WO2002030972A2 - Regulation of nf-at interacting protein nip 45 like protein - Google Patents

Abstract

Disclosed are novel nucleic acid and amino acid sequences of NF-AT interacting protein NIP 45 like proteins. Reagents that bind to NIP45-like protein gene products can be used to treat conditions involving inflammatory processes, such as allergy, asthma, autoimmune diseases, and other chronic inflammatory diseases where an over-activation or prolongation of the activation of the immune system causes damage to tissues.

Description

REGULATION OF NF-AT INTERACTING PROTEIN NIP 45 like PROTEIN

TECHNICAL FIELD OF THE INVENTION

The present invention relates to nucleic acid and amino acid sequences of a novel NF-AT interacting protein NIP 45 like proteins and their use in diagnosis and therapy for human disease.

BACKGROUND OF THE INVENTION

Many immunologically-mediated clinical diseases including autoimmune diseases, allergic diseases, and infectious diseases are reported to be highly relevant to the ratio of CD4+ T helper cell type 1 (Thl) to CD4+ T helper cell type 2 (Th2) (Heinzel, F.P., et al., (1989) J. Exp. Med. 169: 59-72; Pearce, E.J., et al. (1991) J.

Exp. Med. 173: 159-166; Shearer, G.M. and Clerici, M. (1992) Prog. Chem. Immunol. 54: 21-43). To alter or regulate the ratios of Thl and Th 2 cells, therefore, may give a clue to treat or prevent immunologically-mediated diseases.

The mechanisms by which the Thl and Th 2 ratio is determined involve the differentiation of CD4 T helper precursor cells (Thp) to choose to become Thl or Th 2 effector cells. The differentiation is, in turn, partly regulated by the cytokines, such as IL-2, IL-4, and IL-12. To increase or decrease the level of selected cytokines is one way to regulate Thl to Th2 ratio.

Recently, a protein of 45 kDa derived from murine tissue that interact with members of the Nuclear Factor of Activated T cells (NF-AT) protein has been isolated and termed NIP45 (Hodge MR, Chun HJ, Rengarajan J, Alt A, Lieberson R, Glimcher LH. NF-AT-Driven interleukin-4 transcription potentiated by NIP45. Science, 1996 Dec 13; 274(5294): 1903-5; Hodge MR et al., WO 97/39721, USP 5,858,711, and USP 5,958,671). Further, WO 99/21993 discloses human NIP45 polypeptide and polynucleotide sequences.

NIP45 is known to interact with NFATp and to potentiate the transcription of the IL- 4 gene induced by the binding of NFATp to the IL-4 gene promoter. In contrast,

NIP45 has also been found to interact with the TRAF family of proteins, and this interaction leads to a suppression of the NFATp-related potentiation of IL-4 transcription. The nature of the interaction of NIP45 with NFATp is unclear, but one possibility is that NIP45 is an accessory protein that is involved in the transport of NFATp from the cytoplasm into the nucleus where it can bind to the IL-4 gene promoter. In the absence of such an accessory protein, NFATp may be inactive because of its inability to cross through the nuclear membrane on its own. Another possibility is that NIP45 acts as a adaptor molecule between NFAT and other transcription factors, aiding in the formation of a multi-component transcriptional complex. Experiments with NFATp knockout mice have shown that NFATp deficiency leads to the accumulation of peripheral T cells with a preactivated phenotype, enhanced immune responses of T cells after secondary stimulation in vitro, and severe defects in the proper termination of antigen responses (Schuh, K. et al. (1998) Eur. J. Immunol. 28(8): 2456-66).. In some of these mice, large germinal centers develop in the spleen and peripheral lymph nodes and there is a pronounced retardation in the involution of the thymus.

There is a need in the art to identify additional members of the NF-AT interacting protein like protein whose activity can be regulated to provide therapeutic effects, particularly for diseases and conditions involving immunologically-mediated responses. SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods of regulating a NFAT interacting protein NIP 45 like protein. This and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a NIP 45 like protein polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO:5.

Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a NIP 45 like protein polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO:5.

Binding between the test compound and the NIP 45 like protein polypeptide is detected. A test compound which binds to the NIP 45 like protein polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the activity of the NIP 45 like protein.

Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a polynucleotide encoding a NIP 45 like protein polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 2; the nucleotide sequence shown in SEQ ID NO: 2; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO:3; the nucleotide sequence shown in SEQ ID NO:3; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO:4; the nucleotide sequence shown in SEQ ID NO:4; nucleotide sequences which are at least about 50% identical to the nucleotide sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4; and the nucleotide sequence from residue 246 to residue 506 shown in SEQ ID NO: 3 or

4.

Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the

NIP 45 like protein through interacting with the NIP 45 like protein mRNA.

Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a NIP 45 like protein polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO:5; and the amino acid sequence shown in SEQ ID NO:5. A NIP 45 like protein activity of the polypeptide is detected. A test compound which increases NIP 45 like protein activity of the polypeptide relative to NIP 45 like protein activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation. A test compound which decreases NIP 45 like protein activity of the polypeptide relative to NIP 45 like protein activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a NIP

45 like protein product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 2; and the nucleotide sequence shown in SEQ ID NO: 2 nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO : 3 ; the nucleotide sequence shown in SEQ ID NO:3; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO:4; the nucleotide sequence shown in SEQ ID NO:4; nucleotide sequences which are at least about 50% identical to the nucleotide sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4; and the nucleotide sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or

4. Binding of the test compound to the NIP 45 like protein product is detected. A test compound which binds to the NIP 45 like protein product is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a NIP 45 like protein polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50%) identical to the nucleotide sequence shown in SEQ ID NO: 2; the nucleotide sequence shown in SEQ ID NO:2; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO:3; the nucleotide sequence shown in SEQ ID NO:3; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO:4; the nucleotide sequence shown in SEQ ID NO:4; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in from residue 246 to residue 506 shown in SEQ ID NO:3 or 4; and the nucleotide sequence shown in from residue 246 to residue 506 shown in SEQ ID

NO:3 or 4.

NIP 45 like protein activity in the cell is thereby decreased. The present invention thus provides an NIP45-like protein which can be used to identify NIP 45 analogs as well as compounds which may improve immunologically- mediated conditions.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 shows the DNA-sequence encoding a NIP45-like protein polypeptide (SEQ ID NO:l). Fig. 2 shows the DNA-sequence encoding a NIP45-like protein polypeptide (SEQ ID NO:2).

Fig. 3 shows the cDNA-sequence of NIP45-like protein polypeptide transcript 1 (SEQ ID NO: 3). Fig. 4 shows the cDNA-sequence of NIP45-like protein polypeptide transcript 2 (SEQ ID NO: 4). Fig. 5 shows the amino acid sequence of NIP45-like protein polypeptide

(SEQ ID NO:5). Fig. 6 shows the nucleotide alignment of NIP45-like protein polypeptide cDNA against Human NIP45 cDNA. Fig. 7 shows the amino acid alignment of NIP45-like protein polypeptide against Human NIP45 protein.

Fig. 8 shows the alignment of NIP45-like protein polypeptide cDNA

(translated) NIP45 protein. Fig. 9 shows the expression profile of NIP45-like protein polypeptide mRNA, whole body screen. Fig. 10 shows the expression profile of NIP45-like protein polypeptide mRNA, blood-lung screen. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide encoding a NIP45-like protein polypeptide and being selected from the group consisting of:

a) a polynucleotide encoding a NIP45-like protein polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO:5; and the amino acid sequence shown in SEQ ID NO:5. b) a polynucleotide comprising the sequence of SEQ ID NO: 1, 2, 3, 4 or the sequences from residue 246 to residue 506 shown in SEQ ID NO:3 or 4; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

Furthermore, it has been discovered by the present applicant that human NIP 45 like protein activity can be regulated to control autoimmune diseases, allergic diseases, and infectious diseases, and other chronic inflammatory diseases. Such diseases include asthma, allergic rhinitis, atopic dermatitis, hives, conjunctivitis, vernal catarrh, chronic arthrorheumatism, systemic lupus erythematosus, myasthenia gravis, psoriasis, diabrotic colitis, systemic inflammatory response syndrome (SIRS), llymphofollicular thymitis, sepsis, polymyositis, dermatomyositis, polyaritis nodoa, mixed connective tissue disease (MCTD), Sjoegren's syndrome, gout, and the like. NIP45-like PROTEIN is expressed most highly in thymus, spleen, and mitogen- activated CD4⁺ T cells. This expression of NIP45-like PROTEIN in lymphoid tissues indicates that NIP45-like PROTEIN may play an important role in the development or functioning of the immune system. NIP45-like PROTEIN is identical in amino acid sequence to NIP45 over 63 amino acid residues, but lacks the carboxy terminal

137 amino acid residues of the full-length NIP45 protein. Within this carboxy terminal lies a region of homology to Ubiquitin (NIP45 amino acid residues 274-334) and a region of homology to Sentrin molecules (NIP45 amino acid residues 274- 334). Ubiquitins are involved in the regulated turnover of proteins required for controlling cell cycle progression. Sentrins are small ubiquitin-like proteins that are thought to be covalently attached to other proteins to mark them for transport into the nucleus. The strong homology of NIP45-like PROTEIN to NIP45 and the lack of the Ubiquitin and Sentrin-homolgy domains may give NIP45-like PROTEIN the ability to interact with NFATp, but not give it the ability to transport NFATp into the nucleus. This potential interaction of NIP45-like PROTEIN with NFATp may block the interaction of NIP45 with NFATp so that the coexpression of NIP45 and NFATp no longer has a enhanced effect on transcription via the IL-4 promoter. The effect, therefore, of NIP45-like PROTEIN in the cell may be to block the interaction between NIP45 and NFATp and thereby promote the formation of germinal centers, preactivate T cells, enhance secondary immune responses, and delay termination of antigen responses.

The increased formation of germinal centers in the thymus is a property of human patients with lymphofollicular thymitis and is often connected with the development of autoimmune diseases such as myasthenia gravis. Additionally, NFATp deficient mice exhibit a strong tendency toward the development of Th2 type immune responses, with paradoxically strong enhancement of the transcription of several Th2 type genes, including IL-4, IL-5, and IL-13. This preferential development of Th2 type immune responses may lead to overactive allergic responses and Th2-dependent autoimmune diseases and other disorders. Regulation of NIP45-like PROTEIN is accordingly expected to have an effect on NFAT-dependent gene transcription and subsequent production of immunological mediators such as cytokines and would be useful in the treatment of immune mediated disease and immune mediated pathology.

NIP45-like Polypeptides

NIP45-like polypeptides according to the present invention comprise the amino acid sequence shown in SEQ ID NO: 5, a portion of that sequence, or a biologically active variant of that amino acid sequence, as defined below. An NIP45-like polypeptide of the invention therefore can be a portion of an NIP45-like protein, a full-length NIP45-like , or a fusion protein comprising all or a portion of an NIP45-like protein.

Biologically Active Variants NIP45-like polypeptide variants which are biologically active, i.e., retain the ability to interact NF-AT family of protein. Preferably, naturally or non-naturally occurring NIP45-like variants have amino acid sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to the amino acid sequence shown in SEQ ID NO: 5 . Percent identity between a putative NIP45-like polypeptide variant and an amino acid sequence of SEQ ID NO: 5 is determined using the Blast2 alignment program.

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of an NIP45-like polypeptide can be found using computer programs well known in the art, such as DNASTAR software.

Whether an amino acid change results in a biologically active NIP45-like polypeptide can readily be determined by assaying for NIP45-like polypeptide activity, as described, for example, in the specific examples, below.

Fusion Proteins

Fusion proteins can comprise at least 5, 6, 8, 10, 25, or 50 or more contiguous amino acids of an amino acid sequence shown in SEQ ID NO:5. Fusion proteins are useful for generating antibodies against NIP45-like polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of an NIP45-like polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

An NIP45-like polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 5, 6, 8, 10, 25, or 50 or more contiguous amino acids of SEQ ID NO:5 or of a biologically active variant, such as those described above.

The second polypeptide segment can be a full-length protein or a protein fragment.

Proteins commonly used in fusion protein construction include β-galactosidase, β- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSN-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DΝA binding domain (DBD) fusions, GAL4 DΝA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the ΝIP45-like polypeptide-encoding sequence and the heterologous protein sequence, so that the NIP45-like polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:l or 2 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Alternatively, recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO: 3 or 4 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.

Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wl), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs

Species homologs of the NIP45-like polypeptide can be obtained using NIP45-like polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of the NIP45-like polypeptide, and expressing the cDNAs as is known in the art.

NIP45-like Polynucleotides An NIP45-like polynucleotide can be single- or double-stranded and comprise a coding sequence or the complement of a coding sequence for an NIP45-like polypeptide. The coding sequence for human NIP45-like polypeptide is shown in SEQ ID NOS:l, 2, 3 and 4.

Degenerate nucleotide sequences encoding human NIP45-like polypeptides, as well as homologous nucleotide sequences which are at least about 50, preferably about 75, 90, 96, or 98% identical to the nucleotide sequence shown in SEQ ID NO:l, 2, 3 or 4; or to the nucleotide sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4. also are NIP45-like polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as

ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of NIP45-like polynucleotides which encode biologically active NIP45-like polypeptides also are NIP45-like polynucleotides.

Identification of Variants and Homologs ofNIP45-like Polynucleotides

Variants and homologs of the NIP45-like polynucleotides described above also are

NIP45-like polynucleotides. Typically, homologous NIP45-like polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known

NIP45-like polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25%) basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the NIP45-like polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of NIP45-like polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol.

Biol. 81, 123 (1973). Variants of human NIP45-like polynucleotides or NIP45-like polynucleotides of other species can therefore be identified by hybridizing a putative homologous NIP45-like polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or the complement thereof, or polynucleotide having a nucleotide sequence of SEQ ID NO:2 or the complement thereof or polynucleotide having a nucleotide sequence of SEQ ID NO: 3 or the complement thereof, the nucleotide sequence of SEQ ID NO: 4 or the complement thereof; or the nucleotide sequence from residue 246 to residue 506 in SEQ ID NO:3 or 4 to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising transformylase polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to transformylase polynucleotides or their complements following stringent hybridization and/or wash conditions also are

NIP45-like polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51. Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T_m of the hybrid under study. The T_m of a hybrid between an NIP45-like polynucleotide having a nucleotide sequence shown in SEQ ID NO:l, 2, 3 or 4; or the nucleotide sequence shown in from residue 246 to residue 506 in SEQ ID NO:3 or 4. and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962): T_m = 81.5 °C - 16.6(log₁₀[Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 600/0, where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% form- amide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.

Preparation qfNIP45-like Polynucleotides

A naturally occurring NIP45-like polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated NIP45-like polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise NIP45-like nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules. NIP45-like cDNA molecules can be made with standard molecular biology techniques, using NIP45-like mRNA as a template. NIP45-like cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesizes NIP45-like polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode an NIP45-like polypeptide having, for example, an amino acid sequence shown in SEQ ID NO: 5 or a biologically active variant thereof.

Extending NIP45-like Polynucleotides Various PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human NIP45-like polypeptide to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 °C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

Obtaining NIP45-like Polypeptides

NIP45-like polypeptides can be obtained, for example, by purification from human cells, by expression of NIP45-like polynucleotides, or by direct chemical synthesis.

Protein Purification

NIP45-like polypeptides can be purified from any human cell which expresses the protein, including host cells which have been transfected with NIP45-like polynucleotides. Brain, heads and neck, retina, lung, bone marrow, fetal liver, lymph node, peripheral blood leukocytes, spleen, thymus, fetal lung, lymph tissue, and normal germinal center B cells are particularly useful sources of NIP45-like polypeptides. A purified NIP45-like polypeptide is separated from other compounds which normally associate with the NIP45-like polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

A preparation of purified NIP45-like polypeptides is at least 80% pure; preferably, the preparations are 90%), 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. Expression ofNIP45-like Polynucleotides

To express an NIP45-like polypeptide, an NIP45-like polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding NIP45-like polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et ah, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N. Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding an NIP45-like polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector ~ enhancers, promoters, 5' and 3' untranslated regions ~ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding an NIP45-like polypeptide, vectors based on SN40 or EBN can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the ΝIP45-like polypeptide. For example, when a large quantity of an NIP45-like polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the NIP45-like polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β- galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol. 153, 516-544, 1987. Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding NIP45- like polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express an NIP45-like polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera fi-ugiperda cells or in Trichoplusia larvae. Sequences encoding NIP45-like polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of NIP45-like polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which NIP45-like polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be used to express NIP45-like polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding NIP45-like polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing an NIP45-like polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding NIP45-like polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding an

NIP45-like polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed NIP45-like polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post- translational activities (e.g., CHO, HeLa, MDCK, HEK293, B-lymphoma cells and

WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express NIP45-like polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1 -2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced NIP45-like sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.

Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tic or aprt cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Bio . 55, 121-131, 1995).

Detecting Expression ofNIP45-like Polypeptides

Although the presence of marker gene expression suggests that the NIP45-like polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding an NIP45-like polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode an NIP45-like polypeptide can be identified by the absence of marker gene function.

Alternatively, a marker gene can be placed in tandem with a sequence encoding an NIP45-like polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the NIP45-like polynucleotide.

Alternatively, host cells which contain an NIP45-like polynucleotide and which express an NIP45-like polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding an NIP45-like polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding an NIP45-like polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding an NIP45-like polypeptide to detect transformants which contain an NIP45-like polynucleotide.

A variety of protocols for detecting and measuring the expression of an NIP45-like polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on an NIP45-like polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NIP45-like polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Alternatively, sequences encoding an NIP45-like polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like . Expression and Purification ofNIP45-like Polypeptides

Host cells transformed with nucleotide sequences encoding an NIP45-like polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode NIP45-like polypeptides can be designed to contain signal sequences which direct secretion of soluble NIP45-like polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound NIP45-like polypeptide.

As discussed above, other constructions can be used to join a sequence encoding an NIP45-like polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invifrogen, San Diego, CA) between the purification domain and the NIP45-like polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing an NIP45-like polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al, Prot.

Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the NIP45-like polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993. Chemical Synthesis

Sequences encoding an NIP45-like polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, an NIP45-like polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin

Elmer). Optionally, fragments of NIP45-like polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic NIP45-like polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the NIP45-like polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Production of Altered NIP 45-like Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce NIP45-like polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter NIP45-like polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies Any type of antibody known in the art can be generated to bind specifically to an epitope of an NIP45-like polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of an NIP45-like polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of an NIP45-like polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen. Typically, an antibody which specifically binds to an NIP45-like polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to NIP45-like polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate an NIP45-like polypeptide from solution.

NIP45-like polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, an NIP45-like polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Gueriή) and Corynebacterium parvum are especially useful.

Monoclonal antibodies which specifically bind to an NIP45-like polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80,

2026-2030, 1983; Cole et al., Mol. CellBiol. 62, 109-120, 1984).

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in

GB2188638B. Antibodies which specifically bind to an NIP45-like polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to NIP45-like polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206. A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al,

1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol. Meth. 165, 81-91).

Antibodies which specifically bind to NIP45-like polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which an NIP45-like polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of NIP45-like gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.

Modifications of NIP45-like gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the NIP45-like gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of an NIP45-like polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an NIP45-like polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent NIP45-like nucleotides, can provide sufficient targeting specificity for NIP45-like mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular NIP45- like polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to an NIP45-like polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987;

Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of an NIP45-like polynucleotide, such as the nucleotide sequence shown in SEQ ID NO:l, 2, 3 or 4 or the sequence from residue 246 to residue 506 shown in SEQ ID NO: 3 or 4, can be used to generate ribozymes which will specifically bind to mRNA transcribed from the NIP45-like polynucleotide.

Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within an NIP45-like RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate NIP45-like RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NO:l, 2, 3 or 4 or the sequence from residue 246 to residue 506 shown in SEQ ID NO: 3 or 4 and their complements provide a source of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease NIP45-like expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Screening Methods

The invention provides assays for screening test compounds which bind to or modulate the activity of an NIP45-like polypeptide or an NIP45-like polynucleotide. A test compound preferably binds to an NIP45-like polypeptide or polynucleotide.

More preferably, a test compound decreases or increases the effect of NIP45 or an NIP45 analog as mediated via human NIP45-like by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) relative to the absence of the test compound. Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead, one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J.

Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, Biotechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin,

Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409). High Throughput Screening

Test compounds can be screened for the ability to bind to NIP45-like polypeptides or polynucleotides or to affect NIP45-like activity or NIP45-like gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96- well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. Yet another example is described by Salmon et al., Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site of the NIP45-like polypeptide, thereby making the active site inaccessible or accessible to substrate (e.g., NFATp) such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the NIP45-like polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the NIP45-like polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to an NIP45-like polypeptide can be determined without labeling either of the interactants. For example, a microphysio- meter can be used to detect binding of a test compound with an NIP45-like polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and an NIP45-like polypeptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to an NIP45-like polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, an NIP45-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, Biotechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the NIP45-like polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding an NIP45-like polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the NIP45-like polypeptide.

It may be desirable to immobilize either the NIP45-like polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the NIP45-like polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the NIP45-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to an NIP45-like polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the NIP45-like polypeptide is a fusion protein comprising a domain that allows the NIP45-like polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed NIP45-like polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either an NIP45- like polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NIP45-like polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to an NIP45-like polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the NIP45-like polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the NIP45-like polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the NIP45-like polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to an NIP45-like polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises an

NIP45-like polypeptide or polynucleotide can be used in a cell-based assay system. An NIP45-like polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to an NIP45-like polypeptide or polynucleotide is determined as described above.

Functional Assays

Test compounds can be tested for the ability to increase or decrease a biological effect of an NIP45-like polypeptide. Such biological effects can be determined using the functional assays described in the specific examples, below. Functional assays can be carried out after contacting either a purified NIP45-like polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which decreases a functional activity of an NIP45-like by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing NIP45-like activity. A test compound which increases NIP45-like activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing NIP45-like activity.

One such screening procedure involves the use of B-lymphoma cells which are transfected to express an NIP45-like polypeptide. For example, such an assay may be employed for screening for a compound which inhibits activation of the polypeptide by exposing the transfected B-lymphoma cells which comprise the polypeptide with both endogenously interacting proteins or substrates to a test compound to be screened. Inhibition of the activity of the polypeptide indicates that a test compound is a potential antagonist for the polypeptide, i.e., inhibits the function of the protein. The screen may be employed for identifying a test compound which activates the protein by exposing such cells to compounds to be screened and determining whether each test compound activates the protein.

Other screening techniques include the use of cells which express a human NIP45- like polypeptide (for example, transfected T cells) in a system which measures amounts of secreted proteins generated by polypeptide activation. For example, test compounds may be added to cells that express a human NIP45-like polypeptide and the expression of a reporter gene with specific promoter sequences can be measured to determine whether the test compound activates or inhibits the protein.

Details of functional assays, such as those described above, are provided in the specific examples below.

NIP45-like Gene Expression

In another embodiment, test compounds which increase or decrease NIP45-like gene expression are identified. An NIP45-like polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the NIP45-like polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of NIP45-like mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of an NIP45-like polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into an NIP45- like polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses an NIP45-like polynucleotide can be used in a cell- based assay system. The NIP45-like polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions which can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, an NIP45-like polypeptide, NIP45-like polynucleotide, antibodies which specifically bind to an NIP45-like polypeptide, or mimetics, enhancers and inhibitors, or inhibitors of an NIP45-like polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like , for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Therapeutic Indications and Methods

NIP 45 like protein of the present invention is responsible for many biological functions, including many pathologies. Accordingly, it is desirable to find compounds and drugs which stimulate NIP 45 like protein on the one hand and which can inhibit the function of a NIP 45 like protein on the other hand. Compounds which can modulate the function or expression of NIP 45 like protein are useful in treating various allergic diseases, autoimmune diseases, inflammatory deseases, and infectious deseases including asthma, allergic rhinitis, atopic dermatitis, hives, conjunctivitis, vernal catarrh, chronic arthrorheumatism, systemic lupus erythematosus, myasthenia gravis, psoriasis, diabrotic colitis, systemic inflammatory response syndrome (SIRS), llymphofollicular thymitis, sepsis, polymyositis, dermatomyositis, polyaritis nodoa, mixed connective tissue disease (MCTD), Sjoegren's syndrome, gout, and the like.

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or an NIP45-like polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. A reagent which affects NIP45-like protein activity can be administered to a human cell, either in vitro or in vivo, to reduce NIP45-like like activity. The reagent preferably binds to an expression product of a human NIP45-like gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about

30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10^δ cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and

400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell ligand exposed on the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 mnol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using protein-mediated targeted delivery. Protein-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases NIP45-like activity relative to the

NIP45-like activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50%) of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions which exhibit large therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of an NIP45-like gene or the activity of an

NIP45-like polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of an NIP45-like gene or the activity of an NIP45-like polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to NIP45-like -specific mRNA, quantitative RT-PCR, immunologic detection of an NIP45-like polypeptide, or measurement of NIP45-like activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Methods

NIP 45 like protein also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode a NIP 45 like protein. Such diseases, by way of example, are related to various allergic diseases, autoimmune diseases, inflammatory deseases, and infectious deseases including asthma, allergic rhinitis, atopic dermatitis, hives, conjunctivitis, vernal catarrh, chronic arthrorheumatism, systemic lupus erythematosus, myasthenia gravis, psoriasis, diabrotic colitis, systemic inflammatory response syndrome (SIRS),

Uymphofollicular thymitis, sepsis, polymyositis, dermatomyositis, polyaritis nodoa, mixed connective tissue disease (MCTD), Sjoegren's syndrome, gout, and the like.

Differences can be determined between the cDNA or genomic sequence encoding a NIP 45 like protein in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is like ly to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85, 4397- 4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.

In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of a NIP 45 like protein also can be detected in various tissues. Assays used to detect levels of the protein polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1

The murine NIP45 mRNA sequence (GenBank accession number U76759) was used to search the EST subset of the DNA DataBank of Japan (DDBJ) for homologous sequences using the computer program BLAST 2.0 (National Center for

Biotechnology Information). One human EST sequence (GenBank accession number AA300063) was found that had over 78% nucleotide sequence identity with the murine NIP45 mRNA sequence over a region of 109 bases and, when conceptually translated, over 66% amino acid sequence identity and over 77% amino acid homology over a region of 54 amino acids with the translated murine NIP45 mRNA sequence. Comparison with the human NIP45 mRNA and amino acid sequences showed that the EST had 97% identity at the nucleotide level over a stretch of 164 bases and at least 93% identity at the amino acid level over a stretch of 54 amino acids after conceptual translation. This EST was selected as a potential fragment of a gene homologous the NIP45 gene and the new gene was designated

RCK002. To gather further sequence information on the potential NIP45 homolog, the AA300063 sequence was used to search the DDBJ for homologous sequences. As a result of the second search, an additional human EST, GenBank accession number AA974599, was identified which overlapped with AA300063 at 144 bases. Additional searching for overlapping ESTs resulted in the discovery of four more

ESTs, GenBank accession numbers AW591954, AI702744, W21082, and AW378589, that appeared to come from the same gene as AA300063.

In order to determine the full sequence of NIP 45 like protein, rapid amplification of cDNA ends (RACE) was performed using primer Nip-2

(AAAGGGCCCACCCTCCCAGA) to amplify the 3' end of the NIP 45 like protein transcript and Nip-1 (GGGGAGCGTTGGGAAAATGC), Nip-3

(GAGAGGGGAGCGTTGGGAAA), and Nip-5 (CCCTGCTGGACACCACCATC) to amplify the 5' end of the NIP 45 like protein transcript. RACE was carried out with the SMART RACE cDNA amplification kit (Clontech, PaloAlto, CA, USA) according to the manufacturer's protocol using human peripheral blood leukocyte- derived poly- A RNA as the starting material for cDNA synthesis. Successfully amplified fragments were cloned into the Clontech pCRII-TOPO vector and were sequenced on a ABI Prism 377 DNA sequencer (PE Biosystems) according to the manufacturer's standard sequencing protocol using primers complemetary to the SP6 and T7 promoter regions flanking the insert on each vector. After sequences were obtained, the sequences from each clone were aligned using the computer program Sequencher (GeneCodes Corporation, Ann Arbor, MI, USA) to form a contiguous sequence of DNA. The consensus sequence of this contig was considered to represent the 3' or 5 'end of the NIP 45 like gene transcript.

Properties of the NIP45-like PROTEIN polypeptide cDNA nucleotide sequence

At least 2 species of the NIP45-like PROTEIN gene transcript exist. The 3' sequence of transcript 1 (from NIP45-like PROTEIN clone 2) is 1890 bp in length, with 222 bp at bases 1137-1358 and 144 bp at bases 1497-1640 that are not found in transcript 2.

The 3' sequence of transcript 2 (from NIP45-like PROTEIN clone 3) is 1626 bp in length, with 96 bp at bases 1137-1232 and 6 bp at bases 1289-1294 that are not found in transcript 1. The two transcripts are otherwise identical in sequence.

The sequencing analysis of each clone indicates that at least 2 species of the NIP 45 like protein gene transcript exist (transcript 1 and transcript 2). The nucleotide sequence of transcript 1 is shown in SEQ ID NO:l or 3. The nucleotide sequence of transcript 2 is shown in SEQ ID NO:2 or 4.

The openreading frame corresponds to residue 246 to residue 509 (with the stop codon of 507 to 509).

Properties of the NIP45-like PROTEIN polypeptide amino acid sequence Conceptual translation of either of the transcripts of NIP45-like PROTEIN gives an amino acid sequence homologous to residues 220-282 of human NIP45 (SEQ ID NO:5).

EXAMPLE 2

Functional Characterization

The function of NIP 45 like protein is assessed by its ability of specifically interacting with NF-ATp in mammalian cells. The NIP45-like cDNA inserts obtained in Example 1 is subcloned into a mammalian expression vector which fuses the coding region to an epitope tag from a influenza hemagglutinin (HA) peptide, vector pCEP4-HA (Herrscher, R. F. et al. (1995) Genes Dev. 9:3067-3082), to create the expression vector.

The vector is then cotransfected with an NF-ATp expression plasmid into HepG2 cells expressing low levels of NF-ATp. As controls, HepG2 cells also are cotransfected with NIP45-HA along with the expression vector without the NF-ATp insert or with the NF-ATp expression vector along with an out of frame fusion of NIP45 with the epitope tag. Lysates are prepared from the transfected cells and immunoprecipitated with anti-NF-ATp antibody. Western blot analysis are then performed on the immunprecipitated material using either anti-NF-ATp or anti-HA antibodies to see if NF-AT and NIP 45 like protein physically associate in vivo in mammalian cells.

EXAMPLE 3

Tissue Expression of NIP45-like mRNA

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of

RNA from different human tissues was performed to investigate the tissue expression ofNIP45-like RNA. 100 .mu.g of total RNA from various tissues (Human Total RNA Panel I-N, Clontech Laboratories, Palo Alto, CA, USA) was used as a template to synthsize first-strand cDΝA using the SUPERSCRIPT™ First-Strand Syntheswas System for RT-PCR (Life Technologies, Rockville , MD, USA). 10 ng of the first- strand cDΝA was then used as template in a polymerase chain reaction to test for the presence of the ΝIP45-like mRNA transcript. The polymerase chain reaction was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN, USA), in the presence of the DNA-binding fluorescent dye S YBR Green I which binds to the minor groove of the DNA double helix, produced only when double-stranded DNA is successfully synthesized in the reaction, and upon binding, emits light that can be quantitatively measured by the LightCycler machine. The polymerase chain reaction was carried out using oligonucleotide primers Nip-1 (GGGGAGCGTTGGGAAAATGC, also named NIPh-Rl) and Nip-2 (AAAGGGCCCACCCTCCCAGA, also named NIPh-Ll) and measurements of the intensity of emitted light were taken following each cycle of the reaction when the reaction had reached a temperature of 86 degrees C. Intensities of emitted light were converted into copy numbers of the gene transcript per nanogram of template cDNA by comparison with simultaneously reacted standards of known concentration.

To correct for differences in mRNA transcription levels per cell in the various tissue types, a normalization procedure was performed using calculated expression levels in the various tissues of five different housekeeping genes: glyceraldehyde-3- phosphatase (G3PHD), hypoxanthine guanine phophoribosyl transferase (HPRT), beta-actin, porphobilinogen deaminase (PBGD), and beta-2-microglobulin. Except for the use of a slightly different set of housekeeping genes, the normalization procedures was essentially the same as that described in the RNA Master Blot User

Manual, Apendix C (Clontech Laboratories, Palo Alto, CA, USA).

EXAMPLE 4

Functional Activity of NIP45-like protein in Regulating Gene Expression To test for a functional role of NIP45-like protein in NF-AT-driven transcription, NIP45 is expressed at high levels in HepG2 cells expressing low levels of endogenous NF-AT, and ectopic expression of NF-AT family member proteins has been shown to transactivate NF-AT-driven transcription in this cell line in the absence of exogenous stimulation (Hoey, T. et al. (1995) Immunity 2:461-472).

HepG2 cells are transfected with a 3X NF-AT-CAT reporter from the IL-2 gene (Nenkataraman, L. et al. (1994) Immunity 1:189-196) and control or expression plasmids for a ΝIP45 and NF-AT family members (NF-ATp, NF-ATc, NF-AT3, NF-AT4). HepG2 cells are transfected by the DEAE-Dextran method as described in Hoey, T. et al. (1995) supra, and CAT assays were performed according to standard methodologies.

EXAMPLE 5

Endogenous IL-4 Production

Expression of endogenous IL-4 by cells that do not normally produce IL-4 is examined to see if the combination of NIP45-like protein, NF-ATp and c-Maf is sufficient to induce the expression. M12 B lymphoma cells are transiently cotransfected with expression plasmids for NF-ATp and c-Maf together with NIP45 or pCI vector control. Ml 2 cells are transiently transfected by electroporation as previously described (Ho, I. C. et al. (1996) Cell 85:973-983). Levels of IL-4 in the supernatants harvested 72 hours later are measured by a commercially available IL-4 ELISA (Pharmingen), performed according to the manufacturer's instructions except with modification as described (Ho, I. C. et al. (1996) supra).

EXAMPLE 6

Identification of a test compound which binds to a NIP 45 like protein

Purified NIP 45 like polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. NIP 45 like polypeptides comprise any of the amino acid sequence shown of the SEQ ID NO: 5 . The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to an NIP 45 like polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15%) relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to an NIP 45 like polypeptide.

EXAMPLE 7 Identification of a test compound which modulates (increases or decreases) NIP 45 like gene expression

A test compound is administered to a culture of human lymph node cells and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells incubated for the same time without the test compound provides a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled NIP 45 like -specific probe at 65 ° C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO.T, 2, 3 or 4 or the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4. A test compound which modulates the NIP 45 like -specific signal relative to the signal obtained in the absence of the test compound is identified as an modulator of NIP 45 like gene expression. EXAMPLE 8

Screening for a compound which modulates the interaction between NIP 45 like protein and NF-AT can be done with the use of yeast two-hybrid system (s).

EXAMPLE 9

Treatment of immunologically related diseases by modulating the function of a human NIP 45 like protein.

A polynucleotide which expresses a human NIP 45 like protein or a compound, which modulate the function of NIP 45 like protein is administered to a patient. The severity of the patient's inflammation is lessened.

EXAMPLE 10

Quantitative Expression Profiling - NIP45-like PROTEIN polypeptide

Expression profiling is based on a quantitative polymerase chain reaction (PCR) analysis, also called kinetic analysis, first described in Higuchi et al., 1992 and Higuchi et al., 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies. Using this technique, the expression levels of particular genes, which are transcribed from the chromosomes as messenger RNA (mRNA), are measured by first making a DNA copy (cDNA) of the mRNA, and then performing quantitative PCR on the cDNA, a method called quantitative reverse transcription-polymerase chain reaction (quantitative RT-PCR).

Quantitative RT-PCR analysis of RNA from different human tissues was performed to investigate the tissue distribution of NIP45-like PROTEIN. 25 .mu.g of total RNA from various tissues (Human Total RNA Panel I-N, Clontech Laboratories, Palo Alto, CA, USA) was used as a template to synthsize first-strand cDΝA using the SUPERSCRIPT™ First-Strand Synthesis System for RT-PCR (Life Technologies, Rockville , MD, USA). First-strand cDNA synthesis was carried out according to the manufacturer's protocol using oligo (dT) to hybridize to the 3' poly A tails of mRNA and prime the synthesis reaction. 10 ng of the first-strand cDNA was then used as template in a polymerase chain reaction. The polymerase chain reaction was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN, USA), in the presence of the DNA-binding fluorescent dye S YBR Green I which binds to the minor groove of the DNA double helix, produced only when double-stranded DNA is successfully synthesized in the reaction (Morrison et al., 1998). Upon binding to double-stranded DNA, SYBR Green I emits light that can be quantitatively measured by the LightCycler machine. The polymerase chain reaction was carried out using oligonucleotide primers NIPh-Ll (AAAGGGCCCACCCTCCCAGA) and NIPh-Rl

(GGGGAGCGTTGGGAAAATGC) and measurements of the intensity of emitted light were taken following each cycle of the reaction when the reaction had reached a temperature of 86 degrees C. Intensities of emitted light were converted into copy numbers of the gene transcript per nanogram of template cDNA by comparison with simultaneously reacted standards of known concentration.

To correct for differences in mRNA transcription levels per cell in the various tissue types, a normalization procedure was performed using similarly calculated expression levels in the various tissues of five different housekeeping genes: glyceraldehyde-3-phosphatase (G3PDH), hypoxanthine guanine phophoribosyl transferase (HPRT), beta-actin, porphobilinogen deaminase (PBGD), and beta-2- microglobulin. The level of housekeeping gene expression is considered to be relatively constant for all tissues (Adams et al., 1993, Adams et al., 1995, Liew et al.,

1994) and therefore can be used as a gauge to approximate relative numbers of cells per .mu.g of total RNA used in the cDNA synthesis step. Except for the use of a slightly different set of housekeeping genes and the use of the LightCycler system to measure expression levels, the normalization procedure was similar to that described in the RNA Master Blot User Manual, Apendix C (1997, Clontech Laboratories, Palo Alto, CA, USA). In brief, expression levels of the five housekeeping genes in all tissue samples were measured in three independent reactions per gene using the LightCycler and a constant amount (25 .mu.g) of starting RNA. The calculated copy numbers for each gene, derived from comparison with simultaneously reacted standards of known concentrations, were recorded and the mean number of copies of each gene in all tissue samples was determined. Then for each tissue sample, the expression of each housekeeping gene relative to the mean was calculated, and the average of these values over the five housekeeping genes was found. A normalization factor for each tissue was then calculated by dividing the final value for one of the tissues arbitrarily selected as a standard by the corresponding value for each of the tissues. To normalize an experimentally obtained value for the expression of a particular gene in a tissue sample, the obtained value was multiplied by the normalization factor for the tissue tested. This normalization method was used for all tissues except those derived from the Human Blood Fractions MTC Panel, which showed dramatic variation in some housekeeping genes depending on whether the tissue had been activated or not. In these tissues, normalization was carried out with a single housekeeping gene, beta-2-microglobulin.

Results are given in Figs. 9 and 10, showing the experimentally obtained copy numbers of mRNA per 10 ng of first-strand cDNA on the left and the normalized values on the right. RNAs used for the cDNA synthesis, along with their supplier and catalog numbers are shown in tables 1 and 2.

Table 1. Whole-body-screen tissues

Table 2. Blood/lung-screen tissues

References

Higuchi, R., Dollinger, G., Walsh, P.S. and Griffith, R. (1992) Simultaneous amplification and detection of specific DNA sequences. BioTechnology

10:413-417. Higuchi, R., Fockler, C, Dollinger, G. and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. BioTechnology

11:1026-1030. T.B. Morrison, J.J. Weis & C.T. Wittwer .(1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification.

Biotechniques 24:954-962. Adams, M. D., Kerlavage, A. R., Fields, C. & Venter, C. (1993) 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet.

4:256-265. Adams, M. D., et al. (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.

Nature 311 supp:3-174. Liew, C. C, Hwang, D. M., Fung, Y. W., Laurenson, C, Cukerman, E., Tsui, S. & Lee, C. Y. (1994) A catalog of genes in the cardiovascular system as identified by expressed sequence tags. Proc. Natl. Acad. Sci. USA

91 :10145-10649.

References for data interpretation 1. Hodge MR, Chun HJ, Rengarajan J, Alt A, Lieberson R, Glimcher LH. NF-AT-

Driven interleukin-4 transcription potentiated by NIP45. Science. 1996 Dec 13;274(5294):1903-5.

2. Schuh K, Kneitz B, Heyer J, Bommhardt U, Jankevics E, Berberich-Siebelt F, Pfeffer K, Muller-Hermelink HK, Schimpl A, Serfling E. Retarded thymic involution and massive germinal center formation in NF-ATp-deficient mice. Eur J

Immunol. 1998 Aug;28(8):2456-66.

3. Kamitani T, Nguyen HP, Yeh ET. Preferential modification of nuclear proteins by a novel ubiquitin-like molecule. J Biol Chem. 1997 May 30;272(22): 14001-4.

4. Okura T, Gong L, Kamitani T, Wada T, Okura I, Wei CF, Chang HM, Yeh ET. Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J Immunol. 1996 Nov 15;157(10):4277-81.

5. Muller-Hermelink, HK, Marx, A. Kirchner, T. Thymus and mediastinum. In Damjanov, I, Linder, J (eds.), Mosby-Year Book. Mosby, St. Louis 1996, pp.1218-43. 6. Kiani A, Viola JP, Lichtman AH, Rao A. Down-regulation of IL-4 gene transcription and control of Th2 cell differentiation by a mechanism involving

NFAT1. Immunity. 1997 Dec;7(6):849-60. 7. Lieberson R, Mowen KA, McBride KD, Leautaud V, Zhang X, Suh WK, Wu L,

Glimcher LH. Tumor necrosis factor receptor-associated factor (TRAF)2 represses the T helper cell type 2 response through interaction with NFAT-interacting protein (NIP45). J Exp Med. 2001 Jul2;194(l):89-98.

Claims

1. An isolated polynucleotide encoding a NIP45-like protein polypeptide and being selected from the group consisting of: a) a polynucleotide encoding a NIP45-like protein polypeptide comprising an amino acid sequence selected form the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO:5; and the amino acid sequence shown in SEQ ID NO:5. b) a polynucleotide comprising the sequence of SEQ ID NO: 1, 2, 3, 4 or a polynucleotide comprising the the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a to (d).

2. An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

4. A substantially purified NIP45-like protein polypeptide encoded by a polynucleotide of claim 1.

5. A method for producing a NIP45-like protein polypeptide, wherein the method comprises the following steps: a) culturing the host cell of claim 3 under conditions suitable for the expression of the NIP45-like protein polypeptide; and b) recovering the NIP45-like protein polypeptide from the host cell culture.

6. A method for detection of a polynucleotide encoding a NIP45-like protein polypeptide in a biological sample comprising the following steps: a) hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting said hybridization complex.

7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or a NIP45-like protein polypeptide of claim 4 comprising the steps of: contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the NIP45-like protein polypeptide.

9. A diagnostic kit for conducting the method of any one of claims 6 to 8.

10. A method of screening for agents which decrease the activity of a NIP45-like protein, comprising the steps of: contacting a test compound with any NIP45-like protein polypeptide encoded by any polynucleotide of claim 1 ; detecting binding of the test compound to the NIP45-like protein polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a NIP45-like protein.

11. A method of screening for agents which regulate the activity of a NIP45-like protein, comprising the steps of: contacting a test compound with a NIP45-like protein polypeptide encoded by any polynucleotide of claim 1; and detecting a NIP45-like protein activity of the polypeptide, wherein a test compound which increases the NIP45-like protein activity is identified as a potential therapeutic agent for increasing the activity of the NIP45-like protein, and wherein a test compound which decreases the NIP45-like protein activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the NIP45-like protein.

12. A method of screening for agents which decrease the activity of a NIP45-like protein, comprising the steps of: contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of NIP45-like protein.

13. A method of reducing the activity of NIP45-like protein, comprising the steps of: contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any NIP45-like protein polypeptide of claim 4, whereby the activity of NIP45-like protein is reduced.

14. A reagent that modulates the activity of a NIP45-like protein polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.

15. A pharmaceutical composition, comprising: the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the expression vector of claim 2 or the reagent of claim 14 to produce a medicament for modulating the activity of a NIP45-like protein in a disease.

17. Use of claim 16 wherein the disease is an inflammatory or a chronic inflammatory disease where an over-activation or prolongation of the activation of the immune system causes damage to tissues.

18. A cDNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5

19. The cDNA of claim 18 which comprises SEQ ID NO:l, 2, 3, 4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4;.

20. The cDNA of claim 18 which consists of SEQ ID NO:l, 2, 3, 4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4.

21. An expression vector comprising a polynucleotide which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5.

22. The expression vector of claim 21 wherein the polynucleotide consists of

SEQ ID NO:l, 2, 3, 4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4.

23. A host cell comprising an expression vector which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5.

24. The host cell of claim 23 wherein the polynucleotide consists of SEQ ID NO:l, 2, 3, 4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4.

25. A purified polypeptide comprising the amino acid sequence shown in SEQ ID NO:5.

26. The purified polypeptide of claim 25 which consists of the amino acid sequence shown in SEQ ID NO: 5.

27. A fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NO:5.

28. A method of producing a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5, comprising the steps of: culturing a host cell comprising an expression vector which encodes the polypeptide under conditions whereby the polypeptide is expressed; and isolating the polypeptide.

29. The method of claim 28 wherein the expression vector comprises SEQ ID

NO:l, 2, 3, 4 or a polynucleotide comprising the the sequence from residue

246 to residue 506 shown in SEQ ID NO:3 or 4.

30. A method of detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5, comprising the steps of: hybridizing a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NO:l, 2, 3 ,4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO: 3 or 4 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and detecting the hybridization complex.

31. The method of claim 30 further comprising the step of amplifying the nucleic acid material before the step of hybridizing.

32. A kit for detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5, comprising: a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NO:l, 2, 3 or 4; and instructions for the method of claim 30.

33. A method of detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5, comprising the steps of: contacting a biological sample with a reagent that specifically binds to the polypeptide to form a reagent-polypeptide complex; and detecting the reagent-polypeptide complex.

34. The method of claim 33 wherein the reagent is an antibody.

35. A kit for detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5, comprising: an antibody which specifically binds to the polypeptide; and instructions for the method of claim 33.

36. A method of screening for agents which can modulate the activity of a human NIP45-like protein, comprising the steps of: contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO:5 and (2) the amino acid sequence shown in SEQ ID NO:5; and detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the human NIP45-like protein.

37. The method of claim 36 wherein the step of contacting is in a cell.

38. The method of claim 36 wherein the cell is in vitro.

39. The method of claim 36 wherein the step of contacting is in a cell-free system.

40. The method of claim 36 wherein the polypeptide comprises a detectable label.

41. The method of claim 36 wherein the test compound comprises a detectable label.

42. The method of claim 36 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.

43. The method of claim 36 wherein the polypeptide is bound to a solid support.

44. The method of claim 36 wherein the test compound is bound to a solid support.

45. A method of screening for agents which modulate an activity of a human NIP45-like protein, comprising the steps of: contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5 and (2) the amino acid sequence shown in SEQ ID NO:5; and detecting an activity of the polypeptide, wherein a test compound which increases the activity of the polypeptide is identified as a potential agent for increasing the activity of the human NIP45-like protein, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human NIP45-like protein.

46. The method of claim 45 wherein the step of contacting is in a cell.

47. The method of claim 45 wherein the cell is in vitro.

48. The method of claim 45 wherein the step of contacting is in a cell-free system.

49. A method of screening for agents which modulate an activity of a human NIP45-like protein, comprising the steps of: contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ID NO:l, 2, 3 ,4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4; and detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of the human NIP45-like protein.

50. The method of claim 49 wherein the product is a polypeptide.

51. The method of claim 49 wherein the product is RNA.

52. A method of reducing activity of a human NIP45-like protein, comprising the step of: contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO:l, 2, 3 ,4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4, whereby the activity of a human NIP45-like protein is reduced.

53. The method of claim 52 wherein the product is a polypeptide.

54. The method of claim 53 wherein the reagent is an antibody.

55. The method of claim 52 wherein the product is RNA.

56. The method of claim 55 wherein the reagent is an antisense oligonucleotide.

57. The method of claim 56 wherein the reagent is a ribozyme.

58. The method of claim 52 wherein the cell is in vitro.

59. The method of claim 52 wherein the cell is in vivo.

60. A pharmaceutical composition, comprising: a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5; and a pharmaceutically acceptable carrier.

61. The pharmaceutical composition of claim 60 wherein the reagent is an antibody.

62. A pharmaceutical composition, comprising: a reagent which specifically binds to a product of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO:l, 2, 3 ,4 or a polynucleotide comprising the the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4; and a pharmaceutically acceptable carrier.

63. The pharmaceutical composition of claim 62 wherein the reagent is a ribozyme.

64. The pharmaceutical composition of claim 62 wherein the reagent is an antisense oligonucleotide.

65. The pharmaceutical composition of claim 62 wherein the reagent is an antibody.

66. A pharmaceutical composition, comprising: an expression vector encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:5; and a pharmaceutically acceptable carrier.

67. The pharmaceutical composition of claim 66 wherein the expression vector comprises SEQ ID NO:l, 2, 3 ,4 or a polynucleotide comprising the sequence from residue 246 to residue 506 shown in SEQ ID NO:3 or 4.

68. A method of treating a NIP45-like protein dysfunction related disease, wherein the disease is selected from a disease involving an inflammatory process, or a chronic inflammatory disease where an over-activation or prolongation of the activation of the immune system causes damage to tissues comprising the step of: administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a human NIP45-like protein, whereby symptoms of the NIP45-like protein dysfunction related disease are ameliorated.

69. The method of claim 68 wherein the reagent is identified by the method of claim 36.

70. The method of claim 68 wherein the reagent is identified by the method of claim 45.

71. The method of claim 68 wherein the reagent is identified by the method of claim 49.