JP2002510492A - Interleukin-1 receptor antagonist and use thereof - Google Patents

Abstract

(57) Abstract The present invention provides novel nucleic acids, novel polypeptides encoded by these nucleic acids, and uses thereof. The sequences of these novel polynucleotides and polypeptides have been determined to be novel interleukin-1 receptor antagonists.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

1. TECHNICAL FIELD The present invention provides novel polynucleotides, and proteins encoded by those polynucleotides, and further relates to therapeutic, diagnostic, and research uses for those polynucleotides and proteins.

[0002]

2. Description of the Related Art

2. Background Art The technologies targeted at the discovery of protein factors, including, for example, cytokines such as lymphokines, interferons, CSF, chemokines and interleukins, have been maturing rapidly over the last decade. By currently used hybridization cloning and expression cloning techniques, novel polynucleotides can be used to generate information directly related to the discovered protein (ie, in the case of hybridization cloning, the partial DNA / amino acid sequence of the protein; expression). (In the case of cloning, the activity of the protein)
"Directly" cloned. More recently, indirect cloning techniques, such as signal sequence cloning, are techniques that isolate DNA based on the presence of the now well-recognized secretory leader sequence motif. In addition to cloning techniques using PCR or low stringency hybridization cloning techniques, depending on the secretion characteristics of leader sequence cloning, or on the source of cells or tissues in the case of PCR techniques. The state of the art has evolved until numerous DNA / amino acid sequences are available for proteins known to have biological activity.

[0003] The present invention relates to these proteins and the polynucleotides that encode them. In particular, the present invention relates to novel interleukin-1 receptor antagonists.

[0004] Cytokines such as interleukin-1 are known to cause morphological or functional alterations in endothelial cells. Some of these modifications are due to "endothelial cell activation". Other immune mediators such as tumor necrosis factor (TNF), interleukin-1 (interleukin-1) and γ-interferon (IFN) appear to induce different but increased procoagulant activities (Bevilaqua (1986) PNAS,
83: 4533-4537), PGI and 2 production (Rossi (1985), Science, 229: 174-176), HLA
Patterns of endothelial cell activation, including antigen expression (Pober (1987) J. Immunol., 138: 3319-3324) and lymphocyte adhesion molecules (Carender (1987) J. Immunol., 138: 2149-2154) Some of them are duplicated. These cytokines have also been reported to cause hypotension, vascular hemorrhage and ischemia (Goldblum et al., 1989, Trac
ey et al. Science 234: 470, 1986). The major dose limiting toxicities of these and other biological response modifiers are hypotension and vascular leakage (Dvorak (1989) JNCI, 81:49
7-502).

[0005] The ability of IL-1 to modify biological responses has been demonstrated by various studies.
For example, administration of interleukin-1 to rabbits (Wakabayashi et al., FASEB J 1
991; 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 administration of interleukin-1 to primates (Fischer et al., Am J Physiol 1991; 261; R442). Blood pressure, tachycardia, pulmonary edema, and renal failure were observed, and eventually died in a dose-dependent manner. Examination of sera from animals treated with interleukin-1 shows elevated levels of other cytokines, reaching levels similar to human pancreatitis. (Guice et al., J Surg Res 1991; 51: 495-499; Heath et al., Pan
creas 1993; 66: 41-45) However, there is great evidence available. (Din
arello et al., Arch Surg 1992; 127: 1350-1353).

[0006] The cytokine interleukin-1 is a major mediator in the inflammatory response (
For reference, Dinearello (1991) Blood 77: 1627-1652; Dinearello et al (1993) New England J. Med. 328: 106-113; Dinearello (1994) FASEB J. 8: 1314-132.
See 5). The importance of interleukin-1 in inflammation has been demonstrated by the ability of highly specific interleukin-1 receptor antagonists to alleviate inflammatory conditions (for reference, see Dinearello (1991) Blood 77: 1627-1652;
Dinearello et al (1993) New England J. Med. 328: 106-113; Dinearello (1994
) FASEB J. 8: 1314-1325; Dinearello (1993) Immunol. Today 14: 260-264). Many of the proinflammatory effects of interleukin-1 such as upregulation of cell adhesion molecules on the vascular endothelium are seen at the level of transcriptional regulation. Transcriptional activation by interleukin-1 of cell adhesion molecules and other genes involved in the inflammatory response appears to be primarily mediated by NF-κB (Shirakawa et al. (1989) M
olc. 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 e.
etal., (1993) Trends Cardiovasc. Med. 3: 92-97). In response to interleukin-1, the NF-κB inhibitor IκB is degraded, and NF-κB is inactivated to localize in the nucleus where DNA binds and activates transcription. Released from the cytoplasmic state (Liou et al. (1993) Curr.Opin.Cell Biol. 5: 477-487; Beg et al., (199
3) Mol. Cell. Bid. 13: 3301-3310).

[0007] Interleukin-1 is also a mediator in septic shock. Septic shock, a fatal complication of bacterial infection, results in as many as 150,000 to 300,000 patients each year in the United States (Parrillo, JE (1989), Septic Shock in Humans: Clinical
Evaluation, Pathogenesis, and Therapeutic Approach (2nd edition) In: Textbook
of Critical Care Shoemaker, et al., Compiled by Saunders Publishing Co., Philadelphia, PA, pp. 1006). The complex metabolic disruption associated with cardiovascular breakdown and sepsis is primarily due to bacterial endotoxin (ET), which when administered to animals causes a septic shock-like condition (Natanson, et al. 1989), En
dotoxin and Tumor Necrosis Factor Challanges in Dogs Simulate the Cardio
vascular Profile of Human Septic Shock, J. Exp. Med. 169: 823). Thus, interleukin-1 modulators have been awaited.

[0008]

[Means for Solving the Problems]

The compositions of the present invention include a novel isolated polypeptide, particularly a novel interleukin-1 receptor antagonist protein, an isolated polynucleotide encoding the polypeptide, i.e., a recombinant DNA molecule, Included are naturally occurring variants, such as cloned genes or degenerate variants thereof, particularly allelic variants, and antibodies that specifically recognize one or more epitopes present on the polypeptide.

[0009] In addition, the compositions of the present invention include vectors, including expression vectors, containing the polynucleotide of the present invention, and cells genetically designed to contain the polynucleotide.

[0010] Isolated polynucleotides of the present invention include, but are not limited to, polynucleotides that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5.

[0011] An isolated polynucleotide of the invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4 or 6; SEQ ID NO: 1, 2, 4 or 6
A polynucleotide comprising the full length protein coding sequence of SEQ ID NO: 1, 2
, Including but not limited to, polynucleotides comprising the mature protein coding sequence of 4 or 6. An isolated polynucleotide of the invention is a polynucleotide that hybridizes under stringent hybridization conditions to the complement of the nucleotide sequence of SEQ ID NO: 1, 2, 4, or 6; A polynucleotide that is an allelic variant; a polynucleotide that encodes a species homolog of any of the foregoing proteins;
Includes, but is not limited to, a polynucleotide encoding a polypeptide, including a domain unique to the polypeptide of SEQ ID NO: 1, 2, 4, or 6, or a truncated polypeptide thereof.

[0012] The isolated polynucleotide of the present invention is a nucleotide sequence of a genomic clone of SEQ ID NO: 7 or 8; a polynucleotide constructed from one or more exons of SEQ ID NO: 7 or 8; A polynucleotide constructed from one or more introns of SEQ ID NO: 7 or 8; and a polynucleotide constructed from one or more introns of SEQ ID NO: 7 or 8; A polynucleotide comprising the full length protein coding sequence of
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NO: 7 or 8; or further including, but not limited to, a polynucleotide.

[0013] The polynucleotide of the invention is a polynucleotide that hybridizes under stringent hybridization conditions to the complement of the nucleotide sequence of SEQ ID NO: 7 or 8; either an intron or exon of SEQ ID NO: 7 or 8 A polynucleotide that hybridizes under stringent hybridization conditions with one of the complements; a polynucleotide that is an allelic variant of any of the above polynucleotides; a polynucleotide that encodes a species homolog of any of the above proteins Or a polynucleotide encoding a polypeptide including a domain specific to the polypeptide of SEQ ID NO: 7 or 8 or a truncated polypeptide thereof, but is not limited thereto.

[0014] The polynucleotide of the present invention may further comprise an American type culture.
Collection (ATCC, 10801 University Blvd., Manassas, Virginia, 20110-22
09, USA), which encodes the amino acid sequence of SEQ ID NO: 3 or 5, which is a polynucleotide constructed from the cDNA insert of clone pIL-1Hy273 deposited with the cDNA insert of clone pIL-1Hy. A polynucleotide comprising a nucleotide sequence; a polynucleotide comprising a full length protein coding sequence of SEQ ID NO: 3 or 5, which is a polynucleotide constructed from a cDNA insert of clone pIL-1Hy273; or a mature protein coding of SEQ ID NO: 3 or 5 Includes, but is not limited to, a polynucleotide comprising the nucleotide sequence of the sequence.

[0015] The polynucleotides of the present invention additionally include the complement of any of the above polynucleotides.

An isolated polypeptide of the present invention includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5; a full length protein of SEQ ID NO: 3 or 5; a mature protein coding sequence of SEQ ID NO: 3 or 5 Or, but not limited to, a polypeptide encoded by one or more of the exons of SEQ ID NO: 7 or 8.

The polypeptides of the present invention are further encoded by the cDNA insert of clone pIL-1HY273 deposited at the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Virginia, 20110-2209, USA). A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5 constructed from the amino acid sequence encoded by the cDNA insert of clone pIL-1Hy273; or the amino acid encoded by the cDNA insert of clone pIL-1Hy273 Includes, but is not limited to, the mature protein coding sequence of SEQ ID NO: 3 or 5 constructed from the sequence.

[0018] The protein composition of the invention may further comprise an acceptable carrier, such as a hydrophilic, for example , pharmaceutically acceptable carrier.

The present invention also relates to a method for producing a polypeptide, comprising growing a cell culture of the present invention in a suitable culture medium and purifying the protein from the culture. including. Preferred embodiments include those in which the protein produced by such a method is a mature form of the protein.

[0020] The polynucleotides of the present invention can be widely applied by various techniques known to those skilled in the field of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosomes and gene mapping, and antisense DNA or RNA,
Uses in the production of their chemical analogs and the like are included. For example, if the expression of the mRNA is greatly limited to a particular cell or tissue type, the polynucleotide of the invention may be used, for example, in in situ hybridization, to determine the presence of the particular cell or tissue mRNA in the sample. It can be used as a hybridization probe for detection.

As an example of another embodiment, the polynucleotide is used as an expressed sequence tag to identify an expressed gene or as described in Vollath et al., Science , 258, 52-59 (
1992) is used in diagnostics as an expressed sequence tag for physical mapping of the human genome as known in the art.

[0022] The polypeptides of the present invention can be used in a variety of conventional techniques and methods currently applied to other proteins. For example, the polypeptide of the present invention can be used to produce an antibody that specifically binds to the polypeptide. The polypeptide of the present invention can be used as a molecular weight marker and also as a food supplement.

A method for preventing, treating or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier. Provided.

More specifically, the polypeptides and polynucleotides of the present invention include, for example,
Sepsis, acute pancreatitis, endotoxin shock, cytokine-induced shock, rheumatoid arthritis, chronic arthritis, pancreatic cell damage due to type 1 diabetes mellitus, graft-versus-host disease,
In some methods for the prevention and / or treatment of enteritis, pulmonary disease-related inflammation, other autoimmune diseases or diseases associated with inflammatory diseases, and also for early delivery due to acute or chronic myeloid leukemia or intrauterine infection Can be used as an antiproliferative agent for the prevention of germination.

The method of the invention further relates to a method for detecting the presence of a polynucleotide or a polypeptide of the invention in a sample. The method can be used, for example, as part of the predictive and diagnostic evaluation of the disorders described above and to identify subjects who are predisposed to such conditions. In addition, the present invention provides methods for assessing the efficacy of a drug, and for monitoring the progress of a patient's condition, involving clinical trials for the treatment of the aforementioned disorders.

The present invention also provides a method for identifying a compound that modulates the expression of a polynucleotide and / or polypeptide of the present invention. Such a method
For example, it can be used to identify compounds that can alleviate the symptoms of the disorders as described above. Such methods include, but are not limited to, assays for identifying compounds and other substances that interact (eg, bind) with a polypeptide of the invention.

The method of the present invention also includes a method for treating a disorder as described above, including the step of administering the compound to an individual exhibiting the symptoms or constitution associated with the disorder. Good. In addition, the present invention is directed to methods for treating the disease or disorder by administering compounds and other agents that modulate the overall activity of the target gene product. Compounds and other substances are capable of providing such a modulation, either on the expression level of the target gene or on the activity of the target protein.

[0028]

DETAILED DESCRIPTION OF THE INVENTION 6. EMBODIMENTS FOR CARRYING OUT THE INVENTION 6.1. Definitions The term "nucleotide sequence" refers to a heteropolymer of nucleotides or a sequence of these nucleotides. The terms "nucleic acid" and "polynucleotide" refer to a heteropolymer of nucleotides and are used interchangeably herein. In general, the nucleic acid segments provided by the present invention are combined with a genome to provide a synthetic nucleic acid that can be expressed in a microbial or viral operon or a recombinant transcription unit containing regulatory elements derived from eukaryotic genes. It may be assembled from fragments of short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides.

“Oligonucleotide fragment” or “polynucleotide fragment”, “portion”,
Or, the term `` segment '' is of sufficient length to be used in the polymerase chain reaction (PCR) or various hybridization methods to identify or amplify the same or related portions of an mRNA or DNA molecule. A stretch of polypeptide nucleotide residues.

“Oligonucleotides” or “nucleic acid probes” are prepared based on the polynucleotide sequences provided herein. Oligonucleotides are those that comprise a portion of a polynucleotide sequence, such as having at least about 15 nucleotides, and usually at least about 20 nucleotides. Nucleic acid probes include those portions of the polynucleotide that have less than about 6 kb, usually less than about 1 kb. After appropriate testing to rule out false positives, these probes can be used, for example, to determine whether a specific mRNA molecule is present in a cell or tissue, or by Walsh et al (Walsh, PS et al. ., 1992, PCR Methods
Appl 1 , pp. 241-250) may be used to isolate similar nucleic acid sequences from chromosomal DNA.

The term “probe” includes single- or double-stranded nucleic acids, either naturally occurring or recombinantly or chemically synthesized. Probes may be labeled by nick translation, Klenow-fill-in reaction, PCR or other methods well known in the art. Probes of the invention, their preparation and / or labeling are described 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, NY, the disclosure of which is incorporated by reference in its entirety.

The term “stringent” refers to a condition commonly understood in the art as stringent. Stringent conditions include high stringency conditions (ie, 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS),
Hybridize to the bound DNA at 65 ° C with 1 mM EDTA and wash with 0.1 x SSC / 0.1% SDS at 68 ° C), and conditions of moderate stringency (ie, 42 ° C (Wash with 0.2 × SSC / 0.1% SDS).

As regards the hybridization of deoxyoligonucleotides, further exemplary hybridization conditions include 37 ° C. (for a 14 base oligo), 48 ° C. (for a 17 base oligo), 55 ° C. (for a 17 base oligo). Washing with 6 × SSC / 0.05% sodium pyrophosphate at 60 ° C. (for a 23 base oligo) and at 60 ° C. (for a 23 base oligo) is included.

The term “recombinant” when used herein to refer to a polypeptide or protein means that the polypeptide or protein is derived from a recombinant (eg, microbial or mammalian) expression system. I do. “Microorganism” shall refer to a recombinant polypeptide or protein produced in a bacterial or fungal (eg, yeast) expression system. As a product, a "recombinant microorganism" is defined as a polypeptide or protein that is substantially free of native endogenous material and with inherent concomitant glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., cultures of E. coli, should have no glycosylation modifications, and polypeptides or proteins expressed in yeast will be modified in mammalian cells. It should have glycosylation that is generally different from that expressed.

The term “recombinant expression vehicle or vector” shall refer to a plasmid or phage or virus or vector for expressing a polypeptide from a DNA (RNA) sequence. Expression vehicles include (1) promoters, enhancers, etc., one or more genetic elements that have a regulatory role in gene expression, (2) structural sequences that are transcribed into mRNA and translated into proteins, or It can include a transcription unit comprising a combination of a coding sequence, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence which enables extracellular secretion of proteins translated by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not subsequently be excised from the expressed recombinant protein, yielding the final product.

The term “recombinant expression system” refers to a host cell in which the recombinant transcription unit is stably integrated into chromosomal DNA, or a host cell that carries the recombinant transcription unit extrachromosomally. A recombinant expression system as defined herein should express a heterologous polypeptide or protein upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. The term also includes host cells that have stably integrated one or more recombinant genetic elements that have a regulatory role in gene expression, such as, for example, promoters or enhancers. The recombinant expression system as defined herein is an endogenous DN to be expressed.
Upon induction of a regulatory element linked to the A segment or gene, the cell should express an endogenous polypeptide or protein. The cells may be prokaryotic or eukaryotic.

The term ORF of "open reading frame" refers to a sequence of nucleotide triplets that does not contain any termination codons, encodes an amino acid, and is transcribable to a protein.

The term “expression modulating fragment” EMF refers to a series of nucleotides that modulate the expression of an operably linked ORF or other EMF.

As used herein, a sequence is said to “modulate the expression of an operably linked sequence” if the expression of the sequence is altered by the presence of EMF.
EMFs include, but are not limited to, promoters and sequences that modulate the promoter (inducible elements). Certain classes of EMF are operably linked in response to specific modulators or physiological events
ORF or a fragment that induces expression.

As used herein, UMF in “uptake modulating fragment” refers to a series of nucleotides that mediate the uptake of a ligated DNA fragment into a cell. UMF can be easily identified using UMF, known as a target sequence or target motif, in a computer-based system described below.

The presence and activity of UMF can be confirmed by attaching a candidate UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions, and the incorporation of the marker sequence is determined. As mentioned above, UMF should increase the frequency of incorporation of linked marker sequences.

The term “activator” refers to the biological and / or biological activity of various naturally occurring polypeptides.
Alternatively, it refers to a form of the polypeptide that retains immunological activity.

The term “naturally occurring polypeptide” refers to a polypeptide produced by a cell that has not been genetically engineered, and in particular, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and It includes various polypeptides in which post-transcriptional modification of the polypeptide is found, including but not limited to acylation.

The term “derivative” refers to ubiquitin binding, labeling (eg, labeling with radionuclides or various enzymes) that would not normally occur in human proteins.
, Pegylation (derivatization with polyethylene glycol), and chemical synthesis of amino acids such as ornithine.

The term “recombinant variant” refers to various polypeptides created using recombinant DNA technology that differ from naturally occurring polypeptides by amino acid insertions, deletions and substitutions. And The strategy for determining which amino acid residue should be substituted, added or deleted without impairing the target activity such as cell trafficking is to change the sequence of a specific polypeptide to the sequence of a homologous peptide. It can be found by comparing and minimizing the number of amino acid sequence changes made to regions of high homology.

Preferably, the amino acid “substitution” replaces one amino acid with a similar structural and / or
Or the result of substituting another amino acid with chemical properties,
It is preferable that the acid is substituted by no acid. Amino acid substitutions are dependent on the polarity, charge,
It may be based on hydrophobic, hydrophilic and / or amphiphilic properties. For example
, Non-polar (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, and positively charged (basic) amino acids include
Arginine, lysine and histidine are included and negatively charged (acidic) amino
Acids include aspartic acid and glutamic acid. "Insert" or "
"Deletions" typically range from about 1 to 5 amino acids. Using recombinant DNA technology and assaying the activity of the resulting recombinant variants, the polypeptide molecule
Changes allowed by systematic insertion, deletion or substitution of amino acids
May be determined experimentally.

Alternatively, if a modification in function is desired, insertions, deletions or non-conservative changes can be made to produce a modified polypeptide. Such modifications can, for example, alter one or more of the biological functions or biochemical properties of a polypeptide of the invention. For example, such modifications may include ligand binding affinity,
The properties of the polypeptide, such as interchain affinity, or degradation / rotation rates, may be altered.
In addition, such modifications can be selected to produce polypeptides that are more suitable for expression, scale-up, etc., in the host cell selected for expression. For example, to remove disulfide bridges, delete cysteine residues,
Alternatively, it can be replaced with another amino acid.

As used herein, the term “substantially equivalent” refers to the nucleotide sequence and amino acid sequence, for example, a variant sequence, between a reference sequence and a subject sequence. It shall be possible to refer to sequences which differ from the reference sequence by one or more substitutions, deletions or additions, which do not cause a functional disparity in which the overall effect has an adverse effect. Usually, such substantially equivalent sequences will have no more than 20% difference from one of the sequences disclosed herein ( ie , the replacement of an individual residue in a substantially equivalent sequence). , The number of additions and / or deletions divided by the total number of residues in substantially equivalent sequences as compared to the corresponding reference sequence is less than or equal to about 0.2). Such sequences are said to have 80% sequence identity with the sequences listed above. In one embodiment, a substantially equivalent, eg , variant, sequence of the invention has no more than 10% (90% sequence identity) difference from the disclosed sequences, and in a further embodiment, 5%. Not more than (95% sequence identity), and in a further embodiment 2
% (98% sequence identity). A substantially equivalent, eg , variant, amino acid sequence of the present invention will generally have at least about 95% sequence identity with the amino acid sequence set forth above, while a substantially equivalent nucleotide sequence of the present invention will comprise: For example, one may have a lower percentage of sequence identity, taking into account duplication or degeneracy of the genetic code. For purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. To determine equivalence, truncation of the mature sequence (eg, due to mutations creating a false stop codon) should be ignored.

[0049] Nucleic acid sequences encoding such substantially equivalent sequences, eg , sequences having the above-described identity, can be prepared by standard hybridization methods known to those skilled in the art.
It can be isolated and identified as predetermined.

If desired, the expression vector may be designed to include a “signal or leader sequence” that directs the polypeptide through the cell membrane. Such sequences may be naturally occurring in the polypeptides of the invention, or may be supplied from heterologous protein sources by recombinant DNA technology.

A polypeptide “fragment”, “portion” or “segment” is at least about 5 amino acids, optionally at least about 7 amino acids, usually at least about 9-13 amino acids, and many more. In embodiments, the stretch is at least about 17 or more amino acid residues. To be active, any polypeptide must have sufficient chain length to exhibit biological and / or immunological activity.

Alternatively, recombinant variants encoding the same or similar polypeptides may be synthesized or selected by taking advantage of the “degeneracy” of the genetic code. Various codon substitutions, such as silent changes creating various restriction sites, may be introduced to optimize cloning or expression into a plasmid or viral vector in a particular prokaryotic or eukaryotic system. . Mutations in the polynucleotide sequence can be added to a polypeptide to modify the properties of any portion of the polypeptide to alter properties such as ligand binding affinity, interchain affinity, or degradation / rotation rate. And the domain of the polypeptide or other polypeptide to be expressed.

As used herein, “activated” cells are those that are involved in extracellular or intracellular membrane transport, including the transport of neurosecretory or enzymatic molecules that are part of normal or disease processes. is there.

The term “purified,” as used herein, refers to a nucleic acid or polypeptide,
It indicates that other biological macromolecules such as polynucleotides, proteins and the like are substantially absent. In certain embodiments, the polynucleotide or polypeptide is purified to comprise at least 95%, more preferably at least 99.8%, by weight of the biological macromolecule, provided that water, buffer and other Small molecules, especially those having a molecular weight of less than 1000 Daltons may be present).

As used herein, the term “isolated” refers to at least one other component (eg, a nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide in a natural source. Nucleic acids or polypeptides isolated from According to one embodiment, the nucleic acid or polypeptide is a solvent, if any,
It is in the presence of only buffers, ions or other components normally present in the solution. The terms "isolated" and "purified" are not intended to include nucleic acids or polypeptides present in their natural sources.

The term “infection” refers to the use of a virus or viral vector,
Refers to introducing a nucleic acid into a suitable host cell.

The term “transformation” refers to the transfer of DNA into a suitable host cell such that the DNA becomes replicable, either as an extrachromosomal element or by integration into the chromosome.

The term “transfection” refers to the expression vector being carried by a suitable host cell, whether or not any coding sequences are actually being expressed.

The term “intermediate fragment” refers to a nucleic acid having a chain length of 5 to 1000 base pairs, preferably 10 to 40 base pairs.

“Secreted” refers to a protein that, when expressed in a suitable host cell, is transported across or across a membrane, including the resulting transport of its amino acid sequence by a signal sequence. . “Secreted” proteins include, but are not limited to, proteins that are secreted entirely (eg, soluble proteins) or partially (eg, receptors) from the expressing cells. Also, “secreted” proteins are proteins that contain atypical signal sequences (eg, interleukin-1β, Krasney,
PA and Young, PR (1992) Cytokine 4 (2): 134-143), and factors released from damaged cells (eg, interleukin-1 receptor antagonist, Arend, W
.P. Et al. (1998) Annu . Rev. Immunol . 16: 27-55).

Each of the above terms is intended to encompass all of the above unless otherwise stated.

Polynucleotide and nucleic acid of the present invention The nucleotide and nucleic acid sequences of the present invention are shown below. Fragments of the proteins of the present invention that can exhibit biological activity are also encompassed by the present invention. Protein fragments may be single-stranded or, for example, HU Saragovi et al., Bio / Technology 10, 773-778 (1992), which is incorporated herein by reference.
) And RS McDowell et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992). Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of the protein binding site. For example, a fragment of a protein may be fused to the Fc portion of an immunoglobulin via a "linker" sequence. In the case of a bivalent form of the protein, such a fusion may be made to the Fc portion of an IgG molecule. Other immunoglobulin isotypes can also be used to make such fusions. For example, a protein-IgM fusion would be made with a divalent form of the protein of the invention.

The present invention further relates to full-length and mature forms of the disclosed proteins (eg,
Without a signal sequence or precursor sequence). Full length forms of such proteins are set forth in the Sequence Listing, by translation of the nucleotide sequence of each disclosed clone. Mature forms of such proteins are obtained by expressing the disclosed full-length polynucleotides in a suitable mammalian cell or other host cell. Where the proteins of the invention are membrane-bound, the invention also provides soluble forms of such proteins. In such a form, some or all of the region providing the membrane-bound protein is deleted so that the protein completely secretes the protein from its expressing cells.

[0064] The present invention also provides genes corresponding to the cDNA sequences disclosed herein.
The corresponding gene can be isolated according to known methods, using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence information for identification and / or amplifying the gene with an appropriate genomic library or other source of genetic material. Species homologs of the disclosed polynucleotides and proteins are also provided by the invention. Species homologs can be isolated and identified by making suitable probes or primers from the sequences provided herein and screening for a suitable nucleic acid source from the desired species. The present invention furthermore relates to naturally occurring allelic variants of the disclosed polynucleotides or proteins, i.e., isolated polynucleotides which also encode proteins identical, homologous or related to those encoded by the polynucleotide. It also includes alternative forms. Compositions of the invention include recombinant DNA molecules, cloned genes or degenerate variants thereof, particularly naturally occurring variants such as allelic variants, isolated novel polypeptides, and such polypeptides. And antibodies specifically recognizing one or more epitopes present in E. coli. Species homologs of the disclosed polynucleotides and proteins are also provided by the invention. Species homologs can be isolated by making suitable probes or primers from the sequences provided herein and screening for a suitable nucleic acid source from the desired species.

The present invention also provides allelic variants of the disclosed polynucleotides or proteins,
That is, it includes naturally occurring alternative forms of the isolated polynucleotide which also encode a protein identical, homologous or related to that encoded by the polynucleotide.

6.2. Nucleic Acids of the Invention Isolated polynucleotides of the present invention include, but are not limited to, polynucleotides that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5.

An isolated polynucleotide of the present invention includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4 or 6, including the full length protein coding sequence of SEQ ID NO: 1, 2, 4 or 6. Polynucleotide and SEQ ID NO: 1, 2, 4
Or polynucleotides comprising the six mature protein coding sequences, but are not limited thereto. Polynucleotides of the invention include, under stringent hybridization conditions, a polynucleotide that hybridizes to the complement of the nucleotide sequence of SEQ ID NO: 1, 2, 4, or 6, or an allele of any of the preceding polynucleotides. A polynucleotide that is a variant, a polynucleotide that encodes a species homolog of any of the foregoing proteins, or SEQ ID NO: 1, 2
The polypeptides further include, but are not limited to, unique domains of 4 or 6 polypeptides or truncated polypeptides.

An isolated polynucleotide of the present invention is a nucleotide sequence of a genomic clone of SEQ ID NO: 7 or 8; from one or more exons of SEQ ID NO: 7 or 8 (eg, selectively conjugated). Constructed polynucleotide; polynucleotide constructed from one or more introns of SEQ ID NO: 7 or 8; constructed from one or more exons of SEQ ID NO: 7 or 8 and one or more introns of SEQ ID NO: 7 or 8 Further comprising, but not limited to, a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of SEQ ID NO: 7 or 8; Not done.

A polynucleotide of the invention is a polynucleotide that hybridizes under stringent hybridization conditions to the complement of the nucleotide sequence of SEQ ID NO: 7 or 8; either an intron or exon of SEQ ID NO: 7 or 8 A polynucleotide that hybridizes under stringent hybridization conditions with one of the complements; a polynucleotide that is an allelic variant of any of the above polynucleotides; a polynucleotide that encodes a species homolog of any of the above proteins Or, further including, but not limited to, a polynucleotide encoding a polypeptide, including a domain specific to the polypeptide of SEQ ID NO: 7 or 8 or a truncated polypeptide thereof.

The polynucleotide of the present invention may further comprise an American type culture.
Collection (ATCC, 10801 University Blvd., Manassas, Virginia, 20110-22
09, USA), which encodes the amino acid sequence of SEQ ID NO: 3 or 5, which is a polynucleotide constructed from the cDNA insert of clone pIL-1Hy273 deposited with the cDNA insert of clone pIL-1Hy. A polynucleotide comprising a nucleotide sequence; a polynucleotide comprising a full length protein coding sequence of SEQ ID NO: 3 or 5, which is a polynucleotide constructed from the cDNA insert of clone pIL-1Hy273; or a mature protein coding of SEQ ID NO: 3 or 5 Includes, but is not limited to, a polynucleotide comprising the nucleotide sequence of the sequence.

The polynucleotides of the present invention additionally include the complement of any of the above polynucleotides.

The polynucleotide of the present invention also includes a nucleotide sequence substantially equivalent to the aforementioned polynucleotide. A polynucleotide of the present invention is one that can have at least about 80%, more preferably usually at least about 90%, and even more preferably usually at least about 95% sequence identity with the above-described polynucleotides. The invention further includes a nucleotide sequence having at least about 80%, more preferably usually at least about 90%, and even more preferably usually at least about 95% sequence identity with the polynucleotide encoding the polypeptide described above. A complement of the polynucleotide is also provided. A polynucleotide can be DNA (genome, cDNA, amplified or synthesized DNA) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art, and are used, for example, to determine hybridization conditions that can routinely isolate a polynucleotide of the desired sequence. A method may be included.

The polynucleotides of the present invention can be linked to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (Sambrook, J. et al. (1989)).
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,
See NY). Useful nucleotide sequences for linking polypeptides include:
Vectors well known in the art, for example, plasmids, cosmids,
Combinations of lambda phage derivatives, phagemids and the like are included. Accordingly, the present invention also provides a vector comprising the polynucleotide of the present invention, and a host cell comprising the polynucleotide. Generally, the vector will include at least one origin of replication functional in the organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. The vectors of the present invention include expression vectors, replication vectors, vectors for producing probes, and sequencing vectors.
A host cell of the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or a part of a multicellular organism.

The sequences included within the scope of the present invention are not limited to the specific sequences described,
It also includes its allelic variants. Allelic variants are those of SEQ ID NOs: 1, 2,
4, 6, or 7 or a representative fragment thereof, or SEQ ID NO: 1,
Nucleotide sequences that are at least 99.9% identical to 2, 4, 6, or 7 can be readily determined by comparing to sequences from another isolate of the same type. Furthermore, to accommodate codon variability, the invention also encompasses nucleic acid molecules that encode amino acid sequences similar to those encoded by the particular ORFs disclosed herein. In other words, it is clearly contemplated to replace one codon with another codon encoding the same amino acid in the coding region of the ORF. By rearranging (ie, sequencing both strands) specific fragments, such as ORFs, in both directions, any of the specific sequences disclosed herein can be easily screened for errors.

The present invention further provides a recombinant construct comprising a nucleic acid having the sequence of SEQ ID NO: 1, 2, 4, 6, or 7 or a fragment thereof. The recombinant construct of the present invention comprises a vector such as a plasmid or viral vector, wherein the sequence of SEQ ID NO: 1, 2, 4, 6, 7 or 8 or a fragment thereof is forward or reverse in the vector. Is inserted. In the case of a vector containing one of the ORFs of the invention, the vector is O
It may further comprise regulatory sequences operably linked to the RF, including, for example, a promoter. For vectors comprising EMF and UMF of the present invention, the vector may further comprise a marker sequence or heterologous operably linked to EMF or UMF.
It should include an ORF. Many suitable vectors and promoters are known to those of skill in the art and are commercially available for making recombinant constructs of the present invention. The following vectors are given as examples. Bacteria: pBs, phagescri
pt, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stra
tagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Fungi: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene), pSVK3, pBPV, pMSG,
pSVL (Pharmacia).

The isolated polynucleotide of the present invention may be used to produce pMT2 or pED expression as disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), for recombinantly producing proteins. It may be operably linked to an expression control sequence such as a vector. Many suitable expression control sequences are known in the art. General methods for expressing recombinant proteins are also known; R. Kaufman,
Methods in Enzymology, 185, 537-566 (1990). As used herein, the term "operably linked" refers to a linked polynucleotide /
It means that the isolated polynucleotide of the present invention and the expression control sequence are adapted to a vector or a cell so that the protein is expressed by a host cell transformed (transfected) with the expression control sequence. .

The promoter region is a CAT (chloramphenicol transferase) vector
Or other vectors having a selectable marker to provide various desired
Can be selected from genes. Two suitable vectors are pKK232-8 and pCM7
. Particularly preferred bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P _R And trc are included. Eukaryotic promoters include early CMV and HSV thymidine
Kinases, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter
, Within the ordinary skill level of the art. Generally, a recombinant expression vector
Is the origin of replication, for exampleE. coliAmpicillin resistance gene andS. cerevisi ae Selectable markers that enable transformation of host cells, such as the TRP1 gene
Promoters from highly expressed genes that result in direct transcription of downstream structural genes
And Such promoters are, inter alia, 3-phosphoglycerate
Those derived from an operon encoding a glycolytic enzyme such as an enzyme (PGK), a-factor, acid phosphatase or heat shock protein are preferred. The heterologous structural sequence is
Transcription initiation and termination sequences and, preferably, the cell membrane periphery of the translated protein.
With leader sequences that can provide direct secretion into the marginal space or extracellular medium
In the appropriate phase. Optionally, a heterologous sequence may be included in the recombinant product to be expressed.
N-terminal that imparts desirable characteristics, such as to facilitate stabilization or purification thereof.
It is intended to be able to encode a fusion protein including the terminal identification peptide. Bacteria
Useful expression vectors for
Encodes the desired protein, along with suitable transcription initiation and termination signals.
It is constructed by inserting a structural DNA sequence. The vector may have one or more phenotypic selections to ensure its retention and, if desired, provide for amplification in the host.
Includes a selectable marker and an origin of replication. Prokaryotic cells suitable for transformation
Sex hosts include:E. coli,Bacillus subtilis,Salmonella typhimuriumAnd sh
Various species in Domonas, Streptomyces, and Staphylococcus spp.
, But other options are possible.

As a representative but non-limiting example, expression vectors useful for bacteria include selections from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Mention may be made of those containing a possible marker and a bacterial origin of replication. Such commercially available vectors include, for example, pK
K223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Pr
omega Biotec, Madison, WI, USA). These pBR
The 322 "backbone" section is combined with an appropriate promoter and the structural sequence to be expressed. After transforming a suitable host strain and growing the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (eg, temperature shift or chemical induction), and The cells are cultured for an additional period. Usually, cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is collected and further purified.

Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequences that hybridize under stringent conditions to fragments of the DNA sequence shown in FIG. 2, 5, 8 or 9 or the complement thereof. Yes, the fragment is greater than about 10 base pairs, preferably about 20-50 base pairs, or even more than 100 base pairs. In accordance with the present invention, a polynucleotide sequence encoding a novel nucleic acid, or a functional equivalent thereof, is a recombinant DNA that provides for expression of such a nucleic acid, or a functional equivalent thereof, in a suitable host cell.
It may be used to make molecules.

The nucleic acid sequences of the present invention further relate to sequences encoding variants of said nucleic acids.
These mutated amino acid sequences can be prepared by methods known in the art by introducing appropriate nucleotide changes into a natural or mutated polynucleotide. The construction of a mutated amino acid sequence involves two variables:
There is the location of the mutation and the nature of the mutation. The mutated amino acid sequence of the nucleic acid is preferably constructed by mutating a polynucleotide to give an amino acid sequence that does not occur in nature. These amino acid changes may be made at different sites (variable sites) in nucleic acids from different species, or at highly conserved regions (constant regions). Sites at such positions are preferably, for example, first conservatively selected (eg, hydrophobic amino acids are different hydrophobic amino acids) and then more separated (eg, hydrophobic amino acids are charged amino acids). , May be modified sequentially by making substitutions, followed by deletions or insertions at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are preferably contiguous. Amino acid insertions include amino and / or carboxy terminal fusions ranging in chain length from 1 to 100 residues or more, as well as insertion of one or more amino acid residues into the sequence. Insertions into the sequence may range from about 1 to 10 amino acid residues, preferably 1 to 5 residues. Examples of terminal insertions include heterologous signal sequences required for secretion in different host cells or for intracellular targeting.

In a preferred method, the polynucleotide encoding the novel nucleic acid is altered by site-directed mutagenesis. In this method, an oligonucleotide sequence encoding the polynucleotide sequence of the desired mutated amino acid and, in addition, sufficient both sides of the altered amino acid to form a stable duplex on either end of the altered site. Use adjacent nucleotides. In general, techniques for site-directed mutagenesis are well known to those skilled in the art, and this technique is exemplified in publications such as Edelman et al., DNA , 2, 183 (1983). Zoller and Sm
ith, Nucleic Acids Res. 10, pp . 6487-6500 (1982). PCR may be used to generate a mutated amino acid sequence of a novel nucleic acid. When a small amount of mold DNA is used as a starting material, a desired mutant amino acid sequence can be prepared using primers that are slightly different in sequence from the corresponding region in the template DNA. PC
R amplification results in a population of products of the DNA fragment that differ from the template of the polynucleotide encoding the polypeptide at the position specified by the primer. The product of the DNA fragment replaces the corresponding region of the plasmid and results in the desired mutated amino acid sequence.

[0082] As a further technique for producing mutant amino acid sequence, Wells et al, Gene 34, pp. 315 (1985) cassette mutagenesis technique described in; and, for example, in Sambrook et al., Supra, and Current Protocols in Molecular There are other mutagenesis techniques well known in the art, such as those in Biology , Ausubel et al.

Due to the inherent degeneracy of the genetic code, in practicing the present invention for the cloning and expression of these novel nucleic acids, other DNA sequences encoding substantially similar or functionally equivalent amino acid sequences Can also be used. Such DNA sequences include those that are capable of hybridizing under stringent conditions to the appropriate novel nucleic acid sequence.

6.3. Hosts The present invention further provides host cells that are genetically engineered to contain a polynucleotide of the present invention. For example, such a host cell comprises a nucleic acid of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention further provides host cells that are genetically engineered to express the polynucleotides of the present invention, wherein such polynucleotides drive expression of the polynucleotides in the cells. It is in operative association with a heterologous regulatory sequence.

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 a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into host cells can be accomplished by calcium phosphate transfection, DEAE, transfection using dextran or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).
). Host cells containing one of the polynucleotides of the present invention can be used in conventional ways to produce the gene product encoded by the isolated fragment (in the case of ORFs) or can be heterologous under the control of EMF. It can be used to produce proteins.

[0086] A variety of host / vector systems 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 cells, COS cells and Sf9 cells, as well as prokaryotic hosts such as E. coli and B. subtilis. . Most preferred cells are those that do not normally express a given polypeptide or protein, or that express the polypeptide or protein at low native levels. The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of a suitable promoter. To produce such a protein, a cell-free translation system using RNA derived from the DNA construct of the present invention can also be used. Cloning and expression vectors suitable for use in prokaryotic and eukaryotic hosts are described in Samb, incorporated herein by reference.
rook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spr
ing Harbor, New York (1989).

[0087] Various mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian cell expression systems include Gluzman, Cell 23, 175 (1981
COS-7 cells of monkey kidney fibroblasts according to), and, for example, C127, 3T3, CHO, HeLa
And other cell lines capable of expressing the compatible vector, such as the BHK cell line. The mammalian expression vector will include an origin of replication, a suitable promoter, and any necessary ribosome binding proteins, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 'flanking non-transcribed sequences. Like. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splice and polyadenylation sites, may be used to provide the required non-transcribed genetic elements. Recombinant polypeptides and proteins produced in bacterial cultures
Usually, it is first extracted from the cell pellet and then subjected to one or more salting out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can also be used as needed to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for the final purification step. Bacterial cells used for protein expression can be disrupted by a variety of convenient methods, including freeze-thaw cycling, sonication, mechanical disruption, or use of fine lysing agents.

[0088] Many cell types can serve 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 epithelial A431 cells, human Colo205 cells, 3T3 cells,
CV-1 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, primary explants, HeLa cells, mouse L cells, BHK, HL- 60
, U937, HaK or Jurkat cells and the like.

Alternatively, the protein can be produced in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida or various yeast strains capable of expressing heterologous proteins are included. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhim
urium, or various bacterial strains capable of expressing a heterologous protein are included. If the protein is produced in yeast or bacteria, it may be necessary to modify the protein produced there to obtain a functional protein, such as by phosphorylation or glycosylation at the appropriate site. Such covalent attachment can be performed using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising a polynucleotide of the present invention under the control of an inducible regulatory element, wherein The regulatory sequence of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace regulatory regions present in genes with regulatory sequences isolated from different genes or novel regulatory sequences, which are synthesized by genetic engineering techniques. Such regulatory sequences may include promoters, enhancers, scaffold attachment regions, natural regulatory elements, transcription initiation sites, regulatory protein binding sites, or combinations of such sequences. Alternatively, sequences that affect the structure or stability of the RNA or protein produced may include substitutions, removals, additions or polyadenylation signals,
Other modifications by targeting including mRNA stabilizing elements, splice sites, leader sequences to enhance or modify protein transport or secretion, or other sequences that alter or improve the function or stability of the protein or RNA molecule. May be done.

A targeting event is simply the insertion of regulatory sequences, such as inserting a new promoter or enhancer or both upstream of a gene.
It may be to place the gene under the control of a new regulatory sequence. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as a deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may be to replace an existing element, e.g., an enhancer with a broader cell type specificity than the naturally occurring element, or a cell type specificity different from the naturally occurring element Can replace the tissue-specific enhancer. Here, the naturally occurring sequence is deleted and a new sequence is added. In all cases, the identification of the targeting event will require the identification of the targeting DN such that the foreign DNA allows for the selection of cells that have integrated into the host cell genome.
This can be done without difficulty by using one or more selectable marker genes adjacent to A. Identification of the targeting event may also be such that the negative selectable marker is linked to the exogenous DNA, but the negative selectable marker is flanking the targeting sequence, and the sequence contained in the host cell genome. The stable integration of the negatively selectable marker as a result of the correct homologous recombination event with is prevented from occurring, and is easily accomplished by using one or more marker genes that exhibit the characteristics of negative selection. obtain. Markers useful for this purpose include the herpes simplex virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

Gene targeting or gene activation techniques that can be used with such features of the present invention are described in US Pat. No. 5,272,071 to Chappel, US Pat.
5,578,461, Selden et al., International Application No. PCT / US92 / 09627 (WO93 / 09222), Skoultchi
These are described in more detail in International Application No. PCT / US90 / 06436 (WO91 / 06667), the entire disclosure of which is incorporated herein by reference.

6.4. Polypeptide of the Invention SEQ ID NO: 2 encodes the polypeptide sequence of SEQ ID NO: 3. The amino acid alignment of SEQ ID NO: 3 and rat, mouse, rabbit and human interleukin-1 receptor antagonist polypeptides are shown in FIG. SEQ ID NO: 3 shows high amino acid homology (71%) with human interleukin-1 receptor antagonist and is distinct but very related to interleukin-1 receptor antagonist It means a big new molecule. This is the sequence originally cloned in the interleukin-1 receptor antagonist-related gene. This finding of SEQ ID NO: 3 suggests the existence of a family of interleukin-1 receptor antagonist related genes. Members of another family can be identified using SEQ ID NO: 1 or 2 as molecular probes. SEQ ID NO: 3 encodes a protein containing a four amino acid insertion that differs from all of the interleukin-1 receptor antagonist sequences previously characterized (FIG. 1). This insertion will confer a novel, previously unrecognized activity to SEQ ID NO: 3. Similarly, FIG. 7 shows an amino acid alignment of SEQ ID NO: 5 and a cytoplasm of human IL-1Ra (denoted as “HUMIL1RASIC”). This alignment shows high homology between the two, ie, 48% of the amino acids are identical and 54% are conservative amino acid substitutions.

Interleukin-1 has pleiotropic biological activities, many of which have adverse effects on living organisms, and if the molecule is not damaged, it should be tightly regulated It is. In fact, there are several reports of interleukin-1 inhibitors that modulate the action of interleukin-1. Interleukin-1 inhibitory activity has been reported in media prepared for monocyte cells, that is, media prepared so that monocyte cells grow in adherent immune complexes. Arena, WP, et al
., 1985, Journal of Immun., 134: 3868. In addition, inhibitors have been reported to be present in urine. Seckinger, P. et al., 1987, Journal of Immun., 139
: 1546. Finally, purified and cloned protein inhibitors with interleukin-1 receptor antagonist activity have been reported. Hannum, et a
l., 1990, Nature, 343: 336 and Eisenberg, S., et al., 1990, Nature, 343:
341.

Interleukin-1 inhibitors are believed to be present in urine, and
Eisenberg, S., et al., 1990, Nature, 343: 341; and Carter, D., et al (
1990), Nature, 344: 633.
If not identical to the -1 receptor antagonist, a portion is purified and Seckinger, P
et al., and those similar to those characterized by Seckinger, P. et al., 1987, Journal of Immun., 139: 1541.

[0096] Interleukin-1 receptor antagonists are naturally occurring peptides secreted by macrophages in response to many of the same stimuli that lead to the secretion of interleukin-1 itself. Interleukin-1 receptor antagonists are naturally occurring antagonists to cytokines that recognize receptors for various cell types and block interleukin-1 mediated responses by blocking the receptors. (Wakabayashi et al., FASEB J 1991; 5: 338; Okusa
wa et al. J. Clin Invest 1988; 81: 1162; Ohlsson et al., Nature 1990; 348: 5
50; Aiura, et al. Cytokine 1991; 4: 498; Fischer et al. Am J Physiol 1991;
261: R442). In humans, interleukin-1 receptor antagonists belong to a group of naturally occurring molecules whose three forms have been characterized (two are glycosylated and one is glycosylated). It has not been).

[0097] Fischer et al. (Am J Physiol 1991: 261: R442) noted that the administration of a naturally occurring antagonist to interleukin-1 markedly slowed the cytokine cascade, and It was demonstrated that a lethal dose would enhance survival in the baboons administered. Interleukin-1 receptor antagonists significantly reduce mean arterial blood pressure and cardiac output, and also improve the survival of severe acute pancreatitis. (U.S. Pat. No. 5,508,262) The systemic interleukin-1 response observed as a result of bacterial sepsis is also significantly reduced, correlating with a reduced systemic response to bacterial sepsis.

[0098] Studies in Aiura et al., 9 Cytokine 1991; 4: 498 show that interleukin-1 receptor antagonists are protective in a rabbit model of hypotensive gram-negative septic shock. When an interleukin-1 receptor antagonist was administered to this animal model, mean arterial blood pressure was maintained, lung water was reduced, and urinary excretion was maintained, as compared to controls. This study demonstrates the role of interleukin-1 and the protective effects of interleukin-1 receptor antagonists on Gram-negative septic shock. Interleukin-1 is a major vehicle in a patient's clinical response to several different stresses, regardless of etiology (including acute pancreatitis, sepsis, endotoxin shock, and cytokine-induced shock).

[0099] The isolated polynucleotide of the present invention is an amino acid sequence of SEQ ID NO: 3 or 5; a full-length protein coding sequence of SEQ ID NO: 3 or 5;
Or a polypeptide comprising the polypeptide encoded by one or more exons of SEQ ID NO: 7 or 8.

[0100] The polynucleotides of the present invention may also comprise American Type Cultures.
Collection (ATCC, 10801 University Blvd., Manassas, Virginia, 20110-22
09, USA), the polypeptide encoded by the cDNA insert of clone pIL-1Hy273; the full-length protein of SEQ ID NO: 3 or 5, constructed from the amino acid sequence encoded by the cDNA insert of clone pIL-1Hy273; ,
Includes, but is not limited to, the mature protein coding sequence of SEQ ID NO: 3 or 5 constructed from the amino acid sequence encoded by the cDNA insert of clone pIL-1Hy273.

The protein compositions of the present invention further comprise an acceptable carrier, such as a hydrophilic, for example , a pharmaceutically acceptable carrier.

The present invention further relates to a method for producing a polypeptide, comprising growing a cell culture of the present invention in a suitable culture medium and purifying the protein from the culture. including. For example, the method of the present invention comprises producing a polypeptide by culturing a host cell containing a suitable expression vector containing the polynucleotide of the present invention under conditions permitting the expression of the polypeptide of interest. Process is included. The polypeptide is recovered from the culture, conveniently from the culture medium, and can be further purified. Preferred embodiments include those in which the protein produced by such a method is a full-length or mature form of the protein.

The present invention further provides a polypeptide comprising an amino acid sequence substantially equivalent to the amino acid sequence of SEQ ID NO: 3 or 5. A polypeptide according to the present invention can have at least about 95%, and preferably usually at least about 98, sequence identity with SEQ ID NO: 3 or 5.

The invention further provides an isolated polypeptide encoded by a nucleic acid fragment of the invention or by a degenerate variant of a nucleic acid fragment of the invention. The term "degenerate variant" contemplates nucleotide fragments which differ from the nucleic acid fragments of the invention (eg, ORFs) in nucleotide sequence, but which, due to the degeneracy of the genetic code, encode the same polypeptide sequence. . A preferred nucleic acid fragment of the present invention is an ORF encoding a protein. Various methodologies known in the art can be used to obtain either an isolated polypeptide or protein of the present invention. At the simplest level, amino acid sequences can be synthesized using commercially available peptide synthesizers. This is particularly useful for producing small peptides or fragments of larger peptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. In yet another method, the polypeptide or protein is purified from bacterial cells that naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins to obtain one of the isolated polypeptides or proteins of the present invention. These include immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography, immunoaffinity chromatography, and the like,
It is not limited to these. For example, Scopes, Protein Purification: Pr inciples and Practice, Springer-Verlag (1994); Sambrook et al, Molecular Clo ning: A Laboratory Manual ; Ausubel et al., See Current Protocols in Molecular Bio logy.

The polypeptides and proteins of the present invention may be purified from cells that have been modified to express the desired polypeptide or protein. As used herein, when a cell has been genetically engineered to produce a polypeptide or protein that is not normally produced, or is usually produced only at low levels, the desired polypeptide is obtained. It is said to have been modified to express the peptide or protein. One of skill in the art will appreciate methods for introducing or expressing either recombinant or synthetic sequences to produce cells that produce one of the polypeptides or proteins of the invention, either prokaryotic or eukaryotic cells. Can be applied without difficulty. Purified polypeptides can be used in in vitro binding assays, which are known techniques for identifying molecules that bind to a polypeptide. These molecules include, but are not limited to, for example, small molecules, molecules from combinatorial libraries, antibodies or other proteins.
The molecules identified in the binding assays are then tested for antagonists or agonists in in vivo tissue culture or animal models known in the art. Briefly, molecules are titrated in multiple cell cultures or animals and then examined for animal / cell death or animal / cell survival prolongation.

In addition, the binding molecule may be conjugated to a toxin, for example, ricin or cholera toxin, or other compound that has toxicity to cells. The toxin-binding molecule conjugate is then targeted to tumor cells or other cells depending on the specificity of the binding molecule of SEQ ID NO: 3 or 5.

The protein of the present invention may be, for example, a transgenic bovine, goat, pig or sheep milk, characterized by somatic or germ cells containing the nucleotide sequence encoding the protein. It may be expressed as an animal product.

Proteins may be produced by traditional, known chemical synthesis. The method for constructing the protein of the present invention by a synthetic method is well known to those skilled in the art. By sharing primary, secondary or tertiary and / or tertiary structural and / or conformational properties with proteins, protein sequences constructed by synthetic methods may possess common biological properties, including their protein activity. It is. Thus, they can be used as biological or immunological substitutes for naturally occurring purified proteins in screening therapeutic compounds and in immunological processes for producing antibodies.

The proteins provided by the present invention are proteins characterized by an amino acid sequence similar to the purified protein, but also include those in which the modifications are naturally occurring or intentionally manipulated. I do. For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques. Of interest in modifying protein sequences may be alterations, substitutions, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with other amino acids to alter the configuration of the molecule. Techniques for such alterations, substitutions, insertions or deletions are well known in the art (eg, see US Pat. No. 4,51,51).
8,584). Preferably, such alterations, substitutions, insertions or deletions will retain the desired activity of the protein.

Other fragments and derivatives of the protein sequence which are expected to retain all or a portion of the activity of the protein, and which may be useful for screening or other immunological methods, are also described herein. It can be done without difficulty by those skilled in the art based on the contents disclosed in the present document. Such modifications are considered to be encompassed by the present invention.

[0111] The protein may also be produced by operably linking the isolated polynucleotide of the invention to a suitable control sequence in one or more insect expression vectors and utilizing an insect expression system. Can be. Materials and methods for baculovirus / insect cell expression systems are available in kits, such as, for example, the MaxBat. RTM. Kit commercially available from Invitrogen, San Diego, California, USA, and such methods are described in Summers and S, incorporated herein by reference.
mith, well-known in the art, as described in Texas Agricultural Experiment Station Bulletin No. 1555 (1987). As used herein,
An insect cell capable of expressing the polynucleotide of the present invention has been "transformed".

[0112] The proteins of the present invention are prepared by culturing the transformed host cells under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein is then purified from such culture (ie, culture medium or culture extract) using known purification processes such as gel filtration and ion exchange chromatography. Purification of the protein may further comprise an affinity column containing a reagent that binds the protein; one or more steps using an affinity resin such as Concanavalin A agarose, Heparin-Toyopearl.RTM. Or Shibachrome Blue 3GA Sepharose.RTM. One or more steps utilizing hydrophobic interaction chromatography using a resin such as ether, butyl ether, or propyl ether; or may also include immunoaffinity chromatography and the like.

[0113] Alternatively, the proteins of the invention may be expressed in a form that facilitates purification. For example, it may be expressed as a fusion protein such as a maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX) fusion protein. Kits for expressing and purifying such fusion proteins are available from New England BioLab (Beverly, Mass.), Pharmacia (
Piscataway, NJ) and In Vitrogen. Proteins can also be tagged with an epitope and then purified using a specific antibody directed against such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).

Finally, one or more reversed-phase high-performance liquid chromatography (RP-HPLC) steps utilizing a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups. May be performed for further protein purification. Some or all of the above purification steps may be performed in various combinations to obtain a substantially homogeneous isolated recombinant protein. A protein thus purified is substantially free of other mammalian proteins and is defined in the present invention as an "isolated protein".

The polypeptides of the present invention are interleukin-1 receptor antagonists (IL-1 RA
) Including analogs. This also includes fragments of the IL-1RA of the present invention, as well as interleukin-1 receptor antagonists that contain deletions, insertions or substitutions of one or more amino acids. Also, analogs of the interleukin-1 receptor antagonists of the present invention are those wherein the interleukin-1 receptor antagonist or analog is fused to one or more other moieties, such as a targeting moiety or other therapeutic agent. And a fusion product of an interleukin-1 receptor antagonist or a modified form of the interleukin-1 receptor antagonist. Such analogs will improve properties such as activity and / or stability. Examples of subjects that fuse to an interleukin-1 receptor antagonist or analog include, for example, targets that deliver the polypeptide to pancreatic cells, such as antibodies to pancreatic cells, T cells, monocytes, dendritic cells, granulocytes And antibodies and receptors and ligands expressed on pancreatic cells or immune cells. Other subjects that are fused to interleukin-1 receptor antagonists include therapeutic agents used in therapy, such as, for example, cyclosporine, SK506, azathioprine, CD3 antibodies and immunosuppressants such as steroids. Also, interleukin-1 receptor antagonists can be fused to immunostimulants, immune modulators, and other cytokines such as α- or β-interferon.

6.5. Deposit of Clones The following clone, pIL-1Hy273, was deposited with the American Type Culture Collection (ATCC) 10801 University Avenue, Manassas, Virginia on March 12, 1999 under the Budapest Treaty. . The 2,648 base pair cDNA insert of clone pIL-1Hy273 is contained in the vector pSPORT1 and is flanked by Not1 and Sal1 restriction sites. This clone, as described in the examples below,
Plasmid clones are representative.

[0117]

[Table 1]

6.6. Uses and Biological Activities The polynucleotides and proteins of the present invention exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. It is expected. The uses and activities described for the proteins of the invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (eg, gene therapy, or
A suitable vector for the introduction of DNA).

6.6.1. Research Uses and Utility The polynucleotides provided by the present invention can be used by a research community for a variety of purposes. A polynucleotide is used to express a recombinant protein for analysis, characterization or therapeutic use; the corresponding protein is preferentially expressed (constitutive expression or a specific stage of tissue differentiation or development or disease). State) as a marker for tissue; as a molecular weight marker for Southern gels; as a chromosomal marker or tag (if labeled) to identify chromosomes or to map the location of related genes; PCR primers for performing hybridisation and thus finding novel related DNA sequences to compare with the patient's endogenous DNA to identify certain genetic disorders; PCR primers for genetic fingerprinting As a source of guidance; the process of discovering other novel polynucleotides As a probe to "subtract" a known sequence; for the purpose of selecting and producing oligomers for attachment to a "gene chip" or other support, including experiments with expression patterns; using DNA immunization techniques, It can be used to raise anti-protein antibodies; and to raise anti-DNA antibodies or as an antigen to elicit another immune response.

If the polynucleotide binds to another protein or encodes a protein with potential binding (eg, binding at a receptor-ligand interaction), the polynucleotide may further bind to To identify polynucleotides that encode proteins or to identify inhibitors of binding interactions, interaction capture assays (see, e.g., Gyuris et al., Cell 75, 791-803 (
1993)).

The proteins provided by the present invention have high throughput (high throughput)
In a panel of multiple proteins for screening; to produce antibodies or to elicit another immune response; designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid (Including constitutive expression or tissue differentiation or expression at a particular stage of development or disease state) in which the corresponding protein is preferentially expressed as reagents (including labeled reagents) in the assay. As well as in assays to determine biological activity, including various purposes or uses, such as to isolate relevant receptors or ligands, of course. If the protein binds to or has potential binding to another protein (eg, in a receptor-ligand interaction), to identify other proteins where binding occurs, or Proteins can be used to identify inhibitors of the interaction of. Proteins involved in these binding interactions can also be used to screen binding interacting peptides or small molecules for inhibitors or agonists.

Any or all of these research applications can be developed into reagent or kit formats for commercialization as research results.

[0123] Methods for performing the above uses are well known in the art. References disclosing such a method include "Molecular Cloning: A Laboratory Manu
al "Second Edition, Cold Spring Harbor Laboratory Press, Sambrook, J., EF F
Edited by ritsch and T. Maniatis, 1989, and Methods in Enzymology: Guide t
o Molecular Cloning Techniquies, Acadmic Press, Berger, SL and A.R.
Edited by Kimmel, 1987.

6.6.2. Nutritional Utilization The polynucleotides and proteins of the present invention can also be used as a nutritional source or supplement. Such uses include, but are not limited to, use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the invention can be added to the diet of a particular organism, or as a solid, solid or solid form, such as a powder, pill, solution, suspension or capsule. It can also be administered as a liquid preparation.
In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

6.6.3. Cytokines and Cell Proliferation / Differentiation Activity The proteins of the invention may exhibit cytokine, cell proliferation (either induction or inhibition) or cell differentiation (either induction or inhibition) activity, or Other cytokines in specific cell populations can be induced. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Many protein factors discovered to date, including any known cytokines, exhibit activity in one or more factor-dependent cell proliferation assays, which serve as a convenient method of confirming cytokine activity. The activity of the protein of the present invention is 32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (preB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-. 1, Demonstrated by any of a number of conventional factor-dependent cell proliferation assays for cell lines, including but not limited to Mo7e and CMK. The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for T-cell or thymocyte proliferation include Current Protocols in Imm
unology, JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach,
W. Strober, Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Ch.
apter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3494-3
500, 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; including, but not limited to those described in Bowman et al, I. Immunol. 152: 1756-1761, 1994.

Assays for cytokine production and / or spleen cell, lymph node cell or thymocyte proliferation include polyclonal T cell stimulation, Kruisbeek AM and Sh
evach, EM, Current Protocols in Immunology. Edited by JEea Coligan, V
ol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and measurement of mouse and human interleukin-γ., Schreiber, RD, Current Pro.
tocols in Immunology. JE ea Coligan Editing Vol 1 pp. 6.8.1-6.8.8, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include measurement of mouse and human interleukin-2 and interleukin-4, Bottomly
, K., Davis, LS and Lipsky, PE, Current Protocols in Immunology. J
Edited by E. ea Coligan Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toron
to. 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. USA 80: 2931-
2938, 1983, Measurement of mouse and human interleukin-6-Nordan, R., Curr
ent Protocols in Immunology. Edited by JE ea Coligan Vol.1 pp. 6.6.1-6.6.
5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U
.SA 83: 1857-1861, 1986; Measurement of human interleukin-11--Bennett, F., Gi
annotti, J., Clark, SC and Turner, KJ, Current Protocols in Immuno
logy. JE ea Coligan Editing Vol 1 pp. 6. 15.1 John Wiley and Sons, Tor
onto. 1991; Measurement of mouse and human interleukin-9--Ciarletta, A., G
iannotti, J., Clark, SC and Turner, KJ, Current Protocols in Immun
ology. JE ea Coligan Editing Vol 1 pp. 6.13.1, John Wiley and Sons, Tor
including, but not limited to, those described in onto. 1991.

Assays for T-cell clonal responses to antigens, among others identifying proteins that affect APC-T cell interactions, and identifying direct T-cell effects by measuring proliferation and cytokine production Things), Current
Protocols in Immunology, JE Coligan, AM Kruisbeek, DH Margulies
, EM Shevach, W Strober, Greene Publishing Associates and Wiley-I
nterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; C
hapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic
studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091-60.
95, 1980; Weinberger et al., Eur.J. Immun. 11: 405-411, 1981; Takai et al., J. Imm.
unol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988, but are not limited thereto.

6.6.4. Immunostimulatory or Suppressive Activity The protein of the present invention may also exhibit immunostimulatory or immunosuppressive activity in assays, including but not limited to the activities described herein. .
The polynucleotide of the present invention can encode a polypeptide exhibiting such an activity. Proteins are used in the treatment of various immunodeficiencies and disorders, including severe combined immunodeficiency (SCID), for example, in regulating the growth and proliferation of T and / or B lymphocytes (up-regulation or down-regulation). And may also be useful in providing cytolytic activity of NK cells and other cell populations. These immunodeficiencies can be genetic, caused by a virus (eg, HIV), bacterial or fungal infection, or caused by an autoimmune disorder. Is also good. More particularly, infectious diseases caused by viruses, bacteria, fungi, including various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania subspecies, malaria subspecies, and candidiasis , Or other infections can be treated using the proteins of the present invention. Of course, in this regard, the proteins of the present invention may generally be desired to enhance the immune system, ie,
It may also be useful in treating cancer.

Autoimmune disorders that can be treated using the proteins of the present invention include, for example, connective tissue diseases, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome Autoimmune thyroiditis, insulin-dependent diabetes, myasthenia gravis, graft-host disease and autoimmune inflammatory eye diseases. Such proteins of the invention may also be useful in treating allergic reactions and conditions, such as asthma (particularly allergic asthma), or other respiratory disorders. Other conditions in which immunosuppression is desired (including, for example, organ transplantation) may also be treatable using the proteins of the invention.

It may also be possible to use the proteins of the invention to immunoreact in a number of ways. Down-regulation may be in the form of inhibiting or blocking an immune response that is already in progress, or may involve preventing the induction of an immune response. The function of the activated T cells may be to inhibit the function of the activated T cells by suppressing the response of the T cells or by inducing specific tolerance in the T cells, or both. Immunosuppression of a T cell response is usually an active, non-antigen-specific process that requires continuous exposure of the T cell to the inhibitor. Tolerance involves inducing unresponsiveness or inapparentness in T cells and is usually distinguishable from immunosuppression in that it is antigen specific, resulting in termination of exposure to the tolerizing agent. It persists after Operationally, tolerance can be demonstrated by a lack of response of T cells upon exposure to a specific antigen in the absence of a tolerizing agent.

Down-regulating or preventing one or more antigen functions, including but not limited to B lymphocyte antigen function (eg, B7), eg, high level lymphokine synthesis by activated T cells Prevention and the like may be useful in the context of tissue, skin and organ transplants, and in graft versus host disease (GVHD). For example, blocking T cell function should result in reduced tissue destruction in tissue transplantation. Usually, in tissue transplantation, rejection of the transplant is elicited through recognition by the T cells as foreign and subsequent immune reactions that destroy the transplant. Prior to transplantation, a molecule that inhibits or blocks the interaction of B7 lymphocyte antigen with its natural ligand (s) on immune cells (a soluble monomeric form of a peptide having B7-2 activity, alone or separately) Administration of a B lymphocyte antigen (eg, B7-1, B7-3, etc.) in combination with a monomeric form of a peptide having activity, or an inhibitory antibody, etc., without transmitting the corresponding costimulatory signal Can direct the binding of the molecule to the natural ligand (s) on the immune cell. Inhibiting the B lymphocyte antigen function in this way prevents the synthesis of cytokines by immune cells such as T cells, thereby acting as an inhibitor of immunity. In addition, the lack of costimulation is also sufficient to render T cells insensitive, which may induce tolerance in the subject. Induction of long-term tolerance by B lymphocyte antigen inhibitors may avoid the need for repeated administration of these inhibitors. In order to achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to inhibit the function of the combination of multiple B lymphocyte antigens.

The effectiveness of a particular inhibitor in preventing organ transplant rejection or GVHD can be evaluated using animal models that predict predictive efficacy in humans. Examples of suitable systems that can be used include allogeneic heart transplantation in rats and xenogeneic islet cell transplantation in mice, both of which are described in Lenschow et al., Sc.
ience 257: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci. USA, 89:
As described in 11102-11105 (1992), it has been used to examine the immunosuppressive effect of CTLA4Ig fusion protein in vivo. In addition, a mouse model of GVHD (P
aul Editing, Fundamental Immunology, Raven Press, New York, 1989, pp. 846-8
47), the effect of B lymphocyte antigen function on the development of the disease
Can be used to examine in vivo.

Inhibition of antigen function can further be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive to their own tissues and promote the production of cytokines and autoantibodies involved in the pathology of the disease. Preventing activation of autoreactive T cells may reduce or eliminate symptoms. Receptors for B lymphocyte antigen: administration of reagents that inhibit costimulation of T cells by disrupting ligand interactions, to inhibit T cell activation, and to autoantibodies or It can be used to prevent the production of T cell-derived cytokines. In addition, inhibitors may induce antigen-specific tolerance of autoreactive T cells, which may lead to long-term disease relief. The effectiveness of inhibitors in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune disease. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes in NOD and BB rats, and murine experimental autoimmune encephalitis. Myasthenia gravis is included (Paul editing, Fundamental Immunology, Raven Press)
, New York, 1989, pp. 840-856).

Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means of up-regulating the immune response may also be therapeutically useful. Up-regulation of the immune response may take the form of enhancing an existing immune response or eliciting an early immune response. For example, enhancing autoimmunity through stimulating B lymphocyte antigen function may be useful in case of viral infection. In addition, systemic viral diseases such as influenza, cold and encephalitis may be alleviated by systemic administration of a stimulating form of B lymphocyte antigen.

Alternatively, T cells may be removed from an infected patient and combined with APCs pulsed with a viral antigen expressing any of the polypeptides of the invention, or with a stimulated form of a soluble peptide of the invention. By co-stimulating T cells in vitro and reintroducing activated T cells in vitro into the patient, the anti-living immune response can be enhanced in infected patients. Another way to enhance the antiviral immune response is
Isolating infected cells from a patient and transfecting the cells with a nucleic acid encoding a protein of the invention, according to the disclosure herein, such that such cells express the entire protein or a portion thereof on the cell surface. Then, there is a method of introducing the transfected cells back into the patient. The infected cell should then be able to transmit a costimulatory signal to the T cell in vivo and thereby activate the T cell.

The presence of the peptide of the invention having the activity of the B lymphocyte antigen (s) on the surface of the tumor cells provides a costimulatory signal required for T cells, An immune response is elicited by the T cells against the infected tumor cells. In addition, if there is a lack of MHC class I or MHC class II molecules, or sufficient tumor cells can not be re-express MHC class I or MHC class II molecules in an amount, MHC class Iα chain protein and beta _{2-microglobulin} protein, Alternatively, it is transfected with all or part of the nucleic acid of the MHC class II α-chain protein and MHC class II β-chain protein (for example, the part lacking the cytoplasmic domain), so that the MHC class I or MHC class II is present on the cell surface. The protein can be expressed. Expression of appropriate Class I or Class II MHC in combination with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3)
An immune response is elicited by the T cells against the transfected tumor cells. Optionally, a gene encoding an antisense construct that inhibits expression of MHC class II-related proteins, such as an invariant chain, may also be co-transfected with DNA encoding a peptide having B lymphocyte antigen activity. Can enhance the presentation of tumor-associated antigens and elicit tumor-specific immunity.

Thus, by eliciting an immune response by T cells in a human subject, it is possible to sufficiently overcome tumor-specific usage in the subject.

The activity of the protein of the present invention can be measured by the method described below among various means.

Suitable assays for cytotoxicity on thymocytes or spleen cells include Current
Protocols in Imrnunology, JE Coligan, AM Kruisbeek, DH Margulie
s, EM Shevach, edited by W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Func
tion 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Pr.
oc. 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; He.
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; Bowman et al., J. Virology 61: 1992-
1998; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., Cellular Im.
munology 133: 327-341, 1991; including, but not limited to those described in Brown et al., J. Immunol. 153: 3079-3092, 1994.

Assays for T cell-dependent immunoglobulin response and isotope switching, especially those that identify proteins that modulate T cell-dependent antibody responses and affect Th1 / Th2 profiles, include Malizezews
ki, J. Immunol. 144: 3028-3033, 1990; and Assays for B cell function: I
n vitro antibody production, Mond, JJ and Brunswick, M., Current Prot
ocols in Immunology. JE ea Coligan Editing Vol 1 pp. 3.8.1-3.8.16, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994.

[0143] The mixed lymphocyte reaction (MLR) assay, which specifically identifies proteins that preferentially produce Th1 and CTL responses, includes Current Protocols in Immunology, J
E. Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strobe
r Editing, Pub.Green Publishing Associates and Wiley Interscience (Chapter
3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, I
mmunologic 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, but are not limited thereto.

Dendritic cell-dependent assays, among others that identify proteins expressed by dendritic cells that activate natural T cells, include Guery et al., J. Immunol. 134: 5.
36-544, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 199
1; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgador et al., J
ournal of Experimental Medicine 182: 255-260, 1995; Nair et al., Journal of Vi.
rology 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., Jo
urnal of Clinical Investigation 94: 797-807, 1994; and Inaba et al., Journal
of Experimental Medicine 172: 631-640, 1990, including, but not limited to.

Assays for lymphocyte survival / apoptosis, among others identifying proteins that prevent apoptosis following superantigen induction and proteins that regulate lymphocyte homeostasis, include Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyc
a 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., In
Includes, but is not limited to, those described in the ternational Journal of Oncology 1: 639-648, 1992.

Assays for proteins that affect T cell orientation and its early steps include 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. Natl.
Acad. Sci. USA 88: 7548-7551, 1991, including, but not limited to.

6.6.5. Hematopoietic modulatory activity The proteins of the present invention may be useful in regulating hematopoiesis and consequently in treating bone marrow or lymphoid cell defects. Involvement in hematopoietic regulation with minimal biological activity supporting colony forming cells or factor-dependent cell lines, e.g., supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, For example, useful in combination with radiation therapy / chemotherapy to treat various anemias, or to stimulate production of erythroid progenitor cells and / or red blood cells; bone marrows such as granulocytes and monocytes / macrophages Combining cell growth and proliferation (ie, traditional CSF activity) with, for example, chemotherapy to prevent consequent bone marrow suppression; supporting megakaryocyte growth and proliferation, and consequently platelet growth and proliferation Support growth, thereby enabling the prevention or treatment of various platelet disorders, such as thrombocytopenia, and generally replace or supplement platelet infusion A complete use; and / or can mature into any and all of the hematopoietic cells, and thus produce various stem cell disorders, including but not limited to aplastic anemia and paroxysmal nocturnal hemoglobinuria Therapeutic use in disorders that are usually treated by transplantation, including disease, and even as normal cells or cells that have been genetically engineered for gene therapy, either in-vivo or ex-vivo (ie, Involvement in Hematopoietic Stem Cell Growth and Proliferation with Bone Marrow Transplantation or Peripheral Progenitor Cell Transplantation (Allogeneic or Allogeneic) in Repopulation of Stem Cell Compartments Following Radiation / Chemotherapy Things.

The activity of the protein of the present invention can be measured by the following method, among other means.

Suitable assays for proliferation and differentiation of various hematopoietic cell lines are as described above.

Assays for fetal stem cell differentiation, particularly those that identify proteins that affect fetal differentiated hematopoiesis, include Johansson et al. Cellular Biology 15: 141-
151, 1995; Keller et al., Molecular and Cellular Biology 13: 473-486, 1993; Mc
Includes, but is not limited to, those described in Cianahan et al., Blood 81: 2903-2915, 1993.

Assays for stem cell survival and differentiation, especially those that identify proteins that regulate lympho-hematopoiesis, include Methylcellulose colony forming ass
ays, Freshney, MG In Culture of Hematopoietic Cells.RI Freshney, et al. Vol. pp. 265-268, Wiley-Liss, Inc., New York, NY 1994; Hirayama
Natl. Acad. Sci. USA 89: 5907-5911, 1992; Primitive hematopoiet.
ic colony forming cells with high proliferative potential, McNiece, I. K
and Briddell, RA In Culture of Hematopoietic Cells.RI Freshney,
Edit Vol pp. 23-39, Wiley-Liss, Inc., New York, NY 1994; Neben et al., E
xperimental Hematology 22: 353-359, 1994; Cobblestone area forming cell a
ssay, Ploemacher, RE in Culture of Hematopoietic Cells.RI Freshne
y, et al. Edit Vol pp. 1 -21, Wiley-Liss, Inc., New York, NY 1994; Long ter
m bone marrow cultures in the presence of stromal cells, Spooncer, E., D
exter, M. and Allen, T. In Culture of Hematopoietic Cells.RI Freshne
y, et al. Edit Vol pp. 163-179, Wiley-Liss, Inc., New York, NY 1994; Long t
erm culture initiating cell assay, Sutherland, HJ In Culture of Hemat
opoietic Cells. RI Freshney et al. Editing Vol pp. 139-162, Wiley-Liss, Inc.,
Includes, but is not limited to, those described in New York, NY 1994.

6.6.6. Tissue Growth Activity The proteins of the present invention may also be used for the growth or regeneration of bone, cartilage, tendon, ligament and / or neural tissue, as well as for wound healing and tissue repair and replacement, and for burns. , Incisions and ulcers also have utility in compositions used in the treatment.

[0153] Proteins of the invention that induce cartilage and / or bone growth when bone is not formed properly are applicable to the healing of fractures and cartilage damage or defects in humans and other animals. Such preparations using the proteins of the invention have prophylactic use in reducing closed and open fractures, as well as improving fixation of artificial joints. New bone formation induced by osteogenic agents contributes to the repair of cranial defects induced by congenital, traumatic or oncological resection, and is also useful in cosmetic surgery.

The proteins of the present invention may also be used in the treatment of periodontal disease and other dental restoration processes. Such agents may provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells, or induce differentiation of osteogenic cell precursors. The proteins of the present invention may further be through stimulation of bone and / or cartilage repair, or by inhibiting inflammation, or by inhibiting the process of tissue destruction mediated by the inflammatory process (collagenase activity, osteoclast activity, etc.). It is also useful in the treatment of osteoporosis or osteoarthritis.

Another category of tissue regeneration activity that can contribute to the proteins of the invention is tendon / ligament formation. Proteins of the invention that induce the formation of tendon / ligament-like or other tissue under conditions where such tissue is not formed normally include rupture, deformation, and other changes in tendons or ligaments in humans and other animals. For healing tendon or ligament defects. Preparations using proteins that induce tendon / ligament-like tissue may be used in prophylactic applications to prevent damage to tendon or ligament tissue, as well as to improve fixation of tendons or ligaments to bone or other tissue, or tendons or ligaments. It also has applications for repairing defects in ligament tissue. Neoplastic tendon / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of tendon or ligament defects due to congenital, traumatic or other causes, and for attachment or repair of tendons or ligaments. It is also useful in cosmetic surgery. The composition of the present invention provides an environment for inducing tendon-forming cells or ligament-forming cells, an environment for stimulating the growth of tendon-forming cells or ligament-forming cells, an environment for inducing differentiation of a precursor of tendon-forming cells or ligament-forming cells, Alternatively, it may provide an environment to induce the growth of tendon / ligament cells or precursors ex vivo for in vivo return to affect tissue repair. The compositions of the present invention are also useful in treating tendinitis, carcinomatosis and other tendon or ligament defects. These compositions may also contain suitable matrices and / or sequestering agents well known in the art as carriers.

The proteins of the present invention may also be used for the growth of nerve cells and regeneration of nerve and brain tissue, ie, for the treatment of central and peripheral nervous system diseases and neurological disorders, and for mechanical and traumatic disorders, It may also be useful for treating those involving degeneration, death or trauma to nerve tissue. More specifically, proteins are used in diseases of the peripheral nervous system, such as peripheral nerve injury, peripheral and focal neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy. -It may be useful in treating central nervous system diseases such as Dräger syndrome. Additional conditions that can be treated by the present invention include mechanical and traumatic disorders, such as spinal cord disorders, cerebrovascular disorders such as head trauma and stroke. Peripheral neuropathy resulting from chemotherapy or other medical therapy can also be treated using the proteins of the present invention.

The proteins of the present invention may be better or faster in progressive wounds, including but not limited to pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, etc. It can also be used to facilitate blockade.

The proteins of the present invention may be found in organs (eg, including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal, or cardiac) and vascular (including vascular endothelium) tissues. , Or an activity for the development or regeneration of other tissues, or an activity for promoting the growth of cells containing such tissues. Part of the desired effect is due to inhibition or modulation of fibrous scars, which allows normal tissue to regenerate. The proteins of the present invention may also exhibit angiogenic activity.

The proteins of the present invention may also be useful for gut protection or regeneration and lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

The proteins of the present invention may also be useful in promoting or inhibiting the differentiation of said tissue from progenitor cells or cells, or in inhibiting the growth of said tissue.

[0161] The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for tissue producing activity include International Patent Publication No. WO95 / 16035 (bone, cartilage, tendon); International Patent Publication No. WO95 / 05846 (neural, neuron); International Patent Publication Mo.
Including, but not limited to, those described in WO91 / 07491 (skin, endothelium).

Assays for wound healing activity include Winter, Epidermal Wound Healing, pps
71-112 (Maibach, HI and Rovee, DT, Editing), Year Book Medical Publ.
ishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. De
rmatol 71: 382-84 (1978), including, but not limited to.

6.6.7. Activin / Inhibin Activity The proteins of the invention may also exhibit activin-related or inhibin-related activity. The polynucleotide of the present invention preferably encodes a polypeptide exhibiting such properties. Inhibin is characterized by its ability to inhibit the release of follicle stimulating hormone (FSH), while activin is characterized by its ability to promote the release of follicle stimulating hormone (FSH). Thus, the proteins of the invention, alone or in a heterodimer with a member of the inhibin α-family, reduce fertility of inhibin in female mammals and contraception based on the ability to reduce spermatogenesis in dominant mammals. May be useful as a medicine. Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, the proteins of the invention may be fertilized based on the ability of activin molecules to stimulate FSH release from cells of the anterior pituitary in homodimers or in heterodimers with other protein subunits of the inhibin β group. It may also be useful as an induction therapeutic. For example, U.S. Pat.
See 4,798,885. The proteins of the present invention may also be useful for increasing livelihood reproductive capacity of livestock such as cattle, sheep, pigs, etc., and for hastening the onset of fertility in sexually immature mammals.

The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for actin / inhibin activity include 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. Aca
d. Sci. USA 83: 3091-3095, 1986, including, but not limited to.

6.6.8. Chemotaxis / Chemokine Activity The proteins of the present invention include, for example, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial and / or endothelial cells, and the like. It may have chemotactic or chemokine activity (eg, acting as a chemokine) on mammalian cells, including. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Chemotaxis and chemokine proteins can be used to move or attract a desired cell population to a desired site of action. Chemotaxis or chemokine proteins offer particular advantages in the treatment of wounds and other trauma to tissues, as well as in the treatment of local infections. For example, the attraction of lymphocytes, monocytes or neutrophils to the tumor or site of infection can result in an improved immune response to the tumor or infectious agent.

A protein or peptide will have chemotactic activity on a particular cell population if it can directly or indirectly stimulate the directed orientation or movement of that particular cell population. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a population of cells can be determined without difficulty by using such a protein or peptide in various known assays for cell chemotaxis.

The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for chemotactic activity (assays that identify proteins that induce or prevent chemotaxis) are based on the ability of the protein to induce cells to migrate across a membrane, and the ability of one cell population to It consists of an assay that measures the ability of the protein to induce adhesion to the population. Suitable assays for migration and adhesion include Current Protocols in Immunology, JE Coligan. AM Kruisbeek, DH
Edited by Marguiles, EM Shevach, W. Strober, Pub. Greene Publishing Associ
ates 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: 1744-17
48; Gruber et al. J. of Immunol. 152: 5860-5867, 1994; Johnston et at. J. Immu
nol. 153: 1762-1768, 1994, including, but not limited to.

6.6.9. Hemostatic and thrombolytic activities The proteins of the present invention can also exhibit hemostatic or thrombolytic activity. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Such proteins are expected to be useful in the treatment of various coagulation disorders, including genetic diseases such as hemophilia, or in the treatment of wounds such as those caused by trauma, surgery or other cases. It is expected to enhance coagulation and other hemostatic phenomena. The proteins of the present invention are also useful for inhibiting the dissolution or formation of thrombi, and for treating and preventing conditions resulting therefrom, such as infarction of the heart and central nervous system blood vessels, such as stroke. Can be useful.

[0172] The activity of the protein of the present invention may be measured by the following method, among other means.

Assays for hemostatic and thrombolytic activity include those described by Linet et al., J. Clin. Pharmaco.
l. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Humphr.
ey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 467-474, 19
88, including, but not limited to.

6.6.10. Receptor / Ligand Activity Proteins of the present invention may also exhibit activity as receptors, receptor ligands or inhibitors or agonists of receptor / ligand interactions. The polynucleotide of the present invention can encode a polypeptide exhibiting such properties. Examples of such receptors and ligands include cytokine receptors and their ligands,
Receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (such as cell adhesion molecules (such as selectins, integrins and their ligands)) and antigen presentation, antigen recognition and (Including but not limited to, receptor / ligand pairs involved in the development of cellular and humoral immune responses).
Receptors and ligands are also useful for screening potential peptide or small molecule inhibitors of the relevant receptor / ligand interaction. The proteins of the invention, including but not limited to receptor and ligand fragments, may themselves be useful as inhibitors of receptor / ligand interactions.

The activity of the protein of the present invention may be measured by the following method, among other means.

Suitable assays for receptor-ligand activity include Current Protocols in I
mmunology, JE Coligan, AM Kruisbeek, DH Margulies, EM Shevac
h, edited by W. Strober, Pub. Greene Publishing Associates and Wiley-Interscie
nce (Chapter 7.28, Measurement of Cellular Adhesion under static conditi
ons 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84: 6864-6868, 1
987; Bierer et al., J. Exp. Med. 168: 1145-1156, 1988; Rosenstein et al., J. Exp. M.
ed. 169: 149-160; Stoltenborg et al., J. Immunol. Methods 175: 59-68, 1994; Sti.
tt et al., Cell 80: 661-670, 1995, but are not limited thereto.

By way of example, the novel interleukin-1 receptor antagonists of the present invention can be used as ligands for cytokine receptors, thereby modulating the biological activity of that receptor. Examples of cytokine receptors used include, but are not limited to, interleukin-1 type I or interleukin-1 type II receptors. The novel interleukin-1 receptor antagonists of the present invention are
Expresses agonistic activity, partial agonistic activity, antagonistic activity or partial antagonistic activity for a particular receptor, such as a cytokine receptor, and is determined in a particular cell type by conventional techniques well known to those skilled in the art. can do. In one embodiment, one or more cells expressing a cytokine receptor (eg, an interleukin-1 type I or type II receptor) are contacted with a protein of the invention. Examples of cells that are contacted with the proteins of the present invention include, but are not limited to, mammalian cells such as fibroblasts and T cells. Preferably, the novel proteins of the present invention act as antagonists for cytokine receptors (eg, interleukin-1 receptor) such that the biological activity of the receptor is inhibited.

Studies characterizing drugs or proteins as agonists or antagonists or partial agonists have shown that partial agonists require the use of other proteins as antagonistic ligands. The novel protein of the present invention
Shows affinity for interleukin-1 receptor. Thus, the proteins of the invention can be used, for example, as antagonists in assays involving the interleukin-1 receptor (eg, Example 12). Alternatively, the proteins of the invention can be labeled by binding radioisotopes, colorimetric or toxin molecules by conventional methods. ("Guide to Protein Purification" Murray P. Deutsc
her (edited) Methods in Enzymology Vol. 182 (1990) Academic Press, San Diego) and in both in vivo and in vitro binding to the interleukin-1 receptor. Examples of radioisotopes include, but are not limited to, tritium and carbon-14. Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules. An example of a toxin includes, but is not limited to, ricin. According to an example, a protein bound to such a molecule would be an interleukin-1 receptor
Useful in studies involving in vivo or in vitro metabolism.

6.6.11. Screening of Drugs Using the Novel Interleukin-1 Receptor Antagonist Polypeptides The present invention relates to novel interleukin-1 receptor antagonist polypeptides or binding fragments thereof by various drug screening techniques. Is particularly useful in compound screening. The novel interleukin-1 receptor antagonist polypeptide or fragment used in such a test is not contained in solution, but is attached to a solid support and formed on the cell surface or present between cells. I do. One method of drug screening utilizes prokaryotic or eukaryotic host cells that are stably transformed with recombinant nucleic acids that express the polypeptide or fragment. Such cells, whether active or fixed, can be used in standard binding assays. For example, measuring the formation of a complex between a novel interleukin-1 receptor antagonist polypeptide or fragment and the agent to be tested, or using a novel interleukin-1 receptor antagonist polypeptide in the art, The decrease in complex formation with a suitable cell line known in the art is assayed.

6.6.11.1 Anti-Interleukin-1 Receptor Activity Assay According to one embodiment, the interleukin-1 receptor antagonist activity of a polypeptide of the invention is determined by a method, namely: (1) interleukin-1 1, interleukin-1 receptor, interleukin-1 receptor antagonist polypeptide of the present invention, and / or agonist and antagonist thereof (or agonist or antagonist drug candidate substance), and / or interleukin-1 receptor of the present invention Forming a mixture comprising antibodies specific for the body antagonist polypeptide,
(2) The interleukin-1 receptor antagonist polypeptide of the present invention and / or its agonist and antagonist (or agonist or antagonist drug candidate substance), and / or the interleukin-1 receptor antagonist polypeptide of the present invention. Interleukin except for the presence of specific antibodies
Incubating the mixture under conditions such that -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 Is determined using a method including:

6.6.11.2 Antagonist and Agonist Assays Human HepG2 cells were incubated for 18-24 hours at 37 ° C. in Dulbecco's modified Eagle's medium without serum. Separated monolayers of cells were incubated in the same medium supplemented with various concentrations of interleukin-1 and in the same medium supplemented with various concentrations of interleukin-1 receptor antagonists of the invention. The monolayer is carefully rinsed with an isotonic buffer, incubated with (35-S) methionine, 250 μci / ml methionine-free medium, and used to evaluate synthesis.
The substrate was applied over a period of 30 minutes. Discard the cell culture and rinse the monolayer again,
Then, the cells were resuspended in a cell lysis buffer. The newly synthesized radiolabeled liver proteins in these cell lysates are detected by immunoprecipitation, SDS-PAGE and fluorography.

6.6.12. Anti-inflammatory activity The proteins of the present invention may also exhibit anti-inflammatory activity. Anti-inflammatory activity provides stimulation to cells involved in the inflammatory response, inhibits or promotes cell-cell interactions (eg, cell adhesion, etc.), inhibits or promotes chemotaxis of cells involved in the inflammatory process, It can be achieved by inhibiting or promoting cell extravasation, or by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Proteins exhibiting such activity include, but are not limited to, infection suggestions (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemic reperfusion injury, lethal endotoxin, arthritis Chronic or renal inflammation, including complement-mediated hyperacute rejection, nephritis, cytokine- or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or diseases caused by overproduction of sitekines such as TNF or IL-1. It can be used to treat any inflammatory condition, acute. The proteins of the invention may also be useful in treating anaphylaxis and hypersensitivity to antigenic substances or materials. In particular, interleukin-1 receptor antagonists of the invention include, but are not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine-induced shock, rheumatoid arthritis, chronic arthritis, type I diabetes mellitus Acute or chronic as part of a method for the prevention and / or treatment of pancreatic cell damage, graft-versus-host disease, enteritis disease, lung disease-related inflammation, other autoimmune diseases or disorders involving inflammatory conditions It is used as an antiproliferative agent for myeloid leukemia and the like, or in the prevention of preterm labor caused by intrauterine infection, for prevention or treatment of a condition.

6.6.13. Leukemia Leukemia and related disorders can be treated or prevented by administering a therapeutic agent that promotes or inhibits the function of the polynucleotides and / or polypeptides of the invention. Such leukemias and related disorders include acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic leukemia, For a review of myeloid (granulocytic) leukemia and chronic lymphocytic leukemia (disorders listed above, see Fishman et al., 1985, Medicine , 2nd ed.
B. Lippincott Co., Philadelphia).

6.6.14. Nervous System Disorders Nervous system disorders are compounds that modulate the activity of the polynucleotides and / or polypeptides of the invention and involve cell types that can be tested for the efficiency of intervention. Thus, it can be treated by observing such signs of therapeutic use, including injuries to the nervous system, and any disconnection of axons, loss or degeneration of neurons, or demyelination. Includes, but is not limited to, the disease or disorder that causes it. Nervous system injury in patients (including human and non-human mammalian patients) that can be treated according to the present invention includes central (spinal,
(Including but not limited to, the brain) or the peripheral nervous system, including but not limited to: (i) traumatic injuries, including injuries caused by physical injuries, or surgical injuries such as, for example, amputation of a part of the nervous system or compression injuries; (ii) injuries to a part of the nervous system An ischemic injury in which lack of oxygen causes neuropathy or death, including cerebral infarction or cerebral ischemia, or spinal cord infarction or ischemia; Infectious damage in the part of the nervous system that has been destroyed or damaged by infection with herpes zoster or herpes simplex virus, or with Lyme disease, tuberculosis or syphilis; (iv) not intended to be limiting But not as a result of degenerative processes, including Parkinson's disease, Alzheimer's disease, Huntington's chorea or amyotrophic lateral sclerosis,
Degenerative injury in a part of the nervous system that has been destroyed or damaged; (Vi) limited nutritional or nutritional disorders in parts of the nervous system destroyed or damaged by nutritional or metabolic disorders, including primary degeneration of the corpus callosum and alcoholic cerebellar degeneration; Neurological injuries associated with systemic disease, including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vii) alcohol, lead, or certain Injury caused by toxic substances, including neurotoxins; and (viii) without intending to be limiting Demyelinating diseases or various etiologies, including multiple sclerosis, myelopathy associated with human immunodeficiency virus, transverse spinal cord injury, progressive multifocal leukoencephalopathy, and nerves destroyed or damaged by central pontine myelinolysis Demyelination injury in part of the system.

Therapeutic agents of the invention useful for the treatment of nervous system disorders can be selected by testing for biological activities that promote neuronal survival or differentiation. For example,
The following effects: (i) increase the survival time of neurons in culture; (ii) increase the sprouting of neurons in culture or in vivo; (iii) neuron-related molecules such as exercise in culture or in vivo. For neurons, a therapeutic agent that either produces increased production of choline acetyltransferase or acetylcholinesterase; or (iv) reduces the symptoms of neuronal dysfunction in vivo is useful in the present invention, but is not limited thereto. . Such an effect can be measured by methods known in the art. In a preferred non-limiting embodiment, the increase in neuronal survival is
(1990, J. Neurosci. 10: 3507-3515); increased sprouting of neurons was determined by Pestronk et al. (1980, Exp. Neurol. 70: 65-82) or Brown et al.
nn. Rev. Neurosci. 4: 17-42); the increase in production of neuron-associated molecules can be measured by a bioassay, enzyme assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; Motor neuron impairment function can then be measured, for example, by assessing physical symptoms of motor neuron impairment, such as weakness, motor neuron conduction velocity, or impaired functional ability.

In certain embodiments, the motor neuron disorder that can be treated according to the present invention may include infarction, infection, exposure to toxins, trauma, surgical injury, motor neurons and other components of the nervous system. Includes disorders that selectively affect neurons, such as degenerative diseases or malignancies, and amyotrophic lateral sclerosis, and progressive spinal muscular atrophy, progressive medullary palsy, primary amyotrophic lateral sclerosis, childhood and Includes disorders such as juvenile muscular atrophy, progressive medullary palsy in children (Fatio-Ronde syndrome), gray and white poliomyelitis and post-pediatric syndrome, and hereditary motor perception neuropathy (Charcot-Marie-Tooth disease), Not limited to them.

6.6.15. Other Activities The proteins of the present invention may have one or more of the following other activities or effects: bacteria, viruses, fungi, and parasites, without limitation. Inhibiting the growth, infection or function of infectious agents, including insects, or killing with such agents; height, weight, hair color, eye color, skin, leanness, without limitation Causing changes in physical characteristics, including the degree of obesity or other tissue pigmentation, or organ or body part size or appearance (eg, increased or decreased breast, altered bone morphology or appearance, etc.) Inhibiting or enhancing); causing a change in the biorhythm or the caricadic cycle or rhythm; causing a change in the fertility of a male or female subject; Altering the metabolism, catabolism, assimilation, processing, utilization, saving or elimination of fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional factors or components (s) of matter; Unintentionally altering behavioral characteristics, including appetite, sexual impulses, stress, cognition (including cognitive impairment), decline (including depressive disorders) and violent behavior; analgesic effects or other Providing a pain-relieving effect; promoting the differentiation and growth of embryonic stem cells in cell lines other than hematopoietic cell lines; hormonal or endocrine activity; in the case of enzymes, correcting and eliminating the deletion of said enzymes Treating hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activity (eg, the ability to bind antigen or complement) And the ability to act as an antigen in a vaccine composition to generate an immune response to such a protein, or another material or entity that is cross-reactive with such a protein.

6.6. Therapeutic Methods The novel interleukin-1 receptor antagonists of the present invention have myriad applications in a variety of therapeutic methods. Examples of therapeutic applications are illustrated below, but are not limited thereto.

6.6.1. Sepsis In one embodiment of the present invention, an effective amount of an interleukin-1 receptor antagonist polypeptide of the present invention is administered to a patient who is at high risk of developing sepsis or has already developed sepsis. To be administered. An example of the former is a patient who has undergone a surgical procedure. The mode of administration is not particularly important, but parenteral administration is preferred because of the rapid progression of sepsis and the need to spread the inhibitor quickly throughout the body. Therefore,
A preferred mode of administration is to deliver a small amount of pills intravenously before, during, or after the surgical procedure. The dose of the interleukin-1 receptor antagonist polypeptide of the present invention is usually determined by the prescribing physician. This dose will vary depending on the age, weight and response of the patient. Usually, the amount of inhibitor administered per dose will be about 0.1 to 25 mg / kg of body weight, preferably about 0.1 to 25 mg / kg of body weight.
1010 mg / kg body weight. For parenteral administration, the interleukin-1 receptor antagonist polypeptides of the invention are prepared in injectable form in combination with a pharmaceutically acceptable parenteral vehicle. Such excipients are well known in the art, and include, for example, water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of human serum albumin. The excipient contains minor amounts of additives that maintain the isotonicity and stability of the inhibitor. Preparation of such solutions is within the skill of the art. Typically, cytokine inhibitors are prepared to contain excipients at a concentration of about 1-8 mg / ml to 10 mg / ml.

6.6.2. The Immunosuppressive Effect of Interleukin-1 Inhibitors on Arthritis and Inflammatory Rheumatoid Arthritis
Determined in an experimental animal model system. The experimental model system was rats suffering from arthritis induced by adjuvant, and the protocol was described by J. Holoshitz, et al.,
1983, Science, 219: 56 or B. Waksman et al., 1963, Int.Arch. Allergy App.
l. Immunol., 23: 129. Disease induction is generally accomplished by a single injection of a suspension of complete Freund's adjuvant (CFA) containing sterilized Mycobacterium tuberculosis. The route of administration can be varied, but in the case of rats, the adjuvant mixture is injected at the base of the tail. Inhibitor is phosphate buffer
(PBS) at a dose of about 1-5 mg / kg. Subjects will receive PBS only.

The procedure for testing the effect of the interleukin-1 inhibitor consists of injecting CFA containing sterile Mycobacterium tuberculosis intradermally, then administering the inhibitor immediately, and every other day. Treatment consists of up to 24 days. On days 14, 15, 18, 20, 22, and 24 after injection of Mycobacterium CFA, an overall arthritis score is obtained as described in J. Holoskitz, supra. Analysis of the data would indicate that the decrease in arthritis score would indicate that the inhibitor had a sharp effect on joint swelling.

6.6.3. Diabetic Interleukin-1 has been shown to be involved in islet cell destruction in diabetes mellitus (DM) (mandrup-Paulsen, T., K. Bendtzen, J. Nerup, CA Dinare
llo, M. Svenson and JH Nielson [1986] Diabetologia 29: 63-67). The interleukin-1 receptor antagonist polypeptides of the present invention confer on islet cells in early DM cases identified by disease susceptibility based on genetic background and family history,
Limit lymphocyte and macrophage mediated damage. Inflammatory destruction of pancreatic β-islet cells in individual patients with early DM is reduced by parenteral administration of an interleukin-1 receptor antagonist polypeptide of the present invention that exhibits anti-interleukin-1 effects in the pancreas.

6.6.4. Anti- hypertensive Arginine Free Pharmaceutical Parenteral Preparations means about 3-4 g / l isoleucine, about 4-6 g / l together with pharmaceutically acceptable excipients. 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 0.2 g / l
It is defined as containing 5-0.75 g / l tyrosine, about 4-5 g / l glycine and about 2-3 g / l aspartic acid. In another preferred embodiment of the parenteral preparation, the preparation further comprises ornithine, most particularly at a concentration of about 1-2 g / l. In yet another embodiment of the parenteral formulation, the formulation further comprises citrulline, most specifically at a concentration of about 1 to about 2 g / l. In yet another embodiment of the formulation, both citrulline and ornithine are included and used again at the concentrations described above.

The method uses an arginine-free preparation, which contains the already mentioned amino acids in the already mentioned concentrations, together with pharmaceutically acceptable excipients. The formulation may further include ornithine, citrulline, or both, to provide the physiologically required concentration of the animal's urea cycle substrate. Most preferably, the preparation is provided as a parenteral preparation.

Other aspects of the method include a method of treating chemotherapeutic agent-related hypotension.
In a most preferred embodiment, the method monitors the reduction of systolic blood pressure in animals administered a chemotherapeutic agent to about 100 mm Hg or less to detect animals with systemic hypotension, and provides a therapeutically effective concentration. A therapeutic method using a therapeutically effective amount of an arginine-free modulator sufficient to reduce plasma or serum arginine levels administered concurrently with or prior to administration of an interleukin-1 receptor antagonist; Treating the affected animal and continuing treatment of the animal until it can be detected that systolic blood pressure has risen to at least about 100 mmHg. Most preferably,
Arginine-free preparations are parenteral preparations.

In a preferred embodiment, an Interleukin-1 receptor antagonist polypeptide of the invention is used to treat hypotension in an animal, particularly hypotension resulting from exposure to endotoxin or septic shock. Used in combination with hypotensive arginine-free preparations.

Patients exhibiting systolic blood pressure of about 100 mmHg or less were eligible for this treatment. The patient is subjected to a continuous supply of an arginine-free preparation containing a mixture of essential and non-essential amino acids as described in US Pat. No. 5,334,380. The patient is concurrently treated with an interleukin-1 antagonist polypeptide of the invention. A blood sample is drawn from the patient and the level of arginine in the serum or plasma fraction is determined.

6.7. Pharmaceutical Formulations and Routes of Administration The proteins of the present invention, including those derived from recombinant and non-recombinant sources,
Without limitation, from a variety of sources) can be administered to a patient in need thereof at a dose to treat or ameliorate various disorders, by the protein itself or by a suitable carrier or carrier. It may be administered in a pharmaceutical composition mixed with excipient (s). Such compositions may also contain (in addition to proteins and carriers) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Is also good. The term "pharmaceutically acceptable" means a non-toxic material (s) that does not interfere with the effect of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration. Further, the pharmaceutical composition of the present invention, for example, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6
, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg -It may also contain cytokines such as CSF, thrombopoietin, stem cell factor, and erythropoietin, rifokines, or other hematopoietic factors.

Further, the pharmaceutical compositions of the present invention may contain other agents that either enhance the activity of the protein or enhance its activity or use in therapy. Such additional factors and / or agents may be included in pharmaceutical compositions to produce a synergistic effect with the proteins of the invention or to minimize side effects. Conversely, the proteins of the present invention may include certain cytokines, rifokines, other hematopoietic factors, or other hematopoietic factors to minimize the side effects of , A thrombolytic or antithrombotic factor, or an anti-inflammatory agent. A protein of the invention may be active in multimers (eg, heterodimers or homodimers) or complexes with itself or with other proteins. As a result, a pharmaceutical composition of the invention may include a protein of the invention in such a multimerized or complexed form.

Techniques for the formulation and administration of the present compounds are described in “Remington's Pharmaceutical Scienc.
es, "Mack Publishing Co., Easton, PA, latest edition. Further therapeutically effective doses can be found, for example, in treating, curing, preventing or ameliorating the associated medical condition, or ameliorating such a condition, or in such a condition. Reference is made to an amount of the compound that is sufficient to result in an increase in the rate of treatment, cure, prevention or amelioration of the compound. When applied in combination, it refers to the combined amount of the active ingredients that produce the therapeutic effect, whether the therapeutically effective dose is administered sequentially or simultaneously, in combination.

In practicing the methods of treatment or use of the present invention, a therapeutically effective amount of a protein of the present invention is administered to a mammal having the condition to be treated. The protein of the present invention
In accordance with the methods of the present invention, it may be administered either alone or in combination with other therapies, such as treatment with cytokines, rifokines or other hematopoietic factors. When administered with one or more cytokines, rifokines or other hematopoietic factors, the proteins of the invention may be administered with the cytokine (s), rifokine (s), other hematopoietic factor (s), thrombolytic or antithrombotic. At the same time as sex factors,
Alternatively, they may be administered continuously. When administered continuously, the attending physician administers a protein of the present invention in combination with the cytokine (s), rifokine (s), other hematopoietic factor (s), thrombolytic or antithrombotic factors. Will determine the appropriate order to do.

6.7.1. Routes of Administration Suitable routes of administration are, for example, oral, rectal, transmucosal or intestinal; intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal In addition to injection or intraocular injection, parenteral delivery may include intramuscular, subcutaneous, intramedullary injection. The pharmaceutical composition or the protein of the present invention used to carry out the method of the present invention may be used in various forms, such as oral ingestion, inhalation, topical application or dermal injection, subcutaneous injection, intraperitoneal injection, parenteral injection or intravenous injection. Administration can be carried out in any conventional manner. Intravenous administration to a patient is preferred.

Alternatively, the compound may be administered by systemic techniques, for example, in the form of a depot or sustained release formulation, for example, by injection of the compound into an arthritic joint or directly into fibrotic tissue. Rather, it may be administered in a local manner. The compounds may be administered topically, for example, as eye drops, to prevent the scarring process that often occurs as a complication of glaucoma surgery. Furthermore, in drug-targeted drug delivery systems, for example, targeting arthritic or fibrotic tissue,
For example, they may be administered in the form of liposomes coated with specific antibodies.
The liposomes will be targeted and will be selectively absorbed by the affected tissue.

6.7.2. Compositions / Formulations The pharmaceutical compositions used according to the present invention therefore include excipients and auxiliaries which facilitate the processing of the active compounds into pharmaceutically usable preparations. It may be formed in a conventional manner using one or more physiologically acceptable carriers, including. These pharmaceutical compositions may be manufactured in a manner known per se, for example, 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 a protein of the present invention is administered orally, the protein of the present invention can be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical compositions of the invention may additionally include a solid carrier such as gelatin or an adjuvant. The tablets, capsules and powders comprise about 5-95
% Of the protein of the invention, preferably about 25-90% of the protein of the invention. When administered in liquid form, a liquid carrier such as water, petroleum, peanut oil, mineral oil, animal or vegetable oils such as soybean oil or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical preparation may further contain physiological salt solutions, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical preparation contains about 0.5-90% by weight of a protein of the invention, preferably about 1-50% by weight.

A therapeutically effective amount of a protein of the present invention may be injected intravenously, dermally or subcutaneously.
When administered more, the protein of the present invention is pyrogen-free and parenterally acceptable
In the form of an aqueous solution. Well-considered pH, isotonicity, stability, etc.
Such parenterally acceptable protein solution preparations are known in the art.
It exists. Preferred pharmaceutical composition for intravenous, cutaneous or subcutaneous injection
The product is prepared by adding sodium chloride injection, Ringer injection,
Injection of dextrose, dextrose and sodium chloride, Ringer's lactate
Or other isotonic vehicles, or other vehicles known in the art. Departure
The pharmaceutical compositions of the present invention may also include stabilizers, preservatives, buffers, antioxidants, or
And other known additives. For injection, the drug of the present invention is dissolved in water
In solution, preferably such as Hanks' solution, Ringer's solution or physiological saline buffer.
It may be formulated in a physiologically compatible buffer. Should be penetrated for transmucosal administration
Penetrants appropriate to the barrier are used in the formulation. Such penetrants are known in the art.
In the field, it is generally well known.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. With such carriers, the compounds of the invention may be formulated as tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient to be treated. Can be Pharmaceutical preparations for oral use can include solid excipients, milling the resulting mixture to obtain tablets or dragee cores and, if necessary, adding optional auxiliaries. After that, it is processed into a mixture of granules. Suitable excipients are fillers, such as sugars, including, inter alia, lactose, sucrose, mannitol or sorbitol; for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxy. Cellulose preparations such as sodium methylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or an alginate salt such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may be gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Classes are optionally included. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

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 press-fit capsule may contain the active ingredient as a mixture with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and any 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 dosage forms suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For inhaled administration, compounds for use in accordance with the present invention may employ any suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It is conveniently delivered in the form of an aerosol spray jet from a pressurized pack or nebulizer. In the case of a pressurized aerosol the dosage unit may be determined by providing a value to deliver a measured amount. Cartridges and capsules, for example, for gelatin, for use in inhalers or insufflators, 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, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsifiers in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. .

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 that increase the stability of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, eg, 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 depot preparations may be administered (eg, subcutaneously or intramuscularly), by implantation, or by intramuscular injection. Thus, for example, the compound may be a suitable polymer or hydrophilic material (eg, as an emulsifier in an acceptable oil)
Or with an ion exchange resin, or as a slightly sparingly soluble derivative, for example,
It may be formulated as a slightly hardly soluble salt.

The pharmaceutical carrier for the hydrophilic compound of the present invention is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic solvent and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD, as measured in absolute ethanol, is a solution of 65% w / v polyethylene glycol 300, 8% w / v non-polar surfactant polysorbate 80, and 3% w / v benzyl alcohol. VPD co-solvent system (VPD: 5W) is a 1: 1 dilution of VPD in 5% dextrose in aqueous solution
Consists of This co-solvent system dissolves hydrophilic compounds well, and itself produces only low toxicity upon systemic administration. Of course, the proportions of a co-solvent system may be varied without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components is
For example, other low toxicity non-polar surfactants may be used in place of polysorbate 80; the fraction size of polyethylene glycol may be changed; other biocompatible polymers may be polyethylene glycol, For example, it may be replaced with polyvinylpyrrolidone; and dextrose may be replaced with another saccharide or polysaccharide. Alternatively, other delivery systems for hydrophilic pharmaceutical compounds may be used. Liposomes and emulsifiers are well-known examples of delivery vehicles or carriers for hydrophilic drugs. Usually, greater toxicity is at the expense, but certain organic solvents such as dimethyl sulfoxide may be used. In addition, the compounds may be delivered using a depot system, such as semipermeable matrices of solid hydrophilic polymers containing the therapeutic agent. Various long-lasting materials have been established and are well known in the art. Sustained-release capsules may release the compounds from weeks to more than 100 days, depending on their chemical nature. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for protein stabilization may be used.

The pharmaceutical compositions may also include suitable solid or gel phase carriers or excipients. Specific examples of these 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 proteinase inhibiting compounds of the present invention may be provided as salts with pharmaceutically compatible counterions. Such pharmaceutically acceptable base addition salts are salts that retain the biological effectiveness and properties of the free acids, such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamines, dialkylamines. , Monoalkylamines, dibasic amino acids, sodium acetate, potassium benzoate, triethanolamine, and other inorganic or organic bases.

The pharmaceutical compositions of the invention may be in the form of a complex of the protein (s) of the invention and a protein or peptide antigen. Said protein and / or peptide antigen will induce a stimulatory signal for both B and T lymphocytes. B lymphocytes will react with antigen through their surface immunoglobulin receptor. T lymphocytes will react with 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 peptide antigen (s) to T lymphocytes . The antigen component could be supplied as a purified MHC-peptide complex alone or with a costimulatory molecule capable of signaling directly to T cells. Alternatively, antibodies capable of binding to surface immunoglobulins and other molecules on B cells, in addition to antibodies capable of binding to TCRs and other molecules on T cells,
It can be combined with the pharmaceutical composition of the present invention. The pharmaceutical compositions of the present invention may comprise, in addition to other pharmaceutically acceptable carriers, amphipathic agents such as lamellar layers in aqueous solution, liquid crystals, lipids that are present in an aggregated form such as insoluble monolayers or micelles. And the protein of the present invention may be in the form of a lysosome. Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like. For example, U.S. Patent No. 4,235,8, all of which are incorporated herein by reference.
71; 4,501,728; 4,837,028; and 4,737,323, the preparation of such liposome formulations is within the level of skill in the art.

The amount of a protein of the present invention in a pharmaceutical composition of the present invention will depend on the type and severity of the condition to be treated and will depend on the type of prior treatment experienced by the patient. . Ultimately, the attending physician will decide the amount of protein of the present invention to treat each individual patient. First, the attending physician will administer the protein of the invention at a low dose and observe the patient's response. Higher doses of the protein of the present invention may be administered to an amount that produces the optimal therapeutic effect for the patient and does not increase the dosage. Various pharmaceutical compositions used to practice the methods of the present invention may comprise from about 0.01 μg to about 100 mg (preferably from about 0.1 μg to about 10 mg, more preferably from about 0.1 μg to about 1 mg) of the protein of the invention per kg body weight. ) Is contemplated to be included. For compositions of the invention useful for bone, cartilage, tendon or ligament regeneration, methods of treatment include administering the composition locally, systemically, or locally as an implant or device. When administered, the therapeutic composition used in the present invention is, of course, in a pyrogen-free, physiologically acceptable form. In addition, the compositions may be injected or encapsulated in a viscous form, desirably for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Agents useful for the treatment other than the protein of the present invention, which are optionally contained in the composition as described above, may be selectively or additionally administered simultaneously or sequentially with the composition in the method of the present invention. Good. Preferably, for bone and / or cartilage formation, the matrix can deliver the protein-containing composition to the site of bone and / or cartilage damage, provide a structure for developing bone and cartilage, and be optimally resorbed by the body Will be included. Such matrices may be formed of materials currently used for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, physical properties, surface appearance and interface properties. The particular application of the composition will limit the suitable formulation. Possible matrices for the composition are biodegradable,
It may be chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are bioerodible and biologically well defined bone or skin collagen. Further matrices comprise pure proteins or extracellular matrix components. Other possible matrices are non-biodegradable and chemically defined, hydroxyapatite, bioglass,
Such as aluminates or other ceramics. The matrix may comprise a combination of any of the above types of materials, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics may be altered in calcium-aluminate-phosphate composition and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred are 50:50 (molar weight) copolymers of lactic acid and glycolic acid in the form of porous particles having a diameter in the range of 150-800 microns. In some applications, it may be useful to utilize a sequestering agent, such as carboxymethyl cellulose or orthologous clot, to prevent the protein composition from dissociating from the matrix.

A preferred family of said sequestering agents is methylcellulose, ethylcellulose,
Cellulosic materials such as alkylcelluloses (including hydroxyalkylcellulose), including hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, most preferably cationic salts of carboxymethylcellulose (CMC) is there. Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly (
Ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers and poly (vinyl alcohol). The amount of sequestering agent useful herein is from 0.5 to 20% by weight, preferably 1 to 10% by weight, based on the total formulation weight, which prevents desorption of the protein from the polymer matrix, Shows the amount needed to provide proper handling of the composition, but not enough to prevent primary cells from entering the matrix, thereby assisting the protein with the osteogenic activity of the primary cells Opportunities are given. In further compositions, the proteins of the invention may be combined with other agents that are beneficial in treating bone and / or cartilage defects, wounds, or the tissue in question. These drugs include epidermal growth factor
(EGF) platelet-derived growth factor (PDGF), transforming growth factor (TGF-α and
TGF-β) and various growth factors such as insulin-like growth factor (IGF).

At present, the therapeutic compositions are also valuable for veterinary applications. In addition to humans, especially domesticated animals and thoroughbred horses are medical objects for which such treatment with the proteins of the present invention is desired. The dosage regime of the protein-containing pharmaceutical composition to be used for tissue regeneration depends on various factors that modify the behavior of the protein, such as the amount of tissue weight desired to be formed, the site of the injury, the condition of the damaged tissue, Determined by the attending physician considering the size, type of tissue damaged (eg, bone), age, sex, and diet, severity of infection, number of doses, and other clinical factors of the medical subject Will. Dosage may vary with the type of matrix used for reconstitution, and may also vary with the inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF1 (insulin-like growth factor I) to the final composition, may result in a change in dosage. Progress can be monitored by periodic assessment of tissue / bone growth and / or repair, for example, x-ray, histomorphometry, and tetracycline labeling.

The polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced into cells for expression in a mammalian subject, either in vivo or ex vivo. The polynucleotides of the present invention may also be administered by other known methods for introducing nucleic acids into cells or organs, including, but not limited to, in the form of viral vectors or naked DNA. The cells may be cultured ex vivo in the presence of a protein of the present invention to proliferate or to produce the desired effect or activity on the cells. The treated cells can then be introduced in vivo for therapeutic purposes.

6.7.3. Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions that contain an effective amount of the active ingredient to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount that is effective to prevent or alleviate the progression of the existing condition of the subject being treated. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure 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, the dose
It can be formulated in animal models that achieve a circulating concentration that includes the IC ₅₀ as determined in cell culture (ie, the concentration of the test compound that achieves half-maximal inhibition of C-proteinase activity). Such information can be used to more accurately determine useful doses in humans.

[0220] A therapeutically effective dose refers to that amount of the compound that results in an improvement or prolongation of the patient's symptoms. Toxicity and therapeutic efficacy of the compounds are, for example, LD ₅₀ (50% of the population)
Can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the ED ₅₀ (the amount that will kill) and the ED ₅₀ (the amount that produces a therapeutic effect in 50% of the population).
Dose ratio between toxic and therapeutic effects is the therapeutic index can be expressed as the ratio between LD ₅₀ and ED _50. Compounds that exhibit a high therapeutic index 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 humans. Dosage of such compounds lies preferably within a range of circulating concentrations that almost include the ED ₅₀ with absolutely no toxicity or no toxicity. Dosage may vary within this range depending on the dosage form employed and the route of administration used. The exact formulation, route of administration and dosage can be selected by the individual physician in view of the patient's condition. See, for example, Fingl et al., 1975, "The Pha
rmacological Basis of Therapeutics ", Chapter 1, page 1. Dosage and intervals are set to provide plasma levels of the active moiety that are sufficient to maintain a C-proteinase inhibitory effect, or minimal effective concentration (MEC). The MEC will vary for each compound, but the in vitro data; for example, using the assays described herein, inhibit 50-90% inhibition of C-proteinase. The dosage required to achieve MEC will depend on individual characteristics and route of administration, however, HPLC assays or bioassays will determine plasma concentrations. Can be used to

[0221] Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen that maintains plasma levels above the MEC over 10-90% of the time, preferably 30-90%, and most preferably 50-90% of the time. For local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of compound administered will, of course, be dependent on the subject being treated, on the subject's weight,
It will depend on the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

6.7.4. Packaging The composition may, if desired, be contained 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 be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

6.8. Antibodies Another aspect of the invention is antibodies that specifically bind to a polypeptide of the invention.
Such antibodies can be either monoclonal or polyclonal, and of course, such as those produced in transgenic animals.
These fragments and humanized or all human forms are also possible.
The present invention further provides a hybridoma producing an antibody according to the present invention. The antibodies of the present invention are useful in detecting and / or purifying the polypeptide of the present invention.

[0225] The proteins of the present invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies that specifically react with the proteins. Such antibodies may be obtained using either the whole protein or fragments thereof as an immunogen. The peptide immunogen may further contain a cysteine residue at the carboxy terminus and is conjugated to a hapten such as keyhole limpet hemocyanin (KLH). For example, RP Merrifield, J. Amer. Chem. Soc. 85, 2149
Methods for synthesizing such peptides are known in the art, as described in -2154 (1963); JL Krstenansky, et al., FEBS Lett. 211, 10 (1987). A monoclonal antibody that binds to a protein of the invention will be a useful diagnostic agent for immunodetection of said protein. Neutralizing monoclonal antibodies that bind to the protein may also be useful therapeutic agents, both for conditions associated with the protein and for the treatment of some forms of cancer that include aberrant expression of the protein. In the case of cancerous or leukemia cells, neutralizing monoclonal antibodies against the protein may increase the metastatic spread of cancer cells mediated by the protein.
It may be useful for detection and prevention. In general, techniques for preparing polyclonal and monoclonal antibodies, as well as hybridomas that are capable of producing the desired antibody, are well known in the art (Campbell.AM, Monoclon al Antibodies Technology; Laboratory Techniques in Biochemistry and Mole culer Biology, Elsevier Science Publishers, Amsterdam, Netherlands (19
. 84); St Groth et al, J. Immunol 35:. 1-21 ( 1990); Kohler and Milstein, Natur e 256: 495-497 ( 1975)), the trioma technique, the human B cell hybridoma technique (Kozbo
r et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).

Any animal known to produce antibodies (mouse, rabbit, etc.)
It can be immunized with a peptide or polypeptide of the present invention. Methods of immunization are well-known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One of skill in the art will recognize that the amount of protein encoded by the ORF of the present invention used for immunization will vary depending on the animal immunized, the antigenicity of the peptide and the site of injection. The protein used as an immunogen may be administered or modified in an adjuvant to increase the antigenicity of the protein. Methods for increasing the antigenicity of a protein are well known in the art, and include antigens and heterogas proteins (globulins and β
-Galactosidase) or by the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen cells from immunized animals were removed and SP
It is fused with a myeloma cell such as a 2 / 0-Ag14 myeloma cell to obtain a monoclonal antibody-producing hybridoma cell. Hybridoma cells producing antibodies with the desired characteristics can be identified by any one of a number of methods well known in the art. These include screening for hybridomas using ELISA assays, Western blot analysis, or radioimmunoassay (Lutz et al . , Exp. Cell Research. 175: 109-124 (1988)). Hybridomas secreting the desired antibody are cloned and subclass determined using procedures well known in the art (Campbell. AM, Monoclonal Antibodies Technology; Laboratory Techniques in Biochemistry and Moleculer Biology, Elsevier Science Publishers). , Amsterdam, the Netherlands (1984)). Techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to the proteins of the invention.

For polyclonal antibodies, antibodies, including serum, are isolated from the immunized animal and one of the above procedures is used to screen for the presence of antibodies with the desired specificity. The invention further provides the antibody in a detectably labeled form. Antibodies include radioisotopes, affinity labels (such as biotin and avidin), and enzyme labels (such as horseradish peroxidase and alkaline phosphatase)
Fluorescent labels (such as FITC and rhodamine), paramagnetic atoms and the like can be used to detectably label. Such labeling procedures are well known in the art and are described, for example, in (Sternberger, LA et al., J. Histochem. Cytoch em. 18: 315 (1970); Bayer, EA et al ., Meth.Enzym. 62: 308 (1979); Engval, E. et al.
, Immunol. 109: 129 (1972); Goding, JW J. Immunol. Meth. 13: 215 (1976)).

The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues, for expressing fragments of a polypeptide of interest. The antibodies may also be used for direct therapy or other diagnostics. The present invention further provides the antibody immobilized on a solid support. Specific examples of the solid support include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins, and polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, DM et al.,
" Handbook of Experimental Immunology " 4th edition, Blackwell Scientific Publi
cations, Oxford, UK, Chapter 10 (1986);.. Jacoby, WD et al., Meth Enzy m 34 Academic Press, New York (1974)). The immobilized antibody of the present invention can be used for in vitro, in vivo, and in situ assays and for immunoaffinity purification of the protein of the present invention.

6.9. Computer-Readable Sequences In one application of this embodiment, the nucleotide sequences of the present invention can be recorded on a computer-readable medium. As used herein, "computer-readable medium" refers to any medium that can be read and accessed directly by a computer. Such media include magnetic storage media such as floppy disks, hard disk storage media and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and these storage media such as porcelain / optical storage media. Categories include, but are not limited to, hybrids. One of skill in the art will appreciate how to use some of the currently known computer readable media to create products comprising the computer readable media having the nucleotide sequences of the present invention recorded thereon. It can be easily recognized whether or not to use. As used herein, "recorded" refers to the step of storing on a computer-readable medium. One of skill in the art will readily employ any of the currently known methods for storing information on a computer-readable medium to produce a product comprising the nucleotide sequence information of the present invention. Can be.

A variety of data storage structures are available to one of skill in the art to create computer readable media having the nucleotide sequences of the present invention recorded thereon. The choice of data storage structure will generally be based on the means chosen to access the stored information. In addition, various data processor programs and formats can be used to store the nucleotide sequence information of the present invention on a computer-readable medium. The sequence information is WordPe
It can be shown as a word processing text file formatted with commercially available software such as rfect and Microsoft Word, or as an ASCII file stored in a database application such as DB2, Sybase, or Oracle. Those skilled in the art can readily adapt to any number of data processor structural formats (eg, text files or databases) to obtain a computer readable medium having recorded thereon the nucleotide sequence information of the present invention. SEQ ID NO: 1, 2, 4, 6 or 7 nucleotide sequence or a representative fragment thereof, or SEQ ID NO: 1, 2, in computer readable form
By providing a nucleotide sequence that is at least 99.9% or more identical to 4, 5, 6 or 7 or 8, one of ordinary skill in the art can routinely access the sequence information for a variety of purposes. Computer software is publicly available and one skilled in the art can access sequence information provided on computer readable media. According to the examples described below, BLAST (Altschul et al., J. Mol. Biol. 215: 403-41) on the Sybase system to identify open reading frames (ORFs) in nucleic acid sequences .
0 (1990)) and BLAZE (Brutlag et al . , Comp. Chem. 17: 203-207 (1993)). Such ORFs may be fragments-encoding proteins and may be useful in fermentation reactions and in the production of commercially important proteins such as enzymes used in the production of commercially useful metabolites.

As used herein, “computer-based system” refers to hardware means, software means, and data storage means used to analyze nucleotide sequence information of the present invention. The minimum hardware means of the computer-based system of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Those skilled in the art will readily recognize that any one of the currently available computer-based systems is suitable for use in the present invention. As described above, the computer-based system of the present invention comprises a data storage means for storing the nucleotide sequence of the present invention, and hardware means and software means necessary to support and execute the search means. Including.
As used herein, the term "data storage means" refers to a memory capable of storing the nucleotide sequence information of the present invention or a memory access means capable of accessing a product storing the nucleotide sequence information of the present invention.

[0233] As used herein, "search means" refers to one or more of the computer-based systems executed to compare the target sequence or target structural motif with the sequence information stored in the data storage means. Refers to a program. Search means are used to identify fragments or regions of a known sequence that match a particular target sequence or target motif. Various known algorithms are publicly disclosed, and there are various commercially available software for processing search means, which can be used in the computer-based system of the present invention. As a specific example of such software, MacP
attern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). One of ordinary skill in the art will readily recognize that any one of the available algorithms or software packages for performing the homology search can be adapted for use in this computer-based system. As used herein, the “target sequence” can be a nucleic acid or amino acid sequence of 6 or more nucleotides or 2 or more amino acids. One skilled in the art will readily recognize that the longer the target sequence, the less likely the target sequence will be present as a random occurrence in the database. The most preferred sequence length of the target sequence is about 10-100 amino acids or about 30-300 nucleotide residues.
However, it will be appreciated that searches for commercially important fragments, such as sequence fragments related to gene expression and protein processing, may be of shorter length.

As used herein, a “target structural motif or“ target motif ”refers to a sequence (s) selected based on the three-dimensional configuration formed by folding of the target motif. Refers to a sequence or combination of sequences that are selectively selected. There are various target motifs known in the art.
Protein target motifs include, but are not limited to, an enzyme active site and a signal sequence. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures, and inducible expression elements (protein binding sequences).

6.10. Triple Helix In addition, the fragments of the invention can be used to control gene expression by triple helix or antisense DNA or RNA, as described broadly, either method. It is based on the binding of the polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are usually 20-40 bases in length and are designed to be complementary to regions of the gene involved in transcription (triple helix-Lee et al . , Nucl. Acids Res. 6: 3073 (1979); Cooney et al., Science 15241: 456 (1
988); and Dervan et al., Science 251: 1360 (1991)) or mRNA itself (Antisense-Olmno, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression , CRC Press, Boca Raton, Florida (19
88)). While the translation of mRNA molecules into polypeptides is blocked by antisense RNA hybridization, the triple helix structure advantageously results in -blocking of RNA transcription from DNA. Both techniques have proven effective in model systems. The information contained in the sequences of the present invention is necessary for designing antisense or triple helix oligonucleotides.

6.11. Diagnostic Assays and Kits The present invention provides a method for identifying the presence or expression of an ORF of the invention or one of its homologs in a test sample using a nucleic acid probe or antibody of the invention. Provide further.

Generally, the method for detecting a polynucleotide of the present invention comprises the steps of: The method includes a step of detecting the complex, and if the complex is detected, the polynucleotide of the present invention in the sample can be detected.

Such methods also include contacting the sample with a nucleic acid primer that anneals to the polynucleotide of the invention under stringent hybridization conditions, and amplifying the annealed polynucleotide. If the polynucleotide is amplified, the polynucleotide of the present invention can be detected in the sample.

In general, the method for detecting a polypeptide of the present invention comprises the steps of contacting a sample with a compound that binds to the polypeptide to form a complex for a period of time sufficient to form a complex; The method includes the step of detecting the complex, and if the complex is detected, the polypeptide of the present invention in the sample can be detected. In particular, such methods comprise incubating a test sample with one or more antibodies or one or more nucleic acid probes of the invention, and assaying the binding of a component in the test sample to said nucleic acid probe or antibody. The step of performing

[0240] Depending on the test sample, conditions for incubating the nucleic acid probe or antibody may vary. Incubation conditions will depend on the format used in the assay, the detection method used, and the type and nature of the nucleic acid probe or antibody used in the assay. One of skill in the art will recognize that commonly available hybridization, amplification or immunological assay formats can be readily adapted for use with the nucleic acid probes or antibodies of the present invention. Specific examples of these assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands.
(1986); Bullock, GR et al., Techniques in Immunocytochemistry, Academic P.
ress, Orlando, Florida, Volume 1 (1982), Volume 2 (1983), Volume 3 (1985); Tijssen, P., P ractice and Theory of immunoassays: Laboratory Techniques in Biochemistr y and Moleculer Biology, Elsevier Science Publishers, Amsterdam, It can be found in the Netherlands (1985). 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 method will depend on the assay format, the nature of the detection method, and the tissue, cells or extracts used as the sample being assayed. Methods for preparing protein or membrane extracts of cells are well known in the art and can be readily adapted to obtain samples that are compatible with the system utilized.

In another embodiment of the present invention, there is provided a kit comprising the necessary reagents for performing the assays of the present invention. In particular, the present invention provides, under strict monitoring, (a) a first container containing one of the probes or antibodies of the present invention; and (b) one or more other containers containing one or more of the following: That is, a compartment kit for containing one or more containers comprising a washing reagent, a reagent capable of detecting the presence of a bound probe or antibody, is provided.

In particular, compartment kits include any kit that includes the reagents in separate containers. Such containers include small glass containers, plastic containers,
Or including a strip of plastic or paper. Such containers do not cause cross-contamination of sample and reagents, and the efficiency of one compartment to another compartment such that the drug or solution in each container can be added from one compartment to another in a quantitative manner. Transport is possible. Such containers would receive the test sample, containers containing the antibodies used in the assay, containers containing the washing reagents (phosphate buffer, Tris buffer, etc.), and detect bound antibodies or probes. Containers containing the reagents used for this purpose. Types of detection reagent include a labeled nucleic acid probe, a labeled second antibody, or, optionally, an enzyme- or antibody-binding reagent capable of reacting with the labeled antibody if the first antibody is labeled. Probes and antibodies disclosed in the present invention,
One skilled in the art will readily recognize that it can be easily incorporated into one of the established kit formats well known in the art.

6.12. Screening Assays Using the isolated proteins and polynucleotides of the present invention, the present invention
A polypeptide encoded by an ORF obtained from a polynucleotide having the sequence of SEQ ID NO: 1, 2, 4, 6, or 7 or 8, which binds to a specific domain of the polypeptide encoded by such nucleic acid; 1, 2, 4,
Further provided is a method for obtaining and identifying a substance that binds to a nucleic acid having a sequence of 6 or 7. Specifically, the method comprises the following steps: (a) contacting the substance with an isolated protein encoded by the ORF of the present invention or the nucleic acid of the present invention; and (b) Determining whether to bind to the protein or the nucleic acid.

Generally, such methods for identifying compounds that bind to a polynucleotide of the invention include contacting the compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide / compound complex. If the polynucleotide / compound complex is detected, a compound that binds to the polynucleotide of the present invention can be identified.

Similarly, generally, therefore, such a method for identifying a compound that binds to a polypeptide of the present invention comprises the step of attaching the compound to a polypeptide of the present invention to form a polypeptide / compound complex. If the polypeptide / compound complex is detected, the compound that binds to the polynucleotide of the present invention can be identified.

A method for identifying a compound that binds to a polypeptide of the invention comprises contacting a compound of the invention with a polypeptide of the invention in a cell for a period of time sufficient to form a polypeptide / compound complex. The complex drives the expression of the reporter gene sequence in the cell, and then binds to the polynucleotide of the invention if the polypeptide / compound complex is detected by detecting expression of the reporter gene sequence. Detecting the complex so that the compound is identified can also be included.

Compounds identified by such methods include those that modulate the activity of the polypeptide of the invention (ie, increase or decrease the activity relative to the activity observed in the absence of the compound). Can be included. Alternatively, compounds identified by such methods include compounds that modulate the expression of a polynucleotide of the invention (ie, increase or decrease expression relative to the level of expression observed in the absence of the compound). can do. Compounds, such as those identified by the methods of the invention, can be tested for their ability to modulate activity / expression using standard assays well known in the art.

The substances screened in the above assays can include, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical substances. Agents can be randomly selected and screened, or can be rationally selected or designed using protein modeling techniques.

For random screening, substances such as peptides, carbohydrates, pharmaceutical substances are randomly selected and assayed for the ability to bind to the protein encoded by the ORF of the present invention. Alternatively, the materials can be rationally selected or designed. As used herein, a substance is said to be "rationally selected or designed" if it is selected based on the configuration of a particular protein. For example, those skilled in the art will appreciate that rationally designed peptides, such as Hurby et al., Appl.
ication of Synthetic Peptides: Antisense Peptides, " Synthetic Peptides, A User's Guide , WH Freeman, NY (1992), pp. 289-307, and Kaspczak et al.
See Biochemistry 28: 9230-8 (1989), or presently available techniques for making peptides, drug substances, etc., that can bind to specific peptide sequences to make drug substances, etc. Can be applied without difficulty.

In addition to the aforementioned substances, one class of substances of the present invention broadly defines the OR of the present invention.
Can be used to control gene expression through binding to one of F or EMF.
As noted above, such substances may be randomly screened, or have rational design /
You can make a choice. By targeting the ORF and EMF, one skilled in the art can modulate the expression of either a sequence-specific or element-specific substance, either a single ORF or multiple ORFs that are dependent on controlling the expression of the same EMF Can be designed. One class of DNA binding substances are those that contain a basic residue that forms a hybridized or triple helical structure by binding to DNA or RNA. Such materials can be based on traditional phosphodiesters, ribonucleic acid backbones, or various sulfhydryl or polymer derivatives with base attachment ability.

[0251] Suitable materials for use in these methods usually contains 20 to 40 bases, and complementary to a region of the gene involved in transcription (triple helix -Lee et, Nucl. Aci ds Res. 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,
Science 251: 136 (1991)) or complementary to the mRNA itself (antisense-Okano. J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Ant isense Inhibitors of Gene Expression , CRC Press, Boca Raton). , FL (1988))
It is designed to be. Triple helix formation optimally results in a blockade of RNA transcription from DNA, while antisense RNA hybridization inhibits transcription of mRNA molecules into polypeptides. Both techniques have been proven to be effective in model systems. The information contained in the sequences of the present invention is essential for designing antisense or triple helix oligonucleotides, and other DNA binding agents. A substance that binds to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic reagent in controlling bacterial infection by modulating the activity of the protein encoded by the ORF. Can be. A substance that binds to the protein encoded by one of the ORFs of the present invention can be formulated using known techniques for making pharmaceutical compositions.

6.13. Uses of Nucleic Acids as Probes Another feature of the present invention is to provide polypeptide-specific nucleic acid hybridization probes capable of hybridizing to a naturally occurring nucleic acid sequence. The hybridization probe of the present invention comprises SEQ ID NO: 1, 2
It may be derived from 4, 6, or 7 nucleic acid sequences. Since the corresponding gene is expressed only in a limited number of tissues, especially adult tissues, SEQ ID NO: 1,
Hybridization probes derived from 2, 4, 6 or 7 or 8 can be used as an indicator to indicate that RNA of the cell type of such tissue is present in the sample.

[0253] Suitable hybridization techniques, such as in situ hybridization, can be employed. PCR as described in US Pat. Nos. 4,683,195 and 4,965,188 provides an additional use for oligonucleotides based on nucleotide sequences. Probes such as those used in PCR may be of recombinant origin, chemically synthesized, or a combination thereof. Probes comprising separate nucleotide sequences for the detection of matching sequences,
Alternatively, it would include a degenerate pool with potential for the identification of closely related genomic sequences.

Another means for producing specific hybridization probes for nucleic acids involves cloning the nucleic acid sequence into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be prepared in vitro by adding RNA polymerase as a T7 or S6 RNA polymerase and appropriate radioactively labeled nucleotides. Can be used to synthesize probes. The nucleotide sequence can be used to construct a hybridization probe to map the corresponding genomic sequence. The nucleotide sequences provided in the present invention can be mapped to chromosomes or specific regions of chromosomes using well-known genetic and / or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis for known chromosome markers, hybridization screening using libraries or flow-selected chromosome preparations specific for known chromosomes. Techniques for fluorescence in situ hybridization of chromosome seeds are described, inter alia, in Verma et al., (1988) Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York NY.

Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques can correlate with other genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 1981 f). A correlation between the location of a nucleic acid on a physical chromosome map and a specific disease (or predisposition to a specific disease) can help define regions of DNA involved in the genetic disease. The nucleotide sequences of the present invention are used to detect gene sequence differences between normal, carrier or diseased individuals. The nucleotide sequence is recombined
Purified polypeptides can be produced using well known methods in DNA technology. After a gene has been isolated, there are many publications teaching methods for its expression, one of which is described in Goeddel (1990) Gene Expression Technology, Met.
There are hods and Enzymology, Vol 185, Academic Press, San Diego. The polypeptide can be expressed in a variety of host cells, either prokaryotic or eukaryotic. The host cell can be from the same species as the species from which the nucleotide sequence of the particular polypeptide was isolated, or from a different species. Advantages of producing polypeptides by recombinant DNA techniques include the ability to obtain appropriate amounts of protein for purification and the simplification of purification methods.

Each sequence so obtained was developed by Applied Biosystems and
The sequences were compared to GenBank sequences using a search algorithm built into the NHERIT ™ 670 sequence analysis system. In this algorithm, regions of homology were examined using a pattern-specific language (developed by TRW, Los Angeles, CA). The three parameters that determine how to perform a sequence comparison were window size, window correction, and allowable measurement error. Using a combination of these three parameters, DNA databases were searched for sequences containing regions homologous to the query sequence, and appropriate sequences were scored with initial values. Subsequently, these homologous regions were examined using a dot matrix plot to distinguish homologous regions from accidental matches. A Smith-Waterman alignment was used to show the results of the homology search. Peptide and protein sequence homology was confirmed using an INHERIT ™ 670 sequence analysis system in a manner similar to that used for DNA sequence homology. Using a pattern specific language and parameter window, protein databases were searched for sequences containing regions of homology scored by default. Dot-matrix homology plots were examined to distinguish areas of significant homology from chance matches.

Alternatively, local sequence alignment (Altschul SF (1993) J Mol Evol 36: 290-300; Altschul) was performed using BLAST, which represents a basic local alignment search tool.
, SF et al. (1990) J Mol Biol 215: 403-10). BLAST creates an alignment of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignment, BLAST is particularly useful in finding exact matches or in identifying homologs. Matches that do not include gaps are notional to perform a motif-style search because they are conceptual. The basic unit of output of the BLAST algorithm is a high scored segment pair (HSP). An HSP is an optional, but two sequence fragment of the same length, whose alignment is locally maximal and whose alignment score matches or matches the threshold or blockade score set by the user. Consists of more than The BLAST approach looks for HSPs between the query sequence and the database sequence, evaluates the statistical significance of all matches found, and matches those that meet the user-selected significance threshold. Only report. Parameter E establishes a statistically significant threshold for reporting database sequence matches. E is interpreted as an upper limit of the expected frequency of occurrence of an HSP (or a combination of a plurality of HSPs) included in the contents of the entire database search. All database sequences whose match satisfies E are reported in the program output.

In addition, BLAST analysis was used to search for related molecules contained in a library in the LIFESEQ ™ database. This process, an "electronic northern" analysis, is similar to a northern blot analysis in that it uses the sequence of one cell blevin at a time to search for matching or homologous molecules at the set stringency. . Electronic Northern stringency is based on a "product score." The product score is calculated by multiplying the [% of the nucleotide or nucleic acid in Blast [between the query and control sequences] by the maximum% of the BLAST score possible [based on the length of the query and control sequences]). Defined as divided by 100. At a product score of 40, the match is 1-2% accurate, and at 70, the match is accurate. Homologous or related molecules can be identified by selecting those that exhibit a product score between approximately 15-30.

[0259]

【Example】

The present invention is illustrated by the following examples. In view of the disclosure herein,
Those skilled in the art will be able to make various embodiments and modifications to the scope of the present invention. Accordingly, it is understood that broad aspects of the present invention are intended and should not be construed in a limiting sense by the following examples.

7.0. INDUSTRIAL APPLICABILITY 7.1. Example 1 Novel Interleukin-1 Receptor Antagonist Obtained from Fetal Liver-Spleen cDNA Library A number of novel nucleic acids were prepared from human fetal liver-spleen tissue. from b ² HFLS20W cDNA library, Bonaldo et al., Genome Res 6:. as described in 791-806 (1996), were obtained using standard PCR, SBH sequence Sig Nature analysis and Sanger sequencing techniques . Library inserts were amplified by PCR using primers specific for the vector sequence adjacent to the insert. These samples were spotted on a nylon membrane and queried for oligonucleotide probes to give a sequence signature. These clones were clustered into groups of similar or matching sequences, and a single representative clone was selected from each group for gel sequencing. The 5 'sequence of the amplified insert was then estimated using a reverse M13 sequencing primer in a typical Sanger sequencing protocol. The PCR product was purified and subjected to fluorescent termination cycle sequencing. Single flow gel sequencing was performed using a 377 Applied Biosystems (ABI) sequencer. Two of these inserts were previously obtained from this library and were identified as new sequences not previously reported in public databases. These sequences are shown in FIG. 2 as SEQ ID NOs: 1-2.
The polypeptide sequence corresponding to these nucleic acid sequences is shown in FIG. 3 as SEQ ID NO: 3. These amino acid sequences have significant homology to interleukin-1 receptor antagonists.

Example 2 Examination of Expression Using SEQ ID NO: 2 To investigate the role of SEQ ID NO: 2 in regulating inflammatory responses, gene expression was performed using a semi-quantitative polymerase chain reaction-based technique. Was analyzed. The cDNA library was analyzed for relevant tissues (3 leukocyte preparations [2 stimulated, 1 unstimulated], heart, lung, spleen, placenta, testis, fetal liver, adult liver, bone marrow, lymph nodes) , Macrophages, endothelial cells, fetal skin and umbilical cord). A gene-specific primer amplifies a portion of the sequence of SEQ ID NO: 2 from the sample (corresponding to bases 105-772 and 161-690 numbered from the 5 'end of SEQ ID NO: 2) Used for The amplification products are separated on an agarose gel,
Transferred to a nylon filter and chemically bound. Then, the filter, Klenow polymerase, using the random prime method, SEQ ID NO: generated from 2 full-length sequence was hybrida size with radiolabeled ⁽³³ P α-dCTP) double-stranded probe. The filters were washed (high stringency) and used to expose to phosphorescent image screening for several hours. Bands indicate the presence of a cDNA comprising the sequence of SEQ ID NO: 2 in a particular library and the mRNA expression in the corresponding cell type or tissue.

[0262] SEQ ID NO: 2 was expressed in very limited human tissues. Of the 16 human tissues tested, only fetal skin and umbilical cord provided a signal indicating that the expression of SEQ ID NO: 2 was tightly regulated. Expression of both of these IL-1Ras was only limited in a subset of tissues and cell types, and both performed intrinsic expression in the skin, but did not give rise to other tissues or stimuli. The expression was not observed in the non-abundant cell type. Thus, the expression pattern of SEQ ID NO: 2 is similar to that of the IL-1Ra gene, indicating that the novel cytokine encoded by SEQ ID NO: 2 has certain potential in regulating inflammatory response. It indicates that it has fulfilled such a role.

7.3. Example 3 Examination of Chromosome Localization Using SEQ ID NO: 2 Chromosome mapping techniques enable researchers to link genes to specific regions of chromosomes. Chromosome mapping was performed using the Stanford G3 radiation hybrid panel (Rsearch Genetics). This panel contains gene-specific primers (5'-primer: CCCCACTGGATGGTGCT
ACTG; (SEQ ID NO: 15), 3 ′ primer: GGGAAGAGATAGGAAAGGTAG (SEQ ID NO: 16)), and the PCR screening results were obtained from the Stanford Radiation Hybrid Mapping e-
Sent to mail server. Gene location in the radiation hybrid framework map is provided by linking the STS corresponding to SEQ ID NO: 2 with the best linkage to the SHGC marker.

As a result, SEQ ID NO: 2 was located on the long arm of chromosome 2. STS binds to marker SHGC-7020, which has a LOD (probability log) score of 12.25 and a cR-1000 of 5,
This indicates that the STS is located within 120 kb (kilobase) of this marker. In other words, SHGC-7020 is located within 120 kb of the IL-1 Ra gene. Thus, the STS corresponding to SEQ ID NO: 2 is located within about 240 kilobases of the IL-1Ra gene and is very close to the IL-1Ra gene.

Gene family members often bind to specific regions of the chromosome due to the phenomenon of intrachromosomal gene replication that results in a multi-member gene family during the evolutionary process. The interleukin-1 gene has been mapped to the second chromosome. Specifically, all of the interleukin-1 genes (IL-1a, IL-1b) and receptors (IL-1 RI and IL-1 RII), as well as the receptor antagonist IL-1 Ra, are secondarily stained. Recognized on the long arm of the body. By identifying the sequence SEQ ID NO: 2 in the same region,
Produces a physical association of SEQ ID NO: 2 with the interleukin-1 locus, demonstrating that the cytokine encoded by SEQ ID NO: 2 functions as a modulator of the inflammatory response.

7.4. Members within the intron / exon sequence gene family at the locus corresponding to Example 4 SEQ ID NO: 2 well maintain genomic organization in terms of their ability to derive from a common ancestral precursor are doing. IL-1a, IL-1b and IL
The intron / exon sequences of -1 Ra are highly conserved, demonstrating that they may be derived from a common ancestral gene. We have isolated an artificial bacterial chromosome (BAC) containing SEQ ID NO: 2. Using the gene-specific primers, sequencing was performed in the 5 'direction of the clone to outline the coding sequence (same as SEQ ID NO: 2) and intron sequence. The intron / exon structure is identical between IL-1 Ra and the BAC fragment comprising SEQ ID NO: 2, which indicates that these two sequences are members of the same family and that a common gene precursor It proves that it comes from a pair.

7.5. Example 5 Interleukin-1 Receptor Binding Domain and Interleukin-1 Receptor Assay Receptor binding regions of both IL-1β and IL-1Ra were analyzed by site-directed mutagenesis and protein Modification studies mapped a region of 18 amino acids in the carboxy-terminal half of the protein (ie, residues 88-105 of IL-1β). Amino acid alignment of both IL-1β and IL-1Ra with SEQ ID NO: 3 demonstrated that SEQ ID NO: 3 contained a receptor binding region. SEQ ID NO:
3 was 39% identical to IL-1 Ra and 22% identical to IL-1β in this region (39% was conserved). In comparison, IL-1 Ra, known to bind to the IL-1 receptor, was 28% identical and 50% conserved in this region. This region in SEQ ID NO: 3 plus IL-1β and IL-1 R
The striking similarity between a and both receptor binding regions indicates that SEQ ID NO: 3 also contains the IL-1 receptor binding region. An alignment of all three proteins in the receptor binding region is shown in FIG.

Since SEQ ID NO: 3 contains an IL-1 receptor binding region, SEQ ID NO: 3 and the truncated form of SEQ ID NO: 3 containing this receptor binding region are as follows: It is useful as a reagent for identifying cells and tissues that express the IL-1 receptor. Hann
The IL-1 receptor binding assay described in Um et al. Nature 343: 336-340 (1990) is used. In short, the highly radioactive recombinant SEQ ID NO: 3 was grown in M9 medium containing [35S] sulfur to express E. coli expressing SEQ ID NO: 3 and labeled by chromatography on a mono-S column. Prepared by purifying SEQ ID NO: 3. Labeled SEQ ID NO: 3 was incubated with cells or tissues under standard IL-1 binding assay conditions and under [35S] binding conditions. [
Greater binding of [35S] indicates the presence of the IL-1 receptor.

7.6. Example 6 Determination of Nucleotide Sequence Encoding a 155 Amino Acid Protein with Sequence Homology to Human Interleukin-1β and Human Interleukin-1 Receptor Antagonist SEQ ID NO: 4 The nucleotide sequence shown in FIG. 5 labeled with, encodes the translated amino acid sequence of SEQ ID NO: 5 shown in FIG. Extended nucleotide sequences were obtained by isolating PCR products from pools of clones from a fetal skin cDNA library. In short, fetal skin cDNA library,
Approximately 40,000 colony pools were placed on ampicillin-containing plates. These colonies were returned to LB medium and PCR was used to detect the pool containing SEQ ID NO: 2 (FIG. 2). Two pools were identified. PCR using vector and gene specific primers amplified the 5 'portion of the cDNA. Nested primers were used to obtain sequences from the two amplification products. To write out the subsequence and “contig” the full length sequence, the Laser gene
(Registered trademark) software was used.

[0270] SEQ ID NO: 4 encodes a protein of 155 amino acids lacking the common hydrophobic leader peptide, which indicates that this protein is the product of the human IL-1Ra gene product. This suggests that it is retained as a cytoplasmic molecule similar to the cytoplasmic isoform. FIG. 7 shows the amino acid alignment of SEQ ID NO: 5 with the cytoplasmic isoform of human IL-1 Ra (designated "HUMIL1RASIC"). This alignment shows a high degree of homology between the two, where 48% of the amino acids are identical and 54% are conservative amino acid substitutions. The three residues are (
It was shown to be essential for receptor activity by IL-1β (marked with an asterisk in FIG. 7). The corresponding residues in IL-1 Ra are different, conferring IL-1 Ra antagonist activity. These residues are R21, W23 and K152 for IL-1 Ra and T9, R11 and D145 for mature IL-1β. SEQ ID NO: 5 is (IL-
1 includes the combination of R10 (identical to the corresponding residue at Ra) and the residues of both K12 and D148 (identical and identical to the corresponding residue at IL-1β, respectively). Overall homology and agonist / antagonist site-specific sequence homology clearly define SEQ ID NO: 5 as a protein modulator in the inflammatory response.

7.7. Example 7 Three Prime Extensions with SEQ ID NO: 4 SEQ ID NO: 6 (FIG. 8) is an extension of the 3 ′ end of the nucleic acid sequence of SEQ ID NO: 4. SEQ ID NO: 6 provides fetal skin cD as described above for SEQ ID NO: 4.
Obtained from the NA library.

7.8. Example 8 Isolation and Mapping of Genomic Clones Corresponding to SEQ ID NOs: 1, 2 and 4 A human BAC genomic library (Research Genetics) was subjected to a PCR-based assay to identify the genomic A gene-specific primer hybridizing at position 4553 (273-D, 5'-CCCCACTGGATGGTGCTACTG-3 '(SEQ ID NO: 15)), and 273-E, 5'-GGGAAGAGATAGGAAAGGTAG hybridizing at positions 4849 to 4869 -3 '
(SEQ ID NO: 16). Briefly, gene-specific primers were used to amplify BAC DNA as a template using standard PCR conditions. Research continued on BACs producing fragments of DNA corresponding to the expected size.
BAC393-I6 was isolated and its DNA sequenced using a gene-specific primer from SEQ ID NO: 2. The sequence (length 16403 bases)
As shown in FIG. The IL-1 Ra coding sequence was found to be distributed over five exons. Splice donor and acceptor sites according to intron and exon size are shown below.

[0273]

[Table 2]

7.9. Example 9 Expression of SEQ ID NO: 3 in E. coli SEQ ID NO: 3 was expressed in E. coli by subcloning the entire coding region of SEQ ID NO: 2 into a prokaryotic expression vector. The expression vector used (pQE16
) Was obtained from the QIA expression prokaryotic protein expression system (Qiagen). Characteristic of this vector useful for protein expression is an efficient promoter (phage T
5), the expression control obtained by the lac operator system which can be induced by the addition of IPTG (isopropyl-β-D-thiogalactopyranoside), and the encoded His ₆ tag. The latter consist of six histidine amino acid residues that can bind very tightly to the nickel atom. This vector is
The protein can be used to express a recombinant protein having a His ₆ tag fused to its carboxy terminus, and allows rapid and efficient purification of the protein on an affinity column to which nickel is bound.

The coding sequence of SEQ ID NO: 2 (including the start codon but not the stop codon) was obtained by combining the primers for PCR reaction AP1 (5 ′ gaagatctatggtcctgagtggggccctg-3 ′) (SEQ ID NO: 25) and AP2 (5 ′ gaagatctgtcacactgctggaagtagaa). -3 ') (SEQ ID NO: 26). Both primers have at their 5 'ends BglI for cloning purposes.
Has an I restriction site. The PCR fragment obtained during amplification was restricted with BglII in order to obtain a misaligned end for inserting the insert into the vector. The pQE16 plasmid was digested with BglII and BamHI to remove the segment containing the dihydrofolate reductase coding sequence and to obtain an offset end that matched the end of the PCR fragment. This PCR fragment was ligated into the digested pQE16 vector to obtain P16BB-273. The ligations were transformed by electroporation into electroreactive E. coli cells (M15 [pREP4] strain obtained from Qiagen) and the transformed cells were plated on ampicillin-containing plates. Colonies were screened for appropriate inserts with appropriate orientation using PCR reactions using gene-specific and vector-specific primers. Positives were then sequenced to confirm the correct orientation and sequence. To express SEQ ID NO: 3, a colony containing the appropriate recombinant clone was inoculated into L-medium containing 100 μg / ml ampicillin, 25 μg / ml kanamycin, and the culture was incubated at 37 ° C. overnight. Grew up. The saturated culture was then diluted 20-fold in the same medium and grown until the optical density at 600 nm reached 0.5. At this point, IPTG was added to a final concentration of 1 mM to induce protein expression. The culture was grown for an additional 5 hours and the cells were then harvested by centrifugation at 3000 xg for 15 minutes.

The resulting pellet is dissolved or turbid using a non-irritating, non-ionic detergent containing 20 mM Tris-HCl (pH 7.5) (B-PER® reagent from Pierce). The cell suspension was sonicated until translucent. The resulting lysate was further purified under non-denaturing conditions using a nickel-containing column (nickel-NTA spin column from Qiagen). In essence, the lysate was transferred to a system containing 300 mM sodium chloride and 10 mM imidazole, and 700 × g on a rotating column to allow the histidine-tagged recombinant protein to allow binding to the nickel column. Centrifuged. Next, the column was washed with a washing buffer (50 mM NaH ₂ PO ₄ , pH 8.0: 300 mM).
Wash twice with sodium chloride; 20 mM imidazole, and elute buffer (50 mM
M NaH ₂ PO ₄ , pH 8.0: eluted with 300 mM sodium chloride; 250 mM imidazole).

The above procedures were all performed at 4 ° C. The purified protein was confirmed by SDS-PAGE. A strong single band was observed at a molecular weight of 16 kD, which corresponds to the expected size of SEQ ID NO: 3.

7.10. Example 10 Use of SEQ ID NOs: 3 and 5 7.10.1. Medical Imaging The novel interleukin-1 receptor antagonist polypeptides of the present invention include:
It is useful in medical imaging, for example, for imaging infections, inflammation and other sites having interleukin-1 receptor antagonist receptor molecules. For example, Ku
See nkel et al., U.S. Patent No. 5,413,778. Such methods include chemical attachment of a label, administration of a labeled interleukin-1 receptor antagonist polypeptide incorporated into a pharmaceutically acceptable carrier to a patient, and labeling at a target site. Involved in in vivo imaging of interleukin-1 receptor antagonist polypeptides.

7.10.2. Pancreatitis Acute edematous necrotizing pancreatitis was induced in adult male Swiss mice weighing 35 g or more using the analog of cholecystokinin, kaerulein. The mice were divided into four groups, three of which were given 50 mu g / kg frograin by intraperitoneal (IP) injection for 3 hours as described above.
Four doses are administered. (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., Pancrea
s 1993; 66; 41-45; Saluja et al., Amer Physiological Society 1985; G702-G71
0; Manso et al., Digestive Diseases and Sciences 1992; 37; 364-368).

[0280] Group 1 is a control group (n = 9) receiving IP injection of saline only. Group 2 is the untreated control disease group (n = 12). Group 3 is a group (n = 12) that received three injections (10 mg / kg / hr) one hour before pancreatitis was induced. Group 4 is a group (n = 12) that received three injections (10 mg / kg / hour) one hour after induction of pancreatitis.

After an appropriate time, all animals were euthanized, blood was collected, and the pancreas was surgically removed and weighed. Serum was assayed for amylase, lipase, IL-6 and TNF levels. Each pancreas was fixed and stained and blinded and histologically graded for interstitial edema, granulocyte infiltration, vesicle vacuolation and vesicle cells. This determines the serum level of the interleukin-1 receptor antagonist and allows comparison between dose, serum level, systemic cytokine response and degree of pancreatic damage.

Interleukin-6, interleukin-1, interleukin-1 receptor antagonist and TNF are measured by commercially available ELISA kits (Genzyme, Boston, Mass.). All samples were subjected to triplicate measurements. Amylase and lipase serum levels were determined using a Kodak Ectachem 700 automated analyzer (Eastman Kod
AK, Rochester, NY).

Following rapid excision followed by fixation in 10% formalin, tissue strips are prepared as is well known in the art. The tissue is embedded in paraffin and then stained with hematoxylin and eosin by standard methods, as is well known in the art. The strips are assayed and blind graded by an association certified pathologist.

7.10.3. Inhibition of Interleukin-1-Induced Cell Proliferation Mouse D10 T cells are obtained from the American Type Culture Collection (Rockville, Md.). Cells are maintained in Dulbecco's modified 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 were 0.25 as determined by the Limulus Amebocyte Migration Cell Assay.
It contained less than ng / ml endotoxin.

Mouse D10 cells, an interleukin-1-dependent T-cell line, are used to measure interleukin-1 mitogenic activity. In the presence of interleukin-1,
Cell proliferation with and without the interleukin-1 receptor antagonist polypeptide of the present invention is assayed by thymidine (up to 3H) incorporation, as described previously (Bakouche, O., et al., J. Immunol. 138). : 4249-4255, 1987). In a preferred embodiment, the antagonist and agonist of the interleukin-1 receptor antagonist polypeptide of the invention comprises a candidate compound and an interleukin-1 receptor.
And interleukin-1 receptor antagonist polypeptides of the invention are added, and the change in cell proliferation caused by the candidate compound is determined in this assay.

7.10.4. Inhibition of Interleukin-1-Induced Cell Cytotoxicity Inhibition of interleukin-1-induced cytotoxicity was demonstrated , for example, in A375 tumors at a density of 6000 cells / well placed in 96-well microtiter plates. Study using a suitable cell line, such as a cell. After standing overnight, interleukin-1 (3-300 ng / m
l) is added in the presence or absence of NAA or NMA. After incubating the cells for 3 days, thymidine (up to 3H) is added for an additional 2 hours (1 mu).
Ci / well). Cells were harvested on glass fiber discs (PHD cell harvest: Cambridge Technology, Watertown, Mass.). Air dry discs overnight and use Model 1900TR scintillation counter (Packard Inst
rument Division, Downer Grove, Illinois).

7.10.5. Induction of Nitrite Synthesis in Smooth Muscle Cells Aortic smooth muscle cells are cultured by explanting a segment of the middle layer of the aorta of adult male Fischer 344 rats. The aorta is aseptically removed and the outer and endothelial cells are cleaned by scraping both the inner and outer luminal surfaces. The middle layer fragments are kept moist by the growth medium until the cells appear,
Primaria 25-cm sup 2 can be attached in a tissue culture flask (Becton-Dickinson, Lincoln Park, NJ). Cultures contained twice weekly 10% fetal calf serum, 25 mM HEPES buffer (pH 7.4), 2 mM L-glutamine, 40 mu g
Medium 199 containing 1 / ml endothelial cell growth aid (Biomedical Technologies, Stoughton, Mass.) and 10 mug / ml gentamicin (GIBCO BRL, Grand Island, NY) is supplemented. When the first culture becomes confluent,
They are subjected to trypsinization and the explants are discarded. For these studies, cells from passages 12-14 are seeded at a density of 20,000 per well of a 96-well plate and used at confluence (60,000-80,000 cells / well). These cells
It exhibits a typical smooth muscle cell phenotype with an undulating morphology and strongly stains actin in smooth muscle.

Rat aortic smooth muscle cells were purified from 10% calf serum, 25 mM HEPES buffer, pH 7.4, 2 mM glutamine, 80 U / ml penicillin, 80 μg / ml streptomycin, 2 μg / ml.
ml fungi zone, including interleukin-1, IFN-γ and various inhibitors
Incubate in RPMI-1640 medium. At the desired time points, the nitrite concentration in the culture medium is determined using a 96-well microtiter plate reader (Grosss, SS, et al. Bioc.
hem. Biophys. Res. Commun. 178: 823-829, 1991) using a standard Grey's assay (Green, L., et al. Anal. Biochem. 126: 131-138, 1982). I do. Then, add 100 ml of Grey's reagent (0.5% sulfanilic acid, 0.05% naphthalenediamine and 2.5% phosphoric acid) to an equal volume of the culture medium, measure the OD sub550, and refer to the standard curve to determine the nitrite concentration and Let them correlate. The background OD sub550 of the medium incubated in the absence of cells is subtracted from the experimental values.

Rat aortic smooth muscle cells were purified from 10% calf serum, 25 mM HEPES buffer (pH 7.4), 2 mM glutamine, 80 U / ml penicillin, 80 μg / ml streptomycin, 2 μg / ml.
Incubate with RPMI-1640 medium containing ml fungi zone, 30 mu g / ml lipopolysaccharide (E. coli 0111: B4) and 50 U / ml IFN-γ. After 24 hours, collect the cells and prepare a cytosol (Gross, SS, et al. Biochem. Biophys. Res.Commun. 178: 82).
3-829, 1991). Cytosolic NO synthase activity is assayed by the Fesup2 + myoglobin method described previously (Gross, SS, et al. Biochem. Biophys. Res. Comm.
un. 178: 823-829, 1991).

7.10.6 Allo-Reactivity Determined by Increased Lymph Node Weight Systemic administration of an interleukin-1 receptor antagonist polypeptide of the present invention localized to alloantigens provided by allogeneic cells Experiments were performed to show that they suppress T cell-dependent immune responses. The induced allogeneic spleen cells are injected into the footpad of the mouse. Then, the induced syngeneic spleen cells are injected into the opposite footpad of the mouse. Allogeneic reaction (marked by lymphocyte proliferation and inflammation)
Are found in the footpads treated with allogeneic cells, and this response can be determined by increasing the size and weight of popliteal lymph nodes draining the site of antigen deposition relative to controls or by increasing cytoplasm .

Certain 8-12 week old BALB / c (H-2 sup d) and C57BL / 6 (H-2 s
up d) Mice (Jackson Laboratory, Bar Harbor, ME) were used in this experiment. Forty-eight BALB / c mice were divided into 16 groups, and 3 mice were assigned to each group (unless otherwise noted). Each group of mice received a different treatment.
On day 0, C57 in 50 μl RPMI-1640 (Gibco) was used as an antigen on the left footpad of all mice.
107 raised (2500R) syngeneic spleen cells from BL / 6 mice were injected intradermally, and 10 sup 7 from BALB / c mice were raised (2500R) in the opposite right footpad of the same mice.
) Inject syngeneic spleen cells.

Seven days after antigen administration, mice are sacrificed and popliteal lymph nodes (PLN) are removed from the left and right popliteal cavity by surgical resection. The lymph nodes are weighed and the difference (DELTA) between the weight of the lymph node draining the site of allogeneic cell injection (mg) and the weight of the lymph node draining the site of syngeneic cell injection (DELTA) is determined. Lymph nodes draining the site of injection of syngeneic cells weigh about 1 mg, regardless of whether they were obtained from mice treated with MSA or the interleukin-1 receptor antagonist polypeptide of the invention. And no significant difference from the weight of lymph nodes obtained from mice that did not receive cell injections.

7.10.7. Inhibition of Organ Graft Rejection In Vivo The neonatal C57BL / 6 (H-2 sup d) heart was isolated from Trager et al., Transplantation 47:
Using the method of Fulmer et al., Am. J. Anat. 113: 273, 1963, modified in 587, 1989 and Van Buren et al., Transplant. Proc. 15: 2967, 1983, adult Of BALB / c (H-2sup d) recipient mice. The survival of the transplanted heart is visually inspected for graft pulsation. Pulsation is determined 5 or 6 days after transplantation by applying soft reflected light to the ear-heart graft of anesthetized recipient mice and examining them with a dissecting microscope. The time of graft rejection is defined as the number of days after transplantation when contractile activity ends.

Transplanted mice are transplanted on day 0, and from day 0 to day 6 with the interleukin-1 receptor antagonist polypeptide of the invention by either the intraperitoneal route or the subcutaneous route. Inject with MSA (mouse serum albumin, 100 ng) or MSA alone. In a second heart transplant experiment, MSA alone is injected by the intraperitoneal route only, on days 0-2.

7.10.8. Inhibition of Inflammatory Arthritis Twenty rats are divided into four groups GJ, with five rats assigned to each group. On day 21 after immunization with mBSA, an inflammatory arthritis response is elicited. On the same day, the negative control group is injected with a volume of 0.2 ml of saline. The test group is injected with increasing amounts of the interleukin-1 receptor antagonist polypeptide of the invention. Interleukin-1 is injected into the positive control group. The diameter of the largest area of the treated joint is measured with a caliper on days 2, 4, 6, and 8, with the antigen injected intra-articularly on day 0.

7.11. Example 11 Isolation and Mapping of Genomic Clone Corresponding to SEQ ID NO: 6 FIGS. 10A-C show genomic sequences obtained by extending the genomic sequence (SEQ ID NO: 7) shown in FIGS. 9A-C. The clone (SEQ ID NO: 8) is shown. SEQ ID NO: 8 contains the (untranslated 3 major) extension sequence shown in SEQ ID NO: 6. The isolation of this genomic clone (SEQ ID NO: 8) was identical to that described in Example 8. Sequences 7605 nucleotides in length are shown in FIGS. 10A-C. The composition of exons in this clone is shown below.

[0297]

[Table 3]

7.12. Example 12 Expression and Purification of Recombinant Protein An expression vector having an interleukin-1 receptor antagonist coding sequence (SEQ ID NO: 3) lacking an N-terminal hydrophobic sequence was used to construct a pRSET vector (ampicillin-resistant). (Given) after the His ₆ tag (six histidine amino acid residues) and the expression tag (antibody tag). Hi fused to the amino terminus of the recombinant protein
s ₆ tag can makes it possible to rapidly and efficiently purified using an affinity column of nickel is bound.

This construct was transformed into E. coli (BL-21 with the pLYS plasmid conferring chloramphenicol resistance) and induced with IPTG (1 mM final).
The bacterial pellet was suspended in 50 mM Tris pH 7.5. Sonication was used to lyse the cells in the presence of 1 mg / ml lysozyme (10 ml buffer / g wet cells). Cell debris was removed by centrifugation. Imidazole was added to a final concentration of 10 mM using 100 mM 25 sodium chloride. The extract was applied to a nickel chelate column. The protein was eluted from this column using a linear gradient of imidazole from 50 mM to 300 mM. After collecting the peak, the solution was desalted with a Sephadex G-25 column into a 20 mM sodium phosphate buffer. Q for protein
Applied to a Sepharose column and eluted with a gradient from 0 to 350 mM.

The final recombinant protein was filtered and stored at -20 ° C.

7.13. Example 13 Binding of Interleukin-1 Receptor Antagonist to Interleukin-1 Receptor To demonstrate that interleukin-1 receptor antagonists of the present invention bind to interleukin-1 receptor Next, a cell binding assay was performed. in short,
Cell binding of the recombinant protein in the presence and absence of 100-fold or more untagged interleukin-1β (IL-1β) ligand (see Example 12) was demonstrated by interleukin-1 receptor. Using a fluorescent antibody specific to the tag expressed by the recombinant protein of the body antagonist, the analysis was performed by a fluorescence-activated cell sorter (FACS). In each reaction, 10 ⁶ cell NHDFs (normal human dermal fibroblasts) were
Resuspended in μl FACS buffer (diluted PBS and 3% calf serum and 0.01% azide). Cells were bound by adding 5 nM recombinant interleukin-1 receptor antagonist in 100 μl of cell suspension, and 500 nM recombinant IL-1β was also added as a competitor in one reaction. Cells were incubated for 1 hour on ice. The cells were pelleted, 200 μl of 0.2 mM BS3 (crosslinker) was added, and the cells were kept on ice for 30 minutes. Next, 10 μl of 1 M Tris pH 7.5
Was added and the cells were kept on ice for 15 minutes. The cells are pelleted, washed once with FACS buffer, resuspended in 100 μl of FACS buffer, and added with 2 μl of primary antibody (anti-expressed tag antibody 1 mg / ml) and then on ice Incubated for 30 minutes. The cells were pelleted, washed with FACS buffer, and resuspended in FACS buffer (100 μl volume). 2 μl of secondary antibody (phycoerythrin conjugated) of anti-mouse Ig (1 mg / ml) was added and cells were incubated for 3 hours.
Incubated for 0 minutes. The cells were pelleted again, washed twice with FACS buffer, resuspended in 0.5 ml FACS buffer, and analyzed by FACS. A shift in fluorescence was observed in cells treated with the recombinant tagged interleukin-1 receptor antagonist. This binding is to the untagged IL-1β
Specificity was observed because it was out of competition with the protein. These results suggest the binding of the interleukin-1 receptor antagonist protein of the present invention to the IL-1 receptor.

The present invention has been described in terms of the embodiments, which are merely intended to illustrate one aspect of the invention, and functionally equivalent compositions and methods are intended to cover the scope of the invention. And its disclosure should not be construed as limiting its scope. Indeed, many modifications and variations in the practice of the present invention will occur to those skilled in the art in view of the preferred embodiments of the invention. Accordingly, the scope of the invention should be limited only by the claims provided in the appended claims. All references cited herein are hereby incorporated by reference in their entirety.

[0303]

[Sequence list]

 SEQUENCELISTING <110> Hyseq, Inc. <120> A Novel Interleukin-1 Receptor Antagonist and Uses Thereof <130> 20411-733PCT (28110/35844) <140> PCT / US99 / 04291 <141> 1999-04-05 <150 > 09 / 055,010 US <151> 1998-04-03 <150> 09 / 079,909 US <151> 1998-05-15 <150> 09 / 082,364 US <151> 1998-05-20 <150> 09 / 099,818 US <151> 1998-06-19 <150> 09 / 127,698 US <151> 1998-07-31 <150> 09 / 229,591 US <151> 1999-01-13 <150> 09 / 251,370 US <151> 1999- 02-17 <160> 30 <170> PatentIn Ver. 2.0 <210> 1 <211> 338 <212> DNA <213> Homo sapiens <400> 1 ccgactctaa cactagagcc agtgaacatc atggagctct atcttggtgc caaggaatcc 60 aagagcttca ccttctaccg gcgggacgcgccctgcctgcc ggttcctgtg cacggtgcct gaagccgatc agcctgtcag actcacccag 180 cttcccgaga atggtggctg gaatgccccc atcacagact tctacttcca gcagtgtgac 240 tagggcaacg tgccccccag aactccctgg gcagagccag ctcggntgan gggngagngn 300 nnnnnnnnnn ngnnnnnnnn nnnnnnnnnn nnnnnnnn 338 <210> 2 <211> 985 <212> DNA <213> Homo sapiens <400> 2 ccgactctaa cactagag cc agtgaacatc atggagctct atcttggtgc caaggaatcc 60 aagagcttca ccttctaccg gcgggacatg gggctcacct ccagcttcga gtcggctgcc 120 tacccgggct ggttcctgtg cacggtgcct gaagccgatc agcctgtcag actcacccag 180 cttcccgaga atggtggctg gaatgccccc atcacagact tctacttcca gcagtgtgac 240 tagggcaacg tgccccccag aactccctgg gcagagccag ctcgggtgag gggtgagtgg 300 aggagaccca tggcggacaa tcactctctc tgctctcagg acccccacgt ctgacttagt 360 gggcacctga ccactttgtc ttctggttcc cagtttggat aaattctgag atttggagct 420 cagtccacgg tcctccccca ctggatggtg ctactgctgt ggaaccttgt aaaaaccatg 480 tggggtaaac tgggaataac atgaaaagat ttctgtgggg gtggggtggg ggagtggtgg 540 gaatcattcc tgcttaatgg taactgacaa gtgttaccct gagccccgca ggccaaccca 600 tccccagttg agccttatag ggtcagtagc tctccacatg aagtcctgtc actcaccact 660 gtgcaggaga gggaggtggt catagagtca gggatctatg gcccttggcc cagccccacc 720 cccttccctt taatcctgcc actgtcatat gctacctttc ctatctcttc cctcatcatc 780 ttgttgtggg catgaggagg tggtgatgtc agaagaaatg gctcgagctc agaagataaa 840 agataagtag ggtatgctga tcctctttta aaaaccc aag atacaatcaa aatcccagat 900 gctggtctct attcccatga aaaagtgctc atgacatatt gagaagacct acttacaaag 960 tggcatatat ttgcaattaa tttta 985 <210> 3 <211> 80 <212> PRT <213> Homo sapiens <400> 3 Pro Thr Leu Val Glue Lelu Glu Leu Gly 1 5 10 15 Ala Lys Glu Ser Lys Ser Phe Thr Phe Tyr Arg Arg Asp Met Gly Leu 20 25 30 Thr Ser Ser Phe Glu Ser Ala Ala Tyr Pro Gly Trp Phe Leu Cys Thr 35 40 45 Val Pro Glu Ala Asp Gln Pro Val Arg Leu Thr Gln Leu Pro Glu Asn 50 55 60 Gly Gly Trp Asn Ala Pro Ile Thr Asp Phe Tyr Phe Gln Gln Cys Asp 65 70 75 80 <210> 4 <211> 1282 <212> DNA <213> Homo sapiens <400> 4 ccacgcgtcc gcacagctcc cgccaggaga aaggaacatt ctgaggggag tctacaccct 60 gtggagctca agatggtcct gagtggggcg ctgtgcttcc gaatgaagga ctcggcattg 120 aaggtgcttt atctgcataa taaccagctt ctagctggag ggctgcatgc agggaaggtc 180 attaaaggtg aagagatcag cgtggtcccc aatcggtggc tggatgccag cctgtccccc 240 gtcatcctgg gtgtccaggg tggaagccag tgcctgtcat gtggggtggg gcaggagccg 300 actctaacac tagagccagt gaacatcatg gagctct atc ttggtgccaa ggaatccaag 360 agcttcacct tctaccggcg ggacatgggg ctcacctcca gcttcgagtc ggctgcctac 420 ccgggctggt tcctgtgcac ggtgcctgaa gccgatcagc ctgtcagact cacccagctt 480 cccgagaatg gtggctggaa tgcccccatc acagacttct acttccagca gtgtgactag 540 ggcaacgtgc cccccagaac tccctgggca gagccagctc gggtgagggg tgagtggagg 600 agacccatgg cggacaatca ctctctctgc tctcaggacc cccacgtctg acttagtggg 660 cacctgacca ctttgtcttc tggttcccag tttggataaa ttctgagatt tggagctcag 720 tccacggtcc tcccccactg gatggtgcta ctgctgtgga accttgtaaa aaccatgtgg 780 ggtaaactgg gaataacatg aaaagatttc tgtgggggtg gggtggggga gtggtgggaa 840 tcattcctgc ttaatggtaa ctgacaagtg ttaccctgag ccccgcaggc caacccatcc 900 ccagttgagc cttatagggt cagtagctct ccacatgaag tcctgtcact caccactgtg 960 caggagaggg aggtggtcat agagtcaggg atctatggcc cttggcccag ccccaccccc 1020 ttccctttaa tcctgccact gtcatatgct acctttccta tctcttccct catcatcttg 1080 ttgtgggcat gaggaggtgg tgatgtcaga agaaatggct cgagctcaga agataaaaga 1140 taagtagggt atgctgatcc tcttttaaaa acccaagata caatcaaaat cc cagatgct 1200 ggtctctatt cccatgaaaa agtgctcatg acatattgag aagacctact tacaaagtgg 1260 catatatttg caattaattt ta 1282 <210> 5 <211> 155 <212> PRT <213> Homo sapiens <400> 5 Met Val Leu Ser Gly Ala Leu Cys Phe Ars 1 5 10 15 Lys Val Leu Tyr Leu His Asn Asn Gln Leu Leu Ala Gly Gly Leu His 20 25 30 Ala Gly Lys Val Ile Lys Gly Glu Glu Ile Ser Val Val Pro Asn Arg 35 40 45 Trp Leu Asp Ala Ser Leu Ser Pro Val Ile Leu Gly Val Gln Gly Gly 50 55 60 Ser Gln Cys Leu Ser Cys Gly Val Gly Gln Glu Pro Thr Leu Thr Leu 65 70 75 80 Glu Pro Val Asn Ile Met Glu Leu Tyr Leu Gly Ala Lys Glu Ser Lys 85 90 95 Ser Phe Thr Phe Tyr Arg Arg Asp Met Gly Leu Thr Ser Ser Phe Glu 100 105 110 Ser Ala Ala Tyr Pro Gly Trp Phe Leu Cys Thr Val Pro Glu Ala Asp 115 120 125 Gln Pro Val Arg Leu Thr Gln Leu Pro Glu Asn Gly Gly Trp Asn Ala 130 135 140 Pro Ile Thr Asp Phe Tyr Phe Gln Gln Cys Asp 145 150 155 <210> 6 <211> 2648 <212> DNA <213> Homo sapiens <400> 6 cacagctccc gccaggagaa aggaacattc tgaggggagt ctac accctg tggagctcaa 60 gatggtcctg agtggggcgc tgtgcttccg aatgaaggac tcggcattga aggtgcttta 120 tctgcataat aaccagcttc tagctggagg gctgcatgca gggaaggtca ttaaaggtga 180 agagatcagc gtggtcccca atcggtggct ggatgccagc ctgtcccccg tcatcctggg 240 tgtccagggt ggaagccagt gcctgtcatg tggggtgggg caggagccga ctctaacact 300 agagccagtg aacatcatgg agctctatct tggtgccaag gaatccaaga gcttcacctt 360 ctaccggcgg gacatggggc tcacctccag cttcgagtcg gctgcctacc cgggctggtt 420 cctgtgcacg gtgcctgaag ccgatcagcc tgtcagactc acccagcttc ccgagaatgg 480 tggctggaat gcccccatca cagacttcta cttccagcag tgtgactagg gcaacgtgcc 540 ccccagaact ccctgggcag agccagctcg ggtgaggggt gagtggagga gacccatggc 600 ggacaatcac tctctctgct ctcaggaccc ccacgtctga cttagtgggc acctgaccac 660 tttgtcttct ggttcccagt ttggataaat tctgagattt ggagctcagt ccacggtcct 720 cccccactgg atggtgctac tgctgtggaa ccttgtaaaa accatgtggg gtaaactggg 780 aataacatga aaagatttct gtgggggtgg ggtgggggag tggtgggaat cattcctgct 840 taatggtaac tgacaagtgt taccctgagc cccgcaggcc aacccatccc cagttgagcc 900 ttatagggtc agtagctctc cacatgaagt cctgtcactc accactgtgc aggagaggga 960 ggtggtcata gagtcaggga tctatggccc ttggcccagc cccaccccct tccctttaat 1020 cctgccactg tcatatgcta cctttcctat ctcttccctc atcatcttgt tgtgggcatg 1080 aggaggtggt gatgtcagaa gaaatggctc gagctcagaa gataaaagat aagtagggta 1140 tgctgatcct cttttaaaaa cccaagatac aatcaaaatc ccagatgctg gtctctattc 1200 ccatgaaaaa gtgctcatga catattgaga agacctactt acaaagtggc atatatttgc 1260 aattaatttt aattaaaaga tacctattta tatatttctt tatagaaaaa agtctggaag 1320 agtttacttc aattgtagca atgtcagggt ggtggcagta taggtgattt ttcttttaat 1380 tctgttaatt tatctgtatt tcctaatttt tctacaatga agatgaattc cttgtataaa 1440 aataagaaaa gaaattaatc ttgaggtaag cagagcagac atcatctctg attgtcctca 1500 gcctccactt ccccagagta aattcaaatt gaatcgagct ctgctgctct ggttggttgt 1560 agtagtgatc aggaaacaga tctcagcaaa gccactgagg aggaggctgt gctgaagttg 1620 tgtggctgga atctctgggt aaggaactta aagaacaaaa atcatctggt aattctttcc 1680 tagaaggatc acagcccctg ggattccaag gcattggatc cagtctctaa gaaggctgct 1740 gtactg gttg aattgtgtcc ccctcaaatt cacatccttc ttggaatctc agtctgtgag 1800 tttatttgga gataaggtct ctgcagatgt agttagttaa gacaaggtca tgctggatga 1860 aggtagacct aaattcaata tgactggttt ccttgtatga aaaggagagg acacagagac 1920 agaggagacg cggggaagac tatgtaaaga tgaaggcaga gatcggagtt ttgcagccac 1980 aagctaagaa acaccaagga ttgtggcaac catcagaagc ttggaagagg caaagaagaa 2040 ttcttcccta gaggctttag agggataacg gctctgctga aaccttaatc tcagacttcc 2100 agcctcctga acgaagaaag aataaatttc ggctgtttta agccaccaag gataattggt 2160 tacagcagct ctaggaaact aatacagctg ctaaaatgat ccctgtctcc tcgtgtttac 2220 attctgtgtg tgtcccctcc cacaatgtac caaagttgtc tttgtgacca atagaatatg 2280 gcagaagtga tggcatgcca cttccaagat taggttataa aagacactgc agcttctact 2340 tgagccctct ctctctgcca cccaccgccc ccaatctatc ttggctcact cgctctgggg 2400 gaagctagct gccatgctat gagcaggcct ataaagagac ttacgtggta aaaaatgaag 2460 tctcctgccc acagccacat tagtgaacct agaagcagag actctgtgag ataatcgatg 2520 tttgttgttt taaagttgct cagttttggt ctaacttgtt atgcagcaat agataaataa 2580 tatgcagaga a agagaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2640 aaaaaaaa 2648 <210> 7 <211> 5751 <212> DNA <213> Homo sapiens <400> 7 cngttaatcc taccactatg agnatgtctt atcatttagt angaagattt ttgctttttg 60 cacatgaaaa ataggttgga aaaagtatag gttttgtgat ctgtgtatga aagctgtcta 120 tagtacatgt gtatgtgtgg gagaaaaagt gttgtcattg gttttctrat gantcactma 180 gaaaagcaag tattcacatt ttttcttgtg gctgtctgat tttcaggttt ttctacaatg 240 acatgtaggc tgancattcc ctargcagag agtcccacyt ctaacatctc ytgtaggcct 300 ggcaakgcag cagaaasnca gaggaaggaa ggaggragaa ggaaggagtg aagraaggag 360 taaaaaggta aggaagaaag ggaatagggr agraagggag raaatgggaa gggaaaraag 420 gaaaggaagg aaagagggag ggaagaaagr aagggaaaag ggagggagtg agtgaatgaa 480 agatggaaag aagraagaaa gggagggagg cagggaggaa agaaagttgc gcttcccttg 540 asctgccatg ggcactgmyt cttagggtct gaaagcccct gagatgcaaa agcctagtgc 600 tcacaaagag ctggaaagcc tcaaggaagt tcttcaatat ttctggaagg aaactgtctc 660 cagaagcttc cctccccacg acagataatg agcagcaagt gcttctggcg acttagggtg 720 atgtgaaatc acgctgggaa tcctgctcct cctcaggtcc tggcagtttc agggcccctc 780 cctaggcctt acttaaaagg ctgaggcatc cttggaggaa caggcagact ccacagctcc 840 cgccaggaga aaggaacatt ctgaggtatg ctctggggcg ctggtggtac cggagctctc 900 tcctgacccc agacccagaa tctgctccgt ggaggctgtt cacatgctgg ggagctcggt 960 gcagctgctt gctccccaga ccccagccaa ctcagcctct ctctccatga ttttctgttg 1020 tttattccaa aataggggag tctacaccct gtggagctca agatggtcct gagtggggcg 1080 ctgtgcttcc ggtgagtgta tgaggccctg gtttggtggt gtcctccgga ggaagtgagt 1140 tctggataga cccgttgtcc agctctgagc aggagggagg aagggagggg ctgccattgs 1200 rgctgkbaaa ttgtgaccag cacctcattg ctcttagagt tttcccagcc tttttcaaat 1260 aggggcagga ctggggcarg ccatctcaca aggggwccct gatgctgagg gggacaagtg 1320 aacctcccag wctaragctc cagccaagtc tatccaaggt gggaacgggg gccaggatcc 1380 ctgctcagag ctccgccatt gtcccccatc acagtgaatg gatgtaagct cacccactct 1440 gtgcccctac ctycctgcta ctctttgggg ataatwataa aacaaaaacc attaccatca 1500 gccagtytgt mcacccactg gcatgtacca agccagacac tctgccgtgt tctgggctta 1560 acaacagagg atgaaragtg ggcctttctc tcagkctaa t aaagvacttc ccacgatgkg 1620 ttctatggga ctcgattaga ggagkcccca gaggcatcca ggagatgctt tacacagkgg 1680 agctctctga tcaagtaaaw gcagggaatw ctgctttcta catcctctca taagagaacc 1740 acagcccagc tcagcatatg agwgactgag gktttctgaa gkaaggcaac ttgttgaatc 1800 gyattdagct atgcatcgac ccaattttta cactgcatcc ttttccccca tataactttt 1860 ggagaaaccc actttaggat acatcttcca cctcatagga tgccaggaaa tcaactgagt 1920 tcaaagatga gaaacaactt tgaaaagtta aataaaagaa atttaaattt aaagaamctc 1980 ctcacttagt aaggaatata tgaccaaata gaaatmcatg tatcttgaag aattgaagaa 2040 tcaggcttta acgtggaaga ggcctggatg twatccmccc catcatctta gtgtagcaat 2100 ggggaggctm aracccagag tgggcgagag agttgtctcc tgcgactcag cagcttggag 2160 gmatagatgg ggcaagatcc tagggctstg actcaccgsc agcttctctt ccaacagtag 2220 atgggttggg acagaaaagg ttaaataggg tsaagsakct wrccmcasay tccagtgkga 2280 gactgtgrgg tcatcctcct tgtagrgcat gakcccagca gggctgrgag acaargctgt 2340 gctgttactt ctggctacag tagkaagaaa gagagacaaa atgcytgagw ycmgggggyy 2400 cyctggatcc agggcakgct gragtgtcca ccctcctcct aatg tagtcc tcwccccttc 2460 ctgatgtttc agaatgaagg actcggcatt gaaggtgctt tatctgcata ataaccagct 2520 tctagctgga gggctgcatg cagggaaggt cattaaaggt yggtratgaa acatgaccca 2580 ctttcckkgg kctctataca ctctcagggg agggggcctg aagagggctt agaatagtca 2640 tacagattak cataggccta crgagcccag gcattagggc aghacaaacc aggctctaag 2700 caaaggcaaa taaaatacta cacctmtcag caaagtgaag acacacgctc tggggccacc 2760 tgaagcttyt gtgcagaagt gagaatgttt tccaakakgc ttgtcttgty attcccttac 2820 aggtagatwt aggtcaagca ttgcattccc tgggagccag taagtaccaa ggagagaact 2880 aacgtagatt ctctatacct tttttcccat atgggagwgg gtttctgcct ctccaccctg 2940 ggtcccctct gctctctgaa gatcctcagt cacttagagt ggagggaccc agagaacagg 3000 tggcattgwt ggacctcctg cttgctcact mtgcmccatg cactgcaaca ggtccctcts 3060 taaaatagtt ygcacctgcc cacctggggc acccttgctg agcwcagatg ccaggtagat 3120 ccktcagcta ggccatatgt gtatgygtgt gcttactggt gkatgdatgt gtgcatscag 3180 gcatatatgt gtrarcatat gtgtscatgc atgtatctgt atgtaaccat gtatgtgtra 3240 gtgcagktat gtaggtatga scatgtgtgt gaatatgtat atgtgtscat gcatgtatct 3300 gtgcatgtat gatctgatgt atgtgggtgg tgagggratg tacagagagg aatgagaccc 3360 tcttttgctc tcagcarcct cacagggtgt agaaagttgt ccaamcaatt ccaaaggggg 3420 gcttattaag acagggttca gaaaaaggcc tgagacccaa ggggcattaa aggagggggt 3480 tgagtctatt ttgggttgta gaggcttgaa gatttgmccc tgaaytagag ggtggagtgg 3540 aggtggtaca atgtgcttcc atgccttgat gtccactctg ggccagtgga caggagaanc 3600 catgtmatgc cagctgctra gaagcctccc ttctgcccag cctgggggca ggccgtctca 3660 cagcagtcyt gtgccataga rsgcaggaca rggaraaaag aaggaaaggc atccaggccy 3720 tgcatctggc ntttttccca caggtgaaga gatcaagcgt ggtccccaat cggtggctgg 3780 atgccagcct gtcccccgtc atcttgggtg tccagggtgg aagccagtgc ctgtcatgtg 3840 gggtggggca ggagccgact cttaacacta gaggtgagac ttggggcatc ctcactgggg 3900 actcagccac agatgctgag cctactgaag ccgggcagyc cacagccytg gtgctgtggg 3960 acaccctagc aggattctgt tgatggcagc tttgcctcct ccmtaaggat cctgcccagc 4020 cctccctctg cccctgcttc tgccctcacc tgacctcccc tcctctgccg gcagccagtg 4080 aacatcatgg agctctatct tggtgccaag gaatccaaga gcttcacctt ctacc ggcgg 4140 gacatggggc tcacctccag cttcgagtcg gctgcctacc cgggctggtt cctgtgcacg 4200 gtgcctgaag ccgatcagcc tgtcagactc acccagcttc ccgagaatgg tggctggaat 4260 gcccccatca cagacttcta cttccagcag tgtgactagg gcaacgtgcc ccccagaact 4320 ccctgggcag agccagctcg ggtgaggggt gagtggagga gacccatggc ggacaatcac 4380 tctctctgct ctcaggaccc ccacgtctga cttagtgggc acctgaccac tttgtcttct 4440 ggttcccagt ttggataaat tctgagattt ggagctcagt ccacggtcct cccccactgg 4500 atggtgctac tgctgtggaa ccttgtaaaa accatgtggg gtaaactggg aataacatga 4560 aaagatttct gtgggggtgg ggtgggggag tggtgggaat cattcctgct taatggtaac 4620 tgacaagtgt taccctgagc cccgcaggcc aacccatccc cagttgagcc ttatagggtc 4680 agtagctctc cacatgaagt cctgtcactc accactgtgc aggagaggga ggtggtcata 4740 gagtcaggga tctatggccc ttggcccagc cccaccccct tccctttaat cctgccactg 4800 tcatatgcta cctttcctat ctcttccctc atcatcttgt tgtgggcatg aggaggtggt 4860 gatgtcagaa gaaatggctc gagctcagaa gataaaagat aagtagggta tgctgatcct 4920 cttttaaaaa cccaagatac aatcaaaatc cccagatgct ggtctctatt cccatgaaaa 4980 agtgctcatg acatattgag aagacctact tacaaagtgg catatattgc aatttatttt 5040 aattaaaaga tacctattta tatatttctt tatagaaaaa agtctggaag agtttacttc 5100 aattgtagca atgtcagggt ggtggcagta taggtgatwt ttcttttaat tctgttaatt 5160 tatctgtatt tcctaatttt tctacaatga agatgaattc cttgtataaa aataagaaaa 5220 gaaattaatc ttgaggtaag cagagcagac atcatctctg atkgcctcag cctccacttc 5280 cccagagtaa attcaaattg aatcgagctc tgctgctctg gttggttgta gtagtgatca 5340 ggaawcagat ctcagcaaag ccactgagga ggaggctgtg atgagtttgt gtggctggaa 5400 tctctgggta aggaacttaa agaacaaaaa tcatctggta attctttcct agaaggatca 5460 cagcccctgg gattccaagg cattggatcc agtctctaag aaggctgctg tactggttga 5520 attgtgtccc cctcaaattc acatccttct tggaatctca gtctgtgagt ttatttggag 5580 ataaggtctc tgcagatgta gttagttaag acaaggtcat gctggatgaa ggtagaccta 5640 aattyaatat gactggttty cttgtatgaa aaggagagga cacagagaca gaggagacgc 5700 ggggaagact atgtaaagat gaaggcagag atcggagttt tgcagccaca a 5751 <210> 8 <211> 7605 <212> DNA <213> Homo sapiens <400> 8 aatattgaca gtatgcacag tca tagtttc attttactta ttatttattt atttattgag 60 gcagaagtct agctctcttc tgtcatccag ctgggagtac agtggctcaa tcttggctca 120 ccgcaatctc acctccaggt tcaagcaatt ctcacacctc agcctcctga gtagcttgga 180 ttacaaatgt gcactacaac ccggctaatt tttgtgtttt cagtaaagat ggggttttgc 240 catgttggcc aggcttgtct catttcatct tatttctact gtcattccat ttggtcaatt 300 tgtaatttaa taattttgta caaggcagtt tctacattaa ttttattttt ctgaaaagtg 360 tctatcatat tagtggcatt atgaaaatcg tagattattt tctgtgtttt gaacaacttg 420 atattttatt tattcttgaa cacttgtagg acctcttggc tcatatgtct gaataccatt 480 tctttaaaat gtacgcttta acatgtatac atatgtaaca aacctgcaca ttgtacacat 540 gtatcctaaa acttaaagta taataataat aataaaagaa aaaagaaaaa taaaacttaa 600 aaaaataaag tgtatgcttt aacaataaat tattaataaa tagttatctg tgttattcaa 660 gatataatta aattgttttt tgtgcactga cccttacctt tatttcagat ccatcacaga 720 gagtgcatgg aggtatttaa actgcattac tgtttcacaa ttaagtatta tctttatcaa 780 ttaggattca agaaagatca cttacagaat tatagatggc atgagctaga ttttactttc 840 taaagaaata accagataca tgagcaaaga tgttaataca aa gatgtttg tcacaacatg 900 gttttcaata gcaaaaaaag agagaaaaat atataaaaga caaataacag tggataggtt 960 tcaataaata atgttacagt gatacagtta aatactatac agctattgaa gcatgtcatt 1020 attcatattt agtatggaaa gatattttgc tattttgcta catgaaaaaa tgaggttgga 1080 aaaagtatag gttttgtgaa tctgttgtat gaaagctgtc ttatagttac atgtgtatgt 1140 gtgtggagga aaaagtgttg tcattggttt tctgatgatg cactcagaaa agacaagtat 1200 tcacattttt tcttgtggct gatctggatt ttcaggtttt tctacaatga acatgtaggc 1260 tgaacattcc ctaagcagga gagtcccacc tctaacatct cctgtaggcc tggcaatggc 1320 aggcaggaaa gacagaggaa ggaaggaggg agaagggaag gagtgaagga aggagtgaaa 1380 aaggtaagga agaaagggaa taggggagga agggaggaaa tgggaaggga aagaaggaaa 1440 kgaagggaaa gagggagggg aagaaaggaa ggggaaaagg gagggagtga gttgaatgaa 1500 agatggaaag aaggaagaaa gggagggagg cagggaggaa agaaagttgc gcttcccttg 1560 agctgcccat gggcacctga ctcttagggt ctgaaaggcc cctgagatgc aaaagcctag 1620 tgctcacaaa gagctggaaa gcctcaagga agttcttcaa tatttctgga aggaaactgt 1680 ctccagaagc ttccctcccc acgacagata atgagcagca agtgcttctg gcgacttagg 1740 gtgatgtgaa attcacgctg ggaatcctgc tcctcctcag gtcctggcaa gtttcagggc 1800 ccctccctag gccttactta aaaggctgag gcatccttgg aggaacaggc agactccaca 1860 gctcccgcca ggagaaagga acattctgag gtatgctctg gggcgctggt ggtaccggag 1920 ctctctcctg accccagacc cagaatctgc tccgtggagg ctgttcacat gctggggagc 1980 tcggtgcagc tgcttgctcc ccagacccca gccaactcag cctctctctc catgattttc 2040 tgttgtttat tccaaaatag gggagtctac accctgtgga gctcaagatg gtcctgagtg 2100 gggcgctgtg cttccggtga gtgtatgagg ccctggtttg gtggtgtcct ccggaggaag 2160 tgagttctgg atagacccgt tgtccagctc tgagcaggag ggaggaaggg agggggctgc 2220 cattgcagct gggaaattgt gaccagcacc tcattgctct tagagttttc ccagcctttt 2280 tcaaataggg gcaggactgg ggcaggccat ctcacaaggg gtccctgatg ctgaggggga 2340 caagtgaacc tcccagtcta gagctccagc caagtctatc caaggtggga acgggggcca 2400 ggatccctgc tcagagctcc gccattgtcc cccatcacag tgaatggatg taagctcacc 2460 cactctgtgc ccctacctcc ctgctactct ttgggggata ataataaaac aaaaaccatt 2520 accatcagcc aagtctgtcc acccactggc atgtaccaag ccagacactc tgccg tgttc 2580 tgggcttaac aaccagagga tgagagtggt cctttctctc agtctaataa agcacttccc 2640 acgatgtgtt ctatgggact cgattagagg agtcccacag aggcatccag gagatgcttt 2700 acacagtgga gctctctgat caagtaaatg cagggaattc tgctttctac atcctctcat 2760 aagagaacca cagcccagct cagcatatga gtgactgagg ktttctgaag taaggcaact 2820 tgttgaatcg yatttagcta tgcatcgacc caatttttac actgcatcct tttcccccat 2880 ataacttttg gagaaaccca ctttaggata catcttccac ctcataggat gccaggaaat 2940 caactgagtt caaagatgag aaacaacttt gaaaagttaa ataaaagaaa tttaaattta 3000 aagaaactcc tcacttagta aggaatatat gaccaaatag aaatacatgt atcttgaaga 3060 attgaagaat caggctttaa cgtggaagag gcctggatgt tatccaaccc atcatcttag 3120 tgtagcaatg gggaggctca gacccaagag tgggcgagag agttgtctcc tgcgactcag 3180 cagcattgga ggcatagatg gggcaagatc ctagggctct gactcaccga gcagcttctc 3240 ttccaacagg agatgggttg gggcagaaaa ggttgaatag ggtgaaggag caaaccacag 3300 actccagtgg gagactgtgg ggtcatcctc cttgtagggc atgagcccag cagggctggg 3360 agacaaggct gtgctgttac ttctggcaca gtaggaagaa agagagacaa aatgcctgag 3420 atcagggggt tctctggatc cagggcatgc tggagtgtcc accctcctcc taatgtagtc 3480 ctcacccctt cctgatgttt cagaatgaag gactcggcat tgaaggtgct ttatctgcat 3540 aataaccagc ttctagctgg agggctgcat gcagggaagg tcattaaagg ttggtgatga 3600 aacatgaccc actttccttg gtctctatac actctcaggg gagggggcct gaagagggct 3660 tagaatagtc atacagatta gcataggcct acagagccca ggcattaggg cagcacaaac 3720 caggctctaa gcaaaggcaa ataaaatact acacctctca gcaaagtgaa gacacacgct 3780 ctggggccac ctgaagcttc tgtgcagaag tgagaatgtt ttccaagagg cttgtcttgt 3840 cattccctta caggtagatw taggtcaagc attgcattcc ctgggagcca gtaagtacca 3900 aggagagaac taacgtagat tctctatacc ttttttccca tatgggagtg ggtttctgcc 3960 tctccaccct gggtcccctc tgctctctga agatcctcag tcacttagag tggagggacc 4020 cagagaacag gtggcattgt tggacctcct gcttgctcac tctgccccat gcactgcaac 4080 aggtccctct ctaaaatagt tygcacctgc ccacctgggg cacccttgct gagcacagat 4140 gccaggtaga tccttcagct aggccatatg tgtatgtgtg tgcttactgg tgtatgtatg 4200 tgtgcatgca ggcatatatg tgtgagcata tgtgtgcatg catgtatctg tatgtaacca 4260 t gtatgtgtg agtgcaggta tgtaggtatg agcatgtgtg tgaatatgta tatgtgtgca 4320 tgcatgtatc tgtgcatgta tgatctgatg tatgtgggtg gtgaggggat gtacagagag 4380 gaatgagacc ctcttttgct ctcagcaacc tcacagggtg tagaaagttg tccaaacaat 4440 tccaaagggg ggcttattaa gacagggttc agaaaaaggc ctgagaccca aggggcatta 4500 aaggaggggg ttgagtctat tttgggttgt agaggcttga agatttgacc ctgaactaga 4560 gggtggagtg gaggtggtac aatgtgcttc catgccttga tgtccactct gggccagtgg 4620 acaggagaag ccatgtcatg acagctgctg agaagcctcc cttctgccca gcctgggggc 4680 aggccgtctc acagcagtcc tgtgccctag agcccaggac aggggaagaa ggagggaaag 4740 gcatccaggg ccctgcatct ggcctctttc ccacaggtga agagatcagc gtggtcccca 4800 atcggtggct ggatgccagc ctgtcccccg tcatcctggg tgtccagggt ggaagccagt 4860 gcctgtcatg tggggtgggg caggagccga ctcttaacac tagaggtgag acttggggca 4920 tcctcactgg ggactcagcc acagatgctg agcctactga agccgggcag cccacagccc 4980 tggtgctgtg ggacacccta gcaggattct gttgatggca gctttgcctc ctccctaagg 5040 atcctgccca gccctccctc tgcccctgct tctgccctca cctgacctcc cctcctctgc 5100 cggcagc cag tgaacatcat ggagctctat cttggtgcca aggaatccaa gagcttcacc 5160 ttctaccggc gggacatggg gctcacctcc agcttcgagt cggctgccta cccgggctgg 5220 ttcctgtgca cggtgcctga agccgatcag cctgtcagac tcacccagct tcccgagaat 5280 ggtggctgga atgcccccat cacagacttc tacttccagc agtgtgacta gggcaacgtg 5340 ccccccagaa ctccctgggc agagccagct cgggtgaggg gtgagtggag gagacccatg 5400 gcggacaatc actctctctg ctctcaggac ccccacgtct gacttagtgg gcacctgacc 5460 actttgtctt ctggttccca gtttggataa attctgagat ttggagctca gtccacggtc 5520 ctcccccact ggatggtgct actgctgtgg aaccttgtaa aaaccatgtg gggtaaactg 5580 ggaataacat gaaaagattt ctgtgggggt ggggtggggg agtggtggga atcattcctg 5640 cttaatggta actgacaagt gttaccctga gccccgcagg ccaacccatc cccagttgag 5700 ccttataggg tcagtagctc tccacatgaa gtcctgtcac tcaccactgt gcaggagagg 5760 gaggtggtca tagagtcagg gatctatggc ccttggccca gccccacccc cttcccttta 5820 atcctgccac tgtcatatgc tacctttcct atctcttccc tcatcatctt gttgtgggca 5880 tgaggaggtg gtgatgtcag aagaaatggc tcgagctcag aagataaaag ataagtaggg 5940 tatgctgatc ct cttttaaa aacccaagat acaatcaaaa tcccagatgc tggtctctat 6000 tcccatgaaa aagtgctcat gacatattga gaagacctac ttacaaagtg gcatatattg 6060 caatttattt taattaaaag atacctattt atatatttct ttatagaaaa aagtctggaa 6120 gagtttactt caattgtagc aatgtcaggg tggtggcagt ataggtgatt tttcttttaa 6180 ttctgttaat ttatctgtat ttcctaattt ttctacaatg aagatgaatt ccttgtataa 6240 aaataagaaa agaaattaat cttgaggtaa gcagagcaga catcatctct gattgcctca 6300 gcctccactt ccccagagta aattcaaatt gaatcgagct ctgctgctct ggttggttgt 6360 agtagtgatc aggaatcaga tctcagcaaa gccactgagg aggaggctgt gatgagtttg 6420 tgtggctgga atctctgggt aaggaactta aagaacaaaa atcatctggt aattctttcc 6480 tagaaggatc acagcccctg ggattccaag gcattggatc cagtctctaa gaaggctgct 6540 gtactggttg aattgtgtcc ccctcaaatt cacatccttc ttggaatctc agtctgtgag 6600 tttatttgga gataaggtct ctgcagatgt agttagttaa gacaaggtca tgctggatga 6660 aggtagacct aaattcaata tgactggttt ccttgtatga aaaggagagg acacagagac 6720 agaggagacg cggggaagac tatgtaaaga tgaaggcaga gatcggagtt ttgcagccac 6780 aagctaagaa acaccaag ga ttgtggcaac catcagaagc ttggaagagg caaagaagaa 6840 ttcttcccta gaggctttag agggataacg gctctgctga caccttaatc tcagacttcc 6900 agcctcctga acgaagaaag aataaatttc ggctgtttta agccaccaag gataattggt 6960 tatggcagct ctaggaaact aatacagctg ctaaaatgat ccctgtctcc tcgtgtttac 7020 attctgtgtg tgtcccctcc cacaatgtac caaagttgtc tttgtgacca atagaatatg 7080 gcagaagtga tggcatgcca cttccaagat taggttataa aagacactgc agcttctact 7140 tgagccctct ctctctgcca cccaccgccc ccaatctatc ttggctcact cgctctgggg 7200 gaagctagct tccatgctat gagcaggcct ataaagagac ttatgtggta aaaaatgaag 7260 tctcctgccc acagccacat tagtgaacct agaagcagag actctgtgag ataatcaatg 7320 tttgttgttt taagttgctc agttttggtc taacttgtta tgcagcaata gataaataat 7380 atgcagagaa agagaaacaa atgcatttgt tttattattg caattttctc caatattttt 7440 tattttcttt ctcacaatga acaactatcc ttcatttacc caaatattct atttaaaagc 7500 taataataca gcatttgttg agtcatctgg ttctgcaaga ttgagatcct cttgtcctat 7560 gtgccaggaa tgaactccag tgccccaccc aaaccctggg gaatg 7605 <210> 9 <211> 178 <212> PRT <213> Mus mus culus <400> 9 Met Glu Ile Cys Trp Gly Pro Tyr Ser His Leu Ile Ser Leu Leu Leu 1 5 10 15 Ile Leu Leu Phe His Ser Glu Ala Ala Cys Arg Pro Ser Gly Lys Arg 20 25 30 Pro Cys Lys Met Gln Ala Phe Arg Ile Trp Asp Thr Asn Gln Lys Thr 35 40 45 Phe Tyr Leu Arg Asn Asn Gln Leu Ile Ala Gly Tyr Leu Gln Gly Pro 50 55 60 Asn Ile Lys Leu Glu Glu Lys Ile Asp Met Val Pro Ile Asp Leu His 65 70 75 80 Ser Val Phe Leu Gly Ile His Gly Gly Lys Leu Cys Leu Ser Cys Ala 85 90 95 Lys Ser Gly Asp Asp Ile Lys Leu Gln Leu Glu Glu Val Asn Ile Thr 100 105 110 Asp Leu Ser Lys Asn Lys Glu Glu Asp Lys Arg Phe Thr Phe Ile Arg 115 120 125 Ser Glu Lys Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly 130 135 140 Trp Phe Leu Cys Thr Thr Leu Glu Ala Asp Arg Pro Val Ser Leu Thr 145 150 155 160 Asn Thr Pro Glu Glu Pro Leu Ile Val Thr Lys Phe Tyr Phe Gln Glu 165 170 175 Asp Gln <210> 10 <211> 178 <212> PRT <213> rat <400> 10 Met Glu Ile Cys Arg Gly Pro Tyr Ser His Leu Ile Ser Leu Leu Leu 1 5 10 15 Ile Leu Leu Phe Arg Ser Glu Se r Ala Gly His Pro Ala Gly Lys Arg 20 25 30 Pro Cys Lys Met Gln Ala Phe Arg Ile Trp Asp Thr Asn Gln Lys Thr 35 40 45 Phe Tyr Leu Arg Asn Asn Gln Leu Ile Ala Gly Tyr Leu Gln Gly Pro 50 55 60 Asn Thr Lys Leu Glu Glu Lys Ile Asp Met Val Pro Ile Asp Phe Arg 65 70 75 80 Asn Val Phe Leu Gly Ile His Gly Gly Lys Leu Cys Leu Ser Cys Val 85 90 95 Lys Ser Gly Asp Asp Thr Lys Leu Gln Leu Glu Glu Val Asn Ile Thr 100 105 110 Asp Leu Asn Lys Asn Lys Glu Glu Asp Lys Arg Phe Thr Phe Ile Arg 115 120 125 Ser Glu Thr Gly Pro Thr Thr Ser Ser Phe Glu Ser Leu Ala Cys Pro Gly 130 135 140 Trp Phe Leu Cys Thr Thr Leu Glu Ala Asp His Pro Val Ser Leu Thr 145 150 155 160 Asn Thr Pro Lys Glu Pro Cys Thr Val Thr Lys Phe Tyr Phe Gln Glu 165 170 175 Asp Gln <210> 11 <211> 177 <212> PRT < 213> rabbit <400> 11 Met Arg Pro Ser Arg Ser Thr Arg Arg His Leu Ile Ser Leu Leu Leu 1 5 10 15 Phe Leu Phe His Ser Glu Thr Ala Cys Arg Pro Ser Gly Lys Arg Pro 20 25 30 Cys Arg Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe 35 40 45 Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn 50 55 60 Ala Lys Leu Glu Glu Arg Ile Asp Val Val Pro Leu Glu Pro Gln Leu 65 70 75 80 Leu Phe Leu Gly Ile Gln Arg Gly Lys Leu Cys Leu Ser Cys Val Lys 85 90 95 Ser Gly Asp Lys Met Lys Leu His Leu Glu Ala Val Asn Ile Thr Asp 100 105 110 Leu Gly Lys Asn Lys Glu Gln Asp Lys Arg Phe Thr Phe Ile Arg Ser 115 120 125 Asn Ser Gly Pro Thr Thr Thr Phe Glu Ser Ala Ser Cys Pro Gly Trp 130 135 140 Phe Leu Cys Thr Ala Leu Glu Ala Asp Gln Pro Val Ser Leu Thr Asn 145 150 155 160 Thr Pro Asp Asp Ser Ile Val Val Thr Lys Phe Tyr Phe Gln Glu Asp 165 170 175 Gln <210> 12 <211> 18 <212> PRT <213> Homo sapiens <400> 12 Asn Tyr Pro Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile 1 5 10 15 Glu Ile <210 > 13 <211> 18 <212> PRT <213> Homo sapiens <400> 13 Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser 1 5 10 15 Asp Ser <210> 14 <211> 159 < 212> PRT <213> Homo sapiens <400> 14 Met Ala Leu Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser Ser Lys 1 5 10 15 Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu 20 25 30 Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn 35 40 45 Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe 50 55 60 Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly 65 70 75 80 Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser 85 90 95 Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser 100 105 110 Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu 115 120 125 Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro 130 135 140 Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu 145 150 155 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 15 ccccactgga tggtgctact g 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 16 gggaagagat aggaaaggta g 21 <210> 17 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 17 tctgaggtat gc 12 <210> 18 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 18 aaatagggga gt 12 <210> 19 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 19 cttccggtga gt 12 <210> 20 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 20 tttcagaatg aa 12 <210> 21 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 21 ttaaaggttg gt 12 <210> 22 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400 > 22 ccacaggtga ag 12 <210> 23 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 23 ctagaggtga ga 12 <210> 24 <211> 12 < 212> DNA <213> Artificial Sequence <220> <223> Description o f Artificial Sequence: primer <400> 24 cggcagccag tg 12 <210> 25 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 25 gaagatctat ggtcctgagt ggggccctg 29 < 210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 26 gaagatctgt cacactgctg gaagtagaa 29 <210> 27 <211> 12 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 27 ctgtaggcct gg 12 <210> 28 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 28 aaaaaggtaa gg 12 <210> 29 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 29 cctcaggtcc tg 12 <210> 30 <211 > 177 <212> PRT <213> Homo sapiens <400> 30 Met Glu Ile Cys Arg Gly Leu Arg Ser His Leu Ile Thr Leu Leu Leu 1 5 10 15 Phe Leu Phe His Ser Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser 20 25 30 Ser Lys Met Gln Ala Phe Arg I le Trp Asp Val Asn Gln Lys Thr Phe 35 40 45 Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn 50 55 60 Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala 65 70 75 80 Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val Lys 85 90 95 Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp 100 105 110 Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser 115 120 125 Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp 130 135 140 Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn 145 150 155 160 Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp 165 170 175 Glu

[Brief description of the drawings]

FIG. 1: Sequence of SEQ ID NO: 3 and human (SEQ ID NO: 30), mouse (SEQ ID NO: 9), rat (SEQ ID NO: 10) and rabbit interleukin-1 receptor antagonist (SEQ ID NO: 11) Is a sequence alignment. A is alanine, R is arginine, N is asparagine, D is aspartic acid, C is cysteine, E is glutamic acid, Q is glutamine, G is glycine, H is histidine, I is isoleucine, L is leucine, K is lysine, M Is methionine, F is phenylalanine, P is proline, S is serine, T is threonine, W is tryptophan, Y is tyrosine, V is valine, and X is any of these 20 amino acids. Gaps are represented as dashes. Amino acid numbers were assigned in order to all four sequences.

FIG. 2 shows the nucleic acid sequence obtained from the b ² HFLS20W cDNA library using standard PCR, sequencing by hybridization signature analysis, and single-flow gel sequencing techniques. These sequences are SEQ ID NOs: 1 and 2. A is adenosine, C is cytosine, G is guanosine, T is thymidine, and N is any of these four bases.

FIG. 3 shows the amino acid sequence corresponding to nucleotides 1 to 240 of SEQ ID NO: 2. These sequences are shown as SEQ ID NO: 3. A is alanine, R is arginine, N is asparagine, D is aspartic acid, C is cysteine, E is glutamic acid, Q is glutamine, G is glycine, H is histidine, I is isoleucine,
L is leucine, K is lysine, M is methionine, F is phenylalanine, P is proline, S is serine, T is threonine, W is tryptophan, Y is tyrosine, V is valine, and X is any of these 20 amino acids. It is.

FIG. 4: Homologous region of SEQ ID NO: 3 (amino acids at positions 13 to 30 of SEQ ID NO: 3)
And sequence alignment of a human interleukin-1β receptor binding region (SEQ ID NO: 12) and a human interleukin-1 receptor antagonist (SEQ ID NO: 13). Residues conserved in all three domains are in bold.

FIG. 5 shows the nucleic acid sequence of SEQ ID NO: 4 (the nucleotides at positions 297 to 1282 of SEQ ID NO: 4 correspond to SEQ ID NO: 2), which is described in Example 6. I have.

FIG. 6 shows the amino acid sequence encoded by SEQ ID NO: 4 (amino acids at positions 76 to 155 of SEQ ID NO: 5 correspond to SEQ ID NO: 3). This amino acid sequence is set forth as SEQ ID NO: 5 and is described in Example 6.

FIG. 7 presents an amino acid alignment of SEQ ID NO: 5 (amino acids 1-155 of SEQ ID NO: 5). Conversion of the cytoplasmic form of human IL-1 Ra (SEQ ID NO: 14; specified as "HUMIL1RASIC"). The homology between these two sequences is described in Example 6.

FIG. 8 shows SEQ ID NO: 6, an extension (underlined sequence) of the nucleic acid sequence corresponding to SEQ ID NO: 4, which is described in Example 7.

FIG. 9 shows genomic sequences corresponding to SEQ ID NOs: 1, 2 and 46. The isolation of the genomic clone resulting in this sequence (SEQ ID NO: 7) is described in Example 8. A is adenosine, C is cytosine, G is guanosine, and T is thymidine. Unclear parts are described as follows. R is A or G, M is A or C, W is A or T, Y is C or T, S is C or G, K is G or T, V is A or C or G, H is A or C or T, D is A or G or T, B is C or G or T, and N is any of these four bases.

10A to 10C show the genomic sequence shown in FIGS. 9A to 9C (SEQ ID NO: 7).
) Shows a genomic clone (SEQ ID NO: 8), which is an extended part of FIG. SEQ ID NO: 8 contains the extension sequence of SEQ ID NO: 6 for the interleukin-1 receptor antagonist extension portion of SEQ ID NO: 6. The isolation of the genomic clone resulting in this sequence (SEQ ID NO: 7) is described in Example 11. A is adenosine, C is cytosine, G is guanosine, and T is thymidine. Unclear parts are described as follows. R is A or G, M is A or C, W is A or T, Y is C or T, S is C or G, K is G or T, V is A or C or G, H is A or C or T, D is A or G or T, B is C or G or T, and N is any of these four bases.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/15 C12N 1/21 4H045 1/19 C12Q 1/68 A 1/21 G01N 33/50 Z 5 / 10 33/566 C12Q 1/68 33/68 G01N 33/50 C12N 15/00 ZNAA 33/566 A61K 37/02 33/68 C12N 5/00 A (31) Priority claim number 09 / 082,364 (32) Priority date May 20, 1998 (May 20, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 09 / 099,818 (32) Priority date June 19, 1998 Date (June 19, 1998) (33) Priority claim country United States (US) (31) Priority claim number 09 / 127,698 (32) Priority date July 31, 1998 (July 31, 1998) (33) Priority claim country United States (US) (31) Priority claim number 09/22 , 591 (32) Priority Date January 13, 1999 (Jan. 13, 1999) (33) Priority Country United States (US) (31) Priority Number 09 / 251,370 (32) Priority Date Heisei Heisei February 17, 1999 (Feb. 17, 1999) (33) Priority Claimed States United States (US) (81) Designated States EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG ), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, H, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZWF terms (reference) 2G045 AA25 AA34 AA35 AA40 BB20 CB01 DA12 DA36 DA77 FB01 FB02 FB03 FB07 FB08 FB11 GC12 4B024 AA01 BA21 BA44 CA04 CA07 CA09 CA11 DA02 DA05 DA11 EA04 GA11 HA01 HA03 HA12 HA14 4B063 QA01 QA18 QQ42 QQ52 QR08 QR42 QR56 QR62 QS25 QS34 QS36 QX02 4B065 AA01X AA57X AA87X AA93Y AB01 BA02 CA24 CA44 4C084 AA02 AA06 AA07 BA44 CA53 DA59 ZA362 ZA592 ZA662 ZA812 ZA962 ZB072 ZB082 ZB112 ZB152 ZB272 ZB352 ZC352 4H045 AA10 BA10A20A

Claims (26)

[Claims] 1. An isolated polynucleotide, ie; (a) the nucleotide sequence of SEQ ID NO: 1; (b) the nucleotide sequence of SEQ ID NO: 2; (c) the nucleotide sequence of SEQ ID NO: 4; (E) nucleotide sequence of SEQ ID NO: 7; (f) nucleotide sequence encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of clone pIL-1Hy273; (g) clone pIL (H) nucleotide sequence containing the mature protein coding sequence of the cDNA insert of clone pIL-1Hy273; (i) nucleotide sequence of the cDNA insert of clone pIL-1Hy273; (h) j) nucleotide sequence encoding the full length protein sequence of SEQ ID NO: 3 or 5; (k) mature protein of SEQ ID NO: 3 or 5 An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide sequence; or (l) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5. 2. An isolated polynucleotide that hybridizes under stringent hybridization conditions to the complement of the polynucleotide of claim 1. 3. An isolated polynucleotide comprising the complement of the polynucleotide of claim 1 or 2. 4. A vector comprising the isolated polynucleotide according to claim 1 or 2. 5. An expression vector comprising the isolated polynucleotide according to claim 1 or 2. 6. A host cell genetically engineered to contain the polynucleotide of claim 1 or 2. 7. A host cell genetically engineered to contain the polynucleotide of claim 1 or 2, wherein the host cell has been engineered into an operable relationship with a regulatory sequence that controls expression of the polynucleotide in the host cell. Host cells. 8. The isolated polypeptide, ie; (a) the amino acid sequence of SEQ ID NO: 3 or 5; (b) the amino acid sequence encoded by the cDNA insert of clone pIL-1Hy273; (c) the clone pIL- An isolated polypeptide comprising an amino acid sequence comprising the full-length protein encoded by the cDNA insert of 1Hy273; or (d) a polypeptide comprising the mature protein encoded by the cDNA insert of clone pIL-1Hy273. 9. A composition comprising the polypeptide of claim 8 and a carrier. 10. An antibody against the polypeptide according to claim 8. 11. A method for detecting the polynucleotide according to claim 1 or 2 in a sample, comprising the steps of: (a) binding a sample to the polynucleotide to form a complex; Contacting the compound to be formed for a period of time sufficient to form a complex; and (b) detecting the complex, if the complex is detected, the polynucleotide of claim 1. How is detected. 12. A method for detecting the polynucleotide according to claim 1 or 2 in a sample, comprising the steps of: (a) subjecting the sample to stringent hybridization conditions under stringent hybridization conditions. Contacting a nucleic acid primer that anneals to the polynucleotide of claim 1 or 2 under conditions, and (b) amplifying the polynucleotide of claim 1 or 2, wherein the polynucleotide is amplified. A method for detecting the polynucleotide according to claim 1 or 2, for example. 13. The method according to claim 8, wherein the polynucleotide is an RNA molecule encoding the polypeptide of claim 8, and the method comprises the steps of:
13. The method according to claim 12, further comprising the step of reverse transcription into a polynucleotide. 14. A method for detecting a polypeptide according to claim 8 in a sample, comprising: (a) binding a sample to said polypeptide to form a complex; Contacting the compound with the compound for a period of time sufficient to form a complex, and (b) detecting the complex, wherein if the complex is detected, the polypeptide of claim 8 is detected. Way. 15. A method for identifying a compound that binds to the polypeptide according to claim 8, comprising the following steps: (a) adding the compound to the polypeptide according to claim 8; 9. The polypeptide of claim 8, comprising contacting for a period sufficient to form a peptide / compound complex; and (b) detecting the complex, wherein the polypeptide / compound complex is detected. A method wherein a compound that binds to a peptide is identified. 16. A method for identifying a compound that binds to the polypeptide of claim 8, comprising the steps of: (a) converting the compound to the polypeptide of claim 8 in a cell. , Polypeptide /
Contacting for a time sufficient to form a compound complex, wherein the complex drives expression of the reporter gene sequence in the cell, and (b) detecting the complex by detecting reporter gene sequence expression A method for identifying a compound that binds to a polypeptide according to claim 8, comprising detecting a polypeptide / compound complex. 17. A method for producing the polypeptide according to claim 8, wherein
The following steps: (a) culturing the host cell of claim 7 for a period sufficient to express the polypeptide contained therein; and (b) isolating the peptide from the cell of step 1 Separating. 18. The polypeptide according to claim 8, which can bind to an interleukin-1 receptor. 19. The polypeptide of claim 8, which is capable of binding to an interleukin-1 receptor antagonist. 20. A method of modulating the biological activity of an interleukin-1 receptor, comprising the step of contacting the receptor with the polypeptide of claim 8. 21. The polypeptide of claim 8, wherein said polypeptide is labeled with a radiolabel. 22. The polypeptide of claim 8, wherein said polypeptide is conjugated to a color label. 23. A method for labeling an interleukin-1 receptor, comprising the step of contacting with a labeled polypeptide according to claim 21 or 22. 24. The method of claim 23, wherein said method is performed in vitro. 25. The method according to claim 24, wherein said method is performed in vivo. 26. A kit comprising the polypeptide according to claim 8, 21 or 22.