Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists, antagonists and/or inhibitors which are potentially useful m therapy, and to production of such polypeptides and polynucleotides.
• Functional genomics that is, high throughput genome- or gene-based biology This approach is rapidly superseding earlier approaches based on “positional cloning " A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterize further genes and their related polypeptides/protems, as targets for drug discovery.
• Interleukin 1 refers to two proteins (ILl ⁇ and IL1 ⁇ ) which play a key role early in the inflammatory response [see C.A. Dinarello, Blood, 87:2095-2147 (1996)]. Both proteins are made as 31kD intracellular precursor proteins which are cleaved upon secretion to yield mature carboxy- terrmnal 17kD fragments which are biologically active. In the case of IL-1 ⁇ , this cleavage involves an intracellular cysteme protease, known as ICE, which is required to release the active fragment from the inactive precursor. The precursor of IL-1 ⁇ is active.
• IL-1 activates transcription factors such as NF- ⁇ B and AP-1.
• IL-1 IL-1 receptor antagonist
• ERAP IL-1 receptor antagonist protein
• All three proteins, IL-l ⁇ , IL-l ⁇ and IL-lra share approximately 25-30% ammo acid identity and a similar three-dimensional structure consisting of twelve ⁇ -strands folded into a ⁇ -barrel, with an internal thrice repeated structural motif.
• the active receptor complex consists of the type I receptor and ILlRAcP (for IL-1 accessory protein).
• the type I receptor is responsible for binding of the three ligands, and is able to do so m the absence of the ILlRAcP
• signal transduction requires interaction of IL-l ⁇ or ⁇ with the ILlRAcP.
• IL-lra does not interact with the ILlRAcP and hence cannot signal.
• a third receptor subunit, the type II receptor binds IL-l ⁇ and IL-1 ⁇ but cannot signal due to its lack of an intracellular domain. Rather, it acts as a decoy either in its membrane form or an antagonist m a cleaved secreted form, and hence inhibits EL-l activity. It only weakly binds IL-lra.
• IL-lra soluble IL-1R
• IL-l ⁇ or ⁇ transgenic knockouts of these genes
• IL-ls play a key role in a number of pathophysiologies (see C A. Dmarello, Blood 87:2095-2147 (1996)).
• IL-lra has been shown to be effective in animal models of septic shock, rheumatoid arthritis, graft versus host disease, stroke, cardiac ischemia, and is currently in clinical trials for some of these indications.
• IL-l ⁇ and ⁇ have shown some potential as hematopoietic stem cell stimulators with potential as radio- and chemoprotectants.
• IL-1 Like IL-1, it binds to two receptor subunits which belong to the IL-1 family of receptors [To ⁇ goe et al., J Biol. Chem. 272:25737 (1997), Born et al., J. Biol. Chem. 273 29445 (1998)] Summary of the Invention
• the present invention relates to IL-1H4, in particular mat IL-1H4 polypeptides and mat IL- 1H4 polynucleotides, recombinant mate ⁇ als and methods for their production.
• the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of chronic and acute inflammation, septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoriasis, and arth ⁇ tis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, allergies, asthma, restenosis, brain injury, AIDS, bone diseases (e.g. osteoporosis), cancer (e.g.
• the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with IL-1H4 imbalance with the identified compounds.
• the invention relates to diagnostic assays for detecting diseases associated with inappropriate IL-1H4 activity or levels.
• Fig. 1 illustrates the results of cleavage of IL-1H4 to form mat IL-1H4.
• the present invention relates to IL-1H4 polypeptides and mat IL-1H4 polypeptides.
• peptides include isolated polypeptides comprising an ammo acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 or SEQ ID NO:6 over the entire length of SEQ ID NO:2 or SEQ ID NO.6, respectively.
• polypeptides include those comprising the ammo acid of SEQ ID NO.2 or SEQ ID NO:6.
• peptides of the present invention include isolated polypeptides in which the ammo acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NO:2 or SEQ ID NO:6, over the entire length of SEQ ID NO.2 or SEQ ID NO:6, respectively.
• Such polypeptides include the polypeptide of SEQ ID NO:2 or SEQ rD NO:6.
• peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: l .
• Polypeptides of the present invention are believed to be members of the Interleuk ⁇ n-1 family of polypeptides. They are therefore of interest because these proteins play a role in chronic and acute disease and could therefore be therapeutic agents or targets for therapeutic intervention. These properties are hereinafter referred to as "IL-1H4 activity” or “IL-1H4 polypeptide activity” or "biological activity of IL-1H4". Also included amongst these activities are antigenic and lmmunogenic activities of said EL-1H4 polypeptides, m particular the antigenic and lmmunogenic activities of the polypeptides of SEQ ED NO:2 and SEQ ID NO:6. Preferably, a polypeptide of the present invention exhibits at least one biological activity of IL-1H4.
• polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
• the amino acid sequence of SEQ ID NO:2 contains 218 ammo acids.
• the polypeptide of SEQ ID NO:2 (IL-1H4) is cleaved to a 20 ammo acid sequence, SEQ ID NO:5, and a 198 ammo acid sequence, SEQ ID NO:6.
• the ammo acid sequence of SEQ ID NO:6 is a mature IL-1H4 (mat IL-1H4).
• the present invention also includes include variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination.
• Polypeptides of the present invention can be prepared in any suitable manner.
• Such polypeptides include isolated naturally occurring polypeptides, recombmanfly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
• the present invention relates to IL-1H4 polynucleotides and mat IL-1H4 polynucleotides.
• Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6, over the entire length of SEQ ID NO:2 or SEQ ID NO:6, respectively.
• polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
• polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO.l encoding the polypeptide of SEQ ID NO:2.
• polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2 or SEQ ID NO:6, over the entire coding region.
• polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98- 99% identity are more highly preferred, and those with at least 99%> identity are most highly preferred.
• polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ED NO: 1 over the entire length of SEQ ID NO: 1.
• polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identify are more highly preferred, and those with at least 99% identity are most highly preferred.
• Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 as well as the polynucleotide of SEQ ID NO: 1.
• the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
• the nucleotide sequence of SEQ ID NO: 1 shows homology with unannotated EST de ⁇ ved sequences (GenBank Accession No. AI014548, Al 343258) and one genomic sequence (GenBank Accession No. AQ041691).
• the nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 58 to 714) encoding a polypeptide of 218 amino acids, the polypeptide of SEQ ID NO:2.
• the nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained m SEQ ID NO: l or it may be a sequence other than the one contained in SEQ ID NO: 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO.2.
• the polypeptide of SEQ ED NO:2 is structurally related to other proteins of the Interleukm-1 family, having homology and/or structural simila ⁇ ty with bovine IL-lra (GenBank Accession No. AB005148) and IL-lra from other species.
• Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one IL- 1H4 activity
• Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library de ⁇ ved from RNA in cells of human B cells, testes, and fetal lung, using the expressed sequence tag (EST) analysis (Adams, M D. et al., Science (1991) 252: 1651-1656; Adams, M.D. et al., Nature, (1992) 355:632-634; Adams, M.D. et al. , Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
• EST expressed sequence tag
• the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
• a marker sequence which facilitates purification of the fused polypeptide can be encoded.
• the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
• the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
• polypeptide variants which comp ⁇ se the ammo acid sequence of SEQ ID NO:2 or SEQ ID NO:6, and m which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination.
• Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ED NO: 1 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to SEQ ID NO:l .
• these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent.
• the probes or p ⁇ mers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.
• a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an approp ⁇ ate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
• Such hybridization techniques are well known to the skilled artisan.
• Preferred stringent hybridization conditions include overnight incubation at 42°C m a solution comp ⁇ smg: 50% formamid , 5xSSC (150mM NaCl, 15 M t ⁇ sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in O.lx SSC at about 65°C.
• the present invention also includes polynucleotides obtainable by screening an approp ⁇ ate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof.
• an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. This is a consequence of reverse transc ⁇ ptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis.
• cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide primers
• the PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' the known gene sequence)
• the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
• Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems Accordingly, m a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
• polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et a , Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N.Y. (1989).
• Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, catiomc lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
• Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E.
• coli Streptomyces and Bacillus subtihs cells
• fungal cells such as yeast cells and Asperg ⁇ lus cells
• insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
• plant cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells.
• chromosomal, episomal and virus-derived systems e.g., vectors derived from bacterial plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids.
• the expression systems may contain control regions that regulate as well as engender expression
• any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
• the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
• Approp ⁇ ate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
• a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced lntracellularly, the cells must first be lysed before the polypeptide is recovered.
• Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectm chromatography. Most preferably, high performance liquid chromatography is employed for pu ⁇ fication. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or pu ⁇ fication.
• This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characte ⁇ zed by the polynucleotide of SEQ ED NO:l which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over- expression or altered expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
• Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy mate ⁇ al.
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
• RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in compa ⁇ son to the normal genotype. Point mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled E ⁇ -1H4 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures.
• DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (e.g., Myers et al , Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Set USA (1985) 85: 4397-4401 ).
• an array of ohgonucleotides probes comprising IL-1H4 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
• Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
• the diagnostic assays offer a process for diagnosing or determining a susceptibility to the Diseases through detection of mutation in the IL-1H4 gene by the methods described.
• diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides. such as, for example, nucleic acid amplification, for mstance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
• Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample de ⁇ ved from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
• the present invention relates to a diagnostic kit which comprises:
• a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ;
• an antibody to a polypeptide of the present invention preferably to the polypeptide of SEQ ID NO:2 or SEQ ID NO:6.
• kits may comprise a substantial component.
• a kit will be of use m diagnosing a disease or susceptibility to a disease, particularly chronic and acute inflammation, septicemia, autoimmune diseases (e.g. inflammatory bowel disease, pso ⁇ asis, and arthritis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, allergies, asthma, restenosis, brain injury, AJDS, bone diseases (e.g. osteoporosis), cancer (e g. lymphoproliferative disorders), congestive heart failure, atherosclerosis, and Alzheimer's disease, amongst others.
• the nucleotide sequences of the present invention are also valuable for chromosome identification.
• the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
• the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendehan Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (comhe ⁇ tance of physically adjacent genes).
• the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed m some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease
• the polypeptides of the ⁇ m ention or their fragments or analogs thereof, or cells expressing them, can also be used as immunogens to produce antibodies immunospecif ⁇ c for polypeptides of the present invention.
• immunospecif ⁇ c means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides m the prior art.
• Antibodies generated against polypeptides of the present invention may be obtained by admi ste ⁇ ng the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols.
• an animal preferably a non-human animal
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hyb ⁇ doma technique (Kohler, G.
• the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
• Antibodies against polypeptides of the present invention may also be employed to treat the
• the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
• immunoglobulins of various subclasses IgG, IgM, IgA, IgE.
• Prefe ⁇ ed as an lmmunoglobuhn is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region.
• the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
• this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
• a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914.
• Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others.
• Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
• a further aspect of the invention relates to an lmmunological/vaccme formulation (composition) which, when introduced into a mammalian host, induces an immunological response m that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention.
• the vaccine formulation may further comprise a suitable earner Since a polypeptide may be broken down m the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection).
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation isotomc with the blood of the recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents or thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use.
• the vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
• Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide.
• agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.
• Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
• Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology l(2):Chapter 5 (1991)).
• the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor.
• these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide.
• Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
• Constitutively active polypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide.
• the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring IL-1H4 activity in the mixture, and comparing the IL-1H4 activity of the mixture to a standard.
• Fusion proteins such as those made from Fc portion and IL-1H4 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).
• polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide m cells.
• an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known m the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
• the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art.
• ligand binding and crosslmkmg assays include, but are not limited to, ligand binding and crosslmkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids).
• a source of the putative receptor include biophysical techniques such as surface plasmon resonance and spectroscopy.
• polypeptide antagonists include antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
• the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises: (a) a polypeptide of the present invention;
• a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by: (a) determining in the first instance the three-dimensional structure of the polypeptide;
• the present invention provides methods of treating abnormal conditions such as, for instance, chronic and acute inflammation, septicemia, autoimmune diseases (e.g. inflammatory bowel disease, pso ⁇ asis, and arthritis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, allergies, asthma, restenosis, bram injury, ADDS, bone diseases (e.g. osteoporosis), cancer (e.g. lymphoproliferative disorders), congestive heart failure, atherosclerosis, and Alzheimer's disease, related to either an excess of, or an under- expression of, IL-1H4 polypeptide activity. If the activity of the polypeptide is in excess, several approaches are available.
• abnormal conditions such as, for instance, chronic and acute inflammation, septicemia, autoimmune diseases (e.g. inflammatory bowel disease, pso ⁇ asis, and arthritis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, allergies, asthma, reste
• One approach comprises admimste ⁇ ng to a subject in ne d thereof an inhibitor compound (antagonist) as heremabove desc ⁇ bed, optionally m combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition.
• soluble forms of the polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide may be administered.
• Typical examples of such competitors include fragments of the IL-1H4 polypeptide
• expression of the gene encoding endogenous IL-1H4 polypeptide can be inhibited using expression blocking techniques.
• Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
• ohgonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979)
• IL-1 H4 For treating abnormal conditions related to an under-expression of IL-1 H4 and its activity, several approaches are also available.
• One approach comprises administering to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present invention, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition
• gene therapy may be employed to effect the endogenous production of IL-1H4 by the relevant cells in the subject
• a polynucleotide of the invention may be engineered for expression in a replication defective retro viral vector, as discussed above
• the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
• producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
• gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read. BIOS Scientific Publishers Ltd (1996).
• Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical earner.
• the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient
• a pharmaceutically acceptable earner or excipient include, but are not limited to, saline, buffered salme, dextrose, water, glycerol, ethanol, and combinations thereof
• the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention
• Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
• the composition will be adapted to the route of administration, for instance by a systemic or an oral route
• Preferred forms of systemic administration include injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or mtrape ⁇ toneal, can be used
• the dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are in the range of 0 1 -100 ⁇ g/kg of subject Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art
• Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above
• cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced into the subject
• Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology This is most easily facilitated by storing the sequence m a computer readable medium and then using the stored data to search a sequence database using well known searching tools, such as GCC Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ED NO: 1 and/or a polypeptide sequence encoded thereby.
• Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
• Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
• a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is 'isolated", as the term is employed herein.
• Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
• Modified bases include, for example, t ⁇ tylated bases and unusual bases such as inosine.
• polynucleotide embraces chemically, enzymaticaliy or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
• Polynucleotide also embraces relatively short polynucleotides, often refe ⁇ ed to as ohgonucleotides.
• Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide” refers to both short chains, commonly refe ⁇ ed to as peptides, oligopep tides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene-encoded amino acids.
• Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known m the art Such modifications are well described m basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching.
• Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP- ⁇ bosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
• Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties
• a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
• a typical variant of a polypeptide differs m ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
• a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination.
• a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code.
• a variant of a polynucleotide or polypeptide may be a naturally occurnng such as an allel.c variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring vana ⁇ ts of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
• Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
• identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
• Prefe ⁇ ed methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al., Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec Biol.
• the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al , J. Mol Biol 215. 403-410 (1990).
• the well known Smith Waterman algorithm may also be used to determine identity.
• Prefe ⁇ ed parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
• Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap" program from
• the aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
• a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ED NO:l, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence.
• Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups withm the reference sequence.
• the number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:l by the numerical percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO: l, or:
• n n is the number of nucleotide alterations
• x n is the total number of nucleotides in SEQ ID NO: l
• y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
• Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO.2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
• a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO.2, that is be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the % identity is less than 100%.
• Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence.
• the number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids in SEQ ID NO:2 by the numerical percent of the respective percent ⁇ dent ⁇ ty(d ⁇ v ⁇ ded by 100) and then subtracting that product from said total number of amino acids in SEQ ED NO:2, or: n a ⁇ x a - (x a • y), wherein n a is the number of amino acid alterations, x a is the total number of ammo acids in SEQ ED NO:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non- integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
• Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
• EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
• employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262].
• the deduced ammo acid sequence does not encode the signal sequence usually found secreted proteins, but does contain potential caspase and protease cleavage sites in the ammo terminal region, suggesting that the active form of the protein might require such processing for activity, as is found for IL-1 ⁇ and EL-18, two other member of the IL-1 family.
• the clones encoding BL-1H4 were de ⁇ ved from a pooled and subtracted cDNA library made from fetal lung, testes and a B cell line, and from subtracted colon cDNA library.
• IL-1H4 The full-length IL-1H4 sequence was amplified using cDNA as a template by PCR with gene specific primers (SEQ ED NOS: 3 and 4) containing Ndel restriction enzyme sites and cloned into pET16B vector (Novagen) (Madison, Wisconsin).
• pET16B-IL-lH4 plasmid was introduced into E. cob cells and IL-1H4 protein was expressed as an amino-terminal HiSio epitope tag and a proteolytic cleavage site for Factor Xa.
• IL-1H4 was purified from several liters of E. coli culture by Ni-NTA agarose affinity chromatography (Qiagen Inc.) (Valencia, California). The H ⁇ s 10 tag was then removed by Factor Xa cleavage, followed by Superdex 75 gel filtration to generate full-length pro-IL-lH4.
• IL-1 H4 contains a potential caspase and protease cleavage site between ammo acids 20 (D) and 21 (E) in the amino terminal region, suggesting that IL- 1H4 may be a substrate for caspases and that the active form of the protein might require such processing for activity, as is the case for IL-l ⁇ and IL-18, two other members of the IL-1 family.
• Caspase 1 at a ratio of 1 :50 to 1 :5 (caspase:IL-lH4, weight weight) completely cleaved pro- JJL-1H4 to release the mature IL-1H4 (Fig. 1).
• the mature IL-1H4 after caspase cleavage was separated on a SDS polyacrylamide gel and transfe ⁇ ed to polyvmylidene difluo ⁇ de (PVDF) membrane.
• PVDF polyvmylidene difluo ⁇ de
• Lane 5 contains purified pro-IL-lH4 which was not cleaved by caspase 1 due to the presence of Z-VAD- FMK (Enzymes Systems Products) (Livermore, California), a caspase 1 inhibitor.
• Z-VAD- FMK Enzymes Systems Products
• the pro and mature regions of EL-1H4 sequence after caspase 1 cleavage are shown in SEQ ED NO:5 and SEQ ED NO:6, respectively.

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