AU2002328896B2 - Diagnosis and treatment of diseases associated with altered expression of HIPK1 - Google Patents

Description

WO 03/006689 WO 03106689PCT/EPO2/07854 MAETHODS FOR DIAGNOSIS AND TREATMENT OF DISEA5ES ASSOCIATED WITH ALTERED EXPRESSION OF HIPK1 This application is a continuing application of U, S. 96rili Number 09/668.544, filed September 22,2000.

which is expressly incorporated horein by rrference.

FIELD OF ThE INVENTION The present invention relates to methods for use in diagnosis and treatment of diseases, including Iyrnphomna and leuKemia, associated with~ altered gene expression of HIPKI.

BACKGROUND OF THE INVENTION Lymphornas are a collection of cancers involving the lymphatic system and are generally categorized as Hodgkin's disease and Non-Hodgkin lymnphomna. Hodgkin s lymphormas are of 8 lymphocyte origin.

Non-Hodgkin lymphomas are a ccolloatIn of over 30 different types of cancers including T and 8 tyniphomas. Leukemia Is a disease of the blood forming tissues and includes B and T cell lymphocyto leukemias. It is characterized by an abnormal and persistent increae in the number, of leukocytes and the amount of bone marrow, with enlargement of the spleen and lymph nodes.

Oncogene~s are genes that can cause cancer. Carcinogenesis can occur by a wide variety or mechanisms, including infection of cells by viruses containing oncogenes. activation of protooncogenes in the host genome, and mutations of protooncogenes and tumor suppressor genes.

There are a number Of Viruses known to be involved in human cancer as well as in animal cancer, Of pa-tioular interest here are viruses that do not contain oncogenes, themnselves; these are slowtransforming retroviruses. They induce tumors by integrating into the host genome and affecting neighboring protooncogenes in a variety of ways, including promoter insertion, enhancer insertion, WO 03/006689 WO 03/06689PCT/EP02/07854 andfor trurncallo of a protooncogene or tumor suppressor gene. The analyiis of 2equences at or near the Insertion sites has led to the identification of a number of now protocncogenes.

With respect to lymphoma and levkemia, mwjrine leukemia retrovirus (MuLV). suchi as SL3-3 or Akv. is a potent inducer of tumors when inoculated into suscwzptibla newborn mice, or when carried in the geirmline. A number of sequences have been identified as relevant in the induction of lymphoma and leukemia by analyzing the insertion sites: see Sorensen et al,, J. of Virology 74:2161 (2000); Hansen et al., Genome Res. 1 0(2):237-43 (2000); Sorensen et at.. J- Virology 70:4003 (1996); Sorensen et at., J. Virology 67:7118 (1993); Joosten et al., Virology 268:306 (2000); and Lt tal., Nature Genetics 23:348 (19G9); all of which are expressly incorporated by reference herein.

As demonstrated herein, a HIPK1 gene is also implicated In lymphomas and leukemias. HIPK1 is a member of a nave family of nuclear protlein kiriases that act as transcriptional co-repressors for INK class of homeoproteins (K~im YH et 21., J. Bil, Ch~em. 108, 273:25875-25879). Homeoproteins are transcription factors that regulate homeobox genes, which are Involved in various developmental processes, such as pattern formation and organogenesis (Mc~innis, W, and Krumlauf, Cell 1992, 68:283-302).

Homeoproteins may play a role in human dismase. Aberrant expression of the NKX2-5 thomeodomain transcription factor has been found to be involved in a congsnitat heart disease. (Schott, et al., Science 1198, 261: 103-111).

Accordingly, it is an object of the invention to provide methods for detection and screenIng of dru~g candidates for diseases irvolving HItPKI, particularly with rcspect to lymphomas.

SUMMARY OF THE INVENTION In accordance with the objects outlIned above, the present initentlon provides methods for screening for compositions which modulate diseases including lymphomas. Also provided herein are methods of inhibiting proliferation of a cell, preferably a lymnphoma cell. Methods of treatment of diseases including lymphomas, and (heir diagnosis, ore also provided herein.

In one aspect, a method of sorsening drug candidates comprises providIng a cell that expresses a HIPIK1 gene or fragments thereof. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidlate on the expression of a HIPK1 gene.

00 In one embodiment, the method of screening drug candidates includes comparing the c level of expression in the absence of the drug candidate to the level of expression in the _presence of the drug candidate.

Also provided herein is a method of screening for a bioactive agent capable of binding INO to a protein encoded by a HIPKI gene, the method comprising combining a HIPK1 00 protein and a candidate bioactive agent, and determining the binding of the candidate 00 i agent to a HIPKl protein.

Further provided herein is a method for screening for a bioactive agent capable of modulating the activity of a protein encoded by a HIPKI gene. In one embodiment, the method comprises combining a HIPKI protein and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of a HIPKI protein.

Also provided is a method of evaluating the effect of a candidate lymphoma or leukemia drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile of the patient to an expression profile of a healthy individual.

In a further aspect, a method for inhibiting the activity of a protein encoded by a HIPK1 gene is provided. In one embodiment, the method comprises administering to a patient an inhibitor ofa HIPKI protein.

A method of neutralizing the effect of HIPKI protein is also provided. Preferably, the method comprises contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.

Moreover, provided herein is a biochip comprising a nucleic acid segment which encodes HIPK1 protein.

Also provided herein is a method for diagnosing or determining the propensity to disease, including lymphomas or leukemias, by sequencing at least one HIPKI gene of an individual. In yet another aspect of the invention, a method is provided for determining HIPKI gene copy number in an individual.

193906_1 0 The present invention further provides a method of screening candidate agents for anti- Scancer activity comprising contacting a cell that expresses HIPK Iwith a candidate agent Sand detecting a difference between the level of expression of the HIPKI gene in the presence and absence of the candidate agent, wherein a difference in expression in the presence of the candidate agent as compared to the level of expression of the HIPKI gene in the absence of the candidate agent indicates that the candidate agent has anti-cancer activity.

CO

00 00 The present invention further provides a method of screening for a bioactive agent capable of modulating the activity of HIPK1 protein in a cancerous cell, wherein the HIPK1 protein is encoded by a nucleic acid comprising a nucleic acid sequence having at least identity to SEQ ID NO.4, the method comprising combining the HIPKI protein and a candidate agent and determining the effect of the candidate agent on the bioactivity of the HIPK1 protein, wherein a change in the bioactivity of the HIPKI protein in the presence of the candidate agent as compared to the bioactivity of the HIPKI gene in the absence of the candidate agent indicates that the candidate agent is a bioactive agent capable of modulating the activity of HIPK 1 protein.

The present invention further provides a method of evaluating the effect of a candidate lymphoma or leukemia drug comprising detecting alterations in the expression or activation of a gene comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO.4 or a product of the gene, wherein the gene product is a polypeptide or a mRNA, in a sample obtained from a patient who had been administered with the candidate drug, wherein changes in the expression or activity of the gene or gene product indicates that the candidate drug has anti-lymphoma or anti-leukemia activity.

A method of diagnosing lymphoma or leukemia comprising measuring a level of HIPKI mRNA in a first sample, the first sample comprising a first tissue type from an individual and comparing the level of the mRNA in to a level of mRNA in a second sample, the second sample comprising a normal tissue type, wherein a difference in the mRNA levels indicates that the individual has lymphoma or leukemia.

The present invention further provides an in vitro method for inhibiting the activity of a HIPK1 protein in a cancerous cell, wherein the HIPKI protein is encoded by a nucleic acid comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO.4, the method comprising binding an inhibitor to the HIPKI protein.

0 The present invention further provides a method of treating lymphoma or leukemia comprising administering to a patient an inhibitor of HIPKl protein.

¢"1 The present invention further provides an in vitro method of neutralizing the effect of a HIPK1 protein in a cancerous cell, wherein the HIPKI protein is encoded by a nucleic acid comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO.4, the method comprising contacting an agent specific for the HIPK1 protein with the HIPKI protein.

00 00 The present invention further provides a method of diagnosing lymphomas or leukemias or a propensity to lymphomas or leukemias comprising sequencing at least one HIPKI gene of an individual.

The present invention further provides a method of determining HIPKI gene copy number, comprising adding a HIPKI gene probe to a sample of genomic DNA from an individual with cancer under conditions suitable for hybridization.

The present invention further provides a method of treating a cancerous cell, comprising contacting the cell with an inhibitor of an HIPKI protein.

The present invention further provides a method of diagnosing lymphoma or leukemia, comprising detecting the presence of differential expression of a HIPK1 gene in a patient sample, wherein the presence of differential expression of the HIPKI gene indicates that the patient has lymphoma or leukemia.

The present invention further provides an inhibitor of an HIPK1 protein for use in treating lymphoma or leukemia in a patient or for use in treating a cancerous cell.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

I

Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION WO 03/006689 WO 03/06689PCT/EP02/07854 The present invention is directed to a sequence associated with lymphoma. The use of oncdgenic retroviruses. whose sequences insert into the genome of the host organism resultlng in lymphoma, allows the identificationl of host sequen~ces Involved in lympnorna. These sequences may then be used In a number of different ways, including diagnosis, prognoal.m, screening for modulators (including both agonift and antagonists). antibody generation (for immunotherapy and imaging), etc.

Accordingly, the presont invention provides HIPKI nucleic acid and protein -sequences that are associated with lymphoma. HI PK1 nucleic acid and protein sequences as outlnad herein also are known as SGRS29 nucleic acid an~d protein sequences. Association In this context means that the nuciectide or protein sequences gire either differentially expressed or alter'ed in lymphoma as compared to normal lymphold ti1sue. As outlined below, HIPKII sequences may be u~p-regulated (ILe.

expressed at a higher level) In lymnphome, or down-regulated expressed at a lowe r level), in, lymphoma. HIPK1 sequences also include sequences which have been altered (ie. truncated sequences or sequences with a point mutation) and show either the samne expression profile or an altered profile. In a preferred embodiment. the HIPK1 sequences are from humans; hcwever, as will be appreciated by those in the art, HIPK1 sequences from other organisms may be useful in animal models of disease and drug evaluatIon; thus, other HIPKI sequences are provided, from vertebrates, Including mammals, including rodentLs (rats, mice, hamsters, guinea pigs, etc.), primates, farm animala (inrluding sheep, goss pigs, cows. horses, etc). HIPKII sequences from other organisms may be obtained using the techniques outlined below.

Sequences of the invention can include both nucleic; acid and amino acid sequences. In a preferred, embodiment, the HIPKI sequences are recombinant nucleic acids. By the term "recombinant nucleic acid" herein Is meant nucleic: a cid, originally formed In vAtro, In general, by the manipulation of nucleic acid by polymgrases and endonucleases, in a form not normally found in nature, Thus an isolated.

nucleic acid, In a linar form, or an expression vector formed In vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organismt it will replicate non-recombinantly. L~e. using the in vivo cellular machinery or the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly.

although subsequently replicated non-recombinanily, are still considered recombineint for the purposes of the invention.

Similarly, a "recombinant protein" Is a protein made using recombilinont techniques, ise, through the expression of a recombinant nucleic acid as depicted above. A recombinant protein is distingulshed fromn naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally WO 03/006689 WO 03/06689PCT/EP02/07854 associated in its wild type host, and thus may be substantially pure. For example, an isolated protein Is unaccompanied by at least 3ome of the material with which it is normally associated in its natural state. preferably constituting at least about more prefurably at leasnt obout 6% by weight of the toWa protein in a given sample. A substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred. The definition includes tIhe producton of a HIPKI protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a asnlficrinty hlghpr concentration than is normally seen, throuigh the use of an inducible promoter or high expression promoter, such that the protein is made at inoreased concentration levels, Alternatively, the protein mey be In a form rot normally found In nature, as In the addition of an epitope tag or amino acid substitutiong, insertions 2nd deletions, as discussed below.

In a preferreU embodiment, the sequences of the invention are nucleic acids, As will be appreciated by those in the art and is more fully outlined below, HIPK1 sequences are useful in a variety of applications, including diagnostic applications, which will detect neturally occurring nucleic acids, as well as screening applications; for eycample, biocliipt. comprising nucleic acid probes to a lIIPKI sequences can be generated. In the broadest sense, then, by "nucleic acid' or "ollgonucloolin" or gr-ammatical equivalents herein means at least two nucleotides covalently llnlud together. A nuclaic acid of the present invention will generally contain phosphodiester bonds, although In some casea, s outlined below (for example in antisense applications or when a cand~data agent Is a nucleic acid), nucleic acid analogs may be used that have alternate bzckbones, comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron 4D(10): 1926 (1993) and references therein: Letsinger.

J. Org. Chem. 35:3800 (1970); Sprinzl el al.. Eur. J. Biochem, 81:679 (1977): Letslnger et al., Nucl.

Acids Res. 14:3487 (1966); Sawai et al, Chem, Lett. 805 ('1984), Letsingerera)., J. Am. Chem- Soc.

110:4470 (1988): and Pauwels et at., Chemica Scrlpta 26:1419P1986)), phosphorothioate (Mag et al., Nucleic Acids Res- 19:1437 (1991): and U.S. Patent No. 5.644,048), phosphcrodithioat9 (B5riu et al., J.

Amr. Chem. Soc. 111:-2321 (1989), 0-methylphophoroamidile linkages (see Eckstein, Oligonuc~tsotldia and Analoguss; A Practical Approach, Oxford University PrGi;4), and peptide nucleic acid backbones and linkages (see Eghoim, J. AM. Chem. Soc. 114:18P5 (19-92): M~eierot al., Chem. Irit Ed. Engl, 31:1000 (1092): Nielsen, Nature, 355:566 (1993); Cgirlsson et al., Nature 380:207 (1996), all of which are incorporated by reference), Other analog nucleic acids Include those Yyit~h positive backbones (Deripcy et al., Proc. Nati. Acad. Sci. USA 92:6097 (1995); non-Ionic backbones Patent No's.

5,386,023. 5.637,684. 5.602,240, 5.216.141 and 4,465,863: Kledrowshi et al., Angew. Chemn. Intl. Ed- English 30-423 (1991); Letsirnger et al., J. Am. Chem. Soc. 1 10:44TO (1968); Letsinger et al., Nuzleoside Nucleotide 131.l597 (1994):, Chapters 2 and 3, ASO Symposium Serie's 580, "Carbohydrate Modifications In Anlisunle Research', Ed. Y.S. Sang hui and P. Dan Cook-, Mesmaeker et al., Bicorganic Medicinal Chem. Left. 4:395 (1994); Jeffs el al., J. Blorrolecular NMVR 34:17 WO 03/006689 WO 03/06689PCT/EP02/07854 (1994); Tetrahedron Lett. 37:743 (1096)) and non-ribose bockbores. including those described in U.S.

Patent Nos. 5,235,033 and 5,034.50B, end Chapters 6 and 7. ASC Symposium Series 580, 'Carbohydrate ModificationS in Alitiseflse Reseearch", Ed, YS. Sangriui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included withln one deflhition of nucleic acids (see Jenkins et al., Chem, Soc. Rev. (1995) pip 1 6 9 -1 7 Several nucleic acid analogs are descri bed in Rawls, C E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ibose-phosphate backbone may be done for a variety of reasons, for example to increase the stability and half-life of such mnoleculas in physiological environments or as probes on a biochip, As will be appreciated by those In the art, all of these nucleic acid analogs may find use in the present Invention, In addition, mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includes peptide nucleic acid analogs.

These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiestar backbone of naturally occurring nucleic acids. This results in two advantagee. Firt the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the rneltlng temperature (Tm) for mismatched versus perfectly miatched basepairs. DNA and R.NA typically exhibit a 2-4'C drop in Tmn for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9'C. Similarly, due to their non-ionic nature, hybridization of the bases attached to theae backbones Is relatively Insensitive to salt concentration. In addition, PNAs are not degraded by cellular enzymes, and thus can he more stable- The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. As Wvill be appreciated by those in the art, the depiction of a single strand ('Watson') alea; defines the sequence of the other strand ('Crick"); thus Mne sequences described herein also includes the complement of the sequence. The nucleic acid may be DNA. both genomic and cONA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, gunnina, irosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. As used herein, the termi "nuclecslde" Includes nucleotides and nucleoside and nuc ectide analogs, and modified nucleosides such as amino modified nucleosides. In addition, "nucleoside" Includes non-naturally occurring analog structures. Thus for example the Indlvidual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.

WO 03/006689 WO 03/06689PCT/EP02/07854 A HIP K1 sequence can be initially ideniied by substantial nucleic acid and/or amino acid sequence homcooy to the HI PK1 sequences outlined herein. Such homology can be based upon the, overall nucleic acid or amino acid sequence. and im generally determined as outlined below, using either homology programs or hybridization conditions.

Tne HPi sequences of the Invention were identified as follows; basically, infection of mice with muririe leukemia viruses (NMuLV; including SLM-, Akv and mutat& thereof) resulted in iymphoma.

The HIPK1 sequences outlined herein comprise the insertion sites for the virus. In general. the retrovirus can cause lymphomna in three basic ways; first of all, by Inserting upstreamn of a normally silent hos gene and activating it promoter in'sertion); secondly, by truncating a host gene that leads to oncogenesis; or by enhancing the transcription of a neighboring gene. For example, retrovirus enhancers, including 31-3-3, are known to act on genes up to approximately 200 kilobases of the insertion site.

in a prefen-ea embodiment, HIPKI sequences are those that are ulp-regulated in lymphoma: that is, the expression of these genes is higher in lymnphoma as compared to normal lymphoid tissue of the saine differentiation stage. "Up-regulation' as used herein means at least about 50%, more preferably at least about 100%/6 more preferably at leamt about 150%. more preferably, at least about 200%, with from 300 to at least 1000% being especially preferred.

in a preferred embodiment, IiK sequences are those that are down-regulated in lymphomei; that is.

the expression of these genes is lower In lymphoma as compared to normal lymphoid tissue of the same differentiation stage. *own-regulation" as used herein means at least about 50%. mere preferably at least about 100%, more preferably at least about 160%, more preferably, at least about 200% with from 300 to at least 1000% being especially preferred.

In a preferred embodiment, HIPK1 sequences are those thiat are altered but show either the same expression profile or an altered profile as compared to normal lymphoid tissue of the same differentiation stage. 'Altered HiPK sequences as used herein refers to sequences which are truncated, contain insertions or contain point mutations.

In its native form, HIPK1 is an intracallular protein that is localized in the nucleus. In general, intracellular proteins may be found in the cytoplasm and/or in the nucleus. Intracellular proteins are involved in all aspocts of cellular function and replication (including, for example, signaling piathways); aberrant expression of such proteins results in unregulated or aisregulated cellular processes. For example, many Intracellular proteins have enzymatic activity such ns protein kinase activity, phosphatidyl inositol-conjugated lipid klnase activity, protein phosphatase activity, phosphaidyl WO 03/006689 WO 03/06689PCT/EP02/07854 inoeitol-conjugated lipid phosphatase activity. protease activity, nucleotide cyclass activity, polymerasa activity and the like. Irtracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins. or targeting proteins, to various subcellular localizations, and are Involved in maintaining the structural Integrity of arganellas.

intracellular proteins found in the nucleus Include DNA-tiqding transcription regulatory factors, or transcription factors. These proteins typically bind to specific nucleic. acid sequences located in the regulatory regions of target genes and modulate the transcription of these target genes. WiVthout being bound by theory. DNA-binding transcription factors can act, directly or indirectly, on a number of factors associated with the transcriptional apparatus including A~NA polymerases and basal transcription factors. DNA binding transcription factors can also act at a number of stagas during assembly of the transcriptinal, apparatus, initiation of transcription, and transcript elongation.

An Increasingly approclsted concept in characterzng intracellular proteins is the presence in the proteins of one or more motif.5 for which defined functions have been attributed. In addition to the highly conserved sequences found in the enzymatic domain of proteins, highly conserved sequences have been identified in proteinis that are involved In protein-proteini. interaction. For example, Srchomoiogy-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner.

domains, which are distinct from SH2 domains, also bind tyrosins phosphorylated targets. SH-3 domains bind to praline-rich targets. In addition, PH domains, leirntriropeptide repeats and WD domains to name only a few, have been shown to mediate protein-proteini interactions.'Some of these may also be involved in binding to phospholipids or other second messengers. As will be appreciated by one of ordinary skill in the art, these motifs can be identfiled on the basis of primary sequence: thus, an analysis of the sequence or proteins may provide inciglit into both the enzymatic patential of the molecule and~er molecules with which the protein may associate, Common protein motifs have also been identified among transcription factors and have been used to divide these factors into families. These motifs include the basic helix-loop-helix, basic leucine zipper.

zinc finger and homneodommin motifs, HIPKI is known to contain several conserved domains, including a homeoprotein interaction domain, a protein kinase domain, a PEST domain, and ;A YH domain enriched in tyrosine a rd histidine retilues (Kim et al., J. Biol. Chem. 271:25875 (1998). in the mouse HI PK1 amino acid sequence depicted In Table 2 as SEQ 10 NO. 3, the homeoprotein interaction domain is from about iamino acid 190 to about amino acid 518, the protein kinase domain is rrom about amino acid 581 to about amino acid 848, the PEST domain is from about amino acid 890 to about amino acid 974, and the YH domaein is from about amino acid 1057 to about amino acid 1210.

WO 03/006689 WO 03/06689PCT/EP02/07854 It is recognized that through recombinant techniques, Hil sequences can b6 made to be transmembrane proteins. Trmmnembrane proteins are molecules that span the phospliolipid bilayer of a cell. They generally include approximately 20 consecutive hydrophobic amino acids that may be followed by charged amino acids. They msy have an intracellular domain, an extracellular domain, or both. The introcellujar domains of such proteins may have a number of functions includinq those already described for Irtracellular proteins. For example, the Intracelular domain may have enzymatic activity and/or may serve as a binding site for additional proteins. Frequently the Intracellular domain of transmembrane proteins servac both roles. For examole certain receptor tyrosine kinases have both protein kinose activity and SH2 domains. In addition, autophosphorylation of tyrosinies on the receptor moloculo Itself, creates binding sites for additional SH2 domain containing proteins.

It will also be appreciated by those in the art that a fransmembr-ane protein can be mradri soluble by removing tranamembrane sequences, for example through recombinant methods. Furthermore, transmembrane proteins that have been made soluble can be made to be secreted through recombinant meants by adding an appropriate signal sequence, It is further recognized that HiPK1 proteins can be made to be secreted proteins through techiniques well recognized in the art; the secretion of which can ba either constitutive or regulated. Theas proteins have a signal peptlde or signal sequence that targets the molecule to l"r secretory pathway.

In another preferred embodiment, the HI PKI proteins are nuclear proteins, preferably trarnscrption factors. Transcription factors are involved in numerous physiological events and act by regulating gene expression at the transcriptional level. Transcri ption factors often serve as nodal points of regulation controlling multiple genes. They iare capable of effecting a mulitifarious change in gene expression and can integrate many convergent signals to effect such a change. Transcripton factors are often regarded as 'master regulators' of a partcular cellular state or event. Accordingly, transcription factors have often been found to faithfully mark a partcular cell state, a quality which makes them attractive for use as diegnostic markers. In addition, because of their important role as coordinators of patterns of gene expression a sociated with particiular cell states. transcription factors are attractive therapeutic targets. Intervention at the level of trans cript Il regulation allows one to effectively target multiple genes associated with a dysfunction which fail under the regulation of a 'master regulator' or transcription factor.

A HIPKI sequence is initially identified by substantial nocleic acid and/or amino acid sequence homology to the HIPK sequences cutfined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below. using either homology programs or hybridization conditlona.

WO 03/006689 WO 03/06689PCT/EP02/07854 As used hEroin, a nucleic acid is a 'HtPK1 nucleic acid" if the overall homology of the nucleic acid sequence to one of the: nucleic acids of Tables 1, 2, and 3 is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 80%. In some embodiments the homology will be as high as about 93 to 95 or 98%. In a preferred embodiment, the sequences which are used to determine sequence identity or similarity are selected from those of the nucleic acids of SEQ ID NOS: 1, 2, 4. In another embodiment, the seuenceT are naturally occurr ing lilelic variants of the sequences of the nuclaic acids of SEQ I0 NOS: 1, 2, 4. In another embodiment, the sequences are sequence variants as further described herein.

Homology In this context means 8squence similarity or Identity, with identity being preferred. A preferred comparison for homology purposes is to compare the sequence contaliing sequenig errors to the correct sequence. This homology will be determined usinfg standard techniquts known in the art, including, but not limited to, the local homology algorithm of Smith Waterman, Adv. Appi.

Math. 2.A82 (1981). by the homology alignment algorithm of Needleman Wunsch, J. Mol. Biol, 48:443 (1970), by the search for Gimilarity method of Pearson Lipmant, PNAS USA 85:2A44 (1988).

by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI).

the Best Fit sequence program described by Devereux et al., Nuci, Acid Res. 12:387-395 (1 984).

preferably using the default settings, or by Inspection.

One examplG of a useful algorithm is PILEUP, PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng Doclittle, J. Mol. Evol. 36:351-360 (1987); the method is similar to that described by Higgins Sharp CASIOS 5151-163 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described in Altschul el Al, J. Mol, SioL 215, 403-410, (1990) and Karlin et al,, PNAS USA 90:5673-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from. Altmchwl et al.. Methods in Enzymology, 266: 460-480 (1996); http://blest~wust]. WtJ-BLAST-2 uses several search parameters, most of which are set to the default values, The adjustable parameters are set with the following values: overlap span overlap fraction' 0.125, word threshold 11. The lISP S and liSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequance and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

WO 03/006689 WO 03/06689PCT/EP02/07854 A amino acid sequence identity V21lue is determined by the number of matching identical residugg divided by tho total number of residues of the "longer" sequence in the aligned region. The "longer' sequence Is, the one having the most actual residues in the aligned region (gvkps Introduced by WUJ- Blast-2 to maximize the alignment score are ignored).

Thus, "percent nucleic acid seq~ince identity" is deflned as the percentage of n ucleatide residues in a candidate Gequence that are iderilical with the nucleotIde residues of the nucleic acids of the Sea NOS. 1, 2. 4. A preferred method utilizes the BLASTN module of WU-BLAST-2 sat to the default parameters. with overlap span and overlap fraction set to 1 and 0.125, respectivyely; The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequencea which contain either more or fewer nucleotides than those of the nucleic acids of the SEQ NOS. 4, it is understood that the percentage of homology will be determined based on the number of homologous nucleosides in relation to the total number of nucleosildes. Thus, for example, hon'ology of sequences shorter than those of the sequences identified heroin and as discussed below.

will be determined using the number of nudleosides in the Shorter sequence.

In one embodiment, the nucleic acid homology is determined tJirough hybridization studies, Thus, for example. nucleic acids which hybridize under high stringency to the nucleic acids identified in the figures, or their complements, are considered HIPK1 sequences. High stringency conditions are known in the art; see for example Maniatis et al,, Molecular Cloning: A Labo-atoly Manual, 2d Edition, 1989, and Short Protocols in molecular Biology, ed. Ausube, et at., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circurnstartcen. Longer sequence-s hybridize specifically at higher temperatures. An axtensive guide to the hybrldi2ation of nucleic acids is found in Trjssen, Techniques in Biochemistry and Molecular Biology-H-ybridizatlon with Nucleic Acid Probes, "Overview of' principles of hybridizetion and the strategy of nucleic acid assays' (13893). Generally, styingent. conditions are selected to be about 5-10'C lower than the thermal melting point (Tm) for the specific sequence at a defined Ionic strength pH. The Tm Is the temperature (under defined ionic strength, pH and nucleic acid concenitration) at which 50% of the probes complementary to die target hybridize. to the target sequence at equilibrium the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those In which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 1o01.0 M sodium ion concentration (or other salts) at pHto 8.3 and tlie temperature Is at least about 30"C for short probes 10 to 50 nucleotldes) and at (e3si about 60'C for long probes greater than 50 nucleotides). Stringent conditions may alfo be achieved with the addition of destabilizing agents such as formamide.

WO 03/006689 PCT/EP02/07854 In another embodiment, less stringent hybridization conditions are used; for example, moderate or low stringency conditlons may be used, as are known in the art: see Maniatis and Ausubel, supra, and Tijssen, supra.

In addition, the HIPK1 nucleic acid sequences of the Invention include fragments of larger genes, i.e.

they are nucleic acid segments. "Genes" in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, additional sequences of HIPK1 genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences: see Maniatis et al., and Ausubel, et al., supra, hereby expressly incorporated by reference. In general, this is done using PCR, for example; kinetic PCR.

Once a HIPK1 nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire HIPK1 nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vectoror excised therefrom as a linear nucleic acid segment, the recombinant HIPK1 nucleic acid can be further used as a probe to identify and isolate other HIPK1 nucleic acids, for example additional coding regions. It can also be used as a "precursor" nucleic acid to make modified or variant HIPK1 nucleic acids and proteins.

The HIPK1 nucleic acids of the present invention are used In several ways. In a first embodiment, nucleic acid probes to a HIPK1 nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, for example for gene therapy and/or antisense applications. Alternatively, HIPK1 nucleic acids that include coding regions of HIPK1 proteins can be put into expression vectors for the expression of HIPK1 proteins, again either for scraenlng purposes or for administration to a patient.

In a preferred embodiment, nucleic acid probes to HIPK1 nucleic acids (both the nucleic acid sequences outlined in the figures and/or the complements thereof) are made. The nucleic acid probes attached to the blochlp are designed to be substantially complementary to HIPK1 nucleic acids, i.e. the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will Interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, If the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence Is not a complementary target sequence. Thus, by "substantially complementary" herein is WO 03/006689 PCT/EPO2/07854 meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, at outlined herein.

A nucleic acid probe is generally single stranded but can be partially single and partially double stranded. The strandedness of the probe Is dictated by the structure, composition, and properties of the target sequence, In general,the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.

In a preferred embodiment, more than one probe per sequence Is used, with either overlapping probes or probes to different sections of the target being used. That Is. two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular targeL The probes can be overlapping have some sequence in common), or separate.

As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non-covalent. By "non-covalent binding" and grammatical equivalents herein Is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the noncovalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including slgma bonds, pi bonds and coordination bonds, Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules.

Immobilization may also involve a combination of covalent and non-covalent interactions.

In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.

The biochip comprises a suitable solid substrate. By "substrate" or "solid support" or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by those in the art, the number of -13- WO 03/006689 WO 03/06689PCT/EP02/07854 possible sub-strates are very large, and include. but are not limited to, glass and modified or functionalized glass, plastics (including acrylics. polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethantas, Tenlon,!, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glasses, etc, In general, the substrates allow optical detection and do not appreciably fluoresce.

In a preferred embodiment, the surface of the blachlip and the probe may be deriv.atized with chemical functional groups for subsequent attachment of the two. Thus, for example, the blochlp Is derlvattzed with a chemical functional group !ncluding, but not limited to, amino groups, carboxy groups, oxo group2 and thlol groupt. with amino groups being particularly preferred. Using there fonctional groups, the prcbes can be intlached uging functional groups on the probes. For exampla, nucioic acids containing amino groups can be attached to surfaces comprising amino groups, for example using linkers as are known in the art; for example, homno-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference). In addition, in some cases, additionai linkers, such as alkyl group~s (including substituted and heteroalKyl groups) may be used.

In this embodiment, the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5, or 3' terminus may be attached to the solid support, or attchment may be via an internal nucleoiside.

in an additional embodiment, the immobilization to the solid support may be very strong, yet noncovalent. For example, biotinylated oligonucleotldes can be made, which brid to suirfaiiis coyalently coated with streptavidin. resulting in attachmentL Alternalively, the oligonucleotides may be synthesized on the surface, as is known in the art. For example, photoactivation techniques utilizing photopolymerization compounds and techniques are used. in a preferred embodiment, the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such a3 those described in WO 95/25116; WVO 9513,5505: U.S. Patent Nos. 5,700,637 and 5,445,934; and references oiled within, all of which are expressly incorporated by reference; these methods of attachment form the basis of the Affimetrix GeneChip T M technology.

In addition to the soiid-phmae technology represented by 'olochip arrays. geno3 expression can also be quantified using liquid-phas arrays. One such system is kinetic polymnerEse chain reaction (PCR) Kinetic POR allows for the simullaneous amplification and quantification of specific nucleic acid sequences. The specificity 15 derived from synthetic oligonucleotide primers designed to preferentially -14- WO 03/006689 PCT/EP02/07854 adhere to single-stranded nucleic acid sequences bracketing the target site. This pair of oligonucleotide primers form specific, non-covalently bound complexes on each strand of the target sequence. These complexes facilitate in vitro transcription of double-stranded DNA in opposite orientations; Temperature cycling of the reaction mixture creates a continuous cycle of primer binding, transcription, and re-melting of the nucleic acid to individual strands. The result is an exponential increase of the target dsDNA product This product can be quantified in real time either through the use of an intercalating dye or a sequence specific probe. SYBR® Greene I. is an example of an intercalating dye, that preferentially binds to dsDNA resulting in a concomitant increase in the fluorescent signal. Sequence specific probes, such as used with TaqMan' technology, consist of a fluorochrome and a quenching molecule covalently bound to opposite ends of an oligonucleotide. The probe is designed to selectively bind the target DNA sequence between the two primers. When the DNA strands are synthesized during the PCR reaction, the fluorochrome is cleaved from the probe by the exonuclease activity of the polymerase resulting in signal dequenching. The probe signaling method can be more specific than the intercalating dye method, but in each case, signal strength is proportional to the dsDNA product produced. Each type of quantification method can be used In multiwell liquid phase arrays with each well representing primers and/or probes specific to nucleic acid sequences of interest. When used with messenger RNA preparations of tissues or cell lines, and an array of probelprimer reactions can simultaneously quantify the expression of multiple gene products of interest See Germer, et al., Genome Res. 10:258-266 (2000); Heid, C. at al.. Genome Res.

986-994 (1996), In a preferred embodiment, HIPK1 nucleic acids encoding HIPK1 proteins are used to make a variety of expression vectors to express HIPK1 proteins which can then be used in screening assays, as described below. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate Into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding a HIPK1 protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, Include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it Is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence: or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to WO 03/006689 WO 03/06689PCT/EP02/07854 focili'tate translation. Generally, "operably linked" means that the DNA sequences being linked ;re contiguous, and, in the case of a secretory ieaoer, contiguous and. in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. Il'suchi sites do not exist, synthetic ollgonucleotide adaptors or linkers are used in accordance with conventional practice. The transcriptional andl tronslation-al regulatory nucleic acid will generally be oppropriate to the host cell used to express HIPK1 protein: for example, transcriptional and translational regulatory nucleic acid 8equences from Bacillus are preferably used to express a HIPK1 protein in Bacillus. Numerous types of approprrate expression vectors, and suitoble regulatory sequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences. ribosomal bindinp sites, transcriptional start and stop sequences, tr-anslational start and stop sequences, and enhancer or activator aequencea. In a preferred enmbodiment, the regulatory sequences include a promoter and transcriptional 3tart and stop sequences.

Promoter sequences encode elther constitutive or inducible promoters. The promoters may be either nahurally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present inventon, In addition, the expression vector may comprise additionall elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expresrion vector contains at least one sequence homologous to the host cell genomre, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclwsion in the vector. Constructs for integrating vectors are well known in the art.

in addition. in a preferred embodiment the expression Yector contains a selectable marker gone to allow the selection of transformed host calls, Selection genes are well known in) the art and will vary with the host cell used.

The l-IPKII proteins of the present invention are produced by culturing a host ceil transformed with an expression vector containing nucleic acid encoding a HIPKII protein, under the appropriate conditions to Induce or cause expression of HIPK'I protein- The conoitions appropriate for HIPKI protein expression will vary with the choice of the expression vector and the host cell, and will be easily WO 03/006689 WO 03/06689PCT/EP02/07854 ascertained by one skilled inl the art through routine experimentation. For ex -ample, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the approprat growth conditions for induction. In additIofl, In some embodiments, the timing of the horvest is important. For example, the baculoviral systems used In Insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield, Appropriate host calls Include yeast, bacteria, archaebacteria, fungi, aind insect, plant and animal cells, including mammalian cells. Of particular interest are Drosopf7Ua melanogasifer cells, Seccnaronmyces orevi r.iaa and other yeasts, E. coli, Bacilluis subtf1IiS, Sf9 cells, C1 29 cells, 293 cells, f'Jeurospora, BHK, CHO. COS, HeLa calls, THPl cell line (a macrophage cell line) and human cells and cell lines.

In a preferred embodiment, HIPK1 protein ]a expressed in mammalian cells. Marmalian expression systems are also known in the art, and include retrovirst systems. A preferred expression vector system is a retrovirall vector system such as Is generally described in PCTIVS97/0101 Ra nd PcT/us97101048, both of which are hereby expressly incorporated by reference. Of particular usa as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Excamples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter.

and the CMV promoter, Typically. transcription tennination and polyadanylatlon sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elemrents, flank the coding sequence. Examples of transcription terminator and polyndenylatlon signals include those derived form The me thods of Introducing exogenous nucleic acid into mammalian hosts, a-9 well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include deaxtran-mediated transFection, calcium phosphate precipitation, potybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the poly nucleotide(s) in liposomes. and direct mricroinjection of the DNA into nuclei.

In a preferred embodiment. HIPKl proteins are exp ressed in bacterial systems, bg~rtrdal expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art. in addition, synthetic promoters and hybrid promoters are also useful: for &xample, the Wa promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. in addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence WO 03/006689 WO 03/06689PCT/EP02/07854 that provides for secretion of HIPKI protein in bacteria, The proteini is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space. located between the inner and outer membrane of the cell (gram-negative bacteria), The bactenial expression vector may also Include a selectable marker gene to allow far the selection of bacterial stirains that have been transformed, Suitabla selection genes include genes which render the bacteria resistant to drugs such as ampicilliri.

chtoramphenicol, erythromycin, lkanamnycn, neomyi and tetracycline, Selectable markers also include biosynthetic genes, such as those in the histine. tryptophan and leucine biosynthetic pathways. These components are assembled into expression vectors- Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtiis, E. cofi, Streptococcus crernoris, and Streptococcus Iividens. among others. The bacterial expression vectors are trans'formed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, slGotroporation, and others.

In one embodiment, HIMK proteins are produced in insect cells. Expression vectors for the iranrslormation of insect calls, and in particular, baculoviru~s-based expression vectors, are well known in the art.

In a preferred embodiment, HIPK1 protein is produced in yeast cells. Yeast expression systarms are well known in the art, and include expression vectors for Saccharomycas cerevisiae, Candlia alblcans and C, maltosa, Hanisenula polyinorpha, Kluy.'eromyces rragilis and K. lactis, Pichia, guilt erimondii and P. pastonis, Scliizosaccharomyces pombe, and Yarrowie Iipolytlca, HIPK1 protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, for the creation of monoclonal antibodies. If the desired epitope is small, a HIPK1 protein may be fused to a carrier protein to form an irnmunogen, Alternatively, a HIMK protein may be made as a fusion protein to increase expression, or for other reasons. For example, when a HIPK1I protein is a HIPI(1 pepUde, the nucleic acid encoding the paptide may be linked to other nucieic acid for expression purposes, In one embodiment, the HIPIt nucleic acids. Drotains and antibodies of the invention arx labeled. By "labeled" hereln is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic: labels, which may be radioactive or heavy isotopes;, b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. TI-e labels may be incorporated into a HIPK1 nucleic acids, proteins and antibodies at any position. For example, the label should oe capable or producing, either directly of indirectly, a detectable signal, The detectable moiety may be a radioisotope, such as 3H. 14C. 21. 35S, or 1211, a fluorescent or chemilurninescent compound, such as WO 03/006689 WO 03/06689PCT/EP02/07854 fluorescein isoithiocy-an-ate, rhodarle. or luclferin, or an enzyme, such as alkaline phosphetase, betagalactozidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the label may be employed, including those mathods described by Hunter et al., INture. 144:945 01962); David at al,, Biochemistry. 13:1014 (1974); Piain et al., J. Imnmunol. Meth., 40:219 (1981); and Nygren, J. Histochemn. and Cytochein., 30:407 (1l!82).

Accordingly, the present invention also provides HIPKII protein sequences. A HIPKI protein of the present invention may be identified in saver-al ways. 'Protein" in thlis sense includes proteins, polypeptides. and peptides. As will be appreciated by those in the art, the nuclelc acid sequences of the invention can be used to generate protein sequences. There are gi variety of ways to do this, including cloning the entire gene and verifying its frame and amninc acid sequencea, or by comparing it to known SeqLences to search for homology to provide a frame, assuming a HIPI protein has homology to some protein in the database being used. Generally, the nucleic acid sequences are nput into a program that will search all three frames for homology. This Is clone in a preferred embodiment using the following NCBI Advanced BLAST paramnetiers. The program Is blastx or blastn.

The database is nr. The input data is as "Sequence in FASTA format', The organism list is 'none'.

The 'expect" Is 10:1 the filter is default. The "descriptions" Is 500. the "alignments" is 600, and the "alignment view' is pairwise. The 'Query Genetic Codes' is standard The matrix Is BLOSUM62; gap existence cost is 11. per residue gap cost is 1: and the lambda ratio is _B5 default. This results in the generation of a putative protein sequence.

Also included within one embodiment of HIPKI proteins are amino acid variants 6f D~ie naturally occurring sequences, as determined herein. Preferably, the variants are preferably greater than about homologous to the wild-type sequence, more preferably greater than about 80%. even more preferably greater than about 85% and most preferably greater than 90%. in somne embodiments the homology will be as high as ahout 93 to 95 or 98%. As for nucleic: acids. homology in this context means sequence similarity ,or Identity, with identity being preferred. This homology will be determined using standard techniques known in the art as are outlined above for the nucleic, acid homologies.

HIPKI proteins of the present invention may be shorter or longer than the wild type amino acid sequences. Thus, in a preferred embodiment, included within tlhe definition of HJPK1 proteins are portions or fragments of the wild type sequences herein, In additionl, as outlined above, the HIPKI nuclaIc acids of the invention may be used to obtain additional coding regions, and thus additional protein sequence, using techniques known in the art.

In a preferred embodiment, the H-IPKI proteins are derivative or variant HIPK1 proteins as compared to the wild-type sequence. That is, as outlin ed more fully below, the derivative HIPK1 peptidle will WO 03/006689 WO 03/06689PCT/EP02/07854 contain at leAst one amino aoid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. The amino acid substtutiofl, insertion or deletion may occur at any~ residue within a HIPKI peptide.

Aiwo included in an ei-bodiment of HIPK1 proteins of the present Invention are amino acid sequence varints. These variants fiall into one or more of three classes: sibstitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding a HIPKII protein, i~sing cassette or PCR mutagenassis or olher techniques well known In the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant HIPKI protein frwgmnents having up to about 100-1 residues may be prepared by in vitro synthesis using established techniques. Amrino acid sequence variants are characterized by the predetermined nature of the variation. a feature that sets them apart from naturally occunrng allelic or interspecies variation of a HiPi(1 protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid siequence variation is prodetermined, the mutation per so need not be predetermined. For example, in order to optimize the performance of 2 mutation at a given site, random mutagenesis may be conducted at the target ocan or region and te expressed -IlPKI variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites In DNA Oaving a known sequence are well known, for example, M13 primer mutagenesig and LAR mutagenesis. Screening of the mutants is done using assays of HIlPK1 protein activities.

Amino acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although In some ceses deletions may be much larger Substiturfons, dletions, insertions or any combina~on thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances: When small alterations in the characteristics of a iAIPKI protein are desired, substitutions are generally made In accordance with the following chart 20 WO 03/006689 WO 03/06689PCT/EP02/07854 Chart I Exemplary Substitutions Original Re~ldue Ala Arg Asn Asp Cys Gin Giu Gly His Ilie Lou Lys Met Phe Ser Thr Trp Tyr Val Ser Lys Gin, His Glu Ser Asn Asp Pro Asr, Gin Leu, Vai lie, Val Arg, Gin, GIL Leu, Ile Met, Leu, Tyr Tilt Sur Tyr Trp, Pile Ile, Leu Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart 1. For example. substitutions rray be made which more significantly affect: the structure of tihe polypeptide backbonra In the area of the alteration, for example the a'lpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target oite; or the bulk of the Bide chain. The substitutions which in general are expected to produce the greatest changes In the polypeptide's properties are those In which a hydrophilic residue, e.g. seryl or mmronyl is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylaiany. valyl or alanyl; a cysteine or proline is substituted for (or by) any other residue: a residue having an electropositive side chain, e.g. lysyl, arginyl, or histicyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl:* or a residue having a bulky side chain, e.g, phenylalanine, is substituted for (or by) one not htaving a side chain, e~g. glycine.

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the rnaturally-occurrlng antalogue, although variants also are Selecled to modiy the characteristics of the HlPK1 proteins as needed. Alternatively, the variant may be designed such that the biologicel activity of a HIPKI protein is altered. For example, glycosylation ites may be altered or removed, dominant negative mutions created, etc.

Covalent modifications of HIPK1 polypeptides are included within the scope of this nvantion, for example for use in screening. One type of covalent modification includes reacting targeted amino acid residues or a HIPK1 polypeptide with an organic derivatizing agent that is capable of reacting with -21 WO 03/006689 WO 03/06689PCT/EP02/07854 selected side chans or the N-or C-terminal residues of an H-IPK1 polypeptide. Derivatization with bifunctional agents is useful, for Instance, for crossfinking HIPX'I to a water-insoluble support matrix or surface for use in'the method for pur ilfing anti-HIPKiantibodies or screening assays, as is more fully described below. Commonly used crosslinkirig agents include, 1A1-bis(dlazoacetyl.2..

phenylethane, glutaraidehyde, N-hydroxysuccinirnide esters. for example, estars with 4-azidosalicylic acid, homobifi) notional imidloasters, including disuccinimidyl esters such as 3,3'dithio bis (su coin imidyl proplon ate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-1(p-azido ph enyl) dtiolpropioimid ate.

Other modifications include deamidatiion of glutaminyl -and asparaginyl realdues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylationi of praline and lysine, ohosphorylation of hydroxyl groups of seryl, thrmonyl or tyrosyl residues, methylation of the cx-ainino groups of lysine, arginine, and hlatidline side chains Creighton, Proteing' Structure and Molecular Properties, W.H.

Freeman Co.. San Francisco, pp. 7M-6 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of a HIPKI polypepdde included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the natIvo ghycosyiation pattern" is intended for purposgs herein to mean deleting one or more carbohiydrate moieties found in native sequence HIFK1 polypeptide, and/or Gdding one or more glycosylaton sites that are not present in the native sequence HIPK1 polypeptide, Addition of glycosylation sites to HIPKi polypeptides may be accomplished by altering the amino acid se quence thereof. The alteralion may be made, for Axample by th@ addition of. or substitution by, one or more serine or threonine residues to the native sequence HIPK1 polypeptide (for 0-linked glycosylation sites). A HIPK1 amino acid sequence may optionally be altered thr~ugh changes at the ONA level, particularly by mutating the DNA encoding a HfPKI polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

Anolter means of Increasing the number of carbohydrate moieties on a HIP10 polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods Pire described In the art, in WO 07/05330 published I1I September 1987, and in Aplin and Wriston, LA Gric. Rev.

Blochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on a HiPK1 polypeptide may be accomplished chemically or enzymatically or by mulational substitution of codlons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, 22 WO 03/006689 WO 03/06689PCT/EP02/07854 for instance, by Hakimuddin, et al., Arch, Siochem. Biophys.. 259:52 (1987) and by Edge et al., Anal.

Biachiem., 118: 131 (1981). Enzymatic cleavage of carbohydrato molinties on lplypeptides can be achieved by the use of a variety of endlo-and exo-glycosidasee as doucrlbud by Thotakura et al., Meth, Enzymol., 138:350 (1967).

Another type of covalent modification of HIPKI comprises linking a HIPK1 polypeptfde to one of a vArlAty of nonproteinaceous polymers, polyethylene g lycol, polypropylene glycol, or polycxyalkyltones., in the manner set forth in U.S. Patent Nos. 4.640,835: 4,496.689: 4,301,144; 4.670,417;- 4,701,182 or 4, 17'9,337.

HIPI polypeptides of the present invention moy also be modified in a way to form chimeric molecules comprising a HIPK1 polypeptide fusqed to another, heterologous polypeptide or amino adid sequence.

in one embodiment, such a chimeric molecule comprises a fusion of a HIPK1 polypeptide with a tag polypeptide which provides An epitope to which an anti-tag antibody can selectively bind. The epitope lag is generally placed at the amino-or carboxyl-terminus of a HIPKi polypeptlde. although Internal fusions may also be tolerated in some instances. The presence of such epitope-tagged forms of a HIPK1 polypeptide can be detected using an antibody againist the tag polypeptide. Also, provision of the epitope tag enables a HIPK1I polypeptide to be radIly purified by affinity purification using an antitag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of a HIPKI polypephde with an immunoglobulin or a particular region of an immunoglobulln. For a bivalent form of the chimeric molecule, such a fusion could. be to the Fc: region of an lgG molecule.

Various tag polypopUdes and their respective antibodies are well known In the art. Examples include poly-histidine (poly-hIs) orpoly-histidine-glycine (poly-his-gly) tag$; the flu HA tag polypepbde and its antibody 12CA5 [Field at al,, Mcl. Cell.* Biol., 8:2159-2165 (1958)]: the L-myc tag and the 8F9, 3C7.

6610. G4, B7 and 9E1 0 antibodies thereto [Eyan et al., Molecular and Cellular Biology. 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprolein D.(gD) tap and its antibody (Paborsky et al..

Protein Engineering, 3(65).547-553 (1990)). Other tag polypeptides include the Flag-peptids [Hopp et al.. BioTechnology, 6,1204-1210 (1088)]; the KT3 epitope peptide IMartin et al., Science, 255:192-194 (1 992)]; tubulin epitope peptide [Skinner el:al., J1. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptidle tag [Lutz-Freyermulli et al., Proc. Natl. Acad. Sci. USA, 87:U393-839T (1990)].

included with the definition of HIPK1 protein In one embodiment are other HIPK1 proteins of the HIPK family, and HIPKII proteins from other organisms, which are cloned and expressed as outlined below. Thus, probe orcdegeneratle polymerase chain reaoction (PCR) primer sequences may be used to find other related HIPKI proteins from humans or other organisms. As will be appreciated by those 23 WO 03/006689 PCT/EP02/07854 in the art, particulary useful probe and/or PCR primer sequences include the unique areas of a HIPK1 nucleic acid sequence. As is generally knpwn in the art, preferred PCR primers are from about 16 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art.

In addition, as is outlined herein, HIPK1 proteins can be made that are longer than those encoded by the nucleic acids of the figures, for example, by the elucidation of additional sequences, the addition of epitope or purification tags, the addition of other fusion sequences, etc.

HIPK1 proteins may also be identified as being encoded by HIPK1 nucleic aclde, Thus, HIPK1 proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein.

in a preferred embodiment, the invention provides HIPK1 antibodies. In a preferred embodiment, when a HIPK1 protein is to be used to generate antibodies, for example for immunotherapy, a HIPK1 protein should share at least one epitope or determinant with the full length protein. By "epitope" or "determinant" herein is meant a portion of a protein which will generate end/or bind an antibody or Tcall receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller HIPK1 protein will be able to bind to the full length protein. In a preferred embodiment, the epitope Is unique; that Is, antibodies generated to a unique epitope show little or no cross-reactivity.

In one embodiment, the term "antibody" includes antibody fragments, as are known in the art.

Including Fab, Fab,, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.

Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by.one or more Injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. It may be useful to conjugate the Immunizing agent to a protein known to be Immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophospnoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

WO 03/006689 WO 03/06689PCT/EP02/07854 The antibodies may. alternatively, be rmonoclonal antibodies. Monoclonal antibodies m3Y' be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).

In a hybridoma method, a mouse, hamster, or other appropriate hiost animal, Is typically iMmunl~ed with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a polypeptidle encoded by a nucleic acid of Tables 1, 2, and 3 or fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes ('PBLs') are used if cells of human origin are desired, or spleen cells or lymph noda cells sire used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles arnd Practice, Acadric Press. (1086) pp. 59,103).

immortalized cell lines are usually transformed mammalian cells, particularly myelomna colls of rodent, bovine and human origin. Usually, rat or mouse myelomna cell lines are employed. The hybrldoma cells may be cultured in a suitable culture medium that preferaby contain& one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl trunsferase (HGPRT or HPT) the culture medium for the hybridomas typically will include hypoxanthlne, aminopterin, and thymldine ("HAT medium"), which substances prevent the growth of HGPIRT-deflolent cells.

in one embodiment, the antibodies are bispecific antibodies. Bispeciic antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens, In the present case, one of the binding specificities is for a protein encoded by a nucleic acid of the Tables 1. 2, and 3, or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preforably one that is tumor specific.

16 a preferred embodiment, the antibodies to HIPK1 are capable of reducing or eliminating the biological function of HIPK1, as is described below. That Is. the addition of aotl-HIP<1 antibodiec (either polyclonol or preferably Monoclonal) to HIPK1 (or cells containing HiPK1l) may reduce or eliminate a HIPK1 activity, Generally, at least a 2.5% decrease In activity is preferred, with at least about 50% being particularly preferred and about a 65-100%/ decrease being especially preferred.

In o preferred embodiment the aintIbodies to the HIPKI proteins are humnanized antibodies.

Humanized forms of non-human murine) antibodies are chimeric molecules of immunoglobulins.

immunoglobulin chains or fragments thereof (such as Fv. Fab, Fab', or other antigen binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immurioglobulins (recipient antibody) In Wtiich WO 03/006689 WO 03/06689PCT/EP02/07854 ro-sidues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-nurnan -species (donor antibody) such as mouse, rat or rabbit having the dar red specificity, affinity and capacity. In some Instances, Fv framework residues of the human imrnunoglobulin are replaced by corresponding non-human residuoig. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework cequenCeS. In general, the humanized antibody will comprise substentially all of at least one, and typically two, variable domains, in wh~ich all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework~ residues (FIR) regions are those of a human immunoglobuiri consensus sequence. The humanized antibody optimally aloo will compriste at least a portion of an Immirunoglobulin constant region (Fc), typically that of a human immrunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmanneatal., Nature, 332:323-329 (1988): and Presta, Curr. Op. Struct. Biol., .2:693-596 (1992)].

Methods for humanizing non-human antibodies are well known In the art. Generally, a humanized antibody hes one or more amino acid residues introdUced into it from a source which is non-hurman.

These non-human amino acid residues ore often referred to as import residues, whilch are typically taker from an import variable domain. Humanization can be essentlally performed following the method of Winter and co-worke-rs [Jones et al., Nature, 321;:522-525 (1966); Riechmann at al., Nature, 332:323-327 (198B8): Verhoeyen et al., Science, 239:1534-1536 (1 988)J, by substituting rodent, CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies *re chimeric antibodies Patent No. 4,8 16,567), wherein substantally less than an intzct human variable domain has been substituted by tha. corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antbodies in which some CDR residues and possibly some FIR residues are substituted by residues trom analogous sites in rodent antibodies.

Human antibodies ran also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (199 Marks at al,, J, Mci. Bioll., 222i581 (1991)]. The techniques cf. Cole et al. and Boerner et 21. ara also available for the preparation of human monocional antibodies [C-ole et. al.. monoclonal Antibodies and Cancer Therapy, Alan R.

Liss, p. 77 (1985) and Boemar et al., J. I mmunol., 147(1):85-95 (1991)[. Simlaerly, human1 antibodies 0 can be made by introducing human itnmunogiobullfl loci into transgenic animals, mice in which the ondogenousimmunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, Including gene rearrailgement. assembly, and antibody repertoire. This approach is described, for example, in U,5. Patent Nos. 5.545,007; 5,545,006: 5,569,625: 5,625,126: 5.633.425; 5.661,016, and in the following scientific publications: Marks et al., BiorTechnology 10, 779-783 WO 03/006689 WO 03/06689PCT/EP02/07854 (1992); Lonbarg et al., Nature 368 856-859 (1394); Morrison, Nature 368, 812-13 Fishwild et.

al., Nature Biotechnology 14, 845-51 (1996); Neubtrger, Nature Bictechnoloqy 14. 826 (1996): Lonberg and Huszar. Intern. Rev. Immunol. 13 65-93 (1985).

By immunotherapy Is meant treatment of lymphoma with an antibody raised against -a HIPK1 protein.

As used herein, Immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of 2ntlbody to a recipient (patient). Active immunization is the Induction of antibody and/or T-call responses in a recipient (patient). Induction of an immune respornse is the result of providing the recipient with an antigen to which antibodies are raised. As appreciated by one of ordinary &kill in the art, the antigen may be provided by injecting a polypeptlde against which antibodies -are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen 9nd under conditions for expression of the antigen.

In another preferred embodiment, the antibody is conjugated to a tharapeutic moiety. In one aspect the therapeutic moiety is a small molecule that modulates the activity of a HIIPKI protein. In another aspect tne therapeutic moiety modulates the activIty of molecules associated with or in close prox6ity to a HIPK1 protein. The therapeutic molety may inhibit enzymatic activity such as proteate or protein kinase activity associated with lymphoma.

In a preferred embodImrent, the therapeutic moiety may also be a cytgoxic agent. In this method, targeting the cytotoxic agent to tumor tissue or cells, results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with lymphoma. Cytotoxlc agents are numerous and varied and include. but are not. limited to, cytotoxic drugs or toxins or active fragments of such toxins.

Suitable toxins and their correrponding fragments include diphtheria A chain, exotoxin A chain, ricln A chain, abrin A chain, curoln, oretin, phenomycin, enomycin and the like. Cytotoxic agents also Include radiochnemicalo made by conjugating radioisotopes to antibodies raised against HIPKII proteins, or binding of a radlonuclide to a chelating agent that has been covalently attached to the antibody.

Targeting the therapeutic moiety to transmembrano HIPK1 proteins not only serves to increase the local concentration of therapeutic moiety in the lymphoma. but also serves to reduce deleterious side effects that may be ass:ociatad with the therapeutic moiety, In a preferred embodiment, ia HI PK1 protein against which the antibodies are raised is an intracellular protein. In this case, the antibody may be conjugated to a protein which facilitates entry into the cell, In one case, the antibody enters the cell by endocytosis. In another embodiment, a nucleic acid encoding the antibody is administered to the individual or cell. Moreover, wherein a HIPK1 protein can be targeted within a cell, the nucleus, an antibody thereto contains a signal For that target localization, a nuclear localization signal.

-27 WO 03/006689 WO 03/06689PCT/EP02/07854 The HIPK1 antibodies of the invention specifically bind to HIPKI proteins, By "specifically bind" heroin is meant that the antibodies bind to the protein wilth a binding constant in the range of at least IG"V- 10O4 wvith a preferred range being 10-7 10O9 M".

In a preferred embodiment, a KIPK1 protein is purified or isolated after expression. HIPKI proteins may be Isolated or purlfled in a variety of ways known to those skillied in the art depending on whiat other components are present in the sample. Standard p.arificatlon methods include elect rophoretic, mnoieculiar, immunological and chromatog raphic techniques, including Ion exchange, hydrophobic, affinity, and reverse-phase H PLC chromatography, and chromatonfocusing, For example, a HIP KI protein may be purified using a standard antl-G~a antibody column, Ultraflltration and diaflitration techniques, In conjunction with protein concentration, are also useful, For general guidance in suitable purification techniques, see Scopes, Protein Purification, Springer-Verlag, NY (1982), The degree of purification necessary will vary depending on the use of a HIPKI protein. In somie instances no purification will be necessary.

Once expressed and puried If necessary, the HIPK1 proteins and nucieic acids are useful in a number of applications.

in one aspect, the expression levels of genes are determined for different cellular states In the lymphoma phenotype: that is, the expression levels of genes in normal tissue and in lymphoma tissue (and in some cases, (or varying saejities of iyrnphoma !hat ralate to prognosis, as outlined below) are evaluated to. provide expression prolles. An expression profile of a particular cell zitate or point of development Is essentially a "fingerprint' of the state; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a.

gene expression profile that Is unique to the 3tate or the cell. By comparing expression profiles of cells In different states, informatin regarding which genes are important (including both up- and downregulation of genes) in each of these states is obtained. Then, diagnosis may be done or confirmed: does tissuu from a particular patient have the gene expression profile of normal or lymphoma tissue.

'Differential expression,' oir grammatical equivalents as used herein, refers to both qualitative as well as quantitative differences in the genez' temporal and/or cellular expression patterns within and among the cells, Thus, a dlifferentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus lympnomna tissue. That is, genes may be turned on or turned off in a particular state, relative to another state. As Is apparent to the skilled artisan, any comparison of two or more states can be made. Such a quali'tatively regulated gene will exhibit an expression pattern within a stale or cell type wnich is detectable by standard techniclues in one such state or cell type, but Is not detectable in both. Alternatively, the determination -28- WO 03/006689 WO 03/06689PCT/EP02/07854 is quantitative in that expression is increased or decreased; that Ig, ths expression of the gene is either upregulated, resulting in an increased amount of transcript, or downfegulated, resulting in a decreased amount of transript- The degree to which. expression differs need only be large enough to quantity via standard characterilzation techniques as outlined below, such as by use of Afmetrlx GenechipT" I expresvion array~s, Lockhart, Nature Biotechnology, 1,4:1675-1680 (19M6), hereby expressly Incorporated by reference. Other techniques include, but are not limited to, quantitative revoraon transcriptase POR, Northern analysis and RNase protection. As outlined aboyc-, preferably the change in expression upregulation or downregulation) is at least about 60%, more preferably at least about 100%, more preferably at least about 1501/, more preferably, at least ab~out 200%, with, from 300 to at least 1000% being erpelmlly preferrad.

As will be appreciated by those in the art this may be done by evaluaton at either the gene transcript, or the protein level; that is, the amount of gene expression may be monitored using nuclelc acid probes to the DNA or RNA equivalent of Mle gene tronscript, and the quantification of gene exprovslon levels, or, alternatively, the final gene product Itself (protein) can be monitored, for exampla through the use of antibodies to a HIPK1 protein and standard immunoassays (ELISAs, etc,) or other techniques, including mnass. spectroscopy assays, 2D gel electrophoresis assays. etc. Thus, the proteins corresponding to HIPKM genes, i~e. those, Identified as being important in a lymphoma phenotype, can be evaluated in a lyrmphoma diagnostic test.

In a preferred embodiment, gene expression monlitorIng Is done and a number of genes, i.e. an 9expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well. Similarly, these assays may be done on an individual basis as well.

In this embodiment, the HIPKI nucleic acid probes may be attached to biouhipsa w outlined herein for the detection and quantification nf HIPKI sequences in a particular cull. The assays are done as is known in the art. As will be appreciated by those in the art, any number of different HIPK1 sequences may be used as probes, with single sequence assays being used in some cases, and a plurality of the sequences described herein being used in other embodiments. In addition, while solid-phase assays are clescribed, any number of solution based assays may be done as well.

In a preferred embodiment, both solid and solution based assays may be used to detect HIPK1 sequences that are up-regulated or down-regulated In lymphoma as compared to normal lymphoid 0 tissue. In instances where a HIPKI sequence has been altered but shows the same expression profile or an altered expression profle, the protein will be detected as outlined herein -29 WO 03/006689 WO 03/06689PCT/EP02/07854 In a preferred embodiment nucleic acids encoding n HIPK1 protein are diitec .ed. Although DNA or RNA encoding a HIPK1 protein may be detected, of particular interest are methods wherein the rnRNA encoding9 H-IPKI protein is detected. The prese-ice of rnRNA in a sample is an indication thrat a HIPK1 gene has been transcribed to form the mRNA, and suggests theit tihe pr'oteln is expresed. Probes to detect the mRNA c2n be any nucleotldeldfeoxynucleotide probe that is complementary to and base pairs with the mRNA and includes but is not limited to oligonucleotides, cONA or RNA. Probes also should contain a detectable label, as defined herein. In one method the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridlizing the probe with the sample. Following washing to remove the non-specifically bound probe.

the label6 ietected. In another method detection of the mRNA Is performed in situ. In this method permeabilized cells or tissue samples are contacted with a datectably labeled nucleic ofcid probe for sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the non-,specifi~colly bound probe, th'e label is detected. For example a digaxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding HIPK1 protein is detected by binding the digoxygenin with an anti-digoxygenin secondary antibody and developed with nitro blue ltrezollum and 5-bromo-4-chloro-3-indoyl phosphate.

in a preferrec embodiment, the IiPKI proteins, antibodies, nucle;1c acids, modified HIPIK1 proteins and cells containing HIPKI sequences are used in diagnostic assays. This can be done on an individual gene or corrocponding polypeptide evel, or as sets of assays.

As described and defined herein, HIPK1 proteins find use as markers of lymphoma. Detection of these proteins in putative lymphomic tissue or patents allows for a determination or diagnosis of lymphoma. Numerous methods Known to thoase of ordinary skill in the ort 5nd use in detecting lymphoma. In one embodiment, antibodies are used to detect HIP K1 proteins. A preferred mothod separ-ates proteins from a sample or patient by elec trophoresis on a gel (typically a denaturing end reducing protein gel, but may be any other type of gel ineluding isoelectric focusing gels and the like).

Following separation of proteins, a HIPKI protein Is detected by immunobiotting with antibodies raised against a HiPK1 protein. Methods of imrnunobiotting are well known to those of ordinary skill in the art.

In another preferred method, ant Ibodies to a H-IPKI protein find use in in situ imaging techniques. In this method cells are contacted with from one to many antibodies to a HIPK1 protein(s). Following washing to removn non-specific antibody binding, the presence of tha antibody or antibodies is detected. In one embodiment the antibody is detected by incubating wit a secondary antioody that contains a detectable label. In another method the primary antibody to a HIPK1 protain(s) contains a detecrtable label. In another preferred embodiment each one of multiple primary antibodies contains a WO 03/006689 WO 03/06689PCT/EP02/07854 distinct and detectable label, This method finds particular use in simultarteous GCreening [or a plurality o f HIPK1I proteins. As will be appreciated by one of ordinary skill in the ad, numoroua other histological imaging techniques are useful In the invention.

In a preferred embodiment the label Is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths. In addition, a fluorescence activated cell sorter (FAGS) can be used In the method.

in a preferred embodiment, in situ hybridization of labeled HIPK1 nucleic, acid probes to tissue arrays is done. For exampla. arrays of tissue -samples, including leukemle/lymphoma tiscue and/or normal tissue, are made. In .sflu hybridization as is known in the art can then be done.

It is understood that when comparing thle expression fingerprints between an individual and a standard, the skilled artisan can make a diagnosis as well as a prognosis. It Is further understood that the genes which Indicate the diagnosis may differ from those wh~ch Indicate the prognosis.

In 2 praferred embodiment, the HI proteins, antibodies, nucleic acids, modified HIPK1 proteins end cells containing H-IPK1 sequences are used in prognosis assays. As above, gene expression profiles can be generated that correlate to lymphoma severity, in terms of long term prognosis. Again, this may be done on either a protein or sene level, with the use of genes being preferred. As above, the HIPK1 probes are attached to biochips for the detection ond quantification of HIPKI sequences in a tissue or patient. The assays proceed as outlined for diagnosis.

In a preferred ambodlnlent, any of the HIPK1 sequences as described herein are used In drug screening essays. The HIPKI proteinls, antibodies, nucleic acids, modified HIPK proteInls and cells containing HI sequences are used In drug screening assays or by evaluating the effect of drug candidates on a "gene expression profile or expression profile of polypeptides. in one emnbodiment, the expression profiles are used, preferably in conunction with h~gh throughput screening techniques to allow monitoring for expression profile genes after treatmont with a candidate agent, Zlokarnik, et al., Science 279. 84-8 (1998). Heid. et al., Genomre Res., 6:988-q,94 (1 996).

in a preferred embodimnent, the HIPKI protaln3, antibodies, nucleic acids, modified HIPKI proteins and cells containing the native or modified HIPK proteIns are used In screening asaays. That ie, Itoe present invention provides novel methods for screening for compositions which modulate the lymphooma phenotype. AG above, this; can be done by screening for modulators of gene axpi-ession or for modulators of Drotain activity. Simrilarly, this may be done on an individual gene or protein level or by evaluating the effect of drug candldateG on a "gene expression profile'. In a preferred embodiment.

WO 03/006689 WO 03/06689PCT/EP02/07854 the expression profles are used, preferably in conjunctl~on with high throughput screening techniques to allow monitoring for expression profileo genes after treatment with a candidate agent, see Zlokamik,.

supra.

Having identified the HIPK1 genes herein, a variety of assays to evaluate the effects of agents on gene oxpression may be executed. In a preferred embodiment, assays may be run an an indivdual gene or protein level, That is, having identified a particular gene as aberrantly regulated in lymphomna, candidate bioactive agents may be screened to modulate the gene's response, 'Modulation' thus includes both an increase zind a decrease In gene expression or activity. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tumnor tissue, with changes of at least 10%, preferably SD%. more preferably 100-300%, and in some embodiments 300-1 000% or groater. Thus, if a gene exhibits a 4 fold increase in tumor compared to normal tissue.

a decrease oif about four fold Is desired; a 10 fold decrease in turnor compared to normal tissue gives a 10 fold increase In expression for a candidate agent is desired, etc. Alternaively, where a HIPKI sequence has been altered but shows the same expression profile or an altered expression profile, the protein will be detected as outlined herein.

As will be appreciated by those in the art, this may be done by evaluation at either the gene of the protein level: that is, the amount of gene expression may be monitored using nucleic acid probes and the quantlfication of gene expression levels, or, alternatively, the level of the gene product itself can be monitored, for example through the use oftantibodies. to a HIPK1 protein and standard immunoiassays, Alterniztively, binding and bioactlvity assays with the protein may be done as outlined below.

In 2 preferred embodiment, gene expression monitoring is done and a number of genes, I e. an expression profile, Is monitored simultaneously, although m~ltIpie protein expressio n monitoring can be done as well.

In this embodimenrt, the HIPKI nucleic acid probes are attached to blochips as outlined herein for the detection and quantlification of HIPK1 sequences in a particular cell, The assays are further described aelow.

Generally, ifl a preferred embodiment, a candidate bioactive agent is added to the cells prior to analysis. Moreover, screene; are provided to Identify a candidate bioactive agent which modlulates lymphomna, modulates HIPK1 proteins, binds to HIPKi protein, or interferes Detween the bindIng of HIPKI protein and an antibody.

32 WO 03/006689 PCT/EP02/07854 The term "candidate bicactive agent" or"drug candidate" or grammatical equivalents as used herein describes any molecule, protein, oligopeptide, small organic or inorganic molecule, potsaccharide, polynucleotide, etc., to be tested for bloactive agents that are capable of directly or Indirectly altering either the lymphoma phenotype, binding to and/or modulating the bioactivity of an HIPK1 protein, or the expression of a HIPK1 sequence, including both nucleic acid sequences and protein sequences. In a particularly preferred embodiment, the candidate agent suppresses a lymphomalleukemla associated (LA) phenotype, for example to a normal tissue fingerprint. Similarly, the candidate agent preferably suppresses a severe LA phenotype. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations, Typically, one of these concentrations serves as a negative control, at zero concentration or below the level of detection.

In one aspect, a candidate agent will neutralize the effect of a HIPK1 prolein. By "neutralize" is meant that activity of a protein Is either inhibited or counter acted against so as to have substantially no effect on a cell.

Candidate agents encompass numerous chemical classes, though typically they are organic or inorganic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000. or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural Interaction with proteins, particularly hydrogen bonding, and typically incduda at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.

The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptldes.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced, Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterificalion. amidification to produce structural analogs, WO 03/006689 WO 03/06689PCT/EP02/07854 In a prefer-red embcdiment, the candidate bionctlve agents are proteins, By 'protein* herein is meent at least two covalently attached amino acids, which includes protmins, polypeptidas, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic pepbdomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norgleucine are considered amino acids for the purposes of the invention. 'Amnino acid" also Includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the CR) or the configuration. In ihs preferred embodiment, the amino acids are in the or L-Configration.

If non-naturally occurring sido chains are used, non-amino acid substituents may be used, for example to preverfl.or retard in vivo degracdators.

in a preferred embodiment, the adidlate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins- Thus. for example. cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used, In this way libraries of procaryotic and eucaryoti proteins may be made for screening in the methods of the invention, Particularly preferred in this embodirment are libraries of bacterial, "ugal. viral, and mammalian proteins, with the latter being preferred, and humarl protalne being especally preferred.

In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about being particularly preferred. The peplides may be digests of naturally occurring proteins asi is outlined above, random peptides, or "bias5ed random peplides. By "randomized' or gram matical equivalents herein 1rs meant that each nucleic acid and peptide consists of assentially random nuclec~des and amino acids, respectively, Since generally these random peptides (or nucleic acids, discussed below) are chemicolly synthesized, they may incorporate any riucleaoie or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nuceic acids, to allow the tommation of all or most of the possible combinations over the length of the sequence. thus forning a library of randomi.zed candidate bloactive proteinaceous agents.

In one embodiment, the library Is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biaser. That is, somis positions within the sequence are either held constant, or are selected from a limited number of possibiiies. For toxample, in a preferred embodiment, the nuleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic iomino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cystelnes. for cross-linking. proilnes for SH-3 comains. sortrteg, threonines, tyrosines or histidines for phosphorylatlon sites, etc.. or to purines, etc.

WO 03/006689 WO 03/06689PCT/EP02/07854 in a preferred embodiment, the candidate bloactive agents are nucleic acids, as defined above.

described abo gene-rally for protelins, nucleic acid candidate bioaotive agents may be naturally occurring nucleic: acids, random nucleic acids, or "biased" random nucleic. acids. For example, digests of procar-yotic or eucaryotic: genomes may be used as is outlined above for proteins.

In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.

in aaisays for altering the expression profile of one or more H-iPKl genes, after the candidate agent has been added and the cells allowed to incubate for some period of bms, the sample containing the target sequences to be analyzed is added to the blochip. If required, the target sequence is prepared using known techniques, For exam~ple, the sample may be treated to lyse the cells, using k'nown Iyals buffers, etectroporation, etc., with purification and/or amplification such ms PCR occurring ag needed, as will be appreciated by those in the art. For example, an in viribo transcription with labels covaleitly attachied to the nucleosides is done. Generaly, the nucleic acids are labeled with a label as defined herein, with biobn-FITC or PE, cy3 and cy5 being particularly preferr ed.

In a prefearred embodiment, the target sequence is labeled with, for example, a fluorescent, chemrliuminescent. chemical, or r-adioactive signal, to provide a means of detectng the target sequence's specific binding to a probe- The label also can be an enzyme, such as, alkaline phosphzitose or horseradish peroxiclase. which wnien provided with an appropriate substrate produces a product that can be detected. Alternatively, the label can be a labeted compound or small molecule, such as 2n enzyme inhibitor, ftat binds but is not catalyzed or altered by the enzyme. T1he label also can be o rnoiety or compound, such as, an epitope tag or blotin which specifically bindq to streplavidin.

For the example of biotin, the streptavidin is labeled as described above, thereby, providing a delectable signal for the bound target sequence. As known in the art, unbound labeled streptsevidin is removed prior to analysis.

As will be appreciated by those in the -art these assays can be direct hybridization assays or canl comprise 'sandwich assays', which include th~e use of multiple probes, as Is generally outinad in U.S.

Patent Nos. 5,681,702. 5,597,909, 5,545,730. 5,594,117. 5.591,584, 5,571.670. 5,580,731, 5.571.670, 5,591,584, 5,624.802, 5,635,352, 5,594,118, 5.359,100, 5,124,246 end 5,661,697, all of which are hereby incorporated by reference. In this embodiment, in general, the target nucleic acid is prepared as outlined 2bove, and then added to the bicchip comprising a plurality of nucleic, acid probes, under conditions that allow the formation of a hybridization complex.

35 WO 03/006689 PCT/EPO2/07854 A variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allows formation of the label probe hybridization compleK only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as is generally outlined in U.S.

Patent No. 5,681,697. Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.

The reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents may be Included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microblal agents, etc., may be used, depending on the sample preparation methods and purity of the target. In addition, either solid phase or solution based kinetic PCR) assays may be used- Once the assay is run, the data is analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.

In a preferred embodiment, as for the diagnosis and prognosis applications, having identified the differentially expressed gene(s) or mutated gene(s) important in any one state, screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.

In addition screens can be done for novel genes that are induced in response to a candidate agent After identifying a candidate agent based upon its ability to suppress a HIPK1 expression pattern leading to a normal expression pattern, or modulate a single HIPK1 gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated In response to the agent. Comparing expression proflies between normal tissue and agent treated LA tissue reveals genes that are not expressed In 36 WO 03/006689 PCT/EP02/07854 normal tissue or LA tissue, but are expressed in agent treated tissue. These agent specific sequences can be identified and used by any of the methods describedherein for HIPK1 genes or proteins, In particular these sequences and the proteins they encode find use In marking or identifying agent treated cells. In addition, antibodies can be raised against the agent induced proteins and used to target novel therapeutics to the treated LA tissue sample.

Thus. in one embodiment, a candidate agent is administered to a population of LA cells, that thus has an associated HIPK1 expression profile. By "administration' or 'contacting' herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and Intracellular action, or by action at the cell surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent a peptide) may be put into a viral construct such as a retroviral construct and added to the cell, such that expression of the peptide agent is accomplished; see PCT US97/01019, hereby expressly Incorporated by reference.

Once the candidate agent has been administered to the cells, the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein, Thus, for example, LA tissue may be screened for agents that reduce or suppress the LA phenotype.

A change in at least one gene of the expression profile indicates that the agent has an effect on HIPK1 activity. By defining such a signature for the LA phenotype, screens for new drugs that alter the phenotype can be devised. With this approach, the drug target need not be known and need not be represented In the original expression screening platform, nor does the level of transcript for the target protein need to change.

In a preferred embodiment, as outlined above, screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important In a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done. A HIPK1 product may be a fragment, or alternatively, be the full length protein to the fragment encoded by the nucleic acids of the figures. Preferably, a HIPK1 is a fragment. In another embodiment, the sequences are sequence variants as further described herein.

Preferably, a HIPK1 is a fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment. Preferably, the fragment includes a non-transmembrane region. In a preferred embodiment, the fragment has an N-termlnal Cys to aid in solubility. In one embodiment, the c-terminus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, to cysteine.

WO 03/006689 PCT/EP02/07854 In one embodiment, the HIPK1 proteins are conjugated to an immunogenic agent as discussed herein.

In one embodiment a HIPK1 protein is conjugated to BSA.

In a preferred embodiment, screens for agents that alter the biological function of the expression product of a HIPK1 gene are done- Again, having identified the impuitance of a gene in a particular state, screening for agents that bind andlor modulate the biological activity of the gene product can be run as is more fully outlined below.

In a preferred embodiment, screens are designed to first find candidate agents that can bind to HIPK1 proteins, and then these agents may be used in essays that evaluate the. ability of the candidate agent to modulate a HIPK1 activity and the lymphoma phenotype. Thus. as will be appreciated by those in the art, there are a number of different assays which may be run, binding assays and activity assays.

In a preferred embodiment, binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more H)PK1 nucleic acids are made. In general, this Is done as is known In the art. For example, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present. Alternatively, cells comprising the HIPK1 proteins can be used in the assays.

Thus, In a preferred embodiment, the methods comprise combining HIPK1 protein and a candidate bioactlve agent, and determining the binding of the candidate agent to a HIPK1 protein. Preferred embodiments utilize the human or mouse HIPK1 protein, although other mammalian proteins may also be used. for example for the development of animal models of human disease. In some embodiments, as outlined herein, variant or derivative HIPK1 proteins may be used.

Generally, In a preferred embodiment of the methods herein, a HIPK1 protein or the candidate agent Is non-diffusably bound to an insoluble support having Isolated sample receiving areas a microtter plate, an array, etc.). The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insolub e supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic polystyrene), polysaccharides, nylon or nitrocellulose, Teflon

T

M. etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies (which 38- WO 03/006689 PCT/EP02/07854 do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may.then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other molety, In a preferred embodiment, a HIPK1 protein is bound to the support, and a candidate bloactlve agent is added to the assay. Alternatively, the candidate agent is bound to the support and a HIPK1 protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays. etc.) and the like.

The determination of the binding of the candidate bioactive agent to a HIPK1 protein may be done In a number of ways. In a preferred embodiment, the candidate bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of a HIPK1 protein to a solid support. adding a labeled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label Is present on the solid support Various blocking and washing steps may be utilized as is known in the art.

By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies. particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such.as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal In some embodiments, only one of the components is labeled, For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using or with fluorophores.

Alternatively, more than one component may be labeled with different labels; using 1" for the proteins, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to the target molecule HIPK1 protein), such as an antibody, peptide, binding partner, ligand, etc.

WO 03/006689 PCT/EP02/07854 Under certain circumstances, there may be compelitlve binding as between the bloactive agent and the binding moiety, with the binding moiety displacing the bloactive agent.

In one embodiment, the candidate bioactive agent is labeled. Either the candidate bioactive agent, or the competitor, or.both, is added first to the protein for a time sufficient to allow binding, if present Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40'C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component Is then added, and the presence or absence of the labeled component Is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed by the candidate bioectlve agent.

Displacement of the competitor is an Indication that the candidate bloactive agent is binding to a HIPK1 protein and thus is capable of binding to, and potentially modulating, the activity of a HIPK1 protein. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioaclive agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may Indicate that the bloactive agent is bound to a HIPK1 protein with a higher affinity. Thus. if the candidate bioactive agent Is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to a HIPK1 protein.

In a preferred embodiment, the methods comprise differential screening to identity bloactive agents that-are capable of modulating the activity of a HIPK1 proteins. In this embodiment, the methods comprise combining HIPK1 protein and a competitor in a first sample. A second sample comprises a candidate bioactive agent, HIPK1 protein and a competitor. The binding of the competitor is determined for both samples. and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to a HIPK1 protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to a HIPK1 protein.

Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native HIPK1 protein, but cannot bind to modified HIPK1 proteins. The structure of a HIPK1 protein may be modeled, and used in rational drug design to synthesize agents that interact WO 03/006689 PCT/EP02/07854 with that site. Drug candidates that affect HIPKI bloectlvity ere also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.

Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specificaliy bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.

A variety of other reagents may be Included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease Inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added In any order that provides for the requisite binding.

Screening for agents that modulate the activity of HIPK1 proeins may also be done. In a preferred embodiment, methods for screening for a bioactive agent capable of modulating the activlty of HIPK1 proteins comprise the steps of adding a candidate bicactive agent to a sample of HIPK1 proteins, as above, and determining an alteration in the biological activity of HIPK1 proteins. "Modulating the activity of a HIPK1 protein" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate agent should both bind to HIPK1 proteins (although this may riot be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of HIPK1 proteins.

Thus, in this embodiment, the methods comprise combining HIPK1 sample and a candidate bioactlve agent, and evaluating the effect on HIPK1 activity. By "HIPK1 activity" or grammatical equivalents herein is meant one of a HIPK1 protein's biological activities, including, but not limited to, its role in lymphoma. including cell division, preferably in lymphoid tissue, cell proliferation, tumor growth and transformation of cells. In one embodiment, HIPK1 activity includes activation of or by a protein encoded by a nucleic acid of the tables. An inhibitor of HIPK1 activity is the inhibition of any one or more HIPK1 activities.

WO 03/006689 WO 03/06689PCT/EP02/07854 In a preferred embodiment, the activity of a HIP1.' protein is increased: in another preferred embodiment, the activity of a HIMK protein is decreased. Thus, bioactive agents that are antagonists are preferrod In sonme emnbodimreritQ, and bioactive agen1ts that are agonists may be preferred in ot her embodiments.

in a preferred embodiment, the Invention provides methods for screening for bloacive agents capable of modulating the activity of HIPKI protein. The methodo comprise adding a candidate bioactive agent, as defined above, to a cell comprising HIPM'(ioteins. Preferred cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes HIPKI protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells.

In one uspect, the assays are evalueted In the presencr. or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies. peptides, antigens, cytokines, growth factors, action potentials, pharmacological agen is including che moth e rpeutics. radiation, carcinogenics, or other cells cell-cell contacts). In another example, t he determinations are at different stagcs of the cell cycle process, In this way, bioactive agents are Identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of a HIPKI protein, in one embodiment, a method of inhibiting lymphoma cancer call dlvision is provided. The method comprises administration of a lymphomna cancer inhibitor. 'in a preferred embodiment, tie method comnprias administration of a HIPKI Inhibitor, In another embodiment, a method of inhibiting tumor growth is provided. The method comprises administration of a lymphomna cancer inhibitor. In a preferrud emrbodiment, the method comprises administration of a HIPK1 inhibitor.

In a further embodiment, methads of treating cells or individuals with cancer are provided, The method comprises administration of a lymphoma cancer inhibitor. In a preferred emrbodimnent, the method comprises administratIbn of a HIPK1 inhibitor, in one embodiment, a lymphoma cancer inhibitor is an antibody 3s discussed above. In another embodiment, the lymphoma cancer inhibitor is an anitisense molacule. Antisense molecules as used herein include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for lymphoma cancer molecules. Aritisense or sense oiigonuclectdos. according to the present invention, WO 03/006689 WO 03/06689PCT/EP02/07854 comprise a fragmenlt gerifellY at least about UA nucleoitides, preferably from about 14 to nuclootides. The ability to derive an antisense or a eanse oligonucteotide. bosed upon a cDNA sequence encoding a given protein is described Ii. for example. Stein and Cohen. Cancer Res.

48:2659, (1988) and var, der Krol et al., HioTechniques 6:958, (198).

Anklsense molecules may be introduced into a cell containing the target nucleolide sequence by formotion of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the Ilgand binding molecule does rot substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisonse oligonucleolide or its conjugated version into the call. Alternatively, a sense or an anlisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotidelipid complex. as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of troatment.

The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The agents may be administered In a variety of ways, oraty, parenterally subcutaneously, intraperitanealy, intravascularly, etc. Depending uponi the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100% wgt/vol. The agents may be administered alone or in combination with other treatments, rzdiation.

The pharmaceutical compositions can be prepared in various forms, such as grginules, tablets, pills, Buppositories. capaules. suspensions, salves, lotions and th'e like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and 2nlrmal oils and fats. Stabilizing agents, wetting and emulslFYing agents, salts for varying the osmotic pressure or bUffers for secuwing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.

Without being bound by theory, it appears that the various H-IPKI sequences are important in lymphoma. Accordingly, disorders based on mutant or variant HiPKI genies may be determined. In one embodiment, the invention provides methods for Identifying cell; containing varlant HIPK1 genes comprisina determining all or part of the sequence of at least one endogerious HIPKI genes in a cell.

As wilt be appreciated by those In the art, this may be done using any number of sequencing -43- WO 03/006689 WO 03/06689PCT/EP02/07854 techniques. In a preferred embodiment, the invention provides methods of identifying a HIPKII genoty pe of an indivduel comprising determining all or part of the sequence of at least one HiPKI gene of the individual- This is generally done in at least one tissue of thfe individual. and may include the evaluation of a number of tissues or different samples of the same tissue, The mnethod may include comparing the sequence of the sequenced HIPKI gene to a known HIPKI gene, a wildtype gene. As will be appreciated by those In the art alterations Ini the sequence of some oncogenes can be an Indication of uither the presence of the disease, or propensity to dev/elop the disease, or prognosis evaluations, The sequence of all or part of a HIPKI gene can then be comnpared to the sequence of a known H-IPKI gene to determine if any differences exist This can be done using any number of known homology programs, such as Sestfit, etc, In a preferred embodiment, the presence of a difference in the sequence between a HIPK1 gene of the patient and the known HiPK1 gene is indicative of a disease state or a propensity for a disease state, as outlined herein, In a preferred embodiment, the H-IPKI gones are used as probes to determine the number of copies of a HIPKI gene in. the genome. For example, some canicers exhibit chromosomal delellons or Insertions, resulting in an alteration in the copy number of a gene.

In another preferred embodiment HIPKI genes are usec as probes to determine the chromosomal location of the H-IMK genes, Information such as crnromosomat location finds use In providing a diagnosis or prognosis In particular when chiromosomal abnormnalities such as translocatiors, and the like are identified in HIPK1 gene loci.

Thus, in one embnodiment, methods of modulating NIPKA in cealls or organisms are provided. In one embodiment, the methnods comprise administering to a cell an arni-H-IPK1 antibody that reduces or eliminates the biological activity of an endlogerious IIF protein. Altamatively, the methods comprise administering to a cell or organismn a recombinant nucleic acid encoding HIPKI protein. As will be appreciated by those in the art, this may be accomplished in any number of ways. in a preferred embodiment, for example when a HIPK1 sequence is down-regulated in lymphoma, the activity of a HIPK1 gene is increased by increasing the amount of HIPKI in the cell, for example by overexpresslng the endogenous HiPK1 or by administering a gene encoding a HIPK1 sequence, using known geno-therapy techniques, for example. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR). for example as-described in PCMiUSQ3/03868, hereby incorpcroted by reference in Its entirety. Alternatively, for example when a HIPK1 sequence Is up-regulated in WO 03/006689 WO 03/06689PCT/EP02/07854 lyrnphomna, the actvity of the andogenous HIPK1 gone is decreased, for axample by the administration of a HIPI 1 antisense nucleic acid, in one embodiment, the HIPKI proteins of the present invention may be used to generate polyclonal and monoclonlal antibodies to HIPK1 proteins, which are useful as described herein. Similarly, the HIPK1 proteins can be coupled, using standard technology, to affinity chromatography columns, These columns may then be used to purify HIPK1 antibodies. In a preferred embodiment, the antibodies are generated to epitopes unique to HIPKI protein; that is, the antibodles show little or no crotss-reactivity to other proteins. These antibodies find use in a, numter of applications, For example, the HIPK1 antibodies mayr be coupled to standard affirity chromatography columns and used to purify HIPK1 proteins. The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to a HIPKI protein, In one embodimrent, a therapeutically effective dose of HIPK1 or modulator thereof is administered to a patient. By "therapeutically effective dose"~ herein Is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the troatment, and will be ascertainable by one skilled in the art using known techniques. As Is known in the art adjustments for HlPKI degradation, systemic versus localfzed delivery, and rate of new protease synthesi5, as well as the age, body weight, general health, sex, diet, timne of administration, drug interection and the severity of the condition may be necessary, and will be asceirtainable with routine experimentation by those skilled in the art.

A "paUenit" for the purposes of the present Invention includes both humans and other animals, particularly mammals, and org-anisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient Is human, *The administration of the HIPK1 proteins and modulators of the present invention can be done In a variety of ways as discussed above, including, but not limited to, orally, Fubcutaneously, intravenously, intranasally, transdermally, intraperltoneally. intramusculary, intrapulmo nary. vaginally, rectally, or intraoculariy. In some instances, for example, in the treatment of wounds and inflammation, the HIPKI proteins and modulators may be directly applied as a solution or spray.

The pharmaceutical compositions of the present invention comprise HIPKI protein in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are In a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition S3il refers to WO 03/006689 WO 03/06689PCT/EP02/07854 those salts that retain the biological effectiveness of tho free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such ccG hydrochloric acid, hydrobromic acid, sulfuric acid. nitric acid, phosphoric acid arnd the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, OXBIlC acid, maleic acid, malonic acid, succinic acid, fumaric acld.

tartaric acid, citric acid, benzoic acid, Clinnamic acid, mandelic acid, methanesuifonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmeceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the ike.

Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include aalts of primary, secondary, and tertiary amines, cubstituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, dietnylamine, tiethylamine, tripropylamine, and ethanolamine.

The pharmaceutical com'positions may also include one or more of the following; carrier proteins such as serum albumin: buffers: fiers such as microcrystalling cellulose, lactose, corn and other starches: binding agents: sweeteners and other flavoring agents; coloring agents; and polyethylene glycol, Additives are well known in the art, and are used in a variety of formulations.

In a preferred embodiment, HIMK proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above. Similarly, HIPKI genes (including both the full-length gcquence, partial sequences., or regulatory sequences of the HIPKI coding regions) can be administered in gene thes-apy applications, as is known in the art. These HIPKII genes can Include antisense applicains, eiter as gene therapy for incorporation into the ganome) or as antisense compositions, as will be appreciated by those in the art.

in a preferred embodiment, H1PK1 genes are administered as DNA vaccines, either single genes or combinations of HIPI genes. Naked DNA vaccines are generally known in the art. Brewer, Nature Biotechnology, 16:1304-1305 (19ge8).

In one embodiment, HIPKII genes of the present invention are used as DNA vaccines. Methods for the use of genes as DNA vaccInes are well known to one of ordinary skill in the art, and include placing a i-IPKI gene or portion of a HIPKII gene under the control of a promoter for expression in a LA patient. A HIMK gene used for DNA vaccines can encode full-length H-IPKII proteins, but more preferably. encodes portions of a HIPKI proteins including peptides derived from a HIPK1 protein. In a preferred embodiment a patient is immunIzed with a DNA vaccine comprising a plurality of nucleotide sequences derived from a HIPKI gene. Simnilarly, it is possible to immunize a patient with a plurality of WO 03/006689 WO 03/06689PCT/EP02/07854 HIPK1 genes or portions thereof HS defined herein. Without being bound by theory, expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-c-eIIs, helper T-Cells and antlbodies are induced which recognize and destroy or eliminate cells express~ing H-IPK1 proteins.

in a preferred embodiment the DNA vaccines include a gene encoding an adjuvant molecule With the DNA vaccine. Such adjuvant molecules include cytokJrwc that increase the Imtnunogenic response to a HIPIK1 polypeptide encoded by the DNA vaccine. Additional or alternative adjuvantst are known to those of ordinary skill in the art and find tuse in the invention.

In another preferred embodiment HIPK1 genes find Use In generating animal models of Lymphomra.

As is appreciated by one of ordinary skill in the art, when a HIPK1 gene identIfied is repressed or diminished In tissue, gene therapy technology wherein antisense RNA directed to a HIPl(1 gene will also diminish or repress expression of the gene. An animial generaled as such serves as an animal model of lymphoma that finds use in screening bioactive drug candidates. Similarly, gene knocKcul technology, for example as a result of homclogows recombination with 2n appropriate gene targoting vector, will result in the absence of HIPK1 protein, When desired, tissue-spic ific expression or knockout of HIPKI protein may be necessary.

It is also possible that HIPM( protein is overexpressed in lynpliomra. As such, transgenc animals can be generated that overexpress HIPK1 protein. Depending on the desired expression level, promoters of various strengths can be employed to express the transgene. AIao, the number of copies of the integrated transgene ctn be determined and compared for a determination of the expression level of the transgene. AnImalt generated by such methods find use as animal models of HIPK1I and are additionally useful in screening for. bioacliive molecules to treat lymphoma.

A HIPK1 nucleic acid sequence of the invention is depicted in Table 1 as SEQ ID NO. 1. The nucleic acid sequence shown is from mouse.

TABL.E I TAG# SEOl D SEQUENCE

NO.

30001 3 1 CTCC~TGGAGCCNCNTACGGNGTGT (!GA-CCGGTNTcCCAG.CNTCTC:CGCA AACGGTCTCCNAGGTGGTTT)ACCCGGNGTTTGGTGGN~eTCGOTT7CTTACAGrTA.

GAGCNTACATTAACCCAbCATTAXTTCCArA CTCTGAGGGTTTAGAATTCTOAGTTTTTACTT-AAATTGTTTGTGCCATGTCGkTTTC ______AGaCAGCNAGGGOGNATTTAGATGCCTCCCTGT'CCTTNOA A contig assembled from the mouse EST database by the National Center for Biotechnology Information (NOEl) having homology with all or parts of a H-IPK1 nucleic acid sequence of the inventicfl Is depicted in 47 WO 03/006689 PCT/EP02/07854 Table 2 as SEQ ID NO. 2. SEQ ID NO. 3 represents the amino mfICd sequence of a protein encoded bly SEQ ID NO. 2 TABLE 2

MOUSE

SAGRES REF SEQ SEQUENCE TAGO IDO SoOO01 3 F3 2 OCGCCACCAACGCCGGTrAAACCACCTCGGAGACGCTGTCGGAGAGGACTGGGAAACC GGTCCCCACAOACTGTC4CACGCTGGCTCCCCACGGAGGCCCACCCACACCOGCGGCCCGG

GCAAGATGCAOTGATCTCAGCCCTCCCGCTCCTCCGCACTTOCOCCTCAGTATGOC-CTCACA

OCTGcAGGTOmrCGCCCCCATCAG3TGTCGTOoAGTGCCTGCAG7CAAAAAACTGA

AAATAGAGCCCTCTGGCTGGGATGTTC.AGGACAGASCAGCAAC-ACAA.ATACTATACCCACA

GCAAAACCC'rCCCAGCTACACAAGGGCAAGCCAC3CTCCTCTrACCAGGTAGCAAATTTCAATC CTrCTGATAGCTCAGGCAGCOCCGCACAGCAACC1CCAAACAOCAGACCCTcACTCAC AGGAGCAACGTTTCrGCTTGACCATATCAAAATGTGGATTGAAGAGAAGAGAGGAA GTGGAGAGCA'ACc3GTAGCGTGCAGATCATAGAAGAACACCCCCOCTTcA-rGCTGCAGAACAG AACCGTGG7GGGTGCTGCTGCCACGACACCACTGTGACCAOCAAGAOTAGCAGTTOOAGTG

GACAAGGGGATTACCAGCTGGTCCAGCATGAGATCCTGCTCTATGACCAAAGCAGAA

GTCCTGGAGrTCCTAGGCCGGGGOAC-ATTGGACAGGTGGCAAGTGCTGAAGCGGAGCA CCAAGGAAATrGTOGCCATTAGATCTtGAAGAACCACCCTCCTATGCCAGACAGGACAOA TTrGAAGTGAGCATOCTTCCCGCCTAAGCAGTGAAAATGCTGATGATATACrGTCCOrT CTATGAGTGT11T-CAGCACAAGATCATACCTG3C~lrGTGTrGAGATGTGGAGCAGMCTT GTACBA! iCTAAAGCAMCAAGTTACCCACTGCCACTAATACATAGACCAATTT IG CAGCAGGrGGCCACAGOcCTGATAA(CTGAAGAGTCTTOTCTGATC-ATGCTACCT-AA ACCTGMAAACATATGCTAGTCGATCCAGrCrCCAACCC1'ACCGAOTGAAGG7CATTGACTTr TGGTTrCTGCTrATCATOTOAAcCGTGGTTCACCTACC7GCATCACGCTACAC:AG ACTCCTGAAATTrCCrGGArA~CACTGTAAGCTATGACATGGOTCACTGGGCTGT

GTATAGCT-AGCTGTTC;TGGGATGGCCTCTTATCCTGTGCT-CAAATACATCAGATT

CGCTATATTTCACAAACACAAGGCTGCCAGCTGAGTATCTrTC'tCAGTGCCGAACAAAACA ACCAGGTTrrrAACA3AGATCcTAA1TGGGTACCCACTGTO3AGGCT7AAGACACCTG AAGAACATOA~rTGGAAACTGGATAAAGTCAAAAGAAGCTCOGAAGTACA1TflAACT G-TAOAT(ACATGGCTCAGTAATATOTCTACAGAClAGAGGGACAGATATGTAG CAGAGAAAGCAGATCGGAGAGAGTATATTGATC-1TCTAAAGAh.ATGCTGACGATTGATG CAGATAAGAGAATCACOCCTCTGAAGACTC7AACCACCAMTrGTGACGATOAGTCAC C TCCTr.GACTTTCCTCACAGCAGCCACGTTAAGTCCTGMrCCAGMACATGGAGATCTGCA AGCGGAGGTCACATTAToACACACTATCAATCAAACTCCCvrCACTACACATG TCGCTCCAATACAAGCACAAATCAAcCATGAGCTTCAGCACCAGCTCAACACAGIfGC ACAATCAC GCCAGTGTTCTAGCTTCCAGCTCTAC7GCAGCAGCAGCTACCC71TcTCTGG CrATCAGATGTCTCrCTGCTAACTACCAACGGCMrGTACCCATCGTCGGrAGCGC CACTTCCTGGAG-TGCCCAGCAGGOG~TCCTTACAACC7GCAACCACCCAGAltCGCA CTCAGACAGATCCATTCCAGCAAACA7ATAGTATGCCCACCTGC1TTCAGACTOGAC TACAAGCAACAMCMAAGCA1TCTCGAT-TCCCTGTGAGGATGGATh.ATGC7GTCCAATTCG 7ACCCCAGGCGCTGCT3CTrAGCCGCTOCAGA7CCAOTrCAGGA3TACTCACACAGGA

OCTGTACACCACTAATGGTAGCAACTCTCOACCCTCAAGTAGCCACCATCACGCCOCAGCT

ATGCGGTGCCCTTTrACCCTGAGCTGCOGAGCAGGCCGGCCGGCGTGGTTGAACAOACrG WO 03/006689 WO 03/06689PCT/EP02/07854 SAGRES REF SGQ SEQUENCE TAGQ# 1 D9 CTGCTGTACTGCAAr~rOCTGGCrCTGGAGGAACCCAACAAATrCTCCTGCCTtCAGCCTGGC AGCAGCTGCCCGGGGGrAGC7CTGCACAACTCTGTCCAGCCCTGCTCCACTGATTCCACAGG CCATGGGGAGCAGCcAAcAGCTAGCTOACTGGAGGAATGCCCACTCTCATGGCAACCAGT ACAGrCACTATT-IAGCAGCACCCATCMTGCTCACCA.ACr-ATGTGACC7TGGCCACTGCTC AGCCTCTGAATGTTGGTGnGCCCATTTTC4GACMCACAGTCTAGTTCCCTOC CAA AAGAATAAGCAGTCTQCTCCZAGMTCATCCAAATCCrCTCTGGAAGTCCTGCC1T CTCAAGVTATTCTCTrGGTTGOGAOTAGTCCTC71TCGTACCACATCT-rCTATAATTCCC TAGTTCCTGTCCAGACC;AGCATCAGCCATCATCATCCAGATACCCCCAGCCCTrCCTO TGAGTGTCATCACTATCCGTAGTGACACTGA7GAAGAAGAGGACA.ACAAkATAOMGCOCCA ATAGCTCGAGCCTGAAOGCGAGGTCTAATGECCATCAG 'TATGTCACTGTCAATGAIflCTC

CAGACTCTGACTCCTOCCYGAG)CAGCCCACATCCCACAGACACTTTAGTGCTCTGCGOO

GCAACAGTGGGACCCTTCTrGAQGOOACCTGGCACGACCTGrCAGC-AGATGGrATTG3GCACCC GTACTArATrGTGCCTCCTIrGAAAAcrACAGCTTGGCGAC'TGCACTG1AGCAACACAG cCCAGrCTCCTTAGCAGTAAGACCAACCAGTGGCCTCAGTGAGTG GGCAGTCATCTG GATGCTrGTATCACTCCCACCGGGACCGC3(0TCAGCGAGGGGS3AGCCAGCGCGGTGCAGC rOACTCACCTAGCCAGAACCAGCAGTCATCG'CACTCAAC;C:CGCAGCAAGAAG

CA

GCAACCCTGCTOCCCCGCAGACAGCAGGCAfTTGGCCCCOCTCTCCCMAGCCCCCTACG CCTTCC AGCATGGCAGCCCACTGCACTCGACGGGGCACCCACACTTGGCCCAGCCCTG CTCACCTGCCAAGCCAGCCTCACCTGTATACGTACGCTGCCOCCACTTCTGCTGC1GCAT TOGGCTC CACCAGTrrCCATTG CTCATCTGTTCrCC CC COAG 00 CCTCAAGGCATGCTG CAGCTTATACCACACACCCTAGCACTCTGGTCATAG7TCCTGTCAGTGTCGGGCCCA GCCTCCTCArTG~CCAGrGTGGCCCGTGTCATACAACACCAG~rGCCACTCAGT CCTACATCGGTCTCCCGAGGCrACCMTACACTOATACCCGCTGAGTCCTACCA AGA*TCAOTCAGTATCTACTOAGITGATGAGCACAGGAGGCTCGGGCTGT

;G

CTAA3TA0CCCTGAG7-CTTAATGGGCTCTGCAGACACCTCATATCTCTCTGA G~dToCGACC7CGGrGCATA~-(IArCT7CTGA n7TCT~,C~,~~TGcTrAGGT:GAcc~TrTrA; TACCAAGGAGAGATCGTTGMAGTTACCC.TCTGTCATACMlGGTT1mGACT TGGTTCTATAAATGi-i II M.AATGMOCTAAGCTCTTCTTACGAGGGGAAATGCTGA CTGATCGACGTAAAATCr~rma oTxvArTA ACACA.GACTTCCTrGTCTTATTrGAAACAGTTAGCAGGGTGCTATGGCGT ATGGAACAGAATGAfTCATTTATGTCTCG~ccTArCTrGGGCrArGTAAGT TrAGTACAACOAGTCACTGACCTOCAOcTCTOCTCTAGCTGCTGCAGACAGCA CTGAACAGGCAGCCAGCGCTGCTGGGAAOG0AAOOACOG71GGACTOTGCCOACCAGG A'rCATTCTAATGAGACCATGAGrCAGTCCCCTCCCCTC1TAGlTATA TTCTCCTTATAGAAGCAGTiAGGTGG4TAAGTGTATGGTGGTGGTTTGCATACAA-AG TATGCAAAATTCTCTCTAAATAGATACTCATGA7AACATGC'TGATTTCT ATCOMCAATAATACACGTCTGTGTCCTCATCTC-rCCCT-rCTGTTTCATGTGACT TAMnOAGGGAACTAAGAACTAAMACCAGAMTCGTGTATAGC7TATACTT TAAAGTAGCT'TCCMGTATCCACAGCAAATTGAATGCTCTCTTACTAAGACtrATGT AATAAGTGCATGTAGGATGCAGAATAT1AGTTATACTGAATVAAAT A7MAGAAGT-TTAATGTGGTG71AATATTTGCAAATTAAATATGACATAT TGA1AGAMATATAACAATTMCCTCTAACCCAATGTAMTGATCAATGT -49- WO 03/006689 PCT/EP02/07854 SAGRES REF [SEQ TAG# A ID*

MJOUSE

SEQUENCE

rGTAG~rTACACTGATTGTA1AGTGTGTACTGACCTCCAGTGTACCCCGG AGTG-rTT-CArGAMACAATACGTC-GTGMGA GTGACT-rA-GcAAC TATAcAATATTACTGTATCTGAGAGACAGGGCATT GCCTGCAGAGAGGAGACCTTGGGAGTMGCAC GGTGTGTCTGGTGAGGAG~rGr 1

C

AGTGTTr~7cCCTOC-tCTCCTCTCTCCCC1ATGTAGTGCCATATGATA AT OTAGTGGrAATAGA ACAGGAGCrGrAGGATGACCAGCAAGCCCr-GrC AGCCCAGCTGTTCGC1GGGArTAACAGAGCAGuI IAG1AGCTGTGTrTG~TAAGC GOOTGTrCTCAGTCTCCCTACCGACAGTGACAAGTCAAAGCCGCAGCTIT~rCTCCT1A ACGCC~,GCCTr;;--TGT-rAGCG7TAAAGA~, CTAAMTGGCTCTCCACTGTGCClGCTACCGGCTCTGGATAGA3CGG C GAGAAGAGTGACG CCAGTGCAATAGCATATTrGAAGTGTGCATTAC~rAGGr GGG.ATTCCTn-ITC1CTCCTCrCATGTGATATG3ATAGCC=rCTGrCATAGCTGTCGTUTCC TGGTAJAACTTTGCTTGGTT7TTTTT7r II Ii ii IGI I I I TITTAAACATOTAA CAGA7TGGTTATACCAAAGACCCTGTTGTATTGCTTAATATGTCCICATACTACGAGAAG GG7 AACAIGGCA-ACTACGAjG--CTATGGCA TATGAAAAAGAAGCAACCTC7GAATC1rAACACCTGAGGTnCmGGGACAA

CATGTTGTTCTGGGGCCC'TGCACACTGTAAATTGTCCTAGTATCAACCCCTCCATGG

ATrGGTCAA-I-GAAGG-rACTAGGGGTGGGGACATTC'TrGCCCATGAGGGATTTGTGG

COGAGTACTACAAATGTCCTATAATTTAATG

ATOTAGGAnrGTTCTGT-rTAGGTGGTGTCAGGArGTGCAGGATGGAGATGGGCAGAT- 'rCATGGAACCCGTTCAGGAAGCVCTGAACCAOGTGG~oACACCGAGGGGCTTCAACGAA rCTO(GAGTTCTCATCAT(30(GOAGGCAA(3AOTTCCAOCQCAOGOCAO 3TAarCAO711A GCCTGCCGCACGTOGTTT0rVTCTCTTATCATTATATTAAGCTGTGCGT7 CAOCAO C TTGG TGAGATAACCACGCATCA C ~rTAG GMrTcAcTAGTGT-rATAc COrTTATGTCATrCTGTGTGTGTCMGTGTTTC CTTTGCCCCAAGCATTCTCGGGTnT TOTTTATCGTCAT-CrGCAC~rrAC~rCCAA OGGACAAOATTTA1GCTrTATAGOAATGAGATGCAGG(i3AAAACAAACCMACCCTGT OCOCOTACCCATCArACGrA-GAAGrA7TAATr TGGGMAAGCAGGIATGCCTAGAGGCATCGTACCTGAGACGATCTGGCTAGrGGT AC~AAArrA~rCCCGAATAA CGTTC~AAAC

CCATCTGTGTCTGCTGATGTTATCCAACATAGTACATCA'TGGCCI'G

GGCeAACATTCACTGCTTGCCCTOATGCATGGAGAGAGCCCTACGAACATAGrCSCTG ACTAG3TCAQCArrr&CQTGACTT G rGCVI-GCTTGCTAAACC7TCTCTAGAACGi TGCATrCCArITCTCCCTrCCAGGTGAGAGAGGAACTGGAGGGTTGCATAGGCACA CACGAATATATOGGCCATGACIAGGTGTCCGr ,AACCCCAGGAAGAAGAACCCCATrTCAAACAGTTCCGGCCA1TGAGAGCCTGCT1TTGTG G-TTGCTCA7CCGTCA~TATCCGCTAGAGGGGCTAGCCAOGCCAGCACAOTACTGOCTOT CCTA-rrCTGCAT-TAGTATGCAGGAAT-TACTAG1TGAGATGGTTTG7TTAGGATAGGAG ATGAAATTCCTCGTACAGGAATGGCCAAGCCTGCTTTGTGTT1TTAAATGA TGGATGGTGCAGCATOrTCCAAGTTTCCATGGTT'rGTGCTAAAATTATATMTG TGTGTTCAArTCAATnCAGC-T-TGATAATTCACTATATGTAGCAGTAATA7A TGTACATTATATGTATTTAGTATflTCTTGAATCCTTCGATA7TOGCMTGWTTC C7AATTTA'AAA-rGTA-rGATATGCrAAAAAA WO 03/006689 WO 03/06689PCT/EP02/07854 I

MOUSE

SAr3RIS REF 91F0 SEQUEPICE TAG# it !O0A 3 MAQQFPSSSF$KLIPGOVGSNK H~L~QQSSOA FNPYGLLAAEIVADSSAATOSTTRNSLPO~]Kq~~ HTCLVFE=MLEQNLYDFLKQNKFSPLPLg<YIRP LQQVATALMKLKSLGLJHA0LKr'ENIMLVDPVRQ LOCMAQVNMSTDLEGTDMLAEKADRRFYIDLLKKMLTIO)AOKRlTPU(rLNHQFVrMSHL=fFPHS SHVKSCFQNMEICKRR\AHMYDTVSQlKSPFTrHVAPN4TSTNLTMSFSNOLNTVHNCQASVLASSST

AAATSLANSVSLLNYQSALYPSSAPVPVAQQGVSLOPGTTQICTQIOPFQQTFIVCPPAFQ

TGLQATfl(HSGFPVRMDNAVPIVPQ).PAAQPLQIQSGVTQGSCTPLMVATLHPOVATITPQYVv PFSAlArL.VETVLQAWPGTQQILLPSAWQLFGVLHNSVQPAVIPEAMCSSQQ L.ADwRNAH-SHGNQY$TIMQQPSLLTNMVTLATACPLNVGVAHWRQQQSSSLPSKKNKOSAPVS SKSSLEVLPSQVYSLVGSSPLR1TTSSYN4SLJPVQOQQPI'TPSPPVSVITIPSOTOE-EEONKYK PNS SSL.KARS NV IYVTVND SP OSDSSLSSP HPTDTLSALRGNS GTLLEGCPG RFAAfGIGTRTIIVP SSASTS QERS SNPAP RRO QAFVAPLSQAP YA FQHG SPLHSTGHPHLAPAPAHLPSQP NLYIYAA PT-SAALGSTSSIAHLFSPOGSSRHAAACrrNPSLVHVPJSVPSLLTSSVAPAQYQFAT QSYlr>$SRGSTIYTGYPLSP7hJSQYSYL Also suitable for use in the present invention is the sequence provided in Genbank Accession No. AF077658.

A contig assembled from the humzn EST database by the NOBI having homology with all or parts of a HIPKI nucleic adid soquence of the Invenilon Is depicted in Table 3 as SEQ 10 NO. 4. SEQ ID NO. 5 depicts the amino acid seqluence of a open reading frame of SEQ ID NO, 4 which encodes the C-terminal portion of humnan HIPKI proteln.

TABLE 3 HUdMAN SAQRES REF SEQ SEQUENCE TAi i iOft SODOO13 F30 4 CAkCACCGAGTATTGCiCCTTACTCTGA GCTGCGCAGCCGGCC(Q(tC0rGCTGG- TGAACAGACTGCCGCTGTACTGGCG1GGCCTGGAGGGACTCAG;CAAA7-rCTEcCTr.CCTTC AAcMGGCAACAGTTrlCCTGGGGTAGCTCTACACAACTCTGTCAGCCCACArCAAGAT TCA3GCAGGATGC~,cCA -OCOAGAGCATTAG CAACCAGTACAGCAClATATGCAGCAGCCArGCTACAC;TGGACA11-OC CACTGCTCAGCCTCTGPATGmGGTGGCCATrTCAGACACAACAATCCAG-n-C CCTrCCTrVCGAAGAATAG-CTCAGTCCAGTCTCTCAACTCCTCTOAGATGT ________TAATTCC7GQTCC1GTCCAAGATcAOCATCAGCCCATCATCATTOCAGATAC1.CCCAG -51 WO 03/006689 PCT/EPO2/07854

HUMAN

SAGRES REF SEQ SEQUENCE TAG-4 11 I0J# CCCTCCTGT7GAGTGTCATCACTATCCGAAGTGACACTG~ATGAOG~i.AGGACMACMATA CMAGCCCACTAGCTCTGG0ACGAAGCCAAGGTCTMATGTCATOTTATGTCACrGTCAA TGATTCTCCAGACTCTGACTc-IrC-T7GAGCAGCCcTTATrcCACTATACCCTGAGTOC TCTCCAGGAA-AGTGGATCCGTTTrGAGGGCCTGGCAGAGTTGTOOCAGATGGCAC TGG(CA LCCGCACTATCATTGTGCCTCCACTGAAAACTCAGCTTGG-rGAG-TGrACTGTAGC AACCCAGGCCTCAGGTCTCCTGAGCAATAAGACTAACCCACTcCGCAGTGAGTOOQGCA QTCAICTGGATGCTGTATCACCCCCACAOGGiATCGAGCTCAACGCGGGGGGACCAGTGC- AGCACAACCACTCAMTCTTAGCCAGAACCAGCAGTCATCGGCGGCTCCAACCTCAr-AGGA GAG AAGCAGCAACCCAGCCCCCCGCAGGCAGCAGGCGTTGTGGCCCCTCTCTCCCMAGC CCCC'TACACCT1VCAGCATGGCAGCCCGCTACACTCGACAGGGCACCCACACC1GCCCC OOCCCC-rGCTCACCTGCCAAGCCAGOC'CATCTGTATACGTATCCTCCCCCOACTTCTOc TOCTOC-ACrGGCTCAACCAGCTCCATGCTCATCTTTCTCCCCACAGGGTTCCTCAAG GCATGCTC.CAGCCTATACCACrCACCTAGCACmnCGTGCACCAGGTCCCTGTCAGTGT TGGGCCCAGCCTCCTCACTTCTGCCAC;CGYGGCCCCTGCTCAGTACCAAC-ACCA(frTciC CACCCAATCCTACATTGGGTCTTCCCGAQGCTCAACAAmTACACTGGATACCCGCTGAG TCCTACCAAGATCAGCCAGTAfTTCTAOTATAGTTGGTG3AGCATGAGGGAGGAGGMATO ATGGCTACCTOCTGGCCC'rGG-TCTTAATATTGGjGCTATOGAATCATCCTTA CCCTCTTQAmC7TTAGCCAGCACTGTCTCAGGGCCCACTAAGCAGAAGGTT T77CTrCTGrGcGACCTGTCTCAG'TGTTACTCATGrrGTAGTCTrCCCAMAGMGC CCTATrTTMATTCATTATTTGTACATATT-I GTACTGGAAGAGTAGATrD CCCATCT'TCTGCAGTTACCAAGGAAGAGAG3ATTGTCTGAAGTTACCCTCTGAAAATAT 177GTrCTCTCTGACTGAT1-rCTATAAATGC.I i AAWCAAGTGAAGCCCCTC1TrAT TTCATMTGTG'T7ATTGTGATTGCTGGTCAGGA.AAATCTAAGAGGAGTTGWATC TGATGACMAAAA.AATTACrMGTTTTTAAACTCAGACTTGCCTrArr A1tAAkAGCGr.CTTACACAATCTCCCTTTTGTTTATTGGACATTTMACTACAOAG3T 1TAG'rMGTTAATGTCATA~TATACTAATGCAATTGTAnTrGCAAAACTa GrTACG7ArrACTCTGTTTACTATGAGATCTCCATTGCTCCTGTG~rTGrATAA

AGTAGTGTMAAAGCAGCTCACCAMGCTGTAACTAAGTGAGAGATCATATC

TGCGTGAAACACCAA(GTATTCTTTAATOAAGCACCATAA71TTAAATtAT 7M1AAAGTCTTCCTCTCTGATTCAGC77AATTrrATCGAAAAAGCCATAA G m3GGTTATTATTACATGGTGG7GGTGGTTTrTATTATATGCAAAATCTCTGTc-rTAT( AGATACTGGCATTGATGAGCTTTGCCTAAAGATrAGTATGAAITrCATAATACACCTC TG 1rGCTCATCTCTCCCTCTGrrATGATTrrGGAGAAGCTAAAAA CCTGAACCAQATAAGAACArrCTrGTGTATAGCrTAC-rCAA AGTAGCTTCCTT TGTATCCCAGCAOCATTC-,ATCCTOTC7-rTAAACTrATATATAGTGCATGTAC GAATTGCAAATATTAAAAA11-r7TACTGAAMAAAAATATT-rAGAAGTITTG TAATGGTrGTGT-rrAATATTTAcATAATTAMTA'tGTACATAT-GAT-AoAAAAATAT AACAAGCAATrrCCTGCAACCCATGTATAATCAATGGTAGTGATTAC ACTTrGAATGTGTACTrAGGTOATGTGATCCTCCAQTOT-rATCCCGGATOGA1TrGA TGTCTCCATTrGTAMAACCAAAATGA.AC:TGATACrr0GI-GATGTATGTGAACTAAT TGCAATTATATTAGAGCATATACTCTTCTCAATGAGGGGCATTG CCTOCAG AGAGGAGACCCTTGGAATGnTTCACAGGTGTTCGAGGAGnCAGTVGT GTCTCTTCTTCCCmCTTCCTCCTTCCTATTrGTAGTGCGTTATATGATAAm-G GGTTATAGATTTACAGT3AGCTTGCCTTAGGATCGACCAGCAACCCCCCGTGGAOCCT WO 03/006689 PCT/EP02/07854

HUMAI'I

SAGRES RE EG SEQUENCE TAG# I O I AGGTTCACGGGATATCAGAACAGATAGTAGCTGTA-GTGTAATGCATTG-1T TCTACOT7CCTCrCATGA TG G7CTGAGAAr-yrcCCATTG (GCTCTCTCACTTGCG17GCTACC77GCTTCTGTGAGATCAQGAAGCAGTGAGAGGA GTCAAGCCAATATTAATTOATTOTT-17TAGTATTGCATCAC-r-AGATGAAT TA~rTGCTTG1TGAAAAAPAACATGT-rAACAGATGTGTTTATAC.AAGAGCTG-TGTAT TfGCTTACCATGTCCCCATACTATGAGGAGAAGTrI-GTGGTGCCGCTGTGACA(GAAC 7CACAGAAAGG7m-CTTAGCTGGTrAAOAATATAGACAGGAMCCAA.GC-CT-GAGTC ATTGAGGCTFrTGAGG111cTTrnFTAACAGTT3TATAOTCn-GGGGCCCTTCAAGCTG TGAAATTGTCC170TA=CCAGCTCTGCATGCATCTGGGTCAGTAGAA(QGTACTGOG GATGGGACATCTI3CCCATAAAGATGGGGAGAGArATCCTAAATACAGG -rTGTAAt3ATAGGGCTTCCAAATGGGCA~cTAGTccGACAOC CTACCAGrGAACAT GTCAAGATAGCCAAGCAGmTTTTCTTC0CACTATGCATAAG--1CTGGT AACATGTGTGAT IIGTGTCTCCTTGCCAGCACACTAT~r-Cfl-GTTGGAAC ACAGGTCTAGTTCTAAGGACAAArrT~nCCTGTC-IF-YCTGTAAGGGACAA GAMGTTG-rrrTTAAGAAATGAGATCnCAGGAAACGAAAACCAAATCCAT-CcTGOAC CCCAOTCCAATAAGrGAGATACCACTAAATAGGAGTCrAC7CCACAGAA&oOCATMk TAOCAAGAGCTGTATTGrACCTAGTCACGCCTAGCATGTGTGGCTAAACT AGAGArmCAGTCTTAGTCTGCAAACTGGCAmrCCGATCACAT~AAAMTCCA CCTGTCTCTGCTGAATGTGTATOTATOTGOTCACTGTGOCr-TA3A77CTGTCCQCGGGG TTAGCOCTGTTGGCCcrQACAGGAAGGGAoGjAGCCTGGTGAAl-rAGTGAG

CAGCTGGC

G3GCATTrTCCAACTC7CTCCG(TGAG r.AGAAGCGGA ,AGGG1TCAGTGTAGC; CACTCTGGGCTCATAGGGACACTTGGTC-ACTCCAGAGTT77TTTAGC1TCCAGGAGGTG ATA~rTAMCAGTGCrCAOCTATACCAACCAGGAATAAGACCCATTnCAAAC TATGCAAAAA77CACTAGI-rGAGArTG GrTTTAOATAGGAAATGAAATGCCTCTC AGTGACAGGAGTGGCCCGAGCCTGCTrCCTA7MTTATT-I1--TIr-rAACTGTAG

ATGG

7 GCCAGCATGTCrAATGGTG 1- TTGCTAAACrrAATAATGTTGTTCAA AMrGA'TATOCTAGTTATGTGTGCGA-rTAA1-FrGC-1CTCCCTT-Tn-i-GG ?TGTGC3CrCTACAACAGCCTCTAGAAACAGATAM-TCTGAGMUATACTGAGC TAGATATTATM7TACTA.CTATGGGAAAGGTCC-I-rGTGTAAACATAG~m ACTYVTLACAATGTPALVCTA\ AGrrrAAAGTAG DfA~TI j T~ PQYAVPFT CAAGRPALVr:QTAAVL.AVVPGGTnnILLPS r'lI PGIIAI HNSV0P'r- WO 03/006689 PCT/EP02/07854

HUMAN

SAGRES R4E; S EQ SEQUENCE TAG* II SCQOADWRNAHSHGNQYsIMQQPSLLTNH\ITLATAQPLNVGVAHWRQQQsssLPSKKNKQS APVSSK-SSLfJVLPSqvYSLVGSSPLRTTSSYNSLVPVQOQHQPIIIPOTPSPPVSVITIRSOTOEE-ED.

NKYKPISSSG1KPRSNVIsyvtT~oSPSDSSI.SSPYSTDTLSALRONSGsvLErPG~kWADG7GTR TIVPPLKTQLGDCrtVATOASGL.LSNKTKPVA ,VSGQSSGCCITPTGYRAQRGGTSAAQPLNLSQN QQST6~QE-RSSNPAPR1QAFVAPLSAPTFQHSPLHSTHPHLAPAPALPSAHLYTY A~APTSAWL OS7S SIAHLFS P GSS RHAAAYTHPSTL VHQ VPVSV PS LLTS ASV\APAQTQHQFA TOSYIGSSRGS71YTGYPLSPTKISQYSYL All references cited herein are incorporated by reference.

Claims (14)

1. 2. The method according to claim 1, wherein the cancer is lymphoma or leukemia.
2. 3. A method of screening for a bioactive agent capable of modulating the activity of HIPK1 protein in a cancerous cell, wherein the HIPK1 protein is encoded by a nucleic acid comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO.4, the method comprising: a) combining the HIPK1 protein and a candidate agent; and b) determining the effect of the candidate agent on the bioactivity of the HIPK1 protein; wherein a change in the bioactivity of the HIPKI protein in the presence of the candidate agent as compared to the bioactivity of the HIPK 1 gene in the absence of the candidate agent indicates that the candidate agent is a bioactive agent capable of modulating the activity of HIPKI protein.
3. 4. A method of evaluating the effect of a candidate lymphoma or leukemia drug comprising detecting alterations in the expression or activation of a gene comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO.4 or a product of the gene, wherein the gene product is a polypeptide or a mRNA, in a sample obtained from a patient who had been administered with the candidate drug, wherein changes in the expression or activity of the gene or gene product indicates that the candidate drug has anti-lymphoma or anti- leukemia activity. A method of diagnosing lymphoma or leukemia comprising: a) measuring a level of HIPK1 mRNA in a first sample, the first sample comprising a first tissue type from an individual; and 00 b) comparing the level of the mRNA in to a level of mRNA in a second sample, the second sample comprising a normal tissue type; c wherein a difference in the mRNA levels indicates that the individual has lymphoma or leukemia.
4. 6. An in vitro method for inhibiting the activity ofa HIPKI protein in a cancerous ,O cell, wherein the HIPKI protein is encoded by a nucleic acid comprising a 00 nucleic acid sequence having at least 75% identity to SEQ ID NO.4, the method Scomprising binding an inhibitor to the HIPKI protein. (Ni
5. 7. A method of treating lymphoma or leukemia comprising administering to a patient an inhibitor of HIPKI protein.
6. 8. An in vitro method of neutralizing the effect of a HIPKI protein in a cancerous cell, wherein the HIPK1 protein is encoded by a nucleic acid comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO.4, the method comprising contacting an agent specific for the HIPKI protein with the HIPKl protein.
7. 9. A method of diagnosing lymphomas or leukemias or a propensity to lymphomas or leukemias comprising sequencing at least one HIPKI gene of an individual. A method of determining HIPKI gene copy number, comprising adding a HIPKI gene probe to a sample of genomic DNA from an individual with cancer under conditions suitable for hybridization.
8. 11. A method of treating a cancerous cell, comprising contacting the cell with an inhibitor of an HIPK1 protein.
9. 12. The method according to claim 7 or claim 11, wherein the HIPKI protein is encoded by a nucleic acid comprising a nucleic acid sequence having at least identity to SEQ ID NO:4.
10. 13. A method of diagnosing lymphoma or leukemia, comprising detecting the presence of differential expression of a HIPKI gene in a patient sample, wherein 00 the presence of differential expression of the HIPK1 gene indicates that the patient has lymphoma or leukemia.
11. 14. The method according to any one of claims 3, 4, 6, 8 and 12, wherein the nucleic acid of the gene or encoding the HIPK1 protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO.4. C 00 15. The method according to any one of claims 3, 4, 6, 8 and 12, wherein the 00 C nucleic acid of the gene or encoding the HIPKI protein comprises a nucleic acid ~sequence having at least 85% identity to SEQ ID NO.4. S16. The method according to any one of claims 3, 4, 6, 8 and 12, wherein the nucleic acid of the gene or encoding the HIPKI protein comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO.4.
12. 17. The method according to any one of claims 3, 4, 6, 8 and 12, wherein the nucleic acid of the gene or encoding the HIPKI protein comprises a nucleic acid sequence having at least 95% identity to SEQ ID NO.4.
13. 18. An inhibitor of an HIPK 1 protein when used in treating lymphoma or leukemia in a patient or when used in treating a cancerous cell. WO 03/006689 WO 03/06689PCT/EP02/07854 SEQUENCE LISTING <110> PEDERSEN, FINN S SOEREENSEN, ANNETTE. B HERNANDEZ, JAVIER. M <z120> METHODS FOR DIAGNOSIS AND TREATMENT OF DISEASES ASSOCIATED WITH{ ALTERED EXPRESSION OF HIPE(1 <1:30> A -7001S/RrS/DCF <160>O <270> FatentIn veraion 3-1 <210> 1 <211> 331 <212> DNA (213> Mus niusculus <220> <222> uisc-feature <222> <223> at positions 7, 16, 18, 2E, 41, 50, 61, 70, 66, 06, 124, 12!, 299, 306, or 329 can be any base. <400> 1 ctccgtnggg agccancnto ncggtctccn aggtggttta tcanctC~flC taqtqtqaCa cacatcccag cgartggca gggggnattt aqatgcctcc gacggflgtgt ggggaccggt acCggngttt ggtggnqgtc tcaccccaaa ccaqtgtgat gcgcagggag acattgacta acttaaattg tttgctgcca otgtccttng a lt cccagtcn ggqttt--tta ttttccccca cotqgggggat tgzcgatttc tctccgCaaa cagttagatg acatcccadt gactctgagg agggcagcna <210> 2 (211> 7594 <212> DNA (213> Mus musculus <400> 2 ccgccaccaa acgccggtta aaccacctcg gagactgctg tgcggagagg ac-tgggaaac cggtccccac acactqtcca cgctggctcc ccacggaggc ocacocacac ccgcggcCCg gggcaagatg cagtzgatc zc aaccctcccg ctcctccgca cttccgcctc agtatggcct cacagctqca ggtgttttcg CCCCCatcag tgtcgtcgag tgccttctgc agtgcaaaga aactgaaaat agaqcotct qgctaggatg trttoagaca gragcagcaac gacaaatact WO 03/006689 WO 03/06689PCT/EP02/07854 at accoacag caaatttcaz atattgtggt gocagazcat tgaagagaaa cccctctcat cCaccaacag tttgctctat aggtqgoaaa accaoccctc gtgaaaatgc atacctgcct agtttagoccc tgatgaagct tagtcgaicc atgtttcaa ttatccttgg ctgagctgtt atattCaca Ccaggttttt aagaacatga gtttagatga cacagaaago cagataaqag tcct ggactt agcggagggt C c go tcaaa acaat caqgo c taaCtt aga caaaaccccc aacacjct goc gactoccgg gagtgaggaa gctgcagaac tagcagttcc gaccaaoagc gtgctgg~ag ctatgccaga rgatgagtat act gccact c gaagagtctt agttcgocaa agccgtgtgt attaccattc cotgggatgq aacacaaggc taacagagat attggaaact catggctcag agatcggaga aatcacgcct tcctcacagc toaoatgt-at tacaagcaca cagtgttoca tqtcotcgotg cca gotacac tacgaccagg gat agoccagj agcaacgLtot gtggagagca agaaccgtgg a gtggag aag tat gaagt cc oggagcacca caaggacaga aactttgtcc atgttggagc aagtacataa ggtctgatt C Ocotaccgag toaacctacc tgtgaagcta Oct ctttato ctgcCagotg ggaataaagt. gtaaatatgt gagtatattg 6tgaagaotc agccacgtza gacacagtqa aatctaacca got tccagct Otaaactacc aagggcaagc gcctccttct gcagogccgC ct'ctgcttga acggtagcgt tgggtgctgc gggattacca tggagttcct aggaaattgt ttgaagtgag gttcttatma agaacttgta gaccaatett azgctgacct tgaaggtcat tgcaatcacq ttgacat gtg ctggtgcttc agtatcttct ggt acccact o aa a agaago ctacagactt atcttctaaa ttaaccacca agt Oct gttt qtceagatcaa tgagcttcag ctactgoagc aatcggcttt cagotoctct cu-agct Oct tacagcaaco gccatatca gca gatcoat a tgccacgacc got ggtC oa g aggccggggg ggccattaag oat cctt too gtgttttcag cgatttt Cta gcagcaggtg taaacctg~a tgactttggt cta cta caca gtcactgggo agaatacgat cagtgcoqga gtggaggct C tcggaagtac agaggggaca gaaaatgctg Atttgtgaca coagaaoatg gagtcocttc ca accagct c agcagotaco gtacccatcg caccaggtag gocgtggagc ttccaaagca aaatgtggat gaagaaieacc accactgtqa catgagat cc acat ttggac atct tgzaga ogocctaagca cacaagastc aagcagaaca gccacagccc aacataatgc tCtgctagtc gotoctgaaa tgtgta at a; cagattcgot acaaaaacaa aagacacctg atttttaact gatatgttag acgaitgstg at gatca cc gaga tot oa actacacatg aacacagtgc otttctctg tcggcagogo 360 420 480 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 2680 1740 1800 1260 1920 1980 2040 WO 03/006689 WO 03/06689PCT/EP02/07854 cagttcctgg ctcagacaga tacaagcaac ta ccccag gc gctgtacacc atgcggtgcc ct gctgtact agcagctgcc ccatggggag acagcactat agcctctgaa caaagaaga ctcaagttza tagttcctgt tqaytgt cat atagct cgag cagactctga gcaacacgtgg gtactatcat cctcaggtct qatgctgztat cactcaacct gcaaccctgc czttccagca ctcacctgcc tgctccac Caa cct atdc gcctcctcac tccartccag aac aaag cat qcctcgctgc~t actaatggta ctttaccctq gcaaocctg cggggtagct cagccaacag tatgcagcag tgttggtgtt taagCagtct ttctctggtt ccaagaccag cactatccgt cctgaaggcg ctcczccctg gaCcct tctq tgtgcctcct cct ta'oca gt cactcccacg tagccagaac tc-ccgcaga tqgcagccca aagccagcct cagttcCat cacacaccct ttCtgccagt cagggcgtct caaacattta tctggattcc cagccgrctqc gcaactot cc agctgcgcag cctggaggaa ctgcacaact ctagctgact ccatctttgc gcccatqrt g gCtccagttt gggagtagtc cat cagccaa Agtgacactg aggtctaatg agcagcccac gagggacct g ttgaaaacac aagaccaagc gggtaccggg cagcagtcac- cagoaggcat ctg cact cga cacctgt ata gctcatctqc agcactccgg gtggcccctq ccttacaacc taatatgccc ctgtgaggat aqatccagtc accctcuagt caggccggcc cccaacaaat, ctgtcc;agcc agaggaatgc tg3accaacca tcagacaaca cat ccaaatc ctCt tcgtac tcatcattcc atgaagaaga tcatcagtta at ccca cage a ca gacct g c agcttggcga cagtggcctc ct cagcgagg cgtcagcttc ttgtggcccc cggggcaccc cgtacgctgc tctccccca rgcatcaggt ct ca gt acca tgaaccacc acctgctttt gyataatgct aggactactc agccaccatc ggcgct ggtt tctcctgcct tgctgcagtg ccactct cat tqtgaccttg aCagtctagt ctctctggaa Ca cat cttct agataccccc ggacaacaaa tgtcactgtc cactctgagt agcagatggc Ctgaactgta agtgagtggg gggagcrcagc aacctcgcag gctczcccaa acact tggcc ccccacttct gggttcctca tcctgtcamgt acaccagttt cagatccq(ca cagaccggac gtgccaattg acacagggaa acgccgcagt gaacagactg tcagcctggc attccagagg ggcaaccagt gccactgctc tccctccctt tataatt ccc agccrctcctg tacaagccca aatgattctc gctctgcggg attggcaccc gcaacacagg cagtcatctg gcggtqcagc geaagaagca gcCccctacg ccagcccctg gctgctgoat agqcatgcztg g-:cgggccca gccactcagt 2100 2160 2220 2280 2340 2400 2460
14. 2520. 2580 2640 27 0 2-760 2620 2660 2940 3000 3060 3120 3190 3240 3300 3360 3420 3480 3540 3600 3660 3'72 0 WO 03/006689 WO 03/06689PCT/EP02/07854 cctacat cgg agatcagtca ctaagtag~c attectagcc c a c-t ct cgg tttattcttg taccaaggaa tggtttctat cttgaaatca akcacaagact atggaaacacr rtttagtacaa ccqaacagqc att cattcta ttctccttat tatccaaaat at gaatttca tatttgaggg aataagtgca attttagaa t ga t acag gtagtgatta agatgtta gtqaactaat g cc t gca g a agtgtgtgtc atgtaqtggt acC~ccagct gt cttcccga 9tattcttac ctgagttctt agcagcgCgt tgttgactqC tgacmgcatt gzaaagatcgt a a atgttt tt tgtagcagat tcottgtctt aatgatttca cgagtcactg agacagqct aatgaagacc agaaaagcca ctctctctag ataatacacg gaaaactaaa tcctttgcat tgtaggeatt ttt gta 6t~ aaatataaca cacttgaatt *tgtctccatt tgcaattctz faggagacct *ttttcct~c *tzatagagri g ttcgctggc ggctcascaa ttgtagttga aatgggcct.C tctgcggggc attqttgcag tttqgacgtt tctgaagtta aaaatgaagt gagaaggagt ttattttgaa ttttcatgtc aaacctgtgc gctgggaagg atgagttca gtgaggtggt tctlgtgtttt gaaactaaaa gccaacdgca gcacgaaaata gtggtgtttt atttttcctc gtgtatttag gtatttaaac ttagagcata gggattgjttt tcctctcctc tacagtgagc I atttaacaga tttacactgg atacccqgctg agtcctacca tgagcacgag gaqqgctccg tgqctgcctg ggagagcacc tccattatct catcttgaaa ccactgaagc agaaggcttt tccctgggaa tctcccaaqt ctgccctgtt tttttaattc agaagagctc agaagcccat cttctgcagt ccctctgtca tacatttggt ctctttgaCt aaagctcttc tttacqaggg gaaatgctga agcagcttag caagggtgtg cttatggcgt gtgctgtcot tactgggcag ttgttagagt aactgctgct gagctgctcg cagagcagca aaggtgaggg tgaggactgt gcccaccagg gtcctcctcc tctctctagt ttaacttaaa aagtgtatgg tggtggtttg catacaatag tggcactgat aaacattgcc taagatttct cctcatctct cucttctgtt tcatgtgant ccagaltaagt tgtgtatalgc ttttatctt mat r-aatgc tctcttacta agactteitgt ttttaaaagt ttattactga atttaaaaat aatattttgc ataattaaat atgtacatat taacccaaaa tgttatttgt aatcaaatLgt tgtgtatctq atcctccagt gttaccccgg caaaatgaac tgatacttgt tgqaatqt&t ttactgtagt gctgagagag cagggctt tgc~iceggtg tgtctggtcga ggagttqttc tctcccctta ttgtagtgcc ctatatqata ttgccttagg atgaccagca agccccagtg gcaggttgaqg tagctgtgtt qtgtaaatgc 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4060 4920 49G0 5010 5100 5160 5220 5280 5340 5400 5460 WO 03/006689 WO 03/06689PCT/EP02/07854 gt tcgt gtt c act gccacct ctaacgtggc cgaigaagagt tggtaaactt cagatgtgtt ggttt-tgtag tatgaaaaaa catgttgttc at ttgggt ca ggagaaggtt aattctagga tcatggaacc cttggagttt gcctgCCggt zaacagtct q cgtttatgtc tcctatttaa gqgacaagat C CC cctc ct tgggaaaaga actrtacaqa CcGtctgtgt ggcttaacat act aggtceac tgcatttcce tcagtctccc ctgttccgtt tctctcactg gacgccagtg ttctctcctC tgcttggtti; tataccaaag aactactggt esagcaaaae- -tt ggggccct agttgaaggt aaccctaagc ttggttctct cqttcaogaa ctt~catcatg aacgtggtgt ttqgttIaaa attctgtgtg atacagttct ttattgtttt cacctCCCta ggatqcctag ctttgaggtt ctgctgaatg tgactgcttg Icattgcctga kgtttctccct taccgacagt coattttgga tqccttgcta ctaaatatgc catgtgatat tttttttttt aacctqttgt gacaagaagc ctcttgaatc gcacactgta act aggggtg tacagagtgg gtaggtggtg agctctpaac gggaggaaga gtgttgtctt taaccaegoa tgat ctttgt ag tttCt agg tataggaatg atccaacaag gagtt tocat gitcactctgc tgtatgcgcc ccctgetggc ccc tqqaaca tcccaggtga gacaa~tcaa t-Cttcagctc cctggcttct atatttgaag qatagtcctt tgtttgttgt attgcttaat tcacagaaacg tgaacaattC aaattrgtcct gggacattct tccacctgaa tcaggaggtg caggtggaac gtttcCaggg ttccttaatc tcattgtgta gtttcctttc caaacatttt agatgcaggg cagttattga cqttacctga aaactqacat agacatagtt atggaggaga qcttaaggt gagaggaact aqccgcagct agttCtcaca gtgagagttc gtttgtgcat tccqcatagc ttttttttta atgtcccata gtttcttatat ctgaggtttc agtattcaat tgcccatgag ttaaattata caggatcggag accaaggggc cagggcaggt attatattaa gtttgtcacr. ccccaagcat ttttaacctt aaaaaacaaa agatgggagt gacatctggc ttcagatttt ttacattcat qccctacgaa ttaaaccttc ggaagggttc .ttcctCctta gaagcattcc aggaagcaqg tacttagggt tgtcgttt~c a ag cat gta a ctacgagaag tagtgacgaa titgggaCaa cCctccatgg qgatttgtgg tcagagtggt atgggagatt tgt caacgaa agtcagttta gctgtgcgit agtgttatac tctgggtttt ttctctartaa coaaacctgt cttaaattta tag Cagtgtc cctagataac tctggcctgg catagcgctq tcttagaacg cat aig~cac 5520 5580 5640 5700 57 5820 58B0 5940 6000 6060 6120 61.80 6240 9300 6360 64^40 64150 6540 6600 6660 6720 6780 6840 6300 6960 7020 -7080 caccagqaca cttagtcact ccagagtccc cagttgCaac taggagqtgg ttaCcctgtt 74 7140 WO 03/006689 WO 03/06689PCT/EP02/07854 aaccccrgga gttgctcatc cctattctgc atgaaattgc tggatggtgc tgtqgtttcB tgtacattat Ctaatttatt agaagaaccc Oqt catat attagtatgc ctttcggtga agcatgtttc attcaattca atgtaatgtt aaatgtattt catttcaaac cqct aqagqg aqg atit ac cagjgaat gjc catttcca gcttgaaaaa agtatttttg gatatgctaa agttccggCC gcttagccag tacqttgagat caagcctgct taatttcact ctttgaatcc atigagago gccagcacag ggtctgtttt ttgtgttttt ttgctaaaat atatgtagca ttgatattgc tgcttttgtg tactggctgt aggataggag ttttaaatqa ttatataatF gtacattata aatggattc 7200 7260 7320 '738 0 *7 4 '7500 1560 75941 <210> 3 <211> 1210 <212> PRT <2L3> Mus musculus <400> 3 Met Ala Ser Gin Leu 1 5 Gin Val Phe Ser ?ro Ser Val. Ser Sex Ser Ala Phe Cys V;al Sex Gly Ala Lys Lyz Leu Ile Glu Pro Ser Gly Trp ASP Ser Lys Thr Gin Ser Sez A5n Lys Tyr Tyr Thr Leu Pro Ala Thr Gin Gly Ala SeE Ser Ser Gin Val Ala Asn Asn Leu Pro Ala Asp Gin Gly Leu Leu Pro Ala Pro. Val Giu His Ile Val Thr Ala Ala Ser Sex Giy Ser Ala Ala Thr Aia Thz Ser LQU Leu 115 Gin Ser Sex Gin Leu Thr His Arg Ser Azn ValJ 110 Lys Ser Giu Glu Pzo Tyr Gin Cys Gly Leu Lys Clu Val Glu Ser Asn. Gly Sex Val Gin Ile Ule Giu Glu His Pro Pro WO 03/006;689 PCT/EP02/07854 130 Leu Met 140 Leu Gin Asn Arg 150 Val Val Gly Ala Glu Thr Val Thr Thr Leu Val Gin Leu Glu Phe 195 Lys Ser 165 Glu Ile Gly Arg Ser Ser Ser Leu Cys Ser 185 Gly Thr Phe Ala Thr Thr Thr 160 Gly Asp Tyr Gin 175 Ser Tyr Giu Val Thr Asn .190 Gin Val Ala Lys Cys Trp Gly Lys Arg 210 Pro Ser Ser Thr Lys Tyr Ala Arg Ser Glu Asn Ser GIZa l3e Val 215 Gin Gly Gin 230 Ala Atp Glu Asn His Thr Phe Leu Lys 280 Pro Ile Lou Ala Ile Lys Tie Giu Val 235 Tyr Asi Phe Ile Lou Ser Lys Asa His Val Arg Ser 250 CYs Leu Tyr CGiU 255 Cys Phe Gin Gin Asn Leu 275 Leu Lys Tyr His Lys 260 Tyr Asp Ile Arg Val Phe Glu Asn Lys Phe Met Lou Glu 270 Pro Leu Pro Ala Leu met Gin Gin Val 290 Lys Lu Lys Ser Leu Ile Ris Ala Lys Pro Glu Met Leu Val Val Arq Gln Axg Val Lyg Val Ile 335 Asp Phe Gly LGu Gin Ser 335 Ala Ser His Val Tyr Tyx Arg Ala 360 Ala Val Cys Set Thr Tyr 350 Gly Leu Pro Glu Ile Ile WO 03/006689 WO 03/06688PCT/EP02/07854 Phe Cys 370 Glu Ala Ile Asp Trp 5er Leu Gly Val Ile Ala Giu Phe LQU Gly Trp Pro 390 Gin Leu TIyr Pro Gly Ser Glu Tyr Asp Arg Tyr Ile Thr Gin Giy Ala Gl Tyr Leu Leu 415 Ser Ala Gly Sly Tyr Pro 435 Thr Glv Ile Thr Thr Arg 425 1 hr Asn.Arg Asp Trp Arg Leu Pro GJlu Glu Pro Asn Leu. 430 Glu. Leu Glu Asn Cys Leu Lys Ser Lyn Arg Lys Tyr 450 Asp Asp Met Ala Gin met Ser Thr GIlu Gly Thr Leu Ala Glu Asp Arg Arg Ile Asp L.eu Leu Lys 495 Lys Met Leu Leu Asn His 515 Sez Se: *i~s Asp Ala Asp Ile 'lhr Pro Phe Val Thr His Leu Leu Leu Lys Thr 510 Phe Pro His Cys Lys Arg Val Lys Ser Gin Asn Met 530 Arg Val His Met Tyr Val Ser Gin Ser Pro ?he His Val Ala Thr Ser Thr Thr Met Ser Phe Se: 5-75 Ser Ser Aen Gin Leu aHi s aSrVaL4 Val Hi!P Asn Ala Scr Val Le" WO 03/006;689 PCT/EP02/07854 Sez Thr Ala Ala Ala Ala Tnr Ser Leu Ala Asn Ser Asp Val Ser 605 Ala Ala Pro Val Lou Leu 610 Asn Tyr Gin Ser Leu Tyr Pro Sa Gly Val Ala Gn Gly Val Ser Leu Pro Gly Thr Thr 1le Cys Thr Gin Asp Pro Phe Gin Thr Phe Ile Val Cys Prc 655 Pro Ala Phe Pro Val Arg 675 Thr Gly Leu Gin Thr Thr Lys His Ser Gly Phe 670 Ala Pro Ala Met Asp Asn Ala Pro Ile Val Pro Ala Gin 630 Pro Leu Gin Ile Ser Gly Val Leu Thr Gin Gly Ser Cys 700 Val Ala Thr Ile Thr Pro Leu Met Val Thr Leu His Pro Pro Gin Tyr Ala Pro Phe Thr Leu Cys Ala Ala Gly Arg Pro 735 Ala Leu Val Thr Gin Gin 7 55 Gin Thr Ala Ala Leu G.n Ala Trp Pro Gly Gly 750 Ile Leu Leu Pro Ala Trp Gin Gin Leu Pro Gly Va]. 765 Ala LEu 770 His Asn Ser Val ?rc Ala Ala Val Pro Glu Ala Met Gly Ser Ser Gin Cln 785 As Gin Tyr Ser Thr 805 Ala Asp Trp Arg Ala His Ser His rie Met Gin Gin Ser Leu Leu Thr Asn His 815 WO 03/006;689 PCT/EP02/07854 Val Thr Leu Ala Thr Ala Gin Pro Leu Asn Val Gly Val Ala Ais Val 220 825 830 Val Arg Gin Gin Gin Ser Ser Ser Leu Pro Ser Lys Lys Asn Lys Gin 835 840 845 Ser Ala Pro Val Ser Ser Lys Ser.Ser Leu Giu Val Leu Pro Se-r Gin 850 855 860 Val Tyr Ser Leu Val Gly Ser Ser Pro Leu Ara Thr Thr Ser Ser Tyr 865 870 875 880 Asn 5cr Leu Val Pro Val Gin Asp Gin His Gin Pro lie Ile Ile Pro 885 890 895 Asp Thr Pro Ser Pro Pro Val Ser Val Ile Thr Ile Arg Ser Asp Thr 900 905 910 Asp Giu Giu Giu Asp Asn Lys Tyr Lys Pro Asn Ser Ser Ser Leu Lys 915 920 925 Ala Arg Ser Asn Val Ile Ser Tyr Val Thr Vai Asn Asp Ser Pro Asp 930 335 940 Ser Asp SQr Ser Leu Ser Ser Pro His Pro Thr Asp Thr Leu Ser Ala 945 950 955 960 Leu Arg Gly Asn Ser Gly Thr Leu Leu Giu Gly Pro Gly Arg Pro Ala 965 970 975 Ala Asp Gly Ile Gly Tbr Arg Thr Ile Ile Val Pro Pro Leu Lys Thr 980 985 990 Gin Leu Gly Azp Cys Thr Ve Ala Thr Gin Ala 5er Gly Leu Leu Ser 995 1000 2005 Ser Lya Thr Lys Pro Val Ala Ser Val Ser Gly Gin Ser Ser Gly 1010 1015 1020 Cys Cys Ile Thr Pro Thr Gly Tyr Arg Ala Gin Arg Gly Gly Ala 1025 1030 1035 Ser Ala Val Gin Pro-Leu Asn Leu Ser Gin Asn Gin Gin Ser Ser WO 03/006689 PCT/EP02/07854 1040 Ser Ala 1055 Ara Gin 1070 Rhe Gin 1085 Ala Pro 1100 Tyr Ala 1115 l1e Ala 1130 Ala Tyr 1145 Ser Val 1160 Gln Tyr 1175 Arg Gly 1190 Ile Ser 1205 Ser Thr 5cr Gin Gin Ala Phe Val His Gly Ser Pro Ala Pro Ala His Ala Pro Thr Sex His Lau Phe Ser Thr Thr His Pro Gly Pro Ser Leu Gin His Gin Phe Ser Thr Ile Tyr Gin Tyr Ser Tyr 1045 Glu 1060 Al a 1075 Lqku 1090 Lcu 1105 Ala .120 Pro 1135 Ser 1150 Leu 1165 Ala 1180 Thr 1195 Leu 1210 Arq Ser Ser Asn Pro Leu Ser Gin His Ser Thr Gly Pro Ser Gin Pro Ala Ala Leu Gly G.in Gly Ser Ser Thr Leu Val His Thr Ser Ala Ser Thr Gin Ser Tyr Gly Tyr Pa Leu 1050 Pro 1065 Ala 1080 His 1095 His 1110 Ser 1125 Arg 1140 Gin 1155 Val 1170 Ile 2185 Ser 1200 Ala Pro Arq Pro Tyr Ala Pro His Leu Leu Tyr Thr Thr Ser Ser His Aa Ala Vai Pro Val Ala Pro Ala Gly Ser Ser Pro Thr Lys <210> 4 <211> 5761 <212> DNA <213> Homo sapiens <400> 4 cacaccqcag tatgcggtgc cctttactct gagctgcgca gcoggccggc cggcgctggt tgaacagact gccgctgtac tggcgtggcc t9gagggact cagcaaattc tcctgccttc WO 03/006689 WO 03106689PCT/EP02/07854 aacttggcaa tccagaggcc caaccagtac CaCtgjctcag cci :ccttcg tctgcctcc taattccttg ccci cct gtg caagcccaat tgattctcca tt-tccaggc tggcacccgc nacccaggcc St cat Cu gga agc~caacca gagaagcagc CCCCtaCaac ggcCctcrct tgctgcactg gcat gc-tcgca t gggcccagc tcctaccaag atggctacct cctcttgaa tttctctggg cctattttta cccatctt ct tttgtctctc cagttgcctg atggggagtg agcacta ica cctctgaatg Aagaagaata caagtctatt gtccctgtcc agtgtcatca acctctggac gactctgact aatagtggat actatcattg tcaggtctcQ tgctgtatca ctcaatctta aacccagccc ttccagcatg cacctgccaa ggctca acca gcctatacca ctcctcactt tacattgggt atcagccagt tctcctggcc .att tCt t agc ggaacctgtc aactcattat gcagttacca tgacttgact gggtagctret tgcagcagcc ttggtgttgc agcagtcagc Ctctggttgg aagat cagCa ctatccgaag tgaaccaaq cttitttgag cattttgga tgcct CCact tgagcaataa cccccacagg gccagaacca Ccgcaggca gc.Agcccgc-t gccaggctca gctccattgc Ctcaccctag ctqccaqcgt cttcccgngg att cciact ccgcgttctt Cagcaacttg tcac-tgttga ttttgtgaca aggaagaqag tctataaatg acacaactcZ agct gac tgg atcc~ttgctg ceat gttgtc t ccagtctct gagcagtccc tcagcccat c tgacactgat gt ctaatqrtc cagcccttat ggggcctggc gaaaactcag gdctaagcc~l gzatcgagct gcagtcatca gcaggcgttt acactcgaca tctqtatacg tcatcttttc cactttggtg ggcccctgat ctcaacaatt atagttggtg aazettgggu ttctgcaggg Ctgcattgtt gtaaitttgg attgttctga cttttaaaaa gtccaqccca aggaatqccc act aaccatq agacaacaac tCcaagtcct ctccgcacc atcattccag gaggaagagg atczgttatg tccactgata agagttgtgg cttggtgact gtcgct tcag caacgcgggg gcggctccaa gtggcccrctc gggcacccac tatgctgccc tccccac.-qqg caccaggtccc cagia ica ac t-acactqgat agca tgaggg tatggagaga gcccactgaa gtagtcttcc tacttggaag agttaccrctc caagt gaagc cagcaatqat attctcatgg tgact:attggc aarccagttc ctctagatgt catc-ttctta atactcccag acaacaaata tcactgtcaa ccc~gagtc cagatggcac gcactgtagc tgagtgggca ggaccagtgc ctcac agg a t ctcccaagc accttgcccc cgacttctgc gttcctcaakg Ctgtcaqtgt accagtttgc acccgctgag aiggaggaatc tcctccttca gcagaaggtt caaaqt ttgc agt tcagatg tgaaaaai at ccctctttat 180 240 300 360 420- 400 54 0 600 660 720 780 940 900 960 1020 2.060 2140 1200 1260 1320 1390 1440 1500 1560 1620 1680 1740 1800 1860 WO 03/006689 WO 03/06689PCT/EP02/07854 ttCattttqt tgatgaca8aa attttaaaaq gt tacgtatt agtagtgttt tgcgtgaaaa tttttoaaag ggtggttatr. agatactgge tgttttgctc cctgaaacca tgtatgccag gaattgcaaa taatggtggt aacaagcaat acttgaattg cgtctccatt tqcaattata agaggagacc gtctcttcct ggttaltaga aagttgttca ctcagtttcc tctacccctt gctctctcac gtcaagccaa tttttttt~c gttattgtga aaaagaaaaa cggcttacac ttttastgtc Actctgtgtt aaaaggcagc caccaagctt tctttctctc attacatgqt attgatgagc atCt ct cct gataagaaca cagcaaattg aaatatttta gttttaatat ttttctgct tgtacttagt atatttaaac ttagagcata cttggaattg tccctttctt gtttacagtg ccgggattta ctgccaacat tccattttgg tgtgjcgttgc tattaaatat ttttcccatg ttgctggtca aatctccctt atattaract actattgaga tcaccatttg tctttttaaa tctgattcag gqtggtggtt tttgcctaaa tCtgttt tat tttcttatgt aatgctctct aaaatttatt t tt acat aa t aacccaaaat gtgtatgtga caaaatgaac ttactgjtagt ,ttttgc-acag CCtccttccc agcttgcctt tcagaacagg tgaaaaataa attctcggct taccttgctt gcattctttt tggcagtcct ggaaaaatgc tttgtttata ttgtttattg taatgggjcaa ttctctcaalt ctggtaactt toaagcacca cttaaatttt ttattatatg gattagtatg gtgatttgt atagctttta tattaagact actgaattta taaatatgta gttatttgta t cctccagtg tgatacttgt gctgaaztgag gtgtgtctgg ttattgtagt aggatggacc attagtagct aaacagcagc gagtt ctcac ctgtqqgaat aaagtatgtg t cctgcacat tgatagaagg aactcagact gacatttaaa ttgttatttt tigctcctgtg zatgtgagag tgaattcttt tttatcgaaa caaaatctct aattttagt t ggggagaaa tacttcaaag tat ataataa aaaatatttt catattgatt a tcaaat gtg ttatcccgga tggaatgrt caggggcatt tgagqagttt gccttatatg agcaagcccc qtattgtgta ttttctcctt agaagcatit tcaggaaqca caatcacttt agttgacatt agttgaaatrc tgcctatctt cttacagagt tgcaaaactg t t t t tataa aatceatatc tttadattat aagccatta a qtctattatgj aatacacctC gct aaaaaaa' tagcttcctt gtgcat gtag agaagttttg a gaaaa at at tagtgattac gatggattg4 gtgaactaat gcctgcaaqg ttcagtgtgt at aa tgtagt cgtggaccct atgCattgtt taccaccacc. tccccatgtg ;yqtqaagq8g t agaatgaat cctagtaaaa 1920 1980 2040 2100 2160 2220 2280 23 4 0 2400 2460 2520 25B0 2640 2700 2760 2820 2880 2940 3000 3060 3120 3240 3300 3360 3420 3480 3540 WO 03/006689 WO 03/06689PCT/EP02/07854 tatttgcttq ttgaaaaaaa cat9ttaaca gatgtc.Tttta taccaaagag ccto-ttgtat tgcttaccat t cacagaaag attgaggctt tgaaattgtc ga t gggaca tgtgttccat cagatagaga cagccagctc ttg-taaggag gttgtgctttt gtcaaga tag aacatgtgtg acaggtctag gatttgttgt cccagtccaa taccaagagc agagattttt cctgtgtctg ttagccctgt ctgggecace ggcatttcca cactctgggc a gt t gg cc gctaagcttg tat~caaaaa atgacagga gtccrccatac gttccttagc ttgaggtttc Ctrtgtoctct ttcctgccca ccgaattgaa tggtqtcaag tgtaccaggt taaggcttc ctttattttt ccaagcagtt atctttgtgt tttctaaaqg ttttgtaaga taagcagata ttgtattgtt cagtcttagt ctgaatgtgt tggccctgac gtgacctgac aCttCttcc tcataggar cagtgctcag attctgagcc1 actgccctta ttcactagtt gtggcocgag tatgaggaga tgqtgaagaa ttttttaaca Cagctcctgc taaaggattt aatgatatat gaggtgcagg tgaaca~rcaga caagatggg taaa.atcatt tgtataattt ctccttttta acaa&ttttt aatg.agat gc CCacttaaga accttagtca ctcgcaaactg aggaagggag ctcaa accag etccqggtga acttggtcac ctgaaatacc tgcttttgtg 9ccaggcaag gagatqgttr cctgcttcct agttttgtgg .tatagagaag gcttgtatag atggatctgq gggaaagaa ttgagAtata atggagatgg ggagct gtca caggtagtcc atatrgagtt ctgtcactag o ca agca cett tgttccttgi aggaaagaes taggagtcta cttgcctagc gcattt ccga tcactgtggc gaagcctggt cttaaggctt gageagaagc tccagagttt aaccccagga attgctcatc cacagtaatg gttt taggat attttgattt t googat ggt gaaccaaagc tcttggggcc gtcaagteaga gattaatcct attttaggac gagatttcat aagtatttgg gtacagccta gtqttttcag t9tczatacag tctgattttc cttttttotg accaaatccc aactccacag agtgt1gqc ttttcca gca tttagattct gaatttagtg taettcctct ggagaagggt tt~AQagCLC ataagaactc. tgtgttttgt aggaaartgaa tTtttttttt gacaaggaac ctgttgagtc Cttcaagctg aggt-actggg aaaatacagg tggttctgtg ggagcctggt aotttcttca ccaggaacait cactatattg ttttCtggtc ttglttggac taaqqgacaa attcctgcac aaaaggataa tttaaaaact taaaaatcca gtccctgggg agcagctggc ctcagaactt tcagtritagc ccaoigaggtay catttcaaac cactagaggg t ca goat tat attgcctctc taactgatag 3600 3660 T7 3780 3840 3900 3960 4020 40a0 4140 4200 4Z60 4320 4300 4440 4500 4560 4620 4680 4 4800 486D 4920 4980 5040 5100 5160 5220 5280 atggtgcagc acgtctacat ggttgtctgt tqctaaactt tatataatgt gtggtttcaa WO 03/006689 WO 03/06689PCT/EP02/07854 ttcagcttga tgttagtatt, atttqatatg ttgtgcgctt tatgtttgta tagatattta atctttgqat t acataatat aaaataatct tctqc'zttga ctagttattg tcttttacaa atgcagatgt aaatttaata tcraazgtttc ggtaaaat gt caotacatqt acccttgata tgtgcgattt caagcctcta acttagggag ctaactatg tcttt ggtt t agagcaattg agcagtacat ttgcaatgga 8aactttttt gaaacagata tazgtaaaat gaaaagggtc tacaaagtag caatacatca tat atgtaca attoctac-tt tgctttctcc gtttctgaga aatcatttt a cattgtgtaa cttgtatttt atanaatcgg ttatatgtaa tattaaatgjt Ctttttttgg attactqgg acaaaag~aaa 4acatagttt Cagtattttc to aa tt 1tt 5340 5400 5460 5520 5580 5640 5700 5760 5761 (p210> <211> <212> <213> 490 PR' Homvo sapiens <4 00-, Thz Pro Gin Tyr Al~a 1i Val Pro Phe Thr Leu Ser Cys Ala Ala Gly Arg Pro Ala Leu Glu Gin Thr Ala Val. Leu A-la Trp Pro Gly Va. Thr Gin Gin Ile ILeu Leu Pro Thr Trp Gin Gin Ala Leu His Asn Sar Val Pro Thr Ala Met Pro Glu Ala M1et Gly Ser Gly Gin Gin Asn Gin Tyr Ser Thr Ala Asp Trp Arg Ala hi5 Ser Hiis Ile M~et Gin Gin Zer Leu Leu Thr Asn I-Uls Val Thr Leu Val Aizg Gin 115 Thr Ala Gin Pro Asfl Val Gly Val ALs His Val 110 Asn Lys Gin Gin Gln Ser Ser Leu Pro Ser Lys WO 03/006;689 PCT/EP02/07854 Ser Ala Pro Val Ser Ser Lys Ser Ser Leu Asp val Leu Pro Ser Gin Tyr Snr Ser Leu Leu Val Gly 150 Vral Pro Val Ser Ser Pro Let Thr Ser Ser Gin Asp Gin Pro lie Ile Il Pro 175 ASp Thr Pro Asp Giu Glu 195 Pro Arg Ser Pro Val Ser Thr Ile Arg Asp Asn Lys Pro Ser Ser Ser Asp Thr 190 Gly Leu Lys Ser Pro Asp Asn Val Ile Val Thr Val 210 Ser Asp Ser Ser Leu Pro Tyr Ser Arg Gly Asn Ser Val Leu Thr Asp Thr 235 Gly Pro Gly. Val Pro Pro Leu Ser Ala Asp Gly Gin Lau Gly 275 Asn Lys Thr 290 Cyb Ile Thr Thr Arg Thr Arg Val Val 255 Leu Lys Thr 270 Lou Leu Se Ser Gly Cys Cys Thr Val Gin Al-a Ser Lys Pro Val Pro Thr Gly 310 Leu Asn Leu Val Ser Gly Cin Pro Ser Arg Ala Gin Gin Asn Gin 330 Asn Pro Ala 345 Gly Gly Thr Sex Ser Ser Ala Ala 335 Thr Sex Gin Glu Arg Ser Ser Pro Arg Arg Gin Gin Ala 350 WO 03/006689 Phe Val Ala Pro Lau 355 Pro Leu His ser Thr 370 Leu Pro Ser Gin Ala 385 Ala Ala Leu Gly Ser Gly Ser Ser Arq His 420 Va. His Gin Val Pro 435 Ser Val Ale, Pro Ala 450 119e Gly Set Ser Arg 4 S5 Pro Thr Lys Ile Ser 485c Ser G ly Thr Al a Val1 GIn Gly 470 Gin PCT/EP02/07854 Gly Ser Ala is Ser Ala 400 Pro Gin 415 Thr Leu Ser Ala His Gln Phe Gin Ser Pro Lexa Thr Ile Tyr Tyr Ser Tyr