Novel interleukin - 1 Hy2 materials and methods

FIELD OF THE INVENTION

[0002] The present invention relates to a novel polynucleotide encoding a protein called IL-1 Hy2, which is structurally related to interleukin-1 receptor antagonist protein, along with therapeutic, diagnostic and research utilities for these and related products.

BACKGROUND

[0003] Cytokines are involved in inflammation and the immune response, in part through endothelial cell activation. Distinct immune-mediators such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and gamma-interferon (IFN) appear to induce different but partially overlapping patterns of endothelial cell activation including increased procoagulant activity (Bevilaqua (1986) PNAS, 83:4533-4537), PGI and 2 production (Rossi (1985), Science, 229:174-176), HLA antigen expression (Pober (1987) J. Immunol., 138:3319-3324) and lymphocyte adhesion molecules (Carender (1987) J. Immunol., 138:2149-2154). These cytokines are also reported to cause hypotension, vascular hemorrhage, and ischemia (Goldblum et al. 1989, Tracey et al. Science 234:470, 1986). A major toxicity of these and other biological response modifiers is hypotension and vascular leakage (Dvorak (1989) J.N.C.I., 81:497-502).

[0004] IL-1 is produced by a number of cell types, including monocyte and macrophages, Langerhans cells, natural killer cells, B cells, T cell leukemic cell lines, neutrophils, endothelial cells, dendritic cells, melanoma cell lines, mesangial cells, astrocytes, glioma cells, microglial cells, fibroblasts and epithelial cells. Two forms of IL-1 have been isolated; IL-1α and IL-1β. They represent the products of two distinct genes and their mature forms are 159 and 153 amino acid proteins, respectively. These molecules are extremely potent and multi-functional cell activators, with a spectrum that encompasses cells of hematopoietic origin, from immature precursors to differentiated leukocytes, vessel wall elements, and cells of mesenchymal, nervous and epithelial origin. IL-1 also induces production of secondary cytokines, including ,IL-6, colony stimulating factors (CSFs) and chemokines. IL-1 is active as a hematopoietic growth and differentiation factor; activates endothelial cells in a pro-inflammatory and pro-thrombotic manner (including by inducing production of tissue factor and platelet activating factor); stimulates the release of corticotropin-releasing hormone (CRH) that ultimately causes release of corticosteroids by the adrenals; mediates the acute phase response (including by inducing hepatocyte production of acute phase proteins) and is a central mediator of local and systemic inflammatory reactions that can lead to sepsis and septic shock; is the primary endogenous pyrogen (causing fever); induces slow-wave sleep and anorexia; may play a role in destructive joint and bone diseases (including by inducing production of collagenase by synovial cells and metalloproteinases by chondrocytes); stimulates fibroblast proliferation and collagen synthesis; and may play a role in the pathogenesis of insulin-dependent type I diabetes through its toxicity for insulin-producing beta cells in Langerhans islets.

[0005] The IL-1 pathway consists of the two agonists IL-1α and IL-1β, a specific activation system (IL-1 converting enzyme), a receptor antagonist (IL-1Ra) produced in different isoforms and two high affinity receptors. IL-1α and IL-1β bind to two distinct IL-1 receptor types, IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII), both of which are members of the immunoglobulin superfamily of receptors. Both types of receptors are usually coexpressed, although type I is the predominant form in fibroblasts and T cells, while type II is preferentially expressed on B cells, monocytes and neutrophils. IL-1RI and IL-1RII have different affinities for the three ligands of the IL-1 family(IL-1αa, IL-1β and IL-1Ra). In particular, IL-1Ra binds to the type I receptor with an affinity similar to that of IL-1α, while IL-1Ra binds to the type II receptor 100-fold less efficiently than the type I receptor. There is evidence indicating that IL-1 induced activities are mediated exclusively via the type I receptor, whereas the type II receptor has no signaling activity and inhibits IL-1 activities by acting as a decoy for IL-1.

[0006] IL-1 receptor antagonist (IL-1Ra or IRAP) binds to the IL-1 receptor with affinity similar to that of IL-1 but has no IL-1-like activity, even at very high concentrations, and thus inhibits (antagonizes) the activity of IL-1. The purified IL-1Ra molecule has a molecular weight of approximately 22 kD and is believed to be glycosylated. It has limited sequence similarity to IL-1α and IL-1β at the amino acid level (19% and 26%, respectively). There appear to be at least two isoforms of IL-1Ra, including a soluble form and an intracellular form generated by an alternative splicing event. IL-1Ra appears to be produced by monocytes, macrophages, neutrophils and fibroblasts; keratinocytes and cells of epithelial origin produce almost exclusively the intracellular form. In humans, the gene for IL-1Ra has been localized to the long arm of chromosome 2, which is the same region where IL-1α and IL-1β, as well as IL-1RI and IL-1RII, are found.

[0007] The ability of IL-1 to modify biological responses has been demonstrated in a variety of studies. For example, the administration of IL-1 to rabbits (Wakabayashi et al., FASEB J 1991;5:338; Okusawa et al. J Clin Invest 1988;81:1162; Ohlsson et al., Nature 1990;348:550; Aiura, et al. Cytokine 1991;4:498) and primates (Fischer et al. Am J Physiol 1991;261:R442) has been shown to result in hypotension, tachycardia, lung edema, renal failure, and, eventually, death, depending on the dose. When the serum from the IL-1 treated animals is examined, the elevation of other cytokines is evident, mimicking the levels seen in acute pancreatitis in humans. (Guice et al., J Surg Res 1991;51:495-499; Heath et al., Pancreas 1993;66:41-45). There is a large body of evidence currently available which supports the role of IL-1 as a major mediator of the systemic response to diseases such as sepsis and pancreatitis and as an activator of the remaining members of the cytokine cascade. (Dinarello et al., Arch Surg 1992;127:1350-1353).

[0008] IL-1 is a key mediator in the inflammatory response (for reviews, see Dinarello (1991) Blood 77: 1627-1652; Dinarello et al. (1993) New England J. Med. 328:106-113; Dinarello (1994) FASEB J. 8:1314-1325). The importance of IL-1 in inflammation has been demonstrated by the ability of the highly specific IL-1 receptor antagonist protein to relieve inflammatory conditions (for review, see Dinarello (1991) Blood 77: 1627-1652; Dinarello et al. (1993) New England J. Med. 328:106-113; Dinarello (1994) FASEB J. 8:1314-1325; Dinarello (1993) Immunol. Today 14:260-264). Many of the proinflammatory effects of IL-1, such as the upregulation of cell adhesion molecules on vascular endothelia, are exerted at the level of transcriptional regulation. The transcriptional activation by IL-1 of cell adhesion molecules and other genes involved in the inflammatory response appears to be mediated largely by NF-kappa B (Shirakawa et al. (1989) Molc. Cell Biol. 9:2424-2430; Osborn et al., (1989) Proc. Natl. Acad. Sci. USA 86:2336-2340; Krasnow et al., (1991) Cytokine 3:372-379; Collins et al., (1993) Trends Cardiovasc. Med. 3:92-97). In response to IL-1, the NF-kappa B inhibitory factor I kappa B is degraded and NF-kappa B is released from its inactive cytoplasmic state to localize within the nucleus where it binds DNA and activates transcription (Liou et al. (1993) Curr. Opin. Cell Biol. 5:477-487; Beg et al., (1993) Mol. Cell. Bid. 13:3301-3310).

[0009] IL-1 is also a mediator of septic shock. Septic shock, a life-threatening complication of bacterial infections, affects 150,000 to 300,000 patients annually in the United States (Parrillo, J. E. (1989), Septic Shock in Humans: Clinical Evaluation, Pathogenesis, and Therapeutic Approach (2nd ed.) In: Textbook of Critical Care Shoemaker, et al., editors, Saunders Publishing Co., Philadelphia, Pa., pp. 1006). The cardiovascular collapse and multiple metabolic derangements associated with septic shock are due largely to bacterial endotoxin (ET), which has been shown to elicit a septic shock-like condition when administered to animals (Natanson, et al. (1989), Endotoxin and Tumor Necrosis Factor Challenges in Dogs Simulate the Cardiovascular Profile of Human Septic Shock, J. Exp. Med. 169:823). Thus, there is a great need for modulators of IL-1 which may be useful for modulating inflammation and the immune response.

SUMMARY OF THE INVENTION

[0010] The compositions of the present invention include novel isolated polypeptides, in particular, novel human Interleukin-1 Hy2 (IL-1 Hy2) proteins and active variants thereof, isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.

[0011] The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.

[0012] The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. 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: 2, 4 or 13. The isolated polynucleotides of the invention further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of SEQ ID NOS: 1, 12 or 14; a polynucleotide comprising the full length protein coding sequence of SEQ ID NOS: 1, 12 or 14; and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NOS: 1, 12 or 14. The polynucleotides of the present invention also include, but are not limited to, polynucleotides that encode polypeptides with IL-1 Hy2 activity and that hybridize under stringent hybridization conditions to the complement of (a) the nucleotide sequence of SEQ ID NOS: 1, 12 or 14, or (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID 2, 4 or 13; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homologue of any of the proteins recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide having an amino acid sequence of SEQ ID NOS: 2, 4 or 13.

[0013] The polynucleotides of the present invention still further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of the cDNA insert of clone pIL-1Hy2 deposited on May 21, 1999 under Accession No. PTA-96 with the American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, Va., 20110-2209, U.S.A.) or an IL-1 Hy2 protein coding portion thereof, such as the full length protein coding sequence or the mature protein coding sequence.

[0014] The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.

[0015] A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect nucleic acids that are perfectly complementary (full-match) or mismatched to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.

[0016] The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the amino acid sequence of SEQ ID NOS: 2, 4 or 13, or the amino acid sequence encoded by the cDNA insert of clone pIL-1Hy2, or a portion thereof corresponding to the full length or mature protein. Polypeptides of the invention also include polypeptides with IL-1 Hy2 activity that are encoded by (a) polynucleotides encoding SEQ ID NOS: 2 or 13 (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of the IL-1Ra protein sequence of SEQ ID NOS: 2, 4 or 13 and “substantial equivalents” thereof (e.g., with 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity) that retain IL-1 Hy2 activity, preferably IL-1 antagonist activity, are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.

[0017] Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

[0018] The invention also relates to methods for producing polypeptides of the invention comprising growing a culture of the cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein from the cells or the culture medium. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.

[0019] Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.

[0020] In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.

[0021] The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement. Transgenic animals with altered expression of the polypeptides of the invention (i.e. knock out animals or animals overexpressing IL-1 Hy2) are also contemplated.

[0022] Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.

[0023] In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for the prevention and/or treatment of IL-1, IL-18 and/or IL-12 mediated disorders including disorders involving sepsis (and associated conditions such as fever, tachycardia, tachypnea, cytokine overstimulation, increased vascular permeability, hypotension, complement activation, disseminated intravascular coagulation, anemia, thrombocytopenia, leukopenia, pulmonary edema, adult respiratory distress syndrome, intestinal ischemia, renal insufficiency and failure, metabolic acidosis and multiorgan dysfunction syndrome), endotoxic shock, cytokine induced shock, thrombosis, acute pancreatitis, rheumatoid or reactive arthritis, chronic inflammatory arthritis, vasculitis, lupus, immune complex glomerulonephritis, pancreatic cell damage from diabetes mellitus type 1, allograft and xenograft transplantation, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic myelogenous leukemia, ovarian carcinoma, or in the prevention of premature labor secondary to intrauterine infections, bone degenerative diseases such as osteoporosis, and neurodegenerative disorders such as Alzheimer disease.

[0024] Concurrent administration of other agents that inhibit the production or activity of IL-1 (such as GM-CSF, IL-4, IL-10, IL-13 and transforming growth factor-beta) or other anti-inflammatory agents (such as IL-1Ra, IL-1Ra-like IL-1Hy1 proteins described in co-owned, co-pending U.S. application Ser. No. 09/287,210 filed Apr. 5, 1999, incorporated herein by reference, anti-TNF, corticosteroids, immunosuppressive agents) is also contemplated according to the invention.

[0025] The methods of the present invention further relate to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited above and for the identification of subjects exhibiting a predisposition to such conditions. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.

[0026] The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited above. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.

[0027] The methods of the invention also include methods for the treatment of disorders as recited above which may involve the administration of such compounds to individuals exhibiting symptoms or tendencies related to disorders as recited above. In addition, the invention encompasses methods for treating diseases or disorders as recited above by administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene expression or target protein activity.

[0028] The invention further provides a method of treating an inflammatory disease state mediated by IL-18 comprising administering to a subject in need thereof an amount of an IL-1 Hy2 polynucleotide, polypeptide or agonist effective to inhibit IL-18 activity. Also provided are in vitro and in vivo methods of inhibiting IL-18 activity.

[0029] Three-dimensional modeling data has suggested that the predicted three-dimensional structure of IL-1 Hy2 closely resembles the three-dimensional structure of IL-1β. This data indicates that IL-1 Hy2 may function as a low affinity agonist to the IL-1 receptor in the absence of accessory protein. Therefore, IL-1 Hy2 may induce pro-inflammatory physiological effects similar to IL-1β and plays a role in enhancing inflammation related pathological conditions. On the other hand, experimental results indicate that IL-1 Hy2 is an antagonist of the IL-1 receptor and this is supported by the presence of Lys145 which is an important residue for biological activity.

[0030] The invention provides for antagonists, agonists and modulators of IL-1 Hy2, such as antibodies, antisense oligonucleotides, small molecules, peptides and derivatives thereof which reduce IL-1 Hy2 binding interactions with or activation of the IL-1 receptor. The invention also provides for methods of screening for antagonists and modulators of IL-1 Hy2 and methods of treating pathological conditions associated with inflammation by administering IL-1 Hy2 antagonists or modulators thereof.

[0031] The predicted three-dimensional structure of IL-1 Hy2 recited herein provides a basis for rationally designing IL-1 Hy2 modulators (such as antagonists and agonists) which specifically associate with the amino acids predicted to interact with a receptor such as IL-1 receptor. Such residues include Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105, Tyr147 and other amino acids of SEQ ID NO: 2 within about 2-12 Å, preferably within 7 Å, and more preferably within 5 Å, that may interact with these amino acids and/or contribute to the three-dimensional conformation of the receptor binding residues. In addition, the predicted three-dimensional structure will allow for the creation of IL-1 Hy2 polypeptide mutants that have similar, increased, decreased or different biological activity compared to wild type IL-1Hy2.

[0032] Therefore, the invention provides for a polypeptide comprising an amino acid sequence comprising two or more receptor binding residues substantially defined by structural coordinates of amino acids Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105 and Tyr147 of SEQ ID NO: 2 as set forth in Tables II or III, and said polypeptide capable of binding IL-1 receptor. These polypeptides include those which have a root mean squared deviation from the structural coordinates set forth in Tables II or III within 2-12 Å, preferably within 7 Å, or more preferably within 5 Å, those which have an basic residue at the position corresponding to 145 of SEQ ID NO: 2, such as arginine, lysine, and histidine. These polypeptides also include those that are less than 95% identical, more preferably less than 85% identical over the entire length of S ID NO: 2. The invention also provides for polypeptides that comprises two or more receptor binding residues substantially defined by structural coordinates of amino acids Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, , Ser105 and Tyr147 of SEQ ID NO: 2 as set forth in Tables II or III, and said polypeptide capable of binding IL-1 receptor but the portion outside of the IL-1 receptor binding region has a three-dimensional conformation substantially different from that of IL-1 Hy2 of SEQ ID NO: 2. The invention also provides for a method of treating a pathological condition, such as psoriasis, characterized by aberrant expression or activity of IL-1 receptor comprising administering to a patient a therapeutically effective amount any one of these polypeptides.

[0033] The invention further provides for an IL-1 Hy2 polypeptide variant comprising at least one modification wherein an amino acid residue selected from the group consisting of Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55,Gly92, Gly93, Glnl103, Ser105, Lys145 and Tyr147 is replaced with a different amino acid, and wherein said IL-1 Hy2 polypeptide variant exhibits increased or deceased binding to IL-1 receptor compared to IL-1 Hy2 of SEQ ID NO: 2. The modification contemplates replacing at least one amino acid with a conservative substitution.

[0034] Another embodiment of this invention provides a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional structure corresponding to IL-Hy1 (including IL-1 Hy2 variants), particularly as defined herein with reference to receptor binding residues, accessory protein binding residues and other residues important to IL-1 Hy2 biological function. For example, the machine readable storage medium includes a three-dimensional representation which is substantially defined by the structural coordinates of amino acids Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105, Lys145 and Tyr147 of SEQ ID NO: 2 as set forth in Tables II or III.

[0035] The invention provides for a computer comprising memory containing the three-dimensional representation of IL-1 Hy2 or a portion of IL-1 Hy2 that includes the IL-1 receptor binding regions of IL-1 Hy2. These computers include those comprising memory of a three-dimensional representation that is substantially defined by structural coordinates of IL-1 Hy2 amino acids Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105 and Tyr147 of SEQ ID NO: 2 as set forth in Tables II or III or those wherein the IL-1 receptor binding region has a root mean square deviation from the structural coordinates set forth in Tables II or III of amino acids Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105 and Tyr147 within 2-12 Å, preferably within 7 Å, or more preferably within 5 Å. The invention also provides for a computer comprising a computer readable storage medium, which is a data storage material coded with machine readable data, wherein said data includes the three-dimensional representation of IL-1 Hy2 or a portion of IL-1 Hy2 that includes the IL-1 receptor binding regions of IL-1 Hy2 described herein.

[0036] Another embodiment of the invention provides for methods of identifying potential modulators of IL-1 Hy2 biological activity using a three-dimensional structure of IL-1 Hy2 substantially defined by the structural coordinates of two or more IL-1 Hy2 (SEQ ID NO: 2) amino acids Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105 and Tyr147 as set forth in Tables II or III to design or select potential modulators and contacting said modulators with IL-1 Hy2 in the presence of IL-1 receptor to test the ability of said potential modulator to modulate the interaction between IL-1 Hy2 and IL-1 receptor. These methods include selecting modulators using a computer for interaction with the three-dimensional structure of IL-1 Hy2. These methods also include contacting said potential modulator with an IL-1 Hy2 mutant, which exhibits reduced binding to IL-1 receptor compared to wild type IL-1 Hy2 (SEQ ID NO: 2), to test the ability of the modulator to modulate the interaction between the IL-1 Hy2 mutant and IL-1 receptor. These methods will includes the use of mutants which comprise at least one modification wherein an amino acid residue selected from the group consisting of Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105 and Tyr147 is replaced with a different amino acid, and wherein said IL-1 Hy2 polypeptide variant exhibits decreased binding to IL-1 receptor compared to IL-1 Hy2 of SEQ ID NO: 2.

[0037] The invention provides for methods of treating pathological condition characterized by aberrant expression or activity of IL-1 Hy2, comprising administering to a patient a therapeutically effective amount of a non-peptidyl compound that is a biological modulator of IL-1 Hy2 interaction with IL-1 receptor, said compound containing one or more moieties that mimic one or more of the IL-1 Hy2 amino acids of SEQ ID NO: 2 selected from the group consisting of Met 6, Arg 8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105, Lys145 and Tyr147 and as set forth in Tables II or III.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIGS. 1A-1B show an alignment of the amino acid sequence of IL-1 Hy2 (SEQ ID NO. 2) with the sequences of IL-1 Hy1 (described in co-owned, co-pending U.S. Ser. No. 09/287,210 filed Apr. 5, 1999), rat IL-1Ra, pig IL-1Ra, secreted human IL-1Ra (Hu sIL-1Ra) and intracellular human IL-1Ra (Hu icIL-1Ra), SEQ ID NOS: 5-9, respectively. In these figures, A—Alanine; R—Arginine; N—Asparagine; D—Aspartic Acid; C-Cysteine; E—Glutamic Acid; Q—Glutamine; G—Glycine; H—Histidine; I—Isoleucine; L—Leucine; K-Lysine; M—Methionine; F—Phenylalanine; P—Proline; S—Serine; T—Threonine; W—Tryptophan; Y—Tyrosine; V—Valine; X—any of the twenty amino acids. Gaps are presented as dashes. Amino acid numbers for all sequences are labelled accordingly. Boxed residues indicate consensus or conserved sequence.

[0039]
FIG. 2 sets forth SEQ ID NO: 12 which represents the predicted cDNA sequence based on the human genomic sequence of IL-1 Hy2.

[0040]
FIG. 3 sets forth SEQ ID NO: 13 which represents the human amino acid sequence encoded by the longer open reading frame of SEQ ID NO: 12 which is an alternative form of the IL-1 Hy2 polypeptide.

[0041]
FIG. 4 sets forth SEQ ID NO: 14 which represents the cDNA sequence of human IL-1 Hy2 clones which extends the 5′ region of SEQ ID NO: 1.

[0042]
FIG. 5 shows ribbon diagrams of the IL-1 Hy2 predicted three-dimensional structural model superimposed with the IL-1 Ra (top panel) average NMR structure and IL-1 β (low panel) crystal structure. This figure demonstrates that IL-1 Hy2 is more structurally similar to IL-1β than IL-1 Ra.

[0043] FIG. 6 shows the alignment based on secondary structure of the amino acid sequences of IL-1 Ra and IL-1 Hy2 and indicates the residues involved in receptor interaction and critical function. An amino acid symbol between the two sequences indicates identity and “+” indicates similarity. The amino acids in bold are within the receptor binding region. A critical function amino acid is underlined. The arrows indicate the location of the β-strands within the three-dimensional structure.

[0044]
FIG. 7 shows the alignment based on secondary structure of the amino acid sequences of IL-1 β and IL-1 Hy2 and indicates the residues involved in receptor interaction and function. An amino acid symbol between the two sequences indicates identity and “+” indicates similarity. The amino acids in bold are within the receptor binding region. A critical function amino acid is underlined. The arrows indicate the location of the β-strands within the three-dimensional structure.

DETAILED DESCRIPTION OF THE INVENTION

[0045] 1. Definitions

[0046] The term “nucleotide sequence” refers to a heteropolymer of nucleotides or the sequence of these nucleotides. The terms “nucleic acid” and “polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides. Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.

[0047] The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” is a stretch of polypeptide nucleotide residues which is long enough to use in polymerase chain reaction (PCR) or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules.

[0048] The terms “oligonucleotides” or “nucleic acid probes” are prepared based on the polynucleotide sequences provided in the present invention. Oligonucleotides comprise portions of such a polynucleotide sequence having at least about 15 nucleotides and usually at least about 20 nucleotides. Nucleic acid probes comprise portions of such a polynucleotide sequence having fewer nucleotides than about 6 kb, usually fewer than about 1 kb. After appropriate testing to eliminate false positives, these probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).

[0049] The term “probes” includes naturally occurring or recombinant or chemically synthesized single- or double-stranded nucleic acids. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.

[0050] The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA under in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).

[0051] In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

[0052] The term “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. As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli , will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.

[0053] The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0054] The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers. Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cells can be prokaryotic or eukaryotic.

[0055] The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.

[0056] The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.

[0057] As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are fragments which induce the expression or an operably linked ORF in response to a specific regulatory factor or physiological event.

[0058] As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.

[0059] The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.

[0060] The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide. According to the invention, the term “biologically active” with reference to IL-1 Hy2 means that the polypeptide retains at least one of the biological activities, preferably the IL-1 antagonist activity, of human IL-1 Hy2, while the term “immunologically active” with reference to IL-1 Hy2 means that the polypeptide retains at least one of the immunologic or antigenic activities of human IL-1 Hy2.

[0061] The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.

[0062] The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.

[0063] The term “variant” (or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, such as IL-1 antagonistic activity, may be found by comparing the sequence of the particular polypeptide with that of homologous human or other mammalian peptides e.g. IL-1Ra, IL-1Hy1, or IL-1, and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.

[0064] Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

[0065] Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.

[0066] As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 20% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.2 or less). Such a sequence is said to have 80% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 10% (90% sequence identity); in a variation of this embodiment, by no more than 5% (95% sequence identity); and in a further variation of this embodiment, by no more than 2% (98% sequence identity). Substantially equivalent, e.g., mutant, amino acid sequences according to the invention generally have at least 95% sequence identity with a listed amino acid sequence, whereas substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded. Sequence identity may be determined, e.g., using the Jotun Hein method.

[0067] Nucleic acid sequences encoding such substantially equivalent sequences, e.g., sequences of the recited percent identities, can routinely be isolated and identified via standard hybridization procedures well known to those of skill in the art.

[0068] Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.

[0069] A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids. To be active, any polypeptide must have sufficient length to display biologic and/or immunologic activity.

[0070] Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.

[0071] The term “activated” cells as used in this application are those which are engaged in extracellular or intracellular membrane trafficking, including the export of neurosecretory or enzymatic molecules as part of a normal or disease process.

[0072] The term “purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99.8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).

[0073] The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.

[0074] The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.

[0075] The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extra chromosomal element, or by chromosomal integration.

[0076] The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.

[0077] The term “intermediate fragment” means a nucleic acid between 5 and 1000 bases in length, and preferably between 10 and 40 bp in length.

[0078] The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell. “Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. “Secreted” proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum. “Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2): 134-143) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al. (1998) Annu. Rev. Immunol. 16:27-55)

[0079] Each of the above terms is meant to encompasses all that is described for each, unless the context dictates otherwise.

NUCLEIC ACIDS AND POLYPEPTIDES OF THE INVENTION

[0080] Nucleotide and amino acid sequences of the invention are reported below. Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through “linker” sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein-IgM fusion would generate a decavalent form of the protein of the invention.

[0081] The present invention also provides both full-length and mature forms (for example, without a signal sequence or precusor sequence) of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form. Where protein of the present invention is membrane bound, soluble forms of the protein are also provided. In such forms part or all of the regions causing the protein to be membrane bound are deleted so that the protein is fully secreted from the cell in which it is expressed.

[0082] The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides. The compositions of the present invention include isolated polynucleotides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, novel isolated polypeptides, and antibodies that specifically recognize one or more epitopes present on such polypeptides. Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.

[0083] Description of Sequences

[0084] SEQ ID NO: 1 sets forth one preferred nucleotide sequence of IL-1 Hy2 which contains a protein coding region from nucleotides 54 through 509.

[0085] SEQ ID NO: 2 sets forth an amino acid sequence encoded by SEQ ID NO: 1.

[0086] SEQ ID NO: 3 sets forth a nucleotide sequence identical to SEQ ID NO: 1 except the protein coding region spans nucleotides 3 through 509.

[0087] SEQ ID NO: 4 sets forth the amino acid sequence encoded by SEQ ID NO: 3.

[0088] SEQ ID NOS: 5-9 set forth the amino acid sequences of human IL-1 Hy1. rat IL-1Ra, pig IL-1Ra, human IL-1Ra and intracellular human IL-1Ra, respectively.

[0089] SEQ ID NO: 14 sets forth an extended cDNA sequence of a human IL-1 Hy2 clone which is longer than SEQ ID NO: 1 but encodes the same 152 amino acid polypeptide (SEQ ID NO: 2).

[0090] SEQ ID NO: 15 sets forth the genomic DNA sequence of human IL-1 Hy2.

[0091] SEQ ID NO: 12 sets forth the predicted cDNA sequence based on the human genomic sequence of IL-Hy2.(SEQ ID NO: 15) and differs from SEQ ID NO: 14 at position 279 (C→T).

[0092] SEQ ID NO: 13 sets forth the 200 amino acid sequence encoded by the longer open reading frame of SEQ ID NO: 12.

[0093] SEQ ID NO: 16 sets forth the genomic DNA sequence of murine IL-1 Hy2.

[0094] SEQ ID NO: 17 sets forth the predicted murine cDNA sequence based on the mouse genomic sequence of SEQ ID NO: 16.

[0095] SEQ ID NO: 18 sets forth the deduced amino acid sequence of murine IL-1 Hy2 polypeptide.

[0096] 2. Nucleic Acids of the Invention

[0097] 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: 2, 4 or 13. A preferred nucleic acid sequence is set forth in SEQ ID NO: 1 (which is identical to SEQ ID NO: 3 except for the identification of the protein coding region, which is nucleotides 54 through 509 for SEQ ID NO: 1 and nucleotides 3 through 509 for SEQ ID NO: 3).

[0098] There are two alternative open reading frames in SEQ ID NO: 1. Resequencing of the 5′ region of the IL-1 Hy2 cDNA resulted in SEQ ID NO: 14 which includes the shorter open reading frame of SEQ ID NO: 1 and extends its 5′ sequence. The predicted amino acid sequence based upon the shorter open reading frame of SEQ ID NO: 14 is shown in SEQ ID NO: 2. The predicted cDNA sequence based on the genomic DNA sequence is set forth as SEQ ID NO: 12, which contains a C→T change that results in an alternative upstream initiating methionine which extends the open reading frame of SEQ ID NO: 3. The predicted amino acid sequence based on the longer open reading frame is shown in SEQ ID NO: 13.

[0099] The isolated polynucleotides of the invention further include, but are not limited to a polynucleotide comprising the nucleotide sequence of SEQ ID NOS: 1, 12 or 14; a polynucleotide comprising the full length protein coding sequence of SEQ ID NOS: 1, 12 or 14; and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NOS: 1, 12 or 14. The polynucleotides of the present invention also include, but are not limited to, polynucleotides that encode polypeptides with IL-1 Hy2 activity and that hybridize under stringent hybridization conditions to the complement of either (a) the nucleotide sequence of SEQ ID NOS: 1, 12 or 14, or (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 2, 4 or 13; a polynucleotide which is an allelic variant of any polynucleotide recited above; a polynucleotide which encodes a species homologue of any of the proteins recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide of SEQ ID NOS: 2, 4 or 13.

[0100] The polynucleotides of the present invention still further include, but are not limited to, a polynucleotide comprising the nucleotide sequence of the cDNA insert of clone pIL-1Hy2 or an IL-1 Hy2 protein coding portion thereof, such as the full length protein coding sequence or the mature protein coding sequence.

[0101] The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.

[0102] The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have at least about 65%, more typically at least about 70%, 75%, 80%, 85% or 90%, and even more typically at least about 95%, sequence identity to a polynucleotide recited above. The invention also provides the complement of the polynucleotides including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities.

[0103] 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. In general, 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.

[0104] 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. Allelic variations can be routinely determined by comparing the sequence provided in SEQ ID NOS: 1, 12 or 14, or a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NOS: 1, 12 or 14 with a sequence from another isolate of the same species. Example 2 shows that several allelic variants exist, some of which result in changes in the encoded polypeptide sequence.

[0105] To accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another which encodes the same amino acid is expressly contemplated. Any specific sequence disclosed herein can be readily screened for errors by resequencing a particular fragment, such as an ORF, in both directions (i.e., sequence both strands).

[0106] The present invention further provides recombinant constructs comprising a nucleic acid having the sequence of SEQ ID NOS: 1, 12 or 14; or a fragment thereof or any other polynucleotides of the invention. In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having the sequence of SEQ ID NOS: 1, 12 or 14; or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. For vectors comprising the EMFs and UMFs of the present invention, the vector may further comprise a marker sequence or heterologous ORF operably linked to the EMF or UMF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

[0107] The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

[0108] Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lac,I, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. 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. Optionally, 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. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

[0109] As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0110] Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequences that hybridize under stringent conditions to a fragment of the DNA sequence of SEQ ID NOS: 1, 12 or 14, which fragment is greater than about 10 bp, preferably 20-50 bp, and even greater than 100 bp. In accordance with the invention, polynucleotide sequences which encode the novel nucleic acids, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.

[0111] The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation. The amino acid sequence variants of the nucleic acids are preferably constructed by mutating the polynucleotide to give an amino acid sequence that does not occur in nature. These amino acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues. Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells.

[0112] In a preferred method, polynucleotides encoding the novel nucleic acids are changed via site-directed mutagenesis. This method uses oligonucleotide sequences that encode the polynucleotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983). A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasmid and this gives the desired amino acid variant.

[0113] A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.

[0114] Furthermore, knowledge of the DNA sequence provided by the present invention allows for the modification of cells to permit, or increase, expression of endogenous IL-1 Hy2 polypeptides. Cells can be modified (e.g., by homologous recombination) to provide increased IL-1 Hy2 expression by replacing, in whole or in part, the naturally occurring IL-1 Hy2 promoter with all or part of a heterologous promoter so that the cells express IL-1 Hy2 polypeptides at a higher level. The heterologous promoter is inserted in such a manner that it is operatively linked to IL-1 Hy2 encoding sequences. See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO92/20808, and PCT International Publication No. WO91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the IL-1 Hy2 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the IL-1 Hy2 coding sequences in the cells.

[0115] The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express functional IL-1 Hy2 polypeptides or that express a variant of a IL-1 Hy2 polypeptide. Such animals are useful as models for studying the in vivo activities of IL-1 Hy2 polypeptides as well as for studying modulators of IL-1 Hy2 polypeptides.

[0116] Polynucleotides of the invention can also be used to induce immune responses. For example, as described in Fan et al., Nat. Biotech. 17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA. The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.

[0117] 3. Hosts

[0118] The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention. For example, such 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.

[0119] The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)). The host cells containing one of polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.

[0120] Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.

[0121] Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. 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.

[0122] A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

[0123] Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

[0124] In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals. mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

[0125] The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

[0126] The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/,US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

[0127] 4. Polypeptides of the Invention

[0128] SEQ ID NO. 1 encodes the IL-1 Hy2 polypeptide sequence of SEQ ID NOS: 2, 4 and 13. An amino acid alignment of SEQ ID NO. 2 with human secreted IL-1Ra, human intracellular IL-1Ra and human IL-1Hy1, as well as rat and pig IL-1Ra, is shown in FIG. 1. SEQ ID NO. 2 displays significant amino acid homology with human IL-1Ra and IL-1 Hy1 (41.4% and 45% sequence identity, respectively, using the Jotan Hein method), and thus represents a novel molecule within the IL-1Ra family. The sequence similarities among the three proteins and the localization of the IL-1 Hy2 gene to chromosome 2, where other proteins of the IL-1 system are located, indicate that IL-1 Hy2 is involved in the IL-1 system and may play some common biological roles as IL-1Ra and IL-1Hy1, e.g., acting as an IL-1 antagonist. Additional IL-1Hy2 family members can be identified using SEQ ID NOS: 1, 12 or 14 as a molecular probe.

[0129] Interleukin-1 has pleiotropic biological activities many of which adversely affect the organism, it would be expected that the molecule must be tightly regulated if it is not injurious. Indeed, there are several reports of Interleukin-1 inhibitors that regulate the action of Interleukin-1. Interleukin-1 inhibitory activity has been reported in monocyte conditioned medium, wherein the monocytes are grown on adherent immune complexes. Arena, W. P., et al., 1985, Journal of Immun., 134:3868. Additionally, an inhibitor has been reported to be present urine. Seckinger, P., et al., 1987, Journal of Immun., 139:1546. Lastly, a protein inhibitor, purified and cloned, that has interleukin-1 receptor antagonist activity has been reported. Hannum, et at., 1990, Nature, 343:336, and Eisenberg, S., et al., 1990, Nature, 343:341.

[0130] It is thought that the Interleukin-1 inhibitor present in urine, and which has been partially purified and characterized by Seckinger, P. et al., and Seckinger, P., et al., 1987, Journal of Immun., 139:1541 is similar, if not identical to the cloned Interleukin-1 receptor antagonist reported by Eisenberg, S., et al. (1990), Nature, 343:341; and Carter, D., et al (1990), Nature, 344:633.

[0131] Interleukin-1 receptor antagonist is a naturally occurring peptide secreted by macrophages in response to many of the same stimuli which cause the secretion of Interleukin-1 itself. Interleukin-1 receptor antagonist is a naturally occurring antagonist to the cytokines and recognizes receptors on various cell types and blocks Interleukin-1 mediated responses by occupying the receptor. (Wakabayashi et al., FASEB J 1991;5:338; Okusawa et al. J Clin Invest 1988;81:1162; Ohlsson et al., Nature 1990;348:550; Aiura, et al. Cytokine 1991;4:498; Fischer et al. Am J Physiol 1991;261:R442). In humans, Interleukin-1 receptor antagonist is a naturally occurring group of molecules; three forms have been characterized (two glycosylated and one non-glycosylated).

[0132] Fischer et al. (Am J Physiol 1991;261:R442) demonstrated that the administration of a naturally occurring antagonist to Interleukin-1 will significantly blunt the cytokine cascade and improve survival in baboons given a lethal dose of live bacteria. Interleukin-1 receptor antagonist significantly attenuates the decrease in mean arterial pressure and cardiac output and improves survival for severe acute pancreatitis. (U.S. Pat. No. 5,508,262) The systemic Interleukin-1 response observed as a result of bacterial sepsis was also diminished significantly, correlating with a decrease in the systemic response to bacterial sepsis.

[0133] Studies by Aiura et al. (Cytokine 1991;4:498) have shown that Interleukin-1 receptor antagonist is protective in a rabbit model of hypotensive gram-positive septic shock. The administration of Interleukin-1 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-1 and the protective role of Interleukin-1 receptor antagonist in gram-positive septic shock. Interleukin-1 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).

[0134] The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising the amino acid sequence of SEQ ID NOS: 2, 4, or 13, or the amino acid sequence encoded by the cDNA insert of clone pIL-1Hy2, or a portion thereof corresponding to the full length or mature protein. Polypeptides of the invention also include polypeptides with IL-1 Hy2 activity that are encoded by (a) the polynucleotide of SEQ ID NOS: 1, 12 or 14, or (b) polynucleotides encoding SEQ ID NOS: 2, 4, or 13 (b) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions. Biologically active or immunologically active variants of the IL-1Ra protein sequence of SEQ ID NOS: 2, 4 or 13 and “substantial equivalents” thereof (e.g., with 65%, 70%, 75%, 80%, 85%, 90%, typically 95%, more typically 98% or most typically 99% amino acid identity) that retain IL-1 Hy2 activity, preferably IL-1 antagonist activity, are also contemplated. Polypeptides encoded by allelic variants, such as those described in Example 2 below, may have a similar or increased or decreased activity compared to the polypeptides of SEQ ID NOS: 2, 4 or 13.

[0135] Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

[0136] The invention also relates to methods for producing a polypeptide comprising growing a culture of the cells of the invention in a suitable culture medium, and purifying the protein from the culture. For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide. The polypeptide can be recovered from the culture, conveniently from the culture medium, and further purified. Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.

[0137] The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins. A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. This is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.

[0138] The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention. The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.

[0139] In addition, the binding molecules may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NOS: 2, 4 or 13.

[0140] The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.

[0141] The protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

[0142] The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.

[0143] Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.

[0144] The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MAXBAT™. kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.”

[0145] The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, HEPARIN-TOYOPEARL™ or CIBACROM BLUE 3GA SEPHAROSE™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

[0146] Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and In Vitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (“Flag”) is commercially available from Kodak (New Haven, Conn.).

[0147] Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.”

[0148] The polypeptides of the invention include Interleukin-1 Hy2 analogs or variants. This embraces fragments of IL-1 Hy2 of the invention, as well as analogs (variants) of IL-1 Hy2 in which one or more amino acids has been deleted, inserted, or substituted. Analogs of the invention also embrace fusions or modifications of IL-1 Hy2 wherein the IL-1 Hy2 or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability. Examples of moieties which may be fused to IL-1 Hy2 or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells. Other moieties which may be fused to IL-1 Hy2 or an analog include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids, or immunostimulants, immune modulators, and other cytokines such as alpha or beta interferon.

[0149] 5. Deposit of Clone

[0150] The following clone, pIL-1Hy2 was deposited with the American Type Culture Collection (ATCC) 10801 University Avenue, Manassas, Va., on May 21, 1999 under the terms of the Budapest Treaty. The clone represents a plasmid clone as described in the Examples set forth below.

1Microorganism/CloneATCC Accession No.pIL-1Hy2PTA-96

[0151] 6. Uses and Biological Activity

[0152] The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).

[0153] 6.1. Research Uses and Utilities

[0154] The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

[0155] The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

[0156] Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

[0157] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0158] 6.2. Nutritional Uses

[0159] Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

[0160] 6.3. Cytokine and Cell Proliferation/Differentiation Activity

[0161] A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a protein of the invention may, among other means, be measured by the following methods:

[0162] Assays for T-cell or thymocyte proliferation 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-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761, 1994.

[0163] Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin .gamma., Schreiber, R. D. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

[0164] Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

[0165] Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) 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-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. ,Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

[0166] 6.4. Immune Stimulating or Suppressing Activity

[0167] A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A polynucleotide of the invention can encode a polypeptide exhibiting such activities. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer. IL-1 has been indicated to promote tumor cell growth in cancers of various organs including breast adenocarcinoma, brain tumors, melanoma, myeloma, giant cell tumors of bone, acute myelogenous leukemia, oral epidermoid carcinoma, and squamous cell carcinoma; thus treatment of such cancer disease states involving elevated levels of IL-1 with IL-1 Hy2 polypeptides of the present invention is expected to ameliorate signs and symptoms of cancer.

[0168] Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein (or antagonists thereof, including antibodies) of the present invention may also be useful in the treatment of allergic reactions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies) and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention. The therapeutic effects of IL-1 Hy2 polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).

[0169] Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.

[0170] Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

[0171] The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.

[0172] Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

[0173] Upregulation of an antigen (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.

[0174] Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.

[0175] The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I α chain protein and β2 microglobulin protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

[0176] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0177] Suitable assays for thymocyte or splenocyte cytotoxicity 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-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

[0178] Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

[0179] Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) 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-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

[0180] Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.

[0181] Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.

[0182] Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

[0183] 6.5. Hematopoiesis Regulating Activity

[0184] A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of 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 transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.

[0185] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0186] Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

[0187] Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

[0188] Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

[0189] 6.6. Tissue Growth Activity

[0190] 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 bums, incisions and ulcers.

[0191] A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.

[0192] A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.

[0193] Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

[0194] The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.

[0195] Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

[0196] It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.

[0197] A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

[0198] A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.

[0199] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0200] Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium).

[0201] Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

[0202] 6.7. Activin/Inhibin Activity

[0203] A protein of the present invention may also exhibit activin- or inhibin-related activities. A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin α-family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, 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.

[0204] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0205] Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.

[0206] 6.8. Chemptatic/Chemokinetic Activity

[0207] A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

[0208] A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.

[0209] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0210] Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28); Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.

[0211] 6.9. Hemostatic and Thrombolytic Activity

[0212] 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).

[0213] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0214] Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.

[0215] 6.10. Receptor/Ligand Activity

[0216] 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). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.

[0217] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0218] 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-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. ,Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

[0219] By way of example, the IL-1 Hy2 polypeptides of the invention may be used as a ligand for a cytokine receptor thereby modulating (i.e., enhancing or inhibiting) the biological activity of that receptor. Examples of cytokine receptors that may be used include, but are not limited to, the Interleukin-1 Type I or Type II Receptors. Whether the IL-1 Hy2 polypeptides of the invention exhibit agonist, partial agonist, antagonist, or partial antagonist activity for a particular receptor, such as a cytokine receptor, in a particular cell type can be determined by conventional techniques known to those skilled in the art, such as those described below in sections 6.11.1 and 6.11.2 and in the Examples below. In one embodiment, one or more cells expressing a cytokine receptor (e.g., Interleukin-1 Type I or Type II Receptors) are contacted with the protein of the invention. Examples of cells that may be contacted with the protein of the invention include, but are not limited to, mammalian cells such as fibroblasts and T-cells. Preferably the novel protein of the invention acts as an antagonist for a cytokine receptor (e.g.-the Interleukin-I Receptor) so that the biological activities of that receptor are inhibited.

[0220] Studies characterizing drugs or proteins as agonist or antagonist or partial agonists a partial antagonist require the use of other proteins as competing ligands. The polypeptides of the present invention are expected to exhibit an affinity for Interleukin-1 Receptor. Thus, the polypeptides of the present invention may be used, for example, as competitors in assays involving Interleukin-1 Receptors. Alternatively, the polypeptides of the invention may be labelled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods. (“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990) Academic Press, Inc. San Diego) and used in both in vivo and in vitro to bind to the Interleukin-1 Receptor. Examples of radioisotopes include, but are not limited to, tritium and carbon-14 . Examples of calorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other calorimetric molecules. Examples of toxins include, but are not limited, to ricin. By way of example, the proteins coupled to such molecules are useful in studies involving in vivo or in vitro metabolism of the Interleukin-1 Receptor.

[0221] 6.11 Drug Screening with Interleukin-1 Hy2 Polypeptides

[0222] This invention is particularly useful for screening compounds by using the IL-1 Hy2 polypeptides of the invention, particularly binding fragments, in any of a variety of drug screening techniques. The polypeptides employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the desired IL-1 Hy2 polypeptide. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between IL-1 Hy2 polypeptides of the invention and the agent being tested or examine the diminution in complex formation between the IL-1 Hy2 polypeptides and an appropriate cell line, which are well known in the art.

[0223] 6.11.1 Assay for Anti-Interleukin-1 Receptor Activity

[0224] In one embodiment, the Interleukin-1 receptor antagonist activity of the polypeptides of the invention is determined using a method that involve (1) forming a mixture comprising Interleukin-1, the Interleukin-1 receptor, and the IL-1 Hy2 polypeptides of the invention and/or its agonists and antagonists (or agonist or antagonist drug candidates) and/or antibodies specific for the IL-1 Hy2 polypeptides of the invention; (2) incubating the mixture under conditions whereby, but for the presence of said IL-1 Hy2 polypeptide of the invention and/or its agonists and antagonists (or agonist or antagonist drug candidates) and/or antibodies specific for the IL-1 Hy2 polypeptides of the invention, the Interleukin-1 binds to the Interleukin-1 receptor; and (3) detecting the presence or absence of specific binding of Interleukin-1 to the Interleukin-1 receptor.

[0225] 6.11.2 Assay for Anatagonists and Agonists

[0226] Human HepG2 cells are incubated at 37 degree(s) C. for 18-24 hours in serum-free Dulbecco's modified Eagle medium. Separate monolayers of cells are incubated in the same medium supplemented with Interleukin-1 at various concentrations and in the same medium supplemented with a IL-1 Hy2 polypeptide of the invention at various concentrations.

[0227] Monolayers are rinsed vigorously with isotonic buffer and incubated in (35-S) methionine, 250 mu ci/ml methionine-free medium and pulsed for a period of 15-30 minutes to assess net synthesis. Cell culture fluid is discarded and monolayers are again rinsed and resuspended in cell lysis buffer. The newly synthesized radiolabelled hepatic proteins in these cell lysates are detected by immunoprecipitation, SDS-PAGE and fluorography.

[0228] 6.12. Anti-inflammatory Activity

[0229] Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material. In particular, the IL-1 Hy2 polypeptides of this invention may be utilized to prevent or treat condition such as, but not limited to, 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 inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.

[0230] 6.13 Modulation of IL-18, IL-12 and IFN-γ Related Disorders

[0231] Administration of IL-1Hy2 polynucleotides, polypeptides and agonists is also expected to be useful for the treatment of IL-18 and/or IL-12 and/or IFN-γ related disorders. IL-1Hy2 inhibits IL-18 and IL-12 activity, including IL-18 and IL-12 induced IFN-γ production.

[0232] IL-18 has been found to have a variety of biological activities including the stimulation of activated T cell proliferation, enhancement of NK cell lytic activity, induction of IFNγ secretion, enhancement of Fas ligand expression and function, and stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) production by activated T cells. IL-18 has been shown to counteract viral and intracellular infections and suppress tumor formation. However, IL-18 is also involved in the pathogenic progression of chronic inflammatory diseases, including endotoxin-induced shock, liver injury (including endotoxin-induced liver injury, hepatitis, biliary atresia and obesity-related fatty liver) and autoimmune diseases. Other disorders related to IL-18 production include meliodosis, purine nucleoside phosphorylase deficiency, increased susceptibility to Leishmania major and Staphylococcus aureus infection, hemophagocytic lymphohistiocytosis, mononucleosis, viral meningitis/encephalitis, bacterial meningitis/encephalitis and ischemia or ischemia/reperfusion injury.

[0233] Inflammation may result from infection with pathogenic organisms (including gram-positive bacteria, gram-negative bacteria, viruses, fungi, and parasites such as protozoa and helminths), transplant rejection (including rejection of solid organs such as kidney, liver, heart, lung or cornea, as well as rejection of bone marrow transplants including graft versus host disease (GVHD)), or from localized chronic or acute autoimmune or allergic reactions. Autoimmune diseases include acute glomerulonephritis; rheumatoid or reactive arthritis; chronic glomerulonephritis; inflammatory bowel diseases such as Crohn's disease, ulcerative colitis and necrotizing enterocolitis; granulocyte transfusion associated syndromes; inflammatory dermatoses such as contact dermatitis, atopic dermatitis, psoriasis; systemic lupus erythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, some forms of diabetes, or any other autoimmune state where attack by the subject's own immune system results in pathologic tissue destruction. Allergic reactions include allergic asthma, chronic bronchitis, allergic rhinitis, acute and delayed hypersensitivity. Systemic inflammatory disease states include inflammation associated with trauma, burns, reperfusion following ischemic events (e.g. thrombotic events in heart, brain, intestines or peripheral vasculature, including myocardial infarction and stroke), sepsis, ARDS or multiple organ dysfunction syndrome. Inflammatory cell recruitment also occurs in atherosclerotic plaques.

[0234] Endotoxin activation of the systemic inflammatory response leads to a number of disorders including bacterial and/or endotoxin-related shock, fever, tachycardia, tachypnea, cytokine overstimulation, increased vascular permeability, hypotension, complement activation, disseminated intravascular coagulation, anemia, thrombocytopenia, leukopenia, pulmonary edema, adult respiratory distress syndrome, intestinal ischemia, renal insufficiency and failure, and metabolic acidosis.

[0235] Hepatitis represents liver disorders that are characterized by hepatic inflammation and necrosis that can be manifested as an acute or chronic condition. These liver disorders include virus-induced hepatitis such as hepatitis A, hepatitis B, hepatitis C (non-A, non-B hepatitis), hepatitis D, hepatitis E; toxin and drug induced hepatitis such as acetaminophohen hepatotoxicity, halothane hepatotoxicity, mehtyldopa hepatoxicity, iaoniazid hepatoxicity, sodium valproate hepatoxicity, phenytion hepatoxicity, chlorpromazine hepatoxicity, amiodarone hepatoxicity, amioidarone hepatoxicity, erythromycin hepatoxicity, oral contraceptive hepatoxicity, 17, α-alkyl-substituted anabolic steroid hepatoxicity and trimethoprim-sulfamethoxazole hepatoxicity; cholestatic hepatitis; alcoholic hepatitis; autoimmune chronic active hepatitis; and T cell mediated hepatitis. Other conditions that cause liver injury include congenital bilary atresia, obesity-related fatty liver and the autosomal recessive disease heamophagocytic lymphohistocytosis (HLH).

[0236] IL-18 induced IFN-γ plays a role in liver injury. IFNγ has been shown to mediate LPS-induced liver injury following Propionibacterium acnes infection as described in Tsuji et al. (J. Immunol. 162: 1049-55, 1999). Large number of macrophages and lymphocytes infiltrate the portal area in response to P. acnes infection which results in intrahepatic formation of granulomas. IFNγ knock out mice exhibited less macrophage infiltration and a reduction in the number and size of granulomas. Subsequent treatment with low doses of LPS caused massive hepatic necrosis and increased IL-12, IL-18 and TNF-α serum levels in the normal mice, while the knock out mice exhibited drastic decreases in IL-12, IL-18 and TNF-α serum levels. The addition of IFNγ neutralizing antibody also caused a decrease in IL-18 and IL-12 levels. This model of liver injury indicates that LPS-induced liver injury is associated with increased levels of IL-18, IL-12 and IFN-γ. Currently, a role for IL-1β is not known in this liver injury model. Since IL-1β is known to be induced by LPS, it is possible IL-1β also plays a role in the disorder. Treatment with IL-Ra may modulate the severity of liver injury due to IL- 18 induced IFN-γ production and IL-1β.

[0237] IL-18 has also been shown to be involved in the immunomediated hepatitis model where treatment with concavalin A induced hepatitis in mice as described by Fiorucci et al. (Gastroenterology 118: 404-21, 2000). In this model, CD+ T cells and Th1-like cytokines cause Fas mediated liver cell death. Treatment with a nitric oxide derivative of aspirin protected against this cell death by reducing production of IFNγ, IL-18, IL-12, IL-1β and TNF-α. In addition, aneutralizing antibody to IL-18 caused a decrease in IFNγ production and reduced liver injury induced by conA.

[0238] HLH is a fatal autosomal recessive disease that manifests in early childhood. This disease is characterized by fever, hepatosplenomegaly, cytopenia and widespread infiltration of vital organs by activated lymphocytes and macrophages. Patients with HLH exhibit elevated serum levels of IL-18. IL-18 plays an important role in the induction of Th1 cells in HLH patients. (Takada et al., Br. J. Haematol. 106: 182-9, 1999).

[0239] IL-1 Hy2 inhibits IL-18 induced production of IFNγ. In the models described above, the degree of IL-1β activity is not known. Since IL-1β is known to be induced by LPS, it is possible that IL-1β also play a role in the pathogenicity of these conditions. The presence of the appropriate amount of IL-1Hy2 polynucleotides, polypeptides or other agonists may modulate the severity of the disease states due to both IL-18 induced IFNγ production and IL-1β.

[0240] IL-12 is known to potentiate IFNγ production, and the cytolytic activity of NK cells and cytotoxic T lymphocytes. These immunomodulatory effects have implicated a role for IL-12 in therapies for cancer and infectious disease. However, these same therapeutic effects can also promote autoimmune diseases and chronic inflammatory conditions such as multiple sclerosis, transplant rejection and cytotoxicity.

[0241] IL-12 and IFN-γ are involved in the pathogenesis of multiple sclerosis (MS). In the experimental allergic encephalomyelitis animal model (EAE), the demyelinating effect on the central nervous system is carried out similar to that in humans suffering from MS. Currently, IFNβ is used to treat MS. The mechanism of IFNβ treatment may be to decrease the number of IFNγ producing T cells in MS patients. (Rep et al., J. Neuroimmunol. 96:92-100, 1999). In addition, IFNγ production in blood lymphocytes was found to correlate with disability score in MS patients. (Petcreit et al., Mult. Scler. 6: 19-23, 2000). Antibodies against IL-12 were found to prevent superantigen-induced and spontaneous relapses of EAE in mice (Constantineseu et al., J. Immunol. 161: 5097-5104, 1998). All these studies point to the involvement of IL-12 induced IFNγ production in the progression of MS in human patients. Therefore, treatment with IL-1 Hy2 polynucleotides, polypeptides or other agonists to reduce IFNγ production may be an useful therapy for MS patients.

[0242] The combination of IL-12 and IL-2 has synergistic anti-tumor activity in vivo. However, in clinical trials the combination resulted in significant toxicity and subsequently shock and mortality. (Cohen, Science 270: 908 1995). In a murine model investigated by Carson et al. (J. Immunol., 162: 4943-5, 1999) determined that the fatal systemic inflammatory response was NK cell dependent but not related to other effector molecules in the system such as IL-1, TNF-α, and IFNγ. IL-1 Hy2 polynucleotides, polypeptides or other agonists is expected to inhibit IL-12 induced IFN-γ production and is expected to inhibit other biological activities of IL-12 such as NK cell cytolytic activity. Inhibition of NK cell activity, through IL-Ra administration, may reduce toxicity resulting from IL-12 antitumor treatment.

[0243] The effect of IL-1 Hy2 on IL-12 and/or IL-18 activity may be determined by measuring the biological activities of these cytokines. Both IL-12 and IL-18 are known to induce IFNγ production in T cells. In addition to IFN-γ, the combination of IL-12 and IL- 18 increases production of IL-3, IL-6 and TNF. Treatment with IL-1 Hy2 is expected to reduce IFNγ production induced by IL-12 and IL-18. Circulating or local levels of IFNγ in tissue or fluid samples from patients treated with IL-1 Hy2 polynucleotides, polypeptides or other agonists will be an indication of the therapeutic effects of IL-1 Hy2 on the IL-18 and IL-12 related disorders. Tissue samples include tissue samples from an area involved in inflammation or other disease. Fluid samples include, for example, whole blood, plasma, serum, cerebrospinal fluid, synovial fluid, peritoneal fluids (including lavage fluids or exudate), pleural fluids (including lavage fluids or exudate), wound fluids (including lavage fluids or exudate).

[0244] Furthermore, IL-12 is known to activate NK cells and to decrease serum IgE levels. These assays may also be used to measure the effectiveness of IL-1 Hy2 treatment for IL-12 related disorders. The NK cell cytolytic activity in patients treated with IL-1 Hy2 polynucleotides, polypeptides or other agonists can be assayed by measuring patient's blood samples ability to lysis colon carcinoma or lymphoma cells in vitro. (Lieberman et al., J. Sur. Res., 50: 410-415, 1992) In addition, the serum levels of IgE from patients treated with IL-1Hy2 can be measured to determine the effectiveness of treatment for IL-12 related disorders. (Kiniwa et al. J. Clin. Invest., 90 :262-66, 1992)

[0245] 6.14. Leukemias

[0246] Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention. Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

[0247] 6.15. Nervous System Disorders

[0248] Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:

[0249] (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;

[0250] (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;

[0251] (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;

[0252] (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;

[0253] (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;

[0254] (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;

[0255] (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and

[0256] (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

[0257] Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention:

[0258] (i) increased survival time of neurons in culture;

[0259] (ii) increased sprouting of neurons in culture or in vivo;

[0260] (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or

[0261] (iv) decreased symptoms of neuron dysfunction in vivo.

[0262] Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons maybe detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

[0263] In a specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

[0264] 6.16. Other Activities

[0265] 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 and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.

[0266] 6.17 Identification of Polymorphisms

[0267] The demonstration of polymorphisms, for example the T125C, C184T and A205C polymorphisms illustrated in Example 2 below, makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment. Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately. For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.

[0268] Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced. Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labelled nucleotides). In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.

[0269] Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.

[0270] 7. Therapeutic Methods

[0271] The novel IL-1 Hy2 polypeptides (including fragments, analogs and variants) of the invention have numerous applications in a variety of therapeutic methods. Antagonists and agonist of IL-1 Hy2 polypeptides may also have therapeutic applications in these models. Examples of therapeutic applications include, but are not limited to, those exemplified below.

[0272] 7.1 Sepsis

[0273] One embodiment of the invention is the administration of an effective amount of the IL-1 Hy2 polypeptides of the invention or modulators of IL-1 Hy2 polypeptides (such as agonists or antagonists) to individuals that are at a high risk of developing sepsis, or that have developed sepsis. An example of the former category are patients about to undergo surgery. While the mode of administration is not particularly important, parenteral administration is preferred because of the rapid progression of sepsis, and thus, the need to have the inhibitor disseminate quickly throughout the body. Thus, the preferred mode of administration is to deliver an I.V. bolus slightly before, during, or after surgery. The dosage of the IL-1 Hy2 polypeptides of the invention or IL-1 Hy2 modulators will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight and response of the individual patient. Typically, the amount of inhibitor administered per dose will be in the range of about 0.1 to 25 mg/kg of body weight, with the preferred dose being about 0.1 to 10 mg/kg of patient body weight. For parenteral administration, the IL-1 Hy2 polypeptides of the invention or IL-1 Hy2 modulators will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin. The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the inhibitor. The preparation of such solutions is within the skill of the art. Typically, the cytokine inhibitor will be formulated in such vehicles at a concentration of about 1-8 mg/ml to about 10 mg/ml.

[0274] 7.2 Arthritis and Inflamation

[0275] The immunosuppressive effects of the Interleukin-1 inhibitor against rheumatoid arthritis is determined in an experimental animal model system. The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int. Arch. Allergy Appl. Immunol., 23:129. Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture. The inhibitor is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. The control consists of administering PBS only.

[0276] The procedure for testing the effects of the Interleukin-1 inhibitor would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the inhibitor and subsequent treatment every other day until day 24. At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis of the data would reveal that the inhibitor would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.

[0277] 7.3 Diabetes

[0278] Interleukin-1 has been shown to be involved in the destruction of islet cells in diabetes mellitus (DM) (Mandrup-Paulsen, T., K. Bendtzen, J. Nerup, C. A. Dinarello, M. Svenson, and J. H. Nielson [1986] Diabetologia 29:63-67). The IL-1 Hy2 polypeptides of the invention limit lymphocyte and macrophage mediated damage to islet cells in incipient cases of DM identified by disease susceptibility via genetic background and family history. The inflammatory destruction of the pancreatic beta islet cells in such individuals with early DM is reduced by parenterally administering the IL-1 Hy2 polypeptides of the invention or modulators of IL-1 Hy2 polypeptides (such as agonists or antagonists) which have an anti-Interleukin-1 effect in the pancreas.

[0279] 7.4 Anti-hypotensive Arginine-free Formulations

[0280] The parenteral formulation of the therapeutic regimen is defined as including: about 3-4 g/l isoleucine, about 4-6 g/l leucine, about 3-4 g/l lysine, about 1-2 g/l methionine, about 1-2 g/l phenylalanine, about 2-3 g/l threonine, about 0.5-1.5 g/l tryptophan, about 3-4 g/l valine, about 4-5 g/l alanine, about 1-2 g/l histidine, about 3-4 g/l proline, about 1-2 g/l serine, about 0.25-0.75 g/l tyrosine, about 4-5 g/l glycine and about 2-3 g/l aspartic acid, together in a pharmacologically acceptable excipient. In another preferred embodiment of the described parenteral formulation, the formulation may further include ornithine, most particularly at a concentration of about 1-2 g/l. In still another embodiment of the described parenteral formulation, the formulation may include citrulline, most preferably at a concentration of between about 1 g/l and about 2 g/l. Both citrulline and ornithine may be included in still another embodiment of the formulation, again at the concentrations indicated.

[0281] The method includes an arginine-free formulation which comprises the amino acids and concentrations thereof already described herein, together in a pharmacologically acceptable excipient. Again, the formulation may further include ornithine, citrulline, or both, to even further supply physiologically required concentrations of urea cycle substrates in the animal. Most preferably, the formulation is provided as a parenteral formulation.

[0282] Another aspect of the method comprises a method for treating chemotherapeutic agent-related hypotension. In a most preferred embodiment, the method comprises monitoring an animal receiving a chemotherapeutic agent for a decrease in systolic blood pressure to less than about 100 mm Hg to detect an animal with systemic hypotension, treating the animal having systemic hypotension with a therapeutic regimen comprising a therapeutically effective amount of an arginine-free formulation sufficient to reduce plasma or serum arginine concentrations administered concurrently with or followed by the administration of a therapeutically effective concentration of an IL-1 Hy2 polypeptide or modulators of IL-1 Hy2 polypeptides (such as agonists or antagonists), and maintaining the animal on the therapeutic regimen until an increase of systolic blood pressure to at least about 100 mm Hg is detectable. Most preferably, the arginine-free formulation is a parenteral formulation.

[0283] In a preferred embodiment, the IL-1 Hy2 polypeptides of the invention or modulators of IL-1 Hy2 polypeptides (such as agonists or antagonists) are used in combination with the anti-hypotensive arginine free formulation to treat hypotension in an animal, particularly that hypotension caused by exposure to endotoxin or septic shock.

[0284] A patient having a systolic blood pressure of less than about 100 mm Hg will be targeted for the present treatment. Such a patient is to be placed on a continuous feed of an arginine-free formulation which includes a mixture of essential and nonessential amino acids as described in U.S. Pat. No. 5,334,380. The patient is treated concurrently with the interleukin-1 antagonist polypeptides of the invention. Blood samples are to be obtained from the patient and arginine levels in the serum or plasma fraction are determined.

[0285] 7.5 Pharmaceutical Formulations and Routes of Administration

[0286] A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, L-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.

[0287] The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Protein that can be administered with IL-1 Hy2 include other IL-1 receptor antagonist polypeptides such as IL-1Ra and IL-1 Hy1 Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.

[0288] Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. 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. When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, 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.

[0289] In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

[0290] 7.6. Routes of Administration

[0291] Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.

[0292] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation. In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops. Furthermore, one may administer the drug in 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.

[0293] 7.7. Compositions/Formulations

[0294] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.

[0295] When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0296] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained 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, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0297] 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 plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0298] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0299] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may 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. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0300] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0301] A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0302] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the proteinase inhibiting compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.

[0303] The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention. The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.

[0304] The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a 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. Preferably for bone and/or cartilage formation, 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.

[0305] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.

[0306] 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). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growth factor (IGF).

[0307] The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

[0308] Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

[0309] 7.8. Effective Dosage

[0310] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the, IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the C-proteinase activity). Such information can be used to more accurately determine useful doses in humans.

[0311] A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the C-proteinase inhibiting effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; for example, the concentration necessary to achieve 50-90% inhibition of the C-proteinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0312] 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.

[0313] An exemplary dosage regimen for the human IL-1 Hy2 polypeptides of the invention will be in the range of about 0.01 to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.

[0314] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

[0315] 7.9. Packaging

[0316] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition.

[0317] 8. Antibodies

[0318] Another aspect of the invention is an antibody that specifically binds the polypeptide of the invention. Such antibodies can be either monoclonal or polyclonal antibodies, as well fragments thereof and humanized forms or fully human forms, such as those produced in transgenic animals. The invention further provides a hybridoma that produces an antibody according to the invention. Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention.

[0319] 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). Methods for synthesizing such peptides are known in the art, for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein. In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, A. M., Monoclonal Antibodies Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. 35:1-21 (1990); Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).

[0320] Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with a peptide or polypeptide of the invention. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of the protein encoded by the ORF of the present invention used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection. The protein that is used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity. Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or β-galactosidase) or through the inclusion of an adjuvant during immunization.

[0321] For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124 (1988)).

[0322] 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. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention.

[0323] For polyclonal antibodies, antibody containing antiserum is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures. The present invention further provides the above-described antibodies in delectably labeled form. Antibodies can be delectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art, for example, see (Stemberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

[0324] The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed. The antibodies may also be used directly in therapies or other diagnostics. The present invention further provides the above-described antibodies immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press. N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immuno-affinity purification of the proteins of the present invention.

[0325] The three-dimensional structural analysis of IL-1 Hy2 (described in Example 14) demonstrates the IL-1 Hy2 residues involved in IL-1 receptor interactions. Antibodies that specifically bind to these receptor interacting residues are preferred antagonists for IL-1 Hy2 activity. These antibodies will reduce IL-1 Hy2 binding to an IL-1 receptor and thereby inhibit IL-1 Hy2 activity.

[0326] 9. Computer Readable Sequences and Structural Coordinates

[0327] According to one aspect of this invention, a nucleotide sequence, amino acid sequence or three-dimensional structure of the present invention can be recorded on computer readable media. A three-dimensional structure may be represented or displayed using structural coordinates of atoms of amino acids within amino acid sequences of the present invention (including mutant or variant amino acid sequences), particularly amino acids involved in binding to IL-1 receptor or other receptors or IL-1 receptor accessory protein, as well as amino acids involved in other IL-1Hy2 functions.

[0328] As used herein, “computer readable media” or “machine readable storage medium” refers to any medium which can be read and accessed directly by a computer. The term “data storage material” refers to any material on which data can be physically stored. 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. The term “machine readable data” refers to a group of one or more characters, including numbers, representing basic elements of information that can be processed by a computer. A skilled artisan can readily appreciate how any of the presently known computer readable media can be used to create a manufacture comprising a computer readable medium having recorded thereon a nucleotide sequence, amino acid sequence or structural coordinates of the present invention that can be used to render a three-dimensional structure of a polypeptide.

[0329] As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the sequence or structure information of the present invention. A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon sequence or structure information of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the sequence or structure information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the sequence or structure information of the present invention.

[0330] As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein sequence or structure information of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory which can store sequence or structure information of the present invention, or a memory access means which can access manufactures having recorded thereon the sequence or structure information of the present invention.

[0331] Input means can be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input means may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device. Output means may similarly be implemented by conventional devices. By way of example, output hardware may include CRT display terminal for displaying a graphical representation of important functional residues of the invention using a computer program as described herein. Output means might also include a printer, so that hard copy output may be produced, or a disk drive to store system output for later use.

[0332] In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from data storage means including working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of the invention, to form or display a sequence or a three-dimensional structure or representation, or to carry out computational methods of sequence comparison or drug discovery.

[0333] For example, by providing the nucleotide sequence of SEQ ID NOS: 1, 12 or 14 or a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NOS: 1, 12 or 14 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.

[0334] As used herein, “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. Examples of such software includes, but is not limited to, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[0335] As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

[0336] Computational methods of drug discovery may include computational evaluation of a three-dimensional structure for its ability to associate with moieties of chemical compounds. This evaluation may include performing a fitting operation between the structure or a portion thereof and one or more moieties of a chemical compound, and thereby qualitatively or quantitatively judging the proximity and/or extent of interaction between the three-dimensional structure and the chemical moiety(ies). Interaction may take place through, e.g., non-covalent interactions such as hydrogen bonding, van der Waals interactions, hydrophobic interactions and electrostatic interactions, or through covalent bonding. When the structure is displayed in a graphical three-dimensional representation on a computer screen, this allows visual inspection of the structure, as well as visual inspection of the structure's association with chemical moieties.

[0337] Specialized computer programs may be used to assist in a process of selecting chemical moieties or fragments of chemical compounds for further evaluation. These include: 1. GRID (P. J. Goodford, “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules”, J. Med. Chem., 28, pp. 849-857 (1985)). GRID is available from Oxford University, Oxford, UK. 2. MCSS (A. Miranker et al., “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, San Diego, Calif. 3. AUTODOCK (D. S. Goodsell et al., “Automated Docking of Substrates to Proteins by Simulated Annealing”, Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif. 4. DOCK (I. D. Kuntz et al., “A Geometric Approach to Macromolecule-Ligand Interactions”, J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is available from University of California, San Francisco, Calif.

[0338] Assembly of individual chemical moieties or fragments can be assisted by using programs including: 1. CAVEAT (P. A. Bartlett et al, “CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules”, in Molecular Recognition in Chemical and Biological Problems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196 (1989); G. Lauri and P. A. Bartlett, “CAVEAT: a Program to Facilitate the Design of Organic Molecules”, J. Comput. Aided Mol. Des., 8, pp. 51-66 (1994)). CAVEAT is available from the University of California, Berkeley, Calif. 2. 3D Database systems such as ISIS (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Y. C. Martin, “3D Database Searching in Drug Design”, J. Med. Chem., 35, pp. 2145-2154 (1992). 3. HOOK (M. B. Eisen et al, “HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site”, Proteins: Struct., Funct., Genet., 19, pp. 199-221 (1994). HOOK is available from Molecular Simulations, San Diego, Calif.

[0339] Computer programs that assist in designing a chemical compound that potentially interacts with a three-dimensional structure as a whole or “de novo” using either an empty binding site or optionally including some portion(s) of a known modulator(s) include: 1. LUDI (H. -J. Bohm, “The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors”, J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from Molecular Simulations Incorporated, San Diego, Calif. 2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations Incorporated, San Diego, Calif. 3. LeapFrog (available from Tripos Associates, St. Louis, Mo.). 4. SPROUT (V. Gillet et al, “SPROUT: A Program for Structure Generation)”, J. Comput. Aided Mol. Design, 7, pp. 127-153 (1993)). SPROUT is available from the University of Leeds, UK.

[0340] Other molecular modeling techniques may also be employed in accordance with this invention [see, e.g., N. C. Cohen et al., “Molecular Modeling Software and Methods for Medicinal Chemistry”, J. Med. Chem., 33, pp. 883-894 (1990); see also, M. A. Navia and M. A. Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in Structural Biology, 2, pp. 202-210 (1992); L. M. Balbes et al., “A Perspective of Modern Methods in Computer-Aided Drug Design”, in Reviews in Computational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds., VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, “Software For Structure-Based Drug Design”, Curr. Opin. Struct. Biology, 4, pp. 777-781 (1994)].

[0341] Binding affinity may be tested and optimized by computational evaluation, e.g. by minimizing the energy between the bound and free states of the three-dimensional structure (e.g., a small deformation energy of binding, preferably not greater than about 10 kcal/mole and more preferably not greater than 7 kcal/mole).

[0342] Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include: Gaussian 94, revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa.); AMBER, version 4.1 (P. A. Kollman, University of California at San Francisco); QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif.); Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif.); DelPhi (Molecular Simulations, Inc., San Diego, Calif.); and AMSOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be implemented, for instance, using a Silicon Graphics workstation with “IMPACT” graphics. Other hardware systems and software packages will be known to those skilled in the art.

[0343] Such computational drug design may include computer-based screening of small molecule databases for chemical moieties or chemical compounds that can bind in whole, or in part, to the desired three-dimensional structure. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy [E. C. Meng et al., J. Comp. Chem., 13, pp. 505-524 (1992)].

[0344] 10. Triple Helix Formation

[0345] In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.

[0346] 11. Diagnostic Assays and Kits

[0347] The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention.

[0348] In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample. Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.

[0349] In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample. In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.

[0350] Conditions for incubating a nucleic acid probe or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The 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. 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.

[0351] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention. Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.

[0352] In detail, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe. Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

[0353] 12. Medical Imaging

[0354] The novel IL-1 Hy2 polypeptides of the invention are useful in medical imaging, e.g., imaging the site of infection, inflammation, and other sites having Interleukin-1 receptor antagonist receptor molecules. See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778. Such methods involve chemical attachment of a labelling agent, administration of the labelled IL-1 Hy2 polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labelled IL-1 Hy2 polypeptide in vivo at the target site.

[0355] 13. Screening Assays

[0356] Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by the ORF from a polynucleotide with a sequence of SEQ ID NOS: 1, 12 or 14 to a specific domain of the polypeptide encoded by the nucleic acid, or to a nucleic acid with a sequence of SEQ ID NOS: 1, 12 or 14. In detail, said method comprises the steps of:

[0357] (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and

[0358] (b) determining whether the agent binds to said protein or said nucleic acid.

[0359] In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

[0360] Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

[0361] Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.

[0362] Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound). Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound). Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.

[0363] 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.

[0364] For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W. H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.

[0365] In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control. One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.

[0366] 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. 6:3073 (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, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents. Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent, in the control of bacterial infection by modulating the activity of the protein encoded by the ORF. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.

[0367] 14. Use of Nucleic Acids as Probes

[0368] Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences. The hybridization probes of the subject invention may be derived from the nucleotide sequence of the SEQ ID NOS: 1, 12 or 14. Because the corresponding gene is only expressed in a limited number of tissues, especially adult tissues, a hybridization probe derived from SEQ ID NOS: 1, 12 or 14 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.

[0369] Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described U.S. Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences. Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both. The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.

[0370] Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes. Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences. The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.

[0371] Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals. The nucleotide sequence may be used to produce purified polypeptides using well known methods of recombinant DNA technology. Among the many publications that teach methods for the expression of genes after they have been isolated is Goeddel (1990) Gene Expression Technology, Methods and Enzymology, Vol 185, Academic Press, San Diego. Polypeptides may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which a particular polypeptide nucleotide sequence was isolated or from a different species. Advantages of producing polypeptides by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.

[0372] 14.1 Preparation of Sequencing Chips and Arrays

[0373] A basic example is using 6-mers attached to 50 micron surfaces to give a chip with dimensions of 3×3 mm which can be combined to give an array of 20×20 cm. Another example is using 9-mer oligonucleotides attached to 10×10 microns surface to create a 9-mer chip, with dimensions of 5×5 mm. 4000 units of such chips may be used to create a 30×30 cm array. In an array in which 4,000 to 16,000 oligochips are arranged into a square array. A plate, or collection of tubes, as also depicted, may be packaged with the array as part of the sequencing kit.

[0374] The arrays may be separated physically from each other or by hydrophobic surfaces. One possible way to utilize the hydrophobic strip separation is to use technology such as the Iso-Grid Microbiology System produced by QA Laboratories, Toronto, Canada.

[0375] Hydrophobic grid membrane filters (HGMF) have been in use in analytical food microbiology for about a decade where they exhibit unique attractions of extended numerical range and automated counting of colonies. One commercially-available grid is ISO-GRID™ from QA Laboratories Ltd. (Toronto, Canada) which consists of a square (60×60 cm) of polysulfone polymer (Gelman Tuffryn HT-450, 0.45 u pore size) on which is printed a black hydrophobic ink grid consisting of 1600 (40×40) square cells. HGMF have previously been inoculated with bacterial suspensions by vacuum filtration and incubated on the differential or selective media of choice.

[0376] Because the microbial growth is confined to grid cells of known position and size on the membrane, the HGMF functions more like an MPN apparatus than a conventional plate or membrane filter. Peterkin et al (1987) reported that these HGMFs can be used to propagate and store genomic libraries when used with a HGMF replicator. One such instrument replicates growth from each of the 1600 cells of the ISO-GRID and enables many copies of the master HGMF to be made (Peterkin et al., 1987).

[0377] Sharpe et al. (1989) also used ISO-GRID HGMF form QA Laboratories and an automated HGMF counter (MI-100 Interpreter) and RP-100 Replicator. They reported a technique for maintaining and screening many microbial cultures.

[0378] Peterkin and colleagues later described a method for screening DNA probes using the hydrophobic grid-membrane filter (Peterkin et al., 1989). These authors reported methods for effective colony hybridization directly on HGMFs. Previously, poor results had been obtained due to the low DNA binding capacity of the epoxysulfone polymer on which the HGMFs are printed. However, Peterkin et al. (1989) reported that the binding of DNA to the surface of the membrane was improved by treating the replicated and incubated HGMF with polyethyleneimine, a polycation, prior to contact with DNA. Although this early work uses cellular DNA attachment, and has a different objective to the present invention, the methodology described may be readily adapted for Format 3 SBH.

[0379] In order to identify useful sequences rapidly, Peterkin et al. (1989) used radiolabeled plasmid DNA from various clones and tested its specificity against the DNA on the prepared HGMFs. In this way, DNA from recombinant plasmids was rapidly screened by colony hybridization against 100 organisms on HGMF replicates which can be easily and reproducibly prepared.

[0380] Manipulation with small (2-3 mm) chips, and parallel execution of thousands of the reactions. The solution of the invention is to keep the chips and the probes in the corresponding arrays. In one example, chips containing 250,000 9-mers are synthesized on a silicon wafer in the form of 8×8 mM plates (15 uM/oligonucleotide, Pease et al., 1994) arrayed in 8×12 format (96 chips) with a 1 mM groove in between. Probes are added either by multichannel pipette or pin array, one probe on one chip. To score all 4000 6-mers, 42 chip arrays have to be used, either using different ones, or by reusing one set of chip arrays several times.

[0381] In the above case, using the earlier nomenclature of the application, F=9; P=6; and F+P=15. Chips may have probes of formula BxNn, where x is a number of specified bases B; and n is a number of non-specified bases, so that x=4 to 10 and n=1 to 4. To achieve more efficient hybridization, and to avoid potential influence of any support oligonucleotides, the specified bases can be surrounded by unspecified bases, thus represented by a formula such as (N)nBx(N)m.

[0382] 14.2 Preparation of Support Bound Oligonucleotides

[0383] Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.

[0384] Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon. One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers. Immobilization can be achieved using passive adsorption (Inouye & Hondo, 1990); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morriey & Collins, 1989) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.

[0385] Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker. For example, Broude et al. (1994) describe the use of Biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Of course, this same linking chemistry is applicable to coating any surface with streptavidin. Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).

[0386] Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used. Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH. CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling. CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., 1991).

[0387] The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., 1991). In this technology, a phosphoramidate bond is employed (Chu et al., 1983). This is beneficial as immobilization using only a single covalent bond is preferred. The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm. To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group. It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.

[0388] More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7. A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.

[0389] Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well. The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C).

[0390] It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference. This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support. The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support. Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.

[0391] An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed. For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991), incorporated herein by reference. Probes may also be immobilized on nylon supports as described by Van Ness et al. (1991); or linked to Teflon using the method of Duncan & Cavalier (1988); all references being specifically incorporated herein.

[0392] To link an oligonucleotide to a nylon support, as described by Van Ness et al. (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.

[0393] One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994, incorporated herein by reference). These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips). These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner and then used in the advantageous Format 3 sequencing, as described herein.

[0394] 14.3 Preparation of Nucleic Acid Fragments

[0395] The nucleic acids to be sequenced may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps. For example, Sambrook et al. (1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).

[0396] DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods. Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.

[0397] The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.

[0398] Low pressure shearing is also appropriate, as described by Schriefer et al. (1990, incorporated herein by reference). In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures. A lever device allows controlled application of low to intermediate pressures to the cell. The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.

[0399] One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al. (1992). These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing. The present inventor envisions that this will also be particularly useful for generating random, but relatively small, fragments of DNA for use in the present sequencing technology.

[0400] The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. A typical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.

[0401] As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed). These advantages are also proposed to be of use when preparing DNA for sequencing by Format 3.

[0402] Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization. This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip. Phosphate groups must also be removed from genomic DNA by methods known in the art.

[0403] 14.4 Preparation of DNA Arrays

[0404] Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm2, depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones. Each of the subarrays may represent replica spotting of the same samples. In one example, a selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane. Subarrays may contain 64 samples, one from each patient. Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.

[0405] Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g. a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips. A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.

[0406] 14.5 Sequence Comparisons

[0407] Preferred identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990). The BLAST X program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The preferred computer program is FASTA version 3, specifically the FASTy program within the FASTA program package. Another preferred algorithm is the well known Smith Waterman algorithm which can also be used to determine identity.

[0408] Sequences can be compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and incorporated into the INHERIT™ 670 Sequence Analysis System. In this algorithm, Pattern Specification Language (developed by TRW Inc., Los Angeles, Calif.) is used to determine regions of homology. The three parameters that determine how the sequence comparisons run are window size, window offset, and error tolerance. Using a combination of these three parameters, the DNA database can be searched for sequences containing regions of homology to the query sequence, and the appropriate sequences scored with an initial value. Subsequently, these homologous regions are examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith-Waterman alignments can be used to display the results of the homology search. Peptide and protein sequence homologies can be ascertained using the INHERIT™ 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattern Specification Language and parameter windows are used to search protein databases for sequences containing regions of homology that were scored with an initial value. Dot-matrix homology plots can be examined to distinguish regions of significant homology from chance matches.

[0409] Alternatively, BLAST, which stands for Basic Local Alignment Search Tool, is used to search for local sequence alignments (Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol 215:403-10). BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. Whereas it is ideal for matches which do not contain gaps, it is inappropriate for performing motif-style searching. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). 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.

[0410] 15. Gene Therapy

[0411] Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein. The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention. Delivery of a functional genes encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.

[0412] Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art, the removal of the nucleic acids of the present invention such as using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific. Further, the polypeptides of the present invention can be inhibited by the introduction of antisense molecules that hybridize to nucleic acids that encode for the polypeptides of the present invention and by the removal of a gene that encode for the polypeptides of the present invention.

[0413] The present invention still further provides cells genetically engineered in vivo 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. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.

[0414] Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955. It is also contemplated that, in addition to heterologous promoter DNA, ampliflable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.

[0415] In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

[0416] The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

[0417] The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

[0418] 16. Transgenic Animals

[0419] In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122, incorporated herein by reference.

[0420] Transgenic animals can be prepared wherein all or part of a polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

[0421] 17. Three-dimensional Structural Analysis

[0422] The predicted three-dimensional structure of IL-1Hy2, generated by the GeneAtlas™ program (MSI) (as described in Example 28) which includes fold predictions from Fischer and Eisenberg (Protein Science 5: 947-955, 1996) and homology models from Sanchez and Sali (Proc. Natl. Acad. Sci., 95: 13597-13602, 1998), suggests IL-1 Hy2 is structurally related to IL-1 β and IL-1Ra. This analysis can be used to predict residues potentially involved in receptor binding and other residues important to IL-1Hy2 biological function. The three-dimensional structure of IL-1Hy2 will be useful in developing modulators of IL-1Hy2 activity such as antibodies, small molecules, peptides and derivatives thereof.

[0423] The three-dimensional structure of IL-1 Hy2 may be generated using the structural coordinates set forth below in Tables II or III. In addition, it is understood in the art that molecules or molecular complexes that are defined by the structural coordinates of Tables II or III include those plus or minus a root mean square deviation from the conserved backbone atoms of those amino acids of 2-12 Å, preferably not more than about 7 Å, or more preferably not more than about 5 Å, or most preferably not more than about 2 Å.

[0424] Identification of receptor binding residues and other residues important to IL-1 Hy2 biological function will be useful in discovering drugs which may modulate (i.e. increase or decrease) activity of the IL-1 receptor. Small molecules, antibodies and peptides which associate with one or more, or two or more, or three or more, or four or more, or five or more of the receptor binding residues or with other regions of IL-1 Hy2 may modulate IL-1Hy2 activity, e.g., by increasing or decreasing its affinity for the IL-1 receptor. An understanding of the receptor binding residues and associations that occur with these residues will facilitate the development of modulators (including antagonists and agonists) of IL-1Hy2 activity, including receptor binding.

[0425] The “receptor binding residues” of IL-1 Hy2 refer to the amino acid residues of the IL-1Hy2 molecule which interact with the IL-1 receptor or any other receptor to which IL-1Hy2 binds. These amino acids preferably include Met6, Arg8, Gln17, Val27, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, Ser105, and Tyr147 of SEQ ID NO: 2 and other amino acids within 2-12 Å, preferably within 7 Å, or more preferably within 5 Å, that may interact with these listed amino acids and/or contribute to the three-dimensional conformation of these listed amino acids.

[0426] The “accessory protein binding residues” of IL-1 Hy2 refer to the amino acid residues of the IL-1 Hy2 molecule which interact with IL-1 receptor accessory protein. These amino acids preferably include Lys145 of SEQ ID NO: 2 and other amino acids within 2-12 Å, preferably within 7 Å, or more preferably within 5 Å, that may interact with this amino acid and/or contribute to the three-dimensional conformation of this amino acid.

[0427] The IL-1Hy2 three-dimensional structure allows for the generation of polypeptide variants or non-peptidyl compounds that mimic the three-dimensional structure of IL-1Hy2. The IL-1 Hy2 three-dimensional structure also allows for the identification of desirable sites for mutation to create polypeptide or non-peptidyl variants with similar, increased, decreased or different biological activity compared to wild type IL-1 Hy2. Through site-directed mutagenesis, receptor binding residues, accessory protein binding residues or other residues involved in IL-1Hy2 biological function may be mutated to create modulators of IL-1 receptor activity. The mutants may act as antagonists or agonists for the IL-1 receptor. These mutants may be useful in therapeutic compositions directed to modulating the activity of IL-1Hy2 or its receptor. These mutations can be deletions, additions or substitutions of receptor binding residues, accessory protein binding residues or other residues important to IL-1Hy2 biological function. Non-conservative substitutions are expected to be more likely to result in different biological activity compared to wild type IL-1 Hy2. For example, mutations may alter the surface charge of IL-1Hy2. The three-dimensional structure indicates that ,IL-1Hy2 has fewer positively charged molecules on its surface than IL-1β. Therefore, mutations of negatively charged residues on its surface to positively charged residues may alter the biological activity of IL-1Hy2. Other mutations may affect the ability of IL-1Hy2 variants (1) to bind to IL-1 receptor (,IL-1 R) or other receptors to which IL-1 Hy2 binds, (2) to bind to IL-1R accessory protein, or (3) ability to antagonize IL-1R. The effect of various mutations on IL-1 Hy2 activity can be modeled in three-dimensional representations on a computer using any of the computer programs described herein.

[0428] Molecular modeling may be carried out using, e.g., the structural coordinates described herein, and any computer programs known in the art. For example, programs which predict binding sites and aid in designing modulators based on three-dimensional structural models include, but are not limited to, GRID (Oxford University) which aids in determining energetically favorable binding sites (Goodford, J. Med. Chem. 28: 849-857, 1985), MCSS (Molecular Simulations, Burlington, Mass.) which aids in determining functional maps of binding sites (Miranker and Karplus, Proteins, Structure, Function, and Genetics, 11: 29-34, 1991), AUTODOCK (Scripps Research) which aids in automated docking of substrates to proteins (Goodsell and Olsen, Proteins, Structure, Function, and Genetics, 8: 195-202, 1990, DOCK (University of San Francisco) which aids in determining macromolecular-ligand interactions (Kuntz et al., J. Mol. Biol. 161: 269-288, 1982).

[0429] The term “structure coordinates” refers to Cartesian coordinates derived from mathematical equations to generate the three-dimensional model of IL-1 Hy2 as derived from its primary amino acid sequence using, e.g., the GeneAtlas™ program. The model is used to establish the positions of the individual atoms of the IL-1 Hy2 protein.

[0430] Those of skill in the art understand that a set of structure coordinates for a molecule or a portion thereof is a relative set of points that define a structure in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar structure. Moreover, slight variations in the individual coordinates will have little effect on overall shape. Variations in coordinates may be generated by mathematical manipulations of the structural coordinates, e.g., by permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

[0431] Various computational analyses may be done to determine whether a molecule or a portion thereof is sufficiently similar, e.g., using current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, Calif.) version 4.1, and as described in the accompanying User's Guide.

[0432] The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean and is a way to express the deviation or variation from a trend or object. For purposes of the invention, the “root mean square deviation” defines the variation in the backbone of a protein from the polypeptide backbone of IL-1 Hy2 or a portion thereof or selected residues thereof, as substantially defined by the structural coordinates in Tables II or III below.

[0433] The present invention is illustrated in the following examples. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples. Example 1 addresses cloning of IL-1 Hy2 cDNA, Example 2 addresses identification of polymorphisms, Example 3 addresses tissue expression of IL-1Hy2 mRNA and polypeptide, Example 4 addresses chromosomal localization of IL-1Hy2 DNA, Example 5 addresses identification of an IL-1 receptor binding region and binding to IL-1 receptor, Example 6 addresses IL-1Hy2 polypeptide expression in E. coli , Example 7 addresses confirmation of IL-1Hy2 biological activities through assessment of its modulating effect on IL-1 related activities and IL-1 related disorders, Example 8 addresses the sequencing of the IL-1Hy2 human genomic BAC clone, Example 9 addresses the sequencing of IL-1 Hy2 mouse genomic BAC clone, Example 10 addresses inhibition of IL-1 β induced ,IL-6 production by IL-1 Hy2, Example 11 addresses the inhibition of IL-18 activity by IL-1 Hy2, Example 12 addresses IL-1 Hy2 binding to the IL-1 receptor, Example 13 addresses expression of IL-1 Hy2 in mammalian cells. Example 14 addresses the predicted three-dimensional structure of IL-1 Hy2. Example 15 addresses the crystal structure of IL-1 Hy2. Example 16 addresses site directed mutagenesis of IL-1 Hy2 based on the three-dimensional structure. Example 17 addresses expression of IL-1 Hy2 polypeptide in E. coli. Example 18 addresses purification of recombinant IL-Hy2 polypeptide expressed in E. coli.

EXAMPLE 1

Cloning of IL-1 Hy2 cDNA

[0434] A plurality of novel nucleic acids were obtained from the FSK001 cDNA library (prepared from human fetal skin tissue mRNA purchased from Invitrogen, San Diego, Calif.) using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques. The inserts of the library were amplified with PCR using primers specific for pSport1 (GIBCO BRL, Grand Island, N.Y.) vector sequences which flank the inserts. These samples were spotted onto nylon membranes and hybridized with oligonucleotide probes to give sequence signatures. The clones were clustered into groups of similar or identical sequences, and single representative clones were selected from each group for gel sequencing. The 5′ sequence of the amplified inserts was then deduced using the reverse M13 sequencing primer in a typical Sanger sequencing protocol. PCR products were purified and subjected to flourescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer. One cDNA insert was identified by sequencing of several hundred base pairs (approximately 1-386 of SEQ ID NO: 1) as a novel sequence related to IL-1Ra that had not been previously reported in public databases. The remaining sequence of SEQ ID NO: 1 was obtained by further sequencing of the entire cDNA insert of the same clone; the sequence was confirmed in part by sequencing of 5′ RACE PCR products from fetal skin and adult brain cDNA libraries using a Marathon cDNA amplification kit according to the manufacturer's instructions. This sequence and the clone were designated by code name CG149 and clone name RTA00003379F.h.20 (later redesignated pIL-1Hy2 and deposited at the ATCC on May 21, 1999 under Accession No. PTA-96), and the encoded polypeptide was designated IL-1Ra-Hy2 (later redesignated IL-1Hy2).

EXAMPLE 2

Identification of Polymorphisms

[0435] Sequencing of a number of PCR products from various cDNA libraries revealed several potential polymorphisms, which are described with reference to the nucleotide sequence numbering of SEQ ID NO: 1.

[0436] At nucleotide 125 of SEQ ID NO: 1, the “T” may be replaced with a “C”, resulting in a codon change from “GAT” to “GAC” (a silent mutation, as both codons encode the amino acid Asp). At nucleotide 184 of SEQ ID NO: 1, the “C” may be replaced with a “T”, resulting in a codon change from “ACA” (encoding Thr) to “ATA” (encoding Ile). At nucleotide 205 of SEQ ID NO: 1, the “A” may be replaced with a “C”, resulting in a codon change from “GAC” (Asp) to “GCC” (Ala). The changes in the amino acid sequence may be reflected in differences in the biological activities of the molecules, which can be confirmed by testing in any of the activity assays described herein.

EXAMPLE 3

Tissue Expression Study

[0437] 3.1 In situ Hybridization

[0438] Gene expression of human IL-1 Hy2 was analyzed using a semi-quantitative PCR-based technique. A panel of cDNA libraries derived from human tissue (from Clontech and Invitrogen) was screened with IL-1Hy2 specific primers [5′-CCGCACCAAGGTCCCCATTTTC-3′ (nucleotides 206-227), SEQ ID NO: 10 and 3′-GAGCCCACAAGGATAACCCAGG-5′ (nucleotides 728-707), SEQ ID NO: 11] to examine the mRNA expression of IL-1Hy2 in the following human tissues and cell types: heart, kidney, lung, placenta, liver, ovary, lymph node, spleen, testes, thymus, fetal liver, fetal skin, fetal spleen and macrophage. PCR assays (94° C. for 30 sec., 58° C. for 30 sec., 72° C. for 30 sec., for 30 cycles) were performed with 20 ng of cDNA derived from human tissues and cell lines and 10 picomoles of the IL-1Hy2 gene-specific primers. The 522 bp PCR product was identified through gel electrophoresis. Amplified products were separated on an agarose gel, transfered and chemically linked to a nylon filter. The filter was then hybridized with a radioactively labeled (33Palpha-dCTP) double-stranded probe generated from the full-length SEQ ID NO: 1 sequence using a Klenow polymerase, random prime method. The filters were washed (high stringency) and used to expose a phosphorimaging screen for several hours. Bands indicated the presence of cDNA including SEQ ID NO: 1 sequences in a specific library, and thus mRNA expression in the corresponding cell type or tissue.

[0439] IL-1Hy2 mRNA was observed to be expressed in kidney, spleen, and fetal skin. Similar to IL-1Hy2, IL-1Ra and IL-1Hy1 mRNA are also expressed in the human fetal skin tissues, suggesting that this family of proteins may share some physiologic functions. Within the kidney, IL-1 Hy2 mRNA was detected in the distal tubules of the kidney, the glomeruli of the kidney, the Bowman's capsule epithelia, capillary epithelia, and a subset of white blood cells within the blood vessels

[0440] Additional studies were performed to localize IL-1 Hy2 mRNA expression as described by D'Andrea et al. (J. Sur. Path, 1: 191-203,1995). IL-1 Hy2 mRNA was detected in serial sections of human normal tonsil and kidney by DIG-labeled probes consisting of nucleotide 396 to 568 of SEQ ID NO: 14. The slides were hybridized with the IL-iHy2 probes for 2 hours at 54° C. Subsequently, the slides were washed with 2×SSC at room temperature and then washed with 0.1×SSC at 54° C. After the stringency rinses, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) was used a chromagen. For visual detection, the slides were counter-stained with Eosin and examined under a light microscope.

[0441] 3.2 Immunohistochemistry

[0442] The serial sections of normal tonsil were also stained with polyclonal antibodies specific for IL-1 Hy2 prepared by immunizing rabbits with IL-1 Hy2 peptide: 43-56 of SEQ. ID NO.: 2 using conventional methods [see, e.g. Harlow et al., “Antibodies: A Laboratory Manual”. Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (1998)] and control preimmune serum form the immunized rabbits. The resulting anti-IL-1 Hy2 antibodies did not cross react with other IL-1 family members such as IL-1Ra, Il-1β, or IL-1Hy2 on a Western Blot. In addition, the slides were also stained (via double labeling) with antibodies for CD20 (Dako, Carpenteria, Calif.), Ki67 (Coulter Immunotech, Miami Fla.), CD3, CD1a, CD14, CD68, CD45 RO and LN5. immunohistochemistry was performed by QualTek Molecular Systems, Inc. (Santa Barbara, Calif.) using a modified procedure described by (Myers 1995). Antibody binding was detected with biotinylated secondary antibodies and streptavidin-AP. Fast red was used as the chromagen for detection and the slides were counter-stained with hematoxylin. IL-1 Hy2 expression was visually detected under a light microscope. For double labeling, the secondary antibodies were detected using a biotinylated secondary antibody followed by streptavidin-HRP and diaminobenzidine (DAB) was used as a chromagen. For all immunohistochemical studies a negative control was carried out in the absence of primary antibody.

[0443] In the tonsil, IL-1 Hy2 mRNA and protein were detected in a subset of B-cells (CD20 positive) in the germinal center, most of which were proliferating according to Ki67 staining suggesting that IL-1 Hy2 may play a role in regulating immune responses in the tonsil. IL-1 Hy2 was also expressed in the basal squamous epithelial of the skin surrounding the tonsil., lymph node and spleen. In a comparison of psoriatic skin and normal skin, IL-1 Hy2 polypeptide was elevated in the psoriatic skin. Furthermore, the IL-1 Hy2 positive cells did not react with anti-CD45RO (T cell marker) antibody or the anti-CD14 (monocyte marker) antibody suggesting that the IL-1 Hy2 polypeptide was not expressed in T cells or monocytes.

EXAMPLE 4

Chromosomal Localization Study

[0444] Chromosome mapping technologies allow investigators to link genes to specific regions of chromosomes. Chromosomal mapping was performed using the NIGMS human/rodent somatic cell hybrid mapping panel as described by Drwinga, H. L. et al., Genomics, 16, 311-314, 1993 (human/rodent somatic cell hybrid mapping panel #2 purchased from the Coriell Institute for Medical Research, Camden, N.J.). 60 ng of DNA from each sample in the panel was used as template, and 10 picomoles of the same IL-1Hy2 gene-specific oligonucleotides used in Example 3 were used as primers in a PCR assay (94° C. for 3 minutes, followed by 94° C. for 1 minute, 58° C. for 30 sec., 72° C. for 30 seconds, for 30 cycles, then 72° C. for 10 minutes). PCR products were analyzed by gel electrophoresis. The 824 bp genomic PCR product was detected only in the human/rodent somatic cell hybrid DNA containing human chromosome 2.

[0445] The IL-1 Hy2 gene was further localized using the Stanford G3 Human/Hamster Radiation panel as described by Stewart et al., Genome Res. 7: 422-33, (1997)(Research Genetics, Huntsville, Ala.). This analysis was carried out with a PCR assay as described above and localized IL-1 Hy2 gene to the 2q14 region. The gene has a 7 cRs distance from the marker SHGC-7020 and a LOD score of 10.58. The IL-1 Hyl (marker SHGC-7020), IL-1Ra (marker W17030) IL-1β (marker SHGC-10703) were also mapped to the similar location on chromosome 2. See Mulero et al., Biochem. Biophys. Res. Comm. 263: 702-706, 1999; Smith et al., J Biol. Chem., 275: 1169-75, 2000; Kuman et al., J. Biol. Chem., 275: 10308-14, 2000; Busfield et al., Genomics 6: 21-6, 2000; Steinlasserer et al., Genomics 13: 654-7, 1992; Modi et al., Genomics 2: 310-4, 1988; Stockman et al., FEBS Letts. 349: 79-83, 1994.

[0446] Gene family members are often linked to specific regions of chromosomes owing to intrachromosomal gene duplication events that give rise to multimember gene families during the process of evolution. The interleukin-1 gene family has been mapped to chromosome 2. More specifically, all of the interleukin 1 genes (IL-1α, IL-1β) and the receptors (IL-1 RI and IL-1 RII), as well as the receptor antagonist IL-1ra and the newly identified IL-1 Hy2 have been found to be situated in chromosome 2. The identification of IL-1 Hy2 sequences in this same region establishes its physical linkage to the interleukin-1 locus which indicates that IL-1 Hy2 functions as a modulator of the inflammatory response.

EXAMPLE 5

Interleukin-1 Receptor Binding Domain and Interleukin-1 Receptor Assay

[0447] The receptor binding region of both IL-1β and IL-1 Ra have been mapped to an 18 amino acid region in the carboxy terminal half of the proteins (i.e., residues 88-105 of IL-1β) by site-directed mutagenesis and protein modification studies.

[0448] IL-1 Hy2 and fragments thereof that include a receptor binding region are useful as reagents to identify cells and tissues expressing IL-1 receptors. The IL-1 receptor binding assay described in Hannum et al. Nature 343:336-340(1990)may be used. Briefly, highly radioactive recombinant SEQ ID NOS: 2, 4 or 13 is prepared by growing E. coli expressing either of SEQ ID NOS: 2 ,4 or 13 on M9 medium containing [35S] sulphate and purifying the labeled recombinant polypeptide by chromatography on a Mono-S column. The labeled polypeptide 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.

EXAMPLE 6

Expression of IL-1 Hy2 in E. coli

[0449] SEQ ID NOS: 1, 12 or 14 are expressed in E. coli by subcloning the entire coding region into a prokaryotic expression vector. The expression vector (pQE16) used is from the QIAexpression prokaryotic protein expression system (Qiagen). The features of this vector that make it useful for protein expression include: an efficient promoter (phage T5) to drive transcription; expression control provided by the lac operator system, which can be induced by addition of IPTG (isopropyl-β-D-thiogalactopyranoside), and an encoded His6 tag. The latter is a stretch of 6 histidine amino acid residues which can bind very tightly to a nickel atom. The vector can be used to express a recombinant protein with a His6 tag fused to its carboxyl terminus, allowing rapid and efficient purification using Ni-coupled affinity columns.

[0450] PCR is used to amplify the coding region which is then ligated into digested pQE16 vector. The ligation product is transformed by electroporation into electrocompetent E.coli cells (strain M15 [pREP4] from Qiagen), and the transformed cells are plated on ampicillin-containing plates. Colonies are screened for the correct insert in the proper orientation using a PCR reaction employing a gene-specific primer and a vector-specific primer. Positives are then sequenced to ensure correct orientation and sequence. To express IL-1 Hy2, a colony containing a correct recombinant clone is inoculated into L-Broth containing 100 μg/ml of ampicillin, 25 μg/ml of kanamycin, and the culture was allowed to grow overnight at 37° C. The saturated culture is then diluted 20-fold in the same medium and allowed to grow to an optical density at 600 nm of 0.5. At this point, IPTG is added to a final concentration of 1 mM to induce protein expression. The culture is allowed to grow for 5 more hours, and then the cells are harvested by centrifugation at 3000×g for 15 minutes.

[0451] The resultant pellet is lysed using a mild, nonionic detergent in 20 mM Tris HCl (pH 7.5) (B-PER™ Reagent from Pierce), or by sonication until the turbid cell suspension turned translucent. The lysate obtained is further purified using a nickel containing column (Ni-NTA spin column from Qiagen) under non-denaturing conditions. Briefly, the lysate is brought up to 300 mM NaCl and 10 mM imidazole and centrifuged at 700×g through the spin column to allow the His-tagged recombinant protein to bind to the nickel column. The column is then washed twice with Wash Buffer (50 mM NaH2PO4, pH8.0; 300 mM NaCl; 20 mM imidazole) and is eluted with Elution Buffer (50 mM NaH2PO4, pH8.0; 300 mM NaCl; 250 mM imidazole). All the above procedures are performed at 4° C. The presence of a purified protein of the predicted size is confirmed with SDS-PAGE.

EXAMPLE 7

Evaluation of IL-1 Hy2 Activities in vitro and in vivo

[0452] 7.1 Binding to the Interleukin-1 Receptor

[0453] A cell binding assay is carried out to demonstrate that IL-1 Hy2 binds to the Interleukin-1 receptor. Briefly, cell binding of the recombinant protein with and without the presence of 100-fold greater amounts of non tagged Interleukin-1 βeta (IL-1β) ligand is analyzed by using fluorescent antibodies specific for a IL-1 Hy2 polypeptide (e.g. specific for an express tag within the recombinant polypeptide) on the fluorescent activated cell sorter (FACS). In each reaction, 106 cells NHDF (normal human dermal fibroblasts) are resuspended in 100 μl of FACS buffer (distilled PBS and 3% calf serum and 0.01% azide). Cell binding is done by adding 5 nM recombinant IL-1 Hy2 in 100 μl cell suspension and as a competition in one reaction, 500 nM of recombinant IL-1 β is also added. The cells are incubated on ice for 1 hr. The cells are pelleted, 200 μl of 0.2 mM BS3 (crosslinker) is added, and the cells are kept on ice for 30 min. Next, 10 ul 1 M Tris pH 7.5 is added and the cells are incubated for 15 minutes on ice. The cells are pelleted, washed 1 time in FACS buffer, resuspended in 100 μl volume of FACS buffer and 2 μl primary antibody (anti-express tag antibody 1 mg/ml) is added, and incubated on ice for 30 minutes. The cells are pelleted, washed with FACS buffer, and resuspended in FACS buffer (100 ul volume). The secondary antibody (phycoerythrin conjugated) 2 ul of anti-mouse Ig (1 mg/ml) is added and the cells are incubated for 30 minutes on ice. The cells are again pelleted, washed two times with FACS buffer, resuspended in 0.5 ml FACS buffer and analyzed on FACS. A shift in the fluorescence is expected to be observed in the cells treated with the recombinant tagged IL-1 Hy2. This binding is shown to be specific if it is competed off with the non tagged IL-1 β protein. The results will indicate binding of IL-1 Hy2 to the IL-1 receptor.

[0454] 7.2 IL-1 Antagonist Activity

[0455] IL-1 antagonist activity is determined using a prostaglandin E2 (PGE2) based assay as follows. Cells are plated at 20,000 cells per well in a 96 well plate 24 hours before the assay. The cells are then treated with 25 pg/ml recombinant human IL-1β for 7 hours. To evaluate inhibition of IL-1β stimulated PGE2 release by IL-1Hy2 in comparison to IL-1Ra, the cells are pretreated with various amounts of IL-1 Hy2 or IL-1Ra for two hours before the addition of IL-1β. The supernatants are then collected and cell debris is removed by centrifugation. The amounts of PGE2 in the supernatants are determined by ELISA using the PGE2 assay system (R&D Systems) according to the manufacturer's protocol.

[0456] This assay was carried out with IL-1 Hy2 as follows. To stimulate IL-1β induced PGE2 production, human fibroblasts (CCD 1098; accession no. CRL 2127) were plated at 20,000 cells per well in a 96 well plate 24 hours before the assay. The cells were then washed once with fresh media and incubated for 16 hours with fresh media containing 1 ng/ml recombinant human IL-1β. To evaluate inhibition of IL-1β stimulated PGE2 release by IL-1 Hy2 in comparison to IL-1Ra, the cells were treated with various concentrations of IL-2 Hy2 or IL-1Ra together with IL-1β. After the 16 hour stimulation at 37° C. in a 5% CO2 incubator, the culture plates were centrifuged for 5 minutes at 4000 rpm to remove cellular debris. The amounts of PGE2 were determined by assaying 100 μl of supernatant with the PGE2 ELISA assay kit (R & D Systems) according to the manufacturer's protocol.

[0457] The addition of IL-1 Hy2 to the IL-1β stimulated cultures resulted in a dose-dependent partial decrease in PGE2 production. At a concentration of 1000 fold excess, IL-1 Hy2 inhibited IL-1 β induced PGE2 production 40-60%. As a control and a means for comparison, IL-1Ra completely inhibited PGE2 production at a concentration of 100 fold excess. The fact that IL-1 Hy2 only partially inhibits IL-1β activity may be beneficial in the treatment of inflammatory disease states due to fewer side effects. It is possible that more highly purified preparations of IL-1 Hy1 may show complete inhibition in this assay.

[0458] 7.3 Inhibition of Interleukin-1 Induced Cell Proliferation

[0459] Murine D10 T cells are obtained from the American Type Culture Collection (Rockville, Md.). Cells are maintained in Dulbecco's modified Eagle medium and Ham's F-12 medium (1:1) containing 10 mM HEPES buffer (pH 7.4) and 10% fetal bovine serum. All tissue culture reagents contained less than 0.25 ng/mL endotoxin as measured by the limulus amebocyte assay.

[0460] Murine D10 cells, an Interleukin-1 dependent T-cell line, are used to measure Interleukin-1 mitogenic activity. Cell proliferation in the present of Interleukin-1 with and without the IL-1 Hy2 polypeptides of the invention is assessed by incorporation of (3H) thymidine as previously described (Bakouche, O., et al. J. Immunol. 138:4249-4255, 1987). In a preferred embodiment, antagonists and agonist of the IL-1 Hy2 polypeptides of the invention are identified in this assay by adding the candidate compounds with the Interleukin-1 and IL-1 Hy2 polypeptides of the invention and measuring the change in cell proliferation caused by the candidate compound.

[0461] 7.4 Inhibition of Interleukin-1 Induced Cell Cytotoxicity

[0462] Inhibition of Interleukin-1-induced cytotoxicity is studied using an appropirate cell line, such as, for example, A375 tumor cells plated at a density of 6000 cells per well in 96-well microliter plates. After overnight attachment, Interleukin-1(3-300 ng/mL) is added in the presence or absence of NAA or NMA. After cells are incubated for 3 days, (3H) thymidine is added (1 mu Ci per well) for an additional 2 hours. Cells are harvested onto glass fiber disks (PHD Cell Harvested; Cambridge Technology, Inc., Watertown, Ma.) Disks are air dried overnight, and radioactivity is determined with a Model 1900TR Scintillation Counter (Packard Instrument Division, Downers Grove, ,Ill.)

[0463] 7.5 Induction of Nitrite Synthesis in Smooth Muscle Cells

[0464] Aortic smooth muscle cells are cultured by explanting segments of the medial layer of aortas from adult male Fischer 344 rats. Aortas are removed aseptically and freed of adventitial and endothelial cells by scraping both the luminal and abluminal surfaces. Medial fragments are allowed to attach to Primaria 25-cm2 tissue culture flasks (Becton-Dickinson, Lincoln Park, N.J.) which are kept moist with growth medium until cells emerged. Cultures are fed twice weekly with medium 199 containing 10% fetal bovine serum, 25 mM HEPES buffer (pH 7.4), 2 mM L-glutamine, 40 mu g/mL endothelial cell growth supplement (Biomedical Technologies, Inc., Stoughton, Mass.) and 10 mu g/ml gentamicin (GIBCO BRL, Grand Island, N.Y.). When primary cultures become confluent, they are passaged by trypsinization, and explants are discarded. For these studies, cells from passages 12-14 are seeded at 20,000 per well in 96-well plates and are used at confluence (60,000-80,000 cells per well). The cells exhibit the classic smooth muscle cell phenotype with hill and valley morphology, and they stain positively for smooth muscle actin.

[0465] Rat aortic smooth muscle cells are incubated with RPMI-1640 medium containing 10% bovine calf serum, 25 mM HEPES buffer 7.4), 2 mM glutamine, 80 U/mL penicillin, 80 mu g/mL streptomycin, 2 mu g/mL fungizone, and Interleukin-1, IFN-gamma, and various inhibitors. At the desired times, nitrite concentration in the culture medium is measured using the standard Griess assay (Green, L., et al. Anal. Biochem. 126:131-138, 1982) adapted to a 96-well microtiter plate reader (Gross, S. S., et al. Biochem. Biophys. Res. Commun. 178:823-829, 1991). Thus, 100 muL of Griess reagent (0.5% sulfanilic acid, 0.05% naphthalenediamine, and 2.5% phosphoric acid) is added to an equal volume of culture medium, and the OD sub 550 is measured and related to nitrite concentration by reference to a standard curve. The background OD sub 550 of medium incubated in the absence of cells is subtracted from experimental values.

[0466] Rat aortic smooth muscle cells are incubated with RPMI-1640 medium containing 10% bovine calf serum, 25 mM HEPES buffer (pH 7.4), 2 mM glutamine, 80 mu g/mL penicillin, 80 mu g/mL steptomycin, 2 mu g/mL fungizone, 30 mu g/mL lipopolysaccharide (Escherichia coli 0111 :B4), and 50 U/mL IFN-γ. Cells are harvested after 24 hours, and cytosol is prepared (Gross, S. S., et al. Biochem. Biophys. Res. Commun. 178:823-829, 1991). Cytosolic NO synthase activity is assayed by the Fe2+-myoglobin method described previously (Gross, S. S., et al. Biochem. Biophys. Res. Commun. 178:823-829, 1991).

[0467] 7.6 Alloreactivity Determined by Lymph Node Weight Gain

[0468] Experiments are conducted to show that systemic administration of the IL-1 Hy2 polypeptides of the invention suppress a localized, T cell-dependent, immune response to alloantigen presented by allogeneic cells. Mice are injected in the footpad with irradiated, allogeneic spleen cells. The mice are then injected in the contralateral footpad with irradiated, syngeneic spleen cells. An alloreactive response (marked by proliferation of lymphocytes and inflammation) occurs in the footpad receiving the allogeneic cells, which can be measured by determining the increase in size and weight of the popliteal lymph node draining the site of antigen deposition relative to controls or by an increase in cellularity.

[0469] Specific pathogen free 8-12 week old BALB/c (H-2 sup d) and C57BL/6 (H-2 sup b) mice (Jackson Laboratory, Bar Harbor, Me.) are used in this experiment. 48 BALB/c mice are divided into 16 groups, each having 3 mice (unless otherwise indicated). Each group of mice received a different mode of treatment. 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.

[0470] 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. Lymph nodes are weighed and the results expressed as the difference (DELTA) in weight (mg) of the lymph node draining the site of allogeneic cell injection and the weight of the node draining the syngeneic cell injection site. Lymph nodes draining the syngeneic cell injection site weighed approximately 1 mg, regardless of whether they are obtained from mice treated with MSA or IL-1 Hy2 polypeptides of the invention, and did not differ significantly in weight from nodes obtained from mice given no cell injection.

[0471] 7.7 Suppression of Organ Graft Rejection in vivo

[0472] Neonatal C57BL/6 (H-2 sup b) hearts are transplanted into the ear pinnae of adult BALB/c (H-2 sup d) recipients utilizing the method of Fulmer et al., Am. J. Anat. 113:273, 1963, modified as described by Trager et al., Transplantation 47:587, 1989, and Van Buren et al., Transplant. Proc. 15:2967, 1983. Survival of the transplanted hearts is assessed by visually inspecting the grafts for pulsatile activity. Pulsatile activity is determined by examining the ear-heart grafts of anesthetized recipients under a dissecting microscope with soft reflected light beginning on day 5 or 6 post transplant. The time of graft rejection is defined as the day after transplantation on which contractile activity ceases.

[0473] Recipient mice are transplanted on day 0 and injected with either IL-1 Hy2 polypeptides of the invention plus MSA (mouse serum albumin, 100 ng) or with MSA alone on days 0 through 6, alternating i.p. and s.c. routes. In a second heart transplant experiment, the mice are injected with MSA alone on days 0 through 2, i.p. route only.

[0474] 7.8 Suppression of Inflammatory Arthritis

[0475] 20 rats are divided into 4 groups, designated Groups G-J, each having 5 rats. All rats are immunized by subcutaneous injection. On day 21 following immunization with mBSA, an inflammatory arthritis response is elicited. On the same day, a negative control group is injected with a 0.2 ml volume of saline. Groups are injected with increasing amounts of IL-1 Hy2 polypeptides of the invention. Interleukin-1 is injected in one group as a positive control. The diameter of the largest egion of the treated joints is measured using a caliper on days 2, 4, 6 and 8 relative to day 0 intra-articular injection of antigen.

[0476] 7.9 Activity in a Pancreatitis Model

[0477] 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. (Murayama et al., Arch Surg 1990;125:1570-1572; Tani et al., International J Pancreatology 1987;2:337-348; Schoenberg et al., Free Radical Biology & Medicine 1992;12:515-522; Heath et al., Pancreas 1993;66:41-45; Saluja et al., Amer Physiological Society 1985: G702-G710; Manso et al., Digestive Disease and Sciences 1992;37:364-368). Group 1 is a control group (n-9) which receives only IP saline injections. Group 2 (n=12) is an untreated disease control. Group 3 (n=12) receives three injections of drug (10 mg/kg/hr) starting one hour prior to induction of pancreatitis. Group 4 (n=12) receives three injections of drug (10 mg/kg/hr) starting one hour after induction of pancreatitis.

[0478] After a suitable time period, all animals are euthanized, the blood collected, and the pancreata surgically excised and weighed. Serum is assayed for amylase, lipase, IL-6, and TNF levels. Each pancreas is fixed, stained, and graded histologically in a blinded fashion or interstitial edema, granulocyte infiltration, acinar vacuolization, and acinar cell. Additionally, serum levels of IL-1 Hy2 are determined, therefore allowing comparisons between dosage, serum level, systemic cytokine response, and degree of pancreatic damage.

[0479] Interleukin-6, Interleukin-1, Interleukin-1 receptor antagonist, and TNF are measured by commercially available ELISA kits (Genzyme Corp., Boston, Mass.). All specimens are run in triplicate. Serum levels of amylase and lipase are measured on a Kodak Ectachem 700 automated analyzer (Eastman Kodak Company, Rochester, N.Y.).

[0480] Histologic slides are prepared as is known in the art after rapid excision and subsequent fixation in 10% formalin. The tissues are paraffin embedded as is known in the art and then stained with Hematoxylin and Eosin in a standard fashion. These slides are examined and graded in a blinded fashion by a board certified pathologist.

EXAMPLE 8

Sequencing of IL-1 Hy2 Human Genomic BAC Clone

[0481] To understand the genomic organization of the IL-1 Hy2 gene, a commercial human BAC library (Research Genetics) was screened by PCR with primers specific to the full length IL-1 Hy2 cDNA using standard procedures. The BAC39316 clone containing the IL-1 Hy2 gene was partially digested with Sau3A I restriction enzyme. The resulting size-selected restriction fragments were inserted into a BamHI site of pUC18 (Pharmacia) to generate a library for screening. The BAC39316 clone containing the human genomic IL-1 Hy2 gene was sequencedusing M13 forward and reverse primers flanking the inserts. Direct BAC DNA sequencing was also carried out using primers specific to IL-1 HY2 cDNA to confirm exon/intron organization. The sequence of the BAC genomic clone is set out as SEQ ID NO: 15. Based on the sequences, exons of the IL-1 Hy2 gene were predicted using the GenScan software (Stanford University). This analysis indicated that the IL-1 Hy2 cDNA should contain additional sequences at the 5′ end in addition to those set forth in SEQ ID NO: 1.

[0482] The predicted cDNA sequence based on the genomic DNA sequence encoding IL-1 Hy2 was compared to the corresponding cDNA sequence. This analysis indicated that the predicted cDNA sequence based on the human genomic sequence of IL-1 Hy2 (SEQ ID NO: 12) contains a thymidine (T) at nucleotide 279 (see FIG. 2), while the IL-1 Hy2 cDNA sequence (SEQ ID NO: 14; FIG. 4) contains a cytosine (C) at position 279. The change in nucleotides (C→T) would extend the IL-1 Hy2 open reading frame in the 5′ direction, resulting in a 200 amino acid polypeptide, while the cDNA sequence (SEQ ID NO: 14; FIG. 4) encodes a 152 amino acid polypeptide (SEQ ID NO: 2).

[0483] The predicted cDNA sequence (SEQ ID NO: 12) is 1366 nucleotides which contains an open reading frame (nucleotides 278 to 880) that encodes a predicted polypeptide of 200 amino acids (SEQ ID NO: 13; FIG. 2). However, the sequences surrounding the translation initiation codon at nucleotide 422 of SEQ ID NO: 12 are more similar to the Kozak translation start site consensus than the sequences surrounding nucleotide 278. Therefore, it is also possible that the IL-1 Hy2 polypeptide is encoded by a shorter open reading frame between nucleotides 422 and 880 of SEQ ID NO: 12, which encodes a predicted polypeptide of 152 amino acid (SEQ ID NO: 2).

EXAMPLE 9

Sequencing of IL-1 Hy2 Mouse Genomic BAC Clone

[0484] A commercial mouse BAC library (Research Genetics)was screened with the full length IL-1 Hy2 cDNA using standard procedures. The BAC clone containing the mouse IL-1 Hy2 gene was sequenced by conventional methods and is set forth as SEQ ID NO: 17. Based on the sequences, exons of the mouse IL-1 Hy2 gene were predicted using the GenScan software (Stanford University). This analysis indicated that the mouse IL-1Hy2 gene contains 4 exons. The predicted cDNA encoding the mouse IL-1 Hy protein is set forth as SEQ ID NO: 16. The murine IL-1 Hy2 polypeptide translation initiates at nucleotide 1 and terminates at nucleotide 457 of SEQ ID NO: 17. The mouse and human IL-1 Hy2 polypeptide sequences share 81.7% homology. The murine genomic DNA sequence can be used to generate transgenic animals which overexpress the IL-1 Hy2 polypeptide or have the IL-1 Hy2 gene knocked out as described above in Section 16.

EXAMPLE 10

Inhibition of IL-1β Induced IL-6 Production

[0485] Inhibition of interleukin-1β induced IL-6 production was studied using human endothelial cells from umbilical vein (Huvec). Huvec cells were seeded at 2×104 cells per well in a 96-well plate the day before cell stimulation. On the day of stimulation, cells were washed once with fresh medium (F12 medium with 100 μg/ml heparin, 50 μg/ml endothelial growth supplement and 10% fetal bovine serum) and replated with 200 μl of fresh medium [without supplements] in each well. The Huvec cells were then stimulated with 100 pg/ml (final volume) of IL-1β. Although this assay was done with IL-1β any cytokine of interest can be used. To test IL-6 inhibition, different concentrations of IL-1Hy2 (ranging from 10× to 1000× the concentration of IL-1β) or IL-1ra (ranging from 10× to 1000× IL-1β concentration) were added to the wells with the IL-1β.

[0486] After 16 hours of cell stimulation, the culture plate was spun for five minutes at 4000 rpm to remove cell debris. To test for the presence of IL-6, 100 μl of supernatant was removed and assayed with a human IL-6 immunoassay kit (R&D Systems) according to the manufacturer's instructions.

[0487] IL-1 Hy2 partially inhibited IL-1β-stimulated IL-6 production in a dose-dependent manner. In view of the fact that IL-6 blocks production of tumor necrosis factor (TNF), a pro-inflammatory cytokine, the fact that IL-1 Hy2 only partially inhibits of IL-6 production by IL-1 Hy2 may be beneficial in the treatment of inflammatory disease states with IL-1Hy2 due to reduced side effects. It is possible that more highly purified preparations of IL-1 Hy2 may show complete inhibition in this assay.

EXAMPLE 11

Inhibition of IL-18 Activity by IL-1 Hy2

[0488] The following experiment evaluated the ability of IL-1 Hy2 to inhibit IL-18 activity, as measured by induction of IFN-γ. Human lymphocytes (PBMC) were obtained by Ficoll-Hypaque density gradient separation of peripheral blood from healthy volunteer donors. Immediately after isolation, the PBMC were washed two times with growth media, containing RPMI 1640-10% fetal bovine serum, and 3×105 cells/well were seeded in a 96 well plate. The cells were stimulated by adding anti-CD3 antibody (R & D Systems, Minneapolis, Minn.) to all of the samples at a final concentration of 0.5 μg/ml. At the time of stimulation, all but one control well per plate were treated with 100 ng/ml recombinant IL-18 (R&D Systems) for 36 hours at 37° C. at 5% CO2. The untreated well served as a measure of background levels of IFNγ produced by stimulated PBMC cells. IL-18 treatment causes the PBMC cells to increase production of IFN-γ relative to the background levels.

[0489] To assay for IL-1 Hy2 inhibition of IL-18 stimulated IFNγ production, 100× fold to 1000× fold concentration of IL-1 Hy2 (relative to IL-18 concentration) was added to wells together with IL-18 at the time of stimulation. After 36 hours of cell stimulation, the culture plate was centrifuged for 5 minutes at 4000 rpm to remove cell debris. The supernatant was assayed for IFNγ using the Quantikine IFNγ ELISA kit (R & D Systems) according to the manufacturer's suggested protocol.

[0490] Results indicated that IL-18 alone stimulated IFNγ production and that IL-1 Hy2 had some inhibitory activities on the IL-18 stimulation. In order to assess the mechanism by which IL-1 Hy2 reduced IFNγ production, the following assay was carried out.

[0491] Human lymphocytes (PBMC) were obtained, washed, seeded, stimulated with anti-CD3 antibody and treated with a final concentration of 100 ng/ml IL-18 (R & D Systems) as described above. Several blocking antibodies were then used to test inhibition of IFNγ production, including anti-IL 18 receptor antibody, anti-IL-1 receptor accessory protein antibody, anti-IL1 receptor type I antibody and anti-IL-1 receptor type II antibody (all obtained from R & D Systems, Minneapolis, Minn.). Different amounts of each antibody were added to the wells with IL-18, and after 36 hours of cell stimulation, the culture plate was centrifuged for 5 minutes at 4000 rpm to remove cell debris. The supernatant was assayed for IFNγ using the Quantikine IFNγ ELISA kit (R & D Systems) according to manufacturer's instructions.

[0492] In the absence of an antibody, IL-18 stimulated IFNγ production relative to background levels as observed above. However, anti-IL-18 receptor antibody, anti-accessory protein antibody and anti-IL-1 receptor type I, but not type II antibody inhibited IL-18 induced IFNγ production.

[0493] These results indicate that compounds which antagonize the action of the IL-1 receptor inhibit IL-18 activity as measured by induction of IFNγ production.

EXAMPLE 12

Binding of IL-1 Hy2 to the Interleukin-1 Receptor

[0494] A cell binding assay was carried out, in a modification of the procedure as described above in Example 7.1, to determine if IL-1 Hy2 of the invention binds to the interleukin-1 (IL-1) receptor. Briefly, fluorescent activated cell sorting (FACS) was used to measure cell binding of the recombinant protein (see Example 6) using fluorescent antibodies specific for the express tag on the IL-1 Hy2 recombinant protein. In each reaction, 106 cells of human fibroblast cells (CCD 1089) were suspended in 100 μl of FACS buffer (containing distilled PBS, 3% calf serum and 0.01% azide). Cell binding reactions included 5 nM recombinant IL-1 Hy2 in 100 μl cell suspension. The cells were incubated on ice for one hour. The cells were pelleted by centrifugation, 200 μl of 0.2 mM BS3 (crosslinker) was added, and the cells were kept on ice for 30 minutes. Next, 10 μl M Tris pH 7.5 was added and the cells were incubated for 15 minutes on ice. The cells were pelleted by centrifugation, washed one time in FACS buffer, resuspended in 100 μl volume of FACS buffer, 2 μl primary antibody (anti-express tag antibody 1 mg/ml) was added, and incubation continued on ice for an additional 30 minutes. The cells were pelleted by centrifugation, washed with FACS buffer, and resuspended in FACS buffer (100 μl volume). The secondary antibody (phycoerythrin-conjugated), 2 μl of anti-mouse Ig (1 mg/ml), was added and the cells were incubated for 30 minutes on ice. The cells were again pelleted by centrifugation, washed two times with FACS buffer, resuspended in 0.5 ml FACS buffer and analyzed on FACS.

[0495] A shift in the fluorescence was observed for the cells treated with the recombinant tagged IL-1 Hy2. This binding was specific, as binding was not observed with the same molarity of non-related proteins, such as bovine serum albumin (BSA). Specific IL-1 Hy2 binding was also demonstrated in the murine T cell line D10 and the murine monoclonal cell line RAW 264.7. These results indicate binding of the IL-1 Hy2 protein of the invention to the IL-1 receptor.

EXAMPLE 13

Expression of IL-1 Hy2 Polypeptide in Cells

[0496] To express IL-1 Hy2 in mammalian cells, Chinese hamster ovary (CHO) cells were transfected with a mammalian expression vector and IL-1 Hy2 secretion was detected. The protein coding region of IL-1 Hy2 was obtained by PCR. The IL-1 Hy2 cDNA was used as a template for the IL-1 Hy2 specific primers (5′GAGCCGCCATGTGTTCCCTCCCCATGGCAAG 3′ and 5′GCTACCAGC TCTGTTCAAAGT AAAAC3′; SEQ ID NO: 19 and 20 respectively) designed to amplify the shorter ORF. The PCR reaction was run for 30 cycles at 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds. The PCR product was inserted into the pcDNA3.1/V5-His-Topo vector (Invitrogen) per manufacturer's instructions. The resulting expression construct was sequenced to confirm that the inserted IL-1 Hy2 sequence was correct.

[0497] CHO cells were obtained from the ATCC and cultured in F12K media supplemented with 10% FBS and 100 units/ml of penicillin G and 100 μg/ml streptomycin at 37° C. in 5% CO2. The CHO cells were transiently transfected with the mammalian expression vector, pcDNA IL1 Hy2, using the FuGene transfection reagent (Roche Molecular Biochemicals) according to manufacturer's instructions. After transfection, the medium was replaced with serum-free F12K medium The culture medium was then collected 24 hours later and passed through a 0.2 μ filter (Pall Gelman Laboratory) to remove cellular debris.

[0498] The collected conditioned medium was concentrated 10 fold using Microcon YM-1 0 microcolumns (Amicon) according to the manufacturer's instructions and analyzed by electrophoresis on a 15% SDS-polyacrylamide gel followed by Western blot hybridization on Immunobilon-P membrane (Millipore). IL-Hy2 was detected on the Western blot with a polyclonal antibody specific for IL-1 Hy2 using the Supersignal West Pico chemiluminescence detection reagents with goat anti-rabbit IgG conjugated with horseradish peroxidase (Pierce) as a secondary antibody. The polyclonal antibody used for the Western Blot was the IL-1 Hy2 specific antibody described in Example 3.

[0499] The IL-1 Hy2 polypeptide was detected in both the cell culture medium and in the cell lysate, suggesting that IL-1 Hy2 is a secreted polypeptide of the apparent molecular weight 25 kD when expressed in mammalian cells. The IL-1 Hy2 polypeptide expressed in mammalian cells can be sequenced to confirm the amino terminus sequence of the mature protein. Analysis of the amino acid sequence with the SignalP algorithm (Nielsen et al., Int. J. Neural Syst. 8:581-599, 1997) suggested that the IL-1Hy2 does not contain a signal peptide. However, some proteins such as bFGF, TGFβ, IL-1β and IL-18, are known to be secreted in the absence of a signal peptide (Nielsen et al, supra.) similar to IL-1Hy2.

[0500] The recombinant IL-1Hy2 protein expressed in mammalian cells (CHO has two forms, a major for of 25 kDa and a minor form of 17 kDa, which corresponds to the predicted molecular weight (17 kDa). The increase in molecular weight may result from posttranslational modifications of the protein. The IL-1 Hy2 protein lacks N-linked glycosylation consensus sites. Neither N linked glycosylation nor O-linked glycosylation on the recombinant IL-1 Hy2 protein expressed in CHO cells was detected using peptide N-glycosidase F (PNGase F) and O-glycosidase deglycosylation analysis. Thus, the difference between the apparent molecular weight and the predicted molecular weight of IL-1 Hy2 may be due to other posttranslational modifications such as phosphorylation.

EXAMPLE 14

Three-Dimensional Structure of IL-1 Hy2

[0501] The GeneAtlas™ software package (Molecular Simulations Inc. (MSI), San Diego, Calif.) was used to predict the three-dimensional structure models of IL-1 Hy2. Models were generated by (1) PSI-Blast which is the multiple alignment sequence profile-based searching developed by Altschul et al., (Nucl. Acids Res. 25: 3389-3408, 1997), (2) High Throughput Modeling (MSI) which is an automated sequence and structure searching procedure, and (3) SeqFold which is a fold recognition method described by Fischer and Eisenberg (J. Mol. Biol. 209: 779-791, 1998). This analysis was carried out in part by comparing the IL-1 Hy2 amino acid sequence (SEQ ID NO: 2) with the known NMR (nuclear magnetic resonance) and x-ray crystal three-dimensional structures of IL-1 Ra, IL-1 β and IL-1 α as templates. The best structural model prediction for IL-1 Hy2(highest verify score=0.58) was based on the IL-1 β template and the results are summarized in the table I below:

2Amino Acid ResiduesSequenceSequenceStructureProteinof IL-1 Hy2IdentitySimilarityVerify ScoreIL-1 Ra7-150 aa41.1%55.3%0.41IL-1 β5-149 aa21.2%41.5%0.58IL-1 α5-146 aa19.5%37.6%0.38

[0502] IL-1 Hy2 was predicted to exhibit an overall β-barrel structure with a pseudo 3-fold symmetry axis down the center of the barrel. The structural models all consisted of 12 β-strands organized in three trefoil units of four antiparallel β-strands. Six of the β-strands form the barrel and the other six create a triangular array which closes the bottom of the barrel. The 12-β stranded trefoil structure is partially conserved between the IL-1 Hy2 structure models and the IL-1β, IL-1Ra and IL-1α structures . Although the IL-1 Hy2 amino acid sequence is more similar to the IL-1 Ra amino acid sequence; the IL-1 Hy2 structural model has greater structural agreement with the IL-1β structure (0.58 verify-score). The verify score produced by the MSI GeneAtlas™ program indicates the quality of the model. A verify score between 0-1.0, with 1 being the best, represents a good model.

[0503] The predicted three-dimensional structures of IL-1 Hy2 were superimposed with the average NMR structure of IL-1 Ra and the crystal structure of IL-1 β to evaluate their structural differences. The structural alignment overlays of the superimposed ribbon structures are shown in FIG. 5 and display similar interior core β-strands for both overlays. These overlays suggested that IL-1 Hy2 is more structurally similar to IL-1 β than IL-1 Ra. The overlay of the structural alignments also demonstrated major differences within the exterior loop region of the IL-1 Hy2 model based on IL-1 Ra.

[0504] Comparisons of the surface representations of the three-dimensional structures as viewed from surface models of the proteins suggested that IL-1 Hy2 has a similar overall shape to IL-1 β compared to the surface view of IL-1 Ra. This analysis also indicates that IL-1 Hy2 has fewer positively charged surface residues than both IL-1 β and IL-1 Ra.

[0505] Sequence alignment of IL-1 Ra and IL-1 Hy2, based on secondary structure in combination with mutagenesis analysis (See Boraschi et al. Fronteriers in Bioscience 1: 270-308, 1995), was generated as shown in FIG. 6. Residues important for receptor interaction and protein function are identified in this figure with reference to the amino acid numbering of SEQ ID NO: 22 (which is missing the first 6 amino acids compared to SEQ ID NO: 2). This difference in amino acid numbering allows for the figure to correlate with the structural alignment between IL-1Ra and IL-1 Hy2.

[0506] In FIG. 6, the receptor interacting amino acid are Lys7, Gln11, Asp25, Val27 and Tyr 141 and another residue identified as important for biological function is Lys139 of SEQ ID NO: 2. When the amino acid numbering of SEQ ID NO: 2 is used, the residues indicated to be associated with receptor interaction are Lys13, Gln17, Asp31, Val33 and Tyr 147 and another residue identified as important for biological function is Lys145. The corresponding Lys residue in IL-1 Ra is confirmed to be important for IL-1 Ra biological function. (See Boraschi et al., supra.). These results suggest that IL-1 Hy2 may function as an antagonist in the presence of accessory protein.

[0507] The alignment with IL-1β and IL-1 Hy2 is displayed in FIG. 7. Residues important for receptor interaction and protein function are identified in this figure with reference to the amino acid numbering of SEQ ID NO: 24 (which is missing the first 4 amino acids compared to SEQ ID NO: 2). This difference in amino acid numbering allows for the figure to correlate with the structural alignment between IL-1β and IL-1 Hy2.

[0508] In FIG. 7, the receptor interacting amino acid are Met2, Arg4, Lys9 Gln13, Asp27, Val27, Pro42, Val51, Gly88, Gly89, Gln99, and Ser101 and another residue identified as important for biological function is Lys141 of SEQ ID NO: 24. When the amino acid numbering of SEQ ID NO: 2 is used, the residues indicated to be associated with receptor interaction are Met6, Arg8, Lys13, Gln17, Asp31, Val33, Pro46, Val55, Gly92, Gly93, Gln103, and Ser105 and another residue identified as important for biological function is Lys145. In this case, the importance of Lys145 also indicated that IL-1 Hy2 will function as an antagonist. Overall, this analysis suggested that IL-1 Hy2 binds to the same region of the IL-1 receptor as IL-1 β and IL-1 Ra, but IL-1 Hy2 has different atomic interactions. This suggests that IL-1 Hy2 will have a different binding affinity for the IL-1 receptor as compared to IL-1 β or IL-1 Ra.

[0509] The protein database coordinates of the IL-1 Hy2 structural models are shown in Tables II or III below. These structural coordinates were calculated based on the three-dimensional structures of IL-1 Ra and IL-1β. The protein database coordinate output file in this format provided the atom number, atom name, amino acid side chain, amino acid number, the X, Y and Z coordinates, occupancy (Occup.), the B-factor associated and ISG with each atom. The last column indicates the row number. The “Atom Name” refers to the element whose coordinates are measured. The “Amino Acid Side Chain” refers to the name of the amino acid, and the “Amino Acid Number” refers to the position in the IL-1 Hy2 amino acid sequence in the structural model. The “X, Y and Z coordinates” refer to the atomic position measured in Angstroms. An occupancy of 1 indicates the position is fully occupied. The “B-factor” refers to the thermal factor that measures movement of the atom around the atomic center. The term “1SG” indicates the name of the structure file given by the MSI program, and the last column indicates the row number. The MSI GeneAtlas™ program used the known crystal and NMR structures of IL-1β and IL-1 Ra, respectively, as templates to derive the coordinates which can be used to generate the electron density map of IL-1 Hy2. Those of skill in the art will understand the structural coordinates set out in FIGS. 8 and 9 are not without standard error. The verify score produced by the MSI GeneAtlas™ program indicates the quality of the model. A verify score between 0-1.0, with 1 being the best, represents a good model.

[0510] Table II shows protein database coordinates for a IL-1 Hy2 structural models generated by the GeneAtlas™ Program (MSI) using the three-dimensional structure of IL-1 Ra as a template.

3TABLE IIAtomAmino AcidBNo.NameSCNo.XYZOccup.FactorATOM1NALA13.19818.6912.9231.0044.801SG 2ATOM2CAALA12.44017.4432.6861.0044.801SG 3ATOM3CBALA12.64716.9501.2451.0044.801SG 4ATOM4CALA12.89416.3723.6141.0044.801SG 5ATOM5OALA13.29216.6374.7471.0044.801SG 6ATOM6NARG22.83715.1123.1481.0052.081SG 7ATOM7CAARG23.25214.0464.0031.0052.081SG 8ATOM8CBARG22.33912.8113.9191.0052.081SG 9ATOM9CGARG20.89313.0954.3361.0052.081SG 10ATOM10CDARG20.59212.8005.8071.0052.081SG 11ATOM11NEARG20.86214.0366.5941.0052.081SG 12ATOM12CZARG20.30714.1767.8341.0052.081SG 13ATOM13NH1ARG2−0.49113.1888.3341.0052.081SG 14ATOM14NH2ARG20.55215.2998.5701.0052.081SG 15ATOM15CARG24.61913.6433.5601.0052.081SG 16ATOM16OARG24.80513.1312.4581.0052.081SG 17ATOM17NTYR35.61613.8514.4401.0057.091SG 18ATOM18CATYR36.97213.5214.1121.0057.091SG 19ATOM19CBTYR37.98214.5914.5581.0057.091SG 20ATOM20CGTYR37.55315.8773.9501.0057.091SG 21ATOM21CD1TYR37.98316.2422.6991.0057.091SG 22ATOM22CD2TYR36.70016.7154.6301.0057.091SG 23ATOM23CE1TYR37.58317.4302.1371.0057.091SG 24ATOM24CE2TYR36.29517.9064.0761.0057.091SG 25ATOM25CZTYR36.73818.2642.8261.0057.091SG 26ATOM26OHTYR36.32519.4852.2511.0057.091SG 27ATOM27CTYR37.26312.3154.9341.0057.091SG 28ATOM28OTYR37.03912.3176.1431.0057.091SG 29ATOM29NTYR47.76111.2404.2991.0060.921SG 30ATOM30CATYR47.91810.0335.0541.0060.921SG 31ATOM31CBTYR46.8918.9824.5921.0060.921SG 32ATOM32CGTYR46.9827.7185.3701.0060.921SG 33ATOM33CD1TYR46.5157.6496.6631.0060.921SG 34ATOM34CD2TYR47.4876.5894.7741.0060.921SG 35ATOM35CE1TYR46.5896.4707.3651.0060.921SG 36ATOM36CE2TYR47.5605.4145.4691.0060.921SG 37ATOM37CZTYR47.1185.3536.7641.0060.921SG 38ATOM38OHTYR47.2054.1347.4611.0060.921SG 39ATOM39CTYR49.3069.4934.8601.0060.921SG 40ATOM40OTYR49.8539.5523.7591.0060.921SG 41ATOM41NILE59.9158.9805.9561.0062.271SG 42ATOM42CAILE511.2328.3935.9271.0062.271SG 43ATOM43CBILE512.2869.3576.4141.0062.271SG 44ATOM44CG2ILE512.1239.4777.9371.0062.271SG 45ATOM45CG1ILE513.7128.9725.9711.0062.271SG 46ATOM46CD1ILE514.3027.7306.6351.0062.271SG 47ATOM47CILE511.1957.1936.8461.0062.271SG 48ATOM48OILE510.4347.1887.8131.0062.271SG 49ATOM49NILE611.9906.1296.5641.0059.511SG 50ATOM50CAILE611.9884.9617.4191.0059.511SG 51ATOM51CBILE611.3533.7476.8061.0059.511SG 52ATOM52CG2ILE69.8624.0306.6381.0059.511SG 53ATOM53CG1ILE612.0763.3155.5231.0059.511SG 54ATOM54CD1ILE611.6291.9435.0251.0059.511SG 55ATOM55CILE613.3874.5937.8531.0059.511SG 56ATOM56OILE614.3574.7857.1201.0059.511SG 57ATOM57NLYS713.5074.0429.0901.0055.471SG 58ATOM58CALYS714.7773.7389.7001.0055.471SG 59ATOM59CBLYS715.2934.99910.4211.0055.471SG 60ATOM60CGLYS716.8035.10310.6011.0055.471SG 61ATOM61CDLYS717.2686.52910.8991.0055.471SG 62ATOM62CELYS718.7806.65611.0841.0055.471SG 63ATOM63NZLYS719.1648.08611.0881.0055.471SG 64ATOM64CLYS714.5602.62410.7051.0055.471SG 65ATOM65OLYS713.4712.05410.7731.0055.471SG 66ATOM66NTYR815.6122.23211.4711.0050.671SG 67ATOM67CATYR815.4811.20012.4761.0050.671SG 68ATOM68CBTYR816.002−0.16111.9791.0050.671SG 69ATOM69CGTYR815.708−1.22212.9851.0050.671SG 70ATOM70CD1TYR814.449−1.77113.0611.0050.671SG 71ATOM71CD2TYR816.688−1.68813.8321.0050.671SG 72ATOM72CE1TYR814.168−2.75713.9781.0050.671SG 73ATOM73CE2TYR816.413−2.67414.7511.0050.671SG 74ATOM74CZTYR815.150−3.21014.8261.0050.671SG 75ATOM75OHTYR814.864−4.22315.7681.0050.671SG 76ATOM76CTYR816.3081.60613.6701.0050.671SG 77ATOM77OTYR817.3252.28913.5381.0050.671SG 78ATOM78NALA915.8881.14714.8691.0046.421SG 79ATOM79CAALA916.4571.49416.1461.0046.421SG 80ATOM80CBALA915.6910.87317.3281.0046.421SG 81ATOM81CALA917.8811.05116.2571.0046.421SG 82ATOM82OALA918.6901.74316.8751.0046.421SG 83ATOM83NASP1018.227−0.11915.6851.0045.091SG 84ATOM84CAASP1019.555−0.63315.8641.0045.091SG 85ATOM85CBASP1019.643−2.14315.5811.0045.091SG 86ATOM86CGASP1018.842−2.86616.6581.0045.091SG 87ATOM87OD1ASP1018.625−2.26517.7431.0045.091SG 88ATOM88OD2ASP1018.431−4.02916.4061.0045.091SG 89ATOM89CASP1020.5050.05314.9331.0045.091SG 90ATOM90OASP1021.329−0.60114.2961.0045.091SG 91ATOM91NGLN1120.4571.40014.9011.0046.811SG 92ATOM92CAGLN1121.3392.23214.1281.0046.811SG 93ATOM93CBGLN1122.7852.21614.6521.0046.811SG 94ATOM94CGGLN1122.8942.63316.1201.0046.811SG 95ATOM95CDGLN1122.1893.97216.2881.0046.811SG 96ATOM96OE1GLN1122.5334.96215.6441.0046.811SG 97ATOM97NE2GLN1121.1573.99917.1741.0046.811SG 98ATOM98CGLN1121.3751.83812.6791.0046.811SG 99ATOM99OGLN1122.4461.55712.1441.0046.811SG 100ATOM100NLYS1220.2031.80112.0021.0055.601SG 101ATOM101CALYS1220.1731.49410.5961.0055.601SG 102ATOM102CBLYS1219.8700.01110.2991.0055.601SG 103ATOM103CGLYS1220.984−0.92210.7901.0055.601SG 104ATOM104CDLYS1220.628−2.41310.7641.0055.601SG 105ATOM105CELYS1221.764−3.31311.2621.0055.601SG 106ATOM106NZLYS1221.446−4.74111.0221.0055.601SG 107ATOM107CLYS1219.1172.3629.9551.0055.601SG 108ATOM108OLYS1218.1562.73910.6221.0055.601SG 109ATOM109NALA1319.2752.7098.6461.0067.261SG 110ATOM110CAALA1318.3643.6127.9691.0067.261SG 111ATOM111CBALA1318.8805.0627.9161.0067.261SG 112ATOM112CALA1318.1443.1926.5311.0067.261SG 113ATOM113OALA1318.9322.4375.9651.0067.261SG 114ATOM114NLEU1417.0313.6775.9131.0079.421SG 115ATOM115CALEU1416.6743.3994.5381.0079.421SG 116ATOM116CBLEU1415.1673.1704.3401.0079.421SG 117ATOM117CGLEU1414.7792.7912.9011.0079.421SG 118ATOM118CD2LEU1413.2552.7992.7221.0079.421SG 119ATOM119CD1LEU1415.4141.4502.4951.0079.421SG 120ATOM120CLEU1417.0784.6103.7361.0079.421SG 121ATOM121OLEU1416.9505.7394.2131.0079.421SG 122ATOM122NTYR1517.5414.4212.4741.0087.231SG 123ATOM123CATYR1518.2245.5441.8811.0087.231SG 124ATOM124CBTYR1519.5875.4132.5671.0087.231SG 125ATOM125CGTYR1520.6056.4242.2831.0087.231SG 126ATOM126CD1TYR1521.3066.4051.1041.0087.231SG 127ATOM127CD2TYR1520.8897.3433.2581.0087.231SG 128ATOM128CE1TYR1522.2747.3450.8701.0087.231SG 129ATOM129CE2TYR1521.8578.2833.0291.0087.231SG 130ATOM130CZTYR1522.5428.2841.8351.0087.231SG 131ATOM131OHTYR1523.5389.2511.5991.0087.231SG 132ATOM132CTYR1518.3625.3520.3791.0087.231SG 133ATOM133OTYR1518.9514.371−0.0681.0087.231SG 134ATOM134NTHR1617.8656.300−0.4521.0084.171SG 135ATOM135CATHR1617.9286.159−1.8881.0084.171SG 136ATOM136CBTHR1616.8246.887−2.5951.0084.171SG 137ATOM137OG1THR1616.7966.531−3.9691.0084.171SG 138ATOM138CG2THR1617.0668.398−2.4401.0084.171SG 139ATOM139CTHR1619.2246.697−2.4191.0084.171SG 140ATOM140OTHR1619.7257.727−1.9761.0084.171SG 141ATOM141NARG1719.7705.992−3.4291.0078.021SG 142ATOM142CAARG1720.9686.319−4.1491.0078.021SG 143ATOM143CBARG1721.9625.148−4.2391.0078.021SG 144ATOM144CGARG1722.5784.743−2.8971.0078.021SG 145ATOM145CDARG1723.9175.423−2.6001.0078.021SG 146ATOM146NEARG1724.8924.988−3.6401.0078.021SG 147ATOM147CZARG1725.6463.866−3.4521.0078.021SG 148ATOM148NH1ARG1725.4873.110−2.3271.0078.021SG 149ATOM149NH2ARG1726.5693.502−4.3891.0078.021SG 150ATOM150CARG1720.4636.569−5.5311.0078.021SG 151ATOM151OARG1719.5997.421−5.7341.0078.021SG 152ATOM152NASP1821.0785.939−6.5481.0069.341SG 153ATOM153CAASP1820.5026.094−7.8541.0069.341SG 154ATOM154CBASP1821.4385.589−8.9661.0069.341SG 155ATOM155CGASP1820.7615.794−10.3151.0069.341SG 156ATOM156OD1ASP1820.5596.976−10.7051.0069.341SG 157ATOM157OD2ASP1820.4354.771−10.9721.0069.341SG 158ATOM158CASP1819.2055.326−7.9971.0069.341SG 159ATOM159OASP1818.1265.913−8.0581.0069.341SG 160ATOM160NGLY1919.3163.973−8.0671.0063.661SG 161ATOM161CAGLY1918.2593.012−8.3161.0063.661SG 162ATOM162CGLY1917.3712.617−7.1671.0063.661SG 163ATOM163OGLY1916.1702.441−7.3681.0063.661SG 164ATOM164NGLN2017.9142.387−5.9501.0061.511SG 165ATOM165CAGLN2017.0171.871−4.9471.0061.511SG 166ATOM166CBGLN2016.8570.345−4.9681.0061.511SG 167ATOM167CGGLN2016.150−0.087−6.2501.0061.511SG 168ATOM168CDGLN2015.734−1.539−6.1401.0061.511SG 169ATOM169OE1GLN2016.082−2.258−5.2051.0061.511SG 170ATOM170NE2GLN2014.939−1.982−7.1501.0061.511SG 171ATOM171CGLN2017.3722.305−3.5621.0061.511SG 172ATOM172OGLN2018.3742.981−3.3341.0061.511SG 173ATOM173NLEU2116.5011.924−2.5971.0058.631SG 174ATOM174CALEU2116.6772.326−1.2281.0058.631SG 175ATOM175CBLEU2115.3472.472−0.4601.0058.631SG 176ATOM176CGLEU2114.4333.603−0.9811.0058.631SG 177ATOM177CD2LEU2113.2713.871−0.0151.0058.631SG 178ATOM178CD1LEU2113.9513.330−2.4161.0058.631SG 179ATOM179CLEU2117.4971.277−0.5221.0058.631SG 180ATOM180OLEU2117.0290.164−0.2881.0058.631SG 181ATOM181NLEU2218.7481.625−0.1451.0056.391SG 182ATOM182CALEU2219.6540.7160.5121.0056.391SG 183ATOM183CBLEU2221.1401.0770.3391.0056.391SG 184ATOM184CGLEU2221.6270.859−1.1071.0056.391SG 185ATOM185CD2LEU2223.1570.944−1.2041.0056.391SG 186ATOM186CD1LEU2220.9011.793−2.0891.0056.391SG 187ATOM187CLEU2219.3400.6321.9841.0056.391SG 188ATOM188OLEU2218.7261.5342.5531.0056.391SG 189ATOM189NVAL2319.776−0.4702.6481.0053.681SG 190ATOM190CAVAL2319.372−0.6924.0141.0053.681SG 191ATOM191CBVAL2318.712−2.0304.2021.0053.681SG 192ATOM192CG1VAL2318.317−2.2005.6771.0053.681SG 193ATOM193CG2VAL2317.530−2.1323.2271.0053.681SG 194ATOM194CVAL2320.541−0.6154.9721.0053.681SG 195ATOM195OVAL2321.690−0.8914.6271.0053.681SG 196ATOM196NGLY2420.240−0.2296.2361.0053.341SG 197ATOM197CAGLY2421.216−0.1787.2931.0053.341SG 198ATOM198CGLY2422.0891.0397.1581.0053.341SG 199ATOM199OGLY2421.7052.1187.6121.0053.341SG 200ATOM200NASP2523.3360.8886.6381.0054.161SG 201ATOM201CAASP2524.1402.0776.4731.0054.161SG 202ATOM202CBASP2524.8912.5227.7381.0054.161SG 203ATOM203CGASP2525.4753.9027.4491.0054.161SG 204ATOM204OD1ASP2524.9424.5846.5331.0054.161SG 205ATOM205OD2ASP2526.4524.2968.1411.0054.161SG 206ATOM206CASP2525.1941.9205.4001.0054.161SG 207ATOM207OASP2526.3801.8945.7241.0054.161SG 208ATOM208NPRO2624.8401.7784.1461.0053.801SG 209ATOM209CAPRO2625.7881.7203.0511.0053.801SG 210ATOM210CDPRO2623.4982.1003.6921.0053.801SG 211ATOM211CBPRO2624.9291.6371.7901.0053.801SG 212ATOM212CGPRO2623.6442.3862.1881.0053.801SG 213ATOM213CPRO2626.7512.8762.9281.0053.801SG 214ATOM214OPRO2627.9222.7133.2601.0053.801SG 215ATOM215NVAL2726.2854.0652.4741.0051.241SG 216ATOM216CAVAL2727.1935.1722.2871.0051.241SG 217ATOM217CBVAL2728.0955.0121.0951.0051.241SG 218ATOM218CG1VAL2727.2304.971−0.1761.0051.241SG 219ATOM219CG2VAL2729.1236.1581.1051.0051.241SG 220ATOM220CVAL2726.4106.4312.0581.0051.241SG 221ATOM221OVAL2725.3496.4261.4381.0051.241SG 222ATOM222NALA2826.9477.5652.5491.0046.541SG 223ATOM223CAALA2826.3058.8362.3721.0046.541SG 224ATOM224CBALA2825.4779.2673.5951.0046.541SG 225ATOM225CALA2827.3819.8642.2041.0046.541SG 226ATOM226OALA2828.4239.8032.8521.0046.541SG 227ATOM227NASP2927.15610.8481.3161.0046.281SG 228ATOM228CAASP2928.11911.8901.1271.0046.281SG 229ATOM229CBASP2928.44012.177−0.3551.0046.281SG 230ATOM230CGASP2927.17412.563−1.1051.0046.281SG 231ATOM231OD1ASP2926.17311.804−1.0191.0046.281SG 232ATOM232OD2ASP2927.20213.622−1.7881.0046.281SG 233ATOM233CASP2927.57513.1141.7881.0046.281SG 234ATOM234OASP2927.11613.0582.9281.0046.281SG 235ATOM235NASN3027.61814.2631.0951.0048.861SG 236ATOM236CAASN3027.16415.4671.7161.0048.861SG 237ATOM237CBASN3027.22816.6800.7741.0048.861SG 238ATOM238CGASN3028.69216.9930.5101.0048.861SG 239ATOM239OD1ASN3029.59116.4451.1461.0048.861SG 240ATOM240ND2ASN3028.93917.917−0.4541.0048.861SG 241ATOM241CASN3025.73515.2922.1171.0048.861SG 242ATOM242OASN3025.39915.4853.2851.0048.861SG 243ATOM243NCYS3124.84914.9041.1751.0056.831SG 244ATOM244CACYS3123.48214.7391.5821.0056.831SG 245ATOM245CBCYS3122.89615.9822.2761.0056.831SG 246ATOM246SGCYS3121.16715.7512.7861.0056.831SG 247ATOM247CCYS3122.61714.4800.3871.0056.831SG 248ATOM248OCYS3122.85815.003−0.7021.0056.831SG 249ATOM249NCYS3221.58213.6390.5921.0066.991SG 250ATOM250CACYS3220.56813.312−0.3741.0066.991SG 251ATOM251CBCYS3220.97212.185−1.3411.0066.991SG 252ATOM252SGCYS3221.32710.625−0.4801.0066.991SG 253ATOM253CCYS3219.40212.8290.4521.0066.991SG 254ATOM254OCYS3219.62312.3481.5601.0066.991SG 255ATOM255NALA3318.14112.888−0.0511.0071.351SG 256ATOM256CAALA3317.05512.6640.8761.0071.351SG 257ATOM257CBALA3316.26413.9411.2011.0071.351SG 258ATOM258CALA3316.04411.6350.4541.0071.351SG 259ATOM259OALA3315.72111.460−0.7201.0071.351SG 260ATOM260NGLU3415.53310.9401.4971.0072.501SG 261ATOM261CAGLU3414.5669.8731.5841.0072.501SG 262ATOM262CBGLU3414.6689.1012.9121.0072.501SG 263ATOM263CGGLU3413.8207.8282.9551.0072.501SG 264ATOM264CDGLU3414.4896.7812.0751.0072.501SG 265ATOM265OE1GLU3415.3017.1721.1941.0072.501SG 266ATOM266OE2GLU3414.1915.5732.2711.0072.501SG 267ATOM267CGLU3413.13710.3331.4351.0072.501SG 268ATOM268OGLU3412.2429.5031.2811.0072.501SG 269ATOM269NLYS3512.86711.6441.5771.0067.331SG 270ATOM270CALYS3511.53812.2091.5591.0067.331SG 271ATOM271CBLYS3511.60413.7201.2931.0067.331SG 272ATOM272CGLYS3510.25914.4371.2461.0067.331SG 273ATOM273CDLYS3510.41815.9491.0761.0067.331SG 274ATOM274CELYS3510.58516.390−0.3791.0067.331SG 275ATOM275NZLYS3510.68917.864−0.4561.0067.331SG 276ATOM276CLYS3510.68211.6070.4721.0067.331SG 277ATOM277OLYS3511.03411.643−0.7051.0067.331SG 278ATOM278NILE369.51911.0310.8721.0061.391SG 279ATOM279CAILE368.52710.454−0.0081.0061.391SG 280ATOM280CBILE368.5678.950−0.0451.0061.391SG 281ATOM281CG2ILE367.2278.432−0.5911.0061.391SG 282ATOM282CG1ILE369.7658.467−0.8691.0061.391SG 283ATOM283CD1ILE369.5988.822−2.3441.0061.391SG 284ATOM284CILE367.16910.8640.4981.0061.391SG 285ATOM285OILE366.97211.0271.7001.0061.391SG 286ATOM286NCYS376.17811.047−0.4041.0056.421SG 287ATOM287CACYS374.87711.4560.0561.0056.421SG 288ATOM288CBCYS374.17412.446−0.8901.0056.421SG 289ATOM289SGCYS372.56112.996−0.2541.0056.421SG 290ATOM290CCYS374.00910.2370.1791.0056.421SG 291ATOM291OCYS373.9739.390−0.7111.0056.421SG 292ATOM292NILE383.27010.1151.3031.0054.661SG 293ATOM293CAILE382.4558.9401.4691.0054.661SG 294ATOM294CBILE382.6218.2382.7831.0054.661SG 295ATOM295CG2ILE381.5437.1422.8531.0054.661SG 296ATOM296CG1ILE384.0497.7062.9521.0054.661SG 297ATOM297CD1ILE384.2927.1154.3381.0054.661SG 298ATOM298CILE380.9969.2911.3961.0054.661SG 299ATOM299OILE380.53210.1922.0931.0054.661SG 300ATOM300NLEU390.2308.5830.5271.0056.971SG 301ATOM301CALEU39−1.1978.7880.4791.0056.971SG 302ATOM302CBLEU39−1.7109.370−0.8481.0056.971SG 303ATOM303CGLEU39−1.27810.830−1.0831.0056.971SG 304ATOM304CD2LEU39−2.06611.476−2.2341.0056.971SG 305ATOM305CD1LEU390.24410.944−1.2561.0056.971SG 306ATOM306CLEU39−1.8967.4700.7361.0056.971SG 307ATOM307OLEU39−1.5356.4420.1661.0056.971SG 308ATOM308NPRO40−2.8207.4831.6811.0057.041SG 309ATOM309CAPRO40−3.5886.2922.0211.0057.041SG 310ATOM310CDPRO40−2.5208.2642.8741.0057.041SG 311ATOM311CBPRO40−3.5856.2093.5471.0057.041SG 312ATOM312CGPRO40−3.3727.6593.9991.0057.041SG 313ATOM313CPRO40−4.9966.2421.4731.0057.041SG 314ATOM314OPRO40−5.5057.2801.0551.0057.041SG 315ATOM315NASN41−5.6405.0421.4621.0058.991SG 316ATOM316CAASN41−7.0334.9051.0921.0058.991SG 317ATOM317CBASN41−7.5613.4561.1081.0058.991SG 318ATOM318CGASN41−8.9223.4540.4141.0058.991SG 319ATOM319OD1ASN41−9.8384.1820.7941.0058.991SG 320ATOM320ND2ASN41−9.0562.620−0.6521.0058.991SG 321ATOM321CASN41−7.8675.5492.1221.0058.991SG 322ATOM322OASN41−8.7036.4111.8521.0058.991SG 323ATOM323NARG42−7.6105.0913.3521.0061.651SG 324ATOM324CAARG42−8.3325.4314.5281.0061.651SG 325ATOM325CBARG42−9.6504.6614.6581.0061.651SG 326ATOM326CGARG42−10.6454.9723.5401.0061.651SG 327ATOM327CDARG42−11.8994.1023.5861.0061.651SG 328ATOM328NEARG42−11.4612.6933.3861.0061.651SG 329ATOM329CZARG42−12.3731.6813.4581.0061.651SG 330ATOM330NH1ARG42−13.6881.9623.6941.0061.651SG 331ATOM331NH2ARG42−11.9660.3873.2951.0061.651SG 332ATOM332CARG42−7.4544.9555.6341.0061.651SG 333ATOM333OARG42−6.6415.7116.1631.0061.651SG 334ATOM334NGLY43−7.6293.6756.0281.0065.681SG 335ATOM335CAGLY43−6.8463.1137.0901.0065.681SG 336ATOM336CGLY43−5.4332.8616.6471.0065.681SG 337ATOM337OGLY43−4.9471.7336.7131.0065.681SG 338ATOM338NLEU44−4.7413.9106.1701.0075.221SG 339ATOM339CALEU44−3.3313.8865.8851.0075.221SG 340ATOM340CBLEU44−2.4913.2687.0181.0075.221SG 341ATOM341CGLEU44−2.5414.0408.3471.0075.221SG 342ATOM342CD2LEU44−2.1835.5198.1481.0075.221SG 343ATOM343CD1LEU44−1.6753.3579.4171.0075.221SG 344ATOM344CLEU44−3.0113.0794.6571.0075.221SG 345ATOM345OLEU44−2.0853.4293.9281.0075.221SG 346ATOM346NASP45−3.8092.0274.3681.0083.451SG 347ATOM347CAASP45−3.6081.1093.2761.0083.451SG 348ATOM348CBASP45−4.325−0.2383.4731.0083.451SG 349ATOM349CGASP45−3.689−0.9664.6471.0083.451SG 350ATOM350OD1ASP45−2.519−0.6444.9861.0083.451SG 351ATOM351OD2ASP45−4.369−1.8565.2241.0083.451SG 352ATOM352CASP45−4.2871.7672.1301.0083.451SG 353ATOM353OASP45−4.1192.9711.9851.0083.451SG 354ATOM354NARG46−4.9750.9881.2491.0085.551SG 355ATOM355CAARG46−5.6471.4730.0581.0085.551SG 356ATOM356CBARG46−4.9862.742−0.5491.0085.551SG 357ATOM357CGARG46−5.9503.634−1.3101.0085.551SG 358ATOM358CDARG46−5.6485.146−1.4461.0085.551SG 359ATOM359NEARG46−4.1935.443−1.5401.0085.551SG 360ATOM360CZARG46−3.8336.703−1.9211.0085.551SG 361ATOM361NH1ARG46−4.7927.669−2.0261.0085.551SG 362ATOM362NH2ARG46−2.5407.000−2.2261.0085.551SG 363ATOM363CARG46−5.5140.346−0.9351.0085.551SG 364ATOM364OARG46−5.227−0.776−0.5191.0085.551SG 365ATOM365NTHR47−5.7120.588−2.2601.0082.811SG 366ATOM366CATHR47−5.374−0.404−3.2681.0082.811SG 367ATOM367CBTHR47−6.409−1.482−3.4761.0082.811SG 368ATOM368OG1THR47−7.687−0.898−3.6421.0082.811SG 369ATOM369CG2THR47−6.445−2.462−2.2941.0082.811SG 370ATOM370CTHR47−5.0690.262−4.6001.0082.811SG 371ATOM371OTHR47−5.5861.338−4.8981.0082.811SG 372ATOM372NLYS48−4.191−0.377−5.4241.0079.661SG 373ATOM373CALYS48−3.7430.033−6.7481.0079.661SG 374ATOM374CBLYS48−4.7320.809−7.6461.0079.661SG 375ATOM375CGLYS48−4.4360.722−9.1511.0079.661SG 376ATOM376CDLYS48−5.6520.996−10.0391.0079.661SG 377ATOM377CELYS48−6.594−0.209−10.1421.0079.661SG 378ATOM378NZLYS48−7.7500.112−11.0101.0079.661SG 379ATOM379CLYS48−2.6040.962−6.5821.0079.661SG 380ATOM380OLYS48−1.8020.828−5.6631.0079.661SG 381ATOM381NVAL49−2.5491.965−7.4811.0077.691SG 382ATOM382CAVAL49−1.6033.034−7.4101.0077.691SG 383ATOM383CBVAL49−1.9104.144−8.3761.0077.691SG 384ATOM384CG1VAL49−0.9345.309−8.1291.0077.691SG 385ATOM385CG2VAL49−1.8603.567−9.8031.0077.691SG 386ATOM386CVAL49−1.7453.536−6.0111.0077.691SG 387ATOM387OVAL49−0.7723.947−5.4011.0077.691SG 388ATOM388NPRO50−2.9283.481−5.4731.0081.221SG 389ATOM389CAPRO50−3.0933.780−4.0781.0081.221SG 390ATOM390CDPRO50−4.0454.050−6.2311.0081.221SG 391ATOM391CBPRO50−4.5903.799−3.9311.0081.221SG 392ATOM392CGPRO50−5.0734.492−5.1971.0081.221SG 393ATOM393CPRO50−2.2802.994−3.0591.0081.221SG 394ATOM394OPRO50−1.5772.067−3.4621.0081.221SG 395ATOM395NILE51−2.4403.350−1.7441.0085.661SG 396ATOM396CAILE51−1.5873.232−0.6261.0085.661SG 397ATOM397CBILE51−1.3351.9570.0441.0085.661SG 398ATOM398CG2ILE51−0.9542.3381.4861.0085.661SG 399ATOM399CG1ILE51−2.4961.010−0.0391.0085.661SG 400ATOM400CD1ILE51−2.6900.573−1.4561.0085.661SG 401ATOM401CILE51−0.3313.247−1.3541.0085.661SG 402ATOM402OILE510.2242.187−1.6481.0085.661SG 403ATOM403NPHE520.1534.443−1.6671.0083.411SG 404ATOM404CAPHE521.3774.400−2.3851.0083.411SG 405ATOM405CBPHE521.3384.682−3.8881.0083.411SG 406ATOM406CGPHE520.9976.115−4.1201.0083.411SG 407ATOM407CD1PHE52−0.2836.572−3.9291.0083.411SG 408ATOM408CD2PHE521.9556.999−4.5581.0083.411SG 409ATOM409CE1PHE52−0.6027.891−4.1481.0083.411SG 410ATOM410CE2PHE521.6448.320−4.7801.0083.411SG 411ATOM411CZPHE520.3638.771−4.5721.0083.411SG 412ATOM412CPHE522.2385.478−1.8701.0083.411SG 413ATOM413OPHE521.8876.191−0.9331.0083.411SG 414ATOM414NLEU533.4185.585−2.4931.0078.561SG 415ATOM415CALEU534.3726.566−2.0861.0078.561SG 416ATOM416CBLEU535.6125.924−1.4381.0078.561SG 417ATOM417CGLEU535.2974.976−0.2611.0078.561SG 418ATOM418CD2LEU534.3935.6440.7861.0078.561SG 419ATOM419CD1LEU536.5824.3800.3391.0078.561SG 420ATOM420CLEU534.8547.291−3.3171.0078.561SG 421ATOM421OLEU534.9316.694−4.3901.0078.561SG 422ATOM422NGLY545.1788.607−3.2201.0070.601SG 423ATOM423CAGLY545.7199.237−4.4011.0070.601SG 424ATOM424CGLY546.19510.638−4.1351.0070.601SG 425ATOM425OGLY545.40011.486−3.7371.0070.601SG 426ATOM426NILE557.51710.892−4.3531.0062.731SG 427ATOM427CAILE558.19212.176−4.2731.0062.731SG 428ATOM428CBILE558.30112.845−2.9101.0062.731SG 429ATOM429CG2ILE559.02814.185−3.1321.0062.731SG 430ATOM430CG1ILE556.95013.168−2.2721.0062.731SG 431ATOM431CD1ILE556.13814.174−3.0891.0062.731SG 432ATOM432CILE559.61611.941−4.7171.0062.731SG 433ATOM433OILE559.90211.796−5.9041.0062.731SG 434ATOM434NGLN5610.54511.904−3.7281.0054.761SG 435ATOM435CAGLN5611.96911.738−3.8751.0054.761SG 436ATOM436CBGLN5612.41510.369−4.4241.0054.761SG 437ATOM437CGGLN5612.3139.219−3.4201.0054.761SG 438ATOM438CDGLN5612.9047.975−4.0721.0054.761SG 439ATOM439OE1GLN5613.8618.062−4.8401.0054.761SG 440ATOM440NE2GLN5612.3196.788−3.7601.0054.761SG 441ATOM441CGLN5612.57812.795−4.7441.0054.761SG 442ATOM442OGLN5613.28012.494−5.7091.0054.761SG 443ATOM443NGLY5712.28214.073−4.4381.0049.121SG 444ATOM444CAGLY5712.94315.172−5.0791.0049.121SG 445ATOM445CGLY5712.07615.715−6.1611.0049.121SG 446ATOM446OGLY5711.75316.902−6.1671.0049.121SG 447ATOM447NGLY5811.64914.860−7.1071.0045.251SG 448ATOM448CAGLY5810.83215.410−8.1411.0045.251SG 449ATOM449CGLY5810.18214.303−8.9071.0045.251SG 450ATOM450OGLY5810.75413.751−9.8461.0045.251SG 451ATOM451NSER598.92314.013−8.5311.0045.651SG 452ATOM452CASER598.03413.084−9.1751.0045.651SG 453ATOM453CBSER597.52913.586−10.5411.0045.651SG 454ATOM454OGSER598.59213.606−11.4831.0045.651SG 455ATOM455CSER598.58611.699−9.3761.0045.651SG 456ATOM456OSER598.56711.189−10.4961.0045.651SG 457ATOM457NARG609.08811.039−8.3121.0050.201SG 458ATOM458CAARG609.4559.657−8.4671.0050.201SG 459ATOM459CBARG6010.8939.337−8.0181.0050.201SG 460ATOM460CGARG6011.9149.981−8.9591.0050.201SG 461ATOM461CDARG6013.3319.413−8.8701.0050.201SG 462ATOM462NEARG6013.9749.939−7.6351.0050.201SG 463ATOM463CZARG6015.3369.979−7.5561.0050.201SG 464ATOM464NH1ARG6016.0919.573−8.6181.0050.201SG 465ATOM465NH2ARG6015.94410.429−6.4201.0050.201SG 466ATOM466CARG608.4668.869−7.6521.0050.201SG 467ATOM467OARG608.3289.077−6.4491.0050.201SG 468ATOM468NCYS617.7447.923−8.2911.0053.331SG 469ATOM469CACYS616.6897.234−7.5951.0053.331SG 470ATOM470CBCYS615.4257.069−8.4641.0053.331SG 471ATOM471SGCYS614.0656.207−7.6201.0053.331SG 472ATOM472CCYS617.1365.867−7.1591.0053.331SG 473ATOM473OCYS617.6275.070−7.9581.0053.331SG 474ATOM474NLEU626.9645.566−5.8491.0056.921SG 475ATOM475CALEU627.2924.264−5.3341.0056.921SG 476ATOM476CBLEU627.7074.220−3.8481.0056.921SG 477ATOM477CGLEU629.1574.668−3.5731.0056.921SG 478ATOM478CD2LEU629.5804.316−2.1381.0056.921SG 479ATOM479CD1LEU629.3856.146−3.9161.0056.921SG 480ATOM480CLEU626.0993.381−5.5061.0056.921SG 481ATOM481OLEU625.0353.631−4.9371.0056.921SG 482ATOM482NALA636.2872.308−6.3081.0055.921SG 483ATOM483CAALA635.2491.375−6.6571.0055.921SG 484ATOM484CBALA634.6091.687−8.0211.0055.921SG 485ATOM485CALA635.8590.000−6.7641.0055.921SG 486ATOM486OALA637.074−0.166−6.6731.0055.921SG 487ATOM487NCYS645.008−1.042−6.9091.0054.101SG 488ATOM488CACYS645.479−2.389−7.0861.0054.101SG 489ATOM489CBCYS644.550−3.436−6.4521.0054.101SG 490ATOM490SGCYS644.399−3.210−4.6551.0054.101SG 491ATOM491CCYS645.496−2.628−8.5731.0054.101SG 492ATOM492OCYS644.482−2.990−9.1621.0054.101SG 493ATOM493NVAL656.669−2.465−9.2221.0052.491SG 494ATOM494CAVAL656.782−2.513−10.6651.0052.491SG 495ATOM495CBVAL658.032−1.844−11.1631.0052.491SG 496ATOM496CG1VAL658.114−1.972−12.6901.0052.491SG 497ATOM497CG2VAL658.017−0.388−10.6811.0052.491SG 498ATOM498CVAL656.777−3.931−11.1661.0052.491SG 499ATOM499OVAL657.153−4.855−10.4501.0052.491SG 500ATOM500NGLU666.328−4.150−12.4271.0049.661SG 501ATOM501CAGLU666.253−5.496−12.9181.0049.661SG 502ATOM502CBGLU664.897−5.874−13.5491.0049.661SG 503ATOM503CGGLU663.727−5.980−12.5641.0049.661SG 504ATOM504CDGLU663.125−4.595−12.3541.0049.661SG 505ATOM505OE1GLU663.826−3.708−11.7961.0049.661SG 506ATOM506OE2GLU661.946−4.407−12.7551.0049.661SG 507ATOM507CGLU667.281−5.726−13.9671.0049.661SG 508ATOM508OGLU667.236−5.144−15.0511.0049.661SG 509ATOM509NTHR678.259−6.592−13.6521.0047.841SG 510ATOM510CATHR679.128−7.002−14.7001.0047.841SG 511ATOM511CBTHR6710.473−7.508−14.2651.0047.841SG 512ATOM512OG1THR6711.353−7.552−15.3781.0047.841SG 513ATOM513CG2THR6710.310−8.924−13.6941.0047.841SG 514ATOM514CTHR678.355−8.150−15.2391.0047.841SG 515ATOM515OTHR677.242−8.397−14.7791.0047.841SG 516ATOM516NGLU688.852−8.870−16.2501.0046.721SG 517ATOM517CAGLU688.000−9.946−16.6601.0046.721SG 518ATOM518CBGLU688.509−10.736−17.8771.0046.721SG 519ATOM519CGGLU687.553−11.873−18.2591.0046.721SG 520ATOM520CDGLU688.228−12.752−19.3011.0046.721SG 521ATOM521OE1GLU689.049−12.210−20.0881.0046.721SG 522ATOM522OE2GLU687.938−13.979−19.3191.0046.721SG 523ATOM523CGLU687.902−10.940−15.5481.0046.721SG 524ATOM524OGLU686.805−11.305−15.1251.0046.721SG 525ATOM525NGLU699.067−11.369−15.0271.0046.101SG 526ATOM526CAGLU699.118−12.419−14.0511.0046.101SG 527ATOM527CBGLU6910.565−12.836−13.7581.0046.101SG 528ATOM528CGGLU6911.352−13.198−15.0191.0046.101SG 529ATOM529CDGLU6911.792−11.888−15.6641.0046.101SG 530ATOM530OE1GLU6912.402−11.055−14.9391.0046.101SG 531ATOM531OE2GLU6911.527−11.698−16.8801.0046.101SG 532ATOM532CGLU698.528−12.003−12.7421.0046.101SG 533ATOM533OGLU697.571−12.599−12.2511.0046.101SG 534ATOM534NGLY709.034−10.915−12.1461.0046.581SG 535ATOM535CAGLY708.525−10.675−10.8321.0046.581SG 536ATOM536CGLY708.540−9.209−10.5421.0046.581SG 537ATOM537OGLY709.152−8.419−11.2581.0046.581SG 538ATOM538NPRO717.776−8.841−9.5411.0048.891SG 539ATOM539CAPRO717.766−7.452−9.1411.0048.891SG 540ATOM540CDPRO716.437−9.409−9.4811.0048.891SG 541ATOM541CBPRO716.301−7.073−8.9201.0048.891SG 542ATOM542CGPRO715.599−8.411−8.6701.0048.891SG 543ATOM543CPRO718.623−7.095−7.9491.0048.891SG 544ATOM544OPRO718.676−7.874−6.9971.0048.891SG 545ATOM545NSER729.202−5.869−7.9371.0052.251SG 546ATOM546CASER7210.027−5.386−6.8581.0052.251SG 547ATOM547CBSER7211.533−5.496−7.1561.0052.251SG 548ATOM548OGSER7211.900−6.860−7.3051.0052.251SG 549ATOM549CSER729.716−3.925−6.6601.0052.251SG 550ATOM550OSER729.029−3.313−7.4771.0052.251SG 551ATOM551NLEU7310.200−3.332−5.5431.0056.411SG 552ATOM552CALEU739.942−1.944−5.2431.0056.411SG 553ATOM553CBLEU7310.215−1.624−3.7631.0056.411SG 554ATOM554CGLEU739.954−0.167−3.3581.0056.411SG 555ATOM555CD2LEU7310.3840.077−1.9051.0056.411SG 556ATOM556CD1LEU738.4930.230−3.6101.0056.411SG 557ATOM557CLEU7310.809−1.069−6.1071.0056.411SG 558ATOM558OLEU7312.017−1.286−6.2091.0056.411SG 559ATOM559NGLN7410.210−0.035−6.7451.0058.131SG 560ATOM560CAGLN7410.9800.815−7.6111.0058.131SG 561ATOM561CBGLN7410.9640.374−9.0771.0058.131SG 562ATOM562CGGLN7411.7931.311−9.9481.0058.131SG 563ATOM563CDGLN7411.5700.904−11.3861.0058.131SG 564ATOM564OE1GLN7411.127−0.211−11.6551.0058.131SG 565ATOM565NE2GLN7411.8741.835−12.3291.0058.131SG 566ATOM566CGLN7410.4362.215−7.6041.0058.131SG 567ATOM567OGLN749.2542.440−7.3411.0058.131SG 568ATOM568NLEU7511.3173.201−7.8991.0057.601SG 569ATOM569CALEU7510.9224.581−8.0001.0057.601SG 570ATOM570CBLEU7511.8655.553−7.2511.0057.601SG 571ATOM571CGLEU7513.3455.118−7.1201.0057.601SG 572ATOM572CD2LEU7514.0444.945−8.4741.0057.601SG 573ATOM573CD1LEU7513.4863.883−6.2171.0057.601SG 574ATOM574CLEU7510.8614.943−9.4591.0057.601SG 575ATOM575OLEU7511.8635.291−10.0811.0057.601SG 576ATOM576NGLU769.6464.903−10.0421.0053.821SG 577ATOM577CAGLU769.4795.166−11.4451.0053.821SG 578ATOM578CBGLU768.3084.373−12.0641.0053.821SG 579ATOM579CGGLU766.9594.659−11.3941.0053.821SG 580ATOM580CDGLU765.8803.832−12.0831.0053.821SG 581ATOM581OE1GLU765.7472.627−11.7321.0053.821SG 582ATOM582OE2GLU765.1804.388−12.9691.0053.821SG 583ATOM583CGLU769.2076.631−11.6351.0053.821SG 584ATOM584OGLU768.4797.245−10.8571.0053.821SG 585ATOM585NASP779.8057.235−12.6871.0049.181SG 586ATOM586CAASP779.6128.637−12.9401.0049.181SG 587ATOM587CBASP7710.6859.258−13.8531.0049.181SG 588ATOM588CGASP7711.9939.332−13.0771.0049.181SG 589ATOM589OD1ASP7712.0078.894−11.8961.0049.181SG 590ATOM590OD2ASP7712.9949.835−13.6541.0049.181SG 591ATOM591CASP778.2938.804−13.6241.0049.181SG 592ATOM592OASP778.0918.307−14.7311.0049.181SG 593ATOM593NVAL787.3539.521−12.9731.0047.591SG 594ATOM594CAVAL786.0579.685−13.5681.0047.591SG 595ATOM595CBVAL784.9239.505−12.5961.0047.591SG 596ATOM596CG1VAL785.00110.596−11.5141.0047.591SG 597ATOM597CG2VAL783.6019.479−13.3841.0047.591SG 598ATOM598CVAL785.96311.043−14.2011.0047.591SG 599ATOM599OVAL786.09212.078−13.5481.0047.591SG 600ATOM600NASN795.73411.042−15.5281.0047.001SG 601ATOM601CAASN795.65812.196−16.3821.0047.001SG 602ATOM602CBASN795.70011.835−17.8741.0047.001SG 603ATOM603CGASN797.12011.417−18.2261.0047.001SG 604ATOM604OD1ASN797.42110.232−18.3541.0047.001SG 605ATOM605ND2ASN798.02012.421−18.4001.0047.001SG 606ATOM606CASN794.41913.016−16.1611.0047.001SG 607ATOM607OASN794.43214.219−16.4201.0047.001SG 608ATOM608NILE803.30512.405−15.7081.0048.121SG 609ATOM609CAILE802.06613.141−15.6581.0048.121SG 610ATOM610CBILE800.89912.322−16.1271.0048.121SG 611ATOM611CG2ILE800.78811.081−15.2261.0048.121SG 612ATOM612CG1ILE80−0.37313.179−16.1801.0048.121SG 613ATOM613CD1ILE80−1.52412.502−16.9171.0048.121SG 614ATOM614CILE801.74713.618−14.2681.0048.121SG 615ATOM615OILE801.69112.840−13.3191.0048.121SG 616ATOM616NGLU811.57814.950−14.1251.0050.041SG 617ATOM617CAGLU811.24415.589−12.8801.0050.041SG 618ATOM618CBGLU811.53217.100−12.9161.0050.041SG 619ATOM619CGGLU813.00417.437−13.1831.0050.041SG 620ATOM620CDGLU813.86116.850−12.0681.0050.041SG 621ATOM621OE1GLU813.87315.598−11.9251.0050.041SG 622ATOM622OE2GLU814.52317.646−11.3501.0050.041SG 623ATOM623CGLU81−0.20615.397−12.5121.0050.041SG 624ATOM624OGLU81−0.53515.164−11.3501.0050.041SG 625ATOM625NGLU82−1.11415.502−13.5041.0052.621SG 626ATOM626CAGLU82−2.53315.506−13.2581.0052.621SG 627ATOM627CBGLU82−3.34615.831−14.5251.0052.621SG 628ATOM628CGGLU82−4.84016.034−14.2631.0052.621SG 629ATOM629CDGLU82−5.53214.679−14.3041.0052.621SG 630ATOM630OE1GLU82−5.49414.034−15.3861.0052.621SG 631ATOM631OE2GLU82−6.10714.270−13.2601.0052.621SG 632ATOM632CGLU82−3.02714.200−12.7211.0052.621SG 633ATOM633OGLU82−3.77514.175−11.7441.0052.621SG 634ATOM634NLEU83−2.61013.075−13.3261.0056.831SG 635ATOM635CALEU83−3.11211.795−12.9151.0056.831SG 636ATOM636CBLEU83−2.65110.687−13.8861.0056.831SG 637ATOM637CGLEU83−3.0039.238−13.5031.0056.831SG 638ATOM638CD2LEU83−4.5059.074−13.2301.0056.831SG 639ATOM639CD1LEU83−2.1228.732−12.3511.0056.831SG 640ATOM640CLEU83−2.61411.512−11.5341.0056.831SG 641ATOM641OLEU83−1.41011.435−11.2961.0056.831SG 642ATOM642NTYR84−3.54911.364−10.5731.0063.401SG 643ATOM643CATYR84−3.15411.085−9.2241.0063.401SG 644ATOM644CBTYR84−3.21212.343−8.3431.0063.401SG 645ATOM645CGTYR84−2.23812.180−7.2331.0063.401SG 646ATOM646CD1TYR84−2.49111.399−6.1301.0063.401SG 647ATOM647CD2TYR84−1.03612.843−7.3251.0063.401SG 648ATOM648CE1TYR84−1.54411.291−5.1351.0063.401SG 649ATOM649CE2TYR84−0.09012.739−6.3351.0063.401SG 650ATOM650CZTYR84−0.34611.960−5.2361.0063.401SG 651ATOM651OHTYR840.62111.848−4.2151.0063.401SG 652ATOM652CTYR84−4.19810.135−8.7161.0063.401SG 653ATOM653OTYR84−5.35510.206−9.1231.0063.401SG 654ATOM654NLYS85−3.8399.209−7.8141.0072.581SG 655ATOM655CALYS85−4.8568.315−7.3541.0072.581SG 656ATOM656CBLYS85−4.4286.838−7.3651.0072.581SG 657ATOM657CGLYS85−4.2696.258−8.7741.0072.581SG 658ATOM658CDLYS85−5.5376.382−9.6241.0072.581SG 659ATOM659CELYS85−5.4965.585−10.9311.0072.581SG 660ATOM660NZLYS85−4.4506.118−11.8321.0072.581SG 661ATOM661CLYS85−5.1738.680−5.9491.0072.581SG 662ATOM662OLYS85−4.4149.381−5.2841.0072.581SG 663ATOM663NGLY86−6.2938.160−5.4281.0079.591SG 664ATOM664CAGLY86−6.6008.530−4.0861.0079.591SG 665ATOM665CGLY86−7.6127.565−3.5751.0079.591SG 666ATOM666OGLY86−8.5627.929−2.8931.0079.591SG 667ATOM667NGLY87−7.2816.269−3.7141.0082.841SG 668ATOM668CAGLY87−8.1435.212−3.2821.0082.841SG 669ATOM669CGLY87−7.8623.982−4.1041.0082.841SG 670ATOM670OGLY87−7.2913.040−3.5771.0082.841SG 671ATOM671NGLU88−8.1573.975−5.4111.0081.601SG 672ATOM672CAGLU88−7.9802.864−6.3291.0081.601SG 673ATOM673CBGLU88−6.7032.910−7.1681.0081.601SG 674ATOM674CGGLU88−6.6534.106−8.1181.0081.601SG 675ATOM675CDGLU88−7.8984.060−8.9911.0081.601SG 676ATOM676OE1GLU88−8.1263.017−9.6601.0081.601SG 677ATOM677OE2GLU88−8.6455.075−8.9941.0081.601SG 678ATOM678CGLU88−8.2251.460−5.7871.0081.601SG 679ATOM679OGLU88−8.6631.254−4.6571.0081.601SG 680ATOM680NGLU89−8.0130.431−6.6561.0075.521SG 681ATOM681CAGLU89−8.298−0.958−6.3401.0075.521SG 682ATOM682CBGLU89−9.371−1.608−7.2381.0075.521SG 683ATOM683CGGLU89−10.804−1.270−6.8131.0075.521SG 684ATOM684CDGLU89−11.217−2.282−5.7491.0075.521SG 685ATOM685OE1GLU89−10.499−3.307−5.6001.0075.521SG 686ATOM686OE2GLU89−12.257−2.049−5.0761.0075.521SG 687ATOM687CGLU89−7.054−1.811−6.4261.0075.521SG 688ATOM688OGLU89−6.055−1.435−7.0191.0075.521SG 689ATOM689NALA90−7.066−3.024−5.8441.0073.231SG 690ATOM690CAALA90−5.868−3.821−5.8121.0073.231SG 691ATOM691CBALA90−6.075−5.191−5.1421.0073.231SG 692ATOM692CALA90−5.299−4.073−7.1881.0073.231SG 693ATOM693OALA90−5.976−4.582−8.0811.0073.231SG 694ATOM694NTHR91−4.002−3.715−7.3581.0076.691SG 695ATOM695CATHR91−3.218−3.923−8.5541.0076.691SG 696ATOM696CBTHR91−3.348−2.825−9.5651.0076.691SG 697ATOM697OG1THR91−2.778−3.225−10.8021.0076.691SG 698ATOM698CG2THR91−2.613−1.588−9.0241.0076.691SG 699ATOM699CTHR91−1.789−3.897−8.0811.0076.691SG 700ATOM700OTHR91−1.546−3.688−6.8991.0076.691SG 701ATOM701NARG92−0.779−4.097−8.9571.0083.501SG 702ATOM702CAARG920.550−4.076−8.4041.0083.501SG 703ATOM703CBARG921.571−4.950−9.1621.0083.501SG 704ATOM704CGARG921.360−6.457−8.9881.0083.501SG 705ATOM705CDARG922.509−7.298−9.5531.0083.501SG 706ATOM706NEARG922.189−8.736−9.3211.0083.501SG 707ATOM707CZARG922.507−9.327−8.1331.0083.501SG 708ATOM708NH1ARG923.084−8.594−7.1351.0083.501SG 709ATOM709NH2ARG922.253−10.654−7.9381.0083.501SG 710ATOM710CARG921.098−2.673−8.3851.0083.501SG 711ATOM711OARG922.048−2.377−9.1021.0083.501SG 712ATOM712NPHE930.386−1.709−7.7691.0091.531SG 713ATOM713CAPHE930.882−0.395−7.4071.0091.531SG 714ATOM714CBPHE930.1160.754−8.0681.0091.531SG 715ATOM715CGPHE930.7761.072−9.3691.0091.531SG 716ATOM716CD1PHE930.4550.407−10.5301.0091.531SG 717ATOM717CD2PHE931.7432.055−9.4151.0091.531SG 718ATOM718CE1PHE931.0840.726−11.7131.0091.531SG 719ATOM719CE2PHE932.3732.377−10.5951.0091.531SG 720ATOM720CZPHE932.0431.711−11.7501.0091.531SG 721ATOM721CPHE930.997−0.098−5.9181.0091.531SG 722ATOM722OPHE931.7510.783−5.5091.0091.531SG 723ATOM723NTHR940.159−0.778−5.1011.0092.251SG 724ATOM724CATHR94−0.205−0.526−3.7191.0092.251SG 725ATOM725CBTHR94−1.574−1.115−3.5511.0092.251SG 726ATOM726OG1THR94−1.700−1.825−2.3271.0092.251SG 727ATOM727CG2THR94−1.859−2.036−4.7421.0092.251SG 728ATOM728CTHR940.670−1.156−2.6661.0092.251SG 729ATOM729OTHR941.484−2.031−2.9501.0092.251SG 730ATOM730NPHE950.508−0.688−1.3921.0085.341SG 731ATOM731CAPHE951.202−1.286−0.2701.0085.341SG 732ATOM732CBPHE952.422−0.4810.2151.0085.341SG 733ATOM733CGPHE953.449−0.351−0.8541.0085.341SG 734ATOM734CD1PHE954.238−1.418−1.2151.0085.341SG 735ATOM735CD2PHE953.6400.864−1.4701.0085.341SG 736ATOM736CE1PHE955.190−1.277−2.1971.0085.341SG 737ATOM737CE2PHE954.5901.012−2.4511.0085.341SG 738ATOM738CZPHE955.366−0.062−2.8161.0085.341SG 739ATOM739CPHE950.297−1.2870.9361.0085.341SG 740ATOM740OPHE95−0.435−0.3261.1641.0085.341SG 741ATOM741NPHE960.299−2.3531.7701.0074.791SG 742ATOM742CAPHE96−0.453−2.1492.9721.0074.791SG 743ATOM743CBPHE96−1.538−3.1863.3761.0074.791SG 744ATOM744CGPHE96−1.065−4.5043.8931.0074.791SG 745ATOM745CD1PHE96−0.376−4.5925.0831.0074.791SG 746ATOM746CD2PHE96−1.389−5.6703.2341.0074.791SG 747ATOM747CE1PHE960.035−5.8095.5731.0074.791SG 748ATOM748CE2PHE96−0.985−6.8913.7241.0074.791SG 749ATOM749CZPHE96−0.265−6.9654.8921.0074.791SG 750ATOM750CPHE960.555−1.9704.0501.0074.791SG 751ATOM751OPHE961.472−2.7694.2121.0074.791SG 752ATOM752NGLN970.423−0.8634.7931.0062.311SG 753ATOM753CAGLN971.368−0.4995.7991.0062.311SG 754ATOM754CBGLN971.4381.0345.9151.0062.311SG 755ATOM755CGGLN972.4041.6076.9501.0062.311SG 756ATOM756CDGLN972.2633.1236.8621.0062.311SG 757ATOM757OE1GLN973.1563.8336.4021.0062.311SG 758ATOM758NE2GLN971.0803.6337.2991.0062.311SG 759ATOM759CGLN970.849−1.0397.0851.0062.311SG 760ATOM760OGLN97−0.081−0.4787.6581.0062.311SG 761ATOM761NSER981.448−2.1427.5821.0050.811SG 762ATOM762CASER980.973−2.7128.8091.0050.811SG 763ATOM763CBSER981.105−4.2438.8441.0050.811SG 764ATOM764OGSER980.624−4.75410.0771.0050.811SG 765ATOM765CSER981.816−2.1689.9161.0050.811SG 766ATOM766OSER982.841−2.74310.2711.0050.811SG 767ATOM767NSER991.372−1.06110.5371.0043.311SG 768ATOM768CASER992.198−0.48611.5581.0043.311SG 769ATOM769CBSER992.1941.05311.5501.0043.311SG 770ATOM770OGSER993.0321.54912.5841.0043.311SG 771ATOM771CSER991.694−0.93012.8861.0043.311SG 772ATOM772OSER990.573−0.62413.2891.0043.311SG 773ATOM773NSER1002.541−1.67613.6161.0037.371SG 774ATOM774CASER1002.133−2.10814.9131.0037.371SG 775ATOM775CBSER1002.444−3.59315.1641.0037.371SG 776ATOM776OGSER1003.826−3.84514.9521.0037.371SG 777ATOM777CSER1002.899−1.28615.8891.0037.371SG 778ATOM778OSER1003.984−1.66516.3261.0037.371SG 779ATOM779NGLY1012.323−0.14116.2941.0035.211SG 780ATOM780CAGLY1013.0360.71217.1951.0035.211SG 781ATOM781CGLY1014.1001.42516.4101.0035.211SG 782ATOM782OGLY1013.8232.03415.3781.0035.211SG 783ATOM783NSER1025.3491.39616.9211.0038.311SG 784ATOM784CASER1026.4642.09216.3321.0038.311SG 785ATOM785CBSER1027.6912.20917.2601.0038.311SG 786ATOM786OGSER1027.3873.03418.3741.0038.311SG 787ATOM787CSER1026.9761.48715.0441.0038.311SG 788ATOM788OSER1027.5592.21714.2451.0038.311SG 789ATOM789NALA1036.8140.16614.7901.0043.591SG 790ATOM790CAALA1037.451−0.40413.6171.0043.591SG 791ATOM791CBALA1038.435−1.54013.9561.0043.591SG 792ATOM792CALA1036.438−0.97112.6551.0043.591SG 793ATOM793OALA1035.323−1.30213.0521.0043.591SG 794ATOM794NPHE1046.801−1.09011.3441.0052.491SG 795ATOM795CAPHE1045.834−1.60810.4001.0052.491SG 796ATOM796CBPHE1045.040−0.5259.6561.0052.491SG 797ATOM797CGPHE1045.8670.1228.6041.0052.491SG 798ATOM798CD1PHE1046.9780.8588.9361.0052.491SG 799ATOM799CD2PHE1045.536−0.0397.2771.0052.491SG 800ATOM800CE1PHE1047.7281.4547.9511.0052.491SG 801ATOM801CE2PHE1046.2810.5596.2911.0052.491SG 802ATOM802CZPHE1047.3811.3096.6281.0052.491SG 803ATOM803CPHE1046.460−2.5279.3771.0052.491SG 804ATOM804OPHE1047.613−2.9369.5071.0052.491SG 805ATOM805NARG1055.644−2.9098.3541.0061.031SG 806ATOM806CAARG1055.980−3.8327.2931.0061.031SG 807ATOM807CBARG1055.433−5.2327.6021.0061.031SG 808ATOM808CGARG1055.822−5.7298.9971.0061.031SG 809ATOM809CDARG1055.102−7.0139.4051.0061.031SG 810ATOM810NEARG1055.506−7.32610.8051.0061.031SG 811ATOM811CZARG1056.100−8.51911.1011.0061.031SG 812ATOM812NH1ARG1056.333−9.43710.1171.0061.031SG 813ATOM813NH2ARG1056.458−8.79612.3881.0061.031SG 814ATOM814CARG1055.272−3.3726.0211.0061.031SG 815ATOM815OARG1054.242−2.7016.0881.0061.031SG 816ATOM816NLEU1065.792−3.7134.8101.0071.081SG 817ATOM817CALEU1065.107−3.2753.6061.0071.081SG 818ATOM818CBLEU1065.972−2.4462.6341.0071.081SG 819ATOM819CGLEU1066.471−1.0923.1621.0071.081SG 820ATOM820CD2LEU1067.461−1.2854.3141.0071.081SG 821ATOM821CD1LEU1065.308−0.1453.4911.0071.081SG 822ATOM822CLEU1064.592−4.3932.7221.0071.081SG 823ATOM823OLEU1065.344−5.0311.9901.0071.081SG 824ATOM824NGLU1073.264−4.6132.6801.0080.651SG 825ATOM825CAGLU1072.734−5.5981.7661.0080.651SG 826ATOM826CBGLU1071.306−6.0362.0981.0080.651SG 827ATOM827CGGLU1070.903−7.3661.4571.0080.651SG 828ATOM828CDGLU1071.626−8.4962.1871.0080.651SG 829ATOM829OE1GLU1072.414−8.1993.1251.0080.651SG 830ATOM830OE2GLU1071.397−9.6771.8131.0080.651SG 831ATOM831CGLU1072.737−4.9750.3861.0080.651SG 832ATOM832OGLU1072.674−3.7520.2601.0080.651SG 833ATOM833NALA1082.816−5.801−0.6901.0085.771SG 834ATOM834CAALA1082.943−5.291−2.0421.0085.771SG 835ATOM835CBALA1084.261−5.704−2.7211.0085.771SG 836ATOM836CALA1081.817−5.779−2.9071.0085.771SG 837ATOM837OALA1081.309−6.879−2.7001.0085.771SG 838ATOM838NALA1091.421−4.968−3.9231.0087.631SG 839ATOM839CAALA1090.192−5.226−4.6291.0087.631SG 840ATOM840CBALA109−0.072−6.686−5.0431.0087.631SG 841ATOM841CALA109−0.682−4.796−3.5181.0087.631SG 842ATOM842OALA109−0.260−3.864−2.8531.0087.631SG 843ATOM843NALA110−1.960−5.155−3.3461.0080.921SG 844ATOM844CAALA110−2.367−4.822−2.0011.0080.921SG 845ATOM845CBALA110−3.788−4.244−1.9381.0080.921SG 846ATOM846CALA110−2.329−5.995−1.0471.0080.921SG 847ATOM847OALA110−1.480−6.110−0.1631.0080.921SG 848ATOM848NTRP111−3.319−6.916−1.2451.0069.811SG 849ATOM849CATRP111−3.479−8.105−0.4431.0069.811SG 850ATOM850CBTRP111−4.946−8.567−0.2881.0069.811SG 851ATOM851CGTRP111−5.836−7.5550.3921.0069.811SG 852ATOM852CD2TRP111−6.624−6.588−0.3161.0069.811SG 853ATOM853CD1TRP111−6.080−7.3551.7191.0069.811SG 854ATOM854NE1TRP111−6.969−6.3191.8821.0069.811SG 855ATOM855CE2TRP111−7.314−5.8400.6361.0069.811SG 856ATOM856CE3TRP111−6.766−6.348−1.6541.0069.811SG 857ATOM857CZ2TRP111−8.156−4.8320.2651.0069.811SG 858ATOM858CZ3TRP111−7.615−5.330−2.0261.0069.811SG 859ATOM859CH2TRP111−8.297−4.586−1.0831.0069.811SG 860ATOM860CTRP111−2.609−9.224−0.9491.0069.811SG 861ATOM861OTRP111−1.799−9.759−0.1941.0069.811SG 862ATOM862NPRO112−2.728−9.603−2.2121.0062.431SG 863ATOM863CAPRO112−1.865−10.612−2.7631.0062.431SG 864ATOM864CDPRO112−3.976−9.518−2.9601.0062.431SG 865ATOM865CBPRO112−2.652−11.309−3.8721.0062.431SG 866ATOM866CGPRO112−3.722−10.282−4.2681.0062.431SG 867ATOM867CPRO112−0.692−9.863−3.2891.0062.431SG 868ATOM868OPRO112−0.720−8.634−3.2381.0062.431SG 869ATOM869NGLY1130.349−10.556−3.7931.0062.371SG 870ATOM870CAGLY1131.428−9.790−4.3391.0062.371SG 871ATOM871CGLY1132.736−10.297−3.8031.0062.371SG 872ATOM872OGLY1133.147−11.409−4.1261.0062.371SG 873ATOM873NTRP1143.446−9.452−3.0121.0067.981SG 874ATOM874CATRP1144.724−9.793−2.4371.0067.981SG 875ATOM875CBTRP1145.914−9.196−3.2031.0067.981SG 876ATOM876CGTRP1146.075−9.845−4.5561.0067.981SG 877ATOM877CD2TRP1147.053−10.850−4.8661.0067.981SG 878ATOM878CD1TRP1145.355−9.641−5.6971.0067.981SG 879ATOM879NE1TRP1145.810−10.470−6.6911.0067.981SG 880ATOM880CE2TRP1146.858−11.217−6.1981.0067.981SG 881ATOM881CE3TRP1148.032−11.425−4.1041.0067.981SG 882ATOM882CZ2TRP1147.640−12.165−6.7921.0067.981SG 883ATOM883CZ3TRP1148.820−12.380−4.7091.0067.981SG 884ATOM884CH2TRP1148.627−12.743−6.0261.0067.981SG 885ATOM885CTRP1144.774−9.341−0.9911.0067.981SG 886ATOM886OTRP1143.866−8.644−0.5351.0067.981SG 887ATOM887NPHE1155.840−9.745−0.2341.0075.121SG 888ATOM888CAPHE1155.900−9.5421.2011.0075.121SG 889ATOM899CBPHE1156.118−10.8511.9841.0075.121SG 890ATOM890CGPHE1154.938−11.7371.7811.0075.121SG 891ATOM891CD1PHE1153.809−11.5862.5491.0075.121SG 892ATOM892CD2PHE1154.965−12.7260.8251.0075.121SG 893ATOM893CE1PHE1152.722−12.4062.3631.0075.121SG 894ATOM894CE2PHE1153.879−13.5490.6351.0075.121SG 895ATOM895CZPHE1152.753−13.3901.4051.0075.121SG 896ATOM896CPHE1157.005−8.6171.6871.0075.121SG 897ATOM897OPHE1158.185−8.7451.3811.0075.121SG 898ATOM898NLEU1166.589−7.7332.6041.0079.621SG 899ATOM899CALEU1167.128−6.6143.3441.0079.621SG 900ATOM900CBLEU1166.186−6.4534.5201.0079.621SG 901ATOM901CGLEU1165.684−7.8374.9811.0079.621SG 902ATOM902CD2LEU1164.748−7.7226.1811.0079.621SG 903ATOM903CD1LEU1166.835−8.8085.2661.0079.621SG 904ATOM904CLEU1168.481−6.7004.0351.0079.621SG 905ATOM905OLEU1168.505−6.3115.2001.0079.621SG 906ATOM906NCYS1179.637−7.0463.4141.0073.411SG 907ATOM907CACYS11710.854−7.1224.2161.0073.411SG 908ATOM908CBCYS11711.489−8.5254.1881.0073.411SG 909ATOM909SGCYS11710.392−9.8044.8741.0073.411SG 910ATOM910CCYS11711.925−6.1463.7541.0073.411SG 911ATOM911OCYS11711.660−5.2502.9531.0073.411SG 912ATOM912NGLY11813.169−6.2824.3051.0066.611SG 913ATOM913CAGLY11814.312−5.4563.9681.0066.611SG 914ATOM914CGLY11815.571−6.1254.4941.0066.611SG 915ATOM915OGLY11815.574−6.7215.5681.0066.611SG 916ATOM916NPRO11916.642−6.0423.7381.0063.361SG 917ATOM917CAPRO11917.904−6.6054.1651.0063.361SG 918ATOM918CDPRO11916.408−6.4322.3681.0063.361SG 919ATOM919CBPRO11918.784−6.6022.9251.0063.361SG 920ATOM920CGPRO11917.758−6.9411.8261.0063.361SG 921ATOM921CPRO11918.522−6.0935.4311.0063.361SG 922ATOM922OPRO11918.339−4.9325.7811.0063.361SG 923ATOM923NALA12019.201−7.0036.1651.0059.661SG 924ATOM924CAALA12019.788−6.7727.4581.0059.661SG 925ATOM925CBALA12020.233−8.0788.1391.0059.661SG 926ATOM926CALA12020.980−5.8417.5071.0059.661SG 927ATOM927OALA12021.012−4.9658.3681.0059.661SG 928ATOM928NGLU12121.987−5.9756.6111.0062.171SG 929ATOM929CAGLU12123.212−5.2366.8381.0062.171SG 930ATOM930CBGLU12124.431−6.1756.9101.0062.171SG 931ATOM931CGGLU12125.562−5.6617.7971.0062.171SG 932ATOM932CDGLU12125.087−5.7789.2421.0062.171SG 933ATOM933OE1GLU12124.274−6.6969.5351.0062.171SG 934ATOM934OE2GLU12125.532−4.94310.0731.0062.171SG 935ATOM935CGLU12123.442−4.2225.7461.0062.171SG 936ATOM936OGLU12122.727−4.2144.7451.0062.171SG 937ATOM937NPRO12224.392−3.3195.9311.0063.981SG 938ATOM938CAPRO12224.632−2.3114.9271.0063.981SG 939ATOM939CDPRO12224.701−2.8137.2561.0063.981SG 940ATOM940CBPRO12225.479−1.2235.5951.0063.981SG 941ATOM941CGPRO12225.814−1.7886.9921.0063.981SG 942ATOM942CPRO12225.142−2.8033.6161.0063.981SG 943ATOM943OPRO12226.359−2.8723.4561.0063.981SG 944ATOM944NGLN12324.229−3.0372.6441.0062.661SG 945ATOM945CAGLN12324.578−3.5841.3631.0062.661SG 946ATOM946CBGLN12324.968−5.0731.4491.0062.661SG 947ATOM947CGGLN12326.243−5.3312.2571.0062.661SG 948ATOM948CDGLN12326.356−6.8202.5441.0062.661SG 949ATOM949OE1GLN12325.369−7.5542.5341.0062.661SG 950ATOM950NE2GLN12327.604−7.2832.8261.0062.661SG 951ATOM951CGLN12323.366−3.5150.4651.0062.661SG 952ATOM952OGLN12323.283−2.648−0.4041.0062.661SG 953ATOM953NGLN12422.417−4.4700.6321.0062.661SG 954ATOM954CAGLN12421.243−4.591−0.2051.0062.661SG 955ATOM955CBGLN12420.703−6.025−0.3291.0062.661SG 956ATOM956CGGLN12421.649−6.978−1.0611.0062.661SG 957ATOM957CDGLN12420.854−8.233−1.3841.0062.661SG 958ATOM958OE1GLN12419.643−8.168−1.5851.0062.661SG 959ATOM959NE2GLN12421.544−9.402−1.4421.0062.661SG 960ATOM960CGLN12420.102−3.7070.2501.0062.661SG 961ATOM961OGLN12420.093−3.1561.3521.0062.661SG 962ATOM962NPRO12519.158−3.548−0.6611.0062.421SG 963ATOM963CAPRO12517.985−2.698−0.4941.0062.421SG 964ATOM964CDPRO12519.553−3.620−2.0601.0062.421SG 965ATOM965CBPRO12517.688−2.107−1.8721.0062.421SG 966ATOM966CGPRO12518.355−3.080−2.8531.0062.421SG 967ATOM967CPRO12516.746−3.3300.0751.0062.421SG 968ATOM968OPRO12516.795−4.4950.4391.0062.421SG 969ATOM969NVAL12615.621−2.5690.1301.0063.031SG 970ATOM970CAVAL12614.329−3.0060.6181.0063.031SG 971ATOM971CBVAL12613.319−1.8900.6471.0063.031SG 972ATOM972CG1VAL12613.203−1.283−0.7641.0063.031SG 973ATOM973CG2VAL12611.990−2.4331.1991.0063.031SG 974ATOM974CVAL12613.808−4.122−0.2551.0063.031SG 975ATOM975OVAL12613.983−4.108−1.4741.0063.031SG 976ATOM976NGLN12713.156−5.1460.3531.0063.161SG 977ATOM977CAGLN12712.754−6.261−0.4641.0063.161SG 978ATOM978CBGLN12713.705−7.464−0.3291.0063.161SG 979ATOM979CGGLN12713.396−8.616−1.2831.0063.161SG 980ATOM980CDGLN12714.588−9.562−1.2481.0063.161SG 981ATOM981OE1GLN12715.722−9.152−1.4901.0063.161SG 982ATOM982NE2GLN12714.330−10.859−0.9341.0063.161SG 983ATOM983CGLN12711.354−6.734−0.1801.0063.161SG 984ATOM984OGLN12710.978−7.0060.9601.0063.161SG 985ATOM985NLEU12810.555−6.862−1.2641.0062.341SG 986ATOM986CALEU1289.216−7.389−1.2481.0062.341SG 987ATOM987CBLEU1288.348−6.718−2.3261.0062.341SG 988ATOM988CGLEU1288.284−5.182−2.1951.0062.341SG 989ATOM989CD2LEU1287.831−4.749−0.7931.0062.341SG 990ATOM990CD1LEU1287.423−4.561−3.3041.0062.341SG 991ATOM991CLEU1289.422−8.826−1.6601.0062.341SG 992ATOM992OLEU1289.557−9.131−2.8441.0062.341SG 993ATOM993NTHR1299.464−9.728−0.6601.0061.721SG 994ATOM994CATHR1299.832−11.116−0.7621.0061.721SG 995ATOM995CBTHR12910.572−11.5240.4771.0061.721SG 996ATOM996OG1THR12910.953−12.8850.4191.0061.721SG 997ATOM997CG2THR1299.665−11.2631.6921.0061.721SG 998ATOM998CTHR1298.666−12.052−0.9551.0061.721SG 999ATOM999OTHR1297.499−11.690−0.8201.0061.721SG 1000ATOM1000NLYS1308.997−13.307−1.3411.0060.091SG 1001ATOM1001CALYS1308.075−14.390−1.5691.0060.091SG 1002ATOM1002CBLYS1308.684−15.547−2.3801.0060.091SG 1003ATOM1003CGLYS1307.652−16.602−2.7921.0060.091SG 1004ATOM1004CDLYS1308.091−17.469−3.9771.0060.091SG 1005ATOM1005CELYS1308.073−16.707−5.3081.0060.091SG 1006ATOM1006NZLYS1308.478−17.593−6.4221.0060.091SG 1007ATOM1007CLYS1307.532−14.951−0.2861.0060.091SG 1008ATOM1008OLYS1306.398−15.425−0.2441.0060.091SG 1009ATOM1009NGLU1318.333−14.9640.7971.0060.351SG 1010ATOM1010CAGLU1317.828−15.5981.9811.0060.351SG 1011ATOM1011CBGLU1318.414−17.0072.1721.0060.351SG 1012ATOM1012CGGLU1319.943−17.0302.1051.0060.351SG 1013ATOM1013CDGLU13110.389−18.4852.0731.0060.351SG 1014ATOM1014OE1GLU1319.524−19.3732.3011.0060.351SG 1015ATOM1015OE2GLU13111.597−18.7271.8131.0060.351SG 1016ATOM1016CGLU1318.134−14.7763.1971.0060.351SG 1017ATOM1017OGLU1318.917−13.8273.1591.0060.351SG 1018ATOM1018NSER1327.456−15.1274.3101.0060.941SG 1019ATOM1019CASER1327.647−14.5555.6141.0060.941SG 1020ATOM1020CBSER1326.392−14.5946.5051.0060.941SG 1021ATOM1021OGSER1325.399−13.7195.9891.0060.941SG 1022ATOM1022CSER1328.681−15.4546.2301.0060.941SG 1023ATOM1023OSER1329.704−15.7075.5971.0060.941SG 1024ATOM1024NGLU1338.477−15.9287.4831.0059.091SG 1025ATOM1025CAGLU1339.402−16.8758.0591.0059.091SG 1026ATOM1026CBGLU1339.774−18.0167.0901.0059.091SG 1027ATOM1027CGGLU1338.602−18.9466.7651.0059.091SG 1028ATOM1028CDGLU1338.982−19.7775.5461.0059.091SG 1029ATOM1029OE1GLU13310.160−20.2195.4681.0059.091SG 1030ATOM1030OE2GLU1338.098−19.9694.6681.0059.091SG 1031ATOM1031CGLU13310.647−16.1408.4391.0059.091SG 1032ATOM1032OGLU13310.774−14.9468.1741.0059.091SG 1033ATOM1033NPRO13411.560−16.8109.0921.0058.291SG 1034ATOM1034CAPRO13412.814−16.2249.4671.0058.291SG 1035ATOM1035CDPRO13411.329−18.0969.7211.0058.291SG 1036ATOM1036CBPRO13413.546−17.28410.2951.0058.291SG 1037ATOM1037CGPRO13412.749−18.58510.0551.0058.291SG 1038ATOM1038CPRO13413.485−15.8068.2041.0058.291SG 1039ATOM1039OPRO13413.608−16.6317.2991.0058.291SG 1040ATOM1040NSER13513.927−14.5368.1221.0058.091SG 1041ATOM1041CASER13514.472−14.0436.8941.0058.091SG 1042ATOM1042CBSER13513.418−13.8315.7911.0058.091SG 1043ATOM1043OGSER13512.851−15.0715.3931.0058.091SG 1044ATOM1044CSER13515.079−12.6967.1471.0058.091SG 1045ATOM1045OSER13516.161−12.5777.7181.0058.091SG 1046ATOM1046NALA13614.377−11.6416.6801.0060.011SG 1047ATOM1047CAALA13614.857−10.2846.7071.0060.011SG 1048ATOM1048CBALA13614.753−9.5765.3461.0060.011SG 1049ATOM1049CALA13614.091−9.4477.6951.0060.011SG 1050ATOM1050OALA13613.290−9.9598.4761.0060.011SG 1051ATOM1051NARG13714.372−8.1167.7121.0065.191SG 1052ATOM1052CAARG13713.759−7.2518.6871.0065.191SG 1053ATOM1053CBARG13714.663−6.0959.1651.0065.191SG 1054ATOM1054CGARG13715.054−5.0868.0831.0065.191SG 1055ATOM1055CDARG13716.029−4.0178.5831.0065.191SG 1056ATOM1056NEARG13716.251−3.0527.4731.0065.191SG 1057ATOM1057CZARG13715.451−1.9527.3611.0065.191SG 1058ATOM1058NH1ARG13714.476−1.7228.2881.0065.191SG 1059ATOM1059NH2ARG13715.623−1.0816.3241.0065.191SG 1060ATOM1060CARG13712.483−6.6478.1781.0065.191SG 1061ATOM1061OARG13712.486−5.7307.3571.0065.191SG 1062ATOM1062NTHR13811.344−7.1558.6921.0070.421SG 1063ATOM1063CATHR13810.062−6.5968.3731.0070.421SG 1064ATOM1064CBTHR1389.075−7.6347.8981.0070.421SG 1065ATOM1065OG1THR1387.833−7.0257.5831.0070.421SG 1066ATOM1066CG2THR1388.883−8.7198.9761.0070.421SG 1067ATOM1067CTHR1389.548−5.9779.6391.0070.421SG 1068ATOM1068OTHR1388.432−6.24910.0811.0070.421SG 1069ATOM1069NLYS13910.369−5.10210.2511.0075.061SG 1070ATOM1070CALYS13910.010−4.42611.4631.0075.061SG 1071ATOM1071CBLYS13910.275−5.32112.6801.0075.061SG 1072ATOM1072CGLYS13911.555−6.13112.4841.0075.061SG 1073ATOM1073CDLYS13911.916−7.06313.6331.0075.061SG 1074ATOM1074CELYS13912.988−8.08113.2411.0075.061SG 1075ATOM1075NZLYS13914.190−7.38312.7331.0075.061SG 1076ATOM1076CLYS13910.879−3.21211.5391.0075.061SG 1077ATOM1077OLYS13911.910−3.21212.2101.0075.061SG 1078ATOM1078NPHE14010.475−2.12710.8501.0075.241SG 1079ATOM1079CAPHE14011.255−0.91810.8601.0075.241SG 1080ATOM1080CBPHE14012.050−0.6599.5661.0075.241SG 1081ATOM1081CGPHE14011.191−0.9998.4001.0075.241SG 1082ATOM1082CD1PHE14011.076−2.3198.0331.0075.241SG 1083ATOM1083CD2PHE14010.529−0.0447.6671.0075.241SG 1084ATOM1084CE1PHE14010.307−2.6986.9611.0075.241SG 1085ATOM1085CE2PHE1409.758−0.4196.5921.0075.241SG 1086ATOM1086CZPHE1409.646−1.7416.2361.0075.241SG 1087ATOM1087CPHE14010.4010.25111.2491.0075.241SG 1088ATOM1088OPHE1409.1820.21611.0911.0075.241SG 1089ATOM1089NTYR14111.0111.33611.7861.0071.991SG 1090ATOM1090CATYR14110.1292.33112.3301.0071.991SG 1091ATOM1091CBTYR14110.3352.67513.8231.0071.991SG 1092ATOM1092CGTYR14111.5403.50014.0951.0071.991SG 1093ATOM1093CD1TYR14111.4404.87114.0751.0071.991SG 1094ATOM1094CD2TYR14112.7472.91514.3931.0071.991SG 1095ATOM1095CE1TYR14112.5355.65414.3431.0071.991SG 1096ATOM1096CE2TYR14113.8483.69514.6611.0071.991SG 1097ATOM1097CZTYR14113.7405.06514.6351.0071.991SG 1098ATOM1098OHTYR14114.8605.87414.9101.0071.991SG 1099ATOM1099CTYR1419.9963.56311.4931.0071.991SG 1100ATOM1100OTYR14110.8953.95910.7521.0071.991SG 1101ATOM1101NPHE1428.7784.14611.5891.0066.711SG 1102ATOM1102CAPHE1428.2585.28110.8751.0066.711SG 1103ATOM1103CBPHE1426.7605.51511.1141.0066.711SG 1104ATOM1104CGPHE1425.9174.44610.5321.0066.711SG 1105ATOM1105CD1PHE1425.7873.23711.1691.0066.711SG 1106ATOM1106CD2PHE1425.2544.6659.3511.0066.711SG 1107ATOM1107CE1PHE1424.9952.25810.6231.0066.711SG 1108ATOM1108CE2PHE1424.4633.6908.8041.0066.711SG 1109ATOM1109CZPHE1424.3332.4849.4411.0066.711SG 1110ATOM1110CPHE1428.7896.55811.4411.0066.711SG 1111ATOM1111OPHE1428.7576.76812.6531.0066.711SG 1112ATOM1112NGLU1439.2347.46810.5501.0057.451SG 1113ATOM1113CAGLU1439.6018.79810.9421.0057.451SG 1114ATOM1114CBGLU14311.1049.12810.8351.0057.451SG 1115ATOM1115CGGLU14312.0018.32511.7801.0057.451SG 1116ATOM1116CDGLU14312.5237.11811.0121.0057.451SG 1117ATOM1117OE1GLU14312.8187.2789.7981.0057.451SG 1118ATOM1118OE2GLU14312.6486.02611.6271.0057.451SG 1119ATOM1119CGLU1438.9019.6929.9691.0057.451SG 1120ATOM1120OGLU1438.9229.4458.7641.0057.451SG 1121ATOM1121NGLN1448.24010.75610.4641.0049.901SG 1122ATOM1122CAGLN1447.55811.6229.5481.0049.901SG 1123ATOM1123CBGLN1446.05611.7719.8571.0049.901SG 1124ATOM1124CGGLN1445.28212.5128.7631.0049.901SG 1125ATOM1125CDGLN1443.81212.5579.1591.0049.901SG 1126ATOM1126OE1GLN1442.97413.0578.4111.0049.901SG 1127ATOM1127NE2GLN1443.48712.02010.3661.0049.901SG 1128ATOM1128CGLN1448.21312.9929.6511.0049.901SG 1129ATOM1129OGLN1449.43013.0849.3351.0049.901SG 1130ATOM1130OXTGLN1447.51213.96110.0481.0049.901SG 1131

[0511] Table III show protein database coordinates for a IL-1 Hy2 structural models generated by the Protein Data Bank GeneAtlas™ Program (MSI) using the three-dimensional structure of IL-1 β as a template.

4TABLE IIIAtomAmino AcidBNo.NameSCNo.XYZOccup.FactorATOM1NPRO138.534−21.6586.5151.0033.321SG 2ATOM2CAPRO138.607−21.3697.9751.0033.321SG 3ATOM3CDPRO139.877−22.1266.0261.0033.321SG 4ATOM4CBPRO139.976−21.8698.4341.0033.321SG 5ATOM5CGPRO140.848−21.8297.1751.0033.321SG 6ATOM6CPRO138.437−19.8898.0981.0033.321SG 7ATOM7OPRO138.643−19.1857.1111.0033.321SG 8ATOM8NMET238.063−19.3969.2961.0034.271SG 9ATOM9CAMET237.838−17.9939.4951.0034.271SG 10ATOM10CBMET236.412−17.6769.9981.0034.271SG 11ATOM11CGMET235.307−17.8628.9481.0034.271SG 12ATOM12SDMET233.614−17.5959.5641.0034.271SG 13ATOM13CEMET233.251−19.3469.8881.0034.271SG 14ATOM14CMET238.803−17.50610.5351.0034.271SG 15ATOM15OMET239.300−18.27911.3551.0034.271SG 16ATOM16NALA339.133−16.19910.4771.0038.921SG 17ATOM17CAALA339.983−15.60411.4621.0038.921SG 18ATOM18CBALA341.411−15.32910.9481.0038.921SG 19ATOM19CALA339.349−14.30111.8371.0038.921SG 20ATOM20OALA339.000−13.48710.9801.0038.921SG 21ATOM21NARG439.183−14.07413.1501.0046.121SG 22ATOM22CAARG438.537−12.87513.5711.0046.121SG 23ATOM23CBARG437.249−13.14514.3771.0046.121SG 24ATOM24CGARG436.232−12.00314.4621.0046.121SG 25ATOM25CDARG434.842−12.53814.8221.0046.121SG 26ATOM26NEARG433.872−11.42414.6441.0046.121SG 27ATOM27CZARG433.274−11.20413.4351.0046.121SG 28ATOM28NH1ARG433.483−12.05312.3861.0046.121SG 29ATOM29NH2ARG432.496−10.09613.2741.0046.121SG 30ATOM30CARG439.502−12.14514.4301.0046.121SG 31ATOM31OARG440.306−12.72515.1611.0046.121SG 32ATOM32NTYR539.434−10.81714.3001.0053.721SG 33ATOM33CATYR540.226−9.89215.0251.0053.721SG 34ATOM34CBTYR540.325−8.57114.2531.0053.721SG 35ATOM35CGTYR541.299−8.71013.1361.0053.721SG 36ATOM36CD1TYR541.167−9.62312.1091.0053.721SG 37ATOM37CD2TYR542.352−7.83413.1271.0053.721SG 38ATOM38CE1TYR542.123−9.67111.1141.0053.721SG 39ATOM39CE2TYR543.300−7.87812.1411.0053.721SG 40ATOM40CZTYR543.193−8.80111.1351.0053.721SG 41ATOM41OHTYR544.186−8.82810.1331.0053.721SG 42ATOM42CTYR539.479−9.60816.2831.0053.721SG 43ATOM43OTYR538.330−9.16516.2571.0053.721SG 44ATOM44NTYR640.096−9.91117.4291.0062.651SG 45ATOM45CATYR639.472−9.58318.6711.0062.651SG 46ATOM46CBTYR638.524−10.64319.2621.0062.651SG 47ATOM47CGTYR637.110−10.36218.9011.0062.651SG 48ATOM48CD1TYR636.660−9.06318.9311.0062.651SG 49ATOM49CD2TYR636.253−11.36718.5191.0062.651SG 50ATOM50CE1TYR635.357−8.76718.6271.0062.651SG 51ATOM51CE2TYR634.943−11.08018.2181.0062.651SG 52ATOM52CZTYR634.500−9.77918.2761.0062.651SG 53ATOM53OHTYR633.158−9.47017.9761.0062.651SG 54ATOM54CTYR640.493−9.40119.7241.0062.651SG 55ATOM55OTYR641.618−9.89319.6311.0062.651SG 56ATOM56NILE740.112−8.63820.7611.0068.541SG 57ATOM57CAILE740.917−8.67521.9271.0068.541SG 58ATOM58CBILE741.687−7.46322.3461.0068.541SG 59ATOM59CG2ILE740.686−6.40522.8371.0068.541SG 60ATOM60CG1ILE742.697−7.93223.4221.0068.541SG 61ATOM61CD1ILE743.860−6.99823.7051.0068.541SG 62ATOM62CILE739.977−8.97823.0271.0068.541SG 63ATOM63OILE738.778−8.71222.9361.0068.541SG 64ATOM64NILE840.522−9.59624.0811.0069.271SG 65ATOM65CAILE839.740−9.98425.1991.0069.271SG 66ATOM66CBILE839.960−11.41925.5781.0069.271SG 67ATOM67CG2ILE839.168−11.69526.8641.0069.271SG 68ATOM68CG1ILE839.602−12.34424.4031.0069.271SG 69ATOM69CD1ILE838.170−12.17023.9031.0069.271SG 70ATOM70CILE840.221−9.18826.3581.0069.271SG 71ATOM71OILE841.422−8.99026.5371.0069.271SG 72ATOM72NLYS939.268−8.66927.1461.0068.481SG 73ATOM73CALYS939.604−7.99628.3621.0068.481SG 74ATOM74CBLYS939.034−6.57228.4771.0068.481SG 75ATOM75CGLYS939.763−5.54327.6171.0068.481SG 76ATOM76CDLYS938.943−4.27927.3551.0068.481SG 77ATOM77CELYS939.657−2.99727.7701.0068.481SG 78ATOM78NZLYS940.955−2.90327.0701.0068.481SG 79ATOM79CLYS938.959−8.81729.4221.0068.481SG 80ATOM80OLYS937.956−9.48229.1621.0068.481SG 81ATOM81NTYR1039.482−8.79630.6601.0064.111SG 82ATOM82CATYR1038.779−9.62931.5821.0064.111SG 83ATOM83CBTYR1039.569−10.54032.5671.0064.111SG 84ATOM84CGTYR1040.443−9.81533.5321.0064.111SG 85ATOM85CD1TYR1039.929−8.87934.3911.0064.111SG 86ATOM86CD2TYR1041.775−10.14033.6381.0064.111SG 87ATOM87CE1TYR1040.739−8.22735.2891.0064.111SG 88ATOM88CE2TYR1042.596−9.49634.5311.0064.111SG 89ATOM89CZTYR1042.077−8.53035.3551.0064.111SG 90ATOM90OHTYR1042.911−7.86236.2761.0064.111SG 91ATOM91CTYR1037.811−8.75632.2951.0064.111SG 92ATOM92OTYR1037.697−7.57231.9891.0064.111SG 93ATOM93NALA1137.059−9.32833.2451.0060.601SG 94ATOM94CAALA1135.961−8.63733.8521.0060.601SG 95ATOM95CBALA1135.310−9.42534.9971.0060.601SG 96ATOM96CALA1136.426−7.33134.4101.0060.601SG 97ATOM97OALA1135.649−6.38134.4761.0060.601SG 98ATOM98NASP1237.685−7.25334.8721.0058.921SG 99ATOM99CAASP1238.131−6.02535.4601.0058.921SG 100ATOM100CBASP1239.042−6.24936.6731.0058.921SG 101ATOM101CGASP1238.099−6.63237.8051.0058.921SG 102ATOM102OD1ASP1236.965−6.08137.8151.0058.921SG 103ATOM103OD2ASP1238.481−7.47438.6591.0058.921SG 104ATOM104CASP1238.841−5.14434.4731.0058.921SG 105ATOM105OASP1239.777−4.43134.8331.0058.921SG 106ATOM106NGLN1338.384−5.14333.2071.0057.451SG 107ATOM107CAGLN1338.868−4.23832.1991.0057.451SG 108ATOM108CBGLN1338.526−2.77432.5111.0057.451SG 109ATOM109CGGLN1337.023−2.50032.5201.0057.451SG 110ATOM110CDGLN1336.544−2.55531.0771.0057.451SG 111ATOM111OE1GLN1337.229−2.08430.1701.0057.451SG 112ATOM112NE2GLN1335.343−3.15030.8501.0057.451SG 113ATOM113CGLN1340.349−4.33232.0191.0057.451SG 114ATOM114OGLN1341.004−3.34331.6961.0057.451SG 115ATOM115NLYS1440.910−5.53332.2071.0058.161SG 116ATOM116CALYS1442.302−5.77631.9931.0058.161SG 117ATOM117CBLYS1442.758−6.98332.8371.0058.161SG 118ATOM118CGLYS1444.072−7.65332.4431.0058.161SG 119ATOM119CDLYS1443.997−8.49831.1611.0058.161SG 120ATOM120CELYS1442.643−9.19831.0041.0058.161SG 121ATOM121NZLYS1442.479−9.77629.6551.0058.161SG 122ATOM122CLYS1442.470−6.08830.5381.0058.161SG 123ATOM123OLYS1441.623−6.74829.9421.0058.161SG 124ATOM124NALA1543.595−5.63629.9441.0054.241SG 125ATOM125CAALA1543.937−5.86628.5711.0054.241SG 126ATOM126CBALA1544.398−4.59527.8351.0054.241SG 127ATOM127CALA1545.092−6.82228.5771.0054.241SG 128ATOM128OALA1545.767−6.97329.5931.0054.241SG 129ATOM129NLEU1645.325−7.52827.4501.0050.901SG 130ATOM130CALEU1646.381−8.50327.3921.0050.901SG 131ATOM131CBLEU1645.934−9.85226.8011.0050.901SG 132ATOM132CGLEU1644.833−10.54427.6231.0050.901SG 133ATOM133CD2LEU1645.216−10.61829.1051.0050.901SG 134ATOM134CD1LEU1644.458−11.91327.0361.0050.901SG 135ATOM135CLEU1647.460−7.99926.4831.0050.901SG 136ATOM136OLEU1647.176−7.39625.4491.0050.901SG 137ATOM137NTYR1748.737−8.22826.8631.0048.251SG 138ATOM138CATYR1749.853−7.81726.0561.0048.251SG 139ATOM139CBTYR1750.452−6.46926.5101.0048.251SG 140ATOM140CGTYR1751.599−6.10325.6301.0048.251SG 141ATOM141CD1TYR1751.380−5.53524.3961.0048.251SG 142ATOM142CD2TYR1752.893−6.34626.0271.0048.251SG 143ATOM143CE1TYR1752.432−5.19723.5771.0048.251SG 144ATOM144CE2TYR1753.949−6.00825.2131.0048.251SG 145ATOM145CZTYR1753.722−5.43223.9881.0048.251SG 146ATOM146OHTYR1754.810−5.08923.1581.0048.251SG 147ATOM147CTYR1750.902−8.88926.1481.0048.251SG 148ATOM148OTYR1750.844−9.74527.0301.0048.251SG 149ATOM149NTHR1851.884−8.87825.2211.0046.191SG 150ATOM150CATHR1852.927−9.86725.1931.0046.191SG 151ATOM151CBTHR1853.441−10.09723.8011.0046.191SG 152ATOM152OG1THR1852.393−10.55222.9611.0046.191SG 153ATOM153CG2THR1854.575−11.12523.8381.0046.191SG 154ATOM154CTHR1854.072−9.34726.0021.0046.191SG 155ATOM155OTHR1854.883−8.56325.5131.0046.191SG 156ATOM156NARG1954.171−9.77527.2761.0052.231SG 157ATOM157CAARG1955.219−9.26328.1091.0052.231SG 158ATOM158CBARG1955.131−9.77429.5551.0052.231SG 159ATOM159CGARG1956.226−9.20630.4591.0052.231SG 160ATOM160CDARG1956.158−9.71031.9021.0052.231SG 161ATOM161NEARG1957.280−9.07032.6431.0052.231SG 162ATOM162CZARG1958.522−9.63632.6301.0052.231SG 163ATOM163NH1ARG1958.738−10.79831.9471.0052.231SG 164ATOM164NH2ARG1959.551−9.03533.2961.0052.231SG 165ATOM165CARG1956.546−9.68027.5661.0052.231SG 166ATOM166OARG1957.407−8.84627.2871.0052.231SG 167ATOM167NASP2056.735−11.00027.3811.0059.271SG 168ATOM168CAASP2057.964−11.49426.8381.0059.271SG 169ATOM169CBASP2058.963−11.96627.9071.0059.271SG 170ATOM170CGASP2060.296−12.23727.2211.0059.271SG 171ATOM171OD1ASP2060.343−12.17625.9631.0059.271SG 172ATOM172OD2ASP2061.289−12.50027.9501.0059.271SG 173ATOM173CASP2057.571−12.68326.0421.0059.271SG 174ATOM174OASP2058.291−13.67525.9611.0059.271SG 175ATOM175NGLY2156.385−12.59125.4271.0065.031SG 176ATOM176CAGLY2155.889−13.64624.6101.0065.031SG 177ATOM177CGLY2154.895−14.44425.4041.0065.031SG 178ATOM178OGLY2154.020−15.07924.8181.0065.031SG 179ATOM179NGLN2255.002−14.43226.7541.0068.581SG 180ATOM180CAGLN2254.145−15.24327.5811.0068.581SG 181ATOM181CBGLN2254.580−15.29429.0601.0068.581SG 182ATOM182CGGLN2254.492−13.96229.8091.0068.581SG 183ATOM183CDGLN2255.821−13.23429.6741.0068.581SG 184ATOM184OE1GLN2256.058−12.23430.3491.0068.581SG 185ATOM185NE2GLN2256.717−13.75128.7901.0068.581SG 186ATOM186CGLN2252.705−14.81527.5591.0068.581SG 187ATOM187OGLN2251.859−15.63027.2151.0068.581SG 188ATOM188NLEU2352.401−13.53927.8941.0063.111SG 189ATOM189CALEU2351.081−12.93827.9651.0063.111SG 190ATOM190CBLEU2349.859−13.87327.8031.0063.111SG 191ATOM191CGLEU2349.580−14.38726.3741.0063.111SG 192ATOM192CD2LEU2349.467−13.23125.3711.0063.111SG 193ATOM193CD1LEU2348.345−15.30126.3461.0063.111SG 194ATOM194CLEU2350.939−12.33129.3301.0063.111SG 195ATOM195OLEU2351.090−13.01730.3411.0063.111SG 196ATOM196NLEU2450.622−11.02229.3861.0055.201SG 197ATOM197CALEU2450.498−10.32130.6321.0055.201SG 198ATOM198CBLEU2451.666−9.32130.8201.0055.201SG 199ATOM199CGLEU2451.703−8.46732.1081.0055.201SG 200ATOM200CD2LEU2450.544−7.46032.1811.0055.201SG 201ATOM201CD1LEU2453.044−7.72432.2111.0055.201SG 202ATOM202CLEU2449.194−9.58630.6191.0055.201SG 203ATOM203OLEU2448.705−9.18729.5641.0055.201SG 204ATOM204NVAL2548.578−9.43031.8111.0049.241SG 205ATOM205CAVAL2547.336−8.72731.9451.0049.241SG 206ATOM206CBVAL2546.321−9.52732.7151.0049.241SG 207ATOM207CG1VAL2546.013−10.80831.9301.0049.241SG 208ATOM208CG2VAL2546.856−9.81134.1301.0049.241SG 209ATOM209CVAL2547.609−7.47232.7251.0049.241SG 210ATOM210OVAL2548.274−7.49833.7601.0049.241SG 211ATOM211NGLY2647.094−6.32632.2331.0045.561SG 212ATOM212CAGLY2647.263−5.05932.8901.0045.561SG 213ATOM213CGLY2646.128−4.21032.4311.0045.561SG 214ATOM214OGLY2645.360−4.63831.5791.0045.561SG 215ATOM215NASP2745.967−2.99032.9791.0047.301SG 216ATOM216CAASP2744.862−2.19332.5211.0047.301SG 217ATOM217CBASP2744.021−1.59533.6631.0047.301SG 218ATOM218CGASP2742.744−1.01233.0681.0047.301SG 219ATOM219OD1ASP2742.587−1.07531.8191.0047.301SG 220ATOM220OD2ASP2741.905−0.50233.8571.0047.301SG 221ATOM221CASP2745.405−1.05531.7121.0047.301SG 222ATOM222OASP2745.828−0.04632.2701.0047.301SG 223ATOM223NPRO2845.452−1.21030.4121.0051.051SG 224ATOM224CAPRO2845.924−0.12529.5941.0051.051SG 225ATOM225CDPRO2845.902−2.48429.8781.0051.051SG 226ATOM226CBPRO2846.569−0.75128.3581.0051.051SG 227ATOM227CGPRO2846.948−2.16328.8071.0051.051SG 228ATOM228CPRO2844.8370.82029.2001.0051.051SG 229ATOM229OPRO2843.6650.45329.2591.0051.051SG 230ATOM230NVAL2945.2222.04628.7991.0052.421SG 231ATOM231CAVAL2944.3053.00828.2641.0052.421SG 232ATOM232CBVAL2943.6223.84429.3071.0052.421SG 233ATOM233CG1VAL2944.6884.66730.0501.0052.421SG 234ATOM234CG2VAL2942.5504.70228.6151.0052.421SG 235ATOM235CVAL2945.1453.93127.4451.0052.421SG 236ATOM236OVAL2946.3074.13627.7881.0052.421SG 237ATOM237NALA3044.5954.46426.3281.0050.571SG 238ATOM238CAALA3045.2685.42425.4871.0050.571SG 239ATOM239CBALA3046.3546.29926.1481.0050.571SG 240ATOM240CALA3045.8544.74924.2901.0050.571SG 241ATOM241OALA3045.4333.66723.8841.0050.571SG 242ATOM242NASP3146.8525.41723.6861.0046.541SG 243ATOM243CAASP3147.5214.97522.4981.0046.541SG 244ATOM244CBASP3148.6175.96022.0571.0046.541SG 245ATOM245CGASP3149.0845.57820.6611.0046.541SG 246ATOM246OD1ASP3148.5394.59120.1001.0046.541SG 247ATOM247OD2ASP3149.9956.27420.1381.0046.541SG 248ATOM248CASP3148.1753.66022.7881.0046.541SG 249ATOM249OASP3148.3092.81521.9051.0046.541SG 250ATOM250NASN3248.5833.45424.0531.0040.791SG 251ATOM251CAASN3249.2702.26624.4711.0040.791SG 252ATOM252CBASN3249.7722.31025.9281.0040.791SG 253ATOM253CGASN3248.6402.62826.8921.0040.791SG 254ATOM254OD1ASN3248.7743.57627.6641.0040.791SG 255ATOM255ND2ASN3247.5301.84226.8711.0040.791SG 256ATOM256CASN3248.3971.06924.2551.0040.791SG 257ATOM257OASN3248.883−0.05924.1951.0040.791SG 258ATOM258NCYS3347.0801.29324.1221.0037.361SG 259ATOM259CACYS3346.1170.24523.9401.0037.361SG 260ATOM260CBCYS3344.6890.77823.7331.0037.361SG 261ATOM261SGCYS3344.0071.55425.2291.0037.361SG 262ATOM262CCYS3346.486−0.53022.7101.0037.361SG 263ATOM263OCYS3346.129−1.69922.5721.0037.361SG 264ATOM264NCYS3447.2300.09921.7871.0040.371SG 265ATOM265CACYS3447.592−0.50120.5321.0040.371SG 266ATOM266CBCYS3448.5100.40019.6921.0040.371SG 267ATOM267SGCYS3447.7021.95819.2261.0040.371SG 268ATOM268CCYS3448.342−1.77720.7801.0040.371SG 269ATOM269OCYS3448.269−2.70819.9781.0040.371SG 270ATOM270NALA3549.070−1.86021.9061.0048.301SG 271ATOM271CAALA3549.904−2.98522.2341.0048.301SG 272ATOM272CBALA3550.597−2.81223.5951.0048.301SG 273ATOM273CALA3549.073−4.24122.2761.0048.301SG 274ATOM274OALA3549.558−5.33021.9761.0048.301SG 275ATOM275NGLU3647.797−4.11622.6741.0058.231SG 276ATOM276CAGLU3646.849−5.19422.8031.0058.231SG 277ATOM277CBGLU3645.453−4.62323.0951.0058.231SG 278ATOM278CGGLU3645.311−3.97324.4731.0058.231SG 279ATOM279CDGLU3643.946−3.30124.5281.0058.231SG 280ATOM280OE1GLU3643.488−2.81023.4591.0058.231SG 281ATOM281OE2GLU3643.341−3.26725.6311.0058.231SG 282ATOM282CGLU3646.750−5.97721.5061.0058.231SG 283ATOM283OGLU3646.914−5.40320.4301.0058.231SG 284ATOM284NLYS3746.514−7.32421.5701.0065.721SG 285ATOM285CALYS3746.371−8.10620.3481.0065.721SG 286ATOM286CBLYS3747.679−8.17719.5321.0065.721SG 287ATOM287CGLYS3748.888−8.60020.3601.0065.721SG 288ATOM288CDLYS3748.773−10.03220.8691.0065.721SG 289ATOM289CELYS3749.819−10.37421.9241.0065.721SG 290ATOM290NZLYS3749.625−11.76122.3991.0065.721SG 291ATOM291CLYS3745.835−9.52620.5831.0065.721SG 292ATOM292OLYS3745.594−9.89621.7321.0065.721SG 293ATOM293NILE3845.553−10.30119.4691.0067.021SG 294ATOM294CAILE3845.168−11.71819.3591.0067.021SG 295ATOM295CBILE3844.552−12.41420.5471.0067.021SG 296ATOM296CG2ILE3845.618−12.62621.6371.0067.021SG 297ATOM297CG1ILE3843.245−11.73620.9691.0067.021SG 298ATOM298CD1ILE3842.388−12.62821.8651.0067.021SG 299ATOM299CILE3844.222−11.97518.2051.0067.021SG 300ATOM300OILE3843.388−11.13517.8731.0067.021SG 301ATOM301NCYS3944.369−13.14917.5281.0063.201SG 302ATOM302CACYS3943.484−13.56816.4591.0063.201SG 303ATOM303CBCYS3944.215−13.95815.1611.0063.201SG 304ATOM304SGCYS3945.292−15.40615.3781.0063.201SG 305ATOM305CCYS3942.743−14.80516.9141.0063.201SG 306ATOM306OCYS3943.334−15.69817.5191.0063.201SG 307ATOM307NILE4041.420−14.88716.6191.0057.921SG 308ATOM308CAILE4040.582−15.99417.0201.0057.921SG 309ATOM309CBILE4039.397−15.56917.8471.0057.921SG 310ATOM310CG2ILE4038.402−16.73617.9001.0057.921SG 311ATOM311CG1ILE4039.845−15.05519.2281.0057.921SG 312ATOM312CD1ILE4038.738−14.34120.0051.0057.921SG 313ATOM313CILE4040.056−16.67615.7911.0057.921SG 314ATOM314OILE4039.465−16.04314.9161.0057.921SG 315ATOM315NLEU4140.257−18.00915.7071.0055.121SG 316ATOM316CALEU4139.876−18.77414.5521.0055.121SG 317ATOM317CBLEU4141.103−19.53713.9951.0055.121SG 318ATOM318CGLEU4140.948−20.32412.6761.0055.121SG 319ATOM319CD2LEU4139.844−21.38112.7441.0055.121SG 320ATOM320CD1LEU4142.282−20.97612.2801.0055.121SG 321ATOM321CLEU4138.821−19.75114.9731.0055.121SG 322ATOM322OLEU4138.992−20.49815.9331.0055.121SG 323ATOM323NPRO4237.697−19.71714.3081.0050.961SG 324ATOM324CAPRO4236.686−20.69514.6061.0050.961SG 325ATOM325CDPRO4237.122−18.44213.9141.0050.961SG 326ATOM326CBPRO4235.378−20.13214.0581.0050.961SG 327ATOM327CGPRO4235.605−18.60814.0901.0050.961SG 328ATOM328CPRO4237.113−22.00114.0241.0050.961SG 329ATOM329OPRO4237.212−22.09712.8021.0050.961SG 330ATOM330NASN4337.272−23.04414.8571.0045.741SG 331ATOM331CAASN4337.798−24.27714.3541.0045.741SG 332ATOM332CBASN4339.057−24.74815.1021.0045.741SG 333ATOM333CGASN4338.669−25.00216.5521.0045.741SG 334ATOM334OD1ASN4338.046−24.15717.1931.0045.741SG 335ATOM335ND2ASN4339.040−26.19717.0851.0045.741SG 336ATOM336CASN4336.768−25.33514.5201.0045.741SG 337ATOM337OASN4335.690−25.10715.0651.0045.741SG 338ATOM338NARG4437.078−26.52913.9861.0040.331SG 339ATOM339CAARG4436.185−27.63714.0991.0040.331SG 340ATOM340CBARG4436.409−28.70613.0141.0040.331SG 341ATOM341CGARG4437.827−29.27912.9791.0040.331SG 342ATOM342CDARG4438.132−30.23214.1331.0040.331SG 343ATOM343NEARG4437.286−31.44113.9351.0040.331SG 344ATOM344CZARG4437.213−32.38014.9211.0040.331SG 345ATOM345NH1ARG4437.931−32.21316.0701.0040.331SG 346ATOM346NH2ARG4436.421−33.48014.7601.0040.331SG 347ATOM347CARG4436.368−28.24115.4531.0040.331SG 348ATOM348OARG4437.446−28.18216.0421.0040.331SG 349ATOM349NGLY4535.282−28.82315.9921.0032.711SG 350ATOM350CAGLY4535.330−29.41417.2941.0032.711SG 351ATOM351CGLY4533.922−29.72517.6631.0032.711SG 352ATOM352OGLY4533.034−29.73016.8121.0032.711SG 353ATOM353NLEU4633.680−29.99418.9581.0030.211SG 354ATOM354CALEU4632.346−30.31219.3651.0030.211SG 355ATOM355CBLEU4632.285−31.11020.6781.0030.211SG 356ATOM356CGLEU4632.992−32.47720.5991.0030.211SG 357ATOM357CD2LEU4632.496−33.29719.3981.0030.211SG 358ATOM358CD1LEU4632.888−33.23821.9301.0030.211SG 359ATOM359CLEU4631.642−29.01819.5931.0030.211SG 360ATOM360OLEU4632.084−28.18620.3851.0030.211SG 361ATOM361NASP4730.522−28.81318.8751.0032.571SG 362ATOM362CAASP4729.774−27.60519.0291.0032.571SG 363ATOM363CBASP4729.361−26.95817.6941.0032.571SG 364ATOM364CGASP4728.692−25.62117.9881.0032.571SG 365ATOM365OD1ASP4728.576−25.26319.1901.0032.571SG 366ATOM366OD2ASP4728.292−24.93517.0091.0032.571SG 367ATOM367CASP4728.518−27.94819.7551.0032.571SG 368ATOM368OASP4727.733−28.79019.3221.0032.571SG 369ATOM369NARG4828.324−27.29320.9091.0034.941SG 370ATOM370CAARG4827.162−27.43921.7281.0034.941SG 371ATOM371CBARG4827.404−28.21923.0331.0034.941SG 372ATOM372CGARG4827.668−29.71222.8281.0034.941SG 373ATOM373CDARG4827.895−30.47224.1371.0034.941SG 374ATOM374NEARG4828.133−31.90223.7951.0034.941SG 375ATOM375CZARG4827.092−32.78523.7911.0034.941SG 376ATOM376NH1ARG4825.837−32.35724.1161.0034.941SG 377ATOM377NH2ARG4827.305−34.09423.4681.0034.941SG 378ATOM378CARG4826.868−26.04122.1151.0034.941SG 379ATOM379OARG4827.113−25.12121.3361.0034.941SG 380ATOM380NTHR4926.299−25.82923.3111.0039.861SG 381ATOM381CATHR4926.166−24.45823.6741.0039.861SG 382ATOM382CBTHR4925.210−24.22724.8071.0039.861SG 383ATOM383OG1THR4923.915−24.69424.4591.0039.861SG 384ATOM384CG2THR4925.167−22.72125.1191.0039.861SG 385ATOM385CTHR4927.533−24.07524.1491.0039.861SG 386ATOM386OTHR4927.793−24.02025.3501.0039.861SG 387ATOM387NLYS5028.446−23.83423.1811.0043.041SG 388ATOM388CALYS5029.815−23.47323.4001.0043.041SG 389ATOM389CBLYS5030.522−24.41224.3911.0043.041SG 390ATOM390CGLYS5031.765−23.80725.0411.0043.041SG 391ATOM391CDLYS5032.193−24.55426.3071.0043.041SG 392ATOM392CELYS5033.254−23.82027.1281.0043.041SG 393ATOM393NZLYS5033.489−24.53628.4021.0043.041SG 394ATOM394CLYS5030.457−23.60822.0501.0043.041SG 395ATOM395OLYS5030.036−24.44621.2531.0043.041SG 396ATOM396NVAL5131.478−22.78421.7321.0044.031SG 397ATOM397CAVAL5132.054−22.89920.4191.0044.031SG 398ATOM398CBVAL5131.843−21.68319.5651.0044.031SG 399ATOM399CG1VAL5132.582−21.88818.2311.0044.031SG 400ATOM400CG2VAL5130.332−21.44219.4141.0044.031SG 401ATOM401CVAL5133.534−23.08020.5421.0044.031SG 402ATOM402OVAL5134.192−22.43121.3521.0044.031SG 403ATOM403NPRO5234.065−23.97619.7491.0046.941SG 404ATOM404CAPRO5235.488−24.18919.7571.0046.941SG 405ATOM405CDPRO5233.341−25.20319.4551.0046.941SG 406ATOM406CBPRO5235.721−25.55219.1101.0046.941SG 407ATOM407CGPRO5234.409−26.30819.3851.0046.941SG 408ATOM408CPRO5236.201−23.06519.0771.0046.941SG 409ATOM409OPRO5235.729−22.58918.0441.0046.941SG 410ATOM410NILE5337.355−22.64819.6331.0047.211SG 411ATOM411CAILE5338.096−21.54319.1071.0047.211SG 412ATOM412CBILE5337.817−20.28619.8891.0047.211SG 413ATOM413CG2ILE5338.805−19.18419.4831.0047.211SG 414ATOM414CG1ILE5336.340−19.88819.7331.0047.211SG 415ATOM415CD1ILE5335.935−19.58518.2901.0047.211SG 416ATOM416CILE5339.554−21.85519.2551.0047.211SG 417ATOM417OILE5339.958−22.64420.1081.0047.211SG 418ATOM418NPHE5440.371−21.24218.3781.0047.501SG 419ATOM419CAPHE5441.799−21.33018.3791.0047.501SG 420ATOM420CBPHE5442.309−21.80017.0091.0047.501SG 421ATOM421CGPHE5443.788−21.82817.0281.0047.501SG 422ATOM422CD1PHE5444.457−22.91617.5331.0047.501SG 423ATOM423CD2PHE5444.494−20.76116.5281.0047.501SG 424ATOM424CE1PHE5445.827−22.93317.5421.0047.501SG 425ATOM425CE2PHE5445.865−20.77216.5341.0047.501SG 426ATOM426CZPHE5446.522−21.86317.0431.0047.501SG 427ATOM427CPHE5442.258−19.92018.6011.0047.501SG 428ATOM428OPHE5441.680−18.98818.0451.0047.501SG 429ATOM429NLEU5543.290−19.71119.4461.0047.901SG 430ATOM430CALEU5543.731−18.37219.7141.0047.901SG 431ATOM431CBLEU5543.708−18.03421.2141.0047.901SG 432ATOM432CGLEU5542.301−18.05521.8371.0047.901SG 433ATOM433CD2LEU5541.342−17.14021.0611.0047.901SG 434ATOM434CD1LEU5542.349−17.71823.3351.0047.901SG 435ATOM435CLEU5545.158−18.26019.2791.0047.901SG 436ATOM436OLEU5546.036−18.92319.8241.0047.901SG 437ATOM437NGLY5645.440−17.39318.2921.0045.331SG 438ATOM438CAGLY5646.796−17.22717.8611.0045.331SG 439ATOM439CGLY5647.057−15.76217.8641.0045.331SG 440ATOM440OGLY5646.166−14.96118.1441.0045.331SG 441ATOM441NILE5748.299−15.36017.5511.0042.941SG 442ATOM442CAILE5748.540−13.95317.4901.0042.941SG 443ATOM443CBILE5749.902−13.53017.9611.0042.941SG 444ATOM444CG2ILE5750.962−14.15117.0371.0042.941SG 445ATOM445CG1ILE5749.969−11.99618.0501.0042.941SG 446ATOM446CD1ILE5751.202−11.47518.7851.0042.941SG 447ATOM447CILE5748.424−13.60316.0481.0042.941SG 448ATOM448OILE5748.944−14.30915.1871.0042.941SG 449ATOM449NGLN5847.687−12.52615.7251.0039.721SG 450ATOM450CAGLN5847.564−12.23914.3321.0039.721SG 451ATOM451CBGLN5846.525−11.16713.9661.0039.721SG 452ATOM452CGGLN5846.515−10.85012.4661.0039.721SG 453ATOM453CDGLN5846.163−12.11811.6961.0039.721SG 454ATOM454OE1GLN5845.085−12.68711.8591.0039.721SG 455ATOM455NE2GLN5847.104−12.58110.8291.0039.721SG 456ATOM456CGLN5848.888−11.77713.8291.0039.721SG 457ATOM457OGLN5849.555−10.95114.4491.0039.721SG 458ATOM458NGLY5949.309−12.33612.6801.0036.041SG 459ATOM459CAGLY5950.528−11.91012.0641.0036.041SG 460ATOM460CGLY5951.687−12.63912.6601.0036.041SG 461ATOM461OGLY5952.835−12.35312.3251.0036.041SG 462ATOM462NGLY6051.433−13.60913.5561.0035.521SG 463ATOM463CAGLY6052.554−14.29914.1171.0035.521SG 464ATOM464CGLY6052.189−15.73714.1981.0035.521SG 465ATOM465OGLY6051.033−16.08514.4371.0035.521SG 466ATOM466NSER6153.182−16.62314.0051.0038.491SG 467ATOM467CASER6152.858−18.00814.1061.0038.491SG 468ATOM468CBSER6153.770−18.90613.2541.0038.491SG 469ATOM469OGSER6153.623−18.58811.8781.0038.491SG 470ATOM470CSER6153.090−18.36915.5331.0038.491SG 471ATOM471OSER6154.032−19.09115.8571.0038.491SG 472ATOM472NARG6252.209−17.86916.4231.0041.731SG 473ATOM473CAARG6252.295−18.16217.8231.0041.731SG 474ATOM474CBARG6252.729−16.96418.6871.0041.731SG 475ATOM475CGARG6254.095−16.37918.3171.0041.731SG 476ATOM476CDARG6254.091−15.61116.9931.0041.731SG 477ATOM477NEARG6255.406−14.92116.8591.0041.731SG 478ATOM478CZARG6256.461−15.54716.2621.0041.731SG 479ATOM479NH1ARG6256.317−16.81015.7661.0041.731SG 480ATOM480NH2ARG6257.661−14.90516.1571.0041.731SG 481ATOM481CARG6250.903−18.50318.2451.0041.731SG 482ATOM482OARG6249.960−17.78317.9161.0041.731SG 483ATOM483NCYS6350.731−19.60619.0001.0043.441SG 484ATOM484CACYS6349.397−20.00819.3311.0043.441SG 485ATOM485CBCYS6348.996−21.24718.5231.0043.441SG 486ATOM486SGCYS6349.453−21.04716.7731.0043.441SG 487ATOM487CCYS6349.357−20.39620.7751.0043.441SG 488ATOM488OCYS6350.329−20.92321.3141.0043.441SG 489ATOM489NLEU6448.222−20.11621.4471.0042.331SG 490ATOM490CALEU6448.069−20.52022.8161.0042.331SG 491ATOM491CBLEU6446.996−19.72223.5801.0042.331SG 492ATOM492CGLEU6447.413−18.26523.8751.0042.331SG 493ATOM493CD2LEU6446.354−17.54624.7271.0042.331SG 494ATOM494CD1LEU6447.745−17.49522.5881.0042.331SG 495ATOM495CLEU6447.698−21.96422.7921.0042.331SG 496ATOM496OLEU6446.903−22.39921.9601.0042.331SG 497ATOM497NALA6548.290−22.75223.7081.0036.601SG 498ATOM498CAALA6548.009−24.15423.7561.0036.601SG 499ATOM499CBALA6549.046−25.01423.0141.0036.601SG 500ATOM500CALA6548.059−24.54225.1921.0036.601SG 501ATOM501OALA6548.600−23.81026.0211.0036.601SG 502ATOM502NCYS6647.452−25.69925.5181.0033.081SG 503ATOM503CACYS6647.452−26.17726.8661.0033.081SG 504ATOM504CBCYS6646.041−26.44627.4171.0033.081SG 505ATOM505SGCYS6645.034−24.93427.5011.0033.081SG 506ATOM506CCYS6648.185−27.47726.8641.0033.081SG 507ATOM507OCYS6647.890−28.36626.0671.0033.081SG 508ATOM508NVAL6749.186−27.61227.7531.0034.931SG 509ATOM509CAVAL6749.923−28.83827.8081.0034.931SG 510ATOM510CBVAL6751.213−28.79427.0431.0034.931SG 511ATOM511CG1VAL6750.893−28.58925.5531.0034.931SG 512ATOM512CG2VAL6752.101−27.69227.6471.0034.931SG 513ATOM513CVAL6750.267−29.07729.2351.0034.931SG 514ATOM514OVAL6750.301−28.14930.0431.0034.931SG 515ATOM515NGLU6850.498−30.35129.5941.0041.241SG 516ATOM516CAGLU6850.867−30.62430.9481.0041.241SG 517ATOM517CBGLU6850.712−32.10631.3321.0041.241SG 518ATOM518CGGLU6849.262−32.59531.3201.0041.241SG 519ATOM519CDGLU6849.260−34.06531.7151.0041.241SG 520ATOM520OE1GLU6850.363−34.60232.0001.0041.241SG 521ATOM521OE2GLU6848.155−34.67031.7351.0041.241SG 522ATOM522CGLU6852.311−30.27531.0971.0041.241SG 523ATOM523OGLU6853.151−30.69730.3041.0041.241SG 524ATOM524NTHR6952.630−29.46632.1211.0050.561SG 525ATOM525CATHR6953.984−29.15332.4591.0050.561SG 526ATOM526CBTHR6954.315−27.68632.3511.0050.561SG 527ATOM527OG1THR6955.673−27.46432.6981.0050.561SG 528ATOM528CG2THR6953.380−26.86433.2561.0050.561SG 529ATOM529CTHR6954.053−29.57933.8761.0050.561SG 530ATOM530OTHR6953.165−29.21234.6451.0050.561SG 531ATOM531NGLU7055.092−30.36534.2441.0057.231SG 532ATOM532CAGLU7055.110−30.98535.5371.0057.231SG 533ATOM533CBGLU7055.426−30.05236.7401.0057.231SG 534ATOM534CGGLU7054.504−28.85436.9931.0057.231SG 535ATOM535CDGLU7053.404−29.26637.9621.0057.231SG 536ATOM536OE1GLU7053.653−30.18438.7881.0057.231SG 537ATOM537OE2GLU7052.304−28.65437.8981.0057.231SG 538ATOM538CGLU7053.786−31.66635.6221.0057.231SG 539ATOM539OGLU7053.264−32.10434.5991.0057.231SG 540ATOM540NGLU7153.197−31.83636.8061.0058.061SG 541ATOM541CAGLU7151.892−32.40436.7031.0058.061SG 542ATOM542CBGLU7151.569−33.40337.8261.0058.061SG 543ATOM543CGGLU7150.280−34.19137.5931.0058.061SG 544ATOM544CDGLU7149.097−33.28837.9121.0058.061SG 545ATOM545OE1GLU7149.106−32.66839.0091.0058.061SG 546ATOM546OE2GLU7148.172−33.20237.0621.0058.061SG 547ATOM547CGLU7150.927−31.26736.7921.0058.061SG 548ATOM548OGLU7150.757−30.67137.8541.0058.061SG 549ATOM549NGLY7250.276−30.91735.6641.0053.241SG 550ATOM550CAGLY7249.310−29.86035.7311.0053.241SG 551ATOM551CGLY7249.173−29.22834.3831.0053.241SG 552ATOM552OGLY7250.135−29.09633.6261.0053.241SG 553ATOM553NPRO7347.968−28.81834.0851.0047.471SG 554ATOM554CAPRO7347.742−28.16032.8331.0047.471SG 555ATOM555CDPRO7346.807−29.58534.5071.0047.471SG 556ATOM556CBPRO7346.232−28.16432.6181.0047.471SG 557ATOM557CGPRO7345.773−29.42133.3801.0047.471SG 558ATOM558CPRO7348.355−26.80632.8911.0047.471SG 559ATOM559OPRO7348.345−26.18933.9561.0047.471SG 560ATOM560NSER7448.895−26.32531.7601.0042.891SG 561ATOM561CASER7449.528−25.04731.7931.0042.891SG 562ATOM562CBSER7451.040−25.16432.0321.0042.891SG 563ATOM563OGSER7451.587−23.89332.3281.0042.891SG 564ATOM564CSER7449.301−24.41730.4571.0042.891SG 565ATOM565OSER7449.223−25.10829.4411.0042.891SG 566ATOM566NLEU7549.187−23.07430.4341.0039.971SG 567ATOM567CALEU7548.941−22.34529.2241.0039.971SG 568ATOM568CBLEU7548.073−21.09629.4581.0039.971SG 569ATOM569CGLEU7547.778−20.27628.1911.0039.971SG 570ATOM570CD2LEU7547.229−18.88528.5471.0039.971SG 571ATOM571CD1LEU7546.879−21.05227.2151.0039.971SG 572ATOM572CLEU7550.264−21.87428.7051.0039.971SG 573ATOM573OLEU7551.063−21.30929.4501.0039.971SG 574ATOM574NGLN7650.542−22.11127.4061.0039.381SG 575ATOM575CAGLN7651.799−21.66026.8771.0039.381SG 576ATOM576CBGLN7652.863−22.76626.7651.0039.381SG 577ATOM577CGGLN7652.497−23.84725.7441.0039.381SG 578ATOM578CDGLN7653.654−24.83225.6501.0039.381SG 579ATOM579OE1GLN7654.517−24.88526.5241.0039.381SG 580ATOM580NE2GLN7653.675−25.63524.5521.0039.381SG 581ATOM581CGLN7651.578−21.17225.4831.0039.381SG 582ATOM582OGLN7650.531−21.40724.8831.0039.381SG 583ATOM583NLEU7752.575−20.44124.9451.0038.941SG 584ATOM584CALEU7752.534−19.98823.5841.0038.941SG 585ATOM585CBLEU7753.069−18.56023.3721.0038.941SG 586ATOM586CGLEU7752.141−17.46223.9211.0038.941SG 587ATOM587CD2LEU7751.833−17.69325.4051.0038.941SG 588ATOM588CD1LEU7750.862−17.33423.0781.0038.941SG 589ATOM589CLEU7753.432−20.91522.8411.0038.941SG 590ATOM590OLEU7754.532−21.22023.3011.0038.941SG 591ATOM591NGLU7852.973−21.40521.6741.0036.561SG 592ATOM592CAGLU7853.755−22.35520.9401.0036.561SG 593ATOM593CBGLU7853.041−23.71420.8381.0036.561SG 594ATOM594CGGLU7853.820−24.81220.1161.0036.561SG 595ATOM595CDGLU7852.942−26.05620.1311.0036.561SG 596ATOM596OE1GLU7852.261−26.28021.1671.0036.561SG 597ATOM597OE2GLU7852.933−26.79319.1091.0036.561SG 598ATOM598CGLU7853.953−21.82919.5551.0036.561SG 599ATOM599OGLU7853.044−21.24618.9681.0036.561SG 600ATOM600NASP7955.179−21.99319.0161.0034.751SG 601ATOM601CAASP7955.477−21.56917.6771.0034.751SG 602ATOM602CBASP7956.985−21.34117.4541.0034.751SG 603ATOM603CGASP7957.208−20.49916.2041.0034.751SG 604ATOM604OD1ASP7956.794−20.93515.0971.0034.751SG 605ATOM605OD2ASP7957.816−19.40416.3431.0034.751SG 606ATOM606CASP7955.017−22.66916.7701.0034.751SG 607ATOM607OASP7955.001−23.83517.1631.0034.751SG 608ATOM608NVAL8054.622−22.33215.5261.0033.441SG 609ATOM609CAVAL8054.154−23.35214.6331.0033.441SG 610ATOM610CBVAL8052.661−23.46514.6311.0033.441SG 611ATOM611CG1VAL8052.215−23.83216.0571.0033.441SG 612ATOM612CG2VAL8052.069−22.14114.1171.0033.441SG 613ATOM613CVAL8054.573−22.97413.2481.0033.441SG 614ATOM614OVAL8054.881−21.81512.9791.0033.441SG 615ATOM615NASN8154.622−23.95812.3271.0034.971SG 616ATOM616CAASN8154.977−23.61610.9811.0034.971SG 617ATOM617CBASN8155.218−24.83710.0751.0034.971SG 618ATOM618CGASN8155.874−24.3608.7851.0034.971SG 619ATOM619OD1ASN8157.010−24.7238.4841.0034.971SG 620ATOM620ND2ASN8155.146−23.5237.9981.0034.971SG 621ATOM621CASN8153.813−22.85310.4431.0034.971SG 622ATOM622OASN8152.678−23.32610.4791.0034.971SG 623ATOM623NILE8254.064−21.6319.9391.0040.291SG 624ATOM624CAILE8252.968−20.8309.4881.0040.291SG 625ATOM625CBILE8253.380−19.4229.1431.0040.291SG 626ATOM626CG2ILE8254.483−19.4618.0721.0040.291SG 627ATOM627CG1ILE8252.149−18.5838.7621.0040.291SG 628ATOM628CD1ILE8252.436−17.0858.6741.0040.291SG 629ATOM629CILE8252.320−21.4468.2901.0040.291SG 630ATOM630OILE8251.104−21.6258.2631.0040.291SG 631ATOM631NGLU8353.123−21.7977.2711.0052.051SG 632ATOM632CAGLU8352.595−22.3226.0461.0052.051SG 633ATOM633CBGLU8353.650−22.3824.9281.0052.051SG 634ATOM634CGGLU8353.087−22.8023.5681.0052.051SG 635ATOM635CDGLU8352.374−21.6012.9601.0052.051SG 636ATOM636OE1GLU8352.283−20.5543.6541.0052.051SG 637ATOM637OE2GLU8351.912−21.7161.7931.0052.051SG 638ATOM638CGLU8352.074−23.7106.2271.0052.051SG 639ATOM639OGLU8350.987−24.0405.7531.0052.051SG 640ATOM640NGLU8452.828−24.5566.9511.0066.461SG 641ATOM641CAGLU8452.492−25.9496.9901.0066.461SG 642ATOM642CBGLU8453.450−26.7527.8841.0066.461SG 643ATOM643CGGLU8454.885−26.7907.3591.0066.461SG 644ATOM644CDGLU8455.715−27.6048.3401.0066.461SG 645ATOM645OE1GLU8455.137−28.0669.3601.0066.461SG 646ATOM646OE2GLU8456.937−27.7748.0851.0066.461SG 647ATOM647CGLU8451.134−26.1337.5471.0066.461SG 648ATOM648OGLU8450.259−26.7446.9341.0066.461SG 649ATOM649NLEU8550.907−25.5818.7351.0076.271SG 650ATOM650CALEU8549.617−25.7679.2911.0076.271SG 651ATOM651CBLEU8549.541−26.96910.2561.0076.271SG 652ATOM652CGLEU8548.119−27.30510.7501.0076.271SG 653ATOM653CD2LEU8548.145−28.38811.8411.0076.271SG 654ATOM654CD1LEU8547.197−27.6729.5761.0076.271SG 655ATOM655CLEU8549.373−24.52010.0391.0076.271SG 656ATOM656OLEU8550.142−23.5669.9271.0076.271SG 657ATOM657NTYR8648.261−24.49310.7791.0082.941SG 658ATOM658CATYR8647.945−23.39211.6181.0082.941SG 659ATOM659CBTYR8649.143−22.74312.3301.0082.941SG 660ATOM660CGTYR8649.629−23.83913.2121.0082.941SG 661ATOM661CD1TYR8650.528−24.76712.7391.0082.941SG 662ATOM662CD2TYR8649.165−23.96014.5001.0082.941SG 663ATOM663CE1TYR8650.970−25.79013.5431.0082.941SG 664ATOM664CE2TYR8649.603−24.98115.3101.0082.941SG 665ATOM665CZTYR8650.508−25.89814.8311.0082.941SG 666ATOM666OHTYR8650.962−26.94815.6571.0082.941SG 667ATOM667CTYR8647.128−22.41210.8831.0082.941SG 668ATOM668OTYR8647.412−22.0549.7411.0082.941SG 669ATOM669NLYS8746.095−21.91711.5831.0077.481SG 670ATOM670CALYS8745.112−21.11710.9451.0077.481SG 671ATOM671CBLYS8745.706−19.94010.1531.0077.481SG 672ATOM672CGLYS8746.442−18.91611.0221.0077.481SG 673ATOM673CDLYS8745.557−18.23812.0701.0077.481SG 674ATOM674CELYS8745.379−19.05813.3491.0077.481SG 675ATOM675NZLYS8746.642−19.07314.1211.0077.481SG 676ATOM676CLYS8744.428−22.0289.9871.0077.481SG 677ATOM677OLYS8744.182−21.6748.8361.0077.481SG 678ATOM678NGLY8844.127−23.26210.4491.0067.171SG 679ATOM679CAGLY8843.445−24.1659.5761.0067.171SG 680ATOM680CGLY8842.471−24.99910.3471.0067.171SG 681ATOM681OGLY8842.788−25.52111.4151.0067.171SG 682ATOM682NGLY8941.225−25.0349.8261.0057.431SG 683ATOM683CAGLY8940.112−25.89010.1481.0057.431SG 684ATOM684CGLY8940.328−26.70211.3771.0057.431SG 685ATOM685OGLY8940.056−26.27712.4981.0057.431SG 686ATOM686NGLU9040.797−27.94511.1471.0047.611SG 687ATOM687CAGLU9041.017−28.90612.1821.0047.611SG 688ATOM688CBGLU9041.067−30.34411.6401.0047.611SG 689ATOM689CGGLU9041.181−31.43312.7061.0047.611SG 690ATOM690CDGLU9041.212−32.76811.9731.0047.611SG 691ATOM691OE1GLU9041.160−32.74810.7141.0047.611SG 692ATOM692OE2GLU9041.287−33.82312.6581.0047.611SG 693ATOM693CGLU9042.329−28.59812.8131.0047.611SG 694ATOM694OGLU9043.387−28.75212.2051.0047.611SG 695ATOM695NGLU9142.277−28.16214.0811.0042.981SG 696ATOM696CAGLU9143.457−27.79514.7961.0042.981SG 697ATOM697CBGLU9143.326−26.43515.5041.0042.981SG 698ATOM698CGGLU9144.640−25.89616.0701.0042.981SG 699ATOM699CDGLU9145.438−25.32014.9091.0042.981SG 700ATOM700OE1GLU9144.969−25.45713.7481.0042.981SG 701ATOM701OE2GLU9146.523−24.73515.1671.0042.981SG 702ATOM702CGLU9143.646−28.83115.8531.0042.981SG 703ATOM703OGLU9142.695−29.50316.2481.0042.981SG 704ATOM704NALA9244.894−28.99616.3271.0042.311SG 705ATOM705CAALA9245.159−29.97517.3401.0042.311SG 706ATOM706CBALA9246.646−30.09417.7121.0042.311SG 707ATOM707CALA9244.409−29.56118.5691.0042.311SG 708ATOM708OALA9244.121−28.38318.7671.0042.311SG 709ATOM709NTHR9344.086−30.54519.4301.0043.291SG 710ATOM710CATHR9343.271−30.37420.6031.0043.291SG 711ATOM711CBTHR9343.007−31.66521.3181.0043.291SG 712ATOM712OG1THR9342.362−32.58320.4481.0043.291SG 713ATOM713CG2THR9342.111−31.37522.5351.0043.291SG 714ATOM714CTHR9343.901−29.44221.5941.0043.291SG 715ATOM715OTHR9343.196−28.69922.2751.0043.291SG 716ATOM716NARG9445.238−29.44821.7181.0046.171SG 717ATOM717CAARG9445.875−28.63622.7191.0046.171SG 718ATOM718CBARG9447.406−28.81122.7661.0046.171SG 719ATOM719CGARG9448.142−28.42821.4801.0046.171SG 720ATOM720CDARG9449.655−28.65521.5681.0046.171SG 721ATOM721NEARG9450.260−28.23320.2741.0046.171SG 722ATOM722CZARG9450.342−29.10819.2301.0046.171SG 723ATOM723NH1ARG9449.860−30.37819.3631.0046.171SG 724ATOM724NH2ARG9450.904−28.71118.0511.0046.171SG 725ATOM725CARG9445.566−27.19222.4541.0046.171SG 726ATOM726OARG9445.525−26.37523.3741.0046.171SG 727ATOM727NPHE9545.403−26.84321.1681.0049.821SG 728ATOM728CAPHE9545.133−25.51420.6891.0049.821SG 729ATOM729CBPHE9545.411−25.34019.1871.0049.821SG 730ATOM730CGPHE9546.884−25.45718.9771.0049.821SG 731ATOM731CD1PHE9547.718−24.40119.2661.0049.821SG 732ATOM732CD2PHE9547.435−26.62818.5111.0049.821SG 733ATOM733CE1PHE9549.077−24.50519.0771.0049.821SG 734ATOM734CE2PHE9548.792−26.73718.3191.0049.821SG 735ATOM735CZPHE9549.616−25.67518.6011.0049.821SG 736ATOM736CPHE9543.719−25.05020.9341.0049.821SG 737ATOM737OPHE9543.466−23.84520.9131.0049.821SG 738ATOM738NTHR9642.744−25.97121.0911.0048.191SG 739ATOM739CATHR9641.360−25.56821.1311.0048.191SG 740ATOM740CBTHR9640.427−26.62320.6091.0048.191SG 741ATOM741OG1THR9640.747−26.93919.2621.0048.191SG 742ATOM742CG2THR9638.985−26.09620.7021.0048.191SG 743ATOM743CTHR9640.872−25.20622.5021.0048.191SG 744ATOM744OTHR9641.198−25.83523.5091.0048.191SG 745ATOM745NPHE9740.043−24.13922.5371.0046.051SG 746ATOM746CAPHE9739.392−23.66923.7241.0046.051SG 747ATOM747CBPHE9739.817−22.25324.1581.0046.051SG 748ATOM748CGPHE9741.227−22.29624.6391.0046.051SG 749ATOM749CD1PHE9742.279−22.17523.7601.0046.051SG 750ATOM750CD2PHE9741.496−22.45425.9791.0046.051SG 751ATOM751CE1PHE9743.578−22.21324.2121.0046.051SG 752ATOM752CE2PHE9742.792−22.49326.4371.0046.051SG 753ATOM753CZPHE9743.837−22.37325.5531.0046.051SG 754ATOM754CPHE9737.938−23.58823.3881.0046.051SG 755ATOM755OPHE9737.563−23.34822.2411.0046.051SG 756ATOM756NPHE9837.066−23.81224.3841.0042.941SG 757ATOM757CAPHE9835.665−23.70824.1191.0042.941SG 758ATOM758CBPHE9834.816−24.78324.8191.0042.941SG 759ATOM759CGPHE9835.051−26.08924.1431.0042.941SG 760ATOM760CD1PHE9836.159−26.84924.4391.0042.941SG 761ATOM761CD2PHE9834.149−26.55823.2171.0042.941SG 762ATOM762CE1PHE9836.364−28.05523.8121.0042.941SG 763ATOM763CE2PHE9834.350−27.76322.5871.0042.941SG 764ATOM764CZPHE9835.460−28.51522.8851.0042.941SG 765ATOM765CPHE9835.235−22.39024.6561.0042.941SG 766ATOM766OPHE9835.414−22.10825.8401.0042.941SG 767ATOM767NGLN9934.676−21.53723.7751.0039.291SG 768ATOM768CAGLN9934.184−20.27624.2271.0039.291SG 769ATOM769CBGLN9934.032−19.21023.1261.0039.291SG 770ATOM770CGGLN9935.359−18.70022.5631.0039.291SG 771ATOM771CDGLN9935.045−17.66021.4951.0039.291SG 772ATOM772OE1GLN9933.988−17.69320.8671.0039.291SG 773ATOM773NE2GLN9935.988−16.70321.2861.0039.291SG 774ATOM774CGLN9932.822−20.55424.7651.0039.291SG 775ATOM775OGLN9931.971−21.11524.0751.0039.291SG 776ATOM776NSER10032.591−20.17526.0351.0038.031SG 777ATOM777CASER10031.320−20.43026.6391.0038.031SG 778ATOM778CBSER10031.415−21.21727.9571.0038.031SG 779ATOM779OGSER10032.107−20.45328.9331.0038.031SG 780ATOM780CSER10030.714−19.11126.9611.0038.031SG 781ATOM781OSER10031.403−18.17527.3661.0038.031SG 782ATOM782NSER10129.388−19.00526.7761.0036.291SG 783ATOM783CASER10128.752−17.75727.0561.0036.291SG 784ATOM784CBSER10127.779−17.30825.9541.0036.291SG 785ATOM785OGSER10126.695−18.22125.8621.0036.291SG 786ATOM786CSER10127.944−17.93328.2931.0036.291SG 787ATOM787OSER10127.096−18.82028.3751.0036.291SG 788ATOM788NSER10228.214−17.08629.3021.0034.221SG 789ATOM789CASER10227.433−17.09930.4991.0034.221SG 790ATOM790CBSER10228.186−17.66331.7181.0034.221SG 791ATOM791OGSER10229.341−16.88331.9941.0034.221SG 792ATOM792CSER10227.113−15.66730.7581.0034.221SG 793ATOM793OSER10227.991−14.87631.0981.0034.221SG 794ATOM794NGLY10325.835−15.28630.5991.0032.471SG 795ATOM795CAGLY10325.530−13.90030.7671.0032.471SG 796ATOM796CGLY10326.265−13.21229.6631.0032.471SG 797ATOM797OGLY10326.507−13.79428.6091.0032.471SG 798ATOM798NSER10426.641−11.94229.8741.0033.671SG 799ATOM799CASER10427.366−11.22028.8711.0033.671SG 800ATOM800CBSER10427.414−9.70829.1491.0033.671SG 801ATOM801OGSER10428.126−9.04128.1171.0033.671SG 802ATOM802CSER10428.778−11.72528.8501.0033.671SG 803ATOM803OSER10429.528−11.46127.9111.0033.671SG 804ATOM804NALA10529.170−12.45229.9141.0036.181SG 805ATOM805CAALA10530.512−12.93230.1181.0036.181SG 806ATOM806CBALA10530.761−13.35831.5711.0036.181SG 807ATOM807CALA10530.823−14.12729.2741.0036.181SG 808ATOM808OALA10529.941−14.89128.8831.0036.181SG 809ATOM809NPHE10632.129−14.30928.9731.0040.271SG 810ATOM810CAPHE10632.558−15.44728.2171.0040.271SG 811ATOM811CBPHE10632.928−15.09426.7701.0040.271SG 812ATOM812CGPHE10631.607−14.77526.1611.0040.271SG 813ATOM813CD1PHE10631.001−13.56026.3891.0040.271SG 814ATOM814CD2PHE10630.966−15.70125.3721.0040.271SG 815ATOM815CE1PHE10629.775−13.27425.8351.0040.271SG 816ATOM816CE2PHE10629.741−15.42124.8151.0040.271SG 817ATOM817CZPHE10629.144−14.20625.0481.0040.271SG 818ATOM818CPHE10633.720−16.07028.9201.0040.271SG 819ATOM819OPHE10634.522−15.38429.5531.0040.271SG 820ATOM820NARG10733.810−17.41328.8491.0044.061SG 821ATOM821CAARG10734.882−18.12129.4821.0044.061SG 822ATOM822CBARG10734.434−19.06330.6171.0044.061SG 823ATOM823CGARG10733.858−18.33831.8341.0044.061SG 824ATOM824CDARG10732.483−17.71531.5821.0044.061SG 825ATOM825NEARG10731.496−18.83031.5181.0044.061SG 826ATOM826CZARG10730.924−19.30332.6641.0044.061SG 827ATOM827NH1ARG10731.244−18.74533.8681.0044.061SG 828ATOM828NH2ARG10730.030−20.33332.6061.0044.061SG 829ATOM829CARG10735.510−18.97628.4321.0044.061SG 830ATOM830OARG10734.829−19.45927.5271.0044.061SG 831ATOM831NLEU10836.843−19.15528.5241.0043.881SG 832ATOM832CALEU10837.561−19.97227.5881.0043.881SG 833ATOM833CBLEU10838.797−19.27126.9991.0043.881SG 834ATOM834CGLEU10838.462−18.00126.1941.0043.881SG 835ATOM835CD2LEU10839.691−17.48525.4281.0043.881SG 836ATOM836CD1LEU10837.827−16.92427.0891.0043.881SG 837ATOM837CLEU10838.051−21.17028.3331.0043.881SG 838ATOM838OLEU10838.964−21.08729.1541.0043.881SG 839ATOM839NGLU10937.453−22.33528.0381.0041.591SG 840ATOM840CAGLU10937.797−23.55528.7131.0041.591SG 841ATOM841CBGLU10936.562−24.41129.0291.0041.591SG 842ATOM842CGGLU10936.881−25.78129.6241.0041.591SG 843ATOM843CDGLU10935.600−26.59929.5631.0041.591SG 844ATOM844OE1GLU10934.814−26.54530.5461.0041.591SG 845ATOM845OE2GLU10935.384−27.28428.5271.0041.591SG 846ATOM846CGLU10938.633−24.36927.7861.0041.591SG 847ATOM847OGLU10938.373−24.41926.5861.0041.591SG 848ATOM848NALA11039.676−25.02928.3241.0038.021SG 849ATOM849CAALA11040.494−25.85027.4821.0038.021SG 850ATOM850CBALA11041.774−26.35428.1701.0038.021SG 851ATOM851CALA11039.692−27.04927.0781.0038.021SG 852ATOM852OALA11038.991−27.64827.8931.0038.021SG 853ATOM853NALA11139.769−27.40825.7791.0037.701SG 854ATOM854CAALA11139.096−28.56625.2681.0037.701SG 855ATOM855CBALA11139.238−28.71023.7441.0037.701SG 856ATOM856CALA11139.743−29.76125.8871.0037.701SG 857ATOM857OALA11139.080−30.73026.2531.0037.701SG 858ATOM858NALA11241.086−29.71025.9851.0042.331SG 859ATOM859CAALA11241.885−30.78326.4991.0042.331SG 860ATOM860CBALA11243.393−30.50626.3731.0042.331SG 861ATOM861CALA11241.601−31.02627.9501.0042.331SG 862ATOM862OALA11241.457−32.17628.3631.0042.331SG 863ATOM863NTRP11341.523−29.95928.7731.0051.121SG 864ATOM864CATRP11341.267−30.16230.1731.0051.121SG 865ATOM865CBTRP11342.358−29.57231.0771.0051.121SG 866ATOM866CGTRP11343.692−30.26130.9261.0051.121SG 867ATOM867CD2TRP11344.098−31.40831.6881.0051.121SG 868ATOM868CD1TRP11344.725−29.97030.0841.0051.121SG 869ATOM869NE1TRP11345.750−30.86630.2721.0051.121SG 870ATOM870CE2TRP11345.377−31.75731.2571.0051.121SG 871ATOM871CE3TRP11343.456−32.11332.6651.0051.121SG 872ATOM872CZ2TRP11346.038−32.82231.8001.0051.121SG 873ATOM873CZ3TRP11344.125−33.18633.2111.0051.121SG 874ATOM874CH2TRP11345.391−33.53332.7871.0051.121SG 875ATOM875CTRP11339.992−29.44530.4861.0051.121SG 876ATOM876OTRP11339.952−28.21730.5221.0051.121SG 877ATOM877NPRO11438.959−30.18830.7721.0061.831SG 878ATOM878CAPRO11437.661−29.59130.9371.0061.831SG 879ATOM879CDPRO11438.865−31.55030.2711.0061.831SG 880ATOM880CBPRO11436.677−30.75730.9891.0061.831SG 881ATOM881CGPRO11437.359−31.83330.1231.0061.831SG 882ATOM882CPRO11437.472−28.57432.0221.0061.831SG 883ATOM883OPRO11436.697−27.64431.8481.0061.831SG 884ATOM884NGLY11538.097−28.72233.1811.0068.271SG 885ATOM885CAGLY11537.874−27.76934.2291.0068.271SG 886ATOM886CGLY11538.590−26.47834.0111.0068.271SG 887ATOM887OGLY11538.211−25.45834.5811.0068.271SG 888ATOM888NTRP11639.704−26.52233.2631.0068.481SG 889ATOM889CATRP11640.629−25.42633.2001.0068.481SG 890ATOM890CBTRP11642.011−25.93732.7941.0068.481SG 891ATOM891CGTRP11642.440−27.01633.7521.0068.481SG 892ATOM892CD2TRP11643.383−26.85234.8201.0068.481SG 893ATOM893CD1TRP11642.011−28.30933.8071.0068.481SG 894ATOM894NE1TRP11642.618−28.96034.8521.0068.481SG 895ATOM895CE2TRP11643.469−28.07635.4821.0068.481SG 896ATOM896CE3TRP11644.114−25.77035.2191.0068.481SG 897ATOM897CZ2TRP11644.292−28.23736.5601.0068.481SG 898ATOM898CZ3TRP11644.947−25.93736.3031.0068.481SG 899ATOM899CH2TRP11645.034−27.14736.9601.0068.481SG 900ATOM900CTRP11640.205−24.35532.2521.0068.481SG 901ATOM901OTRP11639.756−24.62831.1391.0068.481SG 902ATOM902NPHE11740.367−23.08332.6931.0063.221SG 903ATOM903CAPHE11739.994−21.97131.8691.0063.221SG 904ATOM904CBPHE11738.785−21.16532.3811.0063.221SG 905ATOM905CGPHE11737.636−22.08732.6111.0063.221SG 906ATOM906CD1PHE11737.093−22.83631.5931.0063.221SG 907ATOM907CD2PHE11737.071−22.16833.8621.0063.221SG 908ATOM908CE1PHE11736.028−23.67231.8331.0063.221SG 909ATOM909CE2PHE11736.005−23.00134.1041.0063.221SG 910ATOM910CZPHE11735.481−23.76133.0891.0063.221SG 911ATOM911CPHE11741.128−20.99431.8481.0063.221SG 912ATOM912OPHE11741.981−20.98832.7361.0063.221SG 913ATOM913NLEU11841.165−20.13530.8071.0054.771SG 914ATOM914CALEU11842.161−19.11030.7511.0054.771SG 915ATOM915CBLEU11842.137−18.30529.4401.0054.771SG 916ATOM916CGLEU11843.205−17.19929.3671.0054.771SG 917ATOM917CD2LEU11842.964−16.26628.1701.0054.771SG 918ATOM918CD1LEU11844.623−17.78829.3861.0054.771SG 919ATOM919CLEU11841.804−18.18831.8651.0054.771SG 920ATOM920OLEU11840.636−17.83232.0261.0054.771SG 921ATOM921NCYS11942.797−17.78932.6801.0045.641SG 922ATOM922CACYS11942.468−16.95233.7921.0045.641SG 923ATOM923CBCYS11942.456−17.69235.1411.0045.641SG 924ATOM924SGCYS11941.139−18.94135.2411.0045.641SG 925ATOM925CCYS11943.469−15.85633.9001.0045.641SG 926ATOM926OCYS11944.536−15.88933.2861.0045.641SG 927ATOM927NGLY12043.114−14.83734.7041.0039.831SG 928ATOM928CAGLY12043.986−13.72834.9211.0039.831SG 929ATOM929CGLY12043.942−13.43936.3831.0039.831SG 930ATOM930OGLY12042.993−13.77737.0911.0039.831SG 931ATOM931NPRO12145.027−12.88336.8321.0037.351SG 932ATOM932CAPRO12145.105−12.50438.2171.0037.351SG 933ATOM933CDPRO12146.285−13.43636.3591.0037.351SG 934ATOM934CBPRO12146.571−12.61338.6091.0037.351SG 935ATOM935CGPRO12147.140−13.63737.6141.0037.351SG 936ATOM936CPRO12144.516−11.15638.4961.0037.351SG 937ATOM937OPRO12144.351−10.35837.5751.0037.351SG 938ATOM938NALA12244.183−10.90039.7751.0035.181SG 939ATOM939CAALA12243.625−9.65540.2141.0035.181SG 940ATOM940CBALA12243.194−9.68441.6901.0035.181SG 941ATOM941CALA12244.642−8.56840.0651.0035.181SG 942ATOM942OALA12244.311−7.45239.6691.0035.181SG 943ATOM943NGLU12345.915−8.86640.3971.0037.201SG 944ATOM944CAGLU12346.938−7.85940.3551.0037.201SG 945ATOM945CBGLU12348.206−8.21241.1481.0037.201SG 946ATOM946CGGLU12348.959−9.41940.5911.0037.201SG 947ATOM947CDGLU12350.199−9.62941.4481.0037.201SG 948ATOM948OE1GLU12350.344−8.89842.4631.0037.201SG 949ATOM949OE2GLU12351.018−10.52141.0981.0037.201SG 950ATOM950CGLU12347.349−7.62238.9411.0037.201SG 951ATOM951OGLU12347.181−8.45938.0551.0037.201SG 952ATOM952NPRO12447.857−6.44138.7321.0040.571SG 953ATOM953CAPRO12448.305−6.07037.4211.0040.571SG 954ATOM954CDPRO12447.362−5.29439.4731.0040.571SG 955ATOM955CBPRO12448.254−4.54037.3611.0040.571SG 956ATOM956CGPRO12448.064−4.09638.8221.0040.571SG 957ATOM957CPRO12449.646−6.63537.0751.0040.571SG 958ATOM958OPRO12450.405−7.00337.9691.0040.571SG 959ATOM959NGLN12549.939−6.69435.7631.0045.041SG 960ATOM960CAGLN12551.200−7.10135.2181.0045.041SG 961ATOM961CBGLN12552.344−6.16335.6341.0045.041SG 962ATOM962CGGLN12552.167−4.73335.1181.0045.041SG 963ATOM963CDGLN12553.357−3.91435.5951.0045.041SG 964ATOM964OE1GLN12554.207−4.41036.3321.0045.041SG 965ATOM965NE2GLN12553.419−2.62535.1671.0045.041SG 966ATOM966CGLN12551.600−8.50435.5631.0045.041SG 967ATOM967OGLN12552.794−8.77835.6811.0045.041SG 968ATOM968NGLN12650.655−9.45335.7111.0051.171SG 969ATOM969CAGLN12651.144−10.79235.8851.0051.171SG 970ATOM970CBGLN12650.898−11.45437.2541.0051.171SG 971ATOM971CGGLN12649.458−11.69237.6821.0051.171SG 972ATOM972CDGLN12649.566−12.46138.9951.0051.171SG 973ATOM973OE1GLN12648.972−12.09940.0091.0051.171SG 974ATOM974NE2GLN12650.351−13.57138.9731.0051.171SG 975ATOM975CGLN12650.634−11.61834.7431.0051.171SG 976ATOM976OGLN12649.703−11.22734.0411.0051.171SG 977ATOM977NPRO12751.248−12.74634.5171.0053.741SG 978ATOM978CAPRO12750.915−13.51933.3501.0053.741SG 979ATOM979CDPRO12752.638−12.92534.8931.0053.741SG 980ATOM980CBPRO12752.083−14.48833.1301.0053.741SG 981ATOM981CGPRO12752.971−14.33334.3791.0053.741SG 982ATOM982CPRO12749.574−14.16833.3671.0053.741SG 983ATOM983OPRO12749.026−14.40634.4411.0053.741SG 984ATOM984NVAL12849.021−14.43332.1651.0052.631SG 985ATOM985CAVAL12847.771−15.12532.0651.0052.631SG 986ATOM986CBVAL12847.027−14.90330.7791.0052.631SG 987ATOM987CG1VAL12846.517−13.46030.7431.0052.631SG 988ATOM988CG2VAL12847.962−15.23429.6091.0052.631SG 989ATOM989CVAL12848.111−16.57132.1391.0052.631SG 990ATOM990OVAL12849.112−17.00331.5691.0052.631SG 991ATOM991NGLN12947.295−17.35332.8751.0048.041SG 992ATOM992CAGLN12947.592−18.74633.0101.0048.041SG 993ATOM993CBGLN12948.225−19.12734.3601.0048.041SG 994ATOM994CGGLN12949.603−18.50634.6001.0048.041SG 995ATOM995CDGLN12949.408−17.09935.1431.0048.041SG 996ATOM996OE1GLN12950.374−16.36235.3331.0048.041SG 997ATOM997NE2GLN12948.130−16.71835.4121.0048.041SG 998ATOM998CGLN12946.317−19.51032.9041.0048.041SG 999ATOM999OGLN12945.256−18.94832.6371.0048.041SG 1000ATOM1000NLEU13046.415−20.84133.0841.0042.951SG 1001ATOM1001CALEU13045.265−21.68833.0321.0042.951SG 1002ATOM1002CBLEU13045.526−22.97132.2231.0042.951SG 1003ATOM1003CGLEU13044.261−23.77931.8981.0042.951SG 1004ATOM1004CD2LEU13044.614−25.14131.2801.0042.951SG 1005ATOM1005CD1LEU13043.309−22.96231.0081.0042.951SG 1006ATOM1006CLEU13044.978−22.04834.4591.0042.951SG 1007ATOM1007OLEU13045.856−22.54335.1661.0042.951SG 1008ATOM1008NTHR13143.741−21.78034.9301.0039.851SG 1009ATOM1009CATHR13143.402−22.05736.2981.0039.851SG 1010ATOM1010CBTHR13142.903−20.85637.0481.0039.851SG 1011ATOM1011OG1THR13143.888−19.83337.0451.0039.851SG 1012ATOM1012CG2THR13142.580−21.27638.4921.0039.851SG 1013ATOM1013CTHR13142.298−23.06236.2871.0039.851SG 1014ATOM1014OTHR13141.472−23.07735.3751.0039.851SG 1015ATOM1015NLYS13242.263−23.95037.3011.0042.271SG 1016ATOM1016CALYS13241.251−24.96237.3071.0042.271SG 1017ATOM1017CBLYS13241.817−26.36437.6021.0042.271SG 1018ATOM1018CGLYS13240.802−27.49537.4141.0042.271SG 1019ATOM1019CDLYS13241.420−28.89437.4401.0042.271SG 1020ATOM1020CELYS13241.938−29.30838.8181.0042.271SG 1021ATOM1021NZLYS13242.507−30.67338.7571.0042.271SG 1022ATOM1022CLYS13240.262−24.65038.3761.0042.271SG 1023ATOM1023OLYS13239.497−25.51938.7911.0042.271SG 1024ATOM1024NGLU13340.215−23.39238.8431.0047.601SG 1025ATOM1025CAGLU13339.251−23.13939.8641.0047.601SG 1026ATOM1026CBGLU13339.872−23.01541.2671.0047.601SG 1027ATOM1027CGGLU13338.858−23.13342.4041.0047.601SG 1028ATOM1028CDGLU13338.080−21.83242.4781.0047.601SG 1029ATOM1029OE1GLU13338.641−20.78242.0661.0047.601SG 1030ATOM1030OE2GLU13336.913−21.87342.9511.0047.601SG 1031ATOM1031CGLU13338.567−21.85639.5431.0047.601SG 1032ATOM1032OGLU13339.207−20.84839.2431.0047.601SG 1033ATOM1033NSER13437.225−21.87539.5921.0052.731SG 1034ATOM1034CASER13436.485−20.67339.3831.0052.731SG 1035ATOM1035CBSER13435.282−20.84038.4401.0052.731SG 1036ATOM1036OGSER13434.602−19.60238.2921.0052.731SG 1037ATOM1037CSER13435.958−20.33040.7341.0052.731SG 1038ATOM1038OSER13435.151−21.06241.3041.0052.731SG 1039ATOM1039NGLU13536.440−19.20441.2831.0055.751SG 1040ATOM1040CAGLU13536.079−18.73442.5861.0055.751SG 1041ATOM1041CBGLU13537.055−19.23043.6751.0055.751SG 1042ATOM1042CGGLU13536.567−19.09745.1201.0055.751SG 1043ATOM1043CDGLU13535.508−20.16445.3631.0055.751SG 1044ATOM1044OE1GLU13535.831−21.36945.1931.0055.751SG 1045ATOM1045OE2GLU13534.360−19.78745.7221.0055.751SG 1046ATOM1046CGLU13536.245−17.25942.4561.0055.751SG 1047ATOM1047OGLU13536.592−16.79241.3771.0055.751SG 1048ATOM1048NPRO13635.992−16.48843.4651.0054.051SG 1049ATOM1049CAPRO13636.189−15.07843.3061.0054.051SG 1050ATOM1050CDPRO13634.852−16.74544.3261.0054.051SG 1051ATOM1051CBPRO13635.458−14.40944.4761.0054.051SG 1052ATOM1052CGPRO13634.896−15.57445.3161.0054.051SG 1053ATOM1053CPRO13637.622−14.66143.1431.0054.051SG 1054ATOM1054OPRO13637.845−13.50642.7861.0054.051SG 1055ATOM1055NSER13738.604−15.54043.4311.0051.531SG 1056ATOM1056CASER13739.993−15.16743.3611.0051.531SG 1057ATOM1057CBSER13740.927−16.31243.7811.0051.531SG 1058ATOM1058OGSER13740.713−16.64445.1441.0051.531SG 1059ATOM1059CSER13740.415−14.75441.9801.0051.531SG 1060ATOM1060OSER13740.455−13.56741.6611.0051.531SG 1061ATOM1061NALA13840.745−15.74641.1231.0051.191SG 1062ATOM1062CAALA13841.240−15.48539.7981.0051.191SG 1063ATOM1063CBALA13842.003−16.67139.1841.0051.191SG 1064ATOM1064CALA13840.085−15.17638.9111.0051.191SG 1065ATOM1065OALA13838.958−15.59839.1681.0051.191SG 1066ATOM1066NARG13940.343−14.42237.8251.0054.851SG 1067ATOM1067CAARG13939.256−14.07236.9681.0054.851SG 1068ATOM1068CBARG13939.302−12.61136.4981.0054.851SG 1069ATOM1069CGARG13938.052−12.19735.7291.0054.851SG 1070ATOM1070CDARG13937.915−10.68535.5681.0054.851SG 1071ATOM1071NEARG13937.851−10.09236.9331.0054.851SG 1072ATOM1072CZARG13936.739−10.27837.7031.0054.851SG 1073ATOM1073NH1ARG13935.744−11.10837.2751.0054.851SG 1074ATOM1074NH2ARG13936.627−9.64438.9071.0054.851SG 1075ATOM1075CARG13939.308−14.96035.7741.0054.851SG 1076ATOM1076OARG13940.374−15.20535.2101.0054.851SG 1077ATOM1077NTHR14038.142−15.50735.3861.0060.011SG 1078ATOM1078CATHR14038.109−16.36334.2441.0060.011SG 1079ATOM1079CBTHR14037.824−17.78834.6141.0060.011SG 1080ATOM1080OG1THR14037.863−18.61733.4631.0060.011SG 1081ATOM1081CG2THR14036.450−17.86535.2931.0060.011SG 1082ATOM1082CTHR14037.025−15.87433.3361.0060.011SG 1083ATOM1083OTHR14036.615−16.58232.4201.0060.011SG 1084ATOM1084NLYS14136.566−14.62433.5491.0064.191SG 1085ATOM1085CALYS14135.490−14.05032.7941.0064.191SG 1086ATOM1086CBLYS14134.440−13.40333.7191.0064.191SG 1087ATOM1087CGLYS14133.242−12.74933.0281.0064.191SG 1088ATOM1088CDLYS14133.563−11.46432.2611.0064.191SG 1089ATOM1089CELYS14132.375−10.89331.4871.0064.191SG 1090ATOM1090NZLYS14131.301−10.50032.4261.0064.191SG 1091ATOM1091CLYS14136.051−12.98631.9071.0064.191SG 1092ATOM1092OLYS14136.909−12.20532.3191.0064.191SG 1093ATOM1093NPHE14235.552−12.92230.6541.0064.861SG 1094ATOM1094CAPHE14236.082−11.97429.7191.0064.861SG 1095ATOM1095CBPHE14236.991−12.63628.6661.0064.861SG 1096ATOM1096CGPHE14238.061−13.39829.3761.0064.861SG 1097ATOM1097CD1PHE14239.036−12.74230.0881.0064.861SG 1098ATOM1098CD2PHE14238.108−14.77229.3141.0064.861SG 1099ATOM1099CE1PHE14240.028−13.43830.7391.0064.861SG 1100ATOM1100CE2PHE14239.099−15.47329.9631.0064.861SG 1101ATOM1101CZPHE14240.062−14.80930.6811.0064.861SG 1102ATOM1102CPHE14234.952−11.35828.9501.0064.861SG 1103ATOM1103OPHE14233.832−11.86628.9391.0064.861SG 1104ATOM1104NTYR14335.255−10.22628.2791.0062.771SG 1105ATOM1105CATYR14334.355−9.49127.4321.0062.771SG 1106ATOM1106CBTYR14334.142−8.01827.8521.0062.771SG 1107ATOM1107CGTYR14333.283−7.85129.0651.0062.771SG 1108ATOM1108CD1TYR14333.820−7.82530.3341.0062.771SG 1109ATOM1109CD2TYR14331.922−7.69828.9261.0062.771SG 1110ATOM1110CE1TYR14333.012−7.65531.4371.0062.771SG 1111ATOM1111CE2TYR14331.109−7.52730.0221.0062.771SG 1112ATOM1112CZTYR14331.654−7.50531.2821.0062.771SG 1113ATOM1113OHTYR14330.819−7.32732.4071.0062.771SG 1114ATOM1114CTYR14335.056−9.39426.1101.0062.771SG 1115ATOM1115OTYR14336.284−9.46326.0561.0062.771SG 1116ATOM1116NPHE14434.291−9.25125.0051.0060.391SG 1117ATOM1117CAPHE14434.903−9.10423.7121.0060.391SG 1118ATOM1118CBPHE14434.301−10.00122.6171.0060.391SG 1119ATOM1119CGPHE14434.118−11.38323.1441.0060.391SG 1120ATOM1120CD1PHE14435.166−12.26023.3241.0060.391SG 1121ATOM1121CD2PHE14432.849−11.78723.4841.0060.391SG 1122ATOM1122CE1PHE14434.937−13.52423.8201.0060.391SG 1123ATOM1123CE2PHE14432.614−13.04623.9771.0060.391SG 1124ATOM1124CZPHE14433.661−13.92024.1441.0060.391SG 1125ATOM1125CPHE14434.546−7.71023.2771.0060.391SG 1126ATOM1126OPHE14433.400−7.28823.4251.0060.391SG 1127ATOM1127NGLU14535.504−6.93922.7271.0057.371SG 1128ATOM1128CAGLU14535.130−5.60522.3531.0057.371SG 1129ATOM1129CBGLU14535.800−4.48923.1841.0057.371SG 1130ATOM1130CGGLU14535.176−4.25024.5651.0057.371SG 1131ATOM1131CDGLU14535.786−5.21325.5771.0057.371SG 1132ATOM1132OE1GLU14536.153−6.35025.1821.0057.371SG 1133ATOM1133OE2GLU14535.895−4.81826.7681.0057.371SG 1134ATOM1134CGLU14535.488−5.35520.9001.0057.371SG 1135ATOM1135OGLU14536.283−4.41020.6481.0057.371SG 1136ATOM1136OXTGLU14534.961−6.08920.0231.0057.371SG 1137

EXAMPLE 15

Determination of IL-1 Hy2 Crystal Structure

[0512] Crystallization is used to verify the predicted three-dimensional structure of IL-1 Hy2 using methods known in the are, e.g., as described by Vigers et al. (Nature 386:190-194, 1997). Recombinant IL-1 Hy2 and the IL-1 receptor art incubated under conditions which promote binding. The IL-1 Hy2 receptor complex is then purified and crystallized.

[0513] Crystals consisting of the IL-1 Hy2 alone. IL-1 Hy2/IL-1 receptor complex or IL-1 Hy2/IL-1 receptor accessory protein or IL-1 Hy2/Il-1R/IL-1 receptor accessory protein are grown by methods known in the art. (See Jensen et al., J. Immunol. 15: 5277-86, 2000). For example, hanging-drop diffusion against a salt solution such as an ammonium sulphate solution, an ammonium nitrate solution or an ammonium chloride solution may be used to form IL-1 Hy2 crystals. The resulting crystals are diffracted in a beam of x-rays to determine their quality. Heavy-atom derivatives are created and compared to the native crystal. The crystals are cryoprotected and diffracted to determine the diffraction pattern, the unit cell dimensions and symmetry. Molecular replacement is used to determine the final three-dimensional structure.

EXAMPLE 16

Determination of Critical Residues by Site-Directed Mutagenesis

[0514] Site-directed mutagenesis is carried out to confirm the location of specific amino acids within the IL-1 Hy2 polypeptide sequence such as those residues predicted to interact with the IL-1 receptor and those predicted to be important to biological function. The mutants are designed based on the predicted three-dimensional structure (described in Example 14) or on the crystal structure (described in Example 15).

[0515] Mutants are produced, e.g., via site-directed mutagenesis performed on IL-1 Hy2 cDNA constructs using any method known in the art. For example, uracil-enrichment of single-stranded DNA may be used as described by Kunkel et al. (Proc. Natl. Acad. Sci. U.S.A., 82: 488-492, 1985). Mutagenesis primers are designed based on the (+) coding orientation, and mutagenesis is carried out with a commercially available mutagenesis kit such as the Muta-Gene kit (Biorad) or the Transformer™ Site-Directed Mutagenesis kit (Clontech) according to the manufacturer's instructions.

[0516] The mutants generated will have non-conservative substitutions of amino acids predicted to be critical for IL-1 Hy2 function or IL-1 receptor binding, or IL-1 receptor accessory protein binding. In addition, mutants will have amino acid insertions and/or deletions within functional domains, and a single mutant may contain more than one change within the amino acid sequence.

[0517] The resulting mutants cDNAs are sequenced, and recombinant IL-1 Hy2 mutants are purified and analyzed in functional assays such as the IL-1 receptor binding assay (described in Example 12), inhibition of IL-6 production assay (described in Example 10) and inhibition of IL-18 activity assay (described in Example 11). Both mutants which knock out function and those that increase function are desirable. In addition, these mutants can be crystallized (as described in Example 15) to determine if a change in the amino acid sequence alters the three-dimensional structure of IL-1 Hy2. Mutants lacking IL-1 Hy2 activity, including receptor binding activity, are useful in screening for compounds which bind to the wild type IL-1 Hy2 polypeptide but do not interact with the IL-1 Hy2 mutants, and thus identifying modulators specific for the active site.

EXAMPLE 17

Expression of IL-1Hy2 in E.coli

[0518] The coding region of SEQ ID NO: 2 was expressed in E. coli. First, the coding region was amplified by PCR using the forward primer QB36 (GTCATATGTGTTCCCTCCCCATGGCAAG; SEQ ID NO: 25) and reverse primer QB38 (GTTTTACTTTGAACAGAGCTGGTAGTGATCAAGCTTC; SEQ ID NO: 26). Primer QB36 corresponds to nucleotides 54 to 76 of SEQ ID NO: 1 and Primer QB38 corresponds to the reverse complement of nucleotides 488 to 512 of SEQ ID NO: 1. The underlined nucleotides are restriction sites to assist in cloning. PCR was carried out using Pfu polymerase and the primer QB36 and QB38.

[0519] The PCR product was ligated into the TOPO cloning vector pCRII (Invitrogen) and transformed into electrocompetent E. coli strain Top10 (Invitrogen). The transformed cells were plated on ampicillin-containing plates. Colonies were screened for the correct insert using a PCR reaction employing a gene-specific primer and a vector-specific primer. Positives were then sequenced to ensure correct sequences.

[0520] The pCRII was digested with Nde,l and HindIII and purified with low melting agarose (LMPA) from FHM Bioproducts. The resulting IL-1 Hy2 fragment was subcloned into the prokaryotic expression vector pRSETB (invitrogen). The pRSETB was useful for protein expression because it contains an efficient promoter (phage T7) to drive trancription. In addition, this vector provides gene expression control with the lac operator system, which can be induced by the addition of IPTG (isopropyl-β-D-thiogaloctopyranoside).

[0521] The pRSETB vector containing IL-1 Hy2 was transformed into E. Coli strain DH10B (Invitrogen) The transformed cells were plated on carbenicillin-containing plates. Colonies containing the correct insert were verified by restriction digest. Six clones were selected, the DNA was isolated with a mini-prep (QIAgen) and then transformed into the BL21(DE3)plysS cells (Invitrogen). Cloning between the NdeI and HindIII sites resulted in IL-1 Hy2 gene expression without tags.

[0522] The transformed BL21DE3pLysS cells containing the plasmid pRSETB with IL-1 Hy2 were cultured in LB broth containing 0.1 mg/ml of ampicillin and 20 μg/ml Chloamphenicol. A 200 ml starter culture was innoculated from with BL21DE3pLysS cells and the culture was allowed to grow overnight at 37° C. with constant shaking at 250 RPM. The starter culture was then used to innoculate 4 liters of complex fermenter media containing 0.1 mg/ml of ampicillin. At this point, IPTG was added to a final concentration of 1 mM at 25 OD to induce protein expression. The culture was allowed to grow for 2 more hours, and then the cells were harvested at a final ODA600 of 48.5 and a density of 59.6 grams/liter by centrifugation at 10,000×g for 30 minutes. The cell pellets were stored at −70° C.

EXAMPLE 18

Purification of IL-1 HY2 Polypeptide from E.coli

[0523] For two 5 liter fermentations, 550 grams of wet cell paste were suspended in 5 liters of lysis buffer (20 mM Tris pH 8.0, 1 mM EDTA). The cells were disrupted with two passes through an Avestin C50 homogenizer at 15,000 psi. The temperature after disruption was kept at or below 24° C. using a cooling coil and ice bath. After disruption, the lysate was centrifuged at 13,000×g for 20 minutes to remove cell debris. The lysate supernatant was clarified with 1M BisTris (pH 6.0) to reach a final concentration of 20 mM Bis Tris. The supernatant was then titrated to pH 4.7 using 0.5 HCl to precipitate the proteins. The precipitated proteins were removed by centrifugation at 13,000×g for 30 minutes. The pH of the clarified titrated supernatant was then adjusted to 6.0 using 0.5 M NaOH.

[0524] Q-Sepharose Anion Exchange Chromatography at pH 8.0

[0525] The pH 6.0 supernatent was loaded onto a 500 ml Q-Sepharose FF column (AP Biotech) equilibrated in 20 mM Bis Tris pH 6.0 (Equilibration Buffer A) at 100 cm/hr linear flow rate. After the sample was applied, the column was washed with 2 volumes of Equilibration Buffer A. Subsequently, the column was washed with two volumes of Elution Buffer A (20 mM Tris pH 8.0) followed by two volumes of 20% Elution Buffer B (20 ,mM Tris pH 8.0, 500 mM NaCl). The column was then eluted with a 15 volume linear gradient consisting of 20% to 100% Buffer B. Fractions were collected and evaluated by SDS PAGE. The fractions were collected in two pools based on the SDS PAGE results and a chromatogram.

[0526] Phenyl Sepharose High Sub Hydrophobic Interaction Chromatography

[0527] The main pool generated by Q-Sepharose chromatography (described above) was titrated to pH7.0 using 2 M NaOH. This pool was then divided into two equal alliquots and ammonium sulfate was added to a final concentration of 1.5 M ammonium sulfate using a 4 M solution. The pool was then loaded onto a 180 ml Phenyl Sepharose High Sub Column (AP Biotech), equilibrated in 1.5 M ammonium sulfate, 50 mM sodium phosphate at pH 7.0 (Equilibration Buffer B), in two runs at 100 cm/hr linear flow rate. The column was then washed with five volumes of Equilibration Buffer B. Subsequently, the column was eluted with a 15 volume linear gradient consisting of 1.2 M to 0.45 M ammonium sulfate. Fractions from the two runs which contained the lowest level of impurities as seen on a SDS-PAGE gel were pooled. The side fractions that were not included in the pool were combined and run as a third aliquot under the same conditions to recover more purified IL-1Hy2.

[0528] The combined pools were concentrated approximately 5 fold using a Millipore PrepScale spiral 10 K molecular weight cut off membrane cartridge. Ammonium sulfate was then removed by dilution and diafiltration with 1 L of 50 mM sodium phosphate (pH 7.0).

[0529] DEAE Sepharose Anion Exchange Chromatography

[0530] The desalted pool was loaded onto a 75 ml DEAE Sepharose FF column (AP Biotech) in order to remove endotoxins and additional impurities. The column was first equilibrated in 20 mM sodium phosphate pH 7.0 (Equilibration Buffer C). After loading of the sample at a flow rate of 100 cm/hr, the column was washed with 5 volumes of Equilibration Buffer C. Subsequently, the column was eluted with 4 volumes of 200 mM NaCl. The protein eluted in one peak and was collected in a single pool.

[0531] Q-Sepharose Anion Exchange Chromotography at pH 7.0

[0532] The pool from the DEAE Sepharose column (described above) was diluted approximately two-fold with 20 mM sodium phosphate (pH 7.0) to lower conductivity and was loaded onto a 50 ml Q-Sepharose column (AP Biotech) equilibrated with Equilibration Buffer C. The column was eluted with 10 volume linear gradient consisting of 0 to 350 mM NaCl. The eluted protein was collected in 3 peaks and the fractions from the first two peaks were pooled together.

[0533] This pool was concentrated to 22.5 mg/ml using an Amicon Stircell with a YM10 membrane. Since the concentration of NaCl in the pool was calculated to be approximately 110 mM and no other formulations were needed. The concentrated pool was sterile filtered and stored at −80° C.

[0534] The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims. All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.

Novel interleukin - 1 Hy2 materials and methods

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