragments that retain the ability to bind binding partners or substrates In another embodiment, the polypeptides and fragments advantageously include regions that are conserved in the R3DAK family as described in Example 1

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

Table 1

The present invention provides both full-length and mature forms of R3DAK polypeptides 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 Several 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) can be similar to or significantly different from a native polypeptide in molecular weight and glycosyiation pattern, depending upon the choice of expression system. Expression of polypeptides of the invention in bacterial expression systems, such as E. coll, provides non-glycosylated molecules. Further, 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. As used herein, 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).

Fragments of the 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 Also included in the present invention are 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 preferably at least 15, or more preferably at least 20, or still more preferably at least 30, or most preferably at least 40 contiguous amino acids Such polypeptides and polypeptide fragments may also contain a segment that shares at least 70% 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 any such segment of any of the R3DAK polypeptides, 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 The percent identity can be determined by visual inspection and mathematical calculation Alternatively, the percent identity of two amino acid or two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6 0 described by Devereux et al (Nucl Acids Res 12 387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG) The preferred default parameters for the GAP program include (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non- ldentities) for nucleotides, and the weighted comparison matrix of Gπbskov and Burgess, Nucl Acids Res 14 6745, 1986, as described by Schwartz and Dayhoff, eds , Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp 353-358, 1979, (2) a penalty of 3 0 for each gap and an additional 0 10 penalty for each symbol in each gap, and (3) no penalty for end gaps Other programs used by those skilled in the art of sequence comparison may also be used, such as, for example, the BLASTN program version 2 0 9, available for use via the National Library of Medicine website www ncbi nlm nih gov/gorf/wblast2 cgi, or the UW-BLAST 2 0 algorithm , with standard default parameter settings as described at http //blast wustl edu/blast/README htm 1# References In addition, the BLAST algorithm uses the BLOSUM64 amino acid scoring matix, and optional parameters that may be used are as follows (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993), also see Wootton JC and Federhen S, 1996, Analysis of compositionally biased regions in sequence databases, Methods Enzymol 266 554-71) or segments consisting of short-periodicity internal repeats (as determined by the XNU program of Claveπe & States (Computers and Chemistry, 1993)), and (B) a statistical significance threshold for reporting matches against database sequences, or E-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990), if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported ), preferred E-score threshold values are 0 5, or in order of increasing preference, 0 25, 0 1, 0 05, 0 01, 0 001, 0 0001, le-5, le-10, le-15, le-20, le-25, le-30, le-40, le-50, le-75, or le-100

In another aspect of the invention, preferred polypeptides comprise various combinations of R3DAK polypeptide domains, such as the kinase domain and the C-terminal domain Accordingly, 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 Further modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques 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 and Arg, Glu and Asp, or Gin and Asn, or substitutions of one aromatic residue for another, such as Phe. Trp, or Tyr for one another Other conservative substitutions, e g , involving substitutions of entire regions having similar hydrophobicity characteristics, are well known Similarly, 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 As another example, 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) As another example, 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 polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain Alteration of a single nucleotide, chosen so that Asn is replaced by a different ammo acid, for example, is sufficient to inactivate an N-glycosylation site Alternatively, the Ser or Thr can by replaced with another ammo acid, such as Ala Known procedures for inactivating N-glycosylation sites in polypeptides include those described in U S Patent 5,071,972 and EP 276,846, hereby incorporated by reference Additional variants within the scope of the invention include polypeptides that can be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, pids, phosphate, acetyl groups and the like Covalent derivatives can be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C- terminus of a polypeptide Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the polypeptide or a substantial equivalent thereof One example is a variant that binds with essentially the same binding affinity as does the native form Binding affinity can be measured by conventional procedures, e g , as described in U S Patent No 5,512,457 and as set forth herein Other derivatives include covalent or aggregative conjugates of the polypeptides with other polypeptides or polypeptides, such as by synthesis m recombinant culture as N-terminal or C-terminal fusions Examples of fusion polypeptides are discussed below in connection with oligomers Further, fusion polypeptides can comprise peptides added to facilitate purification and identification Such peptides include, for example, poly-His or the antigenic identification peptides described in U S Patent No 5,01 1,912 and in Hopp et al , Bio/Technology 6 1204, 1988 One such peptide is the FLAG* peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant polypeptide A muπne hybπdoma designated 4E1 1 produces a monoclonal antibody that binds the FLAG^® peptide in the presence of certain divalent metal cations, as described in U S Patent 5,01 1,912, hereby incorporated by reference The 4E1 1 hybπdoma cell line has been deposited with the American Type Culture Collection under accession no HB 9259 Monoclonal antibodies that bind the FLAG^® peptide are available from Eastman Kodak Co , Scientific Imaging Systems Division, New Haven, Connecticut

Encompassed by the invention are 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 In particular embodiments, the oligomers comprise soluble R3DAK polypeptides Oligomers can be in the form of covalently linked or non-covalently-linked multimers, including dimers, tπmers, or higher oligomers In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, tπvalent, etc , binding sites In an alternative embodiment 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 peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below In embodiments where variants of the R3DAK polypeptides are constructed to include a membrane-spanning domain, they will form a Type I membrane polypeptide Membrane-spanning R3DAK polypeptides can be fused with extracellular domains of receptor polypeptides for which the ligand is known Such fusion polypeptides can then be manipulated to control the intracellular signaling pathways triggered by the membrane-spanning R3DAK polypeptide R3DAK polypeptides that span the cell membrane can also be fused with agonists or antagonists of cell-surface receptors, or cellular adhesion molecules to further modulate R3DAK intracellular effects In another aspect of the present invention, interleukins can be situated between the preferred R3DAK polypeptide fragment and other fusion polypeptide domains

Immunoglobulin-based Oligomers 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 For example, fragments of a R3DAK polypeptide may be fused directly or through linker sequences to the Fc portion of an lmmunoglobulin For 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 For example, 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 dimeπzation are also included Preferred Fc polypeptides comprise an Fc polypeptide derived from a human IgGl antibody As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins Preparation of fusion polypeptides comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e g , by Ashkenazi et al (PNAS USA 88 10535, 1991), Byrn et al (Nature 344 677, 1990), and Hollenbaugh and Aruffo ("Construction of lmmunoglobulin Fusion Polypeptides", in Current Protocols in Immunology, Suppl 4, pages 10 19 1 - 10 19 1 1, 1992) Methods for preparation and use of immunoglobulin-based oligomers are well known in the art One embodiment of the present invention is directed to a dimer comprising two fusion polypeptides created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody A gene fusion encoding the polypeptide/Fc fusion polypeptide is inserted into an appropriate expression vector Polypeptide/Fc fusion polypeptides are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules One suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG l antibody Another useful Fc polypeptide is the Fc mutein described in U S Patent 5,457,035 and in Baum et al , (EMBO J 13 3992-4001, 1994) incorporated herein by reference The ammo acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/ 10151 , except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala The mutein exhibits reduced affinity for Fc receptors The above-described fusion polypeptides comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Polypeptide A or Polypeptide G columns In other embodiments, the polypeptides of the invention can be substituted for the variable portion of an antibody heavy or light chain If fusion polypeptides are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four R3DAK extracellular regions

Peptide-linker Based Oligomers Alternatively, the oligomer is a fusion polypeptide comprising multiple R3DAK polypeptides, with or without peptide linkers (spacer peptides) Among the 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 For example, a chemically synthesized o gonucleotide encoding the linker can be ligated between the sequences In particular embodiments, 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. Among 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. Use of leucine zippers and preparation of oligomers using leucine zippers are well known in the art.

Other fragments and derivatives of the sequences of polypeptides which would be expected to retain polypeptide activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.

Nucleic Acids Encoding R3DAK Polypeptides

Encompassed within the invention are nucleic acids encoding R3DAK polypeptides. These nucleic acids 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. The well-known polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding a R3DAK polypeptide or a desired combination of R3DAK polypeptide fragments. Oligonucleotides that define the desired termini of the combination of DNA fragments are employed as 5' and 3' primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego ( 1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et. at, eds., Academic Press, Inc. ( 1990).

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.

An "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. In the case of 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. In one preferred embodiment, 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)). Such 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. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA. 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. Generally, 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. It should be understood that the 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). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to lO.degrees C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C) = 2(# of A + T bases) + 4(# of #G + C bases). For hybrids above 18 base pairs in length, Tm (degrees C) = 81.5 + 16.6(log₁₀ [Na⁺]) + 0.41(% G + C) - (600/N), where N is the number of bases in the hybrid, and [Na⁺] is the concentration of sodium ions in the hybridization buffer ([Na⁺] for lxSSC = 0.165M). Preferably, 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.

Methods for Making and Purifying R3DAK Polypeptides

Methods for making 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. General methods of expressing recombinant polypeptides are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 ( 1990). As used herein "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. As one embodiment of the invention, 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. As another embodiment of the invention, 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. In a further embodiment 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

In addition, a sequence encoding an appropriate signal peptide (native or heterologous) 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 To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for ιnterleukm-7 (IL-7) described in United States Patent 4,965,195, the signal sequence for ιnterleukιn-2 receptor described in Cosman et al , Nature 312 768 (1984), the ιnterleukιn-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 the DNA is initially transcribed, and the mRNA translated, into a fusion polypeptide comprising the signal peptide A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell The skilled artisan will also recognize that the posιtιon(s) at which the signal peptide is cleaved can differ from that predicted by computer program, and can vary according to such factors as the type of host cells employed in expressing a recombinant polypeptide A polypeptide preparation can include a mixture of polypeptide molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site Established methods for introducing DNA into mammalian cells have been described (Kaufman,

R J , Large Scale Mammalian Cell Culture, 1990, pp 15-69) Additional protocols using commercially available reagents, such as Lipofectamine pid reagent (Gibco/BRL) or Lipofectamine-Plus lipid reagent, can be used to transfect cells (Feigner et ai , Proc Natl Acad Sci USA 84 7413-7417, 1987) In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al (Molecular Cloning A Laboratory Manual, 2 ed Vol 1-3, Cold Spring Harbor Laboratory Press, 1989) Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs Kaufman et al , Meth in Enzymology 185 487-51 1, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance A suitable strain for DHFR selection can be CHO strain DX-B1 1, which is deficient in DHFR (Urlaub and Chas , Proc Natl Acad Sci USA 774216-4220, 1980) A plasmid expressing the DHFR cDNA can be introduced to strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media Other examples of selectable markers that can be incorporated mto an expression vector include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B Cells harboring the vector can be selected on the basis of resistance to these compounds Alternatively, gene products can be obtained via homologous recombination, or "gene targeting," techniques Such techniques employ the introduction of exogenous transcription control elements (such as the CMV promoter or the like) in a particular predetermined site on the genome, to induce expression of the endogenous nucleic acid sequence of interest The location of integration mto a host chromosome or genome can be easily determined by one of skill in the art, given the known location and sequence of the gene In a preferred embodiment, the present invention also contemplates the introduction of exogenous transcπptional control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product, again, without the need for isolation of the gene sequence itself from the host cell The practice of homologous recombination or gene targeting is explained by Schimke, et al " Amplification of Genes in Somatic Mammalian cells, " Methods in Enzymology 151 85- 104 ( 1987), as well as by Capecchi, et al , "The New Mouse Genetics Altering the Genome by Gene Targeting," TIG 5 70-76 (1989)

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 Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides Potentially suitable bacterial strains include Escheπchia coll, Bacillus subti s, Salmonella typhimuπum, or any bacterial strain capable of expressing heterologous polypeptides If the polypeptide is made m yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosyiation of the appropriate sites, in order to obtain the functional polypeptide Such covalent attachments may be accomplished using known chemical or enzymatic methods The polypeptide may also be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e g , Invitrogen, San Diego, Calif , U S A (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No 1555 (1987), and Luckow and Summers, Bio/Technology 6 47 (1988), incorporated herein by reference As used herein, an insect cell capable of expressing a nucleic acid of the present invention is "transformed " Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from nucleic acid constructs disclosed herein A host cell that comprises an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell"

The polypeptide of the invention may be prepared by cultuπng 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 Alternatively, 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 binding polypeptide (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX) Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, Mass ), Pharmacia (Piscataway, N J ) and InVitrogen, respectively The polypeptide can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope One such epitope ("Flag") is commercially available from Kodak (New Haven, Conn ) Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e g , silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the polypeptide Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide The polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the present invention as an "isolated polypeptide", such isolated polypeptides of the invention include isolated antibodies that bind to R3DAK polypeptides, fragments, variants, binding partners etc The polypeptide of the invention may also be expressed as a product of transgenic animals, e g , as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the polypeptide

It is also possible to utilize 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 These 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 In this aspect of 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 of the invention to a solid phase contacting surface can be accomplished by any means, for example, magnetic microspheres can be coated with these polypeptide- binding polypeptides and held in the incubation vessel through a magnetic field Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding polypeptides thereon Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding polypeptide and unbound cells then are washed away The polypeptide may also be produced by known conventional chemical synthesis Methods for constructing the polypeptides of the present invention by synthetic means are known to those skilled in the art The synthetically-constructed polypeptide sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with polypeptides may possess biological properties in common therewith, including polypeptide activity Thus, they may be employed as biologically active or immunological substitutes for natural, purified polypeptides in screening of therapeutic compounds and in immunological processes for the development of antibodies.

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. In such a case, 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. Most preferably, 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.

Antagonists and Agonists of R3DAK Polypeptides

Any method which neutralizes R3DAK polypeptides or inhibits expression of the R3DAK genes (either transcription or translation) can be used to reduce the biological activities of R3DAK polypeptides. In particular embodiments, 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. In certain other embodiments of the invention, 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. Such 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). In the case of double- stranded antisense nucleic acids, 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. However, 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 chimeπc 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 receptors in vivo), or agents facilitating transport across the cell membrane (see, e g , Letsmger et al , 1989, Proc Natl Acad Sci U S A 86 6553-6556, Lemaitre et al , 1987, Proc Natl Acad Sci 84 648-652, PCT Publication No WO88/09810, published Dec 15, 1988), or hybridization- triggered cleavage agents or intercalating agents (See, e g , Zon, 1988, Pharm Res 5 539-549) The antisense molecules should be delivered to cells which express the R3DAK transcript in vivo A number of methods have been developed for delivering antisense DNA or RNA to cells, e g , antisense molecules can be injected directly into the tissue or cell derivation site, or modified antisense molecules, designed to target the desired cells (e g , antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense ohgonucleotide is placed under the control of a strong pol III or pol II promoter The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous R3DAK gene transcripts and thereby prevent translation of the R3DAK mRNA For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA Such vectors can be constructed by recombinant DNA technology methods standard in the art Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells

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 πbozymes that can be used in the present invention include hammerhead πbozymes (Haseloff and Gerlach, 1988, Nature, 334 585-591), RNA endoπbonuc leases (hereinafter "Cech-type πbozymes") 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) As in the antisense approach, the πbozymes can be composed of modified oligonucleotides (e g for improved stability, targeting, etc ) and should be delivered to cells which express the R3DAK polypeptide in vivo A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous R3DAK messages and inhibit translation Because πbozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency

Alternatively, endogenous R3DAK gene expression can be reduced by targeting deoxyπbonucleotide 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,

Ann N Y Acad Sci , 660, 27-36, and Maher, 1992, Bioassays 14(12), 807-815)

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 oligodeoxyπbonucleotides and oligoπbonucleotides 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 (Saπn et al , 1988, Proc

Natl Acad Sci U S A 85 7448-7451) Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the

T7 or SP6 polymerase promoters Alternatively, 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) For example, a mutant, non- functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding regions or regulatory regions of the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo 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 generate animal offspring with an inactive target gene (e g , see Thomas and Capecchi, 1987 and Thompson, 1989, supra), or in model organisms such as Caenorhabditis elegans where the "RNA interference" ("RNAi") technique (Gπshok A, Tabara H, and Mello CC, 2000, Genetic requirements for inheritance of RNAi in C elegans, Science 287 (5462) 2494- 2497), or the introduction of transgenes (Demburg AF, Zalevsky J, Colaiacovo MP, and Villeneuve AM, 2000, Transgene-mediated cosuppression in the C elegans germ line, Genes Dev 14 (13) 1578-1583) are used to inhibit the expression of specific target genes However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly admmistered or targeted to the required site in vivo using appropriate viral vectors Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the nucleic acid sequences disclosed herein are provided The desired change in gene expression can be achieved through the use of antisense nucleic acids or πbozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol Sci 15(7) 250-254, Lavarosky et al , 1997, Biochem Mol Med 62(1) 11-22, and Hampel, 1998, Prog Nucleic Acid Res Mol Biol 58 1- 39, all of which are incorporated by reference herein) Transgenic animals that have multiple copies of the gene(s) corresponding to the nucleic acid sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No 0 649 464 Bl , incorporated by reference herein) In addition, organisms are provided in which the gene(s) corresponding to the nucleic acid sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s) Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9) 629-633, Zwaal et al , 1993, Proc Natl Acad Sci USA 90( 16) 7431- 7435, Clark et al , 1994, Proc Natl Acad Sci USA 91(2) 719-722, all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al , 1988, Nature 336 348-352, U S Pat Nos 5,464,764, 5,487,992, 5,627,059, 5,631,153, 5,614,396, 5,616,491 , and 5,679,523, all of which are incorporated by reference herein) These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the polypeptide product(s) of the corresponding gene(s) The R3DAK polypeptides themselves can also be employed in inhibiting a biological activity of

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 For example, 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 Furthermore, antibodies which bind to R3DAK polypeptides or binding partners may inhibit R3DAK activity and act as antagonists For example, 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 to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeπc antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above In an alternative aspect, the invention further encompasses the use of agonists of R3DAK activity to treat or ameliorate the symptoms of a disease for which increased R3DAK activity is beneficial In a preferred aspect, the invention entails administering compositions comprising an R3DAK nucleic acid or an R3DAK polypeptide to cells in vitro, to cells ex vivo, to cells in vivo, and/or to a multicellular organism Preferred therapeutic forms of R3DAK are soluble forms, as described above In still another aspect of the invention, the compositions comprise administering a R3DAK-encodιng nucleic acid for expression of a R3DAK polypeptide in a host organism for treatment of disease Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with decreased endogenous activity of a R3DAK polypeptide Furthermore, the invention encompasses the administration to cells and/or organisms of compounds found to increase the endogenous activity of R3DAK polypeptides One example of compounds that increase R3DAK polypeptide activity are agonistic antibodies, preferably monoclonal antibodies, that bind to R3DAK polypeptides or binding partners, which may increase R3DAK polypeptide activity by causing constitutive intracellular signaling (or " gand mimicking"), or by preventing the binding of a native inhibitor of R3DAK polypeptide activity

Antibodies to R3DAK Polypeptides

Antibodies that are immunoreactive with the polypeptides of the invention are provided herein

Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding) In the present invention, specifically binding antibodies are those that will specifically recognize and bind with R3DAK polypeptides, homologues, and variants, but not with other molecules In one preferred embodiment, the antibodies are specific for the polypeptides of the present invention and do not cross-react with other polypeptides In this manner, the R3DAK polypeptides, fragments, variants, fusion polypeptides, etc , as set forth above can be employed as "immunogens" in producing antibodies immunoreactive therewith More specifically, 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 epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon polypeptide folding (C A Janeway, Jr and P Travers, Immuno Biology 3 9 (Garland Publishing Inc , 2nd ed 1996)) Because folded polypeptides have complex surfaces, the number of epitopes available is quite numerous, however, due to the conformation of the polypeptide and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C A Janeway, Jr and P Travers, Immuno Biology 2 14 (Garland Publishing Inc , 2nd ed 1996)) Epitopes can be identified by any of the methods known in the art Thus, one aspect of the present invention relates to the antigenic epitopes of the polypeptides of the invention Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below Additionally, epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supematants from cultured hybπdomas Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology

As to the antibodies that can be elicited by the epitopes of the polypeptides of the invention, whether the epitopes have been isolated or remain part of the polypeptides. 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 hybπdoma 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 R Liss, Inc , pp 77-96) Hybπdoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein Such hybπdomas can be produced and identified by conventional techniques The hybπdoma producing the mAb of this invention may be cultivated in vitro or in vivo Production of high titers of mAbs in vivo makes this the presently preferred method of production One method for producing such a hybπdoma cell line comprises immunizing an animal with a polypeptide, harvesting spleen cells from the immunized animal, fusing said spleen cells to a myeloma cell line, thereby generating hybπdoma cells, and identifying a hybπdoma cell line that produces a monoclonal antibody that binds the polypeptide For the production of antibodies, various host animals may be immunized by injection with one or more of the following a R3DAK polypeptide, a fragment of a R3DAK polypeptide, a functional equivalent of a R3DAK polypeptide, or a mutant form of a R3DAK polypeptide Such host animals may include but are not limited to rabbits, mice, and rats Various adjutants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Gueπn) and Corynebacteπum parvum The monoclonal antibodies can be recovered by conventional techniques Such monoclonal antibodies may be of any lmmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof

In addition, techniques developed for the production of "chimeπc antibodies" (Takeda et al , 1985, Nature, 314 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used A chimeπc antibody is 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 muπne 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 In one embodiment, a humanized monoclonal antibody comprises the variable region of a muπne antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody Alternatively, a humanized antibody fragment can comprise the antigen bmding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody Procedures for the production of chimeπc and further engineered monoclonal antibodies include those described in Riechmann et al (Nature 332 323, 1988), Liu et al (PNAS 84 3439, 1987), Larrick et al (Bio/Technology 7 934, 1989), and Winter and Harris (TIPS 14 139, Can, 1993) Procedures to generate antibodies transgenically can be found in GB 2,272,440, US Patent Nos 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incorporated by reference herein Preferably, for use in humans, the antibodies are human or humanized, techniques for creating such human or humanized antibodies are also well known and are commercially available from, for example, Medarex Inc (Princeton, NJ) and Abgennix Inc (Fremont, CA) Antigen-bmdmg antibody fragments which recognize specific epitopes may be generated by known techniques For example, such fragments include but are not limited to the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the (ab')2 fragments Alternatively, Fab expression libraries may be constructed (Huse et al , 1989, Science, 246 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity Techniques described for the production of single chain antibodies (U S Pat No 4,946,778, Bird, 1988, Science 242 423-426, Huston et al , 1988, Proc Natl Acad Sci USA 85 5879-5883, and Ward et al , 1989, Nature 334 544-546) can also be adapted to produce single chain antibodies against R3DAK gene products Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide In addition, antibodies to the R3DAK polypeptide can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" the R3DAK polypeptide and that may bind to the R3DAK polypeptide using techniques well known to those skilled in the art (See, e g , Greenspan & Bona, 1993, FASEB J 7(5) 437-444, and Nissmoff, 1991 , J Immunol 147(8) 2429-2438)

Screening procedures by which such antibodies can be identified are well known, and can involve immunoaffinity chromatography, for example 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-medιated stimulatory pathways Also provided herein are 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

Rational Design of Compounds that Interact with R3DAK Polypeptides

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). In one approach, 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.

It is also possible to isolate 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.

Assays of Activities of R3DAK Polypeptides

The purified R3DAK polypeptides of the invention (including polypeptides, polypeptides, fragments, variants, oligomers, and other forms) are useful in a variety of assays. For example, 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. Alternatively, they can be used to identify non-binding-partner molecules or substances that modulate intracellular communication or cell activity.

Assays to Identify Binding Partners. 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. To illustrate, 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). 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.

One example of 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 cm² dishes are transfected with this recombinant expression vector. CV-l/EB A-1 cells (ATCC CRL 10478) 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. After culturing an additional 48 hours, the transfected cells (about 4 x 10⁴ 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 ¹²⁵I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37°C. After extensive washing, cells are released via trypsinization. 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. In all assays, non-specific binding of ^l25I-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. Cell-bound ^l25I- antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N. Y. Acad. Sci. 51 :660, 1949) are generated on RS/1 (BBN Software, Boston, MA) run on a Microvax computer. 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).

Yeast Two-Hybrid or "Interaction Trap" Assays. Where the R3DAK polypeptide binds or potentially binds to another polypeptide (such as, for example, in a receptor-ligand interaction), 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. Polypeptides involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Competitive Binding Assays Another type of suitable binding assay is a competitive binding assay To illustrate, 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 For example, 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 For example, 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 if such signaling or cell stimulation or cell death or survival is altered in the presence of a candidate agonist or antagonist or another R3DAK polypeptide Kinase Assays A hallmark of protein kinases is their ability to phosphorylate other proteins and to auto- phosphorylate Therefore, in one aspect of the invention, the isolated polypeptides with kinase activity can be used in assays to phosphorylate target proteins, radiolabel target proteins with ³²P, and identify proteins having phosphatase activity Exemplary methods of phosphorylation assays set forth above are disclosed in U S Patent No 5,447,860, which is incorporated herein by reference In addition to full length polypeptides, the invention also includes the isolated active kinase domains of kinases which can function as reagents in kinase assays

Kinase assays are typically carried out by combining R3DAK, or an active kinase domain, with radiolabeled ATP (γ³²P-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 , α or β casein, histone HI, myelin basic protein, etc After incubation of this reaction mixture at 20 to 37°C for a suitable time, 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 which interfere ith the rate of substrate phosphorylation Such inhibitory substances could serve as lead compounds in the development of pharmaceuticals for the treatment of autoimmune, inflammatory, infectious or neoplastic diseases in which there is a deregulation of the apoptotic processes mediated by R3DAK It is conceivable that compounds which inhibit R3DAK could have merit as more general inhibitors of the class of protein kinases which mediate death signaling, including (but not limited to) those mentioned above

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

Cellular signaling often involves a molecular activation cascade, during which a receptor propagates a gand-receptor mediated signal by specifically activating intracellular kinases which phosphorylate target substrates, ultimately resulting in the activation of transcription factors and resultant gene activation and/or deactivation These substrates can themselves be kinases which become activated following phosphorylation Alternatively, they can be adaptor molecules that facilitate down stream signaling through protein-protein interaction following phosphorylation Regardless of the nature of the substrate molecule(s), expressed functionally-active versions of R3DAK, for example the R3DAK kinase domain, can be used in assays such as the yeast 2-hybπd assay to identify what substrate(s) are recognized and altered by R3DAK As such, these novel R3DAK polypeptides can be used as reagents to identify novel molecules involved in signal transduction pathways In addition, R3DAK and other down-stream molecules involved in the signal transduction pathway can be targets for therapeutic compound(s) that interfere with the apoptosis

The extent to which a population of cells is undergoing apoptosis in response to an R3DAK polypeptide or an agonist or antagonist thereof may be assayed using a terminal transferase UTP nick end labeling (TUNEL) assay or a DNA fragmentation assay as described in Aggarwal et al , 1999, J Immunol 162(4) 2154-2161

Screening Assays

The 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 Thus, for example, 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 Potential agonists include small molecules, peptides and antibodies which bind to the instant polypeptides and elicit the same or enhanced biological effects as those caused by the binding of the polypeptides of the instant invention

Small molecule agonists and antagonists are usually less than 10K molecular weight and may possess a number of physiochemical and pharmacological properties that enhance cell penetration, resist degradation and prolong their physiological half-lives (Gibbs, J , Pharmaceutical Research in Molecular Oncology, Cell, Vol 79 (1994) ) 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

Specific screening methods are known in the art and many are extensively incorporated in high throughput test systems so that large numbers of test compounds can be screened within a short amount of time 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 Specifically, 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 To identify compounds for therapeutically active agents that mimic the biological activity of a polypeptide, it must first be determined whether a candidate molecule binds to 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

Diagnostic and Other Uses of R3DAK Polypeptides and Nucleic Acids

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-polypeptide antibodies using DNA immunization techniques, as an antigen to raise anti-DNA antibodies or elicit another immune response, and for gene therapy Uses of R3DAK polypeptides and fragmented polypeptides include, but are not limited to, the following purifying polypeptides and measuring the activity thereof, delivery agents, therapeutic and research reagents, molecular weight and lsoelectπc focusing markers, controls for peptide fragmentation, identification of unknown polypeptides, and preparation of antibodies Any or all nucleic acids suitable for these uses are capable of being developed into reagent grade or kit format for commercialization as products Methods for performing the uses listed above are well known to those skilled in the art References disclosing such methods include without limitation "Molecular Cloning A Laboratory Manual", 2d ed , Cold Spring Harbor Laboratory Press, Sambrook, J , E F Fπtsch and T Maniatis eds , 1989, and "Methods in Enzymology Guide to Molecular Cloning Techniques", Academic Press, Berger, S L and A R Kimmel eds , 1987

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 The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al , 1989 and are described in detail above Using knowledge of the genetic code in combination with the amino acid sequences set forth above, 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 In certain embodiments, 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 (express sequence tag) or DNA libraries Homologous sequences identified by this method would then be used as probes to identify non-human R3DAK homologues

Chromosome Mapping The nucleic acids encoding R3DAK polypeptides, and the disclosed fragments and combinations of these nucleic acids, 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) For example, chromosomes can be mapped by radiation hybridization First, PCR is performed using the Whitehead Institute/MIT Center for Genome Research Genebπdge4 panel of 93 radiation hybrids http //www-genome wi mit edu ftp/dιstrιbutιon/human_STS_releases/july97/rhmap/genebπdge4 html Primers are used which lie within a putative exon of the gene of interest and which amplify a product from human genomic DNA, but do not amplify hamster genomic DNA The results of the PCRs are converted into a data vector that is submitted to the Whitehead/MIT Radiation Mapping site on the internet (http //www-seq wi mit edu) The data is scored and the chromosomal assignment and placement relative to known Sequence Tag Site (STS) markers on the radiation hybrid map is provided The following web site provides additional information about radiation hybrid mapping http //www-genome wi mit edu ftp/distribution/ human_STS_releases/july97/ 07-97 INTRO html

Diagnostics and Gene Therapy The 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 In addition, 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

Methods of Screening for Binding Partners The R3DAK polypeptides of the invention each can be used as reagents in methods to screen for or identify binding partners For example, 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 In particular embodiments, a polypeptide is attached to a solid support by conventional procedures As one example, 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) In an alternative, 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 microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field Suspensions of cell mixtures containing potential binding-partner-expressing cells are contacted with the solid phase having the polypeptides thereon Cells expressing the binding partner on the cell surface bind to the fixed polypeptides, and unbound cells are washed away Alternatively, R3DAK polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for binding partner expression After mcubation, unbound labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined In a further alternative, mixtures of cells suspected of express g the binding partner are incubated with biotmylated polypeptides Incubation periods are typically at least one hour in duration to ensure sufficient binding The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads Procedures for using avidin- coated beads are known (see Berenson, et al J Cell Biochem , 10D 239, 1986) Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods In some instances, the above methods for screening for or identifying b ding partners may also be used or modified to isolate or purify such binding partner molecules or cells expressing them

Measuring Biological Activity Polypeptides also find use in measuring the biological activity of R3DAK-bιndιng 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 For example, 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

Carriers and Delivery Agents 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 coloπmetπc or fluorometπc reaction, and the like, with the particular agent being chosen according to the intended application Among the toxins are πcin, abπn, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, πbosomal inactivating polypeptides, mycotoxins such as tπchothecenes, and derivatives and fragments (e g , single chains) thereof Radionuchdes suitable for diagnostic use include, but are not limited to, ^l23I, ^mI, ^{9 m}Tc, "'in, and ⁷⁶Br Examples of radionuchdes suitable for therapeutic use are ^mI, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re, ^l88Re, ²¹²Pb, ²¹²Bι, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu Such agents can be attached to the polypeptide by any suitable conventional procedure The polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example Alternatively, the polypeptide or agent can be deπvatized to generate or attach a desired reactive functional group The deπvatization can involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to polypeptides (Pierce Chemical Company, Rockford, Illinois) A number of techniques for radiolabehng polypeptides are known Radionuclide metals can be attached to polypeptides by using a suitable bifunctional chelating agent, for example Conjugates comprising polypeptides and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared The conjugates are administered or otherwise employed in an amount appropriate for the particular application Treating Diseases with R3DAK Polypeptides and Antagonists Thereof

It is anticipated that the 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. For example, an antagonist of the kinase activity of R3DAK polypeptides may be selected for treatment of conditions involving R3DAK phosphorylation of a substrate, but a particular fragment of a given R3DAK polypeptide may also act as an effective dominant negative antagonist of that activity. Therefore, in the following paragraphs "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.

The disclosed 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. In addition, provided herein is the use of 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. Provided herein is the use of R3DAK polypeptides or antagonists for treating protozoal diseases, including malaria and schistosomiasis. Additionally provided is the use of 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. Provided also herein is the use of 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. In addition, 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. 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

In addition, the subject 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. As a further example, R3DAK polypeptides or antagonists and the compositions and combination therapies of the invention are used to treat post-herpetic pain.

Provided also are methods for using R3DAK polypeptides or antagonists, compositions or combination therapies to treat various disorders of the endocrine system. For example, 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). In addition, 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. Other 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. In addition, 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.

Included also are methods for using the subject 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. Also treatable with the compounds, compositions and combination therapies of the invention are uremic syndrome and its clinical complications (for example, renal failure, anemia, and hypertrophic cardiomyopathy), including uremic syndrome associated with exposure to environmental toxins, drugs or other causes. 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.

Also provided herein are methods for using R3DAK polypeptides or antagonists, compositions or combination therapies to treat various hematologic and oncologic disorders. For example, 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 Additional diseases treatable with the subject 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 In addition, the subject compounds, compositions or combination therapies are useful for treating leukemia, including acute myelogenous leukemia, chronic or acute lymphoblastic leukemia and hairy ceil leukemia Other malignancies with invasive metastatic potential can be treated with the subject compounds, compositions and combination therapies, including multiple myeloma In addition, the disclosed R3DAK polypeptides or antagonists, compositions and combination therapies can be used to treat anemias and hematologic disorders, including anemia of chronic disease, aplastic anemia, including Fanconi's aplastic anemia, ldiopathic thrombocytopenic purpura (ITP), myelodysplastic syndromes (including refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation), myelofibrosis/ yeloid metaplasia, and sickle cell vasocclusive crisis

Various lymphoproliferative disorders also are treatable with the disclosed R3DAK polypeptides or antagonists, compositions or combination therapies These include, but are not limited to 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

In addition, the subject 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

Other conditions treatable by the disclosed 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

In addition, 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 TNFα 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 Also treatable with the disclosed R3DAK polypeptides or antagonists, compositions, and combination therapies is cholesteatoma, a middle ear disorder often associated with hearing loss

In addition, 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 This encompasses osteoclast disorders that lead to bone loss, such as but not limited to osteoporosis, including post-menopausal osteoporosis, peπodontitis 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) 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 TNFα, and may be triggered by a variety of causes, including exposure to toxic chemicals, pancreatitis, trauma or other causes The disclosed compounds, compositions and combination therapies of the invention also are useful for treating broncho-pulmonary dysplasia (BPD), lymphangioleiomyomatosis, and chronic fibrotic lung disease of preterm infants In addition, the compounds, compositions and combination therapies of the invention are used to treat occupational lung diseases, including asbestosis, coal worker's pneumoconiosis, si cosis or similar conditions associated with long-term exposure to fine particles In other aspects of the invention, the disclosed compounds, compositions and combination therapies are used to treat pulmonary disorders, including chronic obstructive pulmonary disease (COPD) associated with chronic bronchitis or emphysema, fibrotic lung diseases, such as cystic fibrosis, ldiopathic pulmonary fibrosis and radiation-induced pulmonary fibrosis, pulmonary sarcoidosis, and allergies, including allergic rhinitis, contact dermatitis, atopic dermatitis and asthma

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 If desired, treatment with R3DAK polypeptides or antagonists may be administered concurrently with corticosteroids, mucus-thinning agents such as inhaled recombinant deoxyπbonuclease 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 number of pathogenic organisms in sputum or lung lavage (such as Haemophilus influenzae, Stapholococcus aureus, and Pseudomonas aerugmosά), by monitoring the patient for weight gain, by detecting an increase in lung capacity or by any other convenient means

The R3DAK polypeptides or antagonists of the invention, optionally combined with the cytokine IFNγ-lb (such as ACTIMMUNE^®, InterMune Pharmaceuticals) 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 In addition, 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 For treating cystic fibrosis, R3DAK polypeptides or antagonists and IFNγ-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 IFNγ-lb may be administered together with other treatments presently used for treating fibrotic lung disease Such additional treatments include glucocorticoids, azathiopπne 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 When treating fibrotic lung disease in accord with the disclosed methods, 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 In addition, patient improvement may be determined through chest radiography results showing that the progression of fibrosis in the patient's lungs has become arrested or reduced

In addition, R3DAK polypeptides or antagonists (including soluble R3DAK polypeptides or antibodies against R3DAK polypeptides) 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-β 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

Other embodiments 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, osteoarthπtis, 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 psoπatic arthritis and chronic Lyme arthritis Also treatable with these compounds, compositions and combination therapies are Still's disease and uveitis associated with rheumatoid arthritis In addition, 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 Moreover, the compounds, compositions ant combinations disclosed herein are useful for treating sporadic inclusion body myositis, as TNFα may play a significant role in the progression of this muscle disease In addition, the compounds, compositions and combinations disclosed herein are used to treat multicentπc reticulohistiocytosis, a disease in which joint destruction and papular nodules of the face and hands are associated with excess production of proinflammatory cytokines by multinucleated giant cells

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 TNFα 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-α

Cervicogenic headache is a common form of headache arising from dysfunction in the neck area, and which is associated with elevated levels of TNFα, 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

(Mar-Apr, 2000)) Cervicogenic headache may be treated by administering R3DAK polypeptides or antagonists as disclosed herein, thereby reducing the inflammatory response and associated headache pam

The R3DAK polypeptides or antagonists, compositions and combination therapies of the invention are useful for treating primary amyloidosis In addition, 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, hypeπmmunoglobu n D and periodic fever syndrome and TNF-receptor associated periodic syndromes (TRAPS)

Disorders associated with transplantation also are treatable with the disclosed R3DAK polypeptides or antagonists, compositions or combination therapies, 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

The 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 endometπosis

In addition, 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 Also, 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 Gehπg's Disease), Parkinson's disease, and tenosynovitis of unknown cause, as well as various autoimmune disorders or diseases associated with hereditary deficiencies The disclosed R3DAK polypeptides or antagonists, compositions and combination therapies furthermore are useful for treating acute polyneuropathy, anorexia nervosa, Bell's palsy, chronic fatigue syndrome, transmissible dementia, including Creutzfeld- Jacob disease, demye nating neuropathy, Guillam- Barre syndrome, vertebral disc disease, Gulf war syndrome, myasthenia gravis, silent cerebral ischemia, sleep disorders, including narcolepsy and sleep apnea, chronic neuronal degeneration, and stroke, including cerebral lschemic diseases

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 Daπer's disease, keratosis fol cularis and pemphigus vulgaπs 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 dermatosis of childhood), loss of skin elasticity, mucosal surface ulcers, neutrophi c dermatitis (Sweet's syndrome), pityπasis rubra pilaπs, psoriasis, pyoderma gangrenosum, and toxic epidermal necrolysis

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 caπcadic 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 analgesic effects or other pam reducing effects, promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages, hormonal or endocrine activity, in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases, treatment of hypeφroliferative disorders (such as, for example, psoriasis), immunoglobulin-hke activity (such as, for example, the ability to bind antigens or complement), and the ability to act as an antigen in a vaccine composition to raise an immune response against such polypeptide or another material or entity which is cross-reactive with such polypeptide

Administration of R3DAK Polypeptides and Antagonists Thereof

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-medιated disorder Such R3DAK-medιated disorders include conditions caused (directly or indirectly) or exacerbated by bindmg between R3DAK and a binding partner For puφoses of this disclosure, 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 For such therapeutic uses, 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 means Compositions of the present invention can contain a polypeptide in any form described herein, such as native polypeptides, variants, derivatives, oligomers, and biologically active fragments In particular embodiments, the composition comprises a soluble polypeptide or an oligomer comprising soluble R3DAK polypeptides Therapeutically Effective Amount In practicing the method of treatment or use of the present invention, a therapeutically effective amount of a therapeutic agent of the present invention is administered to a patient having a condition to be treated, preferably to treat or ameliorate diseases associated with the activity of a R3DAK polypeptide "Therapeutic agent" includes without limitation any of the R3DAK polypeptides, fragments, and variants, nucleic acids encoding the R3DAK polypeptides, fragments, and variants, agonists or antagonists of the R3DAK polypeptides such as antibodies, R3DAK polypeptide binding partners, complexes formed from the R3DAK polypeptides, fragments, variants, and binding partners, etc As used herein, the term "therapeutically effective amount" means the total amount of each therapeutic agent or other active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, I e , treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions When applied to an individual therapeutic agent or active ingredient, administered alone, the term refers to that ingredient alone When applied to a combination, the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously As used herein, the phrase "administering a therapeutically effective amount" of a therapeutic agent means that the patient is treated with said therapeutic agent in an amount and for a time sufficient to induce an improvement, and preferably a sustained improvement, in at least one indicator that reflects the severity of the disorder An improvement is considered "sustained" if the patient exhibits the improvement on at least two occasions separated by one or more weeks The degree of improvement is determined based on signs or symptoms, and determinations may also employ questionnaires that are administered to the patient, such as quality-of-life questionnaires Various indicators that reflect the extent of the patient's illness may be assessed for determining whether the amount and time of the treatment is sufficient The baseline value for the chosen indicator or indicators is established by examination of the patient prior to administration of the first dose of the therapeutic agent Preferably, the baseline examination is done within about 60 days of administering the first dose If the therapeutic agent is being administered to treat acute symptoms, the first dose is administered as soon as practically possible after the injury has occurred Improvement is induced by administering therapeutic agents such as R3DAK polypeptides or antagonists until the patient manifests an improvement over baseline for the chosen indicator or indicators In treating chronic conditions, this degree of improvement is obtained by repeatedly administering this medicament over a period of at least a month or more, e g , for one, two, or three months or longer, or indefinitely A period of one to six weeks, or even a single dose, often is sufficient for treating acute conditions For injuries or acute conditions, a single dose may be sufficient Although the extent of the patient's illness after treatment may appear improved according to one or more indicators, treatment may be continued indefinitely at the same level or at a reduced dose or frequency Once treatment has been reduced or discontinued, it later may be resumed at the original level if symptoms should reappear

Dosing One skilled in the pertinent art will recognize that 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 For example, 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 Such information can be used to more accurately determine useful doses in humans Ultimately, 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 and observe the patient's response Larger doses of polypeptide of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0 01 ng to about 100 mg (preferably about 0 1 ng to about 10 mg, more preferably about 0 1 microgram to about 1 mg) of polypeptide of the present invention per kg body weight In one embodiment of the invention, R3DAK polypeptides or antagonists are administered one time per week to treat the various medical disorders disclosed herein, in another embodiment is administered at least two times per week, and in another embodiment is administered at least three times per week If injected, the effective amount of R3DAK polypeptides or antagonists per adult dose ranges from 1-20 mg/m", and preferably is about 5-12 mg/m² Alternatively, a flat dose may be administered, whose amount may range from 5-100 mg/dose Exemplary dose ranges for a flat dose to be administered by subcutaneous injection are 5-25 mg/dose, 25-50 mg dose and 50-100 mg/dose In one embodiment of the invention, the various indications described below are treated by administering a preparation acceptable for injection containing R3DAK polypeptides or antagonists at 25 mg dose, or alternatively, containing 50 mg per dose The 25 mg or 50 mg dose may be administered repeatedly, particularly for chronic conditions If a route of administration other than injection is used, the dose is appropriately adjusted in accord with standard medical practices In many instances, an improvement in a patient's condition will be obtained by injecting a dose of about 25 mg of R3DAK polypeptides or antagonists one to three times per week over a period of at least three weeks, or a dose of 50 mg of R3DAK polypeptides or antagonists one or two times per week for at least three weeks, though treatment for longer periods may be necessary to induce the desired degree of improvement For incurable chronic conditions, the regimen may be continued indefinitely, with adjustments being made to dose and frequency if such are deemed necessary by the patient's physician The foregoing doses are examples for an adult patient who is a person who is 18 years of age or older For pediatπc patients (age 4- 17), a suitable regimen involves the subcutaneous injection of 0 4 mg/kg, up to a maximum dose of 25 mg of R3DAK polypeptides or antagonists, administered by subcutaneous injection one or more times per week If an antibody against a R3DAK polypeptide is used as the R3DAK polypeptide antagonist, a preferred dose range is 0 1 to 20 mg/kg, and more preferably is 1-10 mg/kg Another preferred dose range for an antι-R3DAK polypeptide antibody is 0 75 to 7 5 mg/kg of body weight Humanized antibodies are preferred, that is, antibodies m which only the antigen-binding portion of the antibody molecule is derived from a non-human source Such antibodies may be injected or administered intravenously

Formulations 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 The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active mgredιent(s) Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπostats 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 acceptable diluents (e g , saline, Tπs-HCl, acetate, and phosphate buffered solutions), preservatives (e g , thimerosal, benzyl alcohol, parabens), emulsifiers, solubi zers, adjuvants and/or carriers Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed 1980, Mack Publishing Company, Easton, PA In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or mcoφorated into polymeric compounds such as polyacetic acid, polyglyco c acid, hydrogels, dextran, etc , or incoφorated into posomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts Suitable lipids for posomal formulation include, without limitation, monoglyceπdes, diglyceπdes, sulfatides, lysolecithin, phosphohpids, saponin, bile acids, and the like Preparation of such hposomal formulations is within the level of skill in the art, as disclosed, for example, U S Pat No 4,235,871 , U S Pat No 4,501 ,728, U S Pat No 4,837,028, and U S Pat No 4,737,323, all of which are incoφorated herein by reference Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the mtended application, so that the characteristics of the carrier will depend on the selected route of administration In one preferred embodiment of the invention, sustained-release forms of R3DAK polypeptides are used Sustained-release forms suitable for use m the disclosed methods include, but are not limited to, R3DAK polypeptides that are encapsulated in a slowly- dissolving biocompatible polymer (such as the alginate microparticles described in U S No 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant

Combinations of Therapeutic Compounds A R3DAK polypeptide of the present invention may be active in multimers (e g , heterodimers or homodimers) or complexes with itself or other polypeptides As a result, pharmaceutical compositions of the invention may comprise a polypeptide of the invention in such multimeπc or complexed form The pharmaceutical composition of the invention may be in the form of a complex of the polypeptιde(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 by the same or different routes of administration Examples of components that may be included in the pharmaceutical composition of the invention are cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1 , IL-2 IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL- 12, IL-13, IL-14, IL-15, IL-17, IL-18, IFN, TNFO, TNF1, TNF2, G- CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin The pharmaceutical composition may further contain other agents which either enhance the activity of the polypeptide or compliment its activity or use in treatment Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with polypeptide of the invention, or to minimize side effects Conversely, a R3DAK polypeptide or antagonist of the present invention may be included in formulations of the particular cytokme, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokme, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent Additional examples of drugs to be administered concurrently include but are not limited to antivirals, antibiotics, analgesics, corticosteroids, antagonists of inflammatory cytokines, non-steroidal anti-inflammatories, pentoxifyllme, tha domide, and disease-modifying antirheumatic drugs (DMARDs) such as azathiopπne, cyclophosphamide, cyclospoπne, hydroxychloroqume sulfate, methotrexate, leflunomide, minocyc ne, penicillamine, sulfasalazine and gold compounds such as oral gold, gold sodium thiomalate, and aurothioglucose Additionally, R3DAK polypeptides or antagonists may be combined with a second R3DAK polypeptide/antagonist, including an antibody against a R3DAK polypeptide, or a R3DAK polypeptide-deπved peptide that acts as a competitive inhibitor of a native R3DAK polypeptide

Routes of Administration Any efficacious route of administration may be used to therapeutically administer R3DAK polypeptides or antagonists thereof, including those compositions comprising nucleic acids Parenteral administration includes injection, for example, via intra-articular, intravenous, intramuscular, mtralesional, lntrapeπtoneal 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 Alternatively, polypeptideaceous R3DAK polypeptides or antagonists may be administered by implanting cultured cells that express the polypeptide, for example, by implanting cells that express R3DAK polypeptides or antagonists Cells may also be cultured ex vivo in the presence of polypeptides of the present invention in order to proliferate or to produce a desired effect on or activity in such cells Treated cells can then be introduced in vivo for therapeutic puφoses In another embodiment, the patient's own cells are induced to produce R3DAK polypeptides or antagonists by transfection in vivo or ex vivo with a DNA that encodes R3DAK polypeptides or antagonists This DNA can be introduced into the patient's cells, for example, by injecting naked DNA or liposome-encapsulated DNA that encodes R3DAK polypeptides or antagonists, or by other means of transfection Nucleic acids of the invention may also be administered to patients by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA) When R3DAK polypeptides or antagonists are administered in combination with one or more other biologically active compounds, these may be administered by the same or by different routes, and may be administered simultaneously, separately or sequentially

Oral Administration When a therapeutically effective amount of polypeptide of the present invention is administered orally, polypeptide of the present invention will be in the form of a tablet, capsule, powder, solution or elixir When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid caσier 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 When administered in liquid form, 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 sacchaπde solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol When administered in liquid form, 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 the present invention

Intravenous Administration 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 The preparation of such parenterally acceptable polypeptide solutions, having due regard to pH, lsotonicity, stability, and the like, is within the skill in the art 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, or other additives known to those of skill in the art The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the polypeptide of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

Bone and Tissue Administration. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, 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. Preferably for bone and/or cartilage formation, 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. 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. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the polypeptide compositions from disassociating from the matrix. 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). Other preferred 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 In further compositions, 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) The therapeutic compositions are also presently valuable for veterinary applications Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with polypeptides of the present invention The dosage regimen of a polypeptide-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the polypeptides, e g , amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e g , bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other polypeptides in the pharmaceutical composition For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomoφhometπc determinations and tetracyc ne labeling Veterinary Uses In addition to human patients, R3DAK polypeptides and antagonists are useful in the treatment of disease conditions in non-human animals, such as pets (dogs, cats, birds, primates, etc ), domestic farm animals (horses cattle, sheep, pigs, birds, etc ), or any animal that suffers from a TNFα- mediated inflammatory or arthritic condition In such instances, an appropriate dose may be determined according to the animal's body weight For example, a dose of 0 2-1 mg/kg may be used Alternatively, the dose is determined according to the animal's surface area, an exemplary dose ranging from 0 1-20 mg/m², or more preferably, from 5-12 mg/m² For small animals, such as dogs or cats, a suitable dose is 0 4 mg/kg In a preferred embodiment, R3DAK polypeptides or antagonists (preferably constructed from genes derived from the same species as the patient), is administered by injection or other suitable route one or more times per week until the animal's condition is improved, or it may be administered indefinitely Manufacture of Medicaments The present invention also relates to the use R3DAK polypeptides, fragments, and variants, nucleic acids encoding the R3DAK polypeptides, fragments, and variants, agonists or antagonists of the R3DAK polypeptides such as antibodies, R3DAK polypeptide binding partners, complexes formed from the R3DAK polypeptides, fragments, variants, and binding partners, etc, in the manufacture of a medicament for the prevention or therapeutic treatment of each medical disorder disclosed herein

The following examples are offered by way of illustration, and not by way of limitation Those skilled m the art will recognize that variations of the invention embodied in the examples can be made, especially in light of the teachings of the various references cited herein EXAMPLE 1: Isolation of the R3DAK Nucleic Acid

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 Coφ. 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).

Table 2:

Conserved regions of the R3DAK amino acid sequence (upper line) when compared to conserved residues of the family of protein serine/threonine kinases (lower line). Invariant residues are shown in UPPER CASE letters, nearly invariant residues as lower case letters, conserved hydrophobic residues as (o), conserved polar residues as (*), and conserved small residues with near neutral polarity as (+).

R3DAK MSSVKLWLNGASSISLVGSEELEN GFVGKGGFGAVFRARHTAWNLDVAVK

I M i l l MM conserved residues o og G g v oaoK R3DAK IVNSKKISREVKA VNLRHE VLLLLGVTENLEWDYVYGPALVTGFMENGS

I I II II I conserved residues o E oo h oo o o R3DAK LSG LQPSCPRPWPLLCRLLEEWLGMCYLHS NPSL HRDLKPSNVLLDP

I M M I I I I I I I I I l l l l l l l l M M conserved residues 00000*00 0 0 0 0 +0 ooh oohrDok+ Nooo R3DAK ELHAKLADFGLSTFQGGSQSGSGSGSRDSGGTLAYLAPELLDNDGKASKAS II I Mill conserved residues oko+Dfgo+ g+ o +pEoo R3DAK DVYSFGVLV TVLAGREAEWDKTSLIRGAVCNRQRRPPLTELPPDSPETP I I conserved residues Doo+ogoooo o po R3DAK GLEGLKELMTHC SSEPKDRPSFQDCESKTNNVYILVQDKVDAAVSKVKHY conserved residues 00 OO R3DAK LSQYRSSDTKLSARESSQKGTEVDCPRETIVYEM DRLH EEPSGSVPERL

I I I I conserved residues O R + O R3DAK TSLTERRGKEASFGHATPAGTSSDTLAGTPQIPHTLPSRGTTPRPAFTETP R3DAK GPDPQRNQGDGRNSNP YTWNAPNPMTGLQSIVLNNCSEVQIGQHNCMSVQ R3DAK PRTAFPKKEPAQFGRGRGW

Amino acid substitutions and other alterations (deletions, insertions, etc.) to the 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 Conversely, if a change is made to the 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 50%, or more preferably at least 55%, or more preferably at least 60%, or more preferably at least 65%, or more preferably at least 70%, or more preferably at least 75%, or more preferably at least 80%, or more preferably at least 85%, or more preferably at least 90%, or more preferably at least 95% or more preferably at least 97 5%, or more preferably at least 99%, or most preferably at least 99 5% am o acid identity with the R3DAK amino acid sequence shown as SEQ ID NO 2

EXAMPLE 2: Measurement of R3DAK-Bιndιng Activity by ELISA

Serial dilutions of R3DAK-containmg samples (in 50 mM NaHC0₃, brought to pH 9 with NaOH) are coated onto Lmbro/ Titertek 96-well flat bottom E I A microtitration plates (ICN Biomedicals Inc , Aurora, OH) at 100 1/well After incubation at 4 degrees C for 16 hours, the wells are washed six times with 200 1 PBS containing 0 05% Tween-20 (PBS-Tween) The wells are then incubated with FLAG^®- labeled R3DAK-bmdιng partner at 1 mg/ml in PBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100 1 per well), followed by washing as above Next, each well is incubated with the anti-FLAG^® monoclonal antibody M2 at 1 mg/ml m PBS-Tween containing 5% FCS for 90 minutes (100 I per well), followed by washing as above Subsequently, wells are incubated with a polyclonal goat anti-mlgGl- specific horseradish peroxidase-conjugated antibody (a 1 5000 dilution of the commercial stock in PBS- Tween containing 5% FCS) for 90 minutes (100 1 per well) The HRP-conjugated antibody is obtained from Southern Biotechnology Associates, Inc (Birmingham, Alabama) Wells are then washed six times, as above

For development of the ELISA, 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 H₂S0₄ (50 1 per well) Color intensity (indicating R3DAK-bιndmg activity) is determined by measuring extinction at 450 nm on a V Max plate reader (Molecular Devices, Sunnyvale, CA)

EXAMPLE 3: Monoclonal Antibodies That Bind R3DAK

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)

Purified 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.

Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of R3DAK in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and 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. The 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. Alternatively, 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. Alternatively, 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.

EXAMPLE 4: Northern Blot Analysis

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

(Clontech, Palo Alto, CA; Biochain, Palo Alto, CA). 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.

Using the protocol described above, it was determined that a single human R3DAK transcript of approximately 4.0 kilobases (kb) was present in the spleen and lung samples.

Example 5: Isolation of R3DAK Nucleic Acids from Homo sapiens and Other Species

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. For example, 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. Optionally, 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. In this way 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). Alternatively, once a positive RNA source has been identified by a method such as the Northern blotting method described above, 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. In such cases, 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.

It is further contemplated that 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 MgCl₂, 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. A region of the gel, corresponding to the predicted size of the R3DAK-encoding DNA fragment, is excised and the specifically amplified DNA fragments contained therein are electroeluted and cloned into a plasmid vector such as pGEM-3 or pBluescript. Alternatively, to recover small fragments of DNA resulting from the restriction digest, the digested DNA product is diluted in 10 mM Tris-HCl pH 8.0, 1 mM EDTA followed by centrifugation through a Centricon™ 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.

Example 6: Chromosome mapping

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). Data from this analysis is then submitted electronically to the MIT Radiation Hybrid Mapper (URL: http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) following the instructions contained therein. This analysis yields specific genetic marker names which, when submitted electronically to the NCBI Genemap browser: (http://www.ncbi.nlm.nih.gov/genemap/map.cgi7CHRH5), yield the specific chromosome interval.

Example 7: Measuring R3DAK Kinase Activity

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(γ³²P-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. Many such general kinase substrates are known, such as α or β casein, histone HI, myelin basic protein, etc. After incubation of this reaction mixture at 20 to 37°C for a suitable time, 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.

All publications and patent applications cited in this specification are herein incoφorated by reference as if each individual publication or patent application were specifically and individually indicated to be incoφorated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is claimed is

1 An isolated polypeptide comprising an amino acid sequence selected from the group consisting of (a) SEQ ID NO 2, (b) ammo acid 1 to amino acid 300 of SEQ ID NO 2,

(c) amino acid 22 to amino acid 338 of SEQ ID NO 2,

(d) amino acid 22 to amino acid 291 of SEQ ID NO 2,

(e) a fragment of the amino acid sequence of SEQ ID NO 2 having kinase activity,

(f) a fragment of the amino acid sequence of SEQ ID NO 2 having kinase activity, the fragment comprising the am o acid sequence of amino acid 103 to amino acid 216 of SEQ ID NO 2,

(g) a fragment of the ammo acid sequence of SEQ ID NO 2 having kinase activity, the fragment comprising the amino acid sequence of amino acid 139 to ammo acid 165 of SEQ ID NO 2,

(h) a fragment of the amino acid sequence of SEQ ID NO 2 having kinase activity, the fragment comprising the amino acid sequence of amino acid 139 to amino acid 194 of SEQ ID NO 2, (I) a fragment of the ammo acid sequence of SEQ ID NO 2 having kinase activity, the fragment comprising the amino acid sequence of ammo acid 184 to amino acid 216 of SEQ ID NO 2,

(j) fragments of the amino acid sequences of any of (a)-(ι) having kinase activity and comprising at least 20 contiguous ammo acids of SEQ ID NO 2,

(k) fragments of the amino acid sequences of any of (a)-(ι) having kinase activity and comprising at least 30 contiguous amino acids of SEQ ID NO 2,

(1) an ammo acid sequence having kinase activity, comprising at least 20 amino acids, and sharing amino acid identity with the ammo 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 (m) an ammo acid sequence of (1), wherein a polypeptide comprising said amino acid sequence of (1) binds to an antibody that also binds to a polypeptide comprising an ammo acid sequence of any of (a)-(k)

2 The polypeptide of claim 1, wherein the polypeptide has a molecular weight of approximately 52 kD as determined by SDS-PAGE

3 The polypeptide according to any of claims 1 through 2 in non-glycosylated form

4 An isolated nucleic acid encoding a polypeptide of any of claims 1 through 3

5 The nucleic acid of claim 4, wherein the nucleic acid comprises the nucleotide sequence of SEQ

ID NO 1 6 An isolated nucleic acid, having a length of at least 17 nucleotides, that hybridizes under conditions of moderate stringency to the nucleic acid of any of claims 4 through 5

7 The nucleic acid of claim 6 further encoding a polypeptide having kinase activity

8 An isolated nucleic acid comprising a nucleotide sequence that shares nucleotide sequence identity with the nucleotide sequences of the nucleic acids of any of claims 4 through 7, 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%

9 An expression vector comprising at least one nucleic acid according to any of claims 4 though 8

10 A recombinant host cell comprising at least one nucleic acid according to any of claims 4 through 9

1 1 The recombinant host cell of claim 10. wherein the nucleic acid is integrated into the host cell genome

12 A process for producing a polypeptide encoded by the nucleic acid of any of claims 4 through 8, comprising culturing a recombinant host cell under conditions promoting expression of said polypeptide, wherein the recombinant host cell comprises at least one nucleic acid according to any of claims 4 through 8

13 The process of claim 12 further comprising purifying said polypeptide

14 The polypeptide produced by the process of claim 12

15 An isolated antibody that binds to the polypeptide of any of claims 1 through 3 or claim 14

16 The antibody of claim 15 wherein the antibody is a monoclonal antibody

17 The antibody of claim 15 wherein the antibody is a human antibody

18 The antibody of claim 15 wherein the antibody is a humanized antibody

19 The antibody of claim 15 wherein the antibody inhibits the activity of the polypeptide of any of claims 1 through 3 or claim 14

20 A method of designing an inhibitor of the polypeptide of any of claims 1 through 3 or claim 14, the method comprising the steps of determining the three-dimensional structure of such polypeptide, analyzmg the three-dimensional structure for the likely bmding sites of substrates, synthesizing a molecule that mcoφorates a predicted reactive site, and determining the poiypeptide-inhibiting activity of the molecule

21 A method for identifying compounds that alter R3DAK kinase activity comprising (a) mixing a test compound with the polypeptide of any of claims 1 through 3 or claim 14, and

(b) determining whether the test compound alters the kinase activity of said polypeptide

22 A method for identifying compounds that inhibit the binding activity of R3DAK polypeptides comprising

(a) mixing a test compound with the polypeptide of any of claims 1 through 3 or claim 14 and a binding partner of said polypeptide, and

(b) determining whether the test compound inhibits the binding activity of said polypeptide

23 The use of a compound, wherein the compound is selected from the group consisting of (a) the polypeptide of any of claims 1 through 3 or claim 14 and (b) an agonist of the kinase activity of said polypeptide, in the manufacture of a medicament

24 The use of a compound that inhibits the kinase activity of the polypeptide of any of claims 1 through 3 or claim 14 in the manufacture of a medicament