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]
  • C07K14/545—IL-1
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the isolated polynucleotides of the invention include, but are not limited to, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NOS:
• the isolated polynucleotides of the invention further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of SEQ ID NOS: 1, 2, 4, or 6; a polynucleotide comprising the full length protein coding sequence of SEQ ID NOS: 1, 2, 4, or 6, and; a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NOS: 1 , 2, 4, or 6.
• polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for the prevention and/or treatment of disorders involving sepsis, acute pancreatitis, endotoxic shock, cytokine induced shock, rheumatoid arthritis, chronic inflamatory arthitis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflamatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflamatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.
• FIG. 4 shows the sequence alignment of receptor binding regions of human Interleukin- 1 beta (SEQ ID NO. 12) and human Interleukin-l receptor antagonist (SEQ ID NO. 13)aligned with a cognate region of SEQ ID NO.3 (amino acids 13 through 30 of SEQ ID NO.3). Residues conserved among all three domains are shown in boldface.
• FIGS 10A-C show a genomic clone (SEQ ID NO: 8) which is a ⁇ extension of the genomic sequence presented in Figure 9A-C (SEQ ID NO: 7).
• SEQ ID NO: S includes the the . extended sequence shown in SEQ ID NO: 6 for the Interleukin-l Receptor Antagonist extensionclothnted in SEQ ID NO: 6.
• the isolation of this genomic clone (SEQ ID NO:7 ) from which this sequence was derived is described in Example 11.
• R indicates A or G
• M indicates A oi C
• W indicates A or T
• Y indicates C or T
• S indicates C or G
• K indicates G 0 or T
• V indicates A or C or G
• H indicates A or C or T
• D indicates A or G 01 T
• B indicates C or G or T
• N indicates any of the four bases.
• recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial or mammalian) expression systems.
• Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.
• recombinant microbial defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.
• nucleic acid sequences encoding such substantially equivalent sequences can routinely be isolated and identified via standard hybridization procedures well known to those of skill in the art.
• the isolated polynucleotides of the invention include, but are not limited to, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NOS: 3 or 5.
• a polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
• Useful nucleotide sequences for joining to polypeptides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
• the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
• Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
• a host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism. The sequences falling within the scope of the present invention are not limited to the specific sequences herein described, but also include allelic variations thereof.
• Such promoters can be derived from operons encoding glycolytic enzymes such as 3 -phosphogly cerate kinase (PGK), a- factor, acid phosphatase, or heat shock proteins, among others.
• PGK 3 -phosphogly cerate kinase
• the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
• the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
• the present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.
• host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.
• the present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.
• DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
• Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
• Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
• Interleukin-l receptor antagonist is protective in a rabbit model of hypotensive gram-positive septic shock.
• the administration of Interleukin-l receptor antagonist in this animal model has been shown to maintain mean arterial pressure compared to control, as well as decreasing lung water and maintaining urine output.
• This work demonstrated the role of Interleukin-l and the protective role of Interleukin-l receptor antagonist in gram-positive septic shock.
• Interleukin-l is the principal mediator in a patient's clinical response to multiple different stresses regardless of the etiology (including acute pancreatitis, sepsis, endotoxin shock, and cytokine induced shock).
• erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
• the activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
• a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
• the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885.
• a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
• a protein of the invention may also exhibit hemostatic or thrombolytic activity.
• a polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
• Such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
• a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
• the activity of a protein of the invention may, among other means, be measured by the following methods:
• a protein of the present invention may also demonstrate activity as receptors , receptor ligands or inhibitors or agonists of receptor/ligand interactions.
• a polynucleotide of the invention can encode a polypeptide exhibiting such characteristics. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses).
• Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
• a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation
• a therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
• a therapeutically effective dose refers to that ingredient alone.
• a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
• the compounds may be administered topically, for example, as eye drops.
• a targeted drug delivery system for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
• the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.
• Topical administration may be suitable for wound healing and tissue repair.
• Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
• the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
• Such matrices may be formed of materials presently in use for other implanted medical applications.
• a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
• CMC carboxymethylcellulose
• Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
• Dosage intervals can also be determined using MEC value.
• Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
• composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
• Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
• the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
• KLH keyhole limpet hemocyanin
• Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)). Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention.
• a nucleotide sequence of the present invention can be recorded on computer readable media.
• computer readable media refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
• magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
• optical storage media such as CD-ROM
• electrical storage media such as RAM and ROM
• hybrids of these categories such as magnetic/optical storage media.
• data storage means refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.
• search means refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.
• test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.
• biological fluids such as sputum, blood, serum, plasma, or urine.
• the test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.
• the agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.
• the agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.
• Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al, Nucl. Acids Res. 3:173 (1979); Cooney et al, Science 241:456 (1988); and Dervan et al, Science 251: 1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
• An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
• the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
• the parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.
• SEQ ID NO.2 To study the role of SEQ ID NO. 2 in the regulation of the inflammatory response, gene expression was analyzed using a semi-quantatative polymerase chain reaction-based technique. cDNA libraries were used as sources of expressed genes from tissues of interest (three leukocyte preparations [two stimulated and one unstimulated], heart, lung, spleen, placenta, testes, fetal liver, adult liver, bone marrow, lymph node, macrophages, endothelial cells, fetal skin, and umbilical cord). Gene specific primers were used to amplify portions of the SEQ ID NO. 2 sequence (corresponding to bases 105-772 and 161-690, as numbered frome the 5' end of SEQ ID NO.
• Chromosome mapping technologies allow investigators to link genes to specific regions of chromosomes. Chromosomal mapping was performed with the Stanford G3 Radiation Hybrid Panel (Research Genetics). The panel was screened with gene-specific primers (5' primer: CCCCACTGGATGGTGCTACTG; (SEQ ID NO. 15), 3' primer: GGGAAGAGATAGGAAAGGTAG) (SEQ ID NO. 16)that generated a sequence tag site (STS), and the results of the PCR screening were submitted to the Stanford Radiation Hybrid mapping email server at the Stanford Human Genome Center (SHGC). The gene position on the radiation hybrid framework map was provided by linking the STS corresponding to SEQ ID NO. 2 with the SHGC marker with best linkage.
• SHGC Stanford Human Genome Center
• Interleukin-l Receptor Binding Domain and Interleukin-l Receptor Assay The receptor binding region of both IL-1 ⁇ and IL-1 Ra have been mapped an 18 amino acid region in the carboxy terminal half of the proteins (i.e., residues 88-105 of IL-l ⁇ ) by site- directed mutagenesis and protein modification studies.
• An amino acid alignment of SEQ ID NO. 3 with both IL-1 ⁇ and IL-1 Ra demonstrates that SEQ ID NO. 3 contains a receptor binding region.
• SEQ ID NO. 3 is 39% identical to IL-lRa and 22% identical (39% conserved) with IL-1 ⁇ in this region.
• SEQ ID NO. 3 is prepared by growing E.coli expressing SEQ ID NO. 3 on M9 medium containing [35S] sulphate and purifying the labeled SEQ ID NO. 3 by chromatography on a Mono-S column. The labeled SEQ ID NO. 3 is incubated with the cells or tissue under standard IL-1 binding assay conditions, and [35S] binding. Significant [35S] binding indicates the presence of IL-1 receptors.
• the extended nucleotide sequence was obtained by isolating PCR products generated from pools of clones from a fetal skin cDNA library. In short, a fetal skin cDNA library was plated on ampicillin containing plates in pools of about 40,000 colonies. The colonies were recovered into LB medium and PCR was used to detect pools which contained SEQ ID NO. 2 ( Figure 2). Two pools were identified. PCR using vector- and gene-specific primers amplified the 5' portion of the cDNA. Nested primers were used to generate sequence from the two amplified products.
• SEQ ID NO. 5 possesses a combination of both residues, RIO (identical to the corresponding residue in IL-1 Ra) and K12 and D148 (similar and identical, respectively, to the corresponding residues in IL-1 ⁇ ).
• RIO identical to the corresponding residue in IL-1 Ra
• K12 and D148 similar and identical, respectively, to the corresponding residues in IL-1 ⁇ .
• the overall homology and agonist/antagonist site-specific sequence homologies clearly define SEQ ID NO. 5 as a protein modulator of the inflammatory response.
• Acute edematous, necrotizing pancreatitis is induced in adult male Swiss mice weighing more than 35 grams using caerulein ⁇ an analog of cholecystokinin. Mice are divided into four groups with three of the groups receiving caerulein 50 mu g/kg by intraperitoneal (IP) injection in four doses over three hours as previously described.
• IP intraperitoneal
• mice On day 0 the left footpads of all mice are injected intracutaneously with 107 irradiated (2500R), allogeneic spleen cells from C57BL/6 mice in 50 ul of RPMI-1640 (Gibco) as antigen and the right contralateral footpads of the same mice are injected with 10 sup 7 irradiated (2500R), syngeneic spleen cells from BALB/c mice. Seven days after antigen administration, the mice are sacrificed and the popliteal lymph nodes (PLN) are removed from the right and left popliteal fossa by surgical dissection.
• PPN popliteal lymph nodes

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