JP2003523943A - IL-1L1 gene and polypeptide products - Google Patents

Abstract

(57) Abstract The present invention relates to the discovery of a novel gene, IL-1L1, which is encoded by the interleukin-1 locus. The gene is present between the gene encoding IL-1β and the gene encoding the IL-1 receptor antagonist, and is homologous to the IL-1 agonist protein and the IL-1 antagonist protein. Encodes a peptide. Therapies, diagnostics and screening assays utilizing these molecules are also disclosed.

Description

Detailed Description of the Invention

1. BACKGROUND OF THE INVENTION The interleukin-1 (IL-1) gene cluster encodes a number of related polypeptides that regulate the inflammatory response. The IL-1 gene cluster exists in the long arm of human chromosome 2 (2q13), and within the 430 kb region, IL-1 α (encoded by IL-1A) and IL-1β (IL- 1B) and IL-
At least three types of interleukin-1 signal transduction ligands including signal receptor antagonists (encoded by IL-1ra and IL-1RN)
gnick) (Nicklin et al., (1994) Genomi
cs 19: 382-4). Agonist molecules, IL-1α and IL-1β, have potent pro-inflammatory activity and trigger a diverse inflammatory cascade. For example, IL-1α and IL-1β cytokines stimulate the production of other cytokines such as IL-6 and IL-8, which results in the activation of leukocytes and their recruitment to damaged tissue, and vasoactive substances. Has triggered the local production of. Inappropriate exertion of IL-1 agonist activity is thought to play a central role in the pathophysiology of autoimmune diseases and inflammatory diseases including variant arthritis, inflammatory bowel disease and psoriasis. In contrast, IL-1 receptor antagonists (IL-1ra), by binding competitively to certain IL-1 receptors without inducing an inflammatory cascade,
Blocks L-1 agonist signal transduction. Thus, lack of IL-1 receptor antagonist activity may also lead to the progression of autoimmune and inflammatory disease conditions, and such IL-1 receptor antagonists are useful for the treatment of such diseases and conditions. It is thought that it is possible to provide various substances.

IL-1 agonists act through at least two types of receptors. The type I IL-1 receptor (IL-1RI) transmits a signal to the inside of the cell, but the type II receptor (IL-1RII) does not transmit a signal. Therefore, it is known as a "bait" receptor. The responsiveness of cells to IL-1 depends on the IL-1RI receptor and IL-1
It is affected by the ratio with the RII receptor, which means that it activates IL-1RI in an IL-1-dependent manner.
tein: IL-1RAcP) appears to compete for interaction with a third membrane protein (Lang et al. (1998) J. Immunol. 161: 6871-7). IL-1
Is expressed on T cells, fibroblasts and epithelial cells (Sims et al. (1988) Science 241: 585-9), whereas the type II receptor for IL-1 is B
Expressed on cells, neutrophils and macrophages (Bomsztyk)
(1989) Proc. Natl. Acad. Sci. USA 86: 8034-8). Both IL-1a and IL-1b ligands bind to the type I and type II IL-1 receptors, and although their biological activities as cytokines are similar, the apparent affinity of IL-1a Is IL
-1RI is high, while the apparent affinity of IL-1b is high for IL-1RII.

IL-1 receptor antagonists are structurally homologous to IL-1a and IL-1b and bind to the IL-1 type I receptor, but are biologically inactive. Acts as a competitive inhibitor of IL-1. In certain cell types, the mRNA of the IL-1 receptor antagonist is alternatively spliced and integrated into two different first exons. One of the alternatively spliced ones is secreted but the other remains in the cytoplasm, and the function of this intracellular retention type IL-1 receptor antagonist is unknown. In response to proinflammatory cytokines as acute proteins, hepatocytes produce secreted, but not cytoplasmic, retained IL-1ra (Gabay et al., (1997) J. Clin. In
vest. 99: 2930-40). With regard to the use of IL-1 receptor antagonists, their use in the treatment of numerous inflammatory diseases and conditions is considered (US patent).

The genetic diversity of the IL-1RN gene is believed to be involved in the pathogenesis of certain inflammatory diseases and conditions. The second intron of the human IL-1RN gene is 86b
variable number of tandem repeats (VNT)
R), which includes polymorphisms (Tarlow et al., (19
93) Hum. Genet. 91: 403-4). In humans, there are at least 5 alleles for this polymorphism, and a particular allele of this IL-1RN VNTR polymorphism (ie IL-1RN VNTR allele 2) is It is associated with more serious clinical symptoms in several diseases with sexual symptoms, such as periodontal disease (US Pat. No. 5,686,246), osteoporosis (US Pat. No. 5,698,399)
, Erythematous lupus (Blakemore et al., (1994) Arthritis Rheum. 37: 1380.
-5), Ulcerative colitis (Mansfield et al., (1994) Gastroentero
logy 106: 637-42) and alopecia areata (Tarlow et al., (1993) Hum. Ge
91.403-4), and diabetic kidney disease (WO 98/15653, Blakemore (Blake
more) et al. (see 1996) Hum. Genet. 97: 369-74). IL-1RN (V
Other polymorphic alleles of IL-1 that bind to NTR) also affect chronic obstructive airway disease (WO 9
It has been found to be associated with various inflammatory diseases or conditions such as 9/24615) and coronary artery disease (WO 98/40517). Blood levels of interleukin-1ra are elevated in patients with various infectious diseases, autoimmune diseases and trauma. Furthermore, characterization of the human IL-1 locus haplotype, which is associated with an increased risk of inflammatory diseases, has been elucidated (WO 98/54359).

2. SUMMARY OF THE INVENTION The present invention relates, in part, to humans encoding interleukin-1 receptor antagonist proteins (IL-1ra) and novel proteins having sequence homology with interleukin-1 (IL-1). Based on the discovery of a novel gene in the mouse. The newly identified proteins and nucleic acids described herein are "IL
-1L1 group ", and is exemplified herein using human and mouse homologues (homologs) of this gene. Human IL-1L1 gene (herein hI
The transcript (referred to as L-1L1) is shown in Figure 1 and consists of a consensus sequence of 2562 nucleotides (SEQ ID NO: 1) and a 5'sequence of 39 nucleotides (SEQ ID NO: 2) and 42 nucleotides. It contains two 5'sequences of the 5'sequence (SEQ ID NO: 3). The hIL-1L1 gene transcript consists of 5'and 3'untranslated regions and 465 nucleotides encoding a hIL-1L1 polypeptide composed of 155 amino acids shown in (A) of FIG. Open reading frame (SEQ ID NO: 4
)including. The hIL-1L1 gene is highly expressed in placenta and slightly expressed in thymus tissue (FIG. 7). cDNA encoding the full-length hIL-1L1 protein
Nucleic acid containing is available on the American Type Culture Collection (Massas, Virginia, 1801 University Street, Zip code 20110-2209; phone number +01 (703) 365- 2700) and the ATCC accession number is XXXXXX. The hIL-1L1 gene transcript has an especially long 3'untranslated region (UTR) of about 2 kb.

A mouse homologue of hIL-1L1 has also been isolated and is herein referred to as mIL-1L1.
Called. The mIL-1L1 gene transcript consisting of 1284 nucleotides is shown in FIG. 2 (SEQ ID NO: 4), 5 ′ and 3 ′ untranslated region, and 155 nucleotides shown in (B) (SEQ ID NO: 6) of FIG. Contains an open reading frame of 465 nucleotides encoding a mIL-1L1 polypeptide composed of the amino acids of mIL-1
The L1 polypeptide sequence showed 90% identity to that of hIL-1L1 (FIG. 4), suggesting that the encoded IL-1L1 product is highly conserved throughout evolution. ing. Nucleic acid containing a cDNA encoding the mouse IL-1L1 polypeptide was prepared on the American Type Culture Collection (
The American Type Culture Collection) (12301 Park Lawn Street, Rockville, MD) has been deposited, and the ATCC accession number is XXXXXX. The mIL-1L1 gene transcript has a 3'UTR of approximately 0.7 kb. 3'UTR of mIL-1L1 is hIL
-1L1 has only partial homology with 3'UTR. 5'-ACAATNAAAANCCC
The conserved consensus sequence consisting of 41 nucleotides corresponding to NGATNCTTGGTCTCTANTCNCATNAAAA-3 '(SEQ ID NO: 12) begins at nucleotide number 1137 in SEQ ID NO: 1 (hIL-1L1) and at SEQ ID NO: 4 (mIL-1L1). Is nucleotide number 1
It has been confirmed that it starts from 146.

BLAST program (Altschul et al., (1990) J. Mol. Biol. 215.
: 403), and some of the amino acid and nucleic acid sequences in the human and mouse IL-1L1 proteins and nucleic acids newly identified by analysis of amino acid and nucleotide sequences It was revealed to have some sequence similarity with the polypeptide encoded by. In particular, the IL-1L1 polypeptides of the present invention include IL1 domains, casein kinase II phosphorylation sites, myristylation sequences, transmembrane segments, and IL-1
Includes human and mouse IL-1L1 polypeptide sequences identified by one or more of the cognate segments of agonists and antagonists.

[0008] 4. DETAILED DESCRIPTION OF THE INVENTION 4.1 General The present invention is directed , in part, to the IL-1 gene encoding IL-1 receptor antagonists (IL-1ra) and proteins homologous to IL-1β polypeptides. Based on the discovery of genes present within cluster loci. Therefore, the IL-1L1 protein is a pro-inflammatory cytokine (IL-1β) as well as an inflammatory cytokine (IL-1ra).
Related to. IL-1L1 gene that name, this gene is IL-1 beta (IL-1 B eta) gene and IL-1 receptor antagonist that encode (IL-1 R eceptor antag
It comes from the fact that exists between (I nterval) the gene encoding the onist). Accordingly, the genes and proteins disclosed herein are labeled IL-1L1
Gene and IL-1L1 protein.

The IL-1L1 gene is a 680 kb CEPH yeast artificial chromosome (YAC containing the IL-1 cluster.
) Clone 766E12 (Nothwang et al., (1997)). YAC
Clone 766E12 was isolated from the yeast strain producing the clone by performing pulsed field electrophoresis and the resulting DNA was used as a driver in selecting the IL-1 encoded transduction code ( Morgan et al. (1992)
Nucleic. Acids Res. 20: 5173-79). The cDNA was obtained from human peripheral blood mononuclear cells cultured for 6 hours in the presence of 10 ng / ml phorbol myristate acetate and 100 ng / ml E. coli lipopolysaccharide.

The selected cDNA was amplified, and the amplified cDNA fragment was subjected to two cycles of affinity selection, further amplified, and then cloned into pNEB193. All 100 samples randomly selected from the obtained clones were sequenced. The IL-1L1 expressed sequence tag (EST) corresponded to one of the overlapping sequences derived from the three overlapping cDNA fragments that made up a continuous sequence of approximately 530 bp. This sequence was found to be homologous to a human placenta cDNA of 374 bp (GenBank accession number R70041) and a human placenta cDNA of 348 bp (GenBank accession number R70089). Was done. When tested, the sequence was found to specifically hybridize with P1 artificial chromosome 131J6 derived from the Gingrich human PAC library. Therefore, the ICRF human P1 library (Nothwang ) Et al. (1996) Genomics 4
1: 370-8) and was located between the IL1B and IL1RN genes flanking ICRF 700G1305. This latter clone contains the IL-1ra gene (IL1RN).
There was no open reading frame in the database EST sequences or in these novel EST sequences, and further analysis revealed that these sequences showed that the 3'untranslated region (UTR) of the gene transduction code. It became clear that it was included in.

5. Example 5.1 Cloning and analysis of human and mouse IL-1L1 Present within the interleukin-1 gene cluster (Nicklin et al., (1996)), which may be involved in the human inflammatory response. For the purpose of discovering a novel gene, the inventors have developed a yeast (Nothwang et al., Which produces a 680 kb CEPH yeast artificial chromosome (YAC) clone 766E12 containing an IL-1 cluster.
(1996)), and the chromosome was isolated by performing pulse field electrophoresis. The chromosome was used as a driver in the cDNA selection method according to the description of Morgan et al. (1992). The cDNA was obtained from human peripheral blood mononuclear cells cultured for 6 hours in the presence of 10 ng / ml phorbol myristate acetate and 100 ng / ml E. coli lipopolysaccharide. The cDNA was amplified as described, and the cDNA fragment was subjected to two cycles of affinity selection and further amplified.
It was cloned into pNEB193. For 100 randomly selected samples,
Everything was sequenced. The sequences were assembled in duplicate units. The IL-1L1 expressed sequence tag (EST) corresponds to one of the overlapping sequences derived from the three overlapping fragments (in our records, e031 and e049) that make up a continuous sequence of approximately 530 bp. It was This sequence is based on two previously reported EST clones R70041 and
It was found to overlap with R70089. When tested, the sequence was found to specifically hybridize with P1 artificial chromosome 131J6 derived from the Gingrich human PAC library. Therefore, the ICRF human P1 library (Nothwang ) Et al., (1996) Genomics 41: 370-8), ICRF 700G1
It was located between the IL1B and IL1RN genes flanking 305. This latter clone contains the IL-1ra gene (IL1RN). No open reading frame was present either in the database EST sequence or in our new EST sequence.

CDNA was isolated from the H9 placenta cDNA plasmid library derived from HGMP using EST DNA as a probe. This clone was only 1.6 kb in length and contained no coding sequences, despite being polyadenylated. By sequencing all clones and using the 5'end sequence, placenta-derived
Primer dependent amplification of the 5'end of IL-1L1 cDNA
tion). The product was approximately 1.1 kb in length and overlapped the sequence of the cDNA clone. By using multiple sets of 5'primers for the amplification reaction, 5 or more distinct isolates (possibly containing two different non-coding 5'ends (
We have obtained at least 2 isolates for each 5'end, which are shown in FIG. The estimated total length of mRNA was calculated to be 2.6 kb. The coding sequence contained up to the first 600 nucleotides. Eliminating the expected cloning error, all cDNA sequences (3 we sequenced in their entirety) encode the same protein, suggesting that there is no leader sequence. . Indeed, a human sequence identical to the sequence we report here is
The cDNA has been previously reported (Mulero et al., (1999) Biochem. Biophys.
Res. Commun. 263: 702-6; Smith et al., (2000) J. Biol. Chem. 275: 116.
9-75; Busfield et al. (2000) Genomics 66: 213-6).

IL-1L1 Genome Sequencing and Mapping: PAC 131J6 subclones were generated from Xba I and EcoR-I digested DNA and sequenced by primer walking. Good analysis results were obtained at least once from both chains. The IL-1L1 gene consists of 6072 nucleotides and is shown in Figure 11. The position of the intron-exon boundary is shown in Figure 10b, which is
As previously described for other genes in the IL-1 structural family, IL-1R
It is very similar to the position of the N intron-exon boundary. All sequenced segments from PAC 131J6 (accession number AJ consisting of 6540 nucleotides
271338) contains 6 exons from 450 nucleotides upstream of exon 1 to 18 nucleotides downstream of the polyadenylation site. Recently, a high-throughput genomic sequence for PAC RP11-339F22 (accession number AC016724) has also been deposited in the EMBL database. Reconfirming the initial data to look for differences, our genomic sequence differed from the sequence in the database at 44 positions (0.67%). PAC
RP11-339F22 contains a 195 kb genomic insert (insert), but 12 non-overlapping fragments have been identified in this insert at this time. Location of known markers (Hilderbrandt et al., (1996) Cytogenet. Cell Genet
73: 235-239), 11 out of 12 fragments were arranged, and further, a position-fixed sequence in the IL-1RN locus was arranged. Restriction maps and hybridization with IL-1L1-derived 5'and 3'probes allowed the remaining fragments to be located and other fragments to be oriented. Therefore, IL-1L1 is located between the STS markers 10H8S and 131P6S and is transcribed in the 131P6S direction. IL-1L1 is considered to be a small sequence excluded from AC016724, as suggested by the three gaps in IL-1RN.
It was concluded that the polyadenylation signal of Escherichia coli was present at a position approximately 53 kb on the 5'side of the first exon of IL-1RN, and the two genes were oriented in the same direction. From our previous mappings, this means that there is a gap of about 270 kb between IL-1L1 and IL-1B.

Due to the disruption between the human and mouse IL-1L clusters (Zahed
i) et al. (1991) J. Immunol. 146: 4228-33), whether the difference in IL-1L1 transcripts between species is due to the division existing in exon 6 of mouse IL-1L1. I considered. Using the mouse IL-1L1 cDNA probe, the 129 / Sv RPCI21 library (Ioannou et al., (1996), edited by Dracopoli et al., “Current Protocols in Human Genetics”, 5
Screening was performed on a mouse PAC clone derived from .15.1-5.15.24, Wiley, New York, and 3 plasmids were obtained.
The individual also contains Il1rn, which indicates that it is adjacent to Il1l1 and Il1rn, and that the break in the homologous region is Il1b1 rather than between Il1l1 and Il1rn.
There is clear evidence that this occurs between the Low stringency hybridization on Zooblots (Clontech) revealed the presence of the IL-1L1 gene in all eutherian species (FIG. 8).

[Brief description of drawings]

[Figure 1]   Nucleic acid sequences of two human IL-1L1 cDNAs having two 5'ends

[Fig. 2]   Nucleic acid sequence of mouse IL-1L1 cDNA sequence

FIG. 3 Putative polypeptide sequence encoded by human IL-1L1 and mouse IL-1L1.

[Figure 4]   Comparison of human and mouse IL-1L1 polypeptide sequences.

FIG. 5: Comparison of human IL-1L1 polypeptide sequence and human pro-IL-1ra polypeptide sequence.

[Figure 6]   IL-1L1 gene product three recombinant polypeptide sequences

[Figure 7]   Northern blot showing tissue distribution of human IL-1L1 transmission code

[Figure 8]   Zooblots showing the conservation of IL-1L1 gene sequences in mammals

FIG. 9: Chromosomal location of the IL-1L1 gene within the IL-1 locus on human chromosome 2 and the intron / exon structure of the IL-1L1 gene

[Figure 10]   Details of IL-1L1 transcript splicing

FIG. 11   Whole genome sequence of IL-1L1 gene

[Fig. 12]   Immunoprecipitation of native IL-1L1 protein from JEG-3 cells

FIG. 13: Alignment of human and mouse IL-1L1 genes with other human IL-1 like gene families

[Procedure amendment]

[Submission date] August 14, 2002 (2002.8.14)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Contents of correction]

Title of invention IL-1L1 gene and polypeptide products

[Claims]

Detailed Description of the Invention

1. BACKGROUND OF THE INVENTION The interleukin-1 (IL-1) gene cluster encodes a number of related polypeptides that regulate the inflammatory response. The IL-1 gene cluster exists in the long arm of human chromosome 2 (2q13), and within the 430 kb region, IL-1 α (encoded by IL-1A) and IL-1β (IL- 1B) and IL-
At least three types of interleukin-1 signal transduction ligands including signal receptor antagonists (encoded by IL-1ra and IL-1RN)
gnick) (Nicklin et al., (1994) Genomi
cs 19: 382-4). Agonist molecules, IL-1α and IL-1β, have potent pro-inflammatory activity and trigger a diverse inflammatory cascade. For example, IL-1α and IL-1β cytokines stimulate the production of other cytokines such as IL-6 and IL-8, which results in the activation of leukocytes and their recruitment to damaged tissue, and vasoactive substances. Has triggered the local production of. Inappropriate exertion of IL-1 agonist activity is thought to play a central role in the pathophysiology of autoimmune diseases and inflammatory diseases including variant arthritis, inflammatory bowel disease and psoriasis. In contrast, IL-1 receptor antagonists (IL-1ra), by binding competitively to certain IL-1 receptors without inducing an inflammatory cascade,
Blocks L-1 agonist signal transduction. Thus, lack of IL-1 receptor antagonist activity may also lead to the progression of autoimmune and inflammatory disease conditions, and such IL-1 receptor antagonists are useful for the treatment of such diseases and conditions. It is thought that it is possible to provide various substances.

IL-1 agonists act through at least two types of receptors. The type I IL-1 receptor (IL-1RI) transmits a signal to the inside of the cell, but the type II receptor (IL-1RII) does not transmit a signal. Therefore, it is known as a "bait" receptor. The responsiveness of cells to IL-1 depends on the IL-1RI receptor and IL-1
It is affected by the ratio with the RII receptor, which means that it activates IL-1RI in an IL-1-dependent manner.
tein: IL-1RAcP) appears to compete for interaction with a third membrane protein (Lang et al. (1998) J. Immunol. 161: 6871-7). IL-1
Is expressed on T cells, fibroblasts and epithelial cells (Sims et al. (1988) Science 241: 585-9), whereas the type II receptor for IL-1 is B
Expressed on cells, neutrophils and macrophages (Bomsztyk)
(1989) Proc. Natl. Acad. Sci. USA 86: 8034-8). Both IL-1α and IL-1β ligands bind to the type I and type II IL-1 receptors, and although their biological activities as cytokines are similar, the apparent affinity of IL-1α Is IL
-1RI is high, while the apparent affinity of IL-1β is high for IL-1RII.

IL-1 receptor antagonists are structurally homologous to IL-1α and IL-1β, bind to the IL-1 type I receptor, but are biologically inactive, Acts as a competitive inhibitor of IL-1. In certain cell types, the IL-1 receptor antagonist mRNA is alternatively spliced and integrated into two different first exons. One of the alternatively spliced ones is secreted but the other remains in the cytoplasm, and the function of this intracellular retention type IL-1 receptor antagonist is unknown. In response to proinflammatory cytokines as acute proteins, hepatocytes produce secreted, but not cytoplasmic, retained IL-1ra (Gabay et al., (1997) J. Clin. In
vest. 99: 2930-40). With regard to the use of IL-1 receptor antagonists, their use in the treatment of numerous inflammatory diseases and conditions is considered (US patent).

The genetic diversity of the IL-1RN gene is believed to be involved in the pathogenesis of certain inflammatory diseases and conditions. The second intron of the human IL-1RN gene is 86b
variable number of tandem repeats (VNT)
R), which involves polymorphisms (Tarlow et al., (19
93) Hum. Genet. 91: 403-4). In humans, there are at least 5 alleles for this polymorphism, and a particular allele of this IL-1RN VNTR polymorphism (ie IL-1RN VNTR allele 2) is It is associated with more serious clinical symptoms in several diseases with sexual symptoms, such as periodontal disease (US Pat. No. 5,686,246), osteoporosis (US Pat. No. 5,698,399)
, Erythematous lupus (Blakemore et al., (1994) Arthritis Rheum. 37: 1380.
-5), Ulcerative colitis (Mansfield et al., (1994) Gastroentero
logy 106: 637-42) and alopecia areata (Tarlow et al., (1993) Hum. Ge
91.403-4), and diabetic kidney disease (WO 98/15653, Blakemore (Blake
more) et al. (see 1996) Hum. Genet. 97: 369-74). IL-1RN (V
Other polymorphic alleles of IL-1 that bind to NTR) also affect chronic obstructive airway disease (WO 9
It has been found to be associated with various inflammatory diseases or conditions such as 9/24615) and coronary artery disease (WO 98/40517). Blood levels of interleukin-1ra are elevated in patients with various infectious diseases, autoimmune diseases and trauma. Furthermore, characterization of the human IL-1 locus haplotype, which is associated with an increased risk of inflammatory diseases, has been elucidated (WO 98/54359).

IL-1α, IL-1β, IL-1ra and IL-18 are IL-1-like cytokines, and their activities have already been reported. Any type of IL-1 (IL-1α and IL-1
β, encoded by the human genes IL-1A and IL-1B, respectively) is also a single domain protein and acts as an inflammatory cytokine through its interaction with the type I IL-1 receptor (IL-1RAcP) To do.

[0006] The IL-1 receptor antagonist (IL-1ra, encoded by the human gene IL-1RN) has 28% identity to IL-1β, but acts as an antagonist and with respect to binding to IL-1R1. , Inhibits IL-1 signaling by competing with IL-1. IL-1R1 and IL-1ra complex is IL-1RAcP
Failed to activate signal transduction because it did not reinforce (G. Feder et al., J. Biol. Chem. 270: 13757-13765 (1995).
). IL-18 is also a homologue of IL-1 (homolog), but it specifically interacts with another receptor pair, IL-18R and AcPL (Torigoe et al., J.
Biol. Chem. 27: 25737-25742 (1997); Born et al., J. Biol. Chem. 273:
29445-29446 (1998)). The two components that make up the IL-18 receptor are IL-1R
It has a domain structure similar to 1, and the signal passes through the Toll-like domain. IL-1 receptor and IL-18 when expressed in similar cells
The signaling cascade downstream of the receptor is similar (Thomsen (Thom
assen), J. Interferon Cytokine Res. 18: 1077-1088 (1998)). The other three IL-1R1-like receptors are T1 / ST2 (IL-1RL1) (Bergers
s) et al., EMBO J. 13: 1176-1188 (1994)), IL1R-rp2 (IL-1RL2) (Lovenberg et al., Neuroimmunol. 70: 113-122 (1996)) and IL1RAPL (Carrie. , Et al., Nat. Genet. 23: 25-31 (1999)), is not known to be a signal-inducing ligand, but the cytoplasmic domain of ST2 can transduce IL-1R1-like signals in transfected cells. (Reikerstorfer et al., J. Biol. Chem. 28: 17645-17648 (1995)). The inventors have isolated a genomic fragment (IL-1 cluster) of about 430 kb from human chromosome 2q13 (including IL-1α, IL-1β and IL-1ra).
Reikerstorfer et al., J. Biol. Chem. 28: 17645-17648 (
1995)). IL-18 is present on another chromosome (Nolan et al., Genomics
51: 161-163 (1998)).

2. SUMMARY OF THE INVENTION The present invention relates, in part, to humans encoding interleukin-1 receptor antagonist proteins (IL-1ra) and novel proteins having sequence homology with interleukin-1 (IL-1). Based on the discovery of a novel gene in the mouse. The newly identified proteins and nucleic acids described herein are "IL
-1L1 group ", and is exemplified herein using human and mouse homologues (homologs) of this gene. Human IL-1L1 gene (herein hI
The transcript (referred to as L-1L1) is shown in Figure 1 and consists of a consensus sequence of 2562 nucleotides (SEQ ID NO: 1) and a 5'sequence of 39 nucleotides (SEQ ID NO: 2) and 42 nucleotides. It contains two 5'sequences of the 5'sequence (SEQ ID NO: 3). The hIL-1L1 gene transcript consists of 5'and 3'untranslated regions and 465 nucleotides encoding a hIL-1L1 polypeptide composed of 155 amino acids shown in (A) of FIG. Open reading frame (SEQ ID NO: 4
)including. The hIL-1L1 gene is highly expressed in placenta and slightly expressed in thymus tissue (FIG. 7). cDNA encoding the full-length hIL-1L1 protein
Nucleic acid containing is available at American Type Culture Collection (Massas, Virginia, 1801 University Street, Zip code 20110-2209, phone number +01 (703) 365- 2700) and the ATCC Designation number is XXXXXX. hIL-1L1
The gene transcript has a particularly long 2 kb 3'untranslated region (UTR).

A mouse homologue of hIL-1L1 has also been isolated and is herein referred to as mIL-1L1.
Called. The mIL-1L1 gene transcript consisting of 1284 nucleotides is shown in FIG. 2 (SEQ ID NO: 4), 5 ′ and 3 ′ untranslated region, and 155 nucleotides shown in (B) (SEQ ID NO: 6) of FIG. Contains an open reading frame of 465 nucleotides encoding a mIL-1L1 polypeptide composed of the amino acids of mIL-1
The L1 polypeptide sequence showed 90% identity to that of hIL-1L1 (FIG. 4), suggesting that the encoded IL-1L1 product is highly conserved throughout evolution. ing. Nucleic acid containing a cDNA encoding the mouse IL-1L1 polypeptide was prepared on the American Type Culture Collection (
The American Type Culture Collection) (12301, Park Lawn Street, Rockville, MD) has been deposited, and the ATCC accession number is XXXXXX. mI
The L-1L1 gene transcript has a 3'UTR of approximately 0.7 kb. 3'UTR of mIL-1L1 is hIL-1L1
Has only partial homology with the 3'UTR of. 5'-ACAATNAAAANCCCNGAT
The conserved consensus sequence consisting of 41 nucleotides corresponding to NCTGGTCTCTANTCNCATNAAAA-3 ′ (SEQ ID NO: 12) starts from nucleotide number 1137 in SEQ ID NO: 1 (hIL-1L1) and in SEQ ID NO: 4 (mIL-1L1). Is nucleotide number 1146
It has been found to begin with.

BLAST program (Altschul et al., J. Mol. Biol. 215: 403 (19
90)) was used to analyze some of the amino acid and nucleic acid sequences in the newly identified human and mouse IL-1L1 proteins and nucleic acids. It was revealed to have some sequence similarity with the polypeptide encoded by. In particular, the IL-1L1 polypeptide of the invention comprises one or more of an IL1 domain, a casein kinase II phosphorylation site, a myristylation sequence, a transmembrane segment, and a segment homologous to an IL-1 agonist and antagonist. Includes human and mouse IL-1L1 polypeptide sequences characterized by the above.

Viewed from one aspect, the present invention relates to an isolated IL-1L1 nucleic acid molecule.
In one embodiment, the IL-1L1 nucleic acid molecule is of vertebrate origin. In a preferred embodiment, the IL-1L1 nucleic acid molecule is of mammalian origin, such as human. In a more preferred embodiment, the nucleic acid is the nucleic acid sequence shown in SEQ ID NO: 1 or a part thereof, or two alternative 5 ′ alternative 5 ′ ends shown in SEQ ID NO: 2 or 3. ends)).
In another embodiment of the invention, the nucleic acid is of murine origin and has the nucleic acid sequence as set forth in SEQ ID NO: 4, or a portion thereof. The disclosed molecules, whether non-coding sequences (eg, probe, antisense or ribozyme molecules, etc.) or functional IL-1L1 polypeptides (eg, human IL-1L1 polypeptide biological activity of at least). It may also encode a polypeptide that specifically regulates a biological activity by acting as an agonist or antagonist for one). In one embodiment, the nucleic acid molecule is IL-1L1 contained in ATCC Accession No. XXXXX (hIL-1L1) or XXXXX (mIL-1L1).
It can hybridize to a gene. In another embodiment, the nucleic acids of the invention can hybridize to a vertebrate IL-1L1 gene or a gene complementary to a vertebrate IL-1L1 gene. In yet another embodiment,
The nucleic acid of the present invention can hybridize with the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1, 2 or 3) or a sequence complementary thereto. In another embodiment, the nucleic acid of the present invention can hybridize with the nucleic acid sequence shown in FIG. 2 (SEQ ID NO: 4) or a sequence complementary thereto. In a preferred embodiment, hybridization is performed under mild stringent conditions or under stringent conditions.

In a more preferred embodiment, the nucleic acid molecule has at least homology with the nucleic acid set forth in SEQ ID NO: 1, 2, 3 or 4 or the complementary sequence of the nucleic acid set forth in SEQ ID NO: 1, 2, 3 or 4. An IL-1L1 nucleic acid that is about 70%, preferably about 80%, more preferably about 85%, even more preferably at least about 90%, or 95%.
In a further embodiment, the nucleic acid molecule has an ATCC accession number XXXXXX or X
An IL having at least about 70%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90% or 95% sequence similarity to the IL-1L1 nucleic acid contained in XXX. -1L1 nucleic acid.

The present invention also provides a probe and a primer comprising a substantially purified oligonucleotide, wherein the nucleotide is at least about 6 of the sequences shown in SEQ ID NO: 1, 2 or 3. Corresponds to a region of nucleotide sequence that hybridizes to at least about 10, at least about 15, at least about 20, or at least about 25 consecutive nucleotides, or SEQ ID NO: 4, or a sequence complementary thereto. , In a preferred embodiment SEQ ID NO: 10 or
It corresponds to the region of the nucleotide sequence which hybridizes to the sequence shown in 11 or to naturally occurring mutants or allelic variants thereof. In a preferred embodiment, the probes / primers additionally have a detectable label group attached to them.

For expression, for example, at least one transcription promoter (eg, for structural or inducible expression) or a transcription control sequence such as a transcription enhancer sequence is operably linked to a target nucleic acid. can do. IL-1
L1 gene transcription control sequences provide a specific method for controlling the sequences encoding the IL-1L1 polypeptides of the invention as well as heterologous polypeptides. For example, IL-1
The gene encoded by the locus has been shown to respond to lipopolysaccharide (LPS) induction in both activation (IL-1RAcP and IL-1RI) and repression (IL-1RII) (penton- Roll (Penton-Rol) J. Immunol. 162: 2931-2938 (1999))
. Such control sequences can be combined with an IL-1L1 nucleic acid molecule to provide a vector useful for gene expression. The invention further provides prokaryotic or eukaryotic host cells transfected with such expression vectors, and in vitro production of IL-1L1 protein using such expression vectors. (Eg, cell culture, etc.) and in vivo (
(Eg transgenic) methods are described.

Viewed from another aspect, the invention provides an isolated polypeptide, preferably a substantially pure preparation, such as a polypeptide purified from plasma or a recombinantly produced polypeptide. Regarding The IL-1L1 polypeptide includes a full-length protein, or has an amino acid length of at least about 6, 10, 25, 50, 75, 100, 125, 130, 135, 140 pieces, 145 pieces, 150 pieces or 155 pieces
It may include small fragments corresponding to one or more of the particular motifs / domains or fragments that are individual. In a particularly preferred embodiment, such a polypeptide has a biological activity of IL-1L1, such as a receptor, in particular the interleukin-1 receptor (IL-1R) family or interleukin-1 receptor accessory protein ( IL-1RAcP)
It may bind to a coreceptor and / or may alter the activity of the receptor.

[0015] In one embodiment, the polypeptide is SEQ ID NO: 1, 2, 3 or 4.
Is encoded by a nucleic acid that hybridizes to the nucleic acid sequence shown in.
In a preferred embodiment, the polypeptide is encoded by a nucleic acid that hybridizes to a nucleic acid sequence derived from the open reading frame (ORF) of the IL-1L1 gene as set forth in SEQ ID NO: 10 or 11. In a more preferred embodiment, the IL-1L1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6. The IL-1L1 protein also includes modified proteins, such as mutations in which the modification site (eg, tyrosine residue, threonine residue, serine residue, or asparagine residue) is altered,
Alternatively, a protein having resistance to post-translational modification due to a mutation that blocks glycosylation of a protein, or a mutation that blocks interaction of a protein with an intracellular protein involved in signal transduction.

The IL-1L1 polypeptides of the present invention can be glycosylated, or conversely, in a reduced form by selecting an expression system or by modifying the protein sequence to prevent glycosylation. Analogs with carbohydrates can also be provided. Glycosylated forms can also be obtained by derivatizing glycosaminoglycan chains. In addition, it lacked an endogenous signal sequence, although it would normally be cleaved even though it exists as a precursor form of the protein.
An IL-1L1 polypeptide can also be produced.

In yet another preferred embodiment, the present invention provides a purified or recombinant polypeptide having regulatory capabilities such as mimicking or antagonizing the activity of wild-type IL-1L1 protein. Regarding Preferably, such a polypeptide comprises an amino acid sequence identical or homologous to the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6.

In another aspect of the invention, a chimeric molecule containing an IL-1L1 protein (eg,
Fusion proteins, etc.). For example, irrelevant to the IL-1L1 polypeptide (
The IL-1L1 polypeptide can be produced as a recombinant fusion protein containing a second polypeptide moiety, such as a second polypeptide having a (heterologous) amino acid sequence. Preferred IL-1L1 fusion proteins include immunoglobulin-IL-1L1 fusion proteins in which the constant region of an immunoglobulin is fused to an IL-1L1 polypeptide.

Another aspect of the invention relates to an immunogen that comprises an IL-1L1 polypeptide in an immunogenic preparation, wherein the immunogen is a specific immune response to the IL-1L1 polypeptide. Those capable of inducing (eg, humoral response, antibody response and / or cellular response). In a preferred embodiment, the immunogen is the protein encoded by the nucleic acid set forth in SEQ ID NO: 1 or 4,
Alternatively, it has an antigenic determinant, such as a particular determinant of a polypeptide sequence as set forth in SEQ ID NO: 5 or 6.

Another aspect of the invention relates to antibodies and antibody preparations that specifically react with an epitope of the IL-1L1 protein.

The invention further comprises a heterologous form of the IL-1L1 gene described herein (more preferably expressed) or mis-expresses an endogenous IL-1L1 gene (eg Non-human transgenic animal in which the expression of one or more IL-1L1 proteins is disrupted. Use of such transgenic animals as animal models in the study of cellular and / or tissue disorders containing mutated or mis-expressed IL-1L1 alleles, or in drug screening You can Alternatively, such transgenic animals can be used to express recombinant IL-1L1 polypeptides.

The present invention further provides for determining whether an IL-1L1 gene and / or protein in an individual is defective or defective (eg, with respect to activity and / or level, etc.), and / or Assays and kits for performing discrimination of IL-1L1 alleles. In one embodiment, such a method comprises measuring the level of IL-1L1 protein, the level of IL-1L1 mRNA and / or the transcription rate of the IL-1L1 gene. In another embodiment, such a method comprises detecting the presence or absence of the genetic modification in the tissue of the subject individual, wherein the genetic modification is characterized by at least one of the following: gene Deletion of one or more nucleotides from, addition of one or more nucleotides to the gene, replacement of one or more nucleotides of the gene, large-scale rearrangement of the gene on the chromosome, gene Changes in the level of messenger RNA transcripts, the presence of a splicing pattern in the messenger RNA transcript of the gene different from wild type, and / or the level of IL-1L1 protein different from wild type.

For example, to detect the presence of a genetic alteration or a specific polymorphic region, the following steps are included: (i) the sense of the IL-1L1 gene or its naturally occurring mutants. An oligonucleotide sequence which hybridizes to a sequence or an antisense sequence, or 5'or 3 which is essentially related to the IL-1L1 gene
'Providing a probe / primer containing an oligonucleotide sequence that hybridizes to a flanking sequence (flanking sequence); (ii) contacting the probe / primer with a suitable nucleic acid containing sample; The presence or absence of a gene change is detected by hybridizing with a nucleic acid. In a particularly preferred embodiment, the following steps are included: 1) at least a portion of the IL-1L1 gene is sequenced, and 2) single chain conformational polymorphism (SSCP) analysis is performed to obtain the mutant. Detecting the difference in electrophoretic mobility between the nucleic acid of Escherichia coli and the wild-type nucleic acid;
3) Detect or quantify IL-1L1 protein levels in an immunoassay using an antibody that specifically causes an immune reaction with a wild-type or mutant IL-11 protein.

From the information obtained using the diagnostic assays described herein (alone or in combination with information regarding other genetic defects involved in the same disease), subject having symptoms Diagnoses or confirms that, for example, it has a genetic defect that causes or is associated with a particular disease or disorder within the IL-1L1 gene or within a gene that regulates the expression of the IL-1L1 gene. To help you. Alternatively, using such information (alone or in combination with information on other genetic defects associated with the same disease), an asymptomatic subject can be treated with IL-1L1.
It may be possible to predict whether a disease or disorder associated with or associated with abnormal activity or protein levels may develop. In particular, this assay can be used to identify the individual probability of causing a condition that is caused by or is associated with a mutation in IL-1L1. Type (single nucleotide polymorphism: SNP). Based on prognostic information, the physician can recommend to each individual a regimen (eg, diet, exercise, etc.) or treatment that is useful to prevent or delay the onset of a particular disease or condition.

In addition, knowledge of a particular alteration in an individual's IL-1L1 gene or protein deficiency or deficiency may include information about other genetic defects, either alone or associated with the same disease (gene profile for a particular disease). ), The therapeutic and pharmacogenomics goals for diseases specific to each individual's genetic profile can be set. For example, if the doctor obtains the IL-1L1 gene profile of each individual, or the gene profile of the disease or condition caused or associated with the change in the IL-1L1 gene, 1) the disease or Drugs that specify symptoms at the molecular level can be more effectively prescribed, and moreover, 2) more appropriate doses can be determined for specific drugs. For example, by measuring the expression level of IL-1L1 protein in a large number of patients at all stages of the disease, either alone or in combination with the expression levels of other genes known to be involved in the same disease, Transcription or expression profiles can be generated.

The expression pattern of an individual patient can then be compared to the expression profile of the disease to determine the appropriate drug and dosage to administer to that patient.

Based on the genetic profile of IL-1L1 or disease, if the population that is predicted to have the highest clinical effect can be targeted, Substitute for over-the-counter drugs, 2) rescue candidate drugs whose clinical development has been discontinued due to limited safety or efficacy in some patient groups, and 3) accelerated drug development and cost Reductions as well as more optimal drug labeling can be performed (eg, using IL-1L1 as a marker is useful for optimizing effective doses, etc.).

Viewed from another aspect, the invention provides methods for identifying compounds that modulate IL-1L1 activity, such as the interaction of an IL-1L1 polypeptide with a target peptide.
In a preferred embodiment, the method comprises the following steps: (a) (i) IL
-1L1 polypeptide, (ii) IL-1L1 binding partner (eg, IL-1L1 receptor or heparin sulfate proteoglycan, etc.), and (iii) a reaction mixture containing a test compound is prepared; and (b) IL-1L1 Detect the interaction between the polypeptide and the IL-1L1 binding protein. As compared with the case where the test compound was not included, the interaction between the IL-1L1 polypeptide and the IL-1L1 binding protein was statistically significantly changed (enhanced or inhibited) in the presence of the test compound, It suggests that the test compound is an agonist (mimetic or enhancer) or antagonist (inhibitor) for the biological activity of IL-1L1. The reaction mixture includes a cell-free protein preparation (eg, reconstituted protein mixture or cell lysate),
Alternatively, recombinant cells containing a heterologous nucleic acid expressing a binding partner for IL-1L1 by recombination can be used.

In a preferred embodiment, the step of detecting the interaction between IL-1L1 and the binding partner of IL-1L1 uses a competitive binding assay. In another preferred embodiment,
At least one of the binding partners of IL-1L1 and IL-1L1 has a detectable label, and by detecting the label in the complex, IL-1L1
To quantify the interaction of IL-1L1 with its binding partner. As the detectable label, for example, a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor can be used. In another embodiment, the complex is detected by immunoassay.

In yet another embodiment, an assay is provided that screens for test compounds to identify agents that modulate the amount of IL-1L1 produced in the cell. In one embodiment, such a screening assay involves contacting cells transfected with a test compound with a reporter gene operably linked to the IL-1L1 promoter and measuring the expression level of the reporter gene. including. The reporter gene can encode, for example, a gene product which emits a detectable signal (such as dye, fluorescence, luminescence, cell viability, alleviation of cell auxotrophy, cell proliferation and drug resistance). For example, the reporter gene can encode a gene product selected from the group including chloramphenicol acetyltransferase, luciferase, β-galactosidase and alkaline phosphatase.

Also within the scope of the present invention is the treatment of diseases or disorders that are associated with abnormal IL-1L1 levels or activities, or which are efficacious by modulating IL-1L1 levels or activities. Methods are also included. Such methods include, for example, locally or systemically administering to the patient a pharmaceutically effective amount of a composition comprising an IL-1L1 therapeutic agent. Depending on the condition of the patient to be treated, the therapeutic agent can be an IL-1L1 agonist or IL-1L1 antagonist.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

3. Brief Description of the Drawings Figure 1 shows the nucleic acid sequences of two human IL-1L1 cDNAs with two 5'ends. The gene transcript of IL-1L1 is polyadenylated at 3'and two different 5'ends consist of 39 nucleotides and 42 nucleotides (respectively,
SEQ ID NOS: 2 and 3), a consensus sequence consisting of 2562 nucleotides (SEQ ID NO: 1)
including. Open reading frame (ORF) of human IL-1L1 (SEQ ID NO: 10)
Is underlined.

FIG. 2 shows the nucleic acid sequence (SEQ ID NO: 4) of the mouse IL-1L1 cDNA. The mouse open reading frame (ORF) (SEQ ID NO: 11) is underlined.

FIG. 3 shows the deduced polypeptide sequence encoded by human IL-1L1 (panel A; SEQ ID NO: 5) and mouse IL-1L1 (panel B; SEQ ID NO: 6).

FIG. 4 compares the polypeptide sequences of human and mouse IL-1L1 with conservative substitutions (+) and showing the consensus polypeptide sequence (SEQ ID NO: 7).

FIG. 5 shows the conservative substitution (+) and also shows the consensus polypeptide (sequence (SEQ ID NO: 9)) human IL-1L1 polypeptide sequence and human pro-IL.
1 is a comparison of -1ra polypeptide sequences (SEQ ID NO: 8).

FIG. 6 shows the polypeptide sequences of three recombinant IL-1L1 gene products differing in amino terminal amino acid sequence.

[0039]   FIG. 7 is a Northern blot showing the tissue distribution of the human IL-1L1 transmission code.

[0040]   FIG. 8 is a Zooblot showing the conservation of the IL-1L1 gene sequence in mammals.

FIG. 9 shows the chromosomal location of the IL-1L1 gene within the IL-1 locus on human chromosome 2 and the intron / exon structure of the IL-1L1 gene.

[0042]   FIG. 10 shows the details of the splicing of the IL-1L1 transcript.

[0043]   FIG. 11 shows the entire genomic sequence of the IL-1L1 gene.

[0044]   FIG. 12 shows immunoprecipitation of native IL-1L1 protein derived from JEG-3 cells.

FIG. 13 is a side-by-side alignment of the human and mouse IL-1L1 genes with other human IL-1 like gene families. Conserved residues are boxed and represent the β-sheet secondary structure of the folded protein. DETAILED DESCRIPTION OF THE INVENTION 4.1 General The present invention is directed , in part, to the IL-1 gene encoding IL-1 receptor antagonists (IL-1ra) and proteins homologous to IL-1β polypeptides. Based on the discovery of genes present within cluster loci. Therefore, the IL-1L1 protein is a pro-inflammatory cytokine (IL-1β) as well as an inflammatory cytokine (IL-1ra).
Related to. IL-1L1 gene that name, this gene is IL-1 beta (IL-1 B eta) gene and IL-1 receptor antagonist that encode (IL-1 R eceptor antag
It comes from the fact that exists between (I nterval) the gene encoding the onist). Accordingly, the genes and proteins disclosed herein are labeled IL-1L1
Gene and IL-1L1 protein.

The IL-1L1 gene is a 680 kb CEPH yeast artificial chromosome (YAC containing the IL-1 cluster.
) Clone 766E12 (Nothwang et al., (1997)). YAC
Clone 766E12 was isolated from the yeast strain producing the clone by performing pulsed field electrophoresis and the resulting DNA was used as a driver in selecting the IL-1 encoded transduction code ( Morgan et al. (1992)
Nucleic. Acids Res. 20: 5173-79). The cDNA was obtained from human peripheral blood mononuclear cells cultured for 6 hours in the presence of 10 ng / ml phorbol myristate acetate and 100 ng / ml E. coli lipopolysaccharide.

The selected cDNA was amplified, and the amplified cDNA fragment was cloned into pNEB193 after two rounds of affinity selection and further amplification. The entire sequence was performed on 100 samples randomly selected from the obtained clones. The IL-1L1 expressed sequence tag (EST) corresponded to one of the overlapping sequences derived from the three overlapping cDNA fragments that made up a continuous sequence of approximately 530 bp. This sequence consists of a 374 bp cDNA derived from human placenta (GenBank (G
enBank) Accession No. R70041) and 348 bp human placental cDNA (GenBank Accession No. R70089) were found to be homologous. When tested, this sequence was found to be Gingri
ch) The ICRF human P1 library (Nothwan (Nothwan) was found to hybridize specifically with P1 artificial chromosome 131J6 derived from human PAC library.
g) et al., Genomics 41: 370-378 (1990)) and adjacent to ICRF 700G1305 IL1B.
It was located between the gene and the IL1RN gene. This latter clone is IL-1r
a gene (IL1RN) is included. These new ESTs are also in the database EST sequence.
There were no open reading frames in the sequences, and further analysis revealed that these sequences were contained within the 3'untranslated region (UTR) of the gene transmission code. .

Northern blot analysis using this 3 ′ fragment as a probe revealed the presence of an approximately 2.6 kb transcript (FIG. 7) in the human placenta. First, this full length cDNA was obtained by using this 3'fragment as a probe, and then 1.6 kb of IL-1L1 from H9 placenta cDNA plasmid library obtained from HGMP (Human Genome Mapping Project).
The cDNA fragment was isolated. This 1.6 kb clone was polyadenylated but did not contain a clear open reading frame. Next, the 5'end of this clone was used to prepare primers for amplifying the full length 5'end of the IL-1L1 transcript obtained from the human placenta-derived cDNA. The resulting product was approximately 1.1 kb in length, overlapping the sequence of the 1.6 kb clone. Subsequent analysis revealed that the IL-1L1 gene encodes two transcripts with slightly different 5 ′ non-coding regions (FIG. 1). The total length of the concatenated transcripts was approximately 2.6 kb, consistent with the placental mRNA detected by Northern analysis. Transcripts of similar size were analyzed by a probe containing the IL-1L1 coding sequence,
It was also detected in human thymus (Fig. 7). The complete human cDNA is GenBank (
GenBank) was found to be homologous with the following EST cDNA fragments: GenBank Accession No. AI040890 (EST derived from human parathyroid tumor consisting of 485 bp: human common shown in Figure 1). Homologous to the 3'UTR of the antisense strand of IL-1L1 present at nucleotide numbers 955 to 1278 in the transcript sequence; GenBank accession number AI469873 (human EST consisting of 442 bp: from nucleotide number 2133) IL-1L1 3'UTR antisense strand present in 2561; GenBank accession number AA722902 (414 bp human fetal heart-derived EST: IL-1L1 present in nucleotide numbers 2141 to 2555)
'UTR antisense strand homology); GenBank accession number AI
67887 (410 bp derived from human parathyroid EST: nucleotide numbers 871 to 1278
Homology with the IL-1L1 3'UTR antisense strand present in
Accession number R70041 (EST consisting of 317 bp derived from human placenta: homologous to IL-1L1 3'UTR antisense strand present in nucleotide numbers 2191 to 2552); GenBank Accession number R70089 (consisting of 348 bp Derived from human placenta
EST: homologous to IL-1L1 3'UTR sense strand present at nucleotide numbers 1828 to 2144; and GenBank accession number W08205 (mouse-derived EST consisting of 382 bp: IL-present at nucleotide numbers 5 to 308) 1L1 homologous to 5'leader sequence and sense strand of coding sequence).

The human IL-1L1 transcript encodes a polypeptide composed of 155 amino acids (A in FIG. 3), and the coding region (underlined portion in FIG. 1) is about 2 kb in the transcript.
It occupies up to the first 600 nucleotides excluding the large 3'untranslated region of. The human IL-1L1 polypeptide sequence is human IL-1ra (SwissProt (SwissPro
t: SP) Accession number P18510, 73 of 155 amino acids are the same, or the overall identity is 47%, see Fig. 5), and the gene encoding IL-1ra It is homologous to a mouse homolog (SP accession number P25085), a rat homolog (SP accession number P25086) and a rabbit homolog (SP accession number P26890). The human IL-1L1 polypeptide sequence is a human IL-1β polypeptide (SwissProt: SP) accession number P01584, 4 out of 155 amino acids.
The two are identical, or have 27% homology overall, and in addition the red deer I
L-1β polypeptide (SP accession number P51754), sheep IL-1β polypeptide (SP accession number P21621), bovine IL-1β polypeptide (SP accession number P09428), mouse IL-1β polypeptide (SP accession number P107
49), homologous to rat IL-1β polypeptide (SP Accession No. Q63264) and other IL-1β protein homologues.

A 1.2 kb mouse IL-1L1 cDNA has also been obtained and sequenced (
See FIG. 2). This transmission code is presumed to encode a polypeptide composed of 155 amino acids, and the identity with human IL-1L1 shown in Fig. 4 is 90% (of the 155 amino acid sequence, 141 or more were the same). 1.2kb mouse I
The nucleic acid sequence of the coding region of L-1L1 cDNA was highly homologous to the sequence of the coding region of human IL-1L1 cDNA of 2.6 kb (with the human IL-1L1 sequence between nucleotide numbers 7 and 500). The identity was 84%). The 1.2 kb mouse IL-1L1 clone is polyadenylated and its 3'untranslated region has a short region homologous to the 3'UTR of human IL-1L1. This region, which shows 83% sequence identity, is common to human IL-1L1.
It is located at nucleotide numbers 1137 to 1177 of the transcript sequence and at nucleotide numbers 1146 to 1186 of the mouse IL-1L1 sequence. Conservative consensus sequence consisting of 41 bp:
5'-ACAATNAAAANCCCNGATNCTGGTCTCTANTCNCATNAAAA-3 '(SEQ ID NO: 12) is
It has been found in human and mouse IL-1L13′UTR and has GATA-1 and GATA-2 binding sites (Merika and Orkin Mol. Cell Biol. 13: 3999-401.
0 (1993)), the Ikaros binding site (Molnar and Georgopoulos Mol. Cell Biol. 14: 8292-8303 (1994)) and a number of nucleic acid sequences including retroviral poly A downstream elements. Has a motif. Such a conservative downstream nucleotide sequence is considered to have an action of controlling initiation and / or termination of RNA polymerase, polyadenylation of mRNA, stability of mRNA, or initiation or termination of translation of IL-1L1. .

Southern blots under low stringency conditions using human IL-1L1 probe were performed on transcribed samples containing genomic DNA cleavage products derived from human, rhesus monkey, rat, mouse, dog, cow, rabbit, chicken and yeast. Hybridization was performed. This Zoo-blot hybridization revealed the presence of one homologous band in all mammals tested. Therefore, it is considered that the IL-1L1 gene was conserved during the evolution of mammals.

Recombinant human IL-1 receptor antagonists and IL-1L1 polypeptides were generated and purified by nickel chelate chromatography followed by elution with imidazole and treatment with thrombin. The polypeptide sequence shown in 6 was obtained. Nuclear magnetic resonance experiments performed on the IL-1L1 protein doubly labeled with N ¹⁵ and C ¹³ suggest that the IL-1L1 protein is largely folded like a β-sheet structure, which It is consistent with the structure of human IL-1ra.

It was also revealed that administration of recombinant IL-1ra to rabbits produced a polyclonal antibody. The choriocarcinoma cell line JEG-3 has been shown to essentially produce IL-1L1 mRNA. Metabolically labeled proteins from this cell line were immunoprecipitated with anti-IL-1L1 rabbit polyclonal antiserum. From these results, it was suggested that a protein having a size of 17 kDa, which matches the estimated molecular weight of IL-1L1, was immunoprecipitated.

4.2 Definitions For convenience, the meanings of certain terms used in the specification, examples and claims are given below.

“Abnormal activity”, when used against the activity of a polypeptide such as IL-1L1, differs from the activity of a wild-type or naturally occurring polypeptide, or in a healthy individual. It refers to an activity different from that of the peptide. It can be abnormal when the activity of a polypeptide is stronger than the activity of its naturally occurring cognate polypeptide. Alternatively, if the activity of a polypeptide is less than or absent from the activity of its naturally occurring cognate polypeptide, it can be abnormal. Aberrant activity can also change activity. For example, the abnormal polypeptide can interact with a different target peptide. Overexpression or underexpression of the gene encoding IL-1L1 results in cells having aberrant IL-1L1 activity.

As used herein, an “agonist” refers to a substance that mimics or up-regulates (eg, potentiates or supplements) the biological activity of IL-1L1. Examples of the IL-1L1 agonist include wild-type IL-1L1 protein and derivatives thereof having at least one of the biological activities of wild-type IL-1L1 (eg, IL-1B receptor binding activity). As the IL-1L1 therapeutic agent, a compound that increases the expression of the IL-1L1 gene or enhances at least one of the biological activities of the IL-1L1 protein can also be used. As the agonist, a compound that enhances the interaction between the IL-1L1 polypeptide and another molecule (for example, IL-1 type I or type II receptor) can also be used. Such agonists are eg IL-1
Binding of IL-1L1 to type I receptors can be enhanced, thereby facilitating IL-1L1-dependent blockade of inflammatory signaling by IL-1α and IL-1β. Alternatively, the IL-1L1 agonist can enhance the binding of IL-1L1 to the IL-1 type II receptor or "bait receptor". The IL-1 type II receptor is present on B cells and has the effect of reducing IL-1α / IL-1β-dependent inflammatory signaling. Therefore, IL-1L1 agonists can indirectly promote inflammatory signaling by IL-1α and IL-1β.

“Allele” is interchangeable herein with “allelic variant” and refers to another form of a gene or portion thereof. Alleles occupy the same locus or position on homologous chromosomes. An individual is said to be homozygous for the gene or allele if it has two identical alleles for the gene. An individual is said to be heterozygous for the gene if it has two different alleles for the gene. Alleles for a particular gene may differ by one or a few nucleotides and may also include nucleotide substitutions, deletions and insertions. Frequent sequence changes include transition mutations (ie, purine to purine and pyrimidine to pyrimidine substitutions, such as A to G or C to T substitutions) and transversions. Mutations (ie, purine to pyrimidine and pyrimidine to purine, eg, A to T, or C to G) and changes in repetitive DNA sequences (eg, trinucleotide repeats) Sequences and other tandem repeat sequences such as extensions and contractions). Alleles of a gene can also form a gene that contains a mutation. "Allelic variant relating to polymorphic region of IL-1L1 gene" means the region of IL-1L1 gene having one or several nucleotide sequences found in a region of IL-1L1 gene in other individuals. Point

As used herein, an “antagonist” refers to a substance that down-regulates (eg, suppresses or inhibits, etc.) at least one of the biological activities of IL-1L1. IL-1L1 antagonists include IL-1L1 protein and other molecules (
For example, a compound that inhibits or attenuates the interaction with a receptor such as IL-1L receptor (IL-1R) and the like can be used. Thus, preferred antagonists are compounds that diminish binding to the IL-B1 receptor, thereby blocking IL-1B receptor activation. A compound that down-regulates the expression of IL-1L1 gene or reduces the abundance of IL-1L1 protein can also be used as the antagonist. As the IL-1L1 antagonist, the most potent negative form of IL-1L1 polypeptide can be used, for example, it can interact with a target peptide such as IL-1 type I receptor but activates the receptor. One type of IL-1L1 polypeptide that is unable to do so. As the IL-1L1 antagonist, use a nucleic acid encoding a strong negative form of IL-1L1 polypeptide, an IL-1L1 antisense nucleic acid, or a ribozyme capable of specifically interacting with IL-1L1 RNA Can also As the other IL-1L1 antagonist, a molecule that binds to the IL-1L1 polypeptide and inhibits its activity can be used. Such molecules include peptides such as certain types of IL-1L1 target peptides, but such peptides have no biological activity and are suitable for IL-1L1 target molecules (such as IL-1L1 receptor, preferably Inhibits binding to the IL-1 type I receptor). As another action, the IL-1L1 antagonist has an activity of inhibiting the IL-1 type II receptor. The IL-1 type II receptor is called the "bait" receptor,
It is present on B cells and binds to IL-1α and IL-1β to prevent their interaction with the IL-1 type I receptor, thereby antagonizing the activity mediated by the IL-1 type I receptor. Certain IL-1L1 polypeptides are able to bind and antagonize the IL-1 type II decoy receptor, thereby promoting IL-1α and IL-1β inflammatory signaling. Thus, certain IL-1L1 antagonists can act to block this situation from the outset, thereby diminishing IL-1α and IL-1β inflammatory signaling. Such IL-1L1 antagonist polypeptides bind to the active site of IL-1L1 and interfere with the interaction of IL-1L1 with target peptides such as IL-1 type I and type II receptors. In another preferred embodiment, the IL-1L1 antagonist is a small molecule capable of inhibiting the interaction of the IL-1L1 polypeptide with the target IL-1L1R. Alternatively, small molecules can antagonize by interacting with sites other than the IL-1L1R binding site, such as the heparin sulfate proteoglycan binding site.

As used herein, “antibody” refers to any isotype (eg,
IgG, IgA, IgM, IgE, etc.), including fragments thereof that specifically react with vertebrate (eg, mammalian, etc.) proteins. Antibodies can be divided into fragments using conventional techniques and, in addition, fragments similar to those described for whole antibodies can be used to screen fragments, depending on the application. Therefore, the term "antibody" refers to
It includes a segment of a part obtained by proteolysis to an antibody molecule capable of selectively reacting with a certain protein, or a segment of a part prepared by recombination. Such proteolytic and / or recombinant fragments include Fab, F (ab ') 2, Fab', Fv, and V [L] and / or V [H] domains linked by a peptide linker. Single chain antibodies (scFv) including, but not limited to. scFvs form covalently or non-covalently bound antibodies which form two or more binding sites. The present invention includes polyclonal antibodies, monoclonal antibodies or other purified antibodies, as well as recombinant antibodies.

Diseases that are “involved in” or “characterized by” aberrant expression of nucleic acids,
A disease or condition refers to a disease, disorder or condition in a subject that is related to or caused by an abnormal expression level of nucleic acid.

As used herein, “biologically active fragment of IL-1L1 polypeptide” refers to a full-length fragment of IL-1L1 polypeptide, wherein such fragment is a wild-type IL-. It specifically mimics or antagonizes the activity of the 1L1 polypeptide. Preferably, the bioactive fragment is interleukin type I or I.
A fragment capable of interacting with the type I receptor.

“Biological activity” or “biological activity” or “activity” or “biological action”
Can be used interchangeably with each other, and in the present specification, IL-1L1 polypeptide (whether naturally occurring or denatured in conformation) or any of their partial sequences directly or indirectly Effector or antigenic effect exerted by the human body. The biological activity includes, for example, binding to a target peptide such as IL-1 receptor (IL-1R), preferably IL-1 type I receptor. In other words, the biological activity of IL-1L1 can be modulated by modulating the level of IL-1L1 polypeptide, such as modulating the expression of the IL-1L1 gene.

“Biomarker” refers to a biological molecule, such as a nucleic acid, peptide, hormone, whose presence or concentration is detectable, and which is associated with a known condition such as a disease.

“Cells”, “host cells” or “recombinant host cells” can be used interchangeably herein. These terms refer not only to the particular test cell, but also to the descendants or progeny of such cells. Such descendant cells are not necessarily identical to the parental cell, although they may not be identical to the parental cell, as mutations or environmental effects may result in certain changes during cell passage. As such, such cells are also included within the term.

A “chimeric polypeptide” or a “fusion polypeptide” is a heterologous version of the first amino acid sequence encoding one of the IL-1L1 polypeptides and any domain of the IL-1L1 polypeptide. And refers to a fusion with a second amino acid sequence defining a substantially non-cognate domain (eg, a portion of a polypeptide, etc.).
The chimeric polypeptide can be present as a heterologous domain found in an organ that also expresses the first polypeptide, despite being a different polypeptide, or can be expressed by a different kind of organ. It may also be an "interspecies" fusion of the polypeptide structures described, an "intergenic" fusion and the like. Generally, a fusion polypeptide can be represented by the general formula X-IL-1L1-Y, where IL-1L1 represents a portion of a polypeptide derived from an IL-1L1 polypeptide, and X and Y are Each is differently vacant or represents an amino acid sequence (including naturally occurring mutants) unrelated to the IL-1L1 sequence in the organ.

“Transport complex” refers to a targeting method (eg, a molecule that binds a gene, protein, polypeptide or peptide to a target cell surface with a strong affinity binding, and / or cellular or nuclear uptake by the target cell. A molecule that is promoted). Examples of targeting methods include sterols (eg, cholesterol), lipids (eg, cationic lipids, virosomes).
me) or liposomes), viruses (eg, adenovirus, adeno-associated virus and retrovirus), or substances that specifically bind to target cells (eg, ligands recognized by receptors specific to target cells). And so on. Preferred complexes are sufficiently stable in vivo to significantly prevent decoupling prior to internalization by target cells. However, the complex is cleavable under appropriate conditions in the cell, whereby a function-producing gene, protein,
The polypeptide or peptide is released.

As is well known, a gene has one copy or multiple copies in each genome. Such overlapping genes are identical or nucleotide substitutions,
Although there are certain variations, including additions or deletions, they all encode polypeptides with substantially the same activity. Thus, "a DNA sequence encoding IL-1L1" refers to one or more genes present in a particular individual. Furthermore, certain differences in nucleotide sequences also exist between individual organs and are called alleles. Such allelic differences are attributed to differences in the amino acid sequences of the encoded polypeptides, even though the polypeptides having the same biological activity are encoded. It's possible, but it may not be.

“Equivalent” includes nucleotide sequences that encode polypeptides that have similar effects. Equivalent nucleotide sequences include sequences that differ by the substitution, addition or deletion of one or more nucleotides (such as allelic variants), and sequences that differ in the nucleotide sequence of the nucleic acid due to the degeneracy of the genetic code (
For example, SEQ ID NOs: 1 and 4).

As used herein, a “haplotype” refers to a set of alleles that are inherited as a group (disequilIL-1L1ium) at a statistically significant level (p _corr <0.05). As used herein, the phrase "IL-1 haplotype" refers to a haplotype at the IL-1 locus and includes polymorphic variants of the IL-1L1 gene sequence.

“Homology”, “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing for a position within each sequence listed for comparison. Two molecules are identical at a position if the same base or amino acid is present at that position within the compared sequences. The percentage of homology or similarity or identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at the positions occupied by the nucleic acid sequences. The percent homology or similarity of amino acid sequences is a function of the number of amino acids (ie, structurally related) at the positions occupied by the amino acid sequences. The identity of one of the IL-1L1 sequences of the invention with an "irrelevant" or "heterologous" sequence is 40% or less, preferably 25% or less.

As used herein, “IL-1L1” refers to the interleukin 1-like gene described herein and encodes the IL-1L1 protein. The IL-1L1 used in the present invention can be interchanged with the “IBR” gene or protein, and IBR means that the gene exists within the IL-1 locus (IL-1L1
Gene IL-1β (IL-1 B eta) and IL-1 receptor antagonist (IL-1 R eceptor
It is an alias for IL-1L1 based on that during (present in the I nterval)) with a gene encoding the Antagonist). Furthermore, the IL-1L1 gene used in the present specification is also referred to as IL1L1 gene.

“IL-1L1 nucleic acid” refers to a nucleic acid encoding an IL-1L1 protein, such as a nucleic acid having SEQ ID NO: 10 or 11, or a fragment thereof, a nucleic acid complementary thereto, And their derivatives.

“IL-1L1 polypeptide” and “IL-1L1 protein” include a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5 or 6, or a fragment thereof, and a homolog thereof, and further, an agonist Includes polypeptides and antagonist polypeptides.

“IL-1L1 receptor” or “IL-1L1R” refers to a variety of membrane-bound protein receptors capable of binding to IL-1L1 and / or transducing signals from IL-1L1. Now, for example, they are IL-1 type I receptors and IL-1
Type II receptors. "IL-1L1 receptor" or "IL-1L1R" means IL
Capable of binding to and / or transducing signals from any or all of -1 related ligands such as IL-1α, IL-1β and IL-1ra (receptor antagonists) Refers to various membrane-bound protein receptors that can Such receptors include IL-1 type I receptors present on T cells, fibroblasts and connective tissue, and IL-1 type II receptors present on B cells. As used herein, IL-1L1R further refers to the IL-1R accessory protein (IL-1R AcP), which is homologous to the Ig superfamily present in the extracellular domain. It is related and is related because it is partially homologous to the L-1 type I receptor and the IL-1 type II receptor. Since IL-1R AcP is thought to form a complex with the IL-1 type I receptor, it transmits both pro-inflammatory signals from IL-1α or IL-1β. As used herein, IL-1L1 receptor (IL-1
L1R) further refers to IL-1 receptor-related genes such as the orphan receptors such as ST2 / T1 and IL-1rrp2. IL-1L1 used herein
Other IL-1 receptor-related genes included in the R category include MyD88 and rsc786.

“IL-1L1 therapeutic agent” refers to various forms of IL-1L1 polypeptides, as well as to mimic, enhance (coact with) or inhibit the effects of naturally occurring IL-1L1 polypeptides. Peptidomimetics, nucleic acids capable of (at the same time) modulating at least one of the activities of IL-1L1 polypeptide (eg, interaction with IL-1L1 receptor and / or IL-1L1 co-receptor) Or it refers to a small molecule. An IL-1L1 therapeutic that mimics or enhances the activity of wild-type IL-1L1 polypeptide is an “IL-1L1 agonist”. Conversely, an IL-1L1 therapeutic agent that inhibits the activity of wild-type IL-1L1 polypeptide is an “IL-1L1 antagonist”.

As used herein, “IL-1 gene cluster” and “IL-1 locus” include all nucleic acids existing in or near the 2q13 region of chromosome 2 and Includes at least IL-1L1 gene, IL-1A gene, IL-1B gene and IL
-1 RN gene and other linked genes are included (Nicklin (Nick
lin) et al., Genomics 19: 382-384 (1994)). As used herein, "IL-1
"A", "IL-1B" and "IL-1RN" refer to the genes encoding IL-1, IL-1 and IL-1 antagonist, respectively. The gene accession numbers for IL-1A, IL-1B and IL-1RN are X03833, X04500 and X64532, respectively.

By “mutation of the action of IL-1” is meant a mutation within the IL-1 gene cluster that results in a distinct phenotype (ie, that affects the action of the IL-1 gene or protein). . For example, IL-1A (+4845) allele 2, IL-1B (+3954)
Allele 2, IL-1B (+6912) allele 2, and IL-1RN (+2018) allele 2 and the like.

“IL-1X (Z) allele Y” refers to a specific allelic form represented by Y occurring at the IL-1 locus polymorphic site within gene X, where X is , IL-1A, IL-1B or IL-1RN or other gene existing in the IL-1 locus, which is present at or near the position of nucleotide Z. Gene X
It is represented by a number based on the major transcription initiation site of N. Further, the "IL-1X allele (Z)" used in the present specification means
Refers to all alleles for the IL-1 polymorphic site within gene X located at or near nucleotide Z. For example, "IL-1RN (+2018) allele" means 2018
Refers to the alternate form of the IL-1RN gene at position # 1. “IL-1RN (+2018) allele 1” refers to the type of IL-1RN gene having cytosine (C) at position 2018 of the sense strand. Clay et al., Hum. Genet. 97: 723-726 (1996). "IL-1RN (+201
8) "Allele 2" means IL-1R having thymine (T) at position 2018 in the plus chain
Refers to the type of N gene. An individual is said to be homozygous or homozygous if it has two identical IL-1RN alleles. An individual is said to be heterozygous or heterozygous if it has two different IL-1RN alleles. "IL-1RN (+2018) allele 2
, 2 ”means that IL-1RN (+2018) allele 2 is homozygous.
On the contrary, “IL-1RN (+2018) allele 1, 1” means that IL-1RN (+2018) allele 1 is homozygous. “IL-1RN (+2018) alleles 1, 2” means that alleles 1 and 2 are heterozygous.

As used herein, “related to IL-1” refers to human chromosome 2 (
2q12-14) is meant to include all genes related to the human IL-1 locus above. These include the IL-1 gene present in the human IL-1 gene cluster on human chromosome 2 (2q13-14). For example, IL-1L1 gene, interleukin-
IL-1A gene encoding 1α, I encoding interleukin-1β
Examples include the L-1B gene and the IL-1RN (or IL-1ra) gene encoding an interleukin-1 receptor antagonist. In addition, these IL-1
Related genes include the human IL-1 receptor on human chromosome 2 (2q12).
Also included are types I and II, and the mouse homologue located at 19.5 cM on mouse chromosome 1. Since interleukin-1α, interleukin-1β and interleukin-1RN are closely related, they are all IL-1 I
Bind to the type I receptor, but interleukin-1α and interleukin-
1β is an agonist ligand that activates the IL-1 type I receptor, and interleukin-1RN is a naturally occurring antagonist ligand. Where "IL
-1 "is used with respect to a gene product or polypeptide, ie, all gene products encoded by the interleukin-1 locus on human chromosome 2 (2q12-14), as well as others. Corresponding homologues from the species or functional variants thereof. Therefore, IL-1 is a secretory polypeptide that promotes an inflammatory response (such as IL-1α and IL-1β), and a secretory polypeptide that antagonizes an inflammatory response (IL-1 receptor antagonist and IL-1 type II). (Bait) receptor etc.

“IL-1 receptor” or “IL-1R” binds to a ligand (such as IL-1α, IL-1β, IL-1ra or IL-1L1) encoded at the IL-1 locus Refers to a variety of cell membrane bound protein receptors capable of and / or capable of transducing signals from such ligands. The term can be used for any protein that can bind to interleukin-1 (IL-1) molecules, and their native conformation as mammalian plasma membrane proteins arises from IL-1. It is thought to be useful in transmitting the signal to the cell. As used herein, the term includes analogs of naturally occurring proteins that have IL-1 binding activity or signaling activity. Examples of such include human and mouse IL-1 receptors, for example:
A 80-kDa polypeptide expressed on T cells, fibroblasts and epithelial cells and called the IL-1 type I receptor (IL-1R type I; Sims et al., Science 214: 585-
589 (1988)), and a 68 kDa polypeptide expressed in B cells, neutrophils, and macrophages and called the IL-1 type II receptor (IL-1R type II; Bomsztyk et al., Proc. Natl. Acad. Sci. USA 86: 8034-8038 (1989)
) And the like, both of which are structurally related to the immunoglobulin (Ig) superfamily of proteins. IL-1 receptors of the present invention include those described in US Pat. Nos. 4,968,607, 5,081,228 and 5,350,683. `` IL-1
"Nucleic acid" refers to a nucleic acid encoding an IL-1 protein.

“IL-1 polypeptide” and “IL-1 protein” include a polypeptide containing an amino acid sequence encoded by the genomic DNA of IL-1 or a fragment thereof, and a homolog thereof. And further includes agonist and antagonist polypeptides. Examples of such an IL-1 protein include IL-1L1, IL-1α, IL-1β and IL-1ra.

“At increased risk” means that an individual carrying a particular polymorphic allele has a disease or condition as compared to the frequency of occurrence of the disease or condition in a population that does not have the particular polymorphic allele. It means that the frequency of expression is statistically significant.

As used herein, “interaction” means a detectable association or relationship between molecules (eg, biochemical interactions, etc.)
Interactions between proteins-proteins, proteins-nucleic acids, nucleic acids-nucleic acids, and proteins-naturally occurring small molecules, or nucleic acids-naturally occurring small molecules are included.

As used herein, "isolated" with respect to nucleic acids, such as DNA or RNA, is separated from other DNA or RNA present in the natural material composed of macromolecules. Refers to a molecule. For example, an isolated nucleic acid encoding one of the IL-1L1 polypeptides of interest is preferably that of a nucleic acid sequence that is immediately adjacent to the IL-1L1 gene in genomic DNA immediately adjacent to it. A length of 10 kb or less, more preferably, a nucleic acid sequence that is naturally flanked so immediately is 5 kb or less, and most preferably is naturally flanked immediately. The length of the nucleic acid sequence is 1.5 kb or less. Isolated, as used herein, is substantially free of cellular material, viral material or culture medium, or chemically synthesized when produced by recombinant DNA technology. When stated, it also refers to nucleic acids or peptides that are substantially free of chemical precursors or other chemicals. In addition, "isolated nucleic acid" also includes nucleic acid fragments that do not naturally occur as fragments and are not found in nature. As used in this specification,
“Isolated” refers to a polypeptide isolated from other cellular proteins and is meant to include purified polypeptides and recombinant polypeptides.

A “knock-in” transgenic animal refers to an animal carrying a modified gene introduced into its genome, such modified gene being exogenous or endogenous.

A “knockout” transgenic animal is one in which expression of an endogenous gene is partially or completely suppressed (eg, deletion of at least a portion of the gene, replacement of at least a portion of the gene with another sequence, (Eg by introducing a stop codon, mutating the bases encoding important amino acids, or removing intron bonds). In a preferred embodiment, the "knockout" locus corresponding to the endogenously modified gene no longer encodes a functionally active polypeptide,
Called the "null" allele. Thus, a knockout transgenic animal of the invention is an animal having a single null mutation for the IL-1 gene (eg, IL-1.
L1 null allele heterozygotes) and animals with two null mutations for the IL-1 gene (such as IL-1 L1 null allele homozygotes).

“Knockout construct” refers to a nucleic acid sequence that can be used for the purpose of reducing or suppressing the expression of a protein encoded by an endogenous DNA sequence in a cell. In a simple example, the knockout construct is IL
-1RN contains a gene in which a significant part of the gene is deleted, whereby the active protein cannot be expressed. As another example, a number of stop codons can be added to a naturally occurring gene to cause premature termination of protein synthesis or intron binding can be inactivated. In a representative knockout construct, some parts of the gene have been replaced by a selectable marker (such as the neogene) and such a gene can be represented as: IL-1RN5 '/ neo / IL-1RN3 '. Here, IL-1RN5 'and IL-1RN3' refer to genomic sequences or cDNA sequences existing upstream and downstream of a position of the IL-1RN gene, respectively, and neo refers to neomycin resistance gene. In another knockout construct, by adding a second selectable marker at the flanking site, it can be represented as: IL-1RN / neo / IL-1RN / TK. Here, TK is the thymidine kinase gene, which can be added to either the IL-1RN5 ′ sequence or the IL-1RN3 ′ sequence of the previously obtained constructs for further selection in appropriate media. Can be done (ie, because TK is a negative selectable marker). This two-marker construct (tw
The o-marker construct) allows the selection of homologous recombination events in which flanking TK markers have been removed from non-homologous recombination events that generally retain the TK sequence. Gene deletions and / or substitutions can occur in regulatory regions such as exons, introns (especially intron binding) and / or promoters.

“Binding disequilibrium” refers to a higher frequency of co-inheritance of two alleles than the expected segregation frequency of occurrence in each allele within a population. The predicted frequency of occurrence of two independently inherited alleles is the frequency of occurrence of the first allele times the frequency of occurrence of the second allele. Alleles that co-occur at the expected frequency are said to be in "binding equilibrium." The cause of binding disequilibrium is often unknown. It may be due to selection for certain allelic combinations, or may have recently occurred in a mixture of genetically heterogeneous populations. Furthermore, in the case of a marker that is tightly bound to a disease gene, there is a possible relationship between the allele (or allele group that is bound) and the disease gene, and the occurrence of mutations related to the disease is very recent. In some cases, equilibrium may not be reached due to recombination events within a particular chromosomal region. For allele patterns containing more than one allele, if all alleles with the first allele pattern are in binding disequilibrium with at least one of the alleles of the second allele pattern. Said the first allelic pattern is in a disequilibrium of binding with the second allelic pattern. Examples of binding disequilibrium include those occurring between alleles at the IL-1RN (+2018) and IL-1RN (VNTR) polymorphic sites. The two alleles present in IL-1RN (+2018) are in 100% binding disequilibrium with the two most alleles present in IL-1RN (VNTR) (allele 1 and allele 2).

“Marker” refers to a sequence within the genome that is known to differ between individuals. For example, the IL-1RN gene is a marker that contains a variable number tandem repeat sequence (VNTR).

As used herein, “modulation” refers to facilitative control (ie, activation or stimulation (eg, by synergism or enhancement, etc.)) as well as inhibitory control (ie, inhibition or suppression (eg, by inhibition or suppression). Antagonism, decrease or inhibition))).

“Mutated gene” refers to an allelic form of a gene and has an mutated gene as compared to an individual who does not have the mutated gene Can change the phenotype of. A mutation is said to be recessive if it must be homozygous for this mutation to alter its phenotype. A mutation is said to be dominant if one copy of the mutated gene is sufficient for genotypic change. A mutation is said to be co-dominant if it has one copy of the mutated gene and the phenotype is intermediate between homozygous and heterozygous for the gene. .

The “non-human animals” of the present invention include rodents, non-human primates, mammals such as sheep, dogs and cows, as well as chickens, amphibians, reptiles and the like. Preferred non-human animals are selected from rodents including rats and mice, most preferably mice, but transgenic amphibians such as Xenopus, and transgenic chickens also affect embryogenesis and tissue formation, etc. It can be an important material for understanding and confirming substances that can. As used herein, a "chimeric animal" refers to an animal in which a recombinant gene is found, or in some, but not all, of the cells of the animal in which the recombinant gene is expressed. "Tissue-specific chimeric animal" refers to the presence and / or expression or disruption of recombinant IL-1L1 gene in one tissue but not in another. .

As used herein, “nucleic acid” refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
The term is equivalent to RNA or D prepared from nucleotide analogues.
Also included are analogs of NA, and single-stranded (sense or antisense strand) and double-stranded polynucleotides, depending on the described embodiment.

“Nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: x” refers to the nucleotide sequence of the complementary strand of the nucleic acid having SEQ ID NO: x. The term "complementary strand" used in the present specification can be used interchangeably with "complement". A nucleic acid strand complement is a coding strand complement or a non-coding strand complement. In the case of a double-stranded nucleic acid, the complement of the nucleic acid having SEQ ID NO: refers to any nucleic acid having the complementary strand of the strand having SEQ ID NO: or the nucleotide sequence of the complementary strand of SEQ ID NO :. Sequence number ×
In the case of a single-stranded nucleic acid having, the complement of this nucleic acid is a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: x. Nucleotide sequences and their complementary sequences are always written in the 5'to 3'direction.

“Percent identity” refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can be determined by comparing at some position in each sequence listed for comparison purposes. If the same base or amino acid is present at the same position in the compared sequences, then the molecules are identical at that position and the same or similar amino acid residues (eg, steric properties and / or electrical properties) at the same position. Of similar physical properties)
The molecules are said to be homologous (similar) at that position. Percentages of homology, similarity or identity are expressed as a function of the number of positions in the compared sequences where the same or similar amino acids are present. A wide variety of sequences and algorithms and / or programs can be used including FASTA, BLAST or ENTREZ and the like. FASTA and BLAST are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) And can also be used with default settings and the like. ENTREZ is the National Center for Biothchnology Info
rmation), the National Library of Medicine, and the National Institute of Health (Bethesda, MD). In one embodiment, the percent identity between two sequences can be determined by using the GCG program with a gap weight of 1, such as a single amino acid or nucleotide between the two sequences. Weight each amino acid gap as if it were a mismatch.

For other side-by-side techniques, see “Methods in Enzymology, vol. 266: Computer-aided methods for sequence analysis of macromolecules”.
.266: Computer Methods for Macromolecular Sequence Analysis) ”((1996
Year), Doolittle Edition, Academic Press
), A division of Harcourt Brace, Inc., San Diego, Calif., USA). Preferably, the sequences are aligned using an alignment program that allows the presence of gaps within the sequences. Smith-Waterman is one of the algorithms that allows the existence of sequences as well as gaps. Meth. Mol. Biol. 70: 173-187 (1997)
checking ... In addition, Needleman and Wunsch
Sequences can also be aligned using the GAP program using the parallel notation of. Another method is scanning with MPSRCH software running on a MASPAR computer. MPSRCH evaluates sequences on a large parallel computer using the Smith-Waterman algorithm. This method improves the ability to pick up distantly related relationships and is also particularly tolerant of small gaps and nucleotide sequence errors. The amino acid sequences encoded by nucleic acids can be used to scan protein and DNA databases.

A database for individual sequences is available in “Methods in E
nzymology, vol.266) "(edited by Doolittle, ibid.). Databases include GenBank, EMBL and Japanese DNA
The database (DDBJ) is an example.

Nucleic acids that have at least 70% identity with the nucleic acid sequences of the sequences set forth in one of SEQ ID NOs: 1-850 are preferred, more preferably 80% and even more preferably 90% , Even more preferably at least 95%. Sequence number 1
At least 90%, more preferably with at least 90% identity with the nucleic acid sequence shown in one of
It will be appreciated that nucleic acid sequences that are 95%, most preferably about 98-99%, are also within the scope of the invention. In a preferred embodiment, the nucleic acid is mammalian. Several alignment tools are available when comparing a novel nucleic acid to a known sequence. An example of such a tool is PileUp, which creates multiple sequences and is described by Feng et al. (J. Mol. Evol. 25). : 351-360 (1987)). Another method, GAP, uses the parallel notation of Needleman et al. (J. Mol. Biol. 48: 443-453 (1970)). GAP is most suitable for comprehensive sequence alignment. The third method is BestFit, which is based on Smith-Waterman's local homology algorithm (Adv. Appl. Math. 2: 482-489 (1
981)) is used to maximize the number of matches by inserting a gap.

“Polymorphism” refers to the coexistence of one or more types of a gene or a part thereof (eg, allelic variant). The portion of a gene in which there are at least two different types (ie, two different nucleotide sequences) is referred to as the "polymorphic region of the gene." A polymorphic region may be a single nucleotide and its identity varies by allele. The polymorphic region may have several nucleotides in length.

“Polymorphic gene” refers to a gene having at least one polymorphic region.
The term “promoter” used in the present specification controls the expression of a selected DNA sequence linked to the promoter so that the selected DNA sequence can be efficiently expressed in a cell. Means a DNA sequence to be expressed in. The term includes "tissue specific" promoters, ie, promoters that allow for the efficient expression of a selected DNA sequence only in a selected cell, such as in a particular tissue. The term also includes "leaky" promoters, ie, promoters that basically regulate the expression of a selected DNA in one tissue but drive expression in other tissues. The term further includes non-tissue specific promoters, as well as promoters that provide constitutive or inducible expression (ie, the level of expression is controllable).

“Predisposed to disease”, as well as “predisposed to disease” or “prone to disease”, as used in the present invention, means that an IL-1 locus polymorphic allele is associated with a particular disease. Means found or predicted to be. Therefore, the frequency of expression of the allele is higher in individuals suffering from the disease when compared to healthy individuals. Therefore, by using these alleles, the disease can be predicted even in the individual before the onset or before the onset of the disease.

As used herein, “protein”, “polypeptide” and “peptide”
The word is used interchangeably.

“Recombinant protein” refers to a polypeptide of the present invention produced by recombinant DNA technology, in which case the DNA encoding the IL-1L1 polypeptide is generally expressed appropriately. It is inserted into a vector, which is then transformed into a host cell to produce the heterologous protein. Furthermore, “derived from” with respect to the recombinant IL-1L1 gene is “recombinant protein”, that is, having the amino acid sequence of a naturally-occurring IL-1L1 polypeptide or a substitution with respect to the naturally-occurring polypeptide. It is meant to include proteins with amino acid sequences similar to those produced by causing mutations, including deletions (including truncations).

As used herein, “small molecule” refers to a composition having a molecular weight of about 5 kDa or less, most preferably about 4 kDa or less. Small molecules include nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic (carbon-containing) or inorganic molecules. Many pharmaceutical companies own vast libraries of chemical and / or biological mixtures, often extracts of fungi, bacteria or algae, and can They can be used to screen for compounds that modulate the biological activity of IL-1L1.

As used herein, “specifically hybridize” or “specifically detect” means at least about 6, about 12 contiguous vertebrates, preferably the IL-1L1 gene. , About 20, about 30, about 50, about 100, about 150, about 200
Refers to the ability of a nucleic acid molecule of the invention to hybridize with about 300, about 350, about 400, or about 425 nucleotides.

“Transcription control sequence” is a general term used herein to refer to DNA sequences such as initiation signals, enhancers and promoters, which are operably linked. And induce or regulate the transcription of protein-encoding sequences. In a preferred embodiment, transcription of one of the IL-1L1 genes is under the control of a promoter sequence (or other transcription control sequence) that controls the expression of the recombinant gene in the cells in which expression is intended. is there. It will also be appreciated that the recombinant gene is under the control of transcriptional control sequences that are the same or different than the transcriptional control sequences that control the transcription of the naturally occurring form of IL-1L1 polypeptide.

As used herein, “transfection” refers to the introduction of a nucleic acid into a recipient cell, such as via an expression vector, by gene transfer via the nucleic acid. As used herein, the term "transformation" refers to a process in which the genotype of a cell is changed by incorporating exogenous DNA or RNA into the cell. In the case of recombinant expression of the peptide or antisense expression of the introduced gene, expression of the native IL-1L1 polypeptide is interrupted.

As used herein, the term “transgene” refers to a nucleic acid sequence introduced into a cell (a sequence encoding one of IL-1L1 polypeptides, or an antisense transcription thereof). Array that encodes the body). The transgene is
Partial or wholly heterologous to the transgenic animal or cell to be introduced, i.e. foreign, can be used, or is the same as the endogenous gene of the transgenic animal or cell to be introduced, Designed or introduced into the animal's genome in such a way as to alter the genome of the introduced cell (eg, introduced at a position different from that of the natural gene, or introduced gene). Introduced at the position where the knockout is obtained).
The transgene can be present in the cell as an episome. The transgene is
It may include one or more transcription control sequences and other nucleic acids such as introns, which are considered necessary for optimal expression of the selected nucleic acid.

“Transgenic animal” refers to any heterologous nucleic acid that has been introduced into one or more cells of the animal body by human intervention (eg, transgenic techniques known in the art). An animal, preferably a non-human mammal,
Refers to birds or amphibians. Nucleic acids are introduced into cells directly or indirectly by introducing them into cell precursors using deliberate genetic engineering methods such as microinjection or infection with recombinant viruses.
The term genetic manipulation does not imply classical mating or in vitro fertilization, but refers to the introduction of recombinant DNA molecules. This molecule either integrates into the chromosome or replicates the DNA extrachromosomally. In the general transgenic animals described herein, the transgene causes cells to express a recombinant form of one of the IL-1L1 polypeptides (eg, agonistic or antagonistic forms, etc.). . However, transgenic animals may also be contemplated in which the recombinant IL-1L1 gene is silent, including, for example, FLP or CRE recombinase dependent constructs as described below. In addition, "transgenic animals" also include recombinant animals in which gene intervention has occurred in one or more IL-1L1 genes due to human intervention (including recombination and antisense methods).

As used herein, “treating” includes curing and alleviating at least one of the symptoms of the condition or disease.

“Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is a nucleic acid that is capable of episome, ie, extrachromosomal replication. Preferred vectors are those in which the nucleic acids linked to them are capable of autonomous replication and / or expression. In the present specification, a vector which is linked to a gene so that it can exert its function and can express the gene is referred to as an “expression vector”. In general, the use of expression vectors in recombinant DNA technology is often carried out in the form of "plasmids," which refers to circular double-stranded DNA loops, in vector form. Is not attached to the chromosome. In the present definition, "plasmid" and "vector" can be used interchangeably as the plasmid is the most widely used form of vector. However, the present invention also includes other expression vectors that serve equivalent functions and are known in the art following this specification.

“Wild-type allele” refers to an allele where the two copies present in an individual exhibit a wild-type phenotype. Because certain nucleotide changes within a gene do not affect the individual phenotype with two copies of the nucleotide-altered gene, there are several different wild-type alleles for a particular gene. there's a possibility that.

4.3 Nucleic Acids of the Invention The invention provides IL-1L1 nucleic acids, homologues thereof, and parts thereof.
A preferred nucleic acid is the nucleotide sequence of the IL-1L1 gene (eg, SEQ ID NOs: 1, 2,
3 or 4, or one of the sequences complementary thereto, or one of the IL-1L1 nucleic acids having ATCC accession number XXXXX or XXXXX), and at least about 60%, 61%, 62 %, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
It is more preferably about 85%, even more preferably about 90%, even more preferably about 95%, and even more preferably about 99%. At least 90 identities with SEQ ID NO: 1, 2, 3 or 4 or one of their complementary sequences
It is self-evident that nucleic acids which are%, more preferably 95% and most preferably about 98-99% are also included in the scope of the present invention. In a preferred embodiment, the nucleic acid is of mammalian origin, and in a particularly preferred embodiment it comprises all or part of the nucleotide sequence corresponding to the coding region such as the nucleic acid set forth in SEQ ID NO: 10 or 11. The coding region corresponds to the human and mouse IL-1L1 ORF sequences contained within the IL-1L1 cDNA sequence of SEQ ID NO: 1 or 4, respectively.

Furthermore, the present invention resides in the 3'UTR (untranslated region) of the IL-1L1 transcript encoded by human and mouse homologues of the IL-1L1 gene and is conserved throughout the process of evolution. A nucleic acid sequence is provided. The consensus sequence consisting of 41 nucleotides corresponding to this conserved region is 5'-ACAATNAAAANCCCGATNCTGGTCTCTANTCNCATNAAAA-3.
'(SEQ ID NO: 12), which starts from nucleotide number 1137 of SEQ ID NO: 1 (hIL-1L1) and nucleotide number 1146 of SEQ ID NO: 4 (mIL-1L1). Generally, the 3'and 5'UTR sequences are not very well conserved throughout evolution, but this is due to the functional polypeptide gene products that make these sequences subject to selection pressure at the biological level. Is not coded. However, some of such non-coding conserved sequences are associated with mRNA processing (
For example, splicing, termination, or polyadenylation), mRNA stability or polypeptide translation initiation, etc., may play an important role. In other cases, such conserved sequences may represent binding sites for transcriptional regulatory proteins (such as transcription activators and repressors) that bind exon sequences and affect upstream transcription initiation sites. is there. This control sequence can affect expression levels or control, such as recombinant gene expression, by inserting it into a heterologous expression system. Alternatively, the newly identified sequence can be removed from a recombinant form of the IL-1L1 gene with the purpose of altering the level and / or pattern of IL-1L1 gene expression.

The present invention also includes isolated nucleic acids that include a nucleotide sequence that encodes an IL-1L1 polypeptide, variants and / or equivalents of such nucleic acids. An equivalent includes a nucleotide sequence that encodes a functionally equivalent IL-1L1 polypeptide or a functionally equivalent peptide having the activity of an IL-1L1 protein as described herein. That is clear. Equivalent nucleotide sequences include sequences that differ by the substitution, addition or deletion of one or more nucleotides (such as allelic variants), and due to the degeneracy of the genetic code, SEQ ID NOs: 1, 2, It also includes sequences that differ from the nucleotide sequence of the IL-1L1 gene shown in 3 or 4.

A preferred nucleic acid is a vertebrate IL-1L1 nucleic acid. Particularly preferred vertebrate IL-1L1 nucleic acids are mammalian. A preferred IL-1L1 nucleic acid, regardless of species, is a vertebrate
At least about 60% similarity or identity with the amino acid sequence of the IL-1L1 protein,
It encodes a polypeptide that is 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95%. In one embodiment, the nucleic acid encodes a polypeptide having at least one of the biological activities of IL-1L1 polypeptide.
It is cDNA. Preferably, the nucleic acid comprises all or part of the nucleotide sequence corresponding to the nucleic acid of SEQ ID NO: 1, 2, 3 or 4.

Yet another preferred nucleic acid of the invention is at least 2, 5, 10, 25, 50
Encodes an IL-1L1 polypeptide composed of 100, 150, or 200 amino acid residues. For example, such nucleic acid can be about 50, 60, 70, 80, 90 or
It can consist of 100 base pairs. Within the scope of the present invention is the use as a probe / primer or antisense molecule (ie non-coding nucleic acid molecule),
Also included are nucleic acid molecules that are at least about 6, 12, 20, 30, 50, 60, 70, 80, 90 or 100 base pairs in length.

Viewed from another aspect, the present invention, under stringent conditions, SEQ ID NO: 1,
Nucleic acid represented by 2, 3 or 4, nucleic acid complementary thereto, or ATCC
Provided is a nucleic acid that hybridizes with a nucleic acid having an accession number of XXXXX or XXXXX. The appropriate stringency conditions that promote DNA hybridization include
For example, treatment with 6.0 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by washing with 2.0 × SSC at 50 ° C., etc., such conditions are known to those of skill in the art, or "The latest protocol in molecular biology (Cu
rrent Protocols in Molecular Biology ”(John Willey & Sons)
Wiley & Sons), New York, 1989, 6.3.1-6.3.6) or "Molecular Cloning: A Laboratory Manual"
(Cold Spring Harbor Press),
1989). For example, the salt concentration in the wash step is about 2.0 at 50 ° C.
It can be selected from low stringency conditions using × SSC to high stringency conditions using about 0.2 × SSC at 50 ° C. Furthermore, the temperature of the washing step is about 22 ° C. at room temperature and the low stringency condition
It can be increased to high stringency conditions at 65 ° C. The temperature and salt concentration can be varied, or other conditions can be varied while keeping the temperature and salt concentration constant. In a preferred embodiment, the IL-1L1 nucleic acids of the invention have SEQ ID NOs: 1, 2, 3 or 4, under moderate stringency conditions (eg, using about 2.0 × SSC and a temperature of about 40 ° C.), Alternatively, it binds to one of the sequences complementary to them. In a particularly preferred embodiment, the IL-1L1 nucleic acid of the invention has SEQ ID NO: 1, under conditions of high stringency.
It binds to 2, 3 or 4 or one of the sequences complementary thereto. In another particularly preferred embodiment, the IL-1L1 nucleic acid sequence of the invention binds under high stringency conditions to one of SEQ ID NOs: 10 or 11 corresponding to the IL-1L1 ORF nucleic acid sequence. .

Due to the degeneracy of the genetic code, a nucleic acid having a sequence different from the nucleotide sequence shown in SEQ ID NO: 1 or 4, or 10 or 11, or a nucleotide sequence complementary thereto is also included in the present invention. Included in the category. Such a nucleic acid encodes a functionally equivalent peptide (ie, a peptide having the biological activity of the IL-1L1 polypeptide) but is listed in the sequence listing due to the degeneracy of the genetic code. The sequence is different from the sequence. For example, there are many amino acids defined by one or more triplet codons. Codons that specify the same amino acid, ie, synonymous codons (eg, CAU and CAC both encode histidine), are "silent" mutations that do not affect the amino acid sequence of the IL-1L1 polypeptide. Become. However, DNA sequence polymorphisms that result in changes in the amino acid sequence of the IL-1L1 polypeptide are believed to exist among mammals. One of ordinary skill in the art will appreciate that one or more nucleotides in a nucleic acid encoding a polypeptide having the activity of an IL-1L1 polypeptide may be due to natural allelic variation between certain individuals (eg, It will be appreciated that there may be changes (up to about 3-5% of the nucleotides).

4.3.1 Probes and Primers IL-1L1 homologues from other cell types (such as from other tissues), depending on the nucleotide sequence determined by cloning the IL-1L1 gene from mammalian tissue. And allows the production of probes and primers designed for use in the identification and / or cloning of IL-1L1 homologues from other mammalian tissues. For example, the invention also provides a probe / primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide is SEQ ID NO: 1, 2, 3, 4, 10 or 11 under stringent conditions. , Or at least about 12, preferably 25, more preferably 40, 50 or 75 contiguous nucleotides in the sense or antisense sequence selected from the group comprising naturally occurring mutants thereof. A region of the nucleotide sequence that hybridizes to
For example, PCR with a primer based on the nucleic acid sequence shown in SEQ ID NO: 10 or 11
It can be used in the reaction and the gene encoding the IL-1L1 polypeptide can be cloned.

In a preferred embodiment, the IL-1L1 primer is designed to optimize specificity and avoid secondary structures that affect priming efficiency. The optimal primers of the present invention are designed such that the "upstream" and "downstream" primers have approximately the same melting point, where the melting point can be determined using the formula:
T _m = 81.5 ° C -16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) -0.63 (formamide%)-(600 / length) or T _m (° C) = 2 (A / T) +4 ( G / C). The optimal IL-1L1 primer is available from the Whitehead Institute for biomedical research.
stitute for Biomedical Research: http://www-genome.wi.mit.edu/cgi-bin/
It is also possible to design using various programs such as "Primer3" provided by primer / primer3.cgi).

In a preferred embodiment, IL-1L1 probe and primers are used to
Human IL-1 locus polymorphisms that occur within and around the L1 gene sequence can be detected. Genetic mutations within the IL-1 locus are thought to be associated with the progression of many diseases and conditions that develop in humans, such as inflammatory and autoimmune diseases, and such circumstances. In, the polypeptide encoded by IL-1 is an important pathogen. Accordingly, the present invention provides human and mouse ILs.
-Probes and primers for IL-1 locus polymorphisms, including polymorphisms involving the -1L1 gene. PCR primers of the invention include those flanking the human polymorphism of IL-1L1 and capable of amplifying and analyzing this region of the genome. Analysis of polymorphic allele identities can be performed, for example, by direct sequencing, or with allele-specific capture probes, or with molecular beacon probes. Alternatively, the polymorphic allele can be directly detected by creating or deleting a restriction endonuclease recognition site in the PCR product, or at least one such that the primer sequence is modified. The individual primers may be subjected to appropriate sequence variations and combined with at least one set of alleles when such sequences are incorporated into the PCR product obtained by amplification of a particular IL-1 polymorphic allele. , Does not combine with another at least one set of alleles of the polymorphism to create a specific restriction site. The IL-1 polymorphism, which corresponds to the variable number tandem repeat (VNTR) polymorphism, can be detected by electrophoretic mobility, and thus the molecular weight of the PCR product can be determined using primers flanking the VNTR. Desired.
For other IL-1 polymorphisms corresponding to restriction fragment length polymorphisms (RFLPs), appropriate IL-1 locus probes and genomic DNA or cDNA obtained from appropriate sample tissues such as human or non-human animals Can be directly detected from the mobility of the band on the Southern blot.

Similarly, probes based on the IL-1L1 sequence can be used to detect transcripts or genomic sequences encoding the same or cognate proteins, which can be used, for example, in prognostic or diagnostic assays. Available (details described below). The present invention provides probes common to variants of the alternatively spliced IL-1L1 transcript, which are complementary to sequences found in, for example, any of SEQ ID NOs: 1, 4, 10 or 11. Corresponding to at least 12 consecutive nucleotides. Furthermore, the present invention provides probes that specifically hybridize to the alternate splice form of the IL-1L1 transcript. For example, using a probe consisting of at least 12 contiguous nucleotides complementary to the sequence found in either SEQ ID NO: 2 or 3, the selective 5'end corresponds to the type of transcript number 1 or number 2. It is possible to detect an alternative spliced form of the human IL-1L1 gene transcript. In a preferred embodiment, the probe further has a label group attached to it so that it can be detected, and examples of such a label group include radioisotopes, fluorescent compounds, enzymes, and enzymes. It is selected from the group including cofactors and the like.

Probes and primers can be prepared and modified as previously described herein for other types of nucleic acids.

4.3.2 Antisense, Ribozymes and Triplex Technology Another aspect of the invention relates to the use of isolated nucleic acids in “antisense” therapy. As used herein, "antisense" therapy refers to cellular mRNA and / or genomic DNA encoding one or more IL-1L1 proteins under cellular conditions. By administering or generating in situ an oligonucleotide molecule or derivative thereof that specifically hybridizes (eg, binds, etc.),
It refers to inhibiting the expression of the protein (eg, by inhibiting transcription and / or translation, etc.). Binding is believed to occur by conventional base pair complementarity or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, “antisense” treatment refers to the scope of the techniques commonly used in the art and includes any treatment based on the specific binding of oligonucleotide sequences.

The antisense construct of the present invention can be delivered, for example, as an expression plasmid, and when the plasmid is transcribed intracellularly, the specific mRNA of the cellular mRNA encoding the IL-1L1 protein can be expressed. Produces RNA that is at least complementary to the target moiety. Alternatively, the antisense construct is an oligonucleotide probe that is produced ex vivo and, when introduced intracellularly, mRNA and / or IL-1L1 gene. The expression is inhibited by hybridizing with the genomic sequence of. Preferably, such oligonucleotides are endogenous endonucleases (eg, exonucleases and / or
Or endonucleases, etc.)
A modified oligonucleotide that is stable in vivo. Examples of nucleic acid molecules used as antisense oligonucleotides include phosphoramidate, phosphothioate, and methylphosphonate analogs of DNA (see also US Pat.Nos. 5,176,996, 5,264,564 and 5,256,775). ). In addition, reviews have been published on general methods for constructing oligomers useful for antisense therapy, see, for example, Van der Krol et al., Bio Techniques 6: 958-976 (1988). ) And stain (St
ein) et al., Cancer Res. 48: 2659-2668 (1988) and the like. Antisense D
Regarding NA, a translation initiation site (for example, − of the IL-1L1 nucleotide sequence of interest −
Oligodeoxyribonucleotides from (such as the region between 10 and +10) are preferred.

For the antisense method, oligonucleotides complementary to IL-1L1 mRNA (DNA or
RNA). Antisense oligonucleotide is IL-1L1 mRNA
It binds to the transcript and blocks translation. Perfect complementarity is preferred, but not necessary. In the case of double-stranded antisense nucleic acid, two pieces of DNA
One of these is tested, or an assay is performed for triplex formation. The ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid is, the more mismatched bases are contained in RNA, but it forms a stable double strand (triplex in some cases). Those skilled in the art can confirm the allowable range of mismatch by using the standard method of measuring the melting point of the hybridized complex.

Sequences complementary to the 5'end of the mRNA (eg, up to the 5'untranslated region including the AUG start codon) should act most effectively at blocking translation. However, recently, a sequence complementary to the 3'untranslated sequence of mRNA was also shown to exert an effect on blocking translation of mRNA (Wagner, R. et al., Nature 372:
333 (1994)). Therefore, oligonucleotides complementary to the 5'or 3'untranslated, noncoding regions of the IL-1L1 gene can be used in the antisense method to prevent translation of endogenous IL-1L1 mRNA. Oligonucleotides complementary to the 5'untranslated region of the mRNA must include a codon complementary to the AUG start codon. Antisense oligonucleotides complementary to the coding region of mRNA have a low translation blocking effect but can also be used according to the present invention. In order to design to hybridize to either 5 ′, 3 ′ or coding region of IL-1L1 mRNA, the antisense nucleic acid needs to be composed of at least 6 nucleotides, and preferably, It has about 100 or less, more preferably about 50, 25, 17 or 10 nucleotides or less.

Regardless of the choice of target sequence, it is preferable to first perform an in vivo experiment to assess the ability of the antisense oligonucleotide to inhibit gene expression. In these experiments, it is preferable to use controls that distinguish between antisense gene inhibition and non-specific biological effects of oligonucleotides. further,
In these experiments, it is preferred to compare the level of target RNA or protein to that of the internal control RNA or protein. In addition, it includes comparing the results obtained with the antisense oligonucleotides with those with the control oligonucleotides. The control oligonucleotide is preferably about the same length as the test oligonucleotide, and the nucleotide sequence of the oligonucleotide is preferably not different from the antisense sequence due to the need to avoid specific hybridization to target cells.

As the oligonucleotide, DNA or RNA, a chimeric mixture thereof or a derivative or modified form thereof can be used, and a single-stranded one or a double-stranded one can be used. Oligonucleotides can be modified at the base site, sugar site or phosphate skeleton for the purpose of improving the stability of the molecule, hybridization and the like. Oligonucleotides may have other addition groups, such as peptides (e.g., used to target host cell receptors), substances that pass through cell membranes (e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989);
Lemaitre et al., Proc. Natl. Acad. Sci. USA 84: 648-652 (1
987); PCT Publication No. WO88 / 09810, etc., published on December 15, 1988) or substances that pass through the cerebrovascular barrier (for example, PCT Publication No. WO89 /, published on April 25, 1988).
10134, etc.), a cleavage agent that is activated by hybridization (see, eg, Krol et al., Bio Techniques 6: 958-976 (1988)), or an intercalating agent (eg, Zon (Zon). ) Et al., Pharm. Res. 5: 539-549 (1
988) and the like) and the like. Finally, the oligonucleotide may be conjugated to other molecules, such as peptides, hybridization-activated cross-linking agents, transport agents, hybridization-activated cleavage agents, and the like.

Antisense oligonucleotides may include at least one modified base site, such sites being 5-fluorouracil, 5-bromouracil, 5
-Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxyethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl cuocosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,
2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil , Β-
D-mannosyl cuocosine, 5'-methoxycarbomethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-
Oxyacetic acid (v), wybutoxosine, pseudouracil, queucin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil -5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w
, And 2,6-diaminopurine, but is not limited thereto.

The antisense oligonucleotides may comprise at least one modified sugar moiety, such a moiety being selected from, but not limited to, the group consisting of arabinose, 2-fluoroarabinose, xylulose and hexose. It is not done.

Antisense oligonucleotides may also have a neutral peptide-like backbone. Such molecules are referred to as peptide nucleic acid (PNA) -oligomers and are described by Perry-O'Keefe et al. (Proc. Natl. Acad. Sci. USA 93: 14670 (19
96)) and Eglom et al. (Nature 365: 566 (1993)). One advantage of PNA oligomers is that they have a neutral DNA backbone and can bind to complementary DNA essentially independently of the ionic strength of the medium. In yet another embodiment, the antisense oligonucleotides may comprise at least one modified phosphate backbone, such backbone being phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramido. It is selected from the group consisting of dates, phosphordiamidates, methylphosphonates, alkylphosphotriesters, and formacetals and analogs thereof.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. The α-anomeric oligonucleotide has a complementary RN
It forms a double-stranded hybrid specifically with A. At this time, the complementary RNA is composed of strands that are parallel to each other in symmetry with the normal β unit (Gauttier (Gauttier
utier) et al., Nucl. Acids Res. 15: 6625-6641 (1987)). Oligonucleotides are 2'-O-methyl ribonucleotides (Inoue et al., Nucl. Acids Res. 1
5: 6131-6148 (1987)) or a chimeric RNA-DNA analog (Inoue et al., F
EBS Lett. 215: 327-330 (1987)).

The oligonucleotides of the present invention may be used in automated DNA synthesizers (Biosearch).
Can be obtained by standard methods known in the art, such as the use of a commercial company, such as Applied Biosystems, Inc.). For example, the phosphorothioate oligonucleotide is a stain (
Stein) et al. (Nucl. Acids Res. 16: 3209 (1988)) and the methylphosphonate oligonucleotides can be prepared using a pore-adjusted glass polymer support (Sarin et al. , Proc. Natl. Acad. Sci.
USA 85: 7448-7451 (1988)).

Although antisense nucleotides complementary to the IL-1L1 coding region sequences can be used, antisense nucleotides complementary to the transcribed untranslated region and the region containing the initiation methionine are most preferred.

Antisense molecules can be delivered to cells expressing IL-1L1 in vivo. Various methods have been developed for the delivery of antisense DNA or RNA to cells, which can, for example, inject the antisense molecule directly into the tissue site or target the desired cells. Modified antisense molecules so designed (eg, antisense linked to a peptide or antibody that specifically binds to a receptor or antigen expressed on the target cell surface) can be administered systemically.

However, in some cases, the intracellular concentration of antisense results in endogenous mR
It may be difficult to reach a concentration sufficient to suppress translation of NA. Therefore,
A preferred method uses a recombinant DNA construct in which the antisense oligonucleotide is under the control of the strong pol III or pol II promoter. By transfecting a target cell of a patient with such a construct to form a base pair complementary to the endogenous IL-1L1 transcript, sufficient transcription of the single-stranded RNA can be achieved. , Thereby blocking the translation of IL-1L1 mRNA. For example, the vector can be introduced so that it is taken up by cells and targets the transcription of antisense RNA. Such a vector remains episomal as long as it can be transcribed to produce the desired antisense RNA,
Or it can be integrated into the chromosome. Such vectors can be constructed by recombinant DNA methods standard in the art. Vectors include plasmids, viruses or others known in the art and can be used for replication and expression in mammalian cells. Expression of the antisense RNA-encoding sequence can be carried out using any promoter known to act in mammalian cells, preferably human cells. Such promoters are inducible or constitutive and include, for example, those of the SV40 early promoter region (Bernoist and Chambon, Nature 290: 304-310 (1981)), the Rous sarcoma virus. A promoter (Yamamoto (Yamamoto)) present in a long 3'terminal repeat
) Et al., Cell 22: 787-797 (1980)), herpes thymidine kinase promoter (
Wagner, R. et al., Proc. Natl. Acad. Sci. USA 78: 1441-144
5 (1981)), regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42 (1982)) and the like, but are not limited thereto. By using any type of plasmid, cosmid, YAC or viral vector, a recombinant DNA construct that can be directly introduced into a tissue site can be prepared. Alternatively, a viral vector that selectively infects a desired tissue can be used, in which case the administration can be performed by another administration route (for example, systemic administration).

Ribozyme molecules designed to enzymatically cleave IL-1L1 mRNA transcripts can also be used to block translation of IL-1L1 mRNA and expression of IL-1L1 (eg, PCT International Publication No. WO 90/11364, issued October 4, 1990; Saver et al., Science 247: 1222-1225 (1990) and US Pat. No. 5,093,24.
See No. 6). Although it is possible to destroy IL-1L1 mRNA with a ribozyme that cleaves mRNA at a site-specific recognition sequence, it is preferable to use a hammerhead ribozyme. Hammerhead ribozymes cleave mRNAs at positions detected by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA consists of two bases with the following sequence: 5'-
UG-3 '. Construction and production of hammerhead ribozymes are known in the art and include Haseloff and Garlac.
ach) (Nature 334: 585-591 (1988)). Human IL-
Within the nucleotide sequences of 1L1 cDNA (FIG. 1) and mouse IL-1L1 cDNA (FIG. 2) are:
There are many sites that can be the cleavage sites for hammerhead ribozymes. Preferably, the cleavage recognition site is located near the 5'end of IL-1L1 mRNA, that is, in order to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts. To operate.

The ribozyme of the present invention also includes an RNA endoribonucleotidease (hereinafter, referred to as “Cech-type ribozyme” in the present specification). Fira (Tetrahymena thermoph
ila) (naturally known as IVS or L-19IVS RNA) and those described in detail by Thomas Cech and co-workers (Zaug et al. Science 224: 574-578 (1984); Zaug and Cech, Science 231: 470-475 (1986); Zaug et al., Nature 32.
4: 429-433 (1986); Published International Patent Publication No. WO88 / 04300 by University Patents; Bean and Cech, Cell 4
7: 207-216 (1986)) and the like. The sec-type ribozyme has an active site composed of 8 base pairs that hybridizes to the target RNA sequence and replaces it after cleavage of the target RNA. The present invention includes the sec-type ribozyme as described above, which targets the 8-base pair active site sequence present in the IL-1L1 gene.

Similar to the antisense method, ribozymes can be composed of modified oligonucleotides (eg, for the purpose of improving stability, targeting accuracy, etc.), and IL-1L1 in vivo. It must be delivered to cells that express the gene. The preferred transport method is pol II, which is a strong constitutive promoter.
Using a DNA construct that "encodes" a ribozyme under the control of I or pol II, which is sufficient to disrupt the endogenous IL-1L1 genetic information and inhibit translation. This is because the ribozyme of is produced in the infected cell. Ribozymes, unlike antisense molecules, are catalytic and require low intracellular concentrations for efficiency.

Expression of the endogenous IL-1L1 gene was determined by targeting homologous recombination.
ination) and inactivating or “knocking out” the IL-1L1 gene or its promoter (eg, Smithies (
Smithies) et al., Nature 317: 230-234 (1985); Thomas and Capecchi, Cell 51: 503-512 (1987); Thompson et al., Cell.
5: See 313-321 (1989). Each of these documents is incorporated herein by reference in its entirety). For example, a selective marker for a mutant non-functional IL-1L1 (or a completely unrelated DNA sequence) flanked by DNA of the same species as the endogenous IL-1L1 gene (coding region or control region of the IL-1L1 gene) IL-1L1 in vivo with and / or without a negative selective marker
Can be transfected into cells expressing. The IL-1L1 gene was inactivated by the insertion of the DNA construct via the targeted homologous recombinant. Such a method is particularly preferable in the agricultural field, and ES (embryonic stem) cells can be used to obtain offspring of animals having inactivated IL-1L1. (Thomas and Capecchi
), (1987) ibid., And Thompson et al., (1989) ibid.). However, this method can also be applied to humans and recombinant DNA
The constructs can be administered directly or delivered to the required site in vivo using a suitable viral vector.

[0143] Alternatively, a targeting deoxyribonucleotide sequence complementary to the regulatory region of the IL-1L1 gene (ie, IL-1L1 promoter and / or enhancer) is used, and the target intracellular deoxyribonucleotide sequence is used. IL-1
By forming a triple helix structure that blocks transcription of the L1 gene, endogenous IL-1
The expression of the L1 gene can be reduced (generally, Helen (He
Lene), C. Anticancer Drug Res. 6 (6): 569-584 (1991); Helene,
C. Ann. NY Acad. Sci. 660: 27-36 (1992); Maher, LJ Bioas
says 14 (12): See 807-815 (1992)).

The nucleic acid molecule used for triple helix formation to inhibit transcription is preferably single-stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides results in the formation of triple helices according to the Hoogsteen base pairing rule that either the purines or pyrimidines present in one of the duplexes generally need to spread significantly. Facilitate. Since the nucleotide sequence is often based on pyrimidines, the three strands of the resulting triple helix will intersect in the TAT and CGC triplets. A pyrimidine-rich molecule provides a base complementary to the purine-rich region of the duplex in a direction parallel to the strand. Furthermore, as the nucleic acid molecule, a purine-rich nucleic acid having a spread of G residues is selected. These molecules form triplexes with GC-pair-rich DNA duplexes, with most of the purine residues present in one of the target duplexes and the triple helix CGC triplets intersect between the three chains.

Alternatively, the sequences that can be targeted for triple helix formation can be increased by preparing so-called "switchback" nucleic acid molecules. The switchback molecule first base pairs to one strand, then another, eliminating the need for a fairly large stretch of purines or pyrimidines present in one of the duplexes. Alternating 5'to form a base pair for one strand
Synthesize in a 3 ', 3'-5' fashion.

The antisense RNA and DNA, ribozymes, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. Such techniques include chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides known in the art, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be prepared by in vitro and in vivo transcription of DNA sequences encoding antisense RNA molecules. Such DNA sequences can be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

In addition, various known modifications to nucleic acid molecules can be introduced as a means of promoting intracellular stability and extending half-life. Possible variations include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5'and / or 3'ends of the molecule, oligodeoxyribo. Use phosphorothioates or 2'O-methyl rather than phosphodiester bonds within the nucleotide backbone.

4.3.3. Vectors Encoding IL-1L1 Protein and Cells Expressing IL-1L1 Further, the present invention provides plasmids and vectors encoding IL-1L1 protein, which can be used in host cells for IL- 1L1 protein can be produced. As a host cell, any prokaryotic cell or eukaryotic cell can be used. Therefore, the recombinant IL-L-derived protein was cloned from a mammalian IL-1L1 protein, and the nucleotide sequence encoding the full-length or selected portion of the protein was used, and the process of utilizing a microbial cell or a eukaryotic cell was performed. A 1L1 polypeptide can be produced. A polynucleotide sequence is ligated into a gene construct such as an expression vector, and a eukaryotic cell (yeast, bird, insect or mammal) or a prokaryotic cell (bacteria) is used as a host to transform such a host or transduce into such a host. Perfecting is a standard method known in the art.

A vector that allows a nucleic acid to express in a cell is called an expression vector. Generally, the expression vector used to express the IL-1L1 protein comprises a nucleic acid encoding an IL-1L1 polypeptide and operably linked to at least one transcription control sequence. Control sequences are well known in the art and are of interest.
Selected to directly express the IL-1L1 protein. For transcription control sequences,
Goeddel, “Gene Expression Technology: Methods in Enzymology (Gene Expressi
on Technology: Methods in Enzymology) 185 ”(Academic Press (Acad
emic Press), San Diego, Calif. (1990)).
In one embodiment, the expression vector comprises a recombinant gene encoding a peptide having agonistic activity for IL-1L1 polypeptide, and conversely IL.
-1 Includes a recombinant gene encoding an antagonist peptide for the 1L1 protein.

Vectors suitable for the production of IL-1L1 polypeptides include pBR322-derived plasmids, pEMBL-derived plasmids, pEMBL-derived plasmids for expression in prokaryotic cells such as E. coli.
Examples include EX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

There are many vectors for recombinant protein expression in yeast. For example, YEP24
, YIP5, YEP51, YEP52, pYES2 and YRP17 are cloning and expression vehicles useful for introducing gene constructs into S. cerevisiae (eg Broach et al., (1983 ) "Experimental manipulation of gene expression (Experimental
mental Manipulation of Gene Expression ”(M. Inouye, Ed., Academic Press, page 83, etc., incorporated herein by reference). These vectors are able to replicate in E. coli due to the presence of pBR322 ori, and due to the presence of the replication determinant of the yeast 2 micron plasmid, S. cerevisiae.
Can be duplicated in. In addition, resistance markers for drugs such as ampicillin can be used. In one embodiment, SEQ ID NO: 1 or 4
An IL-1L1 polypeptide is produced by recombination using an expression vector prepared by subcloning a sequence encoding one of the IL-1L1 genes shown in 1.

Preferred mammalian expression vectors have prokaryotic sequences for the vector to be able to amplify in bacteria, as well as one or more eukaryotic transcription units that are expressed in eukaryotic cells. Examples of suitable mammalian expression vectors for transfection of eukaryotic cells include pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt,
Examples include pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg. Some of these vectors have been modified with sequences from bacterial plasmids such as pBR322 to allow replication in prokaryotic and eukaryotic cells and to allow drug resistance selection. Separately, viral derivatives such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived and p205) are used to transiently transfer proteins in eukaryotic cells. Can be expressed. In the field,
Numerous methods are known for the preparation of plasmids and the transformation of host organisms.
For other expression systems suitable for both prokaryotic and eukaryotic cells, see “Molecular Cloning: Laboratory Manual (Molecular Cloning
: A Laboratory Manual) 2nd Edition "(Sambrook, Frisch (F
ritsch and Maniatis, Cold Spring Harbor Laboratory Press, (1989).
Year)) See Chapters 16 and 17.

In some embodiments, recombinant IL-1 was prepared using a baculovirus expression system.
It is desirable to express the L1 polypeptide. Examples of such baculovirus expression systems include pVL-derived vectors (pVL1392, pVL1393 and pVL941 etc.)
, PAcUW-derived vectors (pAcUW1, etc.) and pBlueBac-derived vectors (β-gal
And pBlueBac III).

If it is desired to express only a portion of the IL-1L1 protein, such as the form lacking the N-terminus, ie a truncation mutation lacking the signal peptide, an oligo containing the sequence desired for expression. Start codon (ATG) in the nucleotide fragment
Need to be added. It is known that methionine at the N-terminal position can be cleaved by using an enzyme called methionine aminopeptidase (MAP). MAP
E. coli (Ben-Bassat et al., J. Bacteriol. 169).
: 751-757 (1987)) and Salmonella typhimurium, and confirmed in vitro activity for recombinant proteins (Miller et al., PNAS 84: 2718-). 2722 (1987)). Thus, if desired, removal of the N-terminal methionine will result in removal of the IL-1L1-derived polypeptide in a host that produces MAP, such as E. coli or CM89 or S. cerevisiae. Expression in vivo (in vivo)
Or in vitro using purified MAP (eg, the method of Miller et al., Ibid.).

Furthermore, using the gene constructs of the invention as part of a gene therapy protocol,
Nucleic acids encoding agonistic or antagonistic forms of one of the IL-1L1 proteins can be delivered. Therefore, from another aspect of the present invention, in vivo IL-1L1 polypeptide in a specific cell type for the purpose of reconstructing or inhibiting the function of IL-1L1 in a tissue. (In vivo)
Or, it relates to a vector for in vitro transfection as well as for expression. Such manipulations are desirable, for example, if the naturally occurring form of the protein is misexpressed or if the naturally occurring form of the protein is mutated and has reduced activity.

In addition to viral delivery methods, non-viral delivery methods can also be used to drive expression of the IL-1L1 polypeptide in animal tissues. The most commonly used non-viral method of gene transfer is based on the normal mechanisms by which mammalian cells take up and transport intracellular macromolecules. In a preferred embodiment, the non-viral targeting method of the present invention is based on the endocytic pathway for the uptake of the IL-1L1 polypeptide gene by target cells. Examples of such modalities of targeting methods include liposome-derived systems, polylysine conjugates, artificial viral envelopes, and the like.

In another embodiment, the transgenic animals described in detail below can be used to produce recombinant proteins.

4.4. Polypeptides of the Invention The invention makes it possible to obtain isolated IL-1L1 polypeptides isolated from other proteins of cell origin or substantially free of other proteins. . "Substantially free of other protein of cell origin" (also referred to herein as "contaminant protein") or "substantially pure or purified preparation" means that the contaminating protein is about 20%. % (Dry weight) or less, preferably less than about 5% contaminating protein is included in the preparation of IL-1L1 polypeptides. By using the cloned genes described herein, the functional polypeptide of interest was first prepared as a purified preparation.

Preferred IL-1L1 proteins of the present invention have at least about 60%, 65%, 66%, 67%, 68% identity or homology with the amino acid sequence set forth in SEQ ID NO: 5 or 6.
%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80
%, 85%, 90% or 95%. A more preferred IL-1L1 protein is a sequence composed of at least 10, 20, 30 or 50 amino acid residues, wherein the sequence is the amino acid sequence shown in SEQ ID NO: 5 or 6. There is at least about 70%, 80%, 90%, 95%, 97%, 98% or 99% identity or homology with the sequence. Such proteins include recombinant proteins, which can be produced in vitro from a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 1, 4, 10 or 11 or a sequence homologous thereto. it can. For example, a preferred recombinant polypeptide according to the invention is SEQ ID NO: 1, 4, 10 or 11.
Encoded by a nucleic acid that has at least 85%, more preferably 90%, and most preferably 95% homology to the nucleotide sequence shown in. Also included within the scope of the invention are polypeptides encoded by nucleic acids that have at least about 98-99% homology to the sequences set forth in SEQ ID NOs: 1, 4, 10 or 11.

In a preferred embodiment, the IL-1L1 protein of the present invention is a mammalian IL-1L1 protein. In a particularly preferred embodiment, the IL-1L1 protein is shown in SEQ ID NO: 5 or SEQ ID NO: 6. In a particularly preferred embodiment,
The IL-1L1 protein has IL-1L1 biological activity. It will be apparent that certain post-translational modifications, such as phosphorylation, may increase the apparent molecular weight of the IL-1L1 protein relative to the unmodified polypeptide chain.

The present invention also relates to isoforms of the protein encoded by the spliced variant of the invention. Such isoforms have a biological activity that is the same as or different from the biological activity possessed by the IL-1L1 protein identified by SEQ ID NO: 5 or 6. For example, such isoforms are generated by splicing at different sites in one or more of the IL-1L1 gene transcripts.

IL-1L1 Polypeptide Functions as an Agonist or Antagonist for at least One of the Biological Activities of Wild-Type (“Authentic”) IL-1L1 Proteins Described in the Attached Sequence Listing Is preferred. "Evolutionally related" with respect to the amino acid sequence of IL-1L1 protein refers to polypeptides having naturally occurring amino acid sequences and mutants of human IL-1L1 polypeptide derived, for example, by combinatorial mutagenesis. .

The entire length of the protein, or one or more specific motifs and / or domains or any size (eg, at least 5, 10, 20, 25, 50 at least in amino acid sequence length). Fragments, such as 1, 75, and 100) are within the scope of the invention.

For example, the isolated IL-1L1 polypeptide is encoded by all or a portion of the nucleic acid sequence set forth in any of SEQ ID NOs: 1, 4, 10 or 11.
The peptidyl portion of the IL-1L1 protein is obtained by screening peptides recombinantly produced from fragments of nucleic acid encoding such peptides. In addition, conventional Merrifield solid-phase f-
Fragments can be chemically synthesized using techniques known in the art such as Moc or t-Boc chemistry. For example, the IL-1L1 polypeptides of the invention can be optionally divided into non-overlapping fragments of the desired length, or, preferably, overlapping fragments of the desired length. The fragments can be produced recombinantly or by chemical synthesis, and further tested to identify peptidyl fragments that can function as agonists or antagonists of the wild-type ("authentic") IL-1L1 protein. You can

IL-1L1 polypeptides can be membrane bound or soluble. Preferred soluble IL-1L1 polypeptides are polypeptides that do not contain a hydrophobic signal sequence chain. Such proteins can be produced by genetic engineering using methods known in the art. The solubility of the recombinant polypeptide is believed to be increased by the loss of the hydrophobic domain of the wild-type protein, such as the putative transmembrane domain. For example, SMART (Simple Modular Architecture R
esearch Tool, v.3.0; Schultz et al., Proc. Natl. Acad. Sci. U. S.
.A. 95: 5857-5864 (1988)), huIL-1L present in amino acid numbers 53 to 73.
One polypeptide sequence was predicted to encode a transmembrane segment having the sequence LSPVILGVQGGSQCLSCGVG. Therefore, the soluble IL-1L1 protein may have an amino acid sequence near amino acid numbers 74 to 155 of SEQ ID NO: 5 or 6. The second IL-1L1 transmembrane segment is GLTS, located at amino acids 105-125.
The sequence is predicted to be SFESAAYPGWFLCTVP. Thus, certain soluble forms of recombinant IL-1L1 may lack this domain, such as an IL-1L1 polypeptide comprising amino acids 1 to 105 of SEQ ID NO: 5 or 6. Is mentioned. Alternatively, the predicted two transmembrane regions can be deleted and / or sequences distant from these domains can be recombined into recombinant form. For example, a recombinant soluble IL-1L1 polypeptide is produced that includes amino acid numbers 1-52, 74-104 and 126-155 of SEQ ID NO: 5 or 6. Leaderless recombinant interleukin (IL-1) receptor antagonists lacking the native signal sequence accumulate in osmosensitive cells when expressed recombinantly in E. coli (Tostenson (IL Thostenson) et al., J. Bacteriol 179.
: 5333-5339 (1997)). Thus, analog recombinant forms of IL-1L1 polypeptides optimized for recovery of soluble native polypeptides are also within the scope of the invention.

Generally, a polypeptide having an IL-1L1 protein activity (eg, “biological activity”, etc.) according to the invention is any of SEQ ID NOs: 1, 4, 10 or 11. It includes an amino acid sequence encoded by the full length or a portion of the nucleic acid sequence shown in one and further mimics or antagonizes all or part of the biological / biochemical activity of the naturally occurring IL-1L1 protein. Refers to a polypeptide. An example of such a biological activity is a conserved structural region called the IL1 domain (Dinarello, Cytokine Growth Factor Rev. 8).
: 253-265 (1997); Patarca et al., Crit. Rev. Oncol. 8: 142-188 (199).
7); Kurzrock et al., Cytokines Mol. Ther. 1: 177-184 (1995))
And the like, which is in the range of amino acid numbers 4 to 152 of SEQ ID NO: 5 or 6. This domain contains a region having a particularly high homology with IL-1β, which is an IL-1 agonist containing the IL-1L1 agonist consensus sequence, and such a consensus sequence includes amino acid numbers of SEQ ID NOs: 5 and 6. LKXL present in 16-21
XL, FESA present in amino acid numbers 111-114 of SEQ ID NOS: 5 and 6, ITDFXXQ present in amino acid numbers 146-152 of SEQ ID NOS: 5 and 6. In addition, the IL-1L1 polypeptides of the present invention include conserved sequences present within the IL-1ra polypeptide, which is an IL-1 antagonist. For example, a conservative IL-1 antagonist polypeptide comprises the following sequence which is an IL-1L1 / IL-1ra consensus sequence; SEQ ID NO: 5 or 6
YLXNNQLXAGXL present in amino acid numbers 20 to 31, LEXVNIXXL present in amino acid numbers 80 to 88 in SEQ ID NO: 5 or 6 and amino acid numbers 108 to 108 in SEQ ID NO: 5 or 6.
TXSFESAAXPGWFLCTXXEADQPV present in 131. A structural comparison of IL-1L1, the agonist IL-1β and the antagonist IL-1ra revealed that amino acid numbers 1-140 of the human interleukin-1β precursor (GenBank Accession No. P01584). The presence of a long amino acid terminal domain was revealed, which is absent in the IL-1ra polypeptide and in the IL-1L1 polypeptide of SEQ ID NO: 5 or 6. Therefore, the absence of this IL-1 agonist domain
-1 Associated with antagonist polypeptides. The recombinant IL-1L1 polypeptide of the invention is a human interleukin-1β precursor fused to an IL-1L1 polypeptide sequence having one or more segments of the IL-1L1 polypeptide of SEQ ID NO: 5 or 6. Amino acid number 1 of GenBank accession number P01584)
Includes an array consisting of ~ 140.

Another structural feature of the IL-1L1 polypeptide is that it contains a glycosylation consensus sequence, such as a myristylation sequence such as: amino acid numbers 29-34 of SEQ ID NO: 5. Present in GLOHA, GLHAGK present in amino acids 30-35 of SEQ ID NO: 5, GVQG present in amino acids 60-65 of SEQ ID NO: 5
GS, GGSQCL present in amino acid numbers 63 to 68 of SEQ ID NO: 5, GQEPTL present in amino acid numbers 73 to 78 of SEQ ID NO: 5, GAKE present in amino acid numbers 91 to 96 of SEQ ID NO: 5
SK, GLTSSF present at amino acid numbers 106-111 of SEQ ID NO: 5. Furthermore, the IL of the present invention
-1L1 polypeptide has the following two casein kinase 2 phosphorylation sites; SSFE present at amino acid numbers 109-112 of SEQ ID NO: 5, amino acid number 12 of SEQ ID NO: 5.
TVPE present in 3-126.

Other biological activities of the IL-1L1 protein are theoretically apparent to those of skill in the art. According to the invention, a polypeptide has biological activity if it is a specific agonist or antagonist for the native form of the IL-1L1 protein.

As an assay to determine whether a compound (eg, a protein such as the IL-1L1 protein or variants thereof) has one or more of the above biological activities. Include assays known to those of skill in the art used to assess IL-1 agonist activity and IL-1 antagonist activity. For example, the ability of recombinant IL-1L1 polypeptides to activate interleukin-6 gene expression in human skin fibroblasts is an indicator of IL-1β-like IL-1 agonist activity. Conversely, the ability of recombinant IL-1L1 polypeptides to interfere with IL-1α or IL-1β-induced interleukin-6 gene expression was determined by IL-1α.
Is a measure of IL-1 antagonist activity. Since the IL-1L1 gene is known to be highly expressed in the human placenta, and IL-1L1 specific receptors are considered to be present in such tissues, such an assay is basically It is desirable to perform on human umbilical vein endothelial cells.

Other preferred proteins of the present invention include those encoded by the nucleic acids described in the section relating to the nucleic acids of the present invention. In particular, the invention provides fusion proteins such as IL-1L1-immunoglobulin fusion proteins. It is considered that such a fusion protein can improve, for example, the stability and solubility of the IL-1L1 protein, and thus can be used for therapy. The fusion protein can also be used to produce an immunogenic fragment of the IL-1L1 protein. For example, the VP6 capsid protein of rotavirus is IL-
It can be used in monomeric or viral particle form as an immunogenic carrier protein for a portion of the 1L1 polypeptide. IL trying to induce antibody
A set of nucleic acid sequences corresponding to a portion of the -1L1 protein incorporated into a fusion gene construct containing a sequence encoding a late vaccinia virus structural protein and expressing a fusion protein containing an IL-1L1 epitope as part of the virion Recombinant virus can be produced. Similar applications of recombinant hepatitis B virions have been demonstrated by utilizing immunogenic fusion proteins with hepatitis B surface antigen fusion proteins. Similarly, chimeric constructs encoding fusion proteins comprising a portion of the IL-1L1 protein and a poliovirus capsid protein can be created to enhance the immunogenicity of a range of polypeptide antigens (eg, EP Publication). No. 0259149, Evans et al., Nature 339: 385 (1989).
), Huang et al., J. Virol. 62: 3855 (1988), Schlienger.
) Et al., J. Virol. 66: 2 (1992), etc.).

Immunogens can also be generated using the multi-antigen peptide approach to peptide-based immunization, where the desired portion of the IL-1L1 polypeptide is the organic chemistry of the peptide on the oligomeric branched lysine core. It can also be obtained directly by synthesis (see, for example, Posnett et al., JBC 263: 1719 (1988), Nard.
elli) et al., J. Immunol. 148: 914 (1992), etc.). The antigenic determinants of the IL-1L1 protein are also expressed and displayed by bacterial cells.

In addition to using fusion proteins to enhance immunogenicity, it is widely recognized that fusion proteins can be used to express proteins and thus have utility in expressing IL-1L1 polypeptides of the invention. There is. For example, the IL-1L1 polypeptide can be produced as a glutathione-S-transferase (GST-fusion) protein. Such GST-fusion proteins can be readily purified from the IL-1L1 polypeptide, eg, by using a glutathione derivatized matrix (eg, “Current Protocols for Molecular Biology (Current Pr
otocols in Molecular Biology) ", edited by Ausubel et al., John Wiley & Sons (New York), 1991, etc.). Furthermore, the use of a fusion of IL-1L1 polypeptide with a small epitope tag (eg, FLAG or hemagglutinin tag sequence) facilitates immunological purification of the resulting recombinant polypeptide. Alternatively, immunological detection in a cell or tissue sample can be performed. In addition, IL-1L1 polypeptides can be localized in living cells and tissues by fusion to commercially available green fluorescent protein and recombinants thereof known in the art.

Furthermore, the present invention relates to a method of producing an IL-1L1 polypeptide of interest.
For example, a host cell transfected with a nucleic acid vector that expresses a nucleotide sequence encoding a polypeptide of interest can be cultured under suitable conditions to allow peptide production. Suitable media for cell culture are known in the art. By using a protein purification technique known in the art (for example, ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis and immunoaffinity purification using a peptide-specific antibody), a culture medium, Recombinant IL-1L1 polypeptides can be isolated from host cells or both. In a preferred embodiment, the recombinant IL-1L1 polypeptide is a fusion protein containing a domain such as a GST fusion protein useful for purification.

In addition, in general, under certain circumstances, an IL-1L1 agonist (for the purpose of promoting or inhibiting only a portion of the biological activity of the native protein) (
IL-1 with limited function to either mimetic) or an IL-1L1 antagonist
A homologue to one of the L1 polypeptides can be provided. Therefore, treatment with functionally restricted homologues can elicit a specific biological effect, using agonists or antagonists for all biological activities of the native IL-1L1 protein. Side effects are reduced as compared to the case of treatment by

Homologues for each IL-1L1 protein can be created by mutagenesis, such as point mutations, or truncation. For example, a homolog can be generated by mutation that retains an activity that is substantially the same as, or a portion of, the biological activity of the original IL-1L1 polypeptide. Alternatively, an antagonistic protein that can inhibit the function of the native protein can be produced by competitively binding to the IL-1L1 receptor.

Recombinant IL-1L1 polypeptides of the present invention also include homologues of wild-type IL-1L1 protein, eg, protein mutations due to mutations that alter ubiquitination or other enzyme targets associated with the protein. Examples include proteins that have acquired resistance to cleavage.

IL-1L1 derivatives are produced by chemically modifying IL-1L1 polypeptides to form covalent or aggregate conjugates with chemical sites such as glycosyl groups, lipids, phosphate groups, acetyl groups, and the like. You can also do it. Covalent derivatives of the IL-1L1 protein can be found on the amino acid side chains of the protein or at the N-terminus or C of the polypeptide.
It can be prepared by linking a chemical site to the terminal functional group.

Modification of the structure of the IL-1L1 polypeptide improves the therapeutic or prophylactic effect and stabilizes the stability (
For example, it can be performed for the purpose of improving ex vivo storage period and resistance to proteolysis, or post-translational modification (for example, changing the phosphorylation pattern of a protein). Such modified peptides, when designed to retain at least one of the activities of the native protein or to produce specific antagonists thereto, are IL-1L1 polypeptides according to the invention. Is considered to be functionally equivalent to. Such modified peptides can be prepared, for example, by amino acid substitution, deletion or addition. Conservative or non-conservative amino acids are substituted in the substitution variants.

For example, among the isolated substitutes, leucine is replaced with isoleucine or valine, aspartic acid is replaced with glutamic acid, and threonine is replaced with serine, or similarly, an amino acid is replaced with a structurally related amino acid. Of course, the substitutions (eg iso-configuration and / or iso-electric mutations etc.) should not be expected to have a significant effect on the biological activity of the resulting molecule.
Conservative substitutions that occur within a family of amino acids are associated with side chains. Typically,
The encoded amino acids can be divided into four families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine. , Proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarity =
Glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Similarly, the amino acid populations can be divided as follows: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) aliphatic = glycine, alanine, valine, leucine. , Isoleucine, serine, threonine, where serine and threonine are further divided into groups having aliphatic hydroxyl groups; (4) aromatic = phenylalanine, tyrosine, tryptophan;
(5) amide = asparagine, glutamine; and (6) sulfur-containing = cysteine, methionine (for example, "Biochemistry Second Edition", L. Stryer (
Stryer) edited by WH Freeman, 1981 etc.). Whether the change in the amino acid sequence within the peptide resulted in a functional IL-1L1 homologue (eg, a function in the sense that the resulting peptide mimics or antagonizes the wild type) is mutated. It can be readily determined by assessing the ability of the somatic peptide to respond intracellularly in a manner similar to the wild-type protein, or to competitively inhibit such response. Polypeptides having one or more substitutions can be similarly examined.

The invention further relates to methods of making a series of combinatorial and truncation mutants of the IL-1L1 protein, and the invention also provides mutant sequences (eg,
It is particularly useful for identifying potentially homologous sequences. The purpose of screening such combinatorial libraries is, for example, to create new IL-1L1 homologs that can act as agonists or antagonists, or alternatively, combine novel activities. Therefore, the homologue derived from the combinatorial can enhance the activity as compared with the native protein.

In one embodiment, the variegated IL-1L1ary among the IL-1L1 variants is encoded by a variegated gene, 1IL-1L1ary, produced by a combinatorial mutation at the nucleic acid level. ing. For example, such that a modified set of candidate IL-1L1 sequences can be expressed as independent polypeptides, or IL-1L1
Enzymes can be used to ligate a mixture of synthetic oligonucleotides within a gene sequence so that it can be expressed as a set of large fusion proteins (eg, phage display, etc.) that include the set of sequences.

There are a number of ways to generate such libraries of candidate IL-1L1 homologues from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automatic DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector. The purpose of using the degenerate set of genes is to provide all the sequences encoding the desired set of candidate IL-1L1 sequences in a single mix. Synthesis of degenerate oligonucleotides is known in the art (eg, Narang, SA, Tetrahed.
ron 39: 3 (1984); Itakura et al., "Recombinant DNA, Proc. 3rd Cleve
land Sympos. Macromolecules) ”(AG Walton et al., Ed., Amsterdam, Elsevier, pp.273-89) (1981); Itakura
Rev. Biochem. 53: 323 (1984); Itakura et al., Scie.
nce 198: 1056 (1984); Ike et al., Nucleic Acids Res. 11: 477 (1983)). Such techniques have been used to induce selective changes in other proteins (eg, Scott et al., Science 249: 386-390 (19).
90); Roberts et al., PNAS 89: 2429-2433 (1992); Devlin.
Et al., Science 249: 404-406 (1990); Cwirla et al., PNAS 87: 6378-63.
82 (1990); and US Pat. Nos. 5,223,409, 5,198,346 and 5,096,815, etc.).

Similarly, a library of coding sequence fragments for IL-1L1 clones can be provided for the purpose of creating a diverse population of IL-1L1 fragments for screening and selection of bioactive fragments. Various techniques are known in the art for creating such libraries, including chemical synthesis. In one embodiment, a library of coding sequence fragments can be generated as follows: (i) the IL-1L1 coding sequence is generated under conditions such that the nick occurs only once per molecule. The double-stranded PCR fragment is treated with nuclease to (ii) denature the double-stranded DNA, and (iii) restore the DNA to its original state, containing a sense / antisense pair derived from another nick product. The single-stranded portion is removed from the regenerated double strand by producing double-stranded DNA and treating with (iv) S1 nuclease, and (v) the resulting fragment library is placed in an expression vector. Link. According to this embodiment, expression libraries can be obtained which encode the N-terminus, C-terminus and internal fragments of various sizes.

Extensive techniques are known in the art for screening gene products of combinatorial libraries created by point mutations or truncations, as well as screening cDNA libraries for gene products that have certain characteristics. . In general, such techniques can be applied to the rapid screening of gene libraries generated by combinatorial mutations of IL-1L1 homologues. The most widely used technique for screening large gene libraries is to clone a gene library into a replicable expression vector and transform the appropriate cells using the library obtained. Furthermore, regarding a vector encoding a gene whose product has been detected, expressing the combinatorial gene under conditions such that the isolation of the vector is relatively easy by detecting the desired activity. including.
Each of the assays described below can be utilized for the high throughput analysis required to screen a large number of degenerate IL-1L1 sequences generated by combinatorial mutagenesis. Combinatorial mutations can create very large libraries of muteins (eg, about 10 ²⁶ ). Combinatorial libraries of this size are technically challenging even with high throughput screening assays. In order to solve this problem, a new technique, recursive ensemble mutagenesis (recrusiv)
e ensemble mutagenesis (REM) has been developed, which eliminates the majority of non-functional proteins present in random libraries and increases only the frequency of functional proteins, hence the effective use of sequence space. There is a reduction in the complexity needed to do the sampling. REM is an algorithm that increases the frequency of functional mutants in a library when using appropriate selection or screening methods (Arkin and Yourvan, PNAS USA 89: 7811-
7815 (1992); Yourvan et al., "Analysis of parallel problems in nature 2
(Parallel Problem Solving from Nature 2) ", edited by Maenner and Manderick, Elsevier (Amsterdam), pp.401-410 (1992); Delgrave et al., Protein Engineerin
g 6 (3): 327-331 (1993).

The invention provides mimetics (eg, peptides or non-peptides) such as small molecules that can reduce the IL-1L1 protein and prevent the IL-1L1 polypeptides of the invention from binding to molecules such as target peptides. Production of peptide agents, etc.). Accordingly, such mutagenesis techniques described above may be used, for example, for the IL-1L1 protein involved in protein-protein interactions involved in, for example, the binding of the IL-1L1 polypeptide to a target peptide. It is also useful for mapping the determinants of. For example, it is possible to identify important residues of IL-1L1 polypeptide that are related to molecular recognition of the receptor, and using this, IL-1L1-derived peptide mimetics,
Alternatively, small molecules can be generated that competitively inhibit the binding of the authentic IL-1L1 protein to its site. For example, by utilizing scanning mutagenesis, the IL-1L1 protein that enables interaction by mapping the amino acid residues of the IL-1L1 protein involved in binding with other proteins. One can create peptidomimetic compounds that mimic such residues in. By using such mimetics, it is possible to interfere with the normal function of the IL-1L1 protein. For example, non-hydrolyzable peptide analogs for such residues are benzodiazepines (eg, Freidinger et al.,
"Peptides: Chemistry and Biology", GR
Marshall, edited by ESCOM Publisher (Leiden, The Netherlands, 1988), Azepine (eg Huffman et al.,
"Peptides: Chemistry and Biology", GR
See Marshall, ESCOM Publisher (Leiden, The Netherlands), 1988, etc., Substituted γ-lactam rings (Garvey)
Et al., "Peptides: Chemistry and Biology",
GR Marshall edition, ESCOM Publisher (Leiden, The Netherlands), 1988), Ketomethylene Pseudopeptides (Evenson (Ew
Enson) et al. (1986) J. Med. Chem. 29: 295; Ewenson et al.
Peptides: Structure and Function ", 9th American Peptide Symposium Abstracts, Pierce Chemical Company (Rockland, IL), 1985, β-turn Dipeptide cores (Nagai et al., Tetrahedron Lett. 26: 647 (1985)
Sato et al., J. Chem. Soc. Perkin Trans 1: 1231 (1986)), and β-
Amino alcohols (Gordon et al., Biochem. Biophys. Res. Commun.
126: 419 (1985); Dann et al., Biochem. Biophys. Res. Commun. 134: 71 (
1986)).

4.5 Anti-IL-1L1 Antibodies and Their Uses Another aspect of the invention is that it specifically binds to mammalian IL-1L1 proteins, such as wild-type or mutant IL-1L1 proteins. It relates to a reactive antibody. For example, an IL-1L1 protein-derived immunogen (eg, based on a cDNA sequence, etc.) can be used to produce anti-protein / anti-peptide antisera or monoclonal antibodies according to standard protocols (eg, "Antibodies: A Laboratory Manual," edited by Harlow and Lane, Cold Spring Harbor Press
Press), see 1988). Immunogenic peptides such as mammalian IL-1L1 polypeptides or antigenic fragments capable of eliciting an antibody response, or immunogenic peptides such as the fusion proteins described above, can be used to immunize mammals such as mice, hamsters or rabbits. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques known in the art. The immunogenic portion of the IL-1L1 protein can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be performed with the antigen as the immunogen to assess antibody levels. In a preferred embodiment, the antibody of interest is an antigenic determinant of a mammalian IL-1L1 protein, such as SEQ ID NO: 5 or 6.
Immunity against antigenic determinants of the proteins described in 1. or homologues highly related thereto (eg, having at least 90% homology, preferably at least 94% homology). It is specific.

Immunization of animals with antigenic preparations of IL-1L1 polypeptide was followed by
IL-1L1 antiserum was obtained and, if desired, polyclonal anti-IL
-1L1 antibody can be isolated. For the purpose of producing a monoclonal antibody, antibody-producing cells (lymphocytes) are collected from an immunized animal, and fused with immortalized cells such as myeloma cells using a standard somatic cell fusion method. Hybridoma cells can be obtained. Such techniques are known in the art and include, for example, the hybridoma technique originally developed by Kohler and Milstein (Nature 256: 495-497 (1975)), human B.
Cell Hybridoma Technology (Kozbar et al., Immunology Today 4: 72 (198
3)), and EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al., "Monoclonal Antibodies and Cancer Therapy (Monoclonal Ant
ibodies and Cancer Therapy) ", Alan R. Liss, pp.77-
96, 1985) and the like. Hybridoma cells can be immunochemically screened for the production of antibodies specifically reactive with the mammalian IL-1L1 polypeptides of the invention, and monoclonal antibodies isolated from media containing such hybridoma cells. . In one embodiment, the anti-human IL-1L1 antibody specifically reacts with the protein encoded by the nucleic acid having the sequence of SEQ ID NO: 1, 4, 10 or 11.

Antibodies as used herein include fragments that specifically react with one of the mammalian IL-1L1 polypeptides. Antibodies can be fragmented by conventional methods and fragments can be screened using methods similar to those described above for whole antibodies. For example, F (ab) ₂ fragments can be prepared by treating the antibody with pepsin. By treating the obtained F (ab) ₂ fragment, the disulfide bond can be reduced to give a Fab fragment. Antibodies of the invention include bispecific, single chain,
And chimeric and human molecules having affinity for the IL-1L1 protein conferred by at least one CDR region of the antibody. In a preferred embodiment, the antibodies have a detectable label attached to them, such labels can be, for example, radioisotopes, fluorescent compounds, enzymes or enzyme cofactors.

By using an anti-IL-1L1 antibody, it is possible to determine whether an individual has a disease or condition associated with abnormal IL-1L1 protein level, and For the purpose of judging the effect of the treatment method being performed against
It becomes possible to monitor the IL-1L1 protein level in the individual body. IL-1L1
Polypeptide levels can be measured from cells in body fluids such as blood samples.

Other uses of the anti-IL-1L1 antibody of the present invention include λgt11, λgt18-23, λZAP.
And immunological screening of cDNA libraries constructed in expression vectors such as λORF18. This type of messenger library with the inserted coding sequences in the correct reading frame and orientation is capable of producing fusion proteins. For example, λgt11 produces a fusion protein containing a β-galactosidase amino acid sequence at the amino terminus and an exogenous polypeptide at the carboxy terminus. An antigenic epitope of the IL-1L1 protein (eg,
Other orthologs of the particular IL-1L1 protein or other paralogs from the same species) are detected using antibodies such as reactive nitrocellulose filters lifted with anti-IL-1L1 antibody from infected plates, for example. can do.
The positive phage detected by this assay can then be isolated from the infected plate. Thus, the presence of the IL-1L1 homologue can be detected and can be cloned from other animals so that it can be exchanged for human-derived isoforms (including splicing variants).

4.6 Transgenic Animals The present invention further provides transgenic animals that can be utilized for a variety of purposes, such as for identifying IL-1L1 therapeutics. The transgenic animal of the present invention includes a non-human animal having a variant IL-1L1 gene or a fragment thereof under the control of the IL-1L1 promoter or under the control of the atypical promoter. Therefore, as the transgenic animal of the present invention, an animal expressing a transgene encoding a wild-type IL-1L1 protein or a fragment thereof, or a mutant including a mutant and a polymorphic mutant thereof is used. be able to. By using such an animal, it is possible to evaluate the effect of differences (such as differences due to polymorphisms) in the amino acid sequence of the IL-1L1 protein derived from the sequence shown in SEQ ID NO: 5 or 6. By using such animals, the effect of the expression of IL-1L1 protein at a specific site can be confirmed, or the identification of IL-1L1 therapeutic agents or their activity in vivo can be confirmed. You can also do

In one aspect, the present invention provides for the evaluation of transgenic non-human animals and cell lines for in vivo screening, as well as agents and other therapeutics useful in the treatment of inflammatory disorders. I will provide a. In one embodiment, the invention relates to transgenic animals in which the interleukin-1 gene has undergone a selective disruption. In particular, the gene is the IL-1L1 gene. The animal is chimeric, heterozygous or homozygous for the disrupted gene. Homozygous knockout IL-1L1 mammals have osteoarthritis, inflammatory bowel disease, type I diabetes, psoriasis, osteoporosis, nephritis in diabetes mellitus, alopecia areata, Graves disease, systemic lupus erythematosus, lichen sclerosis, A model for studying inflammatory conditions such as ulcerative colitis, coronary artery disease, arteritis, diabetic retinitis, low birth weight, complications of pregnancy, severe periodontal disease, psoriasis and insulin-dependent diabetes mellitus. Provided, but especially characterized by arterial injury. Selective disruption can occur anywhere within the gene, but the only requirement is that the disrupted gene inhibits production of functional IL-1L1 protein. In a preferred embodiment, the disruption removes the entire IL-1L1 encoding sequence as set forth in SEQ ID NO: 11. As the transgenic animal, any species other than human can be used, but mammals are preferable. In a preferred embodiment, the non-human animal having the selectively disrupted IL-1L1 gene has an altered response to inflammation, wherein the selective disruption results in wild-type IL-1L1 poly. Since the production of peptides is inhibited,
The non-human mammalian phenotype is homozygous for the selective disruption.

Viewed from another aspect, the invention relates to a cell or cell line having a selectively disrupted IL-1L1 gene. In a preferred embodiment, the cell or cell line is an undifferentiated cell, such as a stem cell, embryonic stem cell, oocyte or embryonic cell.

Viewed from yet another aspect, the invention features a method of producing a non-human mammal in which the interleukin-1 gene is selectively disrupted. For example, an IL-1L1 knockout construct can be prepared having a portion of the IL-1L1 gene in which an internal portion of the IL-1L1 gene has been replaced with a marker. The knockout construct is then transfected into an embryonic stem (ES) cell population. Transfected cells can be selected according to the expression of the marker. Next, the transfected ES cell is introduced into an embryo of the ancestor of the mammal. Embryos are grown until a chimeric mammal with a germline knockout construct is born. By breeding such chimeric mammals, heterozygous mammals in which the IL-1L1 gene is selectively disrupted will be born. By mating heterozygotes, homozygotes can be born.

Viewed from another aspect, the present invention provides IL-1 that can be used to produce the above-mentioned animals.
Regarding the L1 knockout construct. In one embodiment, the IL-1L1 construct is
It contains a portion of the IL-1L1 gene, where a portion within the IL-1L1 gene can be replaced with a selectable marker. Preferably, the marker is the neo gene and the portion of the IL-1L1 gene is at least 2.5 kb to 7.0 or 9.5 kb in length (including the replacement portion and any IL-1L1 flanking sequences). The internal gene preferably comprises at least a portion of an exon, and in some embodiments, IL-1L1
Contains all of the exons that encode the polypeptide.

Viewed from still another aspect, the invention relates to a method of testing a substance for efficacy in treating and / or inhibiting an inflammatory condition. In one embodiment, the method can use the transgenic animals or cell lines described above. For example, a test substance can be administered to a transgenic animal, and the ability of the substance to reduce an inflammatory condition can be evaluated as an effect on the inflammatory condition. These animals can be used to test for any inflammatory condition involving IL-1, but studies are being conducted specifically for conditions characterized by arterial injury. This method can also be applied to test substances effective against IL-1 inflammatory proteins and their downstream compositions.

Transgenic animals can include a transgene (eg, a reporter gene) under the control of the IL-1L1 promoter or fragments thereof. These animals can express IL-1L1 gene, for example, by regulating the expression of IL-1L1 gene.
It is useful, for example, for identifying an IL-1L1 agent that regulates the production of -1L1. The IL-1L1 gene promoter can be isolated, such as by screening a genomic library with the IL-1L1 cDNA fragment and characterizing according to methods known in the art. In a preferred embodiment of the invention, a transgenic animal carrying such an IL-1L1 reporter gene is used to
A population of bioactive molecules known as steroid hormones is screened for their ability to regulate L1 expression. In a further preferred embodiment of the invention IL-1
Steroid hormones screened for L1 expression regulation activity belong to the group known as androgens. In a further preferred embodiment of the invention the steroid hormone is testosterone or an analogue of testosterone. Other non-human animals within the scope of the present invention include endogenous IL-1.
Those in which the expression of the L1 gene is attenuated or “knocked out” are included. A “knockout” animal is an animal that has a deletion of a particular gene or group of genes in a homozygous or heterozygous state. These animals were tested for whether the absence of IL-1L1 expresses a particular phenotype, in particular such mice are associated with a particular disease (
It is useful for determining whether or not developing (or being prone to) heart disease or cancer). In addition, these animals are useful in screening for agents that reduce or ameliorate disease states caused by mutations in the IL-1L1 gene as described below. These animals have IL-1L1
It is also useful in determining the effects of particular amino acid differences or allelic variations within a gene. That is, an IL-1L1 knockout animal can be bred with a transgenic animal that expresses a mutant or allelic variant of IL-1L1, whereby only the mutant protein but not the wild-type IL-1L1 protein is expressed. An expressing animal can be obtained.

In a preferred embodiment of this aspect of the invention, a mutant IL on one or both chromosomes is used as a model system for transgenic or drug treatment of conditions caused by loss of IL-1L1 expression. Use transgenic IL-1L1 knockout mice with the -1L1 locus.

Methods for obtaining transgenic and knockout non-human animals are known in the art. Knockout mice can be obtained by homozygous integration of a "knockout" construct within the mouse embryonic stem cell chromosome encoding a gene that undergoes knockout. In one embodiment, alterations can be introduced into cultured embryonic stem cells by using gene targeting, which is a method of homologous recombination to transform the genome of an animal. Target IL in ES cells
By targeting the -1L1 gene, these changes can be introduced into the germ line of animals to create chimeras. A gene targeting method is a DNA targeting construct that includes a segment homologous to the target IL-1L1 locus and has expected sequence changes (eg, insertions, deletions, point mutations, etc.) to the IL-1L1 genomic sequence. Can be carried out by introducing into a tissue culture cell. The treated cells are then screened for targeting accuracy to identify and isolate correctly targeted cells.

Gene targeting in embryonic stem cells can be achieved by using one or more IL-1L1
A method contemplated by the present invention as a means of interfering with IL-1L1 gene function by using a targeting transgene construct designed to perform homologous recombination with a genomic sequence. The targeting construct can be designed so that the positive selection marker is inserted (or replaced) within the coding sequence of the gene by recombination with a component of the IL-1L1 gene. The inserted sequence functionally inhibits the IL-1L1 gene while exhibiting positive selection properties. Examples of IL-1L1 targeting constructs are described in detail below.

Generally, the embryonic stem cells (ES cells) used for producing knockout animals are cells of the same species as the knockout animals produced. Thus, for example, mouse embryonic stem cells are commonly used to produce knockout mice.

Embryonic stem cells have been described by Doetschman et al. (J. Embryol.
Exp. MoIL-1L1hol. 87: 27-45 (1985)), and produced and maintained using methods known to those skilled in the art. ES cells of any cell line can be used, but in order for the germline of the knockout construct to be inherited, the ability of the cells to integrate into and become part of the germline of the developing embryo , Specifically select the selected cell line. Thus, any ES cell line believed to have this ability can be used in the applications described herein. The mouse strain commonly used for ES cell production is 129J. Another ES cell line is the mouse cell line D3 (American Type Cu Collection).
lture Collection), catalog number CKL 1934). Yet another preferred ES cell line is the WW6 cell line (Ioffe et al., PNAS 92: 7357-7361 (1995)). The cells are cultured and prepared for knockout construct insertion using methods known to those of skill in the art: Robertson, "Teratocarcinomas and Embryonic. Stem Cells: A Pr
actical Approach ”, edited by EJ Robertson, IRL Press (IRL)
Press, Inc., (Washington, DC), 1987; Bradley et al., Current
Topics in Devel. Biol. 20: 357-371 (1986); Hogan et al., "Manipulating the Mouse Embryo: A Laboratory.
Manual) ", Cold Spring Harbor Laboratory Press (Cold Spring H
arbor Laboratory Press), (Cold Spring Harbor, NY,
1986).

Knockout constructs refer to specially formed nucleic acid fragments that can be introduced into a stem cell line and recombine with the genome at the chromosomal locus of the gene to be mutated. Thus, a given knockout construct is specific to a given gene that has been targeted for disruption. Nevertheless, there are many common elements within these constructs, and these elements are known in the art. Typical knockout constructs are from about 0.5 kb to about 10.5 kb from the 5'and 3'ends of the genomic locus encoding the gene to be mutated.
It has a nucleic acid fragment of 0 kb or less. These two fragments are separated by an intervening fragment of nucleic acid encoding a positive selectable marker such as the neomycin resistance gene (neo ^R ). The resulting nucleic acid fragment is 5'of the genomic locus linked to the nucleic acid encoding the positive selectable marker.
The nucleic acid derived from the extreme end is further included, and the marker is linked to the nucleic acid derived from the extreme 3'end of the genomic locus, and most of the IL-1L1 coding sequence or other gene that undergoes knockout is lost. is doing. When the construct obtained at the locus of the chromosome is homozygously recombined, the missing coding sequence that was supposed to be a structural gene disappears from the genomic locus. Stem cells that undergo such rare homozygous recombination can be stably integrated into the genome of the nucleic acid of the gene encoding the positive selectable marker and, in addition, a suitable drug (neomycin in this example). Can be selected by virtue of their selectivity for cells expressing this marker gene in the presence of

Variations on this basic technique also exist and are known in the art. For example, a "knock-in" construct comprises a nucleic acid from the 5'end of the genomic locus linked to a nucleic acid encoding a positive selectable marker, the other of which is a 3'genomic locus fragment. The basic arrangement is that they are linked to the nucleic acid encoding the, but the coding sequence is not missing, and thus the 5'and 3'genomic fragments used are It was continuous and disrupted by introducing a nucleic acid encoding a positive selective marker. This "knock-in" type construct is very useful for constructing mutant transgenic animals when mutating only a limited region of the genomic locus of the gene (eg, one exon) And can be used for cloning and gene amplification. Alternatively, "knockin" constructs can be used to specifically delete one functional domain of the selected gene, resulting in disruption of one function of the encoded polypeptide domain. It is possible to obtain a transgenic animal that expresses a polypeptide of a gene that retains the other functions. This type of "knock-in" mutant often has what is called a "dominant negative" mutant,
This is because it is possible to specifically block (or "toxic") the activity of the polypeptide product of the wild-type gene from which it is derived, especially in the case of proteins that homohomerize. is there. In a variation of the "knock-in" technique, the marker gene is integrated at the genomic locus of interest such that expression of the marker gene is under the control of transcriptional regulators of the selected gene. A marker gene is a gene encoding an enzyme (eg, β-galactosidase) whose activity can be detected, and an enzyme substrate can be added to cells under appropriate conditions to analyze the enzyme activity. . Those skilled in the art are aware of other useful markers and methods of detecting their presence in a given cell. All such markers are within the scope of the invention.

As mentioned above, homologous recombination of the above “knockout” or “knockin” constructs is very infrequent, and such constructs are heterologously inserted into random regions of the genome. Is high, in which case it has no effect on the gene that is the target of the deletion and may recombine to interfere with another gene that does not want to be modified. Such a non-homologous recombination event can be achieved by using a negative selectable marker at one end of either the knockout or knockin constructs described above, in particular by using two allelic variants of the thymidine kinase gene. Modified to be adjacent to a cell line that undergoes expression in a suitable tissue culture medium known in (i.e., a medium containing a drug such as 5-bromodeoxyuridine) for its polypeptide product. Can be selected by Therefore, in a preferred embodiment of the "knockout" or "knockin" constructs of the invention, the nucleic acid encoding the 5'end of the genomic locus that is linked to the nucleic acid of the positive selectable marker is linked to the nucleic acid. Containing a nucleic acid encoding a negative selective marker, wherein the other end of the positive selective marker is linked to the locus encoding the 3'end of the genomic locus above. The other end of the 3'end is linked to a second nucleic acid encoding a negative selectable marker. Non-homologous recombination of the resulting knockout construct with the genome results in stable integration of one or both of these negative selectable marker genes, and thus cells that have undergone non-homologous recombination Selection can be performed by growing in a suitable selection medium (eg, a medium containing a drug such as 5-bromodeoxyuridine). Simultaneous selection for positive and negative selectable markers significantly increases the number of clones in which the knockout construct undergoes homologous recombination at the locus of the gene for which the mutation is intended. To do. The expected occurrence of chromosomal changes at selected loci in the resulting knockout stem cell line can be confirmed by performing Southern blot analysis known to those skilled in the art. Alternatively, PCR can be used.

Each knockout construct inserted intracellularly must initially be linear. Therefore, if the knockout construct was inserted into the vector (described below)
Linearization is performed by cleaving the DNA with an appropriate restriction endonuclease selected to cleave only within the vector sequence and not within the knockout construct sequence.

Regarding insertion, under conditions suitable for the insertion method known to those skilled in the art,
This is done by adding the knockout construct to ES cells. For example, when ES cells are electroporated, the electroporation apparatus is used to irradiate the ES cells and the knockout construct DNA with electric pulses according to the manufacturer's usage guidelines. After electroporation, ES cells are usually recovered under appropriate incubation conditions. The cells are then screened for the presence of the knockout construct, as previously described. When more than one knockout construct is introduced into ES cells, each knockout construct can be introduced simultaneously or one at a time.

The selection techniques previously outlined identify the appropriate ES cells with the knockout construct in place and then insert the cells into the embryo. Insertion can be performed using a variety of methods known to those of skill in the art, but the preferred method is microinjection. To perform microinjection, collect approximately 10 to 30 cells in a micropipette and inject them into embryos at an appropriate developmental stage to transfer exogenous ES cells with a knockout construct to developing embryos. Incorporate. For example, transformed ES cells can be microinjected into undifferentiated embryo cells. The appropriate developmental stage of the embryo used for ES cell insertion varies from species to species, but for mice it is about 3.5 days old. Embryos are obtained by perfusing the uterus of a pregnant female. Suitable methods for obtaining embryos are known to those of skill in the art and are described, for example, by Bradley et al., Ibid.

Although any embryo at the appropriate developmental stage can be used, the preferred embryo is the male. In the mouse, preferred embryos also have genes encoding coat colors that differ from coat colors encoded by ES cell genes. In this way, the presence of the knockout construct in the offspring can be easily screened by looking for mosaic coat color (indicating that ES cells have been integrated into the developing embryo). Thus, for example, if the ES cell line carries the white hair gene, embryos carrying the black or brown gene are selected.

After the ES cells are introduced into the embryo, the embryo is implanted in the uterus of a pseudopregnant foster mother for pregnancy. Any foster mother can be used, but will generally be selected based on childbirth, fertility, and nursery capacity. Such foster mothers are obtained by mating with vasectomized males of the same species. The time of pseudopregnancy of the adoptive mother is important for successful implantation and varies from species to species. In mice, this time is about 2-3 days of pseudopregnancy.

When the coat color selection method (see above and below in the examples) is used, screening of offspring born of adoptive mothers is first carried out on the mosaic coat color. Still alternatively, the DNA of tail tissue can be screened for the presence of the knockout construct using Southern blot and / or PCR as described above. For the purpose of creating homozygous knockout animals, if the pups exhibiting a mosaic coat color are considered to have a knockout construct in the reproductive system, they are crossed with each other. Homozygotes are mice born from this mating,
In addition, it can be confirmed by performing Southern blotting using an equal amount of genomic DNA derived from a mouse and a wild-type mouse that are known to be heterozygotes.

Other methods can be used to confirm the knockout child and to determine its characteristics. For example, using Northern blot to determine the presence or absence of a transcript encoding the gene being knocked out, the marker gene, or both.
NA can be scrutinized. In addition, Western blot can be used to assess the expression level of the knocked-out IL-1L1 gene in various tissues of the offspring, at which time an antibody to a specific IL-1L1 protein, or expression of this gene If so, probe the Western blot with an antibody against the marker gene product. Finally, using an antibody suitable for confirming the presence or absence of the knockout construct gene product, in situ analysis (fixing the cells, labeling with an antibody, etc.) of various cells collected from the offspring and / or FACS (fluorescence activated cell sorter) analysis can be performed.

Other methods for producing knockout or disrupted transgenic animals are also generally known. For example, "Manipulating the Mouse E
mbryo) (Cold Spring Harbour Laboratory Press (Cold Spring Har
bor Laboratory Press), Cold Spring Harbor, New York, 1986) and others). It is also possible to create a knockout animal for a recombinant enzyme by inserting it into a target sequence by homologous recombination, and in such an individual, the recombinant enzyme sequence causes tissue-specific IL-1LI gene inactivation. And / or temporary control may be implemented (see above).

Animals with one or more knockout constructs and / or one or more transgene expression constructs are generated by any method. A preferred method of generation is to generate a series of mammalian groups in which each individual has one of the desired transgenic phenotypes. A series of crosses, backcrosses and selections are made on such animals to ultimately produce a single animal that contains and / or expresses all the desired knockout constructs. Is congenic (genetically identical) with the wild type except that the knockout construct and / or the transgene is present.

All IL-1LI transgenes encode the wild type of the protein or their homologues, including agonists and antagonists and antisense constructs. In a preferred embodiment, expression of the transgene is limited by the particular cell population, tissue or developmental stage used, for example:
Examples include cis-acting sequences that control expression so that the desired pattern is obtained. In the present invention, such mosaic expression of the IL-1LI protein is essential for phylogenetic analysis of various forms, and further, lack of IL-1LI expression due to a large change in development in a small piece of tissue within normal embryos. It is possible to provide a method for evaluating the influence such as. These, as well as tissue-specific and conditional control sequences, can be used to control transgene expression in a positional pattern. Furthermore, a temporary expression pattern can be obtained by a conditional recombination system or a prokaryotic transcription control sequence.

Genetic engineering techniques for expressing transgenes that can be regulated by site-specific genetic engineering in vivo are known to those of skill in the art. For example, a gene system capable of controlling the expression of a recombinant enzyme that catalyzes gene recombination of a target sequence is available. As used herein, "target sequence" refers to a nucleotide sequence that has been genetically modified by a recombinase. The target sequence is flanked by recombinase recognition sequences and is generally deleted or inverted in the cell expressing the recombinase activity. Recombination events catalyzed by recombinase are targeted by recombination of target sequences
It may be contemplated that the expression of one of the IL-1LI proteins is activated or repressed. Activating expression of a recombinant IL-1LI gene, for example by attempting to delete a target sequence that interferes with expression of the gene, such as a target sequence encoding an antagonist homolog or antisense transcript You can Such interference with protein expression is caused by a variety of mechanisms, such as the IL-1LI gene being located remotely from promoter elements or internal stop codons. Furthermore, the transgene can be prepared so that the coding sequence of the gene is adjacent to the recombination enzyme recognition sequence and is first transfected in the cell in the 3 ′ → 5 ′ direction with respect to the promoter factor. In such an embodiment, if the target sequence is reversed, the gene of interest is placed at the 5'end of the coding sequence relative to a promoter element capable of transcriptional activation by promoter control. Rearrange.

All the transgenic animals of the present invention carry the transgene of the present invention in the majority of cells, and the expression of “host cell” relates to the control of cell proliferation, death and / or differentiation by the transgene. The type has been modified. Since one or more of the transgene constructs described herein can be used to produce the transgene microorganisms of the invention, foreign genetic material is generally used to produce transgenic microorganisms. Make a general description. This general description aims to integrate specific transgene sequences into microorganisms using the following methods and materials,
It can be changed by those skilled in the art.

In one embodiment, the cre / loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS 89: 6232-6236 (1992); Orban et al., PNAS
89: 6861-6865 (1992)) or FLP of Saccharomyces cerevisiae
Recombinant enzyme system (O'Gorman et al., Science 251: 1352-1355 (1991); PC
T publication WO 92/15694) can be used to prepare an in vivo site-specific gene recombination system. Cre recombinase catalyzes the site-specific recombination of intervening target sequences located between loxP sequences. The loxP sequence is a 34 base pair nucleotide repeat sequence to which Cre recombinase is bound and is required for Cre recombinase-mediated gene recombination. The orientation of the loxP sequence is determined by excising or reversing the intervening target sequence in the presence of Cre recombinase (Abremski et al., J. Biol. Chem. 259: 1509-1514 ( 1984)), which catalyzes the excision of the target sequence when the loxP sequence points to the tandem repeat, and the reverse orientation of the target sequence when the loxP sequence points to the inverted repeat.

Therefore, the genetic recombination of the target sequence depends on the expression of Cre recombinase. The expression of the recombinant enzyme can be regulated by a promoter factor for regulatory control (eg, tissue-specific regulation, developmental stage-specific regulation, etc.) that can be induced or suppressed by an externally added substance. Due to this regulatory control, gene recombination of the target sequence occurs only in cells in which the expression of the recombinant enzyme is mediated by the promoter factor. Therefore, recombinant IL-1LI protein expression activation can be regulated through control of recombinant enzyme expression.

To regulate expression of recombinant IL-1LI protein using the cre / loxP recombinase system, transgenic animals carrying a Cre recombinase and a transgene encoding the protein of interest were prepared. It needs to be created. Animals carrying the Cre recombinase and the recombinant IL-1LI gene are obtained by constructing “double” transgenic animals. A convenient way to generate such animals is to breed transgenic animals that have one transgene, such as the IL-1LI gene and the recombinant enzyme gene.

One advantage of first constructing transgenic animals carrying the IL-1LI transgene in a recombinant enzyme mediated expression pattern is whether the protein of interest is agonistic or antagonistic. , Is potentially harmful when expressed in the body of transgenic animals. In such cases, a founder population in which the transgene of interest is silent in all tissues can be propagated and maintained. Individual individuals within this founder population can be
It can be bred to an individual expressing the recombinant enzyme in one or more tissues, and / or in a desired temporal pattern. So, for example,
Creating a founder population in which the antagonistic IL-1LI transgene is silent disrupts IL-1LI-mediated induction in certain tissues, or phenotypes, such as death, at certain developmental stages. The offspring from the founder population to present can be studied.

For the purpose of expressing the IL-1LI transgene, a similar conditional transgene can be prepared using a prokaryotic promoter sequence that requires co-expression of prokaryotic proteins. Examples of promoters and corresponding trans-activated prokaryotic proteins are described in US Pat. No. 4,833,080.

In addition, expression of the conditional transgene can be induced by methods similar to gene therapy, where the gene for the transactivating protein (eg, recombinant enzyme or prokaryotic protein, etc.) It is transported to and expressed in tissues in a manner similar to the type-specific manner. According to this method, the IL-1LIA transgene reaches maturity in a silent state until the transactivator is introduced and the expression is switched on.

[0224] In one embodiment, the "non-human transgenic animal" of the present invention is produced by introducing a transgene into the germline of a non-human animal. The transgene is introduced using target embryonic cells at different developmental stages. Different introduction methods are adopted depending on the developmental stage of the target embryonic cell. The particular strain of any animal used in the practice of the present invention is selected for good health, high yield of embryos, visibility of pronuclei within the embryo and good reproductive compatibility. In addition, the haplotype is also an important factor. For example,
When producing transgenic mice, the C57BL / 6 strain or FVB strain is often used (Jackson Laboratory, Bar Harbor, ME). Preferred strains, C57BL / 6-based or DBA / 1 system has an ^{H-2 b, H-2} d or H-2 ^q haplotypes, and the like. The strains used in the practice of the present invention are themselves transgenic and / or knockout (ie, individuals obtained from animals that have one or more genes that are partially or completely repressed).

[0225] In one embodiment, the transgene construct is introduced into a single developing embryo. The most suitable target for microinjection is the zygote. In the mouse, a male pronucleus grown to a size of about 20 μm was used, and 1-2 pl of DNA was used.
The solution can be injected to allow regeneration. The important advantage of using the zygote as a target for gene transfer is that in most cases, before the first division occurs,
The injected DNA is integrated into the host gene (Brins (Brins
ter) et al., PNAS 82: 4438-4442 (1985)). As a result, all cells of the transgenic animal will have the integrated transgene. Germ cell
Since 50% carry the transgene, the above results are also generally reflected in the efficient transfer of the transgene from the founder to the offspring.

Fertilized embryos are usually incubated in a suitable medium until the pronuclei appear.
At this time, the nucleotide sequence containing the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, male pronuclei are preferred. Most preferably, the exogenous genetic material is added before the zygotic male DNA complement is processed by the egg nucleus or the zygotic female pronucleus. The egg nucleus or female pronucleus is believed to release molecules that affect male DNA complement, probably due to the replacement of protamine in male DNA with histones.
It is considered that complement binding occurs and a diploid zygote is formed.

Therefore, preferably, the male D is complemented before the male DNA complement is affected by the female pronucleus.
Add exogenous genetic material to NA complement or any other DNA complement. For example, exogenous genetic material is added to the early male pronucleus as soon as possible after the male pronucleus is formed, at which time the male and female pronuclei are well separated, Both are located near the cell wall. Alternatively, exogenous genetic material can be added to the sperm nucleus after decondensation. The sperm containing the exogenous genetic material can then be added to the egg, or the decondensed sperm can be added as soon as possible after adding the transgene construct to the egg.

The introduction of the transgene nucleotide sequence into the embryo can be performed using any method known in the art such as microinjection, electroporation, lipofection and the like. Following introduction of the transgene nucleotide sequence into the embryo, the embryo can be incubated in vitro for various periods of time, or reimplanted into a surrogate host, or both. In vitro incubation methods for maturation are within the scope of the invention. One common method for in vitro embryo incubation is about 1-7 days, depending on the species, and then reimplanting them into surrogate parents.

In order to achieve the object of the present invention, the zygote needs to form a diploid cell in which the cell can develop into a complete organism. In general, a zygote comprises an egg with a nucleus formed naturally or artificially by the fusion of two haploid (haploid) nuclei from gametes. Therefore, the gamete nucleus must be naturally compatible, ie, a biologically active zygote that can undergo differentiation and development to a functional organism. Generally, euploid zygotes are preferred. When an aneuploid zygote is obtained, its chromosome number must not differ by more than one with respect to the euploid chromosome of the gamete-bearing organism.

In addition to similar biological considerations, the amount of exogenous genetic material (eg, volume, etc.) that can be added to the genetic material forming the zygote nucleus or part of the zygote nucleus is It also depends on physical considerations. If no genetic material is removed, the amount of exogenous genetic material that can be added is limited to that expected to be absorbed without physical disruption. Generally, the volume of exogenous genetic material inserted is less than about 10 pl. The physical effect of the addition should not be so great as to physically destroy the biological activity of the zygote. Biological restrictions on the number and diversity of DNA sequences will depend on the function of the particular zygote and the exogenous genetic material, but the genetic material containing the exogenous genetic material of the resulting zygote will be Since it must be biologically possible to initiate differentiation and development, and continue to reach a functional organism,
It will be obvious to those skilled in the art.

The copy number of the transgene construct added to the zygote depends on the total amount of exogenous genetic material added and is the amount at which gene transformation can occur. Theoretically, only one copy is required, but in general, a large number of copies (for example, 1000 to 20000 copies) of the transgene construct are prepared for the purpose of ensuring that one copy functions properly. To use. According to the invention, it is advantageous to use one or more functional copies for each inserted foreign DNA sequence to promote phenotypic expression of the foreign DNA sequence.

[0232] Exogenous genetic material can be added within the nuclear genetic material using any technique, as long as it does not disrupt the cell, nuclear envelope or other cellular or genetic structure. The foreign genetic material is preferably inserted into the nuclear genetic material by microinjection. Microinjection into cells and cell structures is known and used in the art.

Re-implantation is performed using standard methods. Usually, the surrogate parent is anesthetized and the embryo is inserted into the fallopian tube. The number of embryos transferred to a particular parent varies from species to species, but is usually comparable to the number of offspring the species naturally inhabits.

The transgenic offspring born from the surrogate parent are screened for the presence and / or expression of the transgene using any suitable method. Screening is often performed by Southern or Northern blot analysis using a probe that is complementary to at least a portion of the transgene. Another or additional method for screening for the presence of the transgene product is Western blot analysis using antibodies to the protein encoded by the transgene. Generally, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for transgenes. Alternatively, tissues or cells suspected of expressing the transgene at the highest level are examined for the presence and expression of the transgene by Southern analysis or PCR, in which case any tissue or cell type may be detected. Can be used.

Another or additional method for determining the presence of the transgene includes suitable live assays such as enzyme and / or immunological assays, histological staining for specific markers or enzyme activity, flow cytometric analysis. Examples include, but are not limited to, chemical assays. Blood analysis is also useful for detecting the presence of transgene products in blood and for assessing the effects of transgenes on various types of blood cells and other blood components.

Progeny of transgenic animals may be obtained by crossing the transgenic animal with a suitable partner, or by fertilizing eggs and / or sperms obtained from the transgenic animal in vitro. it can. When mating is performed, the mating partner may be transgenic and / or knockout, or may not be, and in the case of transgenic, may have the same or different transgenes, or both. Individuals can be used. Alternatively, the parental line can be used as a mating partner. When in vitro (in vitro) fertilization is performed, the fertilized embryo is transplanted into a surrogate parent, incubated in vitro (in vitro), or both. Whichever method is used, offspring are assessed for the presence of the transgene using the methods described above or other suitable methods.

The transgenic animals produced according to the present invention have exogenous genetic material. As mentioned above, in one embodiment, the exogenous genetic material is a DNA sequence that produces an IL-1LI protein (agonistic or antagonistic), as well as an antisense transcript or an IL-1LI mutant. . Moreover, in such an embodiment, the sequence is linked to a transcriptional regulatory factor, such as a promoter, which causes the transgene product to be expressed in certain types of cells.

Retroviral infection can also be used to introduce transgenes into non-human animals. Developing non-human embryos are cultured in vitro to the blastocyst stage. During this time, blastomere dividing cells can be targeted for retroviral infection (Jaenich, R., PNAS 73: 1260-1264 (1976)). By removing the zona pellucida by enzymatic treatment, blastomere dividing cells can be efficiently infected (“Manipulating the Mouse Embryo”), Hogan.
), Cold Spring Harbor Laboratory Press (Cold Spring Harbor L
aboratory Press) (Cold Spring Harbor), 1986). In general,
The viral vector system used to introduce the transgene is a replication defective retrovirus carrying the transgene (Jahner et al. PNAS 82: 6927-6931 (1985);
Van der Putten et al., PNAS 82: 6148-6152 (1985)).
Transfection occurs easily and efficiently by culturing blastomere dividing cells on a monolayer of virus-producing cells (Van der Putten
) Et al., Id .; Stewart et al., EMBO J. 6: 383-388 (1987)). Alternatively, infection can occur at a later stage. Virus or virus-producing cells can be injected into the blastocyst cavity (Jahner et al.,
Nature 298: 623-628 (1982)). Most founders are mosaic for the transgene because uptake occurs only in a portion of the cells that make up the transgenic non-human animal. In addition, the founder may have multiple retroviral inserts of the transgene at different positions in the genome that may generally segregate in offspring. In addition, it is also possible to introduce the transgene into the reproductive system by retroviral infection of the midgestational embryo in utero (
Jahner et al., Ibid.).

The third type of target cell for transgene transfer is the embryonic stem cell (ES).
ES cells are obtained from pre-implantation embryos that have been cultured in vitro and fused with the embryo (Evans et al., Nature 292: 154-156 (1981); Bradley et al., Nature 309: 255-258 (1984); Gossler et al., PNAS 83.
: 9065-9069 (1986); Robertson et al., Nature 322: 445-448 (1986).
)). The transgene can be efficiently introduced into ES cells by DNA transfection or retrovirus-mediated transduction. Such transformed ES cells can then be combined with blastocysts from non-human animals. The ES cells then colonize the embryo and contribute to the reproductive system of the resulting chimeric animal. See the article by Jaenisch, R. (Science 240: 1468-1474 (1988)).

4.7 Screening Assays for IL-1LI Therapeutic Agents The present invention further provides screening methods for determining IL-1LI therapeutic agents, such therapeutic agents include, for example, IL-1LI. For the purpose of preventing treatment and / or progression of diseases or conditions which are caused by or involved in abnormal activity or which are effective by regulating IL-1LI activity or IL-1L1 protein level Is mentioned. Examples of such diseases, conditions or disorders include, for example, osteoarthritis, inflammatory bowel disease, type I diabetes, psoriasis, osteoporosis, nephritis in diabetes mellitus, alopecia areata, Graves' disease, and systemic erythema. Examples include lupus, lichen sclerosis, ulcerative colitis, coronary artery disease, arterial disease, diabetic retinopathy, low birth weight, complications of pregnancy, severe periodontal disease and insulin-dependent diabetes mellitus. In particular, but not limited to, arterial injury, cancer (such as breast cancer, prostate cancer or testicular cancer) involving steroid hormone responsive tumor growth, vascular disease or disorder (eg, thrombotic stroke, deficiency). Hematologic stroke, and peripheral vascular disease that has progressed from atherosclerosis and thrombus formation, heart disease (eg, myocardial infarction, congestive heart failure, unstable angina and ischemia) Cardiovascular disease and disorders, as well as cardiovascular diseases and disorders (eg, due to hypertension, hypotension, cardiomyocyte hypertrophy and congestive heart failure), or other diseases due to abnormal or altered IL-1LI-dependent processes Characterized by a condition or disorder.

The IL-1L1 therapeutic agent can be any type of compound including proteins, peptides, peptidomimetics, small molecules and nucleic acids. As the nucleic acid, for example,
Genes, antisense nucleic acids, ribozymes or triads can be used. IL-1L1 therapeutic agents according to the present invention are agonistic or antagonistic. Preferred IL-1L1 agonists include IL-1L1 proteins or their derivatives that mimic at least one of the activities of IL-1L1 (eg, fibroblast growth factor receptor binding activity or heparin sulfate binding activity). Can be mentioned. Other preferred agonists include compounds that can increase intracellular production of IL-1L1 protein (for example, compounds that can promote and control the expression of IL-1L1 gene), and IL-1L1 activity and And / or compounds that can facilitate the interaction of the IL-1L1 protein with other molecules such as target peptides. Preferred IL-1L1 antagonists include, for example, IL-1L1 protein which is a dominant negative protein having a property of being able to bind to fibroblast growth factor receptor but not to heparin sulfate. Other preferable antagonists include compounds that reduce or inhibit intracellular production of IL-1L1 protein, compounds that can suppress and regulate the expression of IL-1L1 gene, and IL-1L1 activity and / or IL -1 Compounds that can suppress and regulate the interaction between the 1L1 protein and other molecules. In another preferred embodiment, the IL-1L1 antagonist is a variant of the target peptide and is capable of interacting with the FGFR binding domain of the IL-1L1 protein but lacking biological activity (eg, itself. Is not a cell surface receptor).

The invention also provides an IL-1L1 protein (eg, a wild-type IL-1L1 protein or
IL-1L1 protein) and thus IL-1
Provided is a screening method for identifying an IL-1L1 therapeutic agent that regulates the growth factor activity of L1 or causes degradation of IL-1L1. For example, as such an IL-1L1 therapeutic agent, an antibody or a derivative thereof that specifically interacts with the IL-1L1 protein (wild type or mutant type) can be used.

Accordingly, the present invention provides methods for identifying agonist and antagonist compounds of IL-1L1, which methods include IL-1L1 protein or IL-1L1.
Molecules that can interact with proteins (eg, FGF receptors and / or
Or a compound capable of modulating the interaction of IL-1L1 protein with other molecules (eg, receptor and / or heparin sulfate) Including doing.
Generally, a molecule capable of interacting with the IL-1L1 protein is referred to as an “IL-1L1 binding partner”.

The compounds of the present invention can be identified using a variety of assays depending on the type and activity of the compound desired. In addition, test compounds can be further tested in animal models, as described herein. Described below are some assays that can be used to identify IL-1L1 therapeutic agents. It is within the skill of one in the art to set up additional assays to identify IL-1L1 therapeutic agents.

4.7.1 Cell-Free Assay A cell-free assay is used to identify compounds capable of interacting with the IL-1L1 protein or binding partner, thereby determining the activity of the IL-1L1 protein or binding partner. Can be changed. Such compounds can affect their activity, for example, by altering the structure of the IL-1L1 protein or binding partner. IL-1L1 using cell-free assay
It is also possible to identify compounds that modulate the interaction of the protein with the IL-1L1 binding partner (such as the target peptide). In a preferred embodiment, essentially, a cell-free assay for identifying such a compound is a reaction mixture comprising an IL-1L1 protein and a test compound or a test compound library in the presence or absence of a binding partner. To use. Examples of test compounds include IL
-1L1 binding partners (such as biologically inactive target peptides) or small molecules can be used.

Therefore, as one example of the screening assay of the present invention, the IL-1L1 protein or a functional fragment thereof or the IL-1L1 binding partner is contacted with a test compound or a test compound library to form a complex. Including the step of detecting. For detection, the molecule can be labeled with a special marker and the test compound or library of test compounds can be labeled with a different marker. The interaction of the test compound with the IL-1L1 protein or fragment thereof or IL-1L1 binding partner can then be detected by measuring the levels of the two labels after the incubation and washing steps. The presence of the two labels after the wash step indicates that the interaction is occurring.

Interaction between molecules is analyzed by real-time BIA (Biomolecular Interaction Analysis).
: Biomolecular interaction analysis (Pharmacia Biose)
nsor), AB), which detects surface plasmon resonance (SPR), which is an optical phenomenon. The detection is done by changing the mass concentration of the macromolecule at the biospecific interface and does not require a label for the interacting substance. In one embodiment, the test compound library is affixed to the sensor surface, forming, for example, a wall composed of microflow cells. Next, IL-1L1 proteins, their functional fragments, IL-1L1
A solution containing the analog or IL-1L1 binding partner is continuously flowed over the surface of the sensor. It is shown that the interaction is caused by the change in resonance angle that appears in the signal recording. This technology is described in, for example, "BIAtechnology Handbook" published by Pharmacia.
) ”In more detail.

In another embodiment of the screening assays of the present invention, (a) (i) IL
Preparing a reaction mixture comprising -1L1 polypeptide, (ii) IL-1L1 binding partner and (iii) test compound; (b) detecting the interaction between IL-1L1 and IL-1L1 binding partner. IL-1L1 polypeptides and IL-1L1 as described herein.
Binding partners can be prepared recombinantly, purified from materials such as plasma, or chemically synthesized. The change in the interaction between IL-1L1 and IL-1L1 binding protein in the presence of the test compound shows a statistically significant difference (enhancement or inhibition) compared with the change in the interaction in the absence of the test compound. Test compound is IL
It is suggested to be a potent agonist (mimetic or enhancer) or antagonist (inhibitor) for -1L1 biological activity. The compounds of this assay can be contacted simultaneously. Alternatively, the IL-1L1 protein is first contacted with the test compound for a suitable time and then the IL-1L1 binding partner is added to the reaction mixture. The potency of a compound can be assessed by constructing a dose-dependent curve from the data obtained with various concentrations of test compound. In addition, control assays can be performed to create a comparative baseline. In a control assay, isolated and purified IL-1L1 polypeptide or binding partner is added to a composition containing IL-1L1 binding partner or IL-1L1 polypeptide, and complex formation in the absence of the test compound is quantified.

The formation of the complex between the IL-1L1 protein and the IL-1L1 binding partner can be detected by various techniques. Changes in complex formation can be detected, for example, by immunoassay or chromatographic detection using a detectable labeled protein (eg, radiolabeled, fluorescently labeled, or enzyme labeled IL-1L1 protein or IL-1L1 binding partner). It can be quantified.

In general, it is desirable to immobilize either IL-1L1 or its binding partner, which will separate the complex from one or both of the uncomplexed proteins and will also automate the assay. Can be achieved. Binding of IL-1L1 to the IL-1L1 binding partner can be done in any vessel suitable for contacting the reactants. Examples of such vessels include microtiter plates, test tubes, microcentrifuge tubes, and the like. In one embodiment, a fusion protein can be provided that adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase / IL-1L1 (GST / IL-1L1) fusion proteins are adsorbed on glutathione sepharose beads (Sigma Chemical Co., St. Louis, Mo.) or glutathione derivatized microtiter plates. And then mixed with an IL-1L1 binding partner (eg, an IL-1L1 binding partner labeled with ³⁵ S) and a test compound to form a complex (a more stringent condition is This mixture is incubated, for example, at physical conditions such as salt or pH, which may be desirable. After incubation, wash the beads to remove any unbound label, immobilize the matrix and either measure the radiolabel directly (eg placing the beads in a luminescent body) or after dissociating the complex. Measure the Qing. Alternatively, the complexes are dissociated from the matrix, separated by SDS-PAGE, and standard electrophoresis techniques as described in the Examples below are used to detect IL-1L1 protein or IL-1L1 protein in bead fractions. The level of IL-1L1 binding partner can be quantified from the gel.

Other techniques for immobilizing proteins on matrices can also be used in this assay. For example, conjugation of biotin and streptavid can be used to immobilize IL-1L1 or its unique binding partner. For example, a biotinylated IL-1L1 molecule can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques known in the art (eg, Biotin kit (Pierce Chemicals, Illinois). Rockford)) and streptavidin-coated 96-well plates (Pierce Chemicals). Alternatively, IL-1L1-reactive antibodies can be immobilized in the wells of the plate and captured by the antibody conjugation. As described above, the amount of the complex captured in the well can be quantified by incubating the preparation containing the IL-1L1 binding protein and the test compound in the well containing IL-1L1. As an example of a method for detecting such a complex, in addition to the method described above for the GST-immobilized complex, an antibody that reacts with an IL-1L1 binding partner, or an IL-1L1 protein that reacts with a binding partner Included are immunodetections of the complex performed by using competing antibodies, as well as enzyme-linked assays based on the detection of endogenous or exogenous enzyme activities involving binding partners. As an example of the latter, the enzyme can be chemically conjugated to the IL-1L1 binding partner or provided as a fusion protein. Specifically, the IL-1L1 binding partner can be chemically cross-linked or genetically fused with horseradish peroxidase to provide a dye substrate for the enzyme (eg, 3,3′-diamino-benzazine-tetrahydrochloride or Four
-Chloro-1-naphthol, etc.) can be used to measure the amount of polypeptide trapped in the complex. Similarly, the polypeptide and glutathione-S
A fusion protein consisting of -transferase and a 1-chloro-2,4-dinitrobenzene can be used to detect GST activity to quantify complex formation (Habig et al. , J. Biol. Ch
em. 249: 7130 (1974)).

Antibodies to proteins such as anti-IL-1L1 antibodies can be used in methods based on immunodetection aimed at quantifying one of the proteins captured in the complex. Alternatively, the protein to be detected present in the complex can be "epitope tagged" in the form of a fusion protein, in which case the fusion protein is added to the IL-1L1 sequence in addition to the IL-1L1 sequence. , A second peptide for which an antibody is available (commercially available). For example, the GST fusion protein described above can also be used for quantification of binding by using an antibody against the GST site. Another useful epitope tag is my
c-epitope (eg, Ellison et al., J. Biol. Chem. 266: 21150-
21157 (1991) and the like), which includes a sequence consisting of 10 residues derived from c-myc and the pFLAG system (International Biotechnology (InterBiotechnology)
national Biotechnologies) or pEZZ protein A system (Pharmacia (
Pharmacia, Inc., NJ).

By using a cell-free assay, the IL-1L1 protein interacts with IL
Compounds that regulate the activity of the -1L1 protein can be identified. Therefore, in one embodiment, the IL-1L1 protein is contacted with a test compound and the IL-1L1 catalytic activity is monitored. In one embodiment, the ability of IL-1L1 to bind a target molecule is measured. The binding affinity of IL-1L1 for the target molecule can be measured according to methods known in the art.

4.7.2 Cell-Based Assay In addition to the cell-free assay as described above, IL-1 provided by the present invention.
The L1 protein can be used in cell-based assays, such as to identify small molecule agonists or antagonists. In one embodiment, cells expressing the IL-1L1 receptor protein on the outer surface of the cell membrane are incubated in the presence of the test compound alone or in the presence of the test compound and the IL-1L1 protein, eg, a microphysiometer ( McConnell et al., Sci
ence 257: 1906 (1992)) and the like, or between test compound-IL-1L1 receptor protein or IL-1L1 protein (preferably tagged IL-1L1 protein) -IL-1
Detect interactions between L1 receptor proteins. The IL-1L1 receptor protein-test compound or IL-1L1 protein interaction is detected by a microphysiometer as a change in acidification of the medium. Therefore, this assay system
Provided is a means for identifying a molecular antagonist that acts such as interfering with an interaction between IL-1L1-IL-1L1 receptors, as well as a molecular agonist that acts such as activating the IL-1L1 receptor.

Modulating the expression of the IL-1L1 gene by using a cell-based assay,
Compounds that modulate the translation of IL-1L1 mRNA or that regulate the stability of IL-1L1 mRNA or IL-1L1 protein can also be identified. Thus, in one embodiment, cells capable of producing IL-1L1 (eg, a chorioepitheloma cell line such as JEG-3) are incubated with a test compound and the IL-produced in cell culture medium is incubated. The amount of 1L1 is measured and compared with the amount of IL-1L1 produced by cells not contacted with the test compound. Various control analyzes, such as measuring the expression of one or more control genes, confirm the specificity between the compound and IL-1L1. Test compounds include small molecules, proteins and nucleic acids. In particular, this assay can be used to validate the IL-1L1 antisense molecule or ribozyme.

[0256] In another embodiment, a test compound for transcription of the IL-1L1 gene by performing transfection experiments using a reporter gene operably linked to at least a portion of the promoter of the IL-1L1 gene. Confirm the effect of. The promoter region of a gene can be isolated from a genomic library or the like according to a method known in the art. As the reporter gene, any gene encoding a quantifiable protein (eg, luciferase or CAT gene) can be used. Such reporter genes are known in the art.

In a preferred embodiment, the invention provides a cell-based assay using the chorioepithelioma cell line JEG-3. Analysis of this cell line has been shown to produce a 17 kDa IL-1L1 polypeptide that immunoprecipitates with a rabbit anti-IL-1L1 polyclonal antiserum. Therefore, this cell line is a screening assay for substances that promote or repress the expression of the IL-1L1 gene, or influence the steady-state level of the IL-1L1 polypeptide, or translation of the IL-1L1 polypeptide. Post-activity (eg, IL-1L1 proteolytic process, IL-1L1 glycosylation, IL-1L1 phosphorylation or
IL-1L1 secretion and the like) can be used in a screening assay for substances that affect the secretion.

In another preferred embodiment, assays for interleukin-1 agonist and antagonist activity can be used. For example, human dermal fibroblasts express interleukin-6 in response to IL-1 like activators such as IL-1α and IL-1β. Therefore, this cell line can be used in an assay for IL-1 agonist activity. In addition, this cell line is used to provide an assay for IL-1 antagonists by screening for compounds that inhibit or attenuate IL-1α or IL-1β activation of the interleukin-6 gene. Furthermore, by using this cell line with an IL-1 receptor antagonist polypeptide, IL-1L1 polypeptide as well as IL-1 receptor antagonist activity (eg, IL-1β-dependent activity of interleukin-6 gene is blocked). IL-1L1 polypeptide agonists and antagonists that affect (eg, the ability of -1ra) can be identified.

Other cell-based screening assays that can be used in the methods of the invention are known or apparent to those of skill in the art. For example, IL-1 agonists and antagonists can be identified by their effect on downstream IL-1-dependent activation of NF-κB (Reddy et al., Mol. Cell Biol. 19: 4798-4805). (1
997)). IL-1-dependent activation is associated with activation of the IκB kinase, particularly the β subunit (
Li et al., J. Exp. Med. 189: 1839-1845 (1999)), where activation of the IκB kinase β subunit results in NF-κB I activation.
It causes phosphorylation of κB inhibitors and subsequent proteolytic proteolysis, or inhibits nuclear localization to NF-κB transcriptional activators. IL-1L1 is NF-mediated by activation of phosphatidylinositol 3 kinase
κB can also be activated, where phosphatidylinositol 3 kinase transduces a signal generated by phosphorylation of the p65 / RelA subunit,
Thereby, NF-κB-dependent transcriptional activation of a downstream gene is performed (for example, Sizemore (
Sizemore) et al., Mol. Cell Biol. 19: 4798-4805 (1997). Thus, an IL-1 inducible inflammatory response gene reporter can be used to assay IL-1 agonist and antagonist activity according to the methods of the invention.

Furthermore, IL-1β is known to induce the NO synthase (iNos) gene and the cyclooxygenase-2 gene in cardiomyocytes (LaPoint (LaPoint)).
inte) et al., Hypertension 33: 276-282 (1999)). Furthermore, what is particularly remarkable regarding the expression of the IL-1L1 gene in the human placenta is that human umbilical artery endothelial cells (HUAE
In C), many genes are induced by interleukin-1 (Ko et al., Mol. Cell. Probes 13: 203-211 (1999)). Such genes include the basic fibroblast growth factor (bFGF) gene and the vascular endothelial growth factor (VEGF) gene, which regulate angiogenesis and are involved in the progression of atherosclerosis. However, it further includes granulocyte colony-stimulating factor (G-CSF) gene, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and stem cell factor (SCF) gene, which are important growth factor effectors in hematopoiesis. . Utilizing other effects of IL-1 on the activation or suppression of specific genes,
The development of cell-based assays for IL-1L1 and agonist and antagonist activity of IL-1L1 can be developed (eg, Rohn et al., J. I.
mmunol. 162: 886-896 (1999) (inhibition of MHC class II expression by IL-1); Pellacani et al., J. Biol. Chem. 274: 1525-1532 (1999) (non-histone high migration). IL-1 activation of group (HMG) gene expression); Borghaei et al., Biochem. Biophys. Res. Commun. 251: 334-338 (1998) (mitogen-induced nuclear orphan receptor (MINoR) gene expression. IL-1 induction)) etc.)
.

The invention further encompasses the novel agents identified by the screening assays described above and their use for the treatments described herein.

4.8 Candidate Agents The present invention further provides novel IL-1L1 genes and intragenic variants that affect the expression level and / or action of the encoded IL-1L1 protein in a subject, and related It relates to candidate agents that are based at least in part on pathway genes.

For example, the information obtained using the diagnostic assays described herein (alone or in combination with information about other genetic defects associated with similar diseases) is symptomatic. Are useful in diagnosing or confirming a subject (eg, a subject presenting with symptoms of inflammatory osteoarthritis), such a subject is a gene (eg, IL).
-1L1 gene or in a gene that controls the expression of the IL-1L1 gene), which causes or is associated with a particular disease or disorder. From another perspective, using the information alone or in combination with information about other genetic defects associated with similar diseases, asymptomatic subjects can be assessed for IL-1L1 activity within the subject. It can be predicted whether an abnormality or an abnormal IL-1L1 protein level causes or develops a disease or disorder in which it is involved. Based on the predictive information, the physician can recommend effective regimens (eg, diet, exercise, etc.) or treatment protocols to prevent or delay the onset of a particular disease or disorder.

In addition, deletion or deletion of the IL-1L1 gene or protein in the subject (IL-1L1
By having knowledge of the specific changes caused by the gene profile (alone or in combination with information about other genetic defects associated with similar diseases (gene profiles for specific diseases)) Personalized treatments for specific diseases can be provided, which is the ultimate goal of "pharmacogenomics". For example, by obtaining an individual's IL-1L1 gene profile or a gene profile for a disease or condition caused or associated with a change in the L-1L1 gene, it is possible for a doctor to: It makes it possible to more effectively prescribe drugs that act at the molecular level, and 2) makes the appropriate amount of a specific drug easier to determine. For example, in a large number of patients at various stages of disease, measuring the expression level of IL-1L1 protein alone or in combination with the expression levels of other genes known to be associated with similar diseases. Gives a transcriptional or expression profile of the disease. The expression pattern in the individual patient can then be compared to the expression pattern for the disease to determine the appropriate drug and dosage to administer to the patient.

By targeting populations predicted to show the highest clinical efficacy based on IL-1L1 or disease genetic profile,
2) Rescue drug candidates whose clinical development has been discontinued due to safety or efficacy restrictions specific to the patient subpopulation, and 3) accelerated development and cheaper development of drug candidates, and more appropriate drugs. Labeling (eg, using IL-1L1 as a marker is useful for optimizing effective doses).

[0266]   These and other methods are described in detail below.

4.8.1 Prognostic and Diagnostic Assays The present method provides for the treatment of diseases, conditions in which a subject is associated with an abnormality in IL-1L1 activity, such as an abnormality in IL-1L1 protein level or an abnormality in IL-1L1 biological activity. Alternatively, a method for determining whether or not a disease is developed (diagnosis) or at risk of developing a disease (prediction) is provided. Examples of such diseases, conditions or disorders include, but are not limited to: Inflammatory diseases such as osteoarthritis, inflammatory bowel disease, psoriasis, lupus erythematosus, ulcerative colitis and alopecia areata and diabetic nephritis, growth factor cytokine or steroid hormone responsive tumors (eg breast cancer, prostate cancer or testis) Cancer, including cancer, vascular diseases or disorders (eg, thrombotic stroke, ischemic stroke, and peripheral vascular disease resulting from atherosclerosis and thrombus formation), heart disease (eg, myocardial infarction, unstable narrowing). Cardiovascular and ischemic heart disease), cardiovascular diseases and disorders (such as those derived from hypertension, hypotension, myocardial hypertrophy and congestive heart failure), wound healing, limb regeneration, nerve injury or disease (eg Alzheimer's disease). Disease, Parkinson's disease, those associated with AIDS-related disorders or cerebral palsy), or abnormalities in IL-1L1-dependent processes. Other diseases, conditions or disorders caused by changes such as collateral growth and remodeling of cardiovascular, angiogenesis, cell transformation caused by autocrine mechanism or paracrine mechanism, cell (eg, endothelium, etc.) Chemotactic stimuli, neural progenitor cells (eg PC
Neurite outgrowth, maintenance of neurophysiology of mature neurons, proliferation of embryonic mesenchymal and limb bud precursor tissues, mesoderm induction and other developmental processes, collagenase and plasminogen activator Secretion stimulation, tumor vascularization, and tumor invasion and metastasis.

Accordingly, the present invention expresses or is likely to develop a disease, condition or disorder that is caused by or associated with abnormal IL-1L1 levels or abnormal IL-1L1 biological activity. A method of determining whether or not there is such a method, for example, IL
-1L1 gene or IL-1L1 protein, measuring the level of biological activity of IL-1L1 and / or determining the presence of a mutation or particular polymorphic variant within the IL-1L1 gene.

In one embodiment, the method comprises Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation, Western blot hybridization, or immunohistochemistry. The subject has an abnormal mRNA, and / or IL-1 by a technique such as chemistry
Including determining whether the L1 protein level is abnormal. According to this method, cells are obtained from a subject and the level of IL-1L1 protein or mRNA is measured and compared to the level of IL-1L1 protein or mRNA in a healthy subject. Abnormal levels of IL-1L1 polypeptide or mRNA indicate abnormal IL-1L1 activity.

[0270] In another embodiment, the method comprises measuring at least one of IL-1L1 activities. For example, the affinity of IL-1L1 for heparin can be measured as described herein. Similarly, binding partners (eg,
The affinity constant of the subject-derived IL-1L1 protein for the IL-1 type I or type II receptor, etc.) can be determined. Comparison of the results obtained with the results of similar analyzes performed on IL-1L1 protein from healthy subjects indicates whether the subject's IL-1L1 activity is aberrant.

In a preferred embodiment, the method of determining whether a disease caused or implicated by an abnormality in IL-1L1 activity is, or is likely to be, is derived from a subject. Gene characterized by at least one of (i) an alteration affecting the integrity of the gene encoding the IL-1L1 polypeptide or (ii) a misexpression of the IL-1L1 gene in a cell sample of It is characterized by detecting the presence or absence of a change. For example, such genetic alterations include (i) deletion of one or more nucleotides from the IL-1L1 gene, (ii) addition of one or more nucleotides to the IL-1L1 gene, (iii ) Substitution of one or more nucleotides of the IL-1L1 gene, (iv) large-scale chromosomal rearrangement of the IL-1L1 gene, (v) I
Significant changes in messenger RNA transcript level of L-1L1 gene, (vi) abnormal transformation of IL-1L1 gene (abnormal transformation of methylation pattern of genomic DNA, etc.), (vii) IL-1
Non-wild type splicing pattern of messenger RNA transcript of L1 gene, (vii
At least one of i) non-wild type level of IL-1L1 polypeptide, (ix) allelic loss of IL-1L1 gene, and / or (x) inappropriate post-translational modification of IL-1L1 polypeptide, etc. Can be detected by confirming the presence of
As described below, the present invention provides a number of assay techniques for detecting changes occurring within the IL-1L1 gene. These methods include sequence analysis, methods involving Southern blot hybridization, restriction enzyme site mapping, and methods involving detecting the absence of nucleotide pairs between the nucleic acid to be analyzed and the probe, etc. It is not limited to these. These and other methods are described in detail below.

Certain diseases and disorders, such as genetic diseases or disorders, involve particular allelic variants to polymorphic regions of certain genes that need not encode mutant proteins. Therefore, when a subject has a specific allelic variant with respect to a polymorphic region of a gene (eg, single nucleotide polymorphism (SNP), etc.), it can be determined that the subject is likely to develop a specific disease or disorder. . Polymorphic regions present in genes such as the IL-1L1 gene can be identified by confirming the nucleotide sequence of the gene in the population. When polymorphic regions such as SNPs are identified, a particular population (eg, congestive heart failure, hypertension, hypotension, or cancer (
For example, a study can be conducted on a population of patients suffering from a particular disease (such as cancer, including the growth of steroid-responsive tumors) to determine the association with a particular disease. A polymorphic region is any region within a gene, such as an exon,
It exists in coding and non-coding regions of exons, introns, and promoter regions.

The IL-1L1 gene is a polymorphic region, ie, a specific allele involved in or associated with an increased likelihood of developing a particular disease or disorder. Is believed to have. Accordingly, the present invention provides a method of characterizing an allele or allelic variant of a polymorphic region of the IL-1L1 gene present in a subject, whereby a subject has a specific polymorphic region Whether or not the disease or disorder in which the allelic variant is involved is at risk of developing.

In certain embodiments, a sense or antisense sequence to the IL-1L1 gene or a naturally occurring mutant thereof, or an IL-1L1 gene or naturally-occurring mutant thereof is naturally associated. 5'or 3 '
Provided is a nucleic acid composition having a nucleic acid probe containing a nucleotide sequence region capable of hybridizing with a flanking sequence or an intronic sequence. The nucleic acid of the cell is capable of accepting hybridization, contacting the probe with the sample nucleic acid and detecting hybridization of the probe to the sample nucleic acid. Using such techniques, it is possible to detect changes or allelic variants, including deletions, substitutions, etc., at the genomic or mRNA level,
In addition, the mRNA transcript level can be confirmed.

[0275] As a preferred detection method, a mutation site or a polymorphism site is duplicated,
And allele-specific hybridization with a probe having about 5, 10, 20, 25 or 30 nucleotides around the mutated or polymorphic region. In a preferred embodiment of the invention, several probes capable of specifically hybridizing to allelic variants (eg single nucleotide polymorphisms) are provided on a solid support such as a "chip". Bind to the body. Oligonucleotides can be attached to a solid support by a variety of methods including lithography. For example, up to 250,000 oligonucleotides can be attached to the chip. Such a chip ("
For mutation detection analysis performed using "DNA probe arrays"), see, for example, Cronin et al., Human Mutation 7:
244 (1996). In one embodiment, the chip contains all allelic variants to at least one polymorphic region of a gene. Next, a test nucleic acid is brought into contact with the solid support to detect hybridization with a specific probe. Therefore, simple hybridization experiments can confirm the nature of multiple allelic variants for one or more genes.

[0276] In certain embodiments, the detection of alterations is accomplished by polymerase chain reaction (PCR), such as anchor PCR or RACE PCR (eg, US Pat. No. 4,683,195 and
No. 4,683,202), or as another method, ligase chain reaction (LCR) (eg, Landegran et al., Science 241: 1077-1080 (198)).
8); Nakazawa et al., PNAS 91: 360-364 (1994)), using a probe / primer, but for detecting point mutations in the IL-1L1 gene
LCR is particularly useful (Abravaya et al., Nuc. Acid Res. 23: 675-
682 (1995)). In one specific embodiment, the method comprises the following steps: (i) taking a sample of cells from the patient and (ii) nucleic acid (eg genomic nucleic acid or mRNA or Etc.), and (ii
i) hybridizes specifically to the IL-1L1 gene under conditions such that the IL-1L1 gene hybridizes (if present) and amplifies 1
The nucleic acid sample is contacted with one or more primers and (iv) the presence or absence of the amplification product is detected, or the size of the amplification product is detected and compared with the length of the control sample. PCR and / or LCR are considered suitable for use as a pre-amplification step in combination with any of the techniques used to detect mutations described herein.

Another amplification method is the self sustained sequence replication.
ication) (Guatelli, JC et al., Proc. Natl. Acad. Sci. USA 87:
1874-1878 (1990), transcription amplification system (Kwoh, DY et al., Proc. Natl. Acad
Sci. USA 86: 1173-1177 (1989)), Q-β replicase (Lizardi
), PM et al., Bio / Technology 6: 1197 (1988)), or any other nucleic acid amplification method, after amplification, the amplified molecule is detected using techniques known to those of skill in the art. These detection procedures are particularly useful for detection when the number of nucleic acid molecules present is very low.

In a preferred embodiment of this assay, mutations or allelic variants of the IL-1L1 gene obtained from sample cells can be identified by altered restriction enzyme cleavage patterns. For example, sample DNA and control DNA can be isolated, grown if necessary, cleaved with one or more restriction endonucleases,
Measure the size of the fragments by gel electrophoresis. Further, by using a sequence-specific ribozyme (see, for example, US Pat. No. 5,498,531),
The presence of specific mutations can be assessed based on the occurrence or loss of ribozyme cleavage sites.

In yet another embodiment, the IL-1L1 gene is directly analyzed using any of a variety of sequencing reactions known in the art to map the sequence of the sample IL-1L1. Mutations can be detected by comparison with the wild type (control) sequence. An example of such a sequencing reaction is the technique developed by Maxim and Gilbert (Proc. Na.
tl. Acad. Sci. USA 74: 560 (1977)), as well as those based on the technique developed by Sanger (Proc. Natl. Acad. Sci. USA 74: 5463 (1977)). When performing this assay, sequencing by mass spectrometry (eg, PCT Publication WO 94/16101; Cohen et al., Adv. Ch.
romatogr 36: 127-162 (1996); Griffin et al., Appl. Biochem. Biot.
echnol. 38: 147-159 (1993), etc.) and includes any of a variety of automated sequencing methods (Biotechniques 19:
448 (1995)). It will be apparent to one of skill in the art that in certain embodiments, the only bases that need to be confirmed in the sequencing reaction are 1, 2, or 3 nucleic acids. For example, A-track or the like, which detects only one nucleic acid, can be performed.

In yet another embodiment, protection against cleaving agents is used (such as with nucleases, hydroxylamines or osmium tetrachloride, with piperidine) and RNA / RNA, RNA / DNA or DNA / Mismatched bases in DNA heteroduplexes can be detected (Myers et al., Science 230: 1242 (198).
Five)). In general, "mismatch cleavage" is labeled with the wild-type IL-1L1 sequence.
RNA or D with the potential for mutation obtained from RNA or DNA from a tissue sample
Begin by providing a heteroduplex formed by hybridizing with NA. The double-stranded complementary bond is treated with a substance that cleaves the single-stranded region of the complementary bond, and such a single-stranded region is generated due to a base pair mismatch between the control strand and the sample strand. . For example, RNA / DNA complementary bonds can be treated with RNase, DNA / DNA hybrids can be treated with S1 nuclease,
The mismatch region can be enzymatically cleaved. In another embodiment, the DNA / DNA or RNA / DNA complementary bonds are treated with hydroxylamine or osmium tetrachloride and further treated with piperidine for the purpose of cleaving the mismatched regions. After cleaving the mismatched regions, the resulting material is separated according to molecular weight on a denaturing polyacrylamide gel to confirm the mutation site. For example, Cotton et al., Proc. Natl. Acad. Sci. USA 85: 4397 (1988); Salee.
Ba) et al., Methods Enzymol. 217: 286-295 (1992). In a preferred embodiment, control DNA or RNA can be labeled for detection.

In yet another embodiment, a system established to detect and map point mutations in IL-1L1 cDNA obtained from a cell sample recognizes mismatched base pairs in double-stranded DNA. One or more proteins (so-called "DNA
A "mismatch repair" enzyme) is used in the mismatch cleavage reaction. For example, E. coli (E
mutY enzyme of A. coli cleaves A in G / A mismatch, and thymidine DNA glycosylase from HeLa cells cleaves T in G / T mismatch (Hsu et al.
Carcinogenesis 15: 1657-1662 (1994)). In one embodiment, the IL-1L1 sequence (
For example, a probe based on the wild-type IL-1L1 sequence) is hybridized to a cDNA or other DNA product derived from the test cell. When the cleavage product is obtained by treating the complementary binding strand with a DNA mismatch repair enzyme, it can be detected by electrophoresis or the like. See, eg, US Pat. No. 5,459,039.

In another embodiment, alterations in electrophoretic mobility are used to detect mutations in the IL-1L1 gene or to detect polymorphic region allelic variants. For example, single-stranded conformational polymorphisms (SSCPs) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al., P.
roc. Natl. Acad. Sci. USA 86: 2766 (1989); Cotton et al., Mutat.
Res. 285: 125-144 (1993); Hayashi et al., Genet. Anal. Tech. Appl. 9
: 73-79) (1992)). Single stranded DNA fragments of sample and control IL-1L1 nucleic acids were denatured and left to revert. The secondary structure of single-stranded nucleic acid varies depending on the sequence,
Due to the changes that appear in the electrophoretic mobility, even a single base change can be detected. The DNA fragment can be labeled and can be detected with a labeled probe. The sensitivity of this assay can be enhanced by using RNA rather than DNA because secondary structure is more susceptible to sequence changes. In a preferred embodiment, the method can be used in heteroduplex analysis to separate heteroduplex molecules based on base changes in electrophoretic mobility (Keen et al. Trends Genet. 7: 5
(1991)).

In yet another embodiment, denaturing gradient gel electrophoresis (DGGE) is used to assay the mobility of mutant or wild type fragments in polyacrylamide gels loaded with denaturants at different denaturing steps. (Myers
) Et al., Nature 313: 495 (1985)). When DGGE is used as the analysis method, the DNA is modified in order to confirm that the DNA is not completely denatured. For example, by PCR, adding a GC clamp composed of approximately 40 bp of GC-rich DNA having a high melting temperature. In a further embodiment, a temperature gradient is used instead of a denaturant concentration gradient to confirm the difference in mobility of control and sample DNA (Rosenbaum and Reissner, Biophys. Chem. 26).
5: 12753 (1987).

Examples of other techniques used to detect point mutations or identify polymorphic region allelic variants include selective oligonucleotide hybridization, selective amplification, or selective primer extension. However, it is not limited to these. For example, oligonucleotide primers are prepared under conditions such that known mutations or nucleotide differences (eg, within an allelic variant) are centrally prepared and then allowed to hybridize only if there is an exact match. Hybridize to the target DNA in (Saiki et al., Na
ture 324: 163 (1986); Saiki et al., Proc. Natl. Acad. Sci. USA 86: 6.
230 (1989)). By using such allele-specific oligonucleotide hybridization techniques, one mutation or polymorphic region is detected per reaction when the oligonucleotide hybridizes to the PCR amplified target DNA, Alternatively, a large number of distinct mutations or polymorphic regions can be detected when the oligonucleotide binds to the hybridizing membrane and hybridizes to the labeled target DNA.

Alternatively, allele-specific amplification techniques based on selective PCR amplification can be used in combination with the present invention. Oligonucleotides used as primers in specific amplifications have a mutation or polymorphic region in the center of the molecule to allow for differential hybridization-based amplifications (Gibbs et al., Nucleic Acids Res. 17: 2437-2448 (1989))
Alternatively, it has a mutation or polymorphic region at the 3'-most end of the primer under appropriate conditions that prevent mismatch or suppress polymerase extension (Prosnner, Tibtech. 11: 238. (1993)). Furthermore, it is desirable to introduce new restriction enzyme sites into the mutated region to create cleavage-based detection sites (Gasparini et al., Mol. Cell Probes. 6: 1 (1992). In certain embodiments. Can also be amplified using Taq ligase for amplification (Barany, Proc. Natl. Acad. Sci. USA 88: 189 (1991).
). In such a case, since the nucleic acid ligation reaction occurs only when the 3'ends of the 5'sequences are completely matched, the presence of a known mutation at a specific site is detected by searching for the presence or absence of amplification. It becomes possible to do.

In another embodiment, confirmation of allelic variants can be performed using an oligonucleotide ligation assay (OLA), eg, US Pat. No. 4,998,617.
And Landegren, U et al., Science 241: 1077-1080 (1988). The OLA protocol uses two oligonucleotides designed to be able to hybridize to adjacent sequences on one strand of the target. One of the oligonucleotides is linked to another marker, such as a biotinylated marker, and the other has a detectable label. If a sequence is predicted to be complementary within the target molecule, the oligonucleotides are flanked and hybridize to create a ligation substrate.
Avidin or other biotin ligand can be used to recover the labeled oligonucleotide by ligation. Nickerson, D.
A. et al. Describe a nucleic acid detection assay that combines the attributes of PCR and OLA (Nickerson, DA et al., Proc. Natl. Acad. Sci. USA 87:
8923-8927 (1990)). In this method, PCR is used to exponentially amplify the target DNA, followed by detection using OLA.

Several methods based on this OLA method have been developed, and these methods can be used to detect specific allelic variants of the polymorphic region of the IL-1L1 gene. For example, US Pat. No. 5,593,826 discloses forming a conjugate having a phosphoramidate linkage by OLA using an oligonucleotide having a 3′-amino group and a 5′-phosphorylated oligonucleotide.
Another variation on OLA is described by Tobe et al. (Nucleic
Acids Res 24: 3728 (1996)), this method allows the discrimination of two alleles in one microtiter well by combining OLA with PCR. Mark allele-specific primers with unique haptens (ie, digoxigenin and fluorescein) to identify hapten-specific antibodies labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. Each by using
OLA reaction can be detected. In this system, detection of two alleles can be performed using a high-throughput format that develops two different colors.

The invention further provides methods for detecting single nucleotide polymorphisms in the IL-1L1 gene. Since a single nucleotide polymorphism constitutes a modification site that is in contact with the invariant sequence region, it is sufficient in the analysis to identify one nucleotide existing in the modification site, and for each patient, It is not necessary to perform complete gene sequencing. Several methods have been developed to perform such single nucleotide polymorphism analysis.

In one embodiment, single nucleotide polymorphisms using specialized exonuclease resistant nucleotides, eg, as disclosed by Mundy, CR (US Pat. No. 4,656,127). Can be detected. According to this method, a primer complementary to the allelic sequence flanking the polymorphic site on the 3'side is hybridized to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule has a nucleotide complementary to the specialized exonuclease resistant nucleotide derivative, then that derivative is incorporated at the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease and becomes detectable. Since the nature of the exonuclease-resistant derivative of the sample is known, the knowledge that the primer acquired resistance to exonuclease indicates that the nucleotide present at the polymorphic site of the target molecule is the nucleotide derivative used in the reaction. It turns out to be complementary to it. The method is useful in that it does not require a large amount of extra sequence data analysis.

In another embodiment of the invention, the solution method is used to determine the nature of the nucleotide at the polymorphic site (Cohen, D. et al., French Patent No. 2,650,840; PCT Application No. WO 91 / 02087). Similar to the Mundy method of US Pat. No. 4,656,127, a primer complementary to the allelic sequence flanking the polymorphic site on the 3'side is used. This method uses a labeled dideoxynucleotide derivative to determine the nature of the nucleotide at the polymorphic site, which is then incorporated at the end of the primer if the derivative is complementary to the nucleotide at the polymorphic site.

Alternatively, Genetic Bit Analysis or GBA ^™
The method known as is described by Goelet, P. et al.
PCT application number 92/15712). The method of Goelet, P. et al. Uses a mixture of labeled terminators and primers complementary to sequences 3'adjacent to the polymorphic site. The labeled terminator is incorporated and determined by the nucleotides present within the polymorphic site of the target molecule being analyzed and is complementary to such nucleotides. In contrast to the method of Cohen, D. et al. (French Patent No. 2,650,840; PCT Application No. WO 91/02087), Goelet,
The method of P. et al. Is preferably a heterogeneous phase assay in which a primer or target molecule is immobilized on a solid phase.

Recently, several publications have been published regarding primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA (Komher, JS et al., Nucl. Acids). Res. 17
: 7779-7784 (1989); Sokolov, BP, Nucl. Acids Res. 18: 3671
(1990); Syvanen, A. -C. Et al., Genomics 8: 684-692 (1990); Kuppuswamy, MN et al., Proc. Natl. Acad. Sci. USA 88: 1143-114.
7 (1991); Pleasant, TR et al., Hum. Mutat. 1: 159-164 (1992);
Ugozzoli, L. et al., GATA 9: 107-112 (1992); Nyren, P.
Et al., Anal. Biochem. 208: 171-175 (1993)). These methods are a method different from the GBA ^{T M} in that distinguish polymorphic sites base captures labeled deoxynucleotides. In such a manner, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms generated in transcription of the same nucleotide give rise to a signal proportional to the length of transcription (Syva
nen), A.-C. et al., Amer. J. Hum. Genet. 52: 46-59 (1993)).

Mutations that cause premature termination of protein translation can be efficiently diagnosed by the protein truncation test (PTT) (Roest et al., Hum. Mol. Genet. 2). : 1719-21 (1993); Van der Luijt et al., Genomics 20: 1-4 (1994)). For PTT,
First, RNA is isolated from available tissue, reverse transcribed, and the segment of interest is amplified by PCR. Next, the reverse transcription PCR product is subjected to nested PCR amplification (nested PCR amplifi
cation), using a primer containing an RNA polymerase promoter and a sequence for initiating eukaryotic translation. After amplifying the region of interest, the
In vitro transcription and translation occur sequentially. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the translation product, trends in the cleaved polypeptide indicate the presence of mutations that cause premature termination of translation. As a modification of this method, when the target region of interest is derived from one exon,
Use DNA (as opposed to RNA) as a PCR template.

The methods described in the present invention use, for example, a pre-packaged diagnostic kit that includes at least one probe nucleic acid, a primer set, and / or the antibody-reactive reagents described herein. For example, in a clinical setting, a patient exhibiting a condition involving IL-1L1 polypeptide, or IL-1
It can be conveniently used when diagnosing a family history of a disease or illness in which the L1 polypeptide is involved.

Any cell type or tissue can be used in the diagnostics described below. In a preferred embodiment, a body fluid such as blood is collected from a subject and IL-1
To characterize the presence of mutations in polymorphic regions of the L1 gene, or the characteristics of allelic variants. Body fluids such as blood can be collected by known techniques such as venipuncture. Alternatively, nucleic acid tests can be performed on dry samples such as hair and skin. For prenatal diagnosis, a fetal nucleic acid sample can be obtained from maternal blood as described in Bianchi et al. International Patent Application WO 91/07660. Alternatively, a sample can be obtained by amniocentesis or villus puncture for prenatal diagnosis.

When RNA or protein is used to detect the presence of a mutation or a specific allelic variant relating to a polymorphic region of the IL-1L1 gene, the cell or tissue used does not express the IL-1L1 gene. There must be. Preferred cells for use in these methods include cardiac cells (see Examples section). When other cells or tissues are used, it can be judged by determining the expression pattern of the specific IL-1L1 gene in the subject by, for example, Northern blot analysis.

The diagnostic method is performed in situ on patient tissue sections (fixed or frozen) obtained by biopsy or excision so that nucleic acid purification may not be performed.
Situ) can also be done directly. In such in situ methods, nucleic acid reaction reagents can be used as probes and / or primers (eg, Nuovo, GJ, “PCR in Situ (in
situ) hybridization: protocols and applications (PCR in situ hybrid
": ization: protocols and applications)", Raven Press
) Company, New York, 1992 and so on). In addition to methods that are basically focused on the detection of a single nucleic acid sequence, profiles can also be predicted in such detection methods. For example, Northern blot analysis and / or differential display methods such as RT-PCR can be used to generate fingerprint profiles.

For the diagnosis and prognosis of disease, antibodies to wild-type or mutant IL-1L1 polypeptides or allelic variants thereof as described above may also be used. By using such a diagnostic method, an abnormality in the expression level of IL-1L1 polypeptide, a structural abnormality in IL-1L1 polypeptide, and / or an abnormal distribution in tissue, cell or cell component can be detected. Structural abnormalities include, for example, mutant IL-1L1 when compared to a normal IL-1L1 polypeptide.
Examples include the size of the polypeptide, electronegativity, or difference in antigenicity. The protein from the tissue or cell line being analyzed can be readily detected or isolated using techniques known to those of skill in the art, such as Western blots. For a detailed description of how to perform Western blot analysis, see Sambro (Sambro
ok) et al. (Id., 1989), Chapter 18. For protein detection and isolation methods used herein, see Harlow and Lane.
(Harlow and Lane), "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Pres.
s) company, Cold Spring Harbor, New York (1988), etc.,
This description is incorporated herein by reference.

For example, this method involves light microscopy detection of fluorescently labeled antibodies (see below),
This can be done by flow cytometry detection or immunofluorescence in combination with fluorometric detection. Furthermore, the antibodies (or fragments thereof) useful in the present invention can be used histologically in immunofluorescence or immunoelectron microscopy for in situ detection of IL-1L1 polypeptides. . In situ detection can be performed by taking a tissue sample from a patient and adding the labeled antibody of the invention to the sample. The antibody (or fragment) is preferably added with a labeled antibody (or fragment) so as to coat the biological sample. By using such a method, not only the presence of IL-1L1 polypeptide but also the distribution of IL-1L1 polypeptide in the test tissue can be confirmed. One of ordinary skill in the art, using this method, may use any of a variety of histological methods (eg, staining, etc.) to perform such in situ detection. It is easy to understand that anything can be modified.

As a support to which an antigen or antibody can be bound, a solid phase support or carrier is often used. Known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
Amylase, natural or modified cellulose, polyacrylamide, gabros (
gabbros) and magnetite. The nature of the carrier is
It may be soluble or insoluble to some extent for the purposes of the present invention. The support can assume virtually any conformation so long as the molecule to which it is linked is capable of binding to an antigen or antibody. Thus, the three-dimensional structure of the support can be spherical, such as beads, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, it can be a flat surface such as a sheet or test strip. Examples of preferable supports include polystyrene beads. One of ordinary skill in the art would be aware of many other carriers suitable for conjugating antibodies and by routine experimentation will be able to confirm that they are equivalent.

One method of labeling anti-IL-1L1 polypeptide-specific antibodies is through binding to an enzyme, which is used in an enzyme immunoassay (EIA) (Voller (Vol
ler), “Enzyme Linked Immunosorbent Assay: ELIS
A) ”, Diagnostic Horizons 2: 1-7 (1978), Microbiological Society quarterly journal (Microbiologic
al Associaties Quarterly Publication), Walkersville, MD; Voller et al., J. Clin. Pathol. 31: 507-520 (1978); Butler (Bu
73) 482-523 (1981); "Enzyme Immunoassay," edited by Maggio, CRC Press, Boca Raton, FL, 1980; Ishikawa. (Ishikawa) et al., “Enzyme Immunoassay (Enzy
me Immunoassay) ", Kogaku Shoin, Tokyo, 1981). The enzyme that binds to the antibody reacts with a suitable substrate, preferably a chromogenic substrate, in such a way as to produce a chemical moiety that can be detected by, for example, a spectrophotometer, fluorometer or visually. Enzymes that can be used to label the antibody detectably include, but are not limited to, malate dehydrogenase, staphylococcus nuclease, δ-5-steroid. Isomerase, yeast alcohol devidrogenase, α-glycerophosphate, dehydrogenase,
Triose phosphate isomerase, horseradish peroxidase,
Alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6
-Phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be done by the dye method, which uses a dye substrate for the enzyme.
The detection can also be carried out visually by visually comparing the amount of enzyme reaction of the substrate with a control prepared in the same manner.

Detection can also be performed using any of a variety of other immunoassays. For example, a radioimmunoassay (RIA) can be performed with a radioactively labeled antibody or antibody fragment to detect the wild-type or mutant peptide of the fingerprint gene (e.g., Weintraub, B. , "Radioimmunoassay Principles: 7th Training Course on Radioligand Assay Technology (Principles Radioimmunoassays: Seventh
Training Course on Radioligand Assay Techniques) ", The Endocrine Sciety, March 1986, etc., which is incorporated herein by reference). The radioactive isotope can be detected by a method using a γ-counter or a scintillation counter, autoradiography or the like.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is irradiated with light of an appropriate wavelength, the presence of the antibody can be detected by emitting fluorescence. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
Examples include allophycocyanin, Q-phthalaldehyde and fluorescamine.

Metals that fluoresce (eg, ¹⁵² Eu or other lanthanoid metals)
Can also be used to label the antibody with a detectable label. These metals can be attached to antibodies by using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody can be confirmed by detecting the light generated during the progress of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Similarly, bioluminescent compounds can be used to label the antibodies of the invention. Bioluminescence is a mode of chemiluminescence found in biological systems, where catalytic proteins enhance the efficiency of chemiluminescent reactions. The presence of bioluminescent proteins is confirmed by detecting light. Bioluminescent compounds that are important as labels include luciferin, luciferase and aequorin.

Furthermore, it is self-evident that any of the above methods for detecting alterations in genes or gene products, or polymorphic variants can also be used to monitor the course of treatment or therapy.

4.8.2 Pharmacogenetics Knowledge of the specific alterations (IL-1L1 gene profile) that result in a deficiency or deletion of the IL-1L1 gene or protein in individuals, alone or in similar diseases. Used in combination with information on other genetic defects involved (gene profile for specific diseases), "drug genetics (
The goal of "Pharmacogenomics" is to establish therapies for diseases specific to an individual's genetic profile. For example, a subject having a particular allele of the IL-1L1 gene may or may not be symptomatic for a particular disease, or is likely or not likely to be symptomatic for a particular disease. Furthermore, it is possible that the control may be symptomatic and may or may not respond to one drug that is effective for IL-1L1 treatment, or to another drug. Therefore, IL-1
IL from a patient population presenting with a disease or condition caused by, or associated with, a deficiency and / or deletion of the L1 gene and / or protein
-1L1 gene profile (eg, classifying changes in the IL-1L1 gene that are associated with the progression of a particular disease) (IL-1L1 gene population profile), and the individual IL-1L1 profile as the population profile By comparison, it is possible to select or design drugs that are considered safe and effective for a particular patient or population of patients (ie, groups of patients with similar genetic alterations).

[0309] For example, the IL-1L1 population profile describes the IL-1L1 gene within a patient population having a disease caused by, or associated with, a deletion or deletion of the IL-1L1 gene. It is obtained by judging the IL-1L1 profile such as the identity of. In addition, the IL-1L1 population profile also includes information on the response of the population to IL-1L1 treatment, obtained using any of a variety of methods, such as 1) depending on IL-1L1 associated disease. Severity of symptoms, 2) IL-1L1 gene expression level, 3
) IL-1L1 mRNA level, and / or 4) a method for monitoring IL-1L1 protein level, and further presenting in a specific gene change or in the IL-1L1 gene or IL-1L1 pathway gene Examples include methods for distinguishing or classifying populations based on changes. In addition, the IL-1L1 gene population profile can also suggest specific alterations in patients who are responsive or non-responsive to a particular treatment. This information or population profile is useful in predicting response to a particular drug based on the individual's IL-1L1 profile.

In a preferred embodiment, the IL-1L1 profile is a transcription or expression level profile, and the first step is that the expression level of IL-1L1 protein alone or involved in similar diseases. It involves measuring in conjunction with the expression levels of other known genes. The IL-1L1 profile can be measured from numerous patients at various stages of the disease.

Drug genetics studies can also be performed using transgenic animals.
For example, transgenic mice can be generated that have specific allelic variants of the IL-1L1 gene as described herein. Such a mouse can be produced, for example, by replacing the wild-type IL-1L1 gene with an allele of the human IL-1L1 gene. The response of such mice to a particular IL-1L1 treatment can be determined.

4.8.3 Monitoring the Effect of IL-1L1 Therapeutic Agents in Clinical Studies Based on IL-1L1 or disease gene profile, target population expected to show the highest clinical effect As a result, repositioning of over-the-counter drug shelves; 2) rescue drug candidates whose clinical development has been discontinued as a result of limited safety or efficacy in some patient populations; 3) drug candidate development Promote and low cost development, and create more appropriate drug labels (eg IL-1L1 as a marker
Is useful in determining the optimal effective dose).

Treatment with an IL-1L1 therapeutic can be performed at IL-1L1 protein levels or activity, IL-1L1
It can be monitored by measuring mRNA levels, and / or characteristics of IL-1L1, such as IL-1L1 transcription levels. This measure determines if the treatment is effective,
Or it can be an indicator of whether it should be adjusted or optimized. Therefore, IL-1L1 can be used as a marker of efficacy during clinical trials.

In a preferred embodiment, the invention provides agents (eg, agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids, small molecules, or others identified by the screening assays described herein). Drug candidate, etc.) for monitoring the effect of the treatment, which comprises i) collecting a pre-dose sample from the subject prior to administration of the substance; ii) IL in the pre-dose sample. -1 detecting the expression level of 1L1 protein, mRNA or genomic DNA; iii) taking one or more post-administration samples from the subject; iv) IL-1L1 protein, mRNA or genomic DNA in the post-administration sample V) the level of expression or activity of IL-1L1 protein, mRNA or genomic DNA in the pre-dose sample after administration Comparing to those of the IL-1L1 protein, mRNA or genomic DNA in the sample; and further comprising vi) varying the dose of the agent depending on the subject. For example, if it is desired to increase the expression or activity level of IL-1L1 above the detected level, that is, to enhance the effect of the substance, it is desirable to increase the dose of the substance. Conversely, if it is desired to reduce the level of IL-1L1 expression or activity below the detected level, ie, to diminish the effect of the substance, it is desirable to reduce the dose of the substance.

Cells were collected from the subject before and after administration of the IL-1L1 therapeutic agent to detect the expression level of genes other than IL-1L1, and the IL-1L1 therapeutic agent enhances or reduces the expression of harmful genes. You can also confirm that it is not. This can be performed, for example, by using a transfer profile method or the like. Therefore, in vivo
), MRNA derived from cells contacted with IL-1L1 therapeutic agent, and IL-1 in allogeneic cells
Expression and untreatment of genes in cells treated with IL-1L1 therapeutic agent by reverse transcribing mRNA derived from those not exposed to L1 treatment and hybridizing to a chip containing DNA obtained from many genes The expression of the gene in the cell can be compared. For example, the use of this specific IL-1L1 therapeutic agent is undesirable if the IL-1L1 therapeutic agent switches on expression of a protooncogene in a subject.

4.9 IL-1L1 Therapeutic Agents and Therapeutic Methods The present invention provides a disease involving abnormal expression or activity of interleukin-1 (eg, inflammatory disease or autoimmune disease). Provide prophylactic and therapeutic treatments for subjects with or at risk of developing. The cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) are important indicators of the inflammatory response and play a central role in the pathogenesis of many chronic inflammatory diseases. It is believed that. Their biological activity in vivo occurs by attracting and activating leukocytes to tissues, and their secretion by stimulating the secretion of other lymphocyte cytokines and metabolic enzymes. Numerous reports have been made of the local inflammation and being sufficient to rekindle the matrix metabolism. High production of these cytokines is also associated with the response to infection, and local induction of IL-1 and TNF induces microbial invasion. However, classical studies have shown that in certain infectious conditions, the level of production of monocyte cytokines is very high, which leads to a series of associated events (eg tissue metabolism, vascular reactivity,
It has been reported that the development of excessive coagulation due to damage received by the host is activated.

For example, cytokines act throughout development and play an important role, especially in human placental development and function (Jokhi et al., Cytokin.
e 9: 126-137 (1997)). A variety of cytokines have been found at the placenta-uterine interface, but the precise intracellular origin for their production has not been confirmed due to the complex tissue local structure of the implantation site. Among cytokines, EGF, interleukin-1β (IL-1β), IL-2, IL-3, interferon-α (IFN-α),
IFN-γ, tumor necrosis factor-α (TNF-α) and transforming growth factor-β
1 (TGF-β1) expression has been assayed in cells isolated from placenta and decidua. Furthermore, cytokine receptors such as IGF-1r and PDGF-r
α / β, IL-1rII, IL-6r, IL-7r, IFN-γr, TNF-rp80, and endoglin from placental and uterine cells have been assayed by immunohistological and flow cytometric methods. There is. These studies revealed a complex and diverse activity of cytokines at the human placenta-uterine interface.

The pro-inflammatory cytokines IL-1, IL-6 and tumor necrosis factor-α (TNF-α
) Is thought to act in association with prenatal intrauterine infection (IUI) and neonatal brain injury. In addition, maternal IUI increases the risk of preterm birth, which is associated with an increased risk of intraventricular hemorrhage, neonatal white matter injury and subsequent cerebral palsy (Dammann et al., Pediatr. Res. 42: 1-8 (199
7)). IL-1, IL-6 and tumor necrosis factor-α (TNF-α) have been found to be associated with IUI, preterm birth, neonatal infections and neonatal brain injury. Due to the presence of such cytokines in three related maternal / fetal sites (uterus, fetal circulation and fetal brain), and the ability of cytokines to cross the interface of these sites (placenta and cerebrovascular barrier), The role of cytokines in the development of intraventricular hemorrhage and neonatal white matter damage due to prenatal fetal maternal infection is suggested. Therefore, it is believed that inhibition of the IL-1 mediated pro-inflammatory cytokine cascade may prevent disorders that occur in offspring born late in the second trimester.

Interleukin-1β (IL-1β) is present in normal amniotic fluid and is produced by human placental macrophages. IL-1β detected in amniotic fluid during the second trimester of pregnancy
Is three times higher than when labor began. IL-1β is a potent stimulator of decidual and amniotic membrane synthesis of prostaglandins. High levels of PGE ₂ and PGF 2α in prostaglandins in amniotic fluid suggest possible preterm labor and intra-amniotic infection. This is explained by the fact that amniotic membranes from women with preterm labor and suffering from systemic chorioamnionitis produce more PGE ₂ than amniotic membranes from women without placental inflammation. can do. Increase of such PGE ₂ levels, even if clinical or subclinical it is not sexually urinary tract infections, are associated with prematurity underweight disease (PLBW), in fact, most of PLBW birth origin It is believed to be caused by an unknown infection. IL-1 is the cytokine first involved in the onset of labor when infected. IL-1 responds to bacterial products and is produced by human decidua in vitro. IL-1 bioactivity and levels in amniotic fluid are elevated in patients who deliver preterm and have bacteria in the amniotic cavities. Injection of recombinant human IL-1β into 12 day pregnant rats resulted in placental necrosis and fetal resorption. In addition, the IL-1β concentration and biological activity in amniotic fluid during labor increased compared to controls. Furthermore, IL-1β is known to stimulate the production of prostaglandins by the amnion and decidua in vitro.

Therefore, the IL-1L1 therapeutic agent according to the present invention is useful for treating interleukin-1 dependent diseases in the human placenta, including intraventricular hemorrhage, neonatal white matter damage and subsequent cerebral palsy, and premature underweight birth. Substances that antagonize are mentioned.

4.9.1 Prophylaxis In one aspect, the present invention provides that IL-1L1 is administered by administering to a subject a substance that regulates at least one of IL-1L1 expression or IL-1L1 activity. -1 provides a method for preventing a disease or condition associated with abnormal expression or activity of -1L1. Subjects at risk of such a disease can be identified using diagnostic or prognostic assays as described herein. Administration of a prophylactic agent to protect against or delay progression of a disease or disorder characterized by an abnormality in IL-1L1
It can be done before the onset of symptoms. Depending on the mode of IL-1L1 abnormality, for example, IL
Prophylactic measures can be taken with -1L1 agonists or IL-1L1 antagonists. The prophylactic methods are similar to the therapeutic methods of the invention and are described in more detail in the next section.

4.9.2 Therapeutic Methods In general, the present invention provides methods of treating diseases or conditions that are caused by or associated with abnormal activity of IL-1L1. Such methods include administering to a subject an effective amount of a compound capable of regulating IL-1L1 activity. Methods used to alleviate disease symptoms including abnormal IL-1L1 activity include, for example, antisense, ribozyme, and the triple helix molecule described above. Examples of suitable compounds include the antagonists, agonists or homologues detailed herein.

[0323]4.9.3 Effective dose   For the toxicity and therapeutic effects of such compounds, see cell culture or laboratory animals.
Can be determined by standard pharmacological methods, such as Ld ₅₀  (Dose that kills 50% of the population) and Ed₅₀ (50% of the population has a therapeutic effect
The prescribed dose) can be mentioned. The dose ratio between toxicity and therapeutic effect
The therapeutic index, LD₅₀ / ED₅₀ Can be expressed as Compounds with large therapeutic index
The thing is preferable. When using compounds that show toxic side effects,
Such compounds are directed to the infected tissue site to minimize the expected damage.
It is necessary to design the transport system to be effective, thereby reducing side effects
be able to.

The data obtained from the cell culture assays and animal studies can be used in preparing dosage forms for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ without toxicity little or no. The dosage will vary within this range depending upon the dosage form and route of administration. For any compound used in the invention, the cell culture assay can be first performed to establish a therapeutically effective dose. Formulations can be made in animal models to reach circulating plasma concentrations in a range that includes the IC50 determined in cell culture (ie, the concentration of the test compound that causes inhibition of symptoms in half). Such information can be used to more accurately determine useful doses in humans. For example, the level in plasma can be measured by high performance liquid chromatography or the like.

4.9.4 Formulation and Use The pharmaceutical composition according to the present invention is formulated according to conventional methods using one or more physically acceptable carriers or excipients. be able to. Therefore, a compound and a physically acceptable salt and a solvent compound are formulated according to the administration method such as injection, inhalation or inhalation (from the mouth or nose), oral, buccal, parenteral or rectal administration. can do.

For such treatment, the compounds of the invention may be formulated for a variety of administration methods, including systemic administration as well as local or localized administration. General techniques and formulation issues are described in Remmington, "Pharmaceutical Sciences" (Meade Publishing, Easton, PA). For systemic administration, injections including intramuscular, intravenous, intraperitoneal and subcutaneous are preferred. For injection, the compounds of the invention can be formulated as liquid solutions, preferably with physically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized formulations are also included.

For oral administration, the pharmaceutical composition can be made into a dosage form such as a tablet or capsule, which is formulated according to a conventional method using a pharmaceutically acceptable excipient. , Such excipients as binders (corn starch before gelatinization, polyvinylpyrrolidone or hydroxypropylmethylcellulose etc.), fillers (eg lactose, microcrystalline cellulose or hydrogenated calcium phosphate), brighteners (Eg, magnesium stearate, talc or silica), disintegrating agent (eg, potato starch or sodium starch glycolate), wetting agent (eg, sodium lauryl sulfate), and the like. The tablets can be coated by methods known in the art. Liquid dosage forms for oral administration can be in the form of, for example, solutions, syrups, suspensions, or dry products that will be dissolved in water or other suitable vehicle when used. Such liquid formulations can be formulated according to conventional methods using pharmaceutically acceptable additives,
Such additives include suspensions (eg sorbitol syrup, cellulose derivatives or hardened edible oils), emulsifiers (eg lecithin or gum arabic), non-aqueous bases (eg Ationed oil). ), Oily esters, ethyl alcohol or fractionated vegetable oils), as well as preservatives such as methyl or propylhydroxybenzoic acid or sorbic acid. Suitable buffer salts, flavors, dyes and sweeteners can also be added to the formulation.

Formulations for oral administration are preferably formulated to give controlled release of the active compound. For buccal administration, the composition can be in the form of tablets or sublingual tablets formulated according to conventional methods. For administration by inhalation, a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas may be used in using the compositions according to the invention. It can be conveniently delivered as an aerosol spray from a pressurized pack or nebulizer. In the case of a pressurized aerosol the dosage unit may be defined by a valve made to deliver a metered amount. Capsules and cartridges such as gelatin for use in inhalers or air inhalers can be formulated containing a composition and powder mixture consisting of a suitable powder base such as lactose or starch.

The composition may be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. Formulations for injection include preservatives,
It can be in unit dose form such as an ampoule or multi-dose container. The composition can be in the form of a suspension, a solution, or an emulsion in an oily or aqueous vehicle, and may employ formulation aids such as suspending, stabilizing and / or dispersing agents. it can. Alternatively, the active material can be powdered and dissolved in a suitable vehicle, such as sterile pyrogen-free water, before use.

The composition may be formulated as a rectal composition such as a suppository or enema fixative, such composition comprising a conventional suppository base such as cocoa butter or other glycerides. Including agents.

In addition to the formulations described previously, the composition may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (eg, as an acceptable emulsion in oil) or ion exchange resins, or as sparingly soluble derivatives (eg, sparingly soluble salts). can do. Other suitable delivery systems include microspheres that are capable of local, non-invasive delivery of drugs over very long periods of time. This technique uses precapillary-sized microspheres that can be injected through a coronary catheter into any selected organ, such as the heart, without inflammation or congestion. Administered therapeutic agents are gradually released from these microspheres, causing the surrounding tissue cells (
(Eg, endothelial cells).

Systemic administration can also be by transmucosal or transdermal methods. For transmucosal or transdermal administration, penetrants appropriate to the barrier that must be permeable are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, a surfactant may be used for permeation. Transmucosal administration can be performed using nasal spray or suppositories. For topical administration, the oligomers of the invention are formulated as ointments, waxes, gels or creams commonly known in the art. Recovery can be promoted by topical use of the wash solution to treat wounds and inflammation.

In the clinical setting, applying a gene delivery system for IL-1L1 gene therapy to a patient by any of a number of methods, which are known in the art. You can For example, a pharmaceutical preparation of a gene delivery system can be introduced systemically by intravenous injection or the like, and the specificity of transfection preferentially causes the specific induction of a protein in a target cell. Transfection specificity is provided by gene delivery vehicles, cell-type or tissue-type expression by the action of transcriptional regulatory sequences that control expression of the receptor gene, or a combination thereof. In another embodiment, the initial delivery of the recombinant gene is more limited as the introduction into the animal is highly localized. For example, gene delivery vehicles include catheter (
See US Pat. No. 5,328,470) or stereotaxic injection (Chen et al., PNAS 91:
3054-3057 (1994)). IL such as SEQ ID NO: 1 or 4, or the other 5'end of IL-1L1 represented by SEQ ID NO: 3 or 3, or one of the sequences represented by the group including sequences homologous thereto The -1L1 gene is described, for example, by Dev et al. (Cancer Treat Re
v 20: 105-115 (1994)) and can be delivered into gene therapy constructs by electroporation.

Pharmaceutical formulations of the gene therapy constructs or compounds of the invention basically have the gene delivery system in an acceptable diluent or include a sustained release matrix, Embedded within this matrix is a gene delivery vehicle or compound. Alternatively, if the entire gene delivery system is produced intact from a recombinant cell such as a retroviral vector, the pharmaceutical preparation may comprise one or more cells producing the gene delivery system. including.

The compositions may, if desired, be packaged in a pack or dispenser device which may contain one or more dosage units containing the active ingredient. For example,
The pack can be made of a thin layer of metal or plastic such as a blister pack. The pack or dispenser device can be accompanied by administration instructions.

4.10 Kits The invention further provides kits for use or treatment of the diagnosis or prognosis of diseases or conditions associated with abnormal IL-1L1 protein. The present invention also provides a kit for determining whether or not an IL-1L1 therapeutic agent should be administered to a subject. The invention includes a kit for detecting the presence of IL-1L1 mRNA or protein in a biological sample, or for identifying the presence of a mutation or the nature of a polymorphic region within the IL-1L1 gene. . For example, such a kit may include a labeled compound or substance capable of detecting IL-1L1 protein or mRNA in a biological sample, means for measuring the amount of IL-1L1 in the sample, and IL in the sample.
-1 Includes means to compare L1 amount to that of the standard. The kit includes IL-1L1
It may also include instructions for detecting mRNA or protein.

In one embodiment, such a kit comprises a pharmaceutical composition containing an effective amount of an IL-1L1 antagonist therapeutic agent, and instructions for use in the treatment or prevention of hypertension. In another embodiment, such a kit comprises a pharmaceutical composition containing an effective amount of an IL-1L1 agonist therapeutic agent and instructions for use in the treatment of insect bites. Generally, the kit comprises a pharmaceutical composition containing an effective amount of an IL-1L1 agonist or antagonist therapeutic agent, and instructions for use as an analgesic agent. For example, the kit comprises a pharmaceutical composition containing an effective amount of an IL-1LI agonist therapeutic agent and a therapeutic description as an analgesic.

Another kit can be used to determine whether a subject develops or is likely to develop a disease or condition in which an abnormality in IL-1L1 activity is involved. Such kits include, for example, one or more nucleic acid probes capable of specifically hybridizing to at least a portion of the IL-1L1 gene or allelic variants thereof, or mutants thereof. I'm out.

4.11 Other Uses of IL-1L1 Protein and Nucleic Acid The IL-1L1 nucleic acid of the present invention can be further used in the following assays. In one embodiment, a human having SEQ ID NO: 1 or a portion thereof.
Using the IL-1L1 nucleic acid or a nucleic acid that hybridizes to them, the correct chromosomal localization of the IL-1L1 gene within the IL-1 locus can be confirmed. Furthermore, IL
The -1L1 gene can be used as a chromosomal marker in studies on gene linkage involving genes other than IL-1L1.

Chromosomal localization of a gene can be demonstrated by several methods known in the art. For example, Southern blot analysis or PCR mapping of somatic cell hybrids can be used to determine on which chromosome or chromosome fragment a particular gene is localized. Other mapping methods that are also used for locating genes in chromosomes or chromosomal regions include:
Examples include in situ hybridization, prescreening using labeled flow-sorted chromosomes, and preselection by hybridization for the purpose of constructing a chromosome-specific cDNA library.

Furthermore, fluorescence in situ hybridization (FISH) of nucleic acids (eg, IL-1L1 nucleic acids, etc.) to metaphase chromosomes can be performed by a one-step method, which allows The exact location of the nucleic acid is revealed. This technique can be used for nucleic acids as short as 500-600 bases in length, but larger clones of 2000 base pairs or more give a signal of sufficient intensity for easy detection and bind to specific positions on the chromosome. Is likely to. Such techniques are described, for example, in Verma et al., "Human Chromosomes: a Manual of Basic Techniques" (Pergamum
Press (Pergamon Press), New York, 1988) and the like.
By using such a technique, the position of a gene in a chromosomal region containing about 50 to about 500 genes can be confirmed.

Between the affected and unaffected individuals, where the IL-1L1 gene is shown to be present in a chromosomal region that distinguishes, or is associated with, a particular disease. Confirm differences in the cDNA or genomic sequence of. If a mutation is present in some or all of the affected individuals, but not in normal individuals, the mutation is responsible for or contributes to the disease. It is possible that

The present invention is further illustrated by the following examples, which are not intended to limit the present invention. The contents of all references (including documents, issued patents, published patent applications) cited by reference are hereby incorporated by reference.

In practicing the present invention, cell biology, cell culture, molecular biology, transgenic biology, microbiology, known to those of skill in the art, unless otherwise indicated.
Utilizes conventional techniques for recombinant DNA and immunology. Such techniques are explained fully in the literature. For example, edited by Sambrook, Fritsch and Maniatis,
"Molecular Cloning: A Lanoratory Ma
nual) 2nd Edition "(Cold Spring Harbor Laboratory Press (Cold Sprin
g Harbor Laboratory Press), 1989); DN Glover, "DNA Cloning," Volumes 1 and 2 (1985); MJ
Gait, “Oligonucleotide Synthesis”
(1984); Mullis et al., U.S. Pat. No. 4,683,195; edited by BD Hmes and SJ Higgins, "Nucleic acid Hybridization (1984);" Transcription and Translation ( Transcriptio
n And Translation ”(1984); RI Freshney,“ Culture Of Animal Cells ”(Alan R. Liss
), 1987); "Immobilized Cells And Enzymes"
(IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1
984); Reference "Methods In Enzymology" (Academic Press, New York); JH Miller and MP Calos, eds., Gene Transfer Vectors for Mammalian Cells. Gen
e Transfer Vectors For Mammalian Cells "(Cold Spring Harbor Laboratory Press, 1987); Wu et al.," Methods In Enzymology, "Volumes 154 and 155. Vol .: Mayer and Walker, "Immunochemical Methods In Cell And Molecul.
ar Biology ”(Academic Press, London, 1987)
); Edited by DM Weir and CC Blackwell,
"Handbook Of Experimental Immunology" Volume I-
Volume IV (1986); "Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratories)
Y Press), Cold Spring Harbor, NY, 1986).

5. Example 5.1 Cloning and analysis of human and mouse IL-1L1 Present within the interleukin-1 gene cluster (Nicklin et al., (1996)), which may be involved in the human inflammatory response. For the purpose of discovering a novel gene, the inventors have developed a yeast (Nothwang et al., Which produces a 680 kb CEPH yeast artificial chromosome (YAC) clone 766E12 containing an IL-1 cluster.
(1996)), and the chromosome was isolated by performing pulse field electrophoresis. The chromosome was used as a driver in the cDNA selection method according to the description of Morgan et al. (1992). The cDNA was obtained from human peripheral blood mononuclear cells cultured for 6 hours in the presence of 10 ng / ml phorbol myristate acetate and 100 ng / ml E. coli lipopolysaccharide. The cDNA is amplified as described, then
After two cycles of affinity selection and further amplification, the cDNA fragment was cloned into pNEB193. All 100 samples randomly selected were sequenced. The sequences were assembled in duplicate units. The IL-1L1 expressed sequence tag (EST) corresponds to one of the overlapping sequences derived from the three overlapping fragments (in our records, e031 and e049) that make up a continuous sequence of approximately 530 bp. It was This sequence is based on two previously reported EST clones R70089.
And R00741 were found to overlap. When tested, the sequence was found to be the P1 artificial chromosome 131J6 from the Gingrich human PAC library.
Since it was found to hybridize specifically with ICR, it was derived from the ICRF human P1 library (Nothwang et al., Genomics 41: 370-378 (1996)).
It was located between the IL1B and IL1RN genes flanking F700G1305. This latter clone contains the IL-1ra gene (IL1RN). No open reading frame was present either in the database EST sequence or in our new EST sequence.

CDNA was isolated from the H9 placental cDNA plasmid library from HGMP using EST DNA as a probe. Although this clone was only 1.6 kb in length, it was polyadenylated and again did not contain the coding sequence. All clones were sequenced and the 5'end sequence was used to perform primer dependent amplification of the 5'end of the placenta-derived IL-1L1 cDNA. The product was approximately 1.1 kb in length and overlapped the sequence of the cDNA clone. By using multiple sets of 5'primers for the amplification reaction, 5 or more distinct isolates (at least 2 for each 5'end) likely to contain two different non-coding 5'ends. Has been obtained) and they are shown in FIG. The estimated total length of mRNA was calculated to be 2.6 kb. The coding sequence contained up to the first 600 nucleotides. Eliminating the expected cloning error, all cDNA sequences (
The present inventors sequenced the whole 3) and encoded the same protein, suggesting that the leader sequence does not exist. Indeed, a human cDNA identical to the sequence we report here has been previously reported (Mulero et al., Biochem. Biophys. Res. Commun. 263: 702-706 (199).
9); Smith et al., J. Biol. Chem. 275: 1169-1175 (2000); Busfield et al., Genomics 66: 213-216 (2000).

IL-1L1 genomic sequencing and mapping: PAC 131J6 subclones were generated from Xba I and EcoR-I digested DNA and sequenced by primer walking. Good analysis results were obtained at least once from both chains. The IL-1L1 gene consists of 6072 nucleotides,
This is shown in FIG. The position of the intron-exon boundary is shown in Figure 10b, which is very similar to the position of the intron-exon boundary of IL-1RN, as already described for other genes in the IL-1 structural family. Is similar to. The entire sequenced segment from PAC 131J6 (6540 nucleotides, accession number AJ271338) contains 6 exons from 450 nucleotides upstream of exon 1 to 18 nucleotides downstream of the polyadenylation site. Recently, a high-throughput genomic sequence of PAC RP11-339F22 (accession number AC016724) has also been deposited in the EMBL database. Reconfirming the initial data to look for differences, our genomic sequence differed from the sequence in the database at 44 positions (0.67%). PAC RP11-339F22 contains a 195 kb genomic insert (insert), which is currently sequenced within 12 non-overlapping fragments. 12 positions for the use of known marker positions (Hilderbrandt et al., (1996) Cytogenet. Cell Genet. 73: 235-239) and IL-1RN locus-located sequences. 11 of the fragments were placed. Was placed. Restriction map and IL-1
Hybridization with the 5'and 3'probes from L1 allowed the localization of the remaining fragments and the orientation of the other fragments. Therefore, IL-1L1 is located between the STS markers 10H8S and 131P6S and is transcribed in the 131P6S direction. AC016724, as suggested by the three gaps in IL-1RN
It is considered that the sequence excluded from is small. Therefore, the polyadenylation signal of IL-1L1 is present at a position approximately 53 kb on the 5'side of the first exon of IL-1RN, and the two genes have the same orientation. We conclude that it is suitable. From our previous mappings, this means that there is a gap of about 270 kb between IL-1L1 and IL-1B.

Due to the break between the human and mouse IL-1L clusters (Zahed
i) et al., J. Immunol. 146: 4228-4233 (1991)), whether the difference in IL-1L1 transcript between species is due to the division existing in exon 6 of mouse IL-1L1. I considered. 129 / Sv RPCI21 library (using mouse IL-1L1 cDNA probe
5.15. From "Current Protocols in Human Genetics", edited by Ioannou et al., Dracopoli et al.
Mouse PAC from 1-5.15.24, Wiley, New York, 1996)
The clones were screened to obtain 3 plasmids, 2 of which also contained the Il1rn ORF, which indicates that Il1l1 and Il1rn are contiguous and that the homologous region is also present. Evidence was obtained that the disruption of A. cerevisiae occurs between Il1b and Il1l1 rather than between Il1l1 and Il1rn. Low stringency hybridization on Zooblots (Clontech) revealed the presence of the IL-1L1 gene in all eutherian species (FIG. 8).

FIG. 9 shows the mapping of the IL-1L1 locus within the IL-1 gene cluster. FIG. 9 (a) shows ILs for the three identified genes IL1A, IL1B and IL1RN.
-1 shows the approximate position of L1. The total length of the cluster is determined from the restriction enzyme positions for the CpG populations X, Y and Z. The positions of the markers and PAC 131J6 are shown. FIG. 9 (b) shows the first exon of IL-1L1 with respect to the intracellular first exon of IL1RN (IL1Rnex1 ′), and flanking markers 10H8S and 1
A more detailed mapping of 31P6S is shown. The size of the known sequence from the IL1RN locus is shown (accession number U65590). (C) Exon distribution within the IL-1L1 gene obtained from the gene sequence. Bold numbers indicate the position of each exon.

An assessment was made of 5'alternative splicing of IL-1L1 mRNA. Six of the seven 5'RACE products we tested were apparently cloned differently due to differences in the first residue (Fig. 10 (a )). The seven products could be divided into five groups, two of which had completely different sequences at the 5'end and were considered to be derived from another first exon (exon 1 And 2, see FIGS. 9 and 10 (c)). The inventors predicted that the two exons have different promoters, both lacking the TATAA box. Exon 1 has an out-of-frame upstream part of the ORF consisting of 16 codons (
10 (a)), followed by a stop codon. Their arrangement is IL-1L
It is considered to be suitable for translation control of 1.

FIG. 10 shows in some detail the splicing of the IL-1L1 transcript.
(A) In mRNA synthesis, two distinct first exons (exons 1 and 2)
) Is used. Seven different cDNA cloning products derived from the 5'RACE of placental mRNA were sequenced. The appearance of the longest cDNA sequences was confirmed in both rare mRNA types (exon 1 start) and general mRNA types (exon 2 start). The arrow indicates the beginning of the cDNA sequence (note that true transcription must start at A or G). The short sequence out-of-frame upstream of the ORF with AUG in good context is contained in exon 1 and the translated one is shown as the one letter code. (B) shows the intron-exon boundary of the IL-1L1 gene. "+1" in the cDNA sequence is defined as the beginning of code exon 3. When the transcription start position is not determined, the position of the genomic sequence is arbitrarily determined with respect to the genomic sequence described in the database appendix AJ271338. "5 '
"Frank" and "3 'flank" represent the respective flanking sequences. "
"Start" and "end" indicate the first and last bases of the exon. “Exon” indicates the exon number. "Start sequence" and "end sequence" indicate the start and end sequences of an exon. "CDNA" means the cDNA sequence encoded within an exon. (C) The conservation status of the splice site between human IL-1L1 and its closest homologue, IL-1ra, is shown by the parallel listing of the translation products of the two genes. The position of the splice site is indicated by the bracketed overriding number, which represents the nucleotide within the preceding triplet codon that acts as a splice donor.

FIG. 11 shows the entire genomic sequence of human IL-1L1 gene. The full-length EMBL accession numbers for the IL-1L1 cDNA sequence are AJ242737 and AJ242738. The accession number of the entire sequence of IMAGE cDNA clone # 332733 encoding mouse IL-1L1 is AJ
It is 250429. The human IL-1L1 genome is shown in Figure 11, and the accession number is
It is AJ271338.

5.2 Tissue distribution of IL-1L1 mRNA Expression of the IL-1L1 gene was confirmed by Northern blot. A commercial blot of poly (A) + tissue mRNA was investigated using a probe corresponding to the IL-1L1 ORF. Only the placenta gave a strong signal, and bands of 2.7 kb and weaker 1.2 kb were confirmed (Fig. 7 (a)). There appeared to be a small amount of mRNA in the thymus. The first EST used to identify IL-1L1 was derived from PBMC stimulated with endotoxin and PHA, so the raw materials were confirmed. White blood cells were fractionated and total RNA was hybridized with IL-1L1 ORF. IL-1L1 mRNA of the expected size was clearly detected in endotoxin-stimulated monocytes and in macrophages differentiated in vitro. It was not detected in NK cells, lymphocytes, and dendritic cells differentiated in vitro (Fig. 7 (b)). Stronger expression of IL-1ra was observed in all fractions of leukocytes. IL-1L1 mRNA was relatively strongly expressed in differentiated macrophages. By using RT-PCR, IL-1L1 ORF expression was detected in human skin, brain, heart, kidney, leukocytes, placenta and spleen, but not in liver Northern blot was detected with IL-1L1 probe. Hybridization revealed an mRNA corresponding to a transcript of approximately 2.7 kb in placenta. FIG. 7 shows IL-1L1 mRNA detected in human tissues. (A) Isolated from 5 μg of poly A + tissue per lane
Northern blot hybridization of IL-1L1 ORF performed using RNA (Clontech). The following tissues were used. Pa = Pancreas; Ki = Kidney; Mu = Skeletal muscle; Li = Liver; Lu = Lung; Pl = Placenta; Br = Brain; He = Heart; Le
= Peripheral blood leukocytes; Co = colon; In = intestine; Ov = ovary, Te = testis; Pr = prostate; Th = thymus; Sp = spleen. The arrow on the left of the main figure indicates the location of the 2.6 Kb IL-1L1 mRNA.
RNA molecular weight markers are shown on the right. The upper panel shows the appearance of the same blot after hybridization with the control β-actin probe. (B) The same IL-1L1 probe as described above was used to hybridize to a Northern blot containing all of the mRNA isolated from the cells. The following were used as cells. Ly + = Lymphocytes stimulated with 5 μg / ml PHA for 48 hours; M _c- =
Unstimulated monocytes; MC + = monocytes activated with 100 ng / ml LPS; Pm- = unstimulated PMN; Pm
+ = PMN stimulated with 100 ng / ml LPS for 4 hours; MF = Macrophage cultured in serum of macrophage origin for 6 days; NK- = unstimulated NK cells; NK + = 48 with IL-2
Time-activated NK cells; DC- = immature dendritic cells; DC + = mature dendritic cells; HE- = unstimulated H
UVEC; HUVEC treated with HEI = 6 nM IL-1b for 4 hours; HUVEC treated with HEL = 100 ng / ml LPS for 4 hours. The image is the blot exposed for 10 days. The blot was then probed with the ORF of IL-1ra, a control gene that responds to inflammation, and β-actin as a loading control. The location of ribosomal RNA is indicated in the main figure.

5.3 Analysis of IL-1L1 Polypeptide Sequence and Structure The protein product of the IL-1L1 gene was predicted to be 47% residue identical to the human interleukin-1 receptor antagonist protein ( (See FIG. 5). Further investigation of sequence identity revealed a partial cDNA sequence from the mouse clone of the IMAGE database (332733). Here, the partial cDNA sequence derived from the mouse clone encodes a partial polypeptide sequence that is considered to be a mouse homologue to the human IL-1L1 protein sequence. We obtained the clone and sequenced all inserts. A final comparison of the putative mouse and human IL-1L1 proteins, and human IL-1L1 and IL-1ra proteins is shown in FIG. The IMAGE cDNA is also polyadenylated, but differs greatly from the human IL-1L1 cDNA sequence in the 3'non-coding region, and the mRNA length is only 1.2 kb.
And much shorter than human mRNA. So far, IL-1L1 mRNA has not been detected in Northern blots of mouse tissues. In human genes, sequence changes occur within the last very long exon 4. Human IL-1L on Southern blots of numerous genomic DNA truncations (mainly from mammals)
Hybridization with a single-specific probe at reduced stringency revealed a single distinct homology band in all tested mammals but not in yeast and in chickens. No signal appeared (see Figure 8). The intron-exon structure is similar to that of the other members of the IL-1 family.

By comparing the novel open reading frame of human IL-1L1, human IL-1L1 was found to match 73 of 155 residues (47%) with the processed secreted form of human IL-1ra. It was revealed that 42 of 155 residues (27%) were in agreement with human IL-1β (FIG. 13). BLAST (Altschul et al., J. Mol. Biol. 215:
403-410 (1990)) also revealed a fragmentary open reading frame (accession number W08205, IM) that clearly contains the ORF of the mouse homologue of IL-1L1.
AGE clone number # 332733) was confirmed. The inventors have completely sequenced the clone. The mouse ORF starts with two AUG codons. From the relationship with the human sequence and analogy, it is considered that the second AUG is involved in initiation.
The identity of the mouse and human IL-1L1 ORF is 90% (Figure 13). However, clone # 332733, despite having an ORF and polyadenylated, is only 1.2 kb in length, and its sequence is 2.7 kb of human cDNA 34n after translation.
is completely different from t. Reverse transcription and mRN from RAW264.7 mouse macrophages
Although the expression of mouse IL-1L1 ORF was detected by PCR of A, the length was not confirmed by Northern blot hybridization.

We prepared the human IL-1 receptor antagonist protein and IL-1L1 precursor using the predicted sequence (see FIG. 6) as follows and isolated by nickel chelate chromatography: Then, elution with imidazole and treatment with thrombin gave the following putative polypeptide. From the nuclear magnetic resonance preliminary experiments about ¹⁵ N + ^{1 3} C double labeled IL-1L1 protein, protein is suggested that is folded greatly β- sheet structure like, also, from the analysis results, the structure , And is very similar to human IL-1ra physically.

The folding of the recombinant IL-1 protein was also inferred by detailed NMR structural studies. The folding pattern of rhIL-1L1 was investigated with the aim of confirming that it was biologically inactive due to incorrect folding of rhIL-1L1.
One-dimensional ¹ H NMR spectra of Form A and Form B suggested a well-folded monomeric protein. It was revealed that Form A does not dissolve sufficiently to be used for multidimensional NMR methods when it does not possess an artificial N-terminal extension. However, the one-dimensional spectrum of the uncleaved A-form precursor IL-1L1 overlapped considerably with that of the cleaved B-form (which was used in all biological experiments), thus giving the same conformation. It was suggested that there is. We, ¹⁵ N
A non-cleavable Form A precursor double labeled with + ¹³ C was prepared (LH, MN and John
Warso (John Waltho), unpublished data). An α ¹³ C-carbon shift was observed. The presence of β-sheet structure is suggested from the cluster of α-carbons shifted to a high magnetic field of 0.7 ppm or more (Wishart et al., NMR 4: 171-178 (1994)), and FIG.
Is indicated by *. These results correlate well with the position of β-sheet residues within IL-1ra and are indicated by> in FIG. The processed B form rhIL-1L1 and the uncleaved A form are folded in a very similar manner, with the folding pattern being essentially similar to that of their closest protein homolog, IL-1ra. is there.

FIG. 13 shows the human and mouse IL-1L1 sequences aligned with the sequences of other known IL-1 family members. Homology block and IL-1β and IL
Alignment was chosen to preserve the conservative structural features present within -1ra. The core region of the conserved β-sheet of IL-1ra is indicated by> above the sequence. * Indicates an IL-1L1 residue in which α carbon labeled with ¹³ C is shifted to a high magnetic field by 0.7 ppm or more.
The aggregation of such residues suggests a β-sheet structure. The open residues are conserved within this region between human IL-1L1 and the target sequence.

5.4 IL-1L1 Antibody and Immunoprecipitation Assay The recombinant IL-1L1 antibody described above can also be used to induce antibodies in rabbits.

The inventors have shown that the choriocarcinoma cell line JEG-3 produces essentially IL-1L1 mRNA. The protein was metabolically labeled and shown to specifically immunoprecipitate the 17 kDa protein with rabbit anti-IL-1L1 polyclonal antiserum. Expression of IL-1L1 protein was detected by immunoprecipitation. The trophoblast cell line JEG-3 (Kohler et al., J. Clin. Endocrinol. 32: 683-687 (1971)) is structurally a 2.7 kb mRN of IL-1L1.
It is considered to have A (FIG. 12 (a)), which is expected when the major cellular components of placenta and villi express IL-1L1. A specific polyclonal serum against recombinant human IL-1L1 (rhIL-1L1) was elicited in rabbits. JEG-3 was metabolically labeled and cell lysates were prepared. 17kDa from immunoprecipitation
Specific radioactivity band corresponding to the mobility of the protein of Escherichia coli was revealed (Fig. 12).
Similar products were not immunoprecipitated in lanes 3 to 5 in (b) and the control whole serum (lane 1 in FIG. 12 (b)). Pre-competition of the antibody with rhIL-1L1 inhibited immunoprecipitation of the 17kDa band, further confirming that this was endogenous IL-1L
It was suggested to be one protein (compare lanes 1 and 2 and lanes 5 and 6 in FIG. 12 (c)). Unlike the IL-1ra protein, IL-1L1 lacks the N-glycosylation signal, and the mobility of endogenous proteins is reduced by E. coli.
It was confirmed that the material was not deformed, since it was in agreement with that of the material prepared in (1).

FIG. 12 shows immunoprecipitation of native IL-1L1 protein from JEG-3 cells. (A)
Detection of constitutive IL-1L1 mRNA in JEG-3 cells. Using a cDNA probe of IL-1L1 ORF, 2
Northern blots of poly A + RNA selected from × 10 ⁷ JEG-3 cells were performed. The arrow indicates a 2.7 kb mRNA. The position of the RNA molecular weight marker is shown on the left. (B) Immunoprecipitation with extracts of metabolically radiolabeled cells. The material used for sedimentation is lane 1
= 20 μl irrelevant antiserum; lane 2 = no serum; lane 3 = 5 μl anti-IL-1L1 antiserum; lane 4 = 10 μl anti-IL-1L1 antiserum; lane 5 = 20 μl anti-IL-1L1 antiserum.
The mobility of protein molecular weight standards is shown on the left. The arrow on the right of the figure indicates what appears to be a band for the IL-1L1 protein. No immunoprecipitation of this band was observed after blocking the antibody with rhIL-1L1. (C) Evidence that IL-1L1 is secreted from JEG-3 cells. Immunoprecipitations were performed on cell lysates (lanes 1-4) and supernatants (lanes 5-8). The material used for sedimentation was lanes 1 and 5 = 40 μl of anti-IL-
1L1 antiserum; lanes 2 and 6 = pre-blocked with 20 μg rhIL-1L1 20
μl anti-IL-1L1 antiserum; lanes 3 and 7 = irrelevant serum; lanes 4 and 8 =
No serum. The gel is partially shown. The arrow indicates the migration position of rhIL-1L1. In the supernatant,
It contained only 15% of the total TCA-precipitable radioactivity associated with the cells.

[0362] IL-1L1 cell based for measuring 5.5 IL-1 activity assay Lee IL-1L1 is the impact on IL-1-like signals were investigated using convenient assay, the IL-1 The effect on the production of IL-6, a type of cytokine that responds well and directly, was measured (Zilberstein et al., EMBO J. 5: 2529-2537 (1
986)). In E. coli, three types of IL-1L1 were produced as 6His-tagged recombinant fusion proteins, which were cleaved with thrombin (Smith (
Smith) et al., Gene 67: 31-40 (1988)) to obtain the desired protein. A type started
It lacked the Met codon and was replaced by Gly-Ser-Ser- (see Figure 6). N
-If the exact nature of the termini was important, we also created a B-type containing Met1 and a C-type beginning with Val2 (see Figure 6). For comparison, IL-1ra was prepared and similarly isolated,
A modification was also made in which the first two residues after cleavage, Pro-Lys-, were removed and Gly-Ser-Ser was added. Preliminary experiments were performed on all three types of IL-1L1 for IL-1-like activity of stimulating primary fibroblasts to secrete IL-6 and secreted IL-6 was detected by ELISA. Detailed experiments were conducted on the effects of B-type IL-1L1 on fibroblasts and endothelial cells. When IL-1L1 is 0.1μM or less, the activity is 0.1n
It was less than 1% of that of M IL-1β. From these data, the upper limit for IL-1L1-like activity of IL-1L1 is 1/105 or less.

Using a similar system, the activity of IL-1L1 was also compared to IL-1ra. Initial dose response experiment (
(Data not shown), 10 nM IL-1ra protein was shown to reduce the amount of IL-6 produced by fibroblasts in response to 0.1 nM IL-1β by more than 95%.
Preliminary experiments were performed again for all three types of IL-1L1. Then 0.1 μM IL-1
Using ra or IL-1L1, 10nM IL-1α or IL-1β (R & D Systems
ms) company). A detailed experiment comparing type B IL-1L1 with IL-1ra was performed again. IL-1ra completely abrogated the production of IL-6, whereas IL-1L1 had no reproducible and potent effect on either type of IL-1 activity. Although the amount of IL-6 released from fibroblasts was slightly reduced to an inconspicuous level, the extent of the reduction did not increase in the preliminary experiment conducted with 1 μM IL-1L1. Before concluding that IL-1L1 is not a receptor antagonist, experiments were performed with supernatants of COS-7 cells transfected with the IL-1L1 expression vector described above, which cells were treated more naturally. Was considered to be the source of the produced protein. Although the experiment was performed twice at the same time, about 20 nM of COS-producing IL-1 for signaling by 0.1 nM of IL-1β to fibroblasts.
No effect of L1 (measured by Western blot on cell conditioned medium) was detectable and no agonist effect was observed. Control experiments used conditioned medium from cells transfected with the vector from. 20 μM IL-1ra was found to eliminate the effects of IL-1β. Therefore, the inventors have concluded that IL-1L1 does not directly affect the IL-1 system.

The activity of rhIL-1L1 in the IL-18 line was tested using the human myelomonocytic cell line KG-1, which produces IFNg in response to IL-18 stimulation. (Konishi et al., J. Immunol. Methods 209: 187-19 (1997)). The inventors used a modified assay in which cells were stimulated with PHA for 2 hours before being stimulated with IL-18. B-type rhIL-1L1 was examined for its action as an IL-18-like agonist and its action as an antagonist. PHA and IL-1 compared to PHA alone
In the presence of 8, cell IFNg production increased 3- to 5-fold. Furthermore, rhIL-1L1 was found in the system to be less than IL-18 in this system when compared to monoclonal antibodies against IL-18.
-18 could not be blocked. 0.1 μM in cell stimulation with PHA alone
It was noticed that the addition of IL-1L1 of IL-1L1 slightly decreased the IFNg production from the cells. IL
Although the -18 assay was less sensitive and more variable than the IL-1 assay, these results suggested that IL-1L1 also did not act directly on the IL-18 system.

Next, the assay was performed for the presence of the IL-1L1 receptor on fibroblasts. The IL-1 receptor abundance on fibroblasts is very low. Develop a highly sensitive binding assay for IL-1L1 and IL-1ra (control) in order to investigate whether the absence of a biological response to IL-1L1 is due to the absence of the receptor There was a need. After thrombin cleavage, N-terminal extension of GSSGLRRA ^* SLGSS (where * represents a serine residue that is a substrate for protein kinase A) was performed.
IL-1L1 and IL-1ra were prepared. The two extension proteins are KA-IL-1ra and KA-
It was named IL-1L1. KA-IL-1ra lacked the first two residues of the processed secretory protein (similar to the functional rhIL-1ra used in biological experiments). KA-I
L-1L1 only lacked the initiation methionine. These proteins were phosphorylated using protein kinase A (New England BioLabs) to increase specific activity (1-2 × 10 ¹⁷ Bq / mol). In a series of three simultaneous experiments performed on fibroblasts, KA-IL-1ra binding was 0.1 μM I
It could be shown to be competed by L-1ra. In general, specific binding was about 100 Bq / 4 × 10 ⁴ , or about 2000 receptors per cell (in previously established cell lines). When compared, KA-I was found on the fibroblasts.
No receptor for L-1L1 was detected (less than 200 receptors per cell) (data not shown).

5.6 Expression of Recombinant IL-1L1 in COS Cells We used the original translation initiation site of IL-1L1 mRNA to produce recombinant human IL-1L1 in mammalian cells. A 1.4 kb IL-1L1 5′RACE cDNA was fused downstream of the SV40 promoter and synthetic intron, and the polyadenylation signal of SV40 (pSG5, Stratagene) was ligated. Proteins of the desired size were obtained from COS7 cells transfected with this construct. Western blot analysis of cell lysates and supernatants with the same anti-IL-1L1 antiserum (data not shown) showed the presence of a specific protein of 17 kDa (untransfected sample). It was not present in the supernatant), but the amount thereof was 3 times or more in the supernatant fraction as compared with the cytosolic fraction. This, like other members of the IL-1 family, suggests that IL-1L1 has an unusual secretory pathway. The inventors confirmed this finding by detecting endogenous IL-1L1 in the cell border and supernatant fractions of cultured JEG-3 cells. Cells were metabolically labeled as described above and the supernatant and cytoplasmic fractions were compared by immunoprecipitation. From FIG. 12 (c), compared with the cell lysate of JEG-3, although the TCA-precipitable radioactivity in the supernatant was 15% or less of the total activity, it was found in the supernatant to be substantially There is a higher amount of IL-1L1 due to
-1L1 is selectively released from cells, suggesting that there is a signal-independent secretory pathway.

Generally, a full-length or soluble IL of human IL-1L1 protein lacking a signal sequence
An expression construct containing a nucleic acid encoding the -1L1 protein can be constructed as follows. The nucleic acid encoding the full-length human IL-1L1 protein or the soluble IL-1L1 protein described above is obtained by reverse transcription (RT-PCR) of mRNA extracted from human cells.
) Can be obtained. PCR primers further contain suitable restriction enzyme sites for introduction into the expression plasmid. Next, the amplified nucleic acid was labeled with pcDNAI / Amp.
(In Vitrogen) and other eukaryotic expression plasmids. Such a plasmid contains 1) an SV40 origin of replication, 2) an ampicillin resistance gene, 3) an E. coli origin of replication, 4) a CMV promoter followed by a polylinker region, an SV40 intron and a polyadenylation site. .
Next, encode the full-length human IL-1L1 in the polylinker region of the HA tag corresponding to the epitope derived from the influenza hemagglutinin protein already described, and the HA tag or myc tag fused to the 3'end of the frame. Cloned the DNA fragment (I. Wilson, H. Niman, R.
Heighten, A. Chereson, M. Connolly
And R. Lerner, Cell 37, 767 (1984)). By fusing the HA tag to IL-1L1, it became possible to easily detect the recombinant protein using an antibody that recognizes the HA epitope.

[0368]   Regarding the expression of recombinant IL-1L1, the DEAE-DEXTRAN method (J. Sambrook
k), E. Fritsch, T. Maniatis, “Molecular claws”
: Laboratory Manual (Molecular Cloning: A Laboratory Manual),
Cold Spring Harbor Laboratories Press
Y Press), 1989) and transfection of COS cells with an expression vector.
I got it. Expression of IL-1L1-HA protein was carried out using an anti-HA antibody, radiolabeled and
It can be detected by immunoprecipitation (E. Harlow, D. Lane (
Lane), "Antibodies: A Laboratory Manual",
Cold Spring Harbor Laborat
ory Press) company, 1988). For this detection, after transfecting the cells, ³⁵  Labeling was performed for 2 days using S-cysteine. Then the cells or another
For this purpose, collect the culture medium (for example, in the case of soluble IL-1L1) and
IL-1L1 was immunoprecipitated using a noclonal antibody. The full length of IL-1L1 is a membrane protein
And / or secreted protein for the purpose of determining
And transfect with a vector encoding the full-length IL-1L1 protein.
The treated cells were treated with a detergent buffer (RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1%).
% SDS 0.5% DOC, 50 mM Tris, pH 7.5 in 1% NP-40 (I. Wilso
(Wilson et al., Ibid., 37: 767 (1984)). Then the precipitated protein
Quality was analyzed on SDS-PAGE gels. Therefore, the presence of IL-1L1 in cells indicates that IL-1L1
It is suggested that the entire length of L1 is membrane-bound, and the presence of IL-1L1 in the supernatant
, Produced as a secreted protein or released by leakage from cells
Either of these suggests that the protein may be in the dissolved form.

5.7 Materials and Methods Identification of novel transcripts: 100 ng / ml E. coli lipopolysaccharide and 100 μg / m
PMBCs were stimulated for 6 or 18 hours with l PHA. After treatment and mixing, RNA was prepared. Basically described (Morgan et al., Nucleic Acids Res. 20: 5
173-5179 (1992); Futreal et al., Hum. Mol. Genet. 3: 1359-13.
64 (1994)), and 680 kb of YAC 766E12 (obtained from CEPH (Paris)) as a driver (Chumakov et al., Nature 377: 175 (1995)) was used to perform two liquid-layer hybridizations to perform cDNA. Was selected. 100 clones were sequenced differently. Sequences containing high copy-number elements were removed.

CDNA and genomic DNA libraries: Human genomic PAC clone 131J6 has been described (Nothwang et al., Genomics 41: 370-378 (1997)).
Human placenta cDNA library H9 (primary fusion mixture of pCDM8) is a resource center HGM
Obtained from P (Cambridge, England). The mouse PAC clone is 1.2 kb from IMAGE clone # 332733 (Lennon et al., Genomics 33: 151-152 (1996)).
Grid mouse 129 PA using the mouse IL-1L1 cDNA probe
After screening the C library (RPCI21), it was obtained from Resource Center HGMP (Cambridge, UK).

Isolation of a cDNA clone: A human H9 cDNA library was prepared using a 474 bp probe (nucleotide numbers 5902 to 6369 of exon 6 genomic sequence (FIG. 2)) derived from the SET clone according to standard procedures. About 5 × 10 ⁵ colonies were screened. The two clones were confirmed for homogeneity and screened, and found to have inserts of almost the same size.

5'Extension of cDNA: The first cDNA strand was prepared from placental mRNA using MMLV-reverse transcriptase and oligo (dT) primers. Synthesis of the second strand was performed using a cocktail of E. coli DNA polymerase I and RNase H according to the manufacturer's instructions (Marathon RACE, Clontech). (E. coli) DNA ligase and cDNA adapter were ligated. 5'RACE is an adapter-specific primer and genomic sequences 5295-5270.
Sequence-specific complementary primer (30 cycles at 68 ° C.), followed by overlapping PCR (adapter nested primer) and primers corresponding to nucleotide numbers 5273 to 5244. nested PCR) was performed (20 cycles at 60 ° C). The 1.4 kb 5'RACE product was cloned directly into the pCRII vector (Invitrogen) and sequenced with specific oligonucleotide primers.

Northern blot analysis: Human multiple tissue blots were purchased from Clontech Laboratory, which contained 5 μg of mRNA in each lane.
Is included. For the purpose of isolating mRNA from JEG-3 cells, 2 × 10 ⁷ cells were added to RNAzol B (Biogenesis), homogenized, and extracted according to the manufacturer's instructions. Affinity chromatography using oligo (dT) -cellulose type 3 (Promega) was performed to obtain total R
JEG-3 poly (A) + RNA was isolated from NA. Standard Concentration-Centrifugation Protocol (Colotta et al., J. Immunol. 132: 936-944 (1984); Bertani (Ber
Tani) et al., Blood 74: 1811-1816 (1989)). NK cells were obtained by removing CD3 + and CD14 + cells from PBMC. Dendritic cells were obtained by treating monocytes with GM-CSF and IL-4 for 7 days (Sozzani (Sozz
ani) et al., J. Immunol. 159: 1993-2000 (1997). Functional maturation proceeded by stimulation with TNF, IL-1β or LPS for 3-4 hours. Total RNA was prepared from leukocytes according to the guanidine thiocyanate method. RNA was analyzed by electrophoresis in a 1.2% formaldehyde / agarose gel and transferred to a charged nylon membrane (Zeta-Probe, Biorad). IL-1L1 O
IL-1L1 mRN by hybridization with a cDNA probe corresponding to RF
Visualized A.

Preparation of recombinant proteins: rhIL-1L1 forms A, B and C, rhIL-1ra, KA-IL-1
L1 and KA-IL-lra are 6-H in BL21 (DE3) cells from the modified pET plasmid.
It was expressed as an is-tagged recombinant protein, which has a 6-His tag added to the N-terminus of the protein, a flexible linker and a thrombin cleavage site. Bacteria are described (Studier et al., Methods Enzymol. 18).
5: 60-89 (1990)), and after cooling, the cells were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and shaken at 25 ° C. for 18 hours. The recovered bacteria were treated with 100 mM NaCl / 20 mM Tris / HCl pH 7.3 / 10% glycerol (
The cells were resuspended in buffer solution A), and 1 mM phenylmethanesulfonyl fluoride was added thereto, followed by ultrasonication. The lysate was centrifuged at 600,000 xg for 20 minutes, and the 6-His-tagged protein obtained from 1000 ml of culture medium was diluted with 5 ml of Ni-NTA agarose column (Qiagen (
Quiagen)) was used for affinity purification. The column was washed with 50 ml of buffer A followed by 50 ml of buffer A / 20 mM imidazole and the tagged protein was eluted with a minimal amount of buffer A / 100 mM imidazole. As a protein, thrombin (3 units per 1 mg of fusion protein) was used to cleave the protein at 30 ° C for 4 hours, and Sephadex G using buffer A / 2 mM dithiothreitol.
Separated from the N-terminal fragment by gel filtration through -75 (Sephadex G-75). Initially, LPS-derived contaminants could be present, so LPS was removed from the protein samples by affinity purification using a polymyxin B-agarose column (Sigma). Later, this last purification step was not performed, as no significant effect from LPS was detected. Purity was confirmed by gel electrophoresis and concentrations were adjusted for the amino acid composition of the protein
Calculated from A280 measurements. Probably due to steric hindrance from the β-branched Val residue, there was a trace amount of uncleaved material in the preparation of Form C of rhIL-1L1 and K
The purified proteins were considered to be identical except that there was evidence of an additional C-terminal treatment product in A-IL-IL1.

Production of anti-IL-1L1 serum: 150 μg of type A rhIL-1L1 was administered to Dwarf rabbits with Freund's adjuvant five times at 4-week intervals. Serum samples were examined by enzyme-linked immunosorbent assay (ELISA) using IL-1L1.

Metabolic labeling and immunoprecipitation of JEG-3 cells: Cells in 75 cm ² culture bottles (80% fusion) were washed with isotonic saline and cysteine / methionine free MEM (
It was placed in Life Technologies / 1 mM glutamine / 1 mM sodium pyruvate / 0.5% bovine serum albumin and left at 37 ° C. for 1 hour. Thereafter, the 50mCi / ml ⁽³⁵ S) - cysteine / methionine was added (Promix (Promix), NEN Life Sciences (NEN Life Sciences) Ltd.) 5 m
l of fresh medium was added and incubated for 4 hours. Further 50 mCi / ml ( ³⁵ S)-
Cysteine / methionine was added and incubation continued for 20 hours. The medium was collected. In FIG. 12 (b), 1.0 ml of RIPA buffer (100 mM NaCl / 50 mM Tris) containing protease inhibitor cocktail (Boehringer Mannheim).
/ HCl pH8.0 / 1% Nonidet P-40 / 0.5% Sodium Deoxycholate / 0.1%
Sodium dodecyl sulphate), lyse the cells, then at 4 ° C, 13000 xg
The cells were centrifuged to remove fine debris from the cells. Using 20 μl (filling volume) of Protein A-Sepharose beads (Sigma), 0.5 ml of the cell lysate in the sample was pre-washed at 4 ° C. for 1 hour to prepare a test serum. And incubate at 4 ° C with gentle shaking for 24 hours, then
Add 20 μl of Protein A-Sepharose and add 4 more
Incubated for hours. The solid matrix was collected, washed 4 times with RIPA buffer, then resuspended in 60 μl sample buffer and heated at 100 ° C. for 2 minutes. 30 μl of sample was used for analysis by gel electrophoresis. In FIG. 12 (c), the method is basically the same, but cells lysed with 2.5 ml cell supernatant or 2.5 ml RIPA buffer were used.

Investigation of the potential of IL-1L1 in human cells to have IL-1-like, IL-1ra-like and IL-18-like activity: Human gingival fibroblasts were plated in 24-well tissue culture plates (2 ×
(10 ⁴ cells / well) were grown in DMEM supplemented with 10% fetal bovine serum for 24 hours. Human umbilical vein endothelial cells (TCS Biologicals) were placed in 24-well tissue culture plates and grown according to the manufacturer's instructions until they were 80% confluent. A suitable amount of IL-1α, IL-1β (R & D Systems)
Cells were stimulated for 24 hours with fresh medium supplemented with rhIL-1ra or rhIL-1L1 (prepared as described above). From a previous experiment (data not shown)
Although a concentration of 0.1 nM was adequate for all IL-1 to fully activate IL-6 secretion, 10 nM of IL-1ra was The response was attenuated by more than 95%. IL-6 production was measured by ELISA within the linear region of the assay according to the manufacturer's instructions (R & D Systems). Regarding the variation of IL-6 production by day in a similar experiment (cell density or IL
-Possibly due to a slight change in the activity of one species), corrected by normalizing each data set to the average yield from the IL-1 treated well panel generated in the same experiment. The changes in IL-6 production per well of fibroblast cultures treated with IL-1 alone, which were observed through a number of experiments, were
For L-1β it was 7-30 ng / ml and the change in endothelial cells was 1.3-7.0 ng / ml.

KG-1 cells (ATCC CCL-246) were placed in a 24-well tissue culture plate at 1 × 10 ⁶ cells / ml, and the medium was Iscove modified with 20% fetal calf serum. 2 using Dulbecco's medium (Life Technologies) with or without 50 mg / ml PHA (Sigma)
Left for hours. PHA alone or IL-18 (3 nM) and / or IL-1L1 (0.1 μM)
Used to stimulate cells for 48 hours. In some studies, neutralizing anti-IL-18 antibody (50 μg was used to demonstrate that IL-18 increased IFNg production.
/ Ml, R & D Systems (R & D Systems). 900x after stimulation time
The cells were removed by centrifuging the supernatant for 5 minutes at g. IFNg production was measured by ELISA (Biosource). The data set was normalized to the average production in 4 wells within the same experiment stimulated with PHA and IL-18. In 2 experiments performed in 4 wells, the range of IFNg produced by PHA / IL-18-stimulated cells was 2.3.
It was ~ 10.8 iu / ml.

Equivalence One of ordinary skill in the art would be able to recognize and ascertain that equivalents to the particular embodiments described herein exist by routine experimentation. is there. Such equivalents are also intended to be covered by the claims of this application.

[Brief description of drawings]

[Figure 1]   Nucleic acid sequences of two human IL-1L1 cDNAs having two 5'ends

[Fig. 2]   Nucleic acid sequence of mouse IL-1L1 cDNA sequence

FIG. 3 Putative polypeptide sequence encoded by human IL-1L1 and mouse IL-1L1.

[Figure 4]   Comparison of human and mouse IL-1L1 polypeptide sequences.

FIG. 5: Comparison of human IL-1L1 polypeptide sequence and human pro-IL-1ra polypeptide sequence.

[Figure 6]   IL-1L1 gene product three recombinant polypeptide sequences

[Figure 7]   Northern blot showing tissue distribution of human IL-1L1 transmission code

[Figure 8]   Zooblots showing the conservation of IL-1L1 gene sequences in mammals

FIG. 9: Chromosomal location of the IL-1L1 gene within the IL-1 locus on human chromosome 2 and the intron / exon structure of the IL-1L1 gene

[Figure 10]   Details of IL-1L1 transcript splicing

FIG. 11   Whole genome sequence of IL-1L1 gene

[Fig. 12]   Immunoprecipitation of native IL-1L1 protein from JEG-3 cells

FIG. 13: Alignment of human and mouse IL-1L1 genes with other IL-1 like gene families in humans 1) Foreign language specification, page 3, line 1 to page 3, line 29, Page 5, line 8 to page 11, line 19, page 13 line 23 to page 112, line 18, line 1
Since the translation of each part from page 14, line 25 to page 127, the last line was missing,
Replenish and correct mistranslations. 2) Claims of foreign language documents. The translation from page 129, line 1 to page 130, last line was missing.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 11/00 A61P 11/00 13/12 13/12 17/00 17/00 17/14 17/14 19 / 10 19/10 29/00 29/00 31/00 31/00 37/02 37/02 C12N 15/09 C12N 15/00 ZNAA ZNA A // A61K 38/00 A61K 37/02 (81) Designated country 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, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, R , TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM. , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZWF F terms (reference) 4B024 AA01 AA11 BA26 CA04 CA11 DA02 DA05 DA11 EA02 EA03 EA04 FA02 GA11 HA01 HA03 4C084 AA07 BA01 BA08 BA22 BA23 DA12 NA14 ZA402 ZA592 ZA662 ZA672 ZA812 ZA892 ZA922 ZA972 ZB072 ZB112 ZB322 ZC352 4H045 AA10 AA2 0 AA30 BA10 CA40 DA03 EA20 EA50 FA72 FA74

Claims (8)

[Claims] 1. An isolated polypeptide, which is composed of at least 50 amino acids, including an amino acid sequence having at least 70% identity with the polypeptide sequence shown in SEQ ID NO: 5 or 6. A characteristic polypeptide. 2. An isolated polypeptide consisting of at least 30 amino acids, including an amino acid sequence having at least 80% identity with the polypeptide sequence shown in SEQ ID NO: 5 or 6. A characteristic polypeptide. 3. An isolated polypeptide consisting of at least 20 amino acids, including an amino acid sequence which is at least 90% identical to the polypeptide sequence shown in SEQ ID NO: 5 or 6. A characteristic polypeptide. 4. An isolated polypeptide, characterized in that it is composed of at least 10 amino acids comprising an amino acid sequence which is identical to the polypeptide sequence shown in SEQ ID NO: 5 or 6. 5. An isolated polypeptide encoded by a nucleic acid having at least 70% identity to the entire nucleotide sequence set forth in SEQ ID NO: 10 or 11 or a nucleotide sequence complementary thereto. A polypeptide characterized by: 6. An isolated polypeptide which, under stringent conditions, has a nucleic acid sequence corresponding to nucleotide numbers 310 to 2561 of SEQ ID NO: 1 and a nucleic acid sequence corresponding to nucleotide numbers 1-29 of SEQ ID NO: 1. , SEQ ID NO: 2, SEQ ID NO: 3,
And a polypeptide encoded by a nucleic acid that hybridizes to a nucleic acid selected from the group comprising the nucleic acid sequence corresponding to nucleotide numbers 390 to 1284 of SEQ ID NO: 4. 7. The isolated polypeptide according to claim 5 or 6, which comprises an amino acid sequence having at least 70% identity with the entire amino acid sequence shown in SEQ ID NO: 5 or 6. 8. The isolated polypeptide according to any one of claims 1 to 6, characterized in that it is encoded by a mammalian nucleic acid sequence.