HK1028773B - Interleukin-18 binding proteins, their preparation and use - Google Patents

Description

Interleukin-18 binding proteins, their preparation and use

Technical Field

The present invention relates to an interleukin-18 (IL-18) binding protein capable of binding IL-18, hereinafter referred to as IL-18 BP. This invention relates to soluble IL-18BP obtained from a body fluid, to soluble IL-18BP obtained by expression in a host cell by means of a suitable DNA vector, to virally encoded homologues of IL-18BP obtainable by expression in a host cell by means of a suitable DNA vector, to vectors for expression of IL-18BP in humans and other mammals, to antibodies against IL-18BP, to the therapeutic use of said IL-18BP by modulating and/or blocking IL-18 activity, to the therapeutic use of said expression vectors in modulating and/or blocking IL-18 activity and to the use of antibodies.

Background

In 1989, exotoxin-induced serum activity induced by interferon gamma (IFN-. gamma.) obtained from splenocytes from mice was disclosed (27). This serum activity functions not as a direct inducer of IFN- γ but as a co-stimulator with IL-2 or mitogen. Purification attempts from the serum activity of mice after exotoxin treatment showed an apparently homologous 50-55 kilodalton (kDa) protein (26). Since other cytokines can act as co-stimulators of IFN- γ production, the failure of neutralizing antibodies to IL-1, IL-4, IL-5, IL-6 or TNF to neutralize serum activity indicates that it is a different factor. In 1995, these scientists also demonstrated that exotoxin-induced co-stimulators of IFN- γ production were present in liver extracts of mice pretreated with p.acnes (31). In this example, the hepatomacrophage population (Kupffer cells) expanded and in these mice, low doses of bacterial Lipopolysaccharide (LPS), which were not lethal in non-pretreated mice, became lethal. Acnes treated mouse liver was purified as a homogenate from 1,200 grams of IFN- γ -induced factor (IGIF) and later named interleukin-18 (IL-18). Mouse IL-18cDNA was cloned using degenerate oligonucleotides derived from the amino acid sequence of purified IL-18 (31). IL-18 is a 157 amino acid 18-19 kilodalton protein with no obvious similarity to any of the peptides in the database. Messenger RNAs for IL-18 and interleukin-12 (IL-12) are readily detectable in Kupffer cells and activated macrophages. Recombinant IL-18 apparently has a greater potential to induce IFN- γ than IL-12 by an independent pathway (31). Similar to the exotoxin-induced serum activity, IL-18 does not induce IFN- γ by itself, but the primary function is to act as a co-stimulator with either mitogen or IL-2. IL-18 apparently enhances T cell proliferation via the IL-2 independent pathway and enhances the production of Th1 cytokine in vitro and shows synergy when IL-12 is combined with enhanced IFN- γ production (24).

Neutralizing antibodies to mouse IL-18 were shown to prevent lethality of low dose LPS in p.acnes pretreated mice. The importance of IFN-gamma as a mediator of LPS lethality has been reported by others in pre-treated mice. For example, neutralizing anti-IFN- γ antibodies protect mice from Shwartzman-like shock (16), and IFN- γ receptor deficient galactosamine treated mice are resistant to LPS-induced death (7). Therefore, neutralizing antibodies to mouse IL-18 could not be expected to protect p.acnes pretreated mice against lethal LPS (31). Anti-mouse IL-18 treatment also protected surviving mice against severe hepatotoxicity.

After the formation of cloned mice, the human cDNA sequence for IL-18 was reported in 1996 (38). Recombinant human IL-18 shows native IL-18 activity (38). Human recombinant IL-18 has no direct IFN- γ inducing activity on human T cells, but acts as a co-stimulator of IFN- γ production with other T helper-1 (Th1) cytokines (38). To date, it is believed that IL-18 may initially act as a co-stimulator of Th1 cytokine production (IFN-. gamma., IL-2 and granulocyte macrophage colony stimulating factor) (20), and also as a co-stimulator of FAS ligand-mediated cytotoxicity that mice naturally kill cell clones (37).

This cytokine was found to be closely associated with autoimmune disease by cloning IL-18 from infected tissues and studying IL-18 gene expression. Non-obese diabetic (NOD) mice spontaneously developed autoimmune insulitis and diabetes, which were accelerated and synchronized when cyclophosphamide was injected alone. IL-18mRNA was demonstrated by reverse transcriptase PCR in the pancreas of NOD mice during early insulitis. IL-18mRNA levels were rapidly increased after cyclophosphamide treatment and prior to this IFN-. gamma.mRNA was elevated and subsequently developed into diabetes. Interestingly, these kinetics mimic IL-12-p40mRNA which results in a close association with individual mRNA levels. Cloning of IL-18cDNA from pancreatic RNA after sequencing revealed the identity of the IL-18 sequence cloned from Kupffer cells and the pre-activated macrophages in vivo. When macrophages from Balb/c were not treated in parallel, NOD mouse macrophages also responded to cyclophosphamide by IL-18 gene expression. Therefore, IL-18 expression is abnormally regulated in autoimmune NOD mice and is closely associated with the development of diabetes (32).

IL-18 plays a potential role in immune regulation or inflammation by increasing the functional activity of Fas ligand on Th1 cells. IL-18 is also expressed in the adrenal cortex and therefore can be a secreted neuro-immune modulator, playing an important role in the immunochemical immune system after stress experiments (9).

In vivo, IL-18 is formed by cleavage of pro-IL-18, and its endogenous activity appears to be dependent on IFN- γ production in P.acnes and LPS-mediated lethality. Because of its activity, blocking the biological activity of IL-18 in human disease is a therapeutic strategy for many diseases. This can be achieved using soluble receptors or antibodies that block cell binding to the IL-18 receptor.

Cytokine binding proteins (soluble cytokine receptors) correspond to the extracellular ligand binding domains of their respective cell surface cytokine receptors. Like cell surface receptors, they can be derived by alternative splicing of pre-mrnas or by proteolytic cleavage of cell surface receptors. Such soluble receptors have been described in the past and include other soluble IL-6 receptors and IFN-gamma (30), TNF (11, 12), IL-1 and IL-4(21), IFN-alpha/beta (28, 29) and others. A cytokine binding protein designated osteoprogenin (OPG, known as osteoclast inhibitory factor-OCIF), a member of the TNFR/Fas family appears to be the first example of a soluble receptor that exists only as a secreted protein (1, 34, 39).

Summary of The Invention

The present invention provides IL-18 binding proteins (IL-18BP) and virally encoded IL-18BP homologues (hereinafter referred to as viral IL-18BP), and fusion proteins, muteins, functional derivatives, active fragments and circularly permutated derivatives thereof, which are capable of binding IL-18. The invention also provides methods for isolating IL-18BP from human body fluids, and methods for obtaining them by recombinant means. The present invention also provides an expression vector for IL-18BP suitable for expressing IL-18BP in humans and other mammals. The specific IL-18BP, virally encoded IL-18BP homologues, fusion proteins, muteins, functional derivatives, active fragments and cyclically altered derivatives of the present invention may be used to modulate and/or block the biological activity of IL-18.

Replicable expression vectors containing DNA suitable for expression of various IL-18 BPs in host cells, transformed host cells of the invention, and proteins and polypeptides produced by expression of such hosts are also provided.

The invention further provides pharmaceutical compositions comprising a suitable vector and IL-18BP or viral IL-18BP or vectors expressing them in humans and other mammals, for use in the treatment of diseases or conditions requiring modulation or blocking of IL-18 activity.

The invention further provides antibodies suitable for affinity purification and immunoassay of their IL-18BP and viral IL-18 BP.

Description of the drawings

FIG. 1 shows SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) of ligand affinity purified IL-18 binding proteins. Crude urine protein (concentrated by ultrafiltration of 500 liters of normal human urine) was loaded onto an IL-18 agarose column. Washed and bound protein eluted at pH 2.2. The eluted fractions were neutralized, analyzed by SDS-PAGE (10% acrylamide) under non-reducing conditions, and silver stained. These lanes are: 1: crude urine protein (1.5 μ g, loaded onto gel); 2-9: 1-8 eluates from IL-18 agarose columns respectively; 10: molecular weight markers, kilodaltons, as indicated on the right. The arrow indicates the band corresponding to IL-18 BP.

FIG. 2 shows a liquid crystal display device¹²⁵Autoradiography of SDS-PAGE (7.5% acrylamide) of complexes of I-IL-18 (apparent molecular weight 19 kilodaltons) crosslinked with preparations of soluble IL-18 binding proteins: lane 1: washes of IL-18 affinity columns. Lane 2: eluate 2 of an IL-18 affinity column. Lane 3: eluate 3 of an IL-18 affinity column. Molecular weight markers are indicated on the right (kilodaltons). The arrow indicates the crosslinked product (58 kDalton))。

FIGS. 3A-3E show that IL-18BP inhibits IL-18-induced IFN- γ production.

FIG. 3A mouse splenocytes were stimulated (24 hr, 37 ℃) with indicated LPS (1. mu.g/ml) and human IL-18(5 ng/ml) either directly added or after premixing with urinary IL-18BP (1 hr, 37 ℃). The level of muIFN- γ in the culture was determined after 24 hours.

FIG. 3B mouse splenocytes were incubated with LPS (1. mu.g/ml) and mouse IL-18(10 ng/ml) premixed (1 hour, 37 ℃) with increasing concentrations of human IL-18BP for 24 hours.

FIG. 3C mouse splenocytes were incubated with LPS (10. mu.g/ml) and increased concentrations of human IL-18BP (24 hours).

FIG. 3D mouse splenocytes were incubated with Con A (1. mu.g/ml) and increased concentrations of human IL-18BP (24 hours).

FIG. 3E human KG-1 cells were stimulated with TNF-. alpha. (20 ng/mL) and huIL-18(25 ng/mL) added alone or after pre-mixing (1 hour, 37 ℃) with urinary IL-18 BP.

FIGS. 4 and 4A show the sequences of human IL-18BPa cDNA and protein, the signal peptide is underlined.

FIGS. 5 and 5A show the sequences of human IL-18BPb cDNA and protein, the signal peptide is underlined.

FIG. 6, 6A to 6E show the sequence of human IL-18BPc cDNA and protein. The signal peptide is underlined.

FIGS. 7 and 7A show the sequences of human IL-18BPd cDNA and protein. The signal peptide is underlined.

FIG. 8, FIG. 8A to FIG. 8F show the sequences of the human IL-18BP gene. The sequence of the human genomic clone (7.1kb) was determined and compared to various cDNA clones isolated from 3 cDNA libraries. The common translation initiation codon is nucleotides 683-685. The NuMA1 gene was located on the negative strand, from nucleotide 3578 to the terminus.

FIGS. 9A-9D show the effect of recombinant IL-18BP on human and mouse IL-18 activity.

His was transiently expressed in COS7 cells₆Labeled IL-18BPa and purified.

FIG. 9A human IL-18(5 ng/ml) with His₆marker-IL-18 BPa or RPMI were premixed and added to mouse splenocytes together with LPS (1. mu.g/ml). IFN- γ production was measured after 24 hours.

FIG. 9B mice IL-18(10 ng/ml) with His₆marker-IL-18 BPa or RPMI were premixed and added to mouse splenocytes together with LPS (1. mu.g/ml). IFN- γ production was measured after 24 hours.

FIG. 9C human IL-18(25 ng/ml) was premixed with COS7-IL-18BPa or RPMI and human PBMC in the presence of IL-12(10 ng/ml) was added.

FIG. 9D human IL-18(25 ng/ml) was premixed with COS7-IL-18BPa or RPMI and human KG-1 cells were added in the presence of TNF- α (20 ng/ml).

Detailed description of the invention

The present invention relates to various IL-18 BPs and viral IL-18 BPs that bind IL-18. Such IL-18BP is capable of modulating and/or blocking the biological activity of IL-18. The terms "IL-18 BP and viral IL-18 BP" include mature proteins (without signal sequence), proteins containing signal sequence, muteins of IL-18BP and viral IL-18BP, derivatives of IL-18BP and truncated forms of viral IL-18BP and viral IL-18BP and salts thereof.

The invention further relates to replicable expression vectors suitable for expressing various IL-18 BPs or viral IL-18 BPs in host cells and host bacteria. The invention further relates to expression vectors suitable for expressing various IL-18 BPs or viral IL-18 BPs in humans and other mammals.

The invention further relates to DNA encoding various IL-18BP, viral IL-18BP, muteins, fusion proteins, functional derivatives, active fractions and mixtures thereof. The DNA may be genomic DNA, cDNA synthetic DNA, PCR products or a combination thereof. These DNAs can be inserted into replicable expression vectors according to the present invention for expression of various IL-18 BPs and viral IL-18 BPs in host cells. Also included in the present invention are DNAs that hybridize to the above DNAs under stringent conditions and encode proteins or polypeptides.

One such DNA encodes a polypeptide comprising SEQ ID NO: 10 and providing at its 3' end a stop codon of IL-18 BP.

Expression vectors suitable for expressing various IL-18 BPs or viral IL-18 BPs in humans and other mammals for gene therapy may be viral vectors or other types of vectors into which IL-18BP gene or IL-18BP cDNA or DNA encoding viral IL-18BP is inserted in such a manner that IL-18BP or viral IL-18BP can be efficiently expressed in humans or other mammals. The present invention also encompasses DNA molecules which hybridize to the above DNA under stringent conditions and which encode proteins or polypeptides capable of binding IL-18.

According to the present invention, the isolation of IL-18BP may be carried out, for example, by passing human body fluids, such as urine or serum, through an IL-18-coupled chromatography column and then eluting the bound IL-18 BP.

Various IL-18 BPs and viral IL-18 BPs can be prepared by recombinant means, i.e., expression of IL-18BP by operably linked promoters, expression enhancers, regulatory sequences and the like suitable for use by a particular host, allowing for expression in the correct orientation in the appropriate host.

Various IL-18 BPs and viral IL-18 BPs and vectors that express IL-18BP in humans and other mammals may be utilized in the treatment and alleviation of diseases caused by IL-18 involvement or excessive exogenous administration or exogenously produced IL-18. Such conditions may be autoimmune diseases, type I diabetes, rheumatoid arthritis, transplant rejection, inflammatory bowel disease, sepsis, multiple sclerosis, ischemic heart disease (including heart attack), ischemic brain injury, chronic hepatitis, psoriasis, chronic pancreatitis, acute pancreatitis, and the like.

According to the invention, IL-18BP is isolated from normal human urine by a chromatographic step. Crude human urine protein preparation concentrated from 500 liters of normal human urine was loaded onto a column containing agarose-bound human IL-18. Washed and bound protein eluted at low pH. The eluted fractions were neutralized and aliquots and silver stained by SDS-PAGE (10% acrylamide) under non-reducing conditions. A protein band of about 40 kilodaltons was specifically obtained in the eluted fraction (FIG. 1).

Such as IL-18 binding proteins, by which to react with¹²⁵The ability of I-IL-18 to specifically crosslink identifies the approximately 40 kilodalton protein obtained in the first step (FIG. 2). Approximately 40 kilodaltons of protein was further identified by N-terminal protein sequence analysis. Aliquots from the eluted proteins were subjected to SDS-PAGE, electroporated onto PVDF membrane, and subjected to protein microsequence analysis. Similarly, aliquots from the eluted proteins were subjected to direct protein microsequence analysis. In both cases, two polypeptide sequences were obtained. Large and small sequences, the latter corresponding to a fragment of human defensin (accession number p11398), starting at amino acid 65. Subtracting the known defensin sequence provides the following sequence:

T-P-V-S-Q-Q-x-x-x-A-A-A

1 ···5····10 ··

wherein x represents an amino acid which has not yet been determined.

To obtain longer or more accurate sequences, and to identify potential cysteine residues, aliquots of the eluted fractions were reduced under denaturing conditions using DTT, reacted with 4-vinylpyridine, desalted by a micro-ultrafiltration device (Ultrafree, cut-off molecular weight 10,000Da, Millipore) and subjected to protein microsequence analysis. After sequence cycle No. 1, the remaining proteins were reacted with o-phthalaldehyde to block all N-terminal polypeptides instead of Pro and then sequencing was resumed. In this method, the following individual protein sequences were obtained:

TPVSQXXXAA XASVRSTKDP CPSQPPVFPA AKQCPALEVT

1 10 20 30 40

(T ═ Thr; P ═ Pro; V ═ Val; S ═ Ser; Q ═ gin; X ═ unknown; a ═ Ala; R ═ Arg; K ═ Lys; D ═ Asp; C ═ Cys; F ═ Phe; L ═ Leu; E ═ Glu)

The resulting sequence is clearly distinct from any other known protein, as determined by studying the protein database. However, the database studies by the tblastn research program genomics institute (TIGR) (HTTP:// www.ncbi.nlm.nih.gov) provided a cDNA document designated THC123801 whose open reading frame (218 codons) contains a sequence that is highly homologous to the N-terminal sequence of IL-18BP when translated.

Homology is shown below:

1.......TPVSQXXXAAXASVRSTKDPCPSQPPVFPAAKQCPALEVT... 40

| | | ||||||||||||||||||||||||||||||||

51 VTLLVRATXVXQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPE 100

[ sequences (1-40) above are IL-18BP isolated according to the invention; the following sequences (51 to 100) were deduced from TIGR document THC123801 cDNA translation ].

However, the cDNA sequence identified as THC123801 is only an EST (expressed sequence tag), a randomly selected cDNA clone. This EST has never been analysed for the presence of an open reading frame, whether the protein is expressed from the gene corresponding to the EST or from the EST itself, nor even for any function of the identified protein encoded by THC 123801. There is no information at all that the THC123801 contains the open reading frame encoding IL-18 BP.

Forming a labeled ligand which remains bound thereto: (¹²⁵I-IL-18), followed by covalent crosslinking of a molecular weight of 58Affinity for the capacity of the kilodalton complex purified urinary IL-18 BP. The molecular weight of this complex corresponds to approximately 40 kilodaltons IL-18BP and 19 kilodaltons IL-18 in a 1: 1 ratio (FIG. 2).

Affinity purified urinary IL-18BP blocks the biological activity of human and mouse IL-18. Therefore, when IL-18BP was added to human or mouse IL-18, it blocked the ability of IL-18 to induce the production of interferon gamma when added to splenocytes from cultured mice along with Lipopolysaccharide (LPS) (FIG. 3).

For the purposes of the present description, the expression "biological activity of IL-18" means in particular at least one of the following biological properties:

(i) inducing IFN-gamma initially as a co-stimulator with mitogens, IL-1, IL-12, TNF-alpha, LPS in various cell types such as monocytes, mouse splenocytes, human peripheral blood mononuclear cells, human KG-1 cell line and T cells,

(ii) the proliferation of the T cells is enhanced,

(iii) enhance the production of Th-1 cytokines in vitro as a primary co-stimulator,

(iv) IL-12 and enhancing IFN-gamma production synergistic effect, IFN-gamma and other T helper cell-1 cytokine production costimulation effect,

(v) the co-stimulatory effect of FAS ligand-mediated cytotoxicity of mice naturally killing cell lines,

(vi) in human KG-1 cells, it is possible to induce activation of NF-. kappa.B by inducing the formation of 50 NF-. kappa.B homodimers and p65/p50 NF-. kappa.B heterodimers,

(vii) IL-8 was induced.

As used herein, the expression "binds to IL-18" refers to the ability of IL-18BP to bind to IL-18 as indicated by its binding to labeled IL-18 when affinity purified as in example 2 of the invention.

As used herein, expression of "modulating the activity of IL-18" refers to the ability of IL-18BP to modulate any IL-18 activity other than blocking, e.g., partially inhibiting, enhancing or the like.

As used herein, the expression "blocking the activity of IL-18" refers to an activity of IL-18BP to block at least one of the above-mentioned biological activities of IL-18. The ability of IL-18BP to block IL-18 associated with IFN- γ expression in mouse splenocytes is an example of the activity of IL-18BP to block IL-18. As will be shown in detail below, modulation or blocking of IL-18BP activity is due in part to the fact that IL-18BP inhibits NF-. kappa.B activity via IL-18. In addition, IL-18BP blocks at least one of the following IL-18 activities, i.e., IFN-. gamma.induction, IL-8 induction and NF-. kappa.B activation in human and mouse cells.

DNA probes for screening cDNA libraries were prepared by reverse transcription PCR using specific sense and antisense primers, and RNA from human Jurkat T cells and primers from TIGR sequences. The resulting PCR product was confirmed by DNA sequence analysis. The PCR products were labeled with 32[ P ] and used as probes for screening four human cDNA libraries derived from peripheral blood mononuclear cells, Jurkat T cell line, PBMC and human spleen. Each of the independent cDNA clones corresponded to four IL-18BP splice mutants (SEQ ID NOS: 1, 3, 5 and 7). All splice mutants encoded putative soluble secreted proteins. The most abundant one (IL-18BPa) has an open reading frame of 192 codons encoding a signal peptide, sometimes referred to herein as a "leader sequence" of 28 amino acid residues, followed by a mature putative IL-18BPa, the first 40 residues of which perfectly match the N-terminal protein sequence of urinary IL-18BP (SEQ ID NO: 2). The position of the cysteine residue indicates that this polypeptide belongs to the immunoglobulin (Ig) superfamily. Interestingly, each of the four Gln residues within mature IL-18BPa is a potential N-glycosylation site. Three other IL-18BP mutants were less abundant than IL-18 BPa. They included a shorter 1kb IL-18BPb cDNA (SEQ ID NO: 4) encoding a 28 amino acid residue signal peptide of the mature protein followed by 85 amino acid residues. The 2.3kbc DNA encoding the 28 amino acid residue signal peptide of the mature IL-18BP followed by 169 amino acid residues represents the third mutant, IL-18BPc (SEQ ID NO: 6). The fourth IL-18BPd is a signal peptide of 28 amino acid residues encoding mature IL-18BP followed by 133 amino acid residues (SEQ ID NO: 8).

To further investigate the possible presence of additional IL-18BP splicing mutants, a human genomic library was screened using probes corresponding to the full length IL-18BP cDNA. 5 genomic clones that differed in length were identified in this library. DNA sequence analysis was performed on these clones using the outer and inner primers. At the same time, a 7.8kb sequence (SEQ ID NO: 9) was assembled from these clones. No exon coding for Transmembrane (TM) receptors was identified within the 7.8kb sequence. All mutants share a common translational start site, encoding the same 28 amino acid residue signal peptide and size-variable soluble mature protein and C-terminal sequences. The IL-18BP locus contains additional genes encoding the nuclear mitotic apparatus protein 1(NUMA1) which is localized to the negative strand. This finding maps the IL-18BP gene to human chromosome 11q13 (36).

Homology studies were performed using the complete protein sequence of IL-18BPa and the GenPept database (HTTP:// www.ncbi.nlm.nih.gov), using the Smith Watermann algorithm. IL-18BP homologues expressed in several poxviruses as secreted proteins of previously unknown function were found. These viruses have been previously reported to encode various cytokine receptors, and the role of such virus-encoded molecules is to suppress the decoy receptors of the immune response by neutralizing their corresponding cytokines (Spriggs, MK, 1994, Current immunological review, 6: 526-529). The invention therefore furthermore relates to virus-encoded homologues of IL-18BP which can act as biologically active blockers or modulators of IL-18. Table 1 provides examples of virally encoded homologs of IL-18 BP.

According to the invention, homologues of virally encoded IL-18BP may be expressed in prokaryotic or eukaryotic hosts. As used herein, the expression "virally encoded IL-18BP homologue" refers to a sequence at least 50% similar in at least 70% of the amino acid residues. More preferably, it has at least 50%, at least 60%, at least 70%, at least 80% or most preferably at least 90% similarity in the sequence of 100 amino acid residues.

Table 1 Virus-encoded proteins show high homology to human IL-18BP

GenPept sequence	Viral types
		MCU60315_54MCU60315_53SWPHLSB_12CV41KBPL_14VVCGAA_5U01161_3 174VVU18340_6VVU18338_7VVU18337_7VARCG_ 7 173MCU60315_51HNABV_1	U60315 contagious molluscum virus subtype 1L22013 Swan pox virus, vaccinia virus, smallpox virus, main virus contagious molluscum virus, hepatitis non-A, non-B related virus

IL-18BPa was expressed in monkey COS7 cells. For this purpose, the cDNA for IL-18BPa was inserted into the mammalian expression vector pEF-BOS. To simplify purification of the recombinant protein, a code (His) was added to the 3' end of the IL-18BP ORF in frame₆A cassette of sequences. COS7 cells were transiently transfected with the expression vector, and serum-free medium (150 ml) of these cells was concentrated and purified by metal chelate chromatography. IL-18BPa as a single band on silver-stained SDS-PAGE under reducing and non-reducing conditions and has the same apparent molecular weight as urinary IL-18 BP. Protein sequence analysis of this preparation revealed an N-terminal sequence identical to urinary IL-18 BP. Analysis of IL-18BPa by immunoblotting with an antibody against urinary IL-18BP indicated the same molecular weight band as urinary protein. In addition, IL-18BPa can be substituted by immunoprecipitation followed by SDS-PAGE and autoradiography¹²⁵I-IL-18BP, bound to an antibody. Therefore, IL-18BPa structurally corresponds to IL isolated from urine-18BP。

Crude and purified IL-18BPa were tested for their ability to inhibit the biological activity of IL-18. IL-18BPa inhibited the activity of human and mouse IL-18 in mouse splenocytes, PBMC and human KG-1 cell lines (FIG. 9). These results confirm the characteristics of the IL-18BPa cDNA as encoding a biologically active IL-18 BP.

The invention also relates to muteins and fragments of IL-18BP, and viral IL-18BP, and to fusion proteins comprising wild-type IL-18BP and viral IL-18BP or muteins or fragments thereof fused to other polypeptides or proteins and capable of binding IL-18 or homologs thereof.

As used herein, the term "mutein" refers to an analogue of IL-18BP or an analogue of viral IL-18BP in which different amino acid residues have been substituted for, deleted from, or added to one or more amino acid residues of the native IL-18BP or viral IL-18BP, without substantial alteration in the ability of the resulting product to bind IL-18 as compared to the wild-type IL-18BP or viral IL-18 BP. These muteins can be prepared by known synthetic and/or site-directed mutagenesis techniques, or any suitable known technique.

Any such mutein preferably has an amino acid sequence sufficient to replicate IL-18BP or sufficient to replicate viral IL-18BP, so as to have substantially similar activity as IL-18 BP. One activity of IL-18BP is its ability to bind IL-18. As long as the mutant protein has IL-18 basic binding activity, it can be used for example by affinity chromatography for purification of IL-18, so can be considered basically with IL-18BP similar activity. Therefore, by routine experimental means including such mutant protein such as simple sandwich competition test to determine whether any given mutant protein basically with IL-18BP activity, such as radioimmunoassay or ELISA test to determine whether it binds to the appropriate marker of IL-18.

In a preferred embodiment, any such mutein has at least 40% identity or homology with the sequence of IL-18BP or a virally encoded IL-18BP homologue. More preferably, it has at least 50%, at least 60%, at least 70%, at least 80% or most preferably at least 90% identity or homology.

Muteins of IL-18BP polypeptides or muteins of viral IL-18BP that can be utilized according to the present invention, or nucleic acids encoding them, including a limited set of sequences that substantially correspond to the replacement peptide or polypeptide, are routinely obtained by one of ordinary skill in the art without experimentation in light of the guidance and instruction presented herein. For a detailed description of the protein chemistry and structure, see Schulz, g.e. et al, principles of protein structure, Springer-Verlag, group convention, 1978; creighton, t.e., protein: structural and molecular characteristics, w.h.freeman and Co, san francisco, 1983, which are incorporated herein by reference. For indications of nucleotide sequence substitutions such as codon preference, see Ausubel et al, supra, in A.1.1-A.1.24, and Sambrook et al, supra, in appendices C and D.

Preferred muteins of the present invention are altered by substitutions known as "conservative". Amino acid substitutions of a conserved IL-18BP polypeptide or protein or viral IL-18BP may include synonymous amino acids within groups having sufficiently similar physicochemical properties that substitutions between members of the group will retain the biological function of the molecule, Grantham, science, volume 185, 862-864 (1974). It is clear that the insertion and deletion of amino acids can be carried out without altering their function in the sequences identified above, in particular if the insertion or deletion comprises only a few amino acids, for example less than 30, and preferably less than 10, and without removing or replacing key amino acids of the functional configuration, for example cysteine residues, Anfinsen, "principle of controlling the folding of the protein chain", science, Vol.181, 223 to 230 (1973). Proteins and muteins produced by such deletions and/or insertions appear within the scope of the present invention.

However, if to avoid the formation of unwanted intramolecular or intermolecular disulfide bonds which may cause a reduction in the activity of IL-18BP, cysteines not required for biological activity may be replaced with other residues.

Preferably, the synonymous amino acid groups have been identified in table I. Preferably, the synonymous amino acid groups have been identified in table II; most preferably the synonymous amino acid groups have been identified in table III.

Preferred groups of synonymous amino acids of Table I

Amino acid synonymous group

Ser Ser，Thr，Gly，Asn

Arg Arg，Gln，Lys，Glu，His

Leu Ile，Phe，Tyr，Met，Val，Leu

Pro Gly，Ala，Thr，Pro

Thr Pro，Ser，Ala，Gly，His，Gln，Thr

Ala Gly，Thr，Pro，Ala

Val Met，Tyr，Phe，Ile，Leu，Val

Gly Ala，Thr，Pro，Ser，Gly

Ile Met，Tyr，Phe，Val，Leu，Ile

Phe Trp，Met，Tyr，Ile，Val，Leu，Phe

Tyr Trp，Met，Phe，Ile，Val，Leu，Tyr

Cys Ser，Thr，Cys

His Glu，Lys，Gln，Thr，Arg，His

Gln Glu，Lys，Asn，His，Thr，Arg，Gln

Asn Gln，Asp，Ser，Asn

Lys Glu，Gln，His，Arg，Lys

Asp Glu，Asn，Asp

Glu Asp，Lys，Asn，Gln，His，Arg，Glu

Met Phe，Ile，Val，Leu，Met

Trp Trp

More preferred groups of synonymous amino acids of Table II

Amino acid synonymous group

Ser Ser

Arg His，Lys，Arg

Leu Leu，Ile，Phe，Met

Pro Ala，Pro

Thr Thr

Ala Pro，Ala

Val Val，Met，Ile

Gly Gly

Ile Ile，Met，Phe，Val，Leu

Phe Met，Tyr，Ile，Leu，Phe

Tyr Phe，Tyr

Cys Cys，Ser

His His，Gln，Arg

Gln Glu，Gln，His

Asn Asp，Asn

Lys Lys，Arg

Asp Asp，Asn

Glu Glu，Gln

Met Met，Phe，Ile，Val，Leu

Trp Trp

TABLE III most preferred groups of synonymous amino acids

Amino acid synonymous group

Ser Ser

Arg Arg

Leu Leu，Ile，Met

Pro Pro

Thr Thr

Ala Ala

Val Val

Gly Gly

Ile Ile，Met，Leu

Phe Phe

Tyr Tyr

Cys Cys，Ser

His His

Gln Gln

Asn Asn

Lys Lys

Asp Asp

Glu Glu

Met Met，Ile，Leu

Trp Met

Examples of protein amino acid substitutions that can be used to obtain muteins of IL-18BP polypeptides or proteins or to produce muteins of viral IL-18BP used in the present invention include any known method steps, such as those described in U.S. Pat. Nos. RE33,653, 4,959,314, 4,588,585 and 4,737,462 to Mark et al; 5,116,943 to Koths et al; 4,965,195 to Namen et al; 4,879,111 to Chong et al; and 5,017,691 to Lee et al; and lysine substituted proteins as shown in U.S. Pat. No. 4,904,584 (Shaw et al).

In another preferred embodiment of the invention, the mutein of IL-18BP or viral IL-18BP has an amino acid sequence which substantially corresponds to IL-18BP or viral IL-18 BP. The term "substantially corresponds to" is understood to mean proteins which have minimal sequence variation from the native protein, the minimal variation not affecting the essential characteristics of the native protein, in particular insofar as they bind IL-18. It is generally believed that this type of variation in the scope of "substantially corresponding" results from conventional mutagenesis techniques of the DNA encoding these proteins, resulting in several small modifications, and the desired activity is screened in the manner discussed above. In addition to binding to IL-18, the mutant protein can also regulate and/or block IL-18 activity.

Muteins of the present invention include proteins encoded by nucleic acids, such as DNA or RNA, that hybridize under stringent conditions to DNA or RNA encoding IL-18BP or encoding viral IL-18BP according to the present invention. The present invention also includes nucleic acids that can be used as probes in the identification and purification of desired nucleic acids. In addition, such a nucleic acid would be a primer candidate for determining whether it encodes a polypeptide that retains the functional activity of the IL-18BP of the invention. The term "stringent conditions" refers to hybridization and subsequent washing conditions referred to as "stringent" by one of ordinary skill in the art. See Ausubel et al, Current protocols in molecular biology, supra, Interscience, New York, chapters 6.3 and 6.4 (1987, 1992), and Sambrook et al, supra. Without limitation, examples of stringent conditions include those that are 12 to 20 ℃ below the calculated Tm for the hybridization under study, e.g., 2 XSSC and 0.5% SDS5 minutes, 2 XSSC and 0.1% SDS15 minutes; washing conditions of 0.1 XSSC and 0.5% SDS at 37 ℃ for 30 to 60 minutes, and then 0.1 XSSC and 0.5% SDS at 68 ℃ for 30 to 60 minutes. Those stringent conditions understood by those skilled in the art also depend on the DNA sequence, the length of the oligonucleotide probe (e.g., 10-40 bases) or the mixed oligonucleotide probes. If a mixed probe is used, tetramethylammonium chloride (TMAC) is preferably used instead of SSC. See Ausubel, supra.

The invention further includes nucleic acids encoding the IL-18BP of the invention, but which differ in codon sequence due to the degeneracy of the genetic code. DNA sequences shown in FIGS. 4 to 7 may not hybridize under stringent conditions, but DNA capable of encoding IL-18BP of the present invention is also included in the present invention.

The term "fusion protein" refers to a polypeptide comprising IL-18BP or viral IL-18BP or muteins thereof fused to another protein having an extended residence time in body fluids. Thus, IL-18BP or viral IL-18BP may be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or fragment thereof. It may also be fused to polyethylene glycol (PEG) to extend the residence time.

The term "salt" of the present invention refers to salts of carboxyl groups and acid-added salts of amino groups of IL-18BP, viral IL-18BP, muteins or fusion proteins thereof. Salts of the carboxyl groups can be formed by means known in the art and include inorganic salts such as, for example, sodium, calcium, ammonium, iron or zinc salts and the like, and salts with organic bases such as those formed with amines such as triethanolamine, arginine, or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as hydrochloric acid, or sulfuric acid, and salts containing organic acids such as acetic acid or oxalic acid. Of course, any such salt must have substantially similar activity to IL-18 BP.

"functional derivatives" as used in the present invention cover derivatives which can be prepared by means known in the art from functional groups present as side chains on the residues or the N-or C-terminal groups and which are included in the IL-18BP, or viral IL-18BP or muteins and fusion proteins thereof of the present invention as long as they retain pharmaceutically acceptable properties, i.e.they do not destroy the activity of the protein substantially similar to that of IL-18BP or viral IL-18BP, and do not confer toxicity to the composition containing it. These derivatives may, for example, include polyethylene glycol side chains that can mimic antigenic sites and prolong residues of IL-18BP or viral IL-18BP in body fluids. Other derivatives include aliphatic esters of carboxyl groups, amides which form carboxyl groups by reaction with amines or with primary or secondary amines, N-acyl derivatives without amino groups of the amino acid residue formed with an acyl component (e.g., an alcarono or carbocyclic aromatic group) or O-acyl derivatives of free hydroxyl groups (e.g., a seryl or threonyl residue) formed with an acyl component.

With respect to "active portions" of IL-18BP, muteins and fusion proteins, the present invention covers fusion fragments or precursors of the polypeptide chains of the protein molecules alone or in conjunction with related molecules or their linked residues, e.g., sugar or phosphate residues, or aggregates of the protein molecules or sugar residues themselves, provided that said portions substantially retain the ability to bind IL-18.

The term "ring-altered derivative" as used herein refers to a linear molecule in which the ends have been linked together either directly or via a linker to produce a circular molecule, which is then opened at another position to produce a new linear molecule, the ends of which differ from the ends of the original molecule. Cyclic variations include those molecules whose structure is equivalent to a molecule that has been cyclized and then opened. Therefore, the circularly altered molecule can be synthesized anew as a linear molecule, and the cyclization and opening steps are never performed. The preparation of the cyclic derivatives is disclosed in WO 95/27732.

IL-18BP or viral IL-18BP may be produced by various recombinant cells, such as prokaryotic cells, e.g., E.coli, or other eukaryotic cells, such as yeast or insect cells. For the production of recombinant IL-18BP or viral IL-18BP, methods for constructing suitable vectors carrying DNA encoding IL-18BP and suitable for transfection (e.g., E.coli, mammalian cells and yeast cells) or infection of insect cells are known in the art. See, e.g., Ausubel et al, "Current protocols in molecular biology," Current protocols, 1993, and Sambrook et al, "molecular cloning, A laboratory Manual," second edition, Kolder Springhouse Press, 1989.

To express the IL-18BP protein or viral IL-18BP, DNA encoding the IL-18BP or viral IL-18BP, fragments, muteins or fusion proteins thereof and operably linked transcriptional and translational regulatory signals are inserted into a vector capable of integrating the desired gene sequences into the host cell chromosome. In order to be able to select cells which have stably integrated the introduced DNA in their chromosome, one or more markers are used which allow the selection of host cells containing the expression vector. The marker may provide prototrophic biocidal resistance to an auxotrophic host, such as an antibiotic, or resistance to heavy metals such as copper or the like. The selectable marker gene may be directly linked to the expressed DNA gene sequence or introduced into the same cell by transfection. Other elements are also required for optimal synthesis of single-stranded binding protein mrnas. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

Preferably, the DNA molecule introduced into the selected cell is incorporated into a plasmid or viral vector capable of self-replication in the recipient host. Preferred prokaryotic plasmids are derivatives of pBr 322. Preferred eukaryotic vectors include BPV, vaccines, SV40, 2-micron rings, and the like, or derivatives thereof. Such plasmids and vectors are known in the art (2-5, 22). Once the construct-containing vector or DNA sequence has been prepared for expression, the expression vector may be introduced into an appropriate host cell by any of a variety of appropriate means, such as transformation, transfection, lipofection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, and the like.

The host cells utilized in the present invention may be prokaryotic or eukaryotic. Preferred prokaryotic hosts include bacteria such as E.coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. Most preferably the host is E.coli. Particularly advantageous bacterial hosts include E.coli K12 strain 294(ATCC 31446), E.coli X1776(ATCC 31537), E.coli W3110(F-, lambda-, phototropic (ATCC 27325). under such conditions, the protein will not be glycosylated.

However, because native IL-18BP is a glycosylated protein, eukaryotic hosts are preferred over prokaryotic hosts. Preferred eukaryotic hosts are mammalian cells, e.g., human, monkey, mouse, and Chinese Hamster Ovary (CHO) cells, as they provide post-translational modifications of the protein molecule, including proper folding, proper disulfide bond formation, and glycosylation at the correct site. Also yeast cells and insect cells can be subjected to post-translational peptide modifications including high mannose glycosylation.

There are many recombinant DNA protocols that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast and insect cells. Yeast and insect cells recognize leader sequences on cloned mammalian gene products and secrete mature IL-18 BP. After introduction of the vector, the host cell may be grown in a selective medium that selects for growth of cells containing the vector. Expression of the cloned gene sequence results in the production of IL-18BP, viral IL-18BP, fusion protein, or a mutein or fragment thereof. The cloning, clone isolation, identification, characterization and sequencing methods mentioned above are described in detail in the examples below.

The expressed protein is then isolated and purified by any conventional means including extraction, precipitation, affinity chromatography, electrophoresis or the like or by affinity chromatography using, for example, an anti-IL-18 BP monoclonal antibody immobilized on a gel matrix contained within a column. The crude preparation containing the recombinant IL-18BP is passed through the column, so that the IL-18BP will pass through the specific antibody binding column, while the impurities pass through the column. After washing, the protein is eluted from the gel under conditions normally used for this purpose, i.e. at high or low pH, e.g. pH11 or pH2.

The invention further relates to vectors for expressing IL-18BP or viral IL-18 or derivatives thereof in mammals, and more particularly in humans. Vectors for the short and long term expression of genes in mammals have been described in the literature. Studies have shown that therapeutic proteins that release genes into, for example, skeletal muscle, vascular smooth muscle and liver are at a systemic level. Skeletal muscle is a useful target because of its large mass, vascularity and accessibility. However, other targets and bone marrow precursors of specific immune cells have been successfully utilized. For example, currently available vectors for expression of proteins in muscle include plasmid DNA, liposomes, protein-DNA conjugates and vectors based on adenovirus, adeno-associated virus and hepatitis virus. Among these, vectors based on adeno-associated virus (AAV) have been the most successful for time and level of gene expression and for safety reasons (Kessler, p.d.1996, journal of the american college of sciences, 93, 14082-4087).

The construction of AAV-based vectors is described in detail in Snyder et al, 1996, Current protocols in human genetics, Chapter 12.1.1-12.1.17, John Wiley and Sons, and is incorporated herein. Briefly, plasmid psub201 containing the wild-type AAV genome is cleaved with the restriction enzyme XbaI and ligated with a construct containing a potent eukaryotic promoter, e.g., the cytomegalovirus promoter, Kozak isogenic sequences, DNA sequences encoding IL-18BP or viral IL-18BP, or muteins or fusion proteins thereof or fragments thereof, the appropriate 3' untranslated region and a polyadenylation signal, e.g., the polyadenylation signal of monkey virus 40. The resulting recombinant plasmids are co-transfected into mammalian cells, such as human T293 cells, using helper AAV plasmids, such as pAAV/Ad. The cultures were then infected with adenovirus as a helper virus and culture supernatants were collected after 48-60 hours. The supernatant was fractionated by ammonium sulfate precipitation, purified on a CsCl density gradient, dialyzed, and then heated at 56 ℃ to disrupt any adenovirus, and the recombinant AAV capable of expressing IL-18BP or viral IL-18BP or muteins or fusion proteins thereof was stabilized at this step.

To date, the physiological role of receptors for soluble cytokines has not been established. Soluble receptors bind their specific ligands and in most cases inhibit their biological activity, as shown in the TNF system (11, 12). In rare cases, such as IL-6, soluble receptors enhance biological activity. The discovery that recombinant soluble TNF receptors, also known as TBP (TNF binding protein), are capable of preventing septic shock in animal models, while soluble forms of IL-1 receptor have been found to have profound inhibitory effects on the development of in vivo alloreactivity in mouse xenograft receptors.

Similarly, the IL-18BP and viral IL-18BP of the invention may find use as modulators of IL-18 activity in type I diabetes, in sepsis, in autoimmune diseases, in transplant rejection, rheumatoid arthritis, inflammatory bowel disease, sepsis, multiple sclerosis, ischemic heart disease, including acute heart attack, ischemic brain injury, chronic hepatitis, psoriasis, chronic hepatitis and acute hepatitis. Therefore, they can be used for any disease, wherein endogenous production or exogenous administration of IL-18 induces disease or aggravates the condition of the patient.

The invention further relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and the IL-18BP or viral IL-18BP of the invention or active muteins, fusion proteins and salts, functional derivatives or active parts thereof.

The invention further relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and, for example, a viral vector such as any of the AAV-based viral vectors described herein or expressing IL-18BP or viral IL-18BP or muteins thereof, fragments or fusion proteins thereof and another vector suitable for gene therapy for administration to humans and other mammals for the purpose of expressing IL-18BP and viral IL-18BP or muteins thereof or fusion proteins of the invention in vivo.

The pharmaceutical compositions of the present invention for administration are prepared by mixing IL-18BP, or viral IL-18BP or derivatives thereof, or vectors expressing them, with physiologically acceptable carriers, and/or stabilizers and/or excipients, and preparing into dosage forms by lyophilization in dosage vials. The method of administration may be any acceptable manner of administration by similar agents and will depend on the condition to be treated, e.g., intravenously, intramuscularly, subcutaneously, by local injection or topical application, or continuous infusion, etc. The amount of active compound administered will depend on the route of administration, the disease to be treated and the patient's symptoms. For example, a local injection will require a lower amount of protein on a weight basis than an intravenous infusion.

Thus, IL-18BP, or viral IL-18BP, or vectors expressing them in vivo, are indicated for use in the treatment of autoimmune diseases, type I diabetes, rheumatoid arthritis, transplant rejection, inflammatory bowel disease, sepsis, multiple sclerosis, ischemic heart diseases including acute heart attack, ischemic brain injury, chronic hepatitis, psoriasis, chronic pancreatitis, acute pancreatitis, and similar conditions in which aberrant expression of IL-18 is present, leading to excessive amounts of IL-18 or complications arising from exogenous administration of IL-18.

The invention also includes antibodies against IL-18BP or viral IL-18BP, as well as muteins, fusion proteins, salts, functional derivatives and active moieties thereof. The term "antibody" refers to antibodies including polyclonal antibodies, monoclonal antibodies (MAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies, which may be labeled in soluble or bound form, as well as humanized antibodies and fragments thereof provided by any of the prior art, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques.

Polyclonal antibodies are a heterogeneous population of antibody molecules derived from the serum of an animal immunized with an antigen. Monoclonal antibodies comprise substantially a homogeneous population of antibodies specific for an antigen, which population comprises substantially similar epitope binding sites. MAbs can be obtained by methods known to those of ordinary skill in the art. See, e.g., Kohler and Milstein, nature, 256: 495 to 497 (1975); U.S. Pat. nos. 4,376,110; ausubel et al, supra, eds, Harlow and Lane, antibodies, A laboratory Manual, Cole de Spulin harbor laboratory, (1988); and Colligan et al, current protocols in immunology, Greene publishing Association, and WileyInterscience, New York (1992, 1993), the contents of which are incorporated herein by reference in their entirety. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. The hybridomas producing the MAb of the present invention can be cultured in vitro, in situ, or in vivo. The generation of high titers of Mab in vivo or in situ makes this the presently preferred method of production.

Chimeric antibodies are molecules whose different parts originate in different animal species, such as those having a variable region originating in a mouse MAb and a human immunoglobulin constant region. Chimeric antibodies were primarily used to attenuate immunogenicity in applications and to enhance production yields, e.g., mouse mabs have higher yields in hybridomas but higher immunogenicity in humans, so that human/mouse chimeric mabs were utilized. Chimeric antibodies and methods for their production are well known in the art (Cabilly et al, Proc. Nature, 312: 643-646 (1984)), Cabilly et al, European patent application 125023 (published 1984 on 11/14), Neuberger et al, Nature, 314: 268-270 (1985), Taniguchi et al, European patent application 171496 (published 1985 on 2/19), Morrison et al, European patent application 173494 (published 1986 on 3/1735), Neuberger et al, PCT application WO8601533 (published 1986 on 3/13), Kudo et al, European patent application 184494 (published 1986 on 6/173187), European patent application WO 1736/17311/976, European patent application WO 0267: 1986/1065 (published 1986/1065), journal of american academy of sciences, 84: 3439 to 3443 (1987); sun et al, Proc. Natl. Acad. Sci. USA 84: 214-218 (1987); better et al, science, 240: 1041 to 1043 (1988); and Harlow and Lane, antibodies, laboratory manuals, supra. These references are fully incorporated into the present invention.

Anti-idiotypic (anti-Id) antibodies are antibodies that recognize unique determinants normally associated with the antigen-binding site of the antibody. Id antibodies can be prepared by immunizing the same species of animal and genotype (e.g., mouse strain) from which the MAb was derived with the anti-Id MAb being prepared. The immunized animal recognizes and responds to the idiotypic determinants of the immunizing antibody by producing antibodies against these idiotypic determinants (anti-Id antibodies). See, for example, U.S. patent No. 4,699,880, which is incorporated herein in its entirety.

The anti-Id antibody may also be used as an "immunogen" to induce an immune response in another animal that produces a so-called anti-Id antibody. The epitope for anti-Id is identical to the original MAb that induced anti-Id. Therefore, by means of antibodies derived from the idiotypic determinants of MAbs, it is possible to identify other clones expressing antibodies of the same specificity.

Thus, anti-Id antibodies can be induced in appropriate animals, such as BALB/c mice, using anti-IL-18 BP-producing MAbs and related proteins of the invention. Splenocytes from such immunized mice were used to generate anti-Id hybridomas that secrete anti-Id MAbs. Alternatively, anti-Id mabs can be conjugated to a carrier such as Keyhole Limpet Hemocyanin (KLH) and used to immunize other BALB/c mice. Sera from these mice will contain anti-Id antibodies with binding properties specific for the original MAb of the IL-18BP epitope or of the viral IL-18BP epitope.

Therefore, anti-Id MAbs have their own idiotypic epitopes, or "idiosyncrasies" with a structure similar to the epitope being evaluated, such as IL-18BP or viral IL-18 BP.

The term "humanized antibody" is meant to include, for example, antibodies obtained by engineering mouse antibodies so as to be more compatible with the human body. Such humanized antibodies have reduced immunogenicity and improved pharmacokinetics in humans. They can be prepared by techniques well known in the art, for example as described for humanized anti-TNF antibodies in molecular immunology Vol.30, No. 16, 1443-1453, 1993.

The term "antibody" is also meant to include intact molecules capable of binding antigen as well as fragments thereof such as Fab and F (ab') 2. Fab and F (ab') 2 fragments lack the Fc fragment of intact antibodies that cycle significantly more rapidly and can have less non-specific tissue binding than intact antibodies [ Wahl et al, journal of nucleic acid methods, 24: 316 to 325 (1983). It is desirable that Fab and F (ab') 2 and other fragments of the antibodies used in the invention can detect and quantify IL-18BP or viral IL-18BP according to the methods disclosed for the intact antibody molecules of the invention. Such fragments are typically produced by cleavage of the protein using an enzyme such as papain (to produce Fab fragments) or trypsin [ to produce F (ab') 2 fragments ].

An antibody is considered "capable of binding" a molecule if it is capable of specifically reacting with the molecule to bind the molecule to the antibody. The term "epitope" refers to the portion of any molecule that is capable of binding by an antibody that is also recognized by that antibody. Epitopes or "antigenic determinants" generally contain chemically active surface groups of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or portion of a molecule that is capable of binding by an antibody that is otherwise capable of inducing an animal to produce an antibody that is capable of binding an epitope of the antigen. An antigen may have one or more epitopes. Specific reaction in the above indicates that an antigen will react in a highly selective manner with its corresponding antibody and not with other antibodies raised against a large number of other antigens.

Antibodies, including fragments of antibodies, can be used in the quantitative or qualitative detection of IL-18BP or viral IL-18BP or related proteins in a sample, or in the detection of the presence of cells expressing such proteins of the invention. This detection can be accomplished by immunofluorescence techniques using a fluorescently labeled antibody (see below) coupled with detection by light microscopy, flow cytometry or fluorimetry.

The antibodies (or fragments thereof) used in the present invention may be used histologically, e.g., in immunofluorescence or immunoelectron microscopy, for in situ detection of the IL-18BP or viral IL-18BP and related proteins of the present invention. In situ detection may be accomplished by removing a tissue sample from a patient and providing such sample with a labeled antibody of the invention. The antibody (or fragment) is preferably provided by applying or overlaying the labeled antibody (or fragment) in a biological sample. By using such a method, not only the presence of IL-18BP or viral IL-18BP or related proteins, but also its distribution on the tissue under examination can be determined. Those of ordinary skill in the art will readily recognize that any variety of histological methods (e.g., staining methods) may be modified to accomplish such in situ detection using the present invention.

Such testing of the IL-18BP or viral IL-18BP, or related protein, of the invention generally comprises incubating a biological sample such as a biological fluid, a tissue extract, freshly collected cells such as lymphocytes or leukocytes, or cells that have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying the IL-18BP or related protein, and detecting the antibody by any of a number of techniques well known in the art.

The biological sample may be treated with a solid support or carrier such as cellulose, or other solid support or carrier capable of immobilizing cells, cell particles or soluble proteins. The support or carrier can then be washed with an appropriate buffer, followed by treatment with the detectably labeled antibody of the invention. The solid support or carrier is then washed a second time with buffer to remove unbound antibody. The amount of bound label on the solid support or carrier is then detected by conventional means.

By "solid support", "solid carrier", "support" or "carrier" is meant any support or carrier capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylase, natural and modified cellulose, polyacrylamide, gabbros and magnetite (magnetite). The nature of the carrier may be such that it is soluble to some extent or insoluble for the purposes of the present invention. In fact, the support material may have any possible structural configuration, as long as the conjugated molecule is capable of binding to an antigen or an antibody. Thus, the support or carrier configuration may be spherical, as in a bead or cylinder, as in the interior surface of a test tube, or the exterior surface of a rod. Alternatively, the surface may be flat, as in paper, test strips, and the like. Preferred supports or carriers include polystyrene beads. Those of ordinary skill in the art will know of many other suitable carriers to bind antibodies or antigens, or will be able to determine them by using routine experimentation.

The binding activity of a given plurality of antibodies of the invention can be determined according to well known methods. One of ordinary skill in the art can determine the operable and optimal test conditions for each test by using routine experimentation.

Other such steps may be added to the test as is conventional or desired under particular conditions, such as washing, stirring, shaking, filtering, etc.

One method of detectably labeling an antibody of the invention is to attach it to an enzyme and use it in an Enzyme Immunoassay (EIA). When subsequently contacted with a suitable substrate, the enzyme in turn reacts with the substrate in such a way as to produce a chemical component which can be detected by means of a spectrophotometer, fluorometer or by means of macroscopic methods. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be accomplished by a colorimeter method that utilizes a chromogen substrate for the enzyme. In comparison with similar preparative standards, the detection can also be accomplished by visual comparison of the extent of enzymatic reaction of the substrate.

Detection may be accomplished by any other immunoassay. For example, by radiolabelling the antibody or antibody fragment, IL-18BP or viral IL-18BP can be detected by using Radioimmunoassay (RIA). A good description of RIA can be found in laboratory techniques and biochemistry in molecular biology, Work, T.S. et al, North Netherlands publishing Co., New York (1978), entitled "introduction to radioimmunoassay and related technology" Chard, T., (incorporated herein by reference). Radioactive isotopes can be detected when using gamma counters or scintillation counters or by autoradiography.

It is also possible to label the antibodies of the invention with a fluorescent compound. When the fluorescently labeled antibody is contacted with light of the appropriate wavelength, its presence can then be detected due to fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Using fluorescent radiation of metals, e.g.¹⁵²Eu, or other lanthanide series species can also detectably label the antibody. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to biotin. The biotinylated antibody can then be detected by avidin or streptavidin coupled to a fluorescent compound or an enzyme such as peroxidase or a radioisotope and the like.

The antibody may also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemofluorescently labeled antibody is then determined by detecting the presence of fluorescence generated during the chemical reaction. Examples of specific chemiluminescent labeling compounds utilized are luminol, isoluminol, thermal acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, bioluminescent compounds may also be used to label the antibodies of the present invention. Bioluminescence is the type of chemiluminescence found in biological systems that catalyze proteins to enhance the efficiency of a chemiluminescent reaction. The presence of the bioluminescent protein is determined by detecting the presence of fluorescence. Important bioluminescent compounds for labeling are luciferin, luciferase and aequorin.

The antibody molecules of the invention may be suitable for use in an immunometric assay, also known as a "2-site" or "sandwich" assay. In a typical immunoassay test, an amount of unlabeled antibody (or antibody fragment) is bound to a solid support or carrier, and the addition of an amount of detectably labeled soluble antibody allows the detection and/or quantification of the ternary complex formed between solid-phase antibody, antigen and labeled antibody.

Typically, and preferably, the immunoassay comprises a "forward" test in which the antibody bound to a solid phase is first contacted with the sample to be tested in order to extract the antigen from the sample by forming a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove residues from the liquid sample, including any unreacted antigen, and then contacted with a solution containing an unknown amount of labeled antibody (which functions as a "receptor molecule"). After a second incubation period to allow complexes of labeled antibody and antigen bound to the solid support or carrier by unlabeled antibody, the solid support or carrier is washed a second time to remove unreacted labeled antibody.

In another type of "sandwich" test, which can also be used for the antigens of the present invention, so-called "simultaneous" and "reverse" tests are utilized. The "simultaneous" test comprises a single incubation step, as the antibody binds to the solid support or carrier and the labeled antibody added simultaneously to the sample to be tested. After the incubation is complete, the solid support or carrier is washed to remove residues of the liquid sample and uncomplexed labeled antibody. The presence of labeled antibody bound to the solid support or carrier is then determined as it is in a conventional "forward" sandwich test.

In the "reverse phase" assay, after an appropriate incubation period is utilized, a solution of labeled antibody is first added stepwise to the liquid sample, followed by addition of unlabeled antibody bound to a solid support or carrier. After the second incubation, the solid phase is washed in a conventional manner to free it from residues of the sample to be tested and from unreacted labeled antibody solution. The test for labeled antibody bound to the solid support or carrier is then determined as in the "simultaneous" and "forward" tests.

As identified above, the present invention also provides DNA molecules encoding any of the proteins of the present invention, replicable expression vectors containing any such DNA molecules, host cells transformed with any such expression vectors including prokaryotes, eukaryotes, and host cells, preferably CHO cells. The invention also includes methods of producing expression vectors encoding any of the proteins of the invention for expression purposes in humans and other mammals.

The invention also includes methods of producing any of the proteins of the invention by culturing the transformed cells of the invention, and recovering the DNA molecules and proteins encoded by the expression vectors in such transformed host cells.

In addition to using IL-18BP or viral IL-18BP in modulating IL-18 activity, they may of course also be used to purify IL-18BP itself. For this purpose, IL-18BP or viral IL-18BP is coupled to an affinity column and passed through crude IL-18. IL-18 is then recovered from the column, for example by elution at low pH.

The invention can be illustrated by the following non-limiting examples:

example 1: isolation of IL-18 binding proteins

Coli IL-18(2.5 ml, Peprotech, NJ) was coupled to affinity gel-10 (0.5 ml, BioRad) and packed into the column according to the manufacturer's instructions. Crude urine protein (100 fold concentrated, 500 ml) was loaded onto the column at a flow rate of 0.25 ml/min. The column was washed with 0.5 mol/l NaCl in 250 ml Phosphate Buffered Saline (PBS). Then immediately using 1 mol/l Na₂CO₃The neutralized 25 mmol/l citric acid, pH2.2 and benzamidine (1 mmol/l) eluted the bound protein. Collecting 1 moleFraction of moles/liter. The fractions were analyzed by SDS-PAGE and silver stained. In fractions 2-8, the IL-18 binding protein eluted as approximately 40,000 daltons protein (FIG. 1). A band of approximately 40 kilodaltons corresponding to IL-18BP exhibited a distinct yellow coloration upon silver staining. By reacting as described in example 2 with¹²⁵I-IL-18 crosslinking, SDS-PAGE and autoradiography analysis of various fractions. IL-18 binding proteins were found in fractions 2-8 and eluted from an IL-18 agarose column (FIG. 2).

Example 2: cross-linking of affinity purified IL-18BP with tagged IL-18

Samples (40. mu.l) of IL-18BP from the affinity purification step were combined with¹²⁵I-IL-18(5,000,000cpm) was incubated (70 min, 4 ℃). Then adding the solution dissolved in dimethyl sulfoxide (Me)₂SO, 20 mmol/l) to a final concentration of 2 mmol/l and the mixture is left at 4 ℃ for 20 minutes. The reaction was stopped by adding 1 mol/l Tris-HCl and 1 mol/l NaCl at pH7.5 to a final concentration of 100 mmol/l. Sample buffer containing dithiothreitol (DTT, 25 mmol/L last) was added and the mixture was analyzed by SDS-PAGE (7.5 acrylamide) followed by autoradiography (FIG. 2).

It was observed that fractions eluted from the IL-18 affinity column (lanes 2 and 3), but not in the column wash containing all other crude urine proteins (lane 1), may contain approximately 20 kilodaltons¹²⁵I-IL-18 crosslinked approximately 40 kilodalton protein molecular weight of 58 kilodalton specific band.

Example 3: protein sequence analysis

Fractions eluted from the affinity column of example 1 were resolved by SDS-PAGE (10% acrylamide) under non-reducing conditions and electrophoresed on a PVDF membrane (Pro-Blot, applied biosystems, USA). Using a Coomassie blue stained membrane, a band of about 40 kilodaltons was cut out, and protein sequence analysis was performed by a Procise micro sequencer (applied biosystems, USA). The following large sequences were obtained:

T-P-V-S-Q-Q-x-x-x-A-A-A

1···5····10 ··

wherein x represents an amino acid which has not yet been determined.

In addition, a small sequence was obtained:

A-x-Y-x-R-I-P-A-x-A-I-A

1···5···· 10 ··

because of this diploid sequence, it is not possible to obtain longer sequence data. A fragment with the small sequence human defensin was identified (accession number p11398), beginning at amino acid 65 of defensin. The large sequence was unable to bind to any other known protein, as determined by the blastp and tblastn research programs studying all available databases in NCBI and TIGR.

To obtain a longer and more accurate sequence, and to identify potential cysteine residues, another aliquot of the fraction eluted from the IL-18 sepharose column was reduced with DTT in 6 moles/l guanidine hydrochloride, reacted with 4-propenylpyridine, desalted by a micro-ultrafiltration device (Ultrafree, cut-off molecular weight 10,000 daltons, Millipore) and subjected to analysis of the protein microsequences. After cycle 1 of sequencing, the filter paper was reacted with o-phthalaldehyde to block the N-terminal polypeptide instead of Pro. In this way, only the following is obtained

The sequence is as follows:

TPVSQXXXAA XASVRSTKDP CPSQPPVFPA AKQCPALEVT

1 10 20 30 40

(T ═ Thr; P ═ Pro; V ═ Val; S ═ Ser; Q ═ gin; X ═ unknown; a ═ Ala; R ═ Arg; K ═ Lys; D ═ Asp; C ═ Cys; F ═ Phe; L ═ Leu; E ═ Glu)

In cycles 6, 7, 8 and 11, a low level of Thr signal was obtained. Because of this low level, we believe that it is more prudent to not assign specific amino acid residues in the cycles described.

The resulting sequence is clearly distinct from any other known protein, as determined by studying the protein database. However, the study of the TIGR database by the tblastn research program provided a cDNA document, designated THC123801, whose open reading frame (218 codons) when translated contained a sequence highly homologous to the N-terminal sequence of IL-18 BP. Homology is shown below:

1.......TPVSQXXXAAXASVRSTKDPCPSQPPVFPAAKQCPALEVT... 40

| | | || |||||||||||||||||||||||||||||

51 VTLLVRATXVXQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPE 100

[ sequences (1-40) above are IL-18BP isolated according to the invention; the following sequences (51 to 100) were deduced by translation of cDNA of TIGR document THC123801 ].

At residues 2 and 4 of IL-18BP, the putative protein sequence obtained by translation of the document THC123801 is ambiguous. It demonstrates the characteristic of amino acid residues 6, 7 and 8 of IL-18BP as Thr, and seems to be the same for residue 11.

Example 4: IL-18BP is a glycoprotein

An aliquot (0.3 ml) of the eluted fractions from example 1 was further purified by exclusion size chromatography on a Superose12 column (1X 30 cm, Pharmacia, Sweden). The column was pre-equilibrated and eluted using phosphate buffer and sodium azide (0.02%) at a flow rate of 0.5 ml/min. Fractions were collected for 1 minute. IL-18BP binding protein was eluted as approximately 40,000 daltons protein in fractions 20-25 as determined by SDS-PAGE and silver staining. Samples containing approximately 40,000 daltons of protein (23, 50 microliter fractions, approximately 50 nanograms of protein) were reacted with N-glycosidase F (PNGase F, Biolab) according to the manufacturer's instructions. Briefly, aliquots were denatured by boiling in 5% SDS for 10 min, 10 XG 7 buffer (2.5. mu.l), 10% NP-40 (2.5. mu.l), and PNGase F (1. mu.l) at 37 ℃ for 1 hour. Samples were analyzed by SDS-PAGE (10% acrylamide) under non-reducing conditions and compared to undigested IL-18BP from the same Superose12 column. It was found that in the PNGase-treated fraction, the IL-18BP band disappeared at about 40 kilodaltons. New bands corresponding to 30 kilodaltons (as above the PNGase band) and 20 kilodaltons were obtained. The disappearance of the band of about 40 kilodaltons indicates that the band is an N-glycosylated protein.

Example 5: blocking IL-18 bioactivity by IL-18BP

The ability of IL-18BP isolated from urine to block IL-18 activity was determined by measuring IL-18-induced IFN- γ production in monocytes. IL-18 induces IFN- γ when added with low concentrations of LPS, IL-12, IL-2 or other stimuli. IL-18 activity was tested on mouse splenocytes, on human Peripheral Blood Mononuclear Cells (PBMC) and on the human KG-1 cell line. Splenocytes were prepared from healthy mice, washed and cultured at 5X 10 in RPMI1640 supplemented with 10% fetal bovine serum⁶Cells/ml suspension. 1.0 ml cultures were stimulated with LPS (0.5. mu.g/ml or 1. mu.g/ml) and recombinant human or mouse IL-18(0.5 ng/ml or 5 ng/ml). Human IL-18 binding protein (0, 5 or 50 ng/ml) was added to recombinant IL-18 prior to the addition of splenocytes. After 24 hours of culture, the splenocytes were subjected to three cycles of freezing (-70 ℃) and thawing (room temperature), cell debris was removed by centrifugation, and the supernatants were tested for IFN-. gamma. (endogenous) for mouse using an ELISA kit. As shown in FIG. 3A, IL-18BP blocked huIL-18 activity in mouse splenocytes in a dose-dependent manner. In contrast, as a control, soluble interferon- α/β receptors had no effect. The activity of recombinant mouse IL-18 was similarly inhibited by human IL-18BP, indicating that human IL-18BP recognizes mouse IL-18 (FIG. 3B). Endogenous IL-18 was induced in mouse splenocytes by high concentrations of LPS, resulting in IFN- γ production. Indeed, urinary IL-18BP also inhibited LPS (10. mu.g/ml) induction of IFN-. gamma. (FIG. 3C). Concanavalin A (conA) activates T cells to produce IFN- γ in the absence of IL-18 (13). Indeed, IL-18BP is even highConcentration did not inhibit ConA induction of IFN- γ (FIG. 3D). This observation demonstrates that IL-18BP is a specific inhibitor of IL-18 bioactivity and not a non-specific inhibitor of IFN- γ production. IL-18BP also inhibited the activity of human IL-18 in human KG-1 cells induced by the combination of IL-18 and TNF- α (FIG. 3E).

The above data demonstrate that urinary IL-18BP inhibits the activity of human and mouse IL-18, as determined by co-induction of IFN- γ in human and mouse monocytes. The concentration of IL-18BP that attenuates IL-18 activity by > 90% is comparable to IL-18 itself, indicating a high affinity interaction between the two proteins.

Example 6: isolation of cDNA clones encoding IL-18BP

Using SuperScript RNase H^-Total RNA from Jurkat T cells (CRL 8163, American type culture Collection) was reverse transcribed by reverse transcriptase (Gibco-BRL) and random primers (Promega, Madison Wis.). Then, the resulting cDNA fragments were amplified by PCR using TaqDNA polymerase (Sigma) and primers corresponding to nucleotides 24 to 44 (sense) and 500 to 481 (antisense) of TIGR clone THC 123801. Amplification was performed in 30 cycles of annealing (55 ℃,2 min) and extension (70 ℃,1 min). The PCR products were resolved by agarose (1%) gel electrophoresis, eluted and cloned into pGEM-Teasy TA cloning vector (Promega). DNA from a single clone was sequenced using the T7 and SP6 primers.

By random bootstrapping³²The resulting 477bp fragment was P-tagged. This probe was used to screen various human cDNA and genomic libraries. Duplicate nitrocellulose filters were removed and hybridized with probes at 60 ℃ in a buffer containing 6 XSSC, 10 XDenhardt's solution, 0.1% SDS and 100. mu.g/ml salmon sperm DNA. Filter paper was washed and exposed overnight at-80 ℃ to Kodak XAR film. Duplicate positive clones were purified from plaques. Plasmids were excised from the lambda pCEV9 clone and ligated to themselves. cDNA clones from other libraries were isolated according to the manufacturer's instructions. Automated DNA sequence analysis of isolated clones was performed using sense and antisense primers and 373A and 377 sequencers (applied biosystems) (33). This is achieved byThese cloning procedures used standard protocols.

The following libraries were screened: miki friendly, human monocyte cDNA library constructed in λ pCEV9 cloning vector (15); human Jurkat leukemia T cell cDNA library, human peripheral blood leukocyte cDNA library, and human spleen cDNA library, all from Clontech (Palo Alto, CA). The human placental genomic library in the lambda FIXII vector was from Stratagene (La Jolla, CA).

All cDNA clones corresponding to four different splicing variants of IL-18BP were obtained and identified. All splice variants encode putative soluble secreted proteins. The most abundant one (IL-18BPa) has an open reading frame of 192 codons encoding a signal peptide of 28 amino acid residues followed by a mature putative IL-18BPa, the first 40 residues of which (SEQ ID NO: 10) perfectly match the N-terminal protein sequence of urinary IL-18BP (SEQ ID NO: 2). The position of the cysteine residue indicates that this polypeptide belongs to the immunoglobulin (Ig) superfamily. Each of the four Gln residues within mature IL-18BPa is a potential N-glycosylation site. The other three splice variants of IL-18BP are significantly less abundant.

Another 1kb IL-18BPb cDNA encodes the mature protein of 85 amino acid residues (SEQ ID NO: 4). The 2.3kb cDNA encoding the mature IL-18BP of 169 amino acid residues (SEQ ID NO: 6) represents the third variant IL-18BPc, the fourth variant, IL-18BPd encodes the mature IL-18BP of 133 amino acid residues (SEQ ID NO: 8). The splice within the exon is present at two sites along the pre-mRNA. These events and the additional 5 'exon in IL-18BPd produced 3 different 5' UTRs in various cDNA clones. Therefore, it is possible to generate different IL-18BP variants in response to different transcriptional regulatory signals.

To date, no cDNA encoding a receptor containing a transmembrane region has been found.

Example 7: construction of mammalian expression vectors, production of recombinant IL-18BP Evaluation of biological Activity of raw and recombinant IL-18BP

By PCR, using sense primer 5' TATATCTAGAGCCACCATGAGACACAACTGGACACCA and antisense primer: 5' ATATCTAGATTAATGATGATGATGATGATGACCCTGCTGCTGTGGACTGC the coding region of the IL-18BPa cDNA was amplified. The PCR product was cleaved with XbaI and cloned into the XbaI site of pEF-BOS expression vector (25) to generate pEF-BOS-IL-18 BPa. The constructs were verified by DNA sequencing.

At room temperature, 6X 10 in 1.4 ml of TD buffer containing pEF-BOS-IL-18BPa plasmid DNA (10. mu.g) and DEAE-dextran (120. mu.g) were incubated as described (35)⁷COS7 cells were incubated for 30 min. Then, cells were washed with DMEM-10% FBS, plated in DMEM-10 for 4 hours, washed and incubated in serum-free DMEM for 3-5 days. The medium was collected, concentrated 6-fold by ultrafiltration (10 kilodalton cut off molecular weight) and on a Talon column (Clontech). Isolation of IL-18BP-His Using imidazole as eluent according to manufacturer's instructions₆。

The immunological cross-linking reactivity of urine and IL-18BP expressed in COS7 was evaluated as follows: will utilize¹²⁵I urinary IL-18BP (5. mu.g) was labeled by the chloramine T method. The supernatant of COS7 cells (250. mu.l) was mixed with an antibody to urinary IL-18BP (1 hour, room temperature, final volume 500. mu.l) and diluted 1: 1000 in Phosphate Buffered Saline (PBS), 0.05% Tween 20 and 0.5% bovine serum albumin (wash buffer). Then add into¹²⁵I-labelled urinary IL-18BP (10)⁶cpm), after 1 hour, protein G-agarose (20. mu.l) was added. The mixture was suspended (1.5 h, 4 ℃), then the beads were separated, washed in 3 × wash buffer, and washed again with PBS. The beads were then eluted with sample buffer and resolved by SDS-PAGE (10% acrylamide under reducing conditions followed by autoradiography).

IL-18BP ran as a single band under reducing and non-reducing conditions with silver staining and had the same apparent molecular weight as urinary IL-18BP (data not shown). Protein sequence analysis of this preparation revealed the same N-terminal sequence as urinary IL-18BP, indicating that the latter was not degraded at the N-terminus.

Analysis of IL-18BPa by immunoblotting with an antibody against urinary IL-18BP indicated the same molecular weight as urinary protein. In addition, IL-18BPa can replace urine using immunoprecipitation followed by SDS-PAGE and autoradiography¹²⁵I-IL-18BP binding antibodies. Therefore, IL-18BPa corresponds in structure to urinary IL-18 BP.

Crude and purified IL-18BPa were tested for their ability to inhibit the biological activity of IL-18. IL-18BPa inhibited IFN- γ in a dose-dependent manner in mouse splenocytes, PBMC and human KG-1 cell lines in order to induce human and mouse IL-18 activity (FIG. 9).

The results of the various biological tests and the migration shift assay (example 8) confirm that it is the cloned IL-18BP that inhibits IL-18 activity, and not any incidental impurities of urinary IL-18BP, such as the intrinsic properties of the co-eluted defensin fragment.

Example 8: electrophoretic migration shift test

The effect of urine and recombinant IL-18BP on IL-18-induced NF-. kappa.B activity in human KG-1 cells was also investigated. Stimulation of human KG-1 cells (4X 10) with huIL-18(10 ng/ml) or with huIL-18 premixed with IL-18BP⁶In 1 ml of RMPI) (20 min, room temperature). After 20 minutes at 37 ℃, the cells were washed three times with ice-cold PBS and immediately frozen in liquid nitrogen. Cell debris was resuspended in buffer A [20 mmol/l Tris pH7.6, 0.4 mol/l NaCl, 0.2 mmol/l EDTA, glycerol (20% by volume), 1.5 mmol/l MgCl₂2 mmole/l Dithiothreitol (DTT), 0.4 mmole/l PMSF, 1 mmole/l Na₃VO₄2. mu.g/ml of each of leupeptin, pepstatin, and aprotinin]In 3 times the compact cell volume. Cell debris was removed by centrifugation (15,000 Xg, 15 min). Aliquots of the supernatant were frozen in liquid nitrogen and stored at-80 ℃. Protein concentrations were determined by Bradford assay (Bio-Rad) using calf serum albumin as a standard. Use of³²P]dCTP (300 Curie/millimole) and T4 Polynucleotide kinase (New England laboratories) labeled double-stranded oligonucleotides corresponding to NF-. kappa.B binding elementsNucleotide (10 picomolar, pralmeger). Free nucleotides were removed by spin column. Extracts of cells treated with IL-18 or IL-18+ IL-18BP (10. mu.g protein) were mixed with labeled probes (3X 10)⁴cpm) and poly dl.dc (500 ng, Pharmacia) were incubated together (15 min, room temperature) and contained HEPES (ph7.5, 10 mmol/l), 60 mmol/l KCl, 1 mmol/l MgCl in 20 μ l₂Salmon sperm DNA (100 ng, Sigma) was denatured in a buffer of 2 mM EDTA, 1 mM DTT and glycerol (5% vol). The mixture was then loaded on a 5% native polyacrylamide gel. Electrophoresis was performed at 185V in 0.5 XTBE (40 mmol/l Tris HCl, 45 mmol/l boric acid, and 2.5 mmol/l EDTA). The gel was dried in vacuo and autoradiographed overnight at-80 ℃. IL-18 was found to induce the formation of p 50-NF-. kappa.B homodimers and p65/p50 NF-. kappa.B heterodimers. Urine as well as recombinant IL-18BP inhibited NF- κ B activation by IL-18 as determined by electrophoretic migration shift assays using KG-1 cell extracts conjugated with radiolabeled oligonucleotides corresponding to NF- κ B isozymes.

Example 9: expression of IL-18BP in E.coli, yeast and insect cells

IL-18BP may also be produced by other recombinant cells such as prokaryotic cells, e.g., E.coli, or other eukaryotic cells such as yeast and insect cells. Known methods for constructing appropriate vectors carrying DNA encoding IL-18BP and suitable for transforming E.coli and yeast cells or infecting insect cells so as to produce recombinant IL-18BP are available. For expression in yeast cells, the DNA encoding IL-18BP was excised (example 6) and inserted into an expression vector suitable for transfection into yeast cells. For expression in insect cells, DNA encoding IL-18BP is inserted into baculovirus, and insect cells are infected with the recombinant baculovirus. For expression in E.coli, DNA encoding IL-18BP was subjected to site-directed mutagenesis using appropriate oligonucleotides such that the initial ATG codon was inserted just before the first codon of mature IL-18 BP. Alternatively, such DNA can be prepared by PCR using appropriate sense and antisense primers. The resulting cDNA construct is then inserted into an appropriately constructed prokaryotic expression vector by techniques well known in the art (23).

Example 10: construction of an in vivo expression vector for the expression of IL-18BPa

A functional gene encoding IL-18BPa (InVitrogen, san Diego, Calif.) was constructed from plasmid pcDNA 3. An IL-18BP cDNA containing a Kozak homology sequence at the 5' end was ligated into the XbaI site of pcDNA3 in such a way as to destroy the restriction site. A new XbaI site was inserted by site-directed mutagenesis before the neomycin cassette (base 2151 of the original pcDNA3 sequence) and after the SV40 polyadenylation signal (domain base 3372 of the original pcDNA3 sequence). The construct was then cleaved with XbaI and the resulting 4.7kb minigene inserted at the XbaI site of plasmid psub201 as described above (Snyder et al, 1996, Current protocols in human genetics, Chapter 12.1.1-12.1.17, John Wiley and Sons). The resulting recombinant plasmids were co-transfected into human T293 cells using helper AAV plasmid pAAV/Ad. Then, the culture was infected with adenovirus as a helper virus, and the cells were collected after 48 to 60 hours of incubation. Cells were subjected to 3 freeze-thaw cycles, cell debris was removed by centrifugation, and the supernatant was saturated with ammonium sulfate 33%. The mixture was then centrifuged and rAAV was precipitated from the supernatant by saturating 50% with ammonium sulfate. The virus was further purified by CsCl and heated for a final 15 minutes at 56 ℃ to destroy any adenovirus.

Example 11: construction of recombinant fusion proteins of IL-18BP

Production of a protein containing IL-18BP fused to the constant region of the heavy chain of IgG2 can be performed as follows: the DNA of IL-18BP is subjected to site-directed mutagenesis using appropriate oligonucleotides so that unique restriction sites are introduced immediately before and after the coding sequence. Plasmids containing the constant region of the heavy chain of IgG2, such as pRKCO42FcI (6), were subjected to the same site-directed mutagenesis to introduce the same unique site as close as possible to the Asp 216 of the heavy chain of IgG1 in a manner that allows translation during the time period of fusion of the protein. The dsDNA fragment containing the 5' untranslated sequence and encoding IL-18BP can be prepared by digestion at unique restriction sites, or alternatively by PCR using appropriately designed primers. Similar digestion of mutant pRKCD42Fc1 produced large fragments containing plasmid and IgG1 sequences. The two fragments were then ligated to produce a new plasmid encoding a precursor polypeptide containing IL-18BP and approximately 227C-terminal amino acids of the IgG heavy chain (hinge region and CH2 and CH3 regions). The DNA encoding the fusion protein can be isolated from the plasmid by digestion with appropriate restriction enzymes and then inserted into an efficient prokaryotic or eukaryotic expression vector.

Example 12: production of chemically modified IL-18BP

To enhance the half-life of IL-18BP in the plasmid, IL-18BP chemically modified with polyethylene glycol (PEG) can be made. The modification can be performed by cross-linking PEG to cysteine residues of the IL-18BP molecule. Mutant IL-18BP may be constructed which contain an amino-terminal exocysteine residue, glycosylation site, and carboxyl-terminal end of IL-18 BP. Mutagenesis can be performed by PCR using oligonucleotides containing the desired mutation. These muteins are expressed in a conventional manner known in the art. Pegylation of these proteins will be performed and activity will be assessed.

Example 13: preparation of polyclonal antibody against IL-18BP

Subcutaneous injection of rabbits was initiated with 5 μ g of neat formulation of urinary IL-18BP emulsified in complete Freund's adjuvant. After 3 weeks they were re-injected subcutaneously with 5 μ g of the IL-18BP formulation in incomplete Freund's adjuvant. Two additional injections of IL-18BP as a solution in PBS were given at 10 day intervals. Rabbit blood was taken 10 days after the last immunization. Radioimmunoassay was performed after antibody levels had developed. Will be provided with¹²⁵I-labeled IL-18BP (166,000cpm) was mixed with various dilutions (1: 50, 1: 500, 1: 5,000 and 1: 50,000) of rabbit serum. A suspension of protein-G sepharose beads (20. mu.l, Pharmacia) was added to a total volume of 200. mu.l. The mixture was left at room temperature for 1 hour, then the beads were washed three times and the bound radioactivity was counted. Rabbit antiserum to human Ieptin was used as a negative control. The titer of the antiserum to IL-18R was between 1: 500 and 1: 5,000, whereas the titer of the negative control was less than 1: 50.

Example 14: preparation of monoclonal antibody to IL-18BP

Female Balb/C mice (3 months old) were first injected with 2. mu.g of purified IL-18BP in an emulsion of complete Freund's adjuvant and 3 weeks later, subcutaneously in incomplete Freund's adjuvant. Three additional injections were given subcutaneously in PBS at 10 day intervals. Mice showing the highest binding titers as determined by IRIA (see below) were given a final intraperitoneal boost 4 and 3 days prior to fusion. Fusions were performed using the NSO/1 melanoma cell line and lymphocytes prepared from the spleen and lymph nodes of animals as fusion participants. The fused cells were dispensed in a microculture plate and hybridomas were selected in DMEM supplemented with HAT and 15% horse serum. Hybridomas that were found to produce antibodies to IL-18BP were subcloned by limiting dilution and injected into Balb/C mice that had been induced to produce ascites using pristane. The isotype of the antibody was determined using a commercially available ELISA kit (Amersham, uk).

Screening of hybridomas producing monoclonal antibodies against IL-18BP was performed as follows: hybridoma supernatants were tested for the presence of anti-IL-18 BP antibodies by a reverse solid phase radioimmunoassay (IRIA). Purification of IL-18BPa-His using Talon-₆(10. mu.g/ml, 100. mu.l/well) coated ELISA plates (Dynatech laboratories, Alexandria, Va.). After overnight incubation at 4 ℃, the plates were washed twice with PBS containing BSA (0.5%) and tween 20 (0.05%) and blocked in the wash solution for at least 2 hours at 37 ℃. Hybridoma culture supernatant (100. mu.l/well) was added and the plates were incubated at 37 ℃ for 4 hours. The plates were washed three times and goat anti-mouse horseradish peroxidase conjugate (HRP, Jackson laboratory, 1: 10,000, 100. mu.l/well) was added at 2 hours at room temperature. The plate was washed four times with H as substrate₂O₂ABTS [2 ', 2' -azino-bis (3-ethylbenzothiazole-6-sulfonic acid, Sigma)]And (5) color spreading. By automatic ELIThe SA reader reads the plate. Samples with readings at least 5 times higher than the value of the negative control were considered positive.

Example 15: affinity chromatography of IL-18BP Using monoclonal antibodies

Antibodies against IL-18BP were used for the purification of IL-18BP by affinity chromatography. The ascites fluid containing the monoclonal antibody secreted by the hybridoma was purified by precipitation of ammonium sulfate at 50% saturation followed by extensive dialysis against PBS. As specified by the manufacturer, approximately 10 mg of immunoglobulin was bound to 1 ml of affinity gel 10(BioRad USA).

250 ml of human urine protein (equivalent to 250 l of crude urine) was loaded on a 0.5 ml anti-IL-18 BP antibody column at 4 ℃ at a flow rate of 0.25 ml/min. The column was washed with PBS until no protein was detected in the wash. IL-18BP was eluted using 25 mmol/l citrate buffer, pH2.2 (8X 1 column volume fraction) over 1 mol/l Na₂CO₃And (4) immediately neutralizing. Further purification of this preparation was obtained by size exclusion chromatography.

Example 16: ELISA test

Microtiter plates (Dynatech or Maxisorb, Nunc) were coated with anti-IL-18 BP monoclonal antibodies (serum-free hybridoma supernatant or ascites immunoglobulin) overnight at 4 ℃. The plates were washed with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked in the same solution at 37 ℃ for at least 2 hours. The test samples were diluted in blocking solution and added to the wells (100. mu.l/well) at 37 ℃ for 4 hours. The plates were then washed three times with PBS containing Tween 20 (0.05%), followed by addition of rabbit anti-IL-18 BP serum (1: 1,000, 100. mu.l/well) for further incubation at 4 ℃ overnight. The plates were washed three times and goat anti-rabbit horseradish peroxidase conjugate (HRP, Jackson laboratory, 1: 10,000, 100. mu.l/well) was added at 2 hours at room temperature. Wash the plate four times with H₂O₂ABTS [2 ', 2' -azino-bis (3-ethylbenzothiazole-6-sulfonic acid, Sigma) as substrate]And (5) color spreading. Plates were read by an automated ELISA reader.

Example 17: the non-glycosylated human IL-18BP is biologically active

Purified recombinant IL-18BPa was tested for its ability to inhibit the biological activity of IL-18. IL-18BPa inhibits IFN-gamma-induced human and mouse IL-18 activity in mouse splenocytes, PBMCs and human KG-1 cell line in a dose-dependent manner.

Will have His at the C-terminus₆The pH of the labeled purified IL-18BPa (1.5. mu.g, 50. mu.l) was adjusted to 7.5 and mixed with N-glycosidase F (3. mu.l, 500,000 units/ml, PNGase F, New England laboratories). The mixture was incubated at 37 ℃ for 24 hours under non-denaturing conditions. Analysis of samples from non-digested IL-18BP-His by SDS-PAGE under non-reducing conditions, followed by immunoblotting with antibodies to IL-18BP₆An aliquot of (a). Discovery of IL-18BP-His₆The band of about 40 kilodaltons disappears in the PNGase treated composition, and a new band of about 20 kilodaltons is obtained. The molecular weight and specificity of the product of PNGase F indicates IL-18BP-His₆And (3) complete glycosylation.

Separately, the PNGase-treated fraction in buffer, undigested IL-18BP-His, was absorbed onto Talon beads₆And a control sample containing PNGase, washed with phosphate buffer and eluted with imidazole (100 mmol/l). The eluted fractions were subjected to bioassay using human IL-18(20 ng/ml), LPS (2. mu.g/ml) and mouse splenocytes. The results are shown in the following table:

sample (I)	IFN-gamma ng/ml (nanogram/ml)
		Control	7.5
Non-digestible IL-18BP-His6	0
		PNGase-treated IL-18BP-His6	0

Therefore, it was concluded that glycosylated IL-18BP is biologically active as a modulator of IL-18 activity.

The foregoing description of the specific embodiments reveals the general nature of the invention so that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the present embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

Reference to the literature

Anderson, d.m. et al, homologs of the TNF receptor and its ligands enhance T cell growth and dendritic cell function, nature, 1997.390 (6656): pages 175-179.

Bollon, D.P. et al (1980) J.Clinical hematological carcinostics, 10: 39 to 48.

Botstein, d, et al, (1982) Miami wing.symp.19: 265 to 274.

Brooch, j.r., "molecular biology of brewer's yeast: life cycle and inheritance "Kord Springs laboratory, Kord Springs, New York, 445-470 (1981).

Broach, J.R. (1982) cell 28: 203 to 204.

Byrn r.a. et al, 1990, nature (london) 344: 667 ~ 670.

Car, b.d., v.m.eng, b.schnyder, l.ozmen, s.huang, p.gallay, d.heumann, m.aguet, and b.ryffel.1994. interferon gamma receptor deficient mice are resistant to endotoxin shock, journal of experimental methods, 179: 1437-44 issn: 0022-1007.

Chater, K.F. et al, in "International seminar on the sixth actinomycete biology", Akadeniai Kaido, Budapest, Hungary (1986), pp.45-54.

9.Conti, b, j.w.jahng.c.tinti, j.h.son, and t.h.joh.1997, induction of interferon gamma-inducing factors in the adrenal cortex, journal of biochemistry, 272: 2035 to 2037.

Dao, t.k.ohashi, t.kayano, m.kurimoto, and h.okamura.1996, interferon gamma inducible factor, novel cytokine, enhance Fas ligand-mediated cytotoxicity of mouse T helper 1 cells. Cellular immunology, 173: 230-5 issn: 0008 to 8749.

Engelmann, h., d.aderka, m.rubinstein, d.rotman, and d.wallach.1989. purification of tumor necrosis factor binding proteins from human urine to homogeneity protects cells from tumor necrosis factor toxicity, journal of biochemistry, 264: 11974 to 11980.

Engelmann, h., d.novick, and d.wallach.1990, two tumor necrosis factor-binding proteins purified from human urine. Evidence of immunological cross-linking reactivity with cell surface tumor necrosis factor receptors, journal of biochemistry, 265: 1531 to 1536.

Fantuzzi, G, et al, as demonstrated in mice deficient in interleukin 1b convertase, IL-18 regulates IFN-g production and cell proliferation. Blood, 1998, 91: 2118 to 2125.

Gryczan, T., "academic Press of Bacillus molecular biology," group York, (1982), 307-329.

Gutkind, J.S. et al, a novel c-fgr exon for Epstein-Barr virus infected B lymphocytes but not normal monocytes. Molecular cell biology, 1991, 11: 1500-1507.

Hermans, h., j.van Damme, c.dillen, r.dijkmans, and a.billiau.1990. interferon γ, mediators of lethal lipopolysaccharide-induced Shwartzman-like shock response in mice, journal of experimental methods, 171: 1853 to 69 issn: 0022 to 1007.

Izaki, k. (1978) journal of japanese bacteriology, 33: 729 to 742.

Review of infectious diseases by John, j.f. et al (1986), 8: 693 to 704.

Kendall, k.j. et al (1987), journal of bacteriology 169: 4177-4183.

20, Kohno, k., j.kataoka, t.ohtsuki, y.suemoto, i.okamoto, m.usui, m.ikeda, and m.kurimoto.1997 IFN- γ -inducible factor (IGIF) is a costimulatory factor in the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12, journal of immunology, 158: 1541 to 1550.

Maliszewski, c.r., t.a.sato, t.vanden Bos, s.waugh, s.k.dower, j.slack, m.p.beckmann, and k.h.grabstein.1990. cytokine receptors and B-cell function I recombinant soluble receptors specifically inhibit IL-1 and IL-4 induced B-cell activity in vitro, journal of immunology, 144: 3028 to 3033.

Maniotis, t., in "cell biology: general paper, Vol.3, Gene expression ", academic Press, New York, 563-608 (1980).

Maniatis et al, molecular cloning: a laboratory Manual, Cole de Spulin harbor laboratory, group, 1982.

Micallef, m.j., t.ohtsuki, k.kohno.f.tanabe, s.ushio, m.namba, t.tanimoto.k.torigoe, m.fujii, m.ikeda, s.fukuda, and m.kurimoto.1996. interferon gamma-induced factors enhance production of T helper 1 cytokines by stimulating human T cells: acts synergistically with interferon 12 for interferon gamma production. Current journal of immunology, 26: 1647-51 issn: 0014 to 2980.

Mizushima, S. and Nagata, S. (1990), pEF-BOS, robust mammalian expression vectors, nucleic acid reviews, 18: 5322 to 5328.

Purification of factors that provide co-stimulatory signals for gamma interferon production, infection of immunology 61: 64 to 70 issn: 0019 to 9567.

Nakamura, k., h.okamura, m.wada, k.nagata, and t.tamura.1989. endotoxin-induced serum factors stimulating gamma interferon production, infection immunology, 57: 590-5 issn: 0019 to 9567.

Novick, d., b.cohen and m.rubinstein.1994. human interferon alpha/beta receptor-identification and molecular cloning, cell 77: 391 to 400.

Novick, d.d., b.cohen, and m.rubinstein.1992, the presence of soluble interferon alpha receptor molecules in body fluids, FEBS communication, 314: 445 to 448.

Novick, d.h. engelmann, d.wallach, and m.rubinstein.1989. presence of soluble cytokine receptors in human normal urine, journal of experimental methods, 170: 1409 to 1414.

Okamura, h., h.tsutsutsutsu, t.komatsu, m.yutsudo, a.hakura, t.tanimoto, k.torigoe, t.okura, y.nukada, k.hattori, k.akita, m.namba, f.tanabe, k.konishi, s.fukuuda, and m.kurimoto.1995, cloning of new cytokines induced by T cells to IFN- γ production, nature 378: 88 to 91.

The active stages of Rothe, h.n.a. jenkins, n.g. copeland and h.kolb.1997. autoimmune diabetes are associated with the expression of a novel cytokine, IGIF, localized adjacent to Idd 2. Journal of clinical research 99: 469 to 74 issn: 0021 to 9738.

Molecular cloning, Sambrook, j., e.f. fritsch, and m.t., molecular cloning: a laboratory Manual, second edition, 1989, Keldswan harbor, N.Y., Keldswan harbor laboratory.

Simonet.w.s., et al, osteoprogenin: a novel secreted protein involved in the regulation of bone density. Cell, 1997, 89 (2): 309 to 319.

Sompayrac, l.h. and k.l.danna, efficient infection of monkey cells with the DNA of monkey virus 40, proceedings of the american college of sciences, 1981, 78: 7575 to 7578.

Sparks, c.a., et al, assigned the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q 13. Genome, 1993, 17: 222 to 224.

Tsutsui, H., K.Nakanishi, K.Matsui, K.Higashino.H.Okamura, Y.Miyazawa, and K.Kaneda.1996.IFN- γ inducing factor up-regulates Fas ligand-mediated cytotoxic activity of mice that naturally kills cell clones. Journal of immunology, 157: 3967-73 issn: 0022 to 1767.

The cDNA clones of human IFN- γ inducible factor, expression in e.coli, studies on the biological activity of proteins, 38.Ushio, s.m.namba, t.okura, k.hattori, y.nukada, k.akita, f.tanabe, k.konishi, m.micellef, m.fujii.k.torigoe, t.tanimoto, s.fukukuda, m.ikeda, h.okamura, and m.kurimoto.1996. Journal of immunology, 156: 4274-4279.34, Okayama, H., and Berg, P. (1983) cDNA cloning vectors that allow expression of cDNA inserts in mammals. Molecular cell biology, 3: 280 to 289.

Characteristics of osteoclastogenesis inhibitory factor (OCIF) and Osteoprogenitor (OPG) in Yasuda, H.et al: the mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro, endocrinology, 1998, 139: pages 1329 to 1337.

Sequence listing

<110>Novick，Daniela

Dinarello，Charles

Rubinstein，Menachem

Kim，Soo Hyun

Yeda research and development Co., Ltd

<120> interleukin-18 binding protein, preparation and use thereof

Use

<130>IL-18 Rubinstein

<140>

<141>

<150>125463

<151>1998-07-22

<150>122134

<151>1997-11-06

<150>121869

<151>1997-09-29

<150>121639

<151>1997-08-27

<150>121554

<151>1997-08-14

<160>10

<170>PatentIn Ver.2.0

<210>1

<211>1348

<212>DNA

<213>Homo sapiens

<400>1

gagaagagga cgttgtcaca gataaagagc caggctcacc agctcctgac gcatgcatca 60

tgaccatgag acacaactgg acaccagacc tcagcccttt gtgggtcctg ctcctgtgtg 120

cccacgtcgt cactctcctg gtcagagcca cacctgtctc gcagaccacc acagctgcca 180

ctgcctcagt tagaagcaca aaggacccct gcccctccca gcccccagtg ttcccagcag 240

ctaagcagtg tccagcattg gaagtgacct ggccagaggt ggaagtgcca ctgaatggaa 300

cgctgagctt atcctgtgtg gcctgcagcc gcttccccaa cttcagcatc ctctactggc 360

tgggcaatgg ttccttcatt gagcacctcc caggccgact gtgggagggg agcaccagcc 420

gggaacgtgg gagcacaggt acgcagctgt gcaaggcctt ggtgctggag cagctgaccc 480

ctgccctgca cagcaccaac ttctcctgtg tgctcgtgga ccctgaacag gttgtccagc 540

gtcacgtcgt cctggcccag ctctgggctg ggctgagggc aaccttgccc cccacccaag 600

aagccctgcc ctccagccac agcagtccac agcagcaggg ttaagactca gcacagggcc 660

agcagcagca caaccttgac cagagcttgg gtcctacctg tctacctgga gtgaacagtc 720

cctgactgcc tgtaggctgc gtggatgcgc aacacacccc ctccttctct gctttgggtc 780

ccttctctca ccaaattcaa actccattcc cacctaccta gaaaatcaca gcctccttat 840

aatgcctcct cctcctgcca ttctctctcc acctatccat tagccttcct aacgtcctac 900

tcctcacact gctctactgc tcagaaacca ccaagactgt tgatgcctta gccttgcact 960

ccagggccct acctgcattt cccacatgac tttctggaag cctcccaact attcttgctt 1020

ttcccagaca gctcccactc ccatgtctct gctcatttag tcccgtcttc ctcaccgccc 1080

cagcagggga acgctcaagc ctggttgaaa tgctgcctct tcagtgaagt catcctcttt 1140

cagctctggc cgcattctgc agacttccta tcttcgtgct gtatgttttt tttttccccc 1200

ttcactctaa tggactgttc cagggaaggg atgggggcac cagctgcttc ggatccacac 1260

tgtatctgtg tcatccccac atgggtcctc ataaaggatt attcaatgga aaaaaaaaaa 1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1348

<210>2

<211>192

<212>PRT

<213>Homo sapiens

<220>

<221> Signal

<222>(1)..(28)

<400>2

Met Arg His Asn Trp Thr Pro Asp Leu Ser Pro Leu Trp Val Leu Leu

1 5 10 15

Leu Cys Ala His Val Val Thr Leu Leu Val Arg Ala Thr Pro Val Ser

20 25 30

Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr Lys Asp Pro

35 40 45

Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys Gln Cys Pro Ala

50 55 60

Leu Glu Val Thr Trp Pro Glu Val Glu Val Pro Leu Asn Gly Thr Leu

65 70 75 80

Ser Leu Ser Cys Val Ala Cys Ser Arg Phe Pro Asn Phe Ser Ile Leu

85 90 95

Tyr Trp Leu Gly Asn Gly Ser Phe Ile Glu His Leu Pro Gly Arg Leu

100 105 110

Trp Glu Gly Ser Thr Ser Arg Glu Arg Gly Ser Thr Gly Thr Gln Leu

115 120 125

Cys Lys Ala Leu Val Leu Glu Gln Leu Thr Pro Ala Leu His Ser Thr

130 135 140

Asn Phe Ser Cys Val Leu Val Asp Pro Glu Gln Val Val Gln Arg His

145 150 155 160

Val Val Leu Ala Gln Leu Trp Ala Gly Leu Arg Ala Thr Leu Pro Pro

165 170 175

Thr Gln Glu Ala Leu Pro Ser Ser His Ser Ser Pro Gln Gln Gln Gly

180 185 190

<210>3

<211>1038

<212>DNA

<213>Homo sapiens

<400>3

gagaagagga cgttgtcaca gataaagagc caggctcacc agctcctgac gcatgcatca 60

tgaccatgag acacaactgg acaccagacc tcagcccttt gtgggtcctg ctcctgtgtg 120

cccacgtcgt cactctcctg gtcagagcca cacctgtctc gcagaccacc acagctgcca 180

ctgcctcagt tagaagcaca aaggacccct gcccctccca gcccccagtg ttcccagcag 240

ctaagcagtg tccagcattg gaagtgacct ggccagaggt ggaagtgcca ctgagctggg 300

ctgagggcaa ccttgccccc cacccaagaa gccctgccct ccagccacag cagtccacag 360

cagcagggtt aagactcagc acagggccag cagcagcaca accttgacca gagcttgggt 420

cctacctgtc tacctggagt gaacagtccc tgactgcctg taggctgcgt ggatgcgcaa 480

cacaccccct ccttctctgc tttgggtccc ttctctcacc aaattcaaac tccattccca 540

cctacctaga aaatcacagc ctccttataa tgcctcctcc tcctgccatt ctctctccac 600

ctatccatta gccttcctaa cgtcctactc ctcacactgc tctactgctc agaaaccacc 660

aagactgttg atgccttagc cttgcactcc agggccctac ctgcatttcc cacatgactt 720

tctggaagcc tcccaactat tcttgctttt cccagacagc tcccactccc atgtctctgc 780

tcatttagtc ccgtcttcct caccgcccca gcaggggaac gctcaagcct ggttgaaatg 840

ctgcctcttc agtgaagtca tcctctttca gctctggccg cattctgcag acttcctatc 900

ttcgtgctgt atgttttttt tttccccctt cactctaatg gactgttcca gggaagggat 960

gggggcacca gctgcttcgg atccacactg tatctgtgtc atccccacat gggtcctcat 1020

aaaggattat tcaatgga 1038

<210>4

<211>113

<212>PRT

<213>Homo sapiens

<220>

<221> Signal

<222>(1)..(28)

<400>4

Met Arg His Asn Trp Thr Pro Asp Leu Ser Pro Leu Trp Val Leu Leu

1 5 10 15

Leu Cys Ala His Val Val Thr Leu Leu Val Arg Ala Thr Pro Val Ser

20 25 30

Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr Lys Asp Pro

35 40 45

Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys Gln Cys Pro Ala

50 55 60

Leu Glu Val Thr Trp Pro Glu Val Glu Val Pro Leu Ser Trp Ala Glu

65 70 75 80

Gly Asn Leu Ala Pro His Pro Arg Ser Pro Ala Leu Gln Pro Gln Gln

85 90 95

Ser Thr Ala Ala Gly Leu Arg Leu Ser Thr Gly Pro Ala Ala Ala Gln

100 105 110

Pro

<210>5

<211>7063

<212>DNA

<213>Homo sapiens

<400>5

gaattcgcgg ccgcgtcgac gccagagggg ctaggatgag agacagaggg tgtgatggtg 60

ggtgctggga aatgtacccg accttggggc tggtggctgg gggagtgggt agcctgggaa 120

aggccaggat gtggacggac tggtatggca ttgagcctga agtggtccaa cttggggttc 180

cccagtgcct aggaaagttg tccccttgaa tgtcagtgtg aaggtgaagg aggaagcaga 240

tgcctgttca tatggaaaca aagacctggc tgtgaagagg ggaggcggac accaaagtcc 300

tgacacttgg gcgggacaga attgatctgt gagagactca tctagttcat accctaggtg 360

accctggggg tggcatgggg gtagattaga gatcccagtc tggtatcctc tggagagtag 420

gagtcccagg agctgaaggt ttctggccac tgaactttgg ctaaagcaga ggtgtcacag 480

ctgctcaaga ttccctggtt aaaaagtgaa agtgaaatag agggtcgggg cagtgctttc 540

ccagaaggat tgctcggcat cctgcccttc ccagaagcag ctctggtgct gaagagagca 600

ctgcctccct gtgtgactgg gtgagtccat attctctctt tgggtctcaa ttttgccttc 660

cctaatgaag gggtaagatt ggactaggta agcatcttac aaccatttgt ggtcatgaga 720

gctggggtgg ggaaggattg tcacttgacc cccccagctc tgtttctaag tgctgaaaga 780

gctccaggct atgctacggg aggagaagcc agctactgag gaaaagccag ctactgagaa 840

aaagcgggag tggtttacca ttctcctccc ccacctttca ccagagaaga ggacgttgtc 900

acacataaag agccaggctc accagctcct gacgcatgca tcatgaccat gagacacaac 960

tggacaccag acctcagccc tttgtgggtc ctgctcctgt gtgcccacgt cgtcactctc 1020

ctggtcagag ccacacctgt ctcgcagacc accacagctg ccactgcctc agttagaagc 1080

acaaaggacc cctgcccctc ccagccccca gtgttcccag cagctaagca gtgtccagca 1140

ttggaagtga cctggccaga ggtggaagtg ccactgaatg gaacgctgag cttatcctgt 1200

gtggcctgca gccgcttccc caacttcagc atcctctact ggctgggcaa tggttccttc 1260

attgagcacc tcccaggccg actgtgggag gggagcacca gccgggaacg tgggagcaca 1320

ggtacgcagc tgtgcaaggc cttggtgctg gagcagctga cccctgccct gcacagcacc 1380

aacttctcct gtgtgctcgt ggaccctgaa caggttgtcc agcgtcacgt cgtcctggcc 1440

cagctctggg tgaggagccc aaggagaggc ctccaggaac aggaggagct ctgcttccat 1500

atgtggggag gaaagggtgg gctctgccag agcagcctgt gaactaatgc ccagcattcc 1560

tcaaggtcag ccagacaaaa aggaacttag gtcttgggca gaggaggtgt agcctggggc 1620

aaagtgatga gatgtccctc ctttccttgg cctgatcctt gtctgccttc acttccctag 1680

gctgggctga gggcaacctt gccccccacc caagaagccc tgccctccag ccacagcagt 1740

ccacagcagc agggttaaga ctcagcacag ggccagcagc agcacaacct tgaccagagc 1800

ttgggtccta cctgtctacc tggagtgaac agtccctgac tgcctgtagg ctgcgtggat 1860

gcgcaacaca ccccctcctt ctctgctttg ggtcccttct ctcaccaaat tcaaactcca 1920

ttcccaccta cctagaaaat cacagcctcc ttataatgcc tcctcctcct gccattctct 1980

ctccacctat ccattagcct tcctaacgtc ctactcctca cactgctcta ctgctcagaa 2040

accaccaaga ctgttgatgc cttagccttg cactccaggg ccctacctgc atttcccaca 2100

tgactttctg gaagcctccc aactattctt gcttttccca gacagctccc actcccatgt 2160

ctctgctcat ttagtcccgt cttcctcacc gccccagcag gggaacgctc aagcctggtt 2220

gaaatgctgc ctcttcagtg aagtcatcct ctttcagctc tggccgcatt ctgcagactt 2280

cctatcttcg tgctgtatgt tttttttttc ccccttcact ctaatggact gttccaggga 2340

agggatgggg gcagcagctg cttcggatcc acactgtatc tgtgtcatcc ccacatgggt 2400

cctcataaag gattattcaa tggaggcatc ctgacatctg ttcatttagg cttcagttcc 2460

actcccagga actttgcctg tcccacgagg gagtatggga gagatggact gccacacaga 2520

agctgaagac aacacctgct tcaggggaac acaggcgctt gaaaaagaaa agagagaaca 2580

gcccataatg ctccccggga gcagaggcca ctaatggaga gtgggaagag cctggaaaga 2640

tgtggcctca ggaaaaggga tgagagaaag gaggtggtat ggaagactca gcaggaacaa 2700

ggtaggcttc aaagagccta tattcctctt tttcccacac cgatcaagtc aactcagtac 2760

tcacgggaga aaaatagact ttatttacaa gtaataacat ttagaaaaga tccatccccg 2820

gcccttaaaa accttcccat cactccaaat cccaccccag tgcaagtctg gggaaggtag 2880

ggtgtgagct gctgctgaag gctgtccccc aaccccactc ctgagacaca gggcccatcc 2940

gtcctgggaa agagcatcct ctggcaggtg ctcccaccag gtcagaccca gtcctggact 3000

tcaagagtga gggcccctgc tgggcccagc caccaggaca gcaggaacca gggcctactc 3060

ctcttatggt cccttctaga tccagaggct aagaggaaga ctggccaggc ccaaggaccc 3120

agccatcaaa accagcctca aatctggttg tgatggagaa gtgactttgc tttaagaaaa 3180

aaggaggcaa ggtagggaga gcgcccacac tgtccatgct ccaggccccc tgggccagct 3240

ccgagaaggc gccagtgaag gaccagggac caggccaggg tgcgggcagg catcactgtc 3300

tctaggggtt tggctactgt tggcctggga gctgagagaa ggcactgaga gggacagtag 3360

gcggaggacc aggtgacggc agcatcgggg acacaggtgg ggccactcac tggtactggc 3420

cctttagtgc tttgcctgaa agagacacag tcacatggcc agatgagaac ttgcgatact 3480

agcctgcacc cactggctgg gaagatctct tcctgctccc acgcccctgt ctggatcccc 3540

tcccttgtga gccccagggt tatcagttgc tggctgtgcc tgagcagctc tgggtgctct 3600

ccatgagaat ggggccatct gtcttctctc cttggagagg agctaccagg acagggacac 3660

ctcttacccc acaccctcca gcagcctggc gtggccccat cttggatgct acttggtggg 3720

gcggtctggg gggtgcccat gctctcatcg ggtttccctc ccccatcctg ccagtgcctc 3780

taccttgccc ttggctcgag gggtggcacc aatggcggca gcagtggcgg cgctggctgt 3840

ggtggtggca atgcgcggag aacggcgggt tccactgcga gtgttggggg aagccttgga 3900

cagggccttc tttgaggctc cccgccgcag aaggctgttc cctagcttct tgggtgtgtt 3960

gaggatgctg aaggccatcg actggcgccg gtcagcctgc aaggaagggc tgtcagaccg 4020

ggagacccaa tgctgccttc ccaggccagc gtgctgtgcc acgctgtacc agcaaggtcc 4080

cgccagggcg tcgcttcatc ccccttcagc cccagcctca cctgtttagt agaagctgga 4140

gctgctttct tctgggcctc agtagtgctc tgtttgcgcc cttcatgtcg gtctcgggga 4200

gtcatggggc gtgggaaaca gctggtggcc ttcttagact atggagaaga ggacagttag 4260

gcagacagta gcaagaggag tcacatctga agccaggtgt cttgtcctct cagagctgag 4320

tggaccttgt aagtcaacgt gcaacctgct ccccttccca actctgggcc agatccttcc 4380

cttcccaaca gttcccatcc atgggtcagg cccttggaga gagggaaaga gagggggaag 4440

tgagggaagg agagagaagg ctccctttag tccttggtga gctgggcctg acctgagcac 4500

agtgctggag taacacccag gagccaccgc gcctacctca ggagttccag ggccctggtg 4560

gggctctagg gagacccgtt tgcgctgctg ccgggtggtg atgccagtgc cctcggctat 4620

ctggattggc tgcatgctgg ctcggcgcag ggtctcttgg gggtctccag ttttcatctc 4680

ctcatctgtg atggtgccca ggctcaggga aggctgcatg ggtggaagag gtggtcagtg 4740

gaccatagct gtatggagat ggaggaggac ctggggctgt tccagaactc tacactcgcc 4800

cgacacttat ggtcgggacc cttcctgcct acgaggtaga aagacacaag cctcctttcc 4860

tgttctgctt tctacctaag ccctgggcaa atggcacaag cagtgcagtc ctgaccagat 4920

tcctctctga gctcctgcct acccccaggg acttcacccc tgagtgccct ccagctgtct 4980

gttccacctg gaacatgaga aggtcacccc ttcccctctt cggccagtca gtgatccagg 5040

gccctagtgc tcaggctaga tcagcaggtg ggattccaag gaagggcagg gatgggaggc 5100

cctgcacagt gaccccaggc ctcaccctgg actccaggga tagcaggtct tcagatgtgg 5160

ggggcacact cgattgcgct gctgcagctc tgcaatgcgg ttccagtcat ccagctgctc 5220

aggctcatcc tggcaagtgc ccatgtagaa gctgttcctt cctgtggaag gcagggaagt 5280

gggaacaaat gagcctggag tcggcaggtc acctcctggc cctggcatct tgccagcctt 5340

tgctgccacc taccccataa acttgaagcc cggcacacca gtctgattca gtgccgcagg 5400

tgcaggagta cggcacacag actatttcta tcctaggggc ttgctcacca ccttctccct 5460

ggagagggca gaagaggtca cacgcagaga ctgctactac atcttattca cctgccaagg 5520

cttggtggcc aacacccaga ggaacaaatt aaggaccggg aattaattcc caggggctcc 5580

ctggtgccca aaggacaaga gcttccaaga agagtctggc cagcctggcc tttccagcag 5640

cccatcaccg cctgagaagg gcatggagga ctccccacag ctaagtgtca caattgtgct 5700

gggaatcccg ggcccttaac tctggctaag agtgccccca acacagccag cccctagatg 5760

ggcaggtaag gaaggccctg aggctgcagg aaggaggggc aggtggagct ggatggtagc 5820

aaggaggcca gccttggatt tttaaaaagc tttcctcttt tccctgtgcc acgatccacc 5880

ttccagtcta attttggggt atagtaagtc cctgtagtcc cctcacctgg aggggcccca 5940

ctggacaccc cggcctggga acgacgagca gaactgcgag tggtggggcg gtagccaggc 6000

aagctgagca gggctgagtt gccataatcg ggagaaccca ggcgagctag agactgagta 6060

gaggaggtgg ctcgcaggct agcctgggaa gcaggagcag accgcgtgct gtagaacgat 6120

gagttggcgc tgtctggctc ttccacatct agcttctgga agacagagtg aatctgttgc 6180

agtgtacagt ccctggcact gtacagaagc ttcccattcc cttccgaagc cctcagatcc 6240

cacggcacat ccatgtattc ccaactgctt tgcaaaggtc cttaaagtgt gtgtctgcaa 6300

gaaatgggcc ttgtcgacag aagccctcac aaggtggtgc tgatgttgtc aagactcttc 6360

tacgcatttt tttcatggag tctattcata atgctttgag gtagggaatg cagagtgttt 6420

atcggcccat tttggagatg aagtgcaaag aaataaagtg actagcccca aatcacactg 6480

ctaggaagta tcagagctgg ggctaggccc catgtctcct gactagtcag gctcatccca 6540

cagcctctgc tgtccctcag tccaaacttc cagggccctt accatgttcc agaacttccc 6600

ccaacttctt ggtagcaggg ggcaccctaa acacacaggt cccccctgct gtaccagggg 6660

ccccctctcc cctcctccca aacctcccct tcaagatgtg gaaacaaagg caagggcctg 6720

cagcctgtca ggcagtccac tgggcagcaa caatgcctct cagctgcatg gggcatgctg 6780

ggaggcacag gatgggctgc agcttcgcca cgttctctcc cttcaccctg cacaggctca 6840

gtgctacgca tggagagaat gctagcctta gtcaggaggc agggatctaa tcctagccct 6900

gcctttttct tcagaagtgc ccttaaccaa gtcactgccc tttttaagac ctctcagctt 6960

tcccactgta acatggactg gctgctcatc cctccctgct cctgactgag tgcccagtgc 7020

aaagatgccc ttgagaggaa gtgggaattg ctgacctgtc gac 7063

<210>6

<211>197

<212>PRT

<213>Homo sapiens

<220>

<221> Signal

<222>(1)..(28)

<400>6

Met Arg His Asn Trp Thr Pro Asp Leu Ser Pro Leu Trp Val Leu Leu

1 5 10 15

Leu Cys Ala His Val Val Thr Leu Leu Val Arg Ala Thr Pro Val Ser

20 25 30

Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr Lys Asp Pro

35 40 45

Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys Gln Cys Pro Ala

50 55 60

Leu Glu Val Thr Trp Pro Glu Val Glu Val Pro Leu Asn Gly Thr Leu

65 70 75 80

Ser Leu Ser Cys Val Ala Cys Ser Arg Phe Pro Asn Phe Ser Ile Leu

85 90 95

Tyr Trp Leu Gly Asn Gly Ser Phe Ile Glu His Leu Pro Gly Arg Leu

100 105 110

Trp Glu Gly Ser Thr Ser Arg Glu Arg Gly Ser Thr Gly Thr Gln Leu

115 120 125

Cys Lys Ala Leu Val Leu Glu Gln Leu Thr Pro Ala Leu His Ser Thr

130 135 140

Asn Phe Ser Cys Val Leu Val Asp Pro Glu Gln Val Val Gln Arg His

145 150 155 160

Val Val Leu Ala Gln Leu Trp Val Arg Ser Pro Arg Arg Gly Leu Gln

165 170 175

Glu Gln Glu Glu Leu Cys Phe His Met Trp Gly Gly Lys Gly Gly Leu

180 185 190

Cys Gln Ser Ser Leu

195

<210>7

<211>1360

<212>DNA

<2l3>Homo sapiens

<400>7

gcggccgcgt cgaccacgca gctaaacaca gctaacttga gtcttggagc tcctaaaggg 60

aagcttctgg aaaggaaggc tcttcaggac ctcttaggag ccaaagaaga ggacgttgtc 120

acagataaag agccaggctc accagctcct gacgcatgca tcatgaccat gagacacaac 180

tggacaccag acctcagccc tttgtgggtc ctgctcctgt gtgcccacgt cgtcactctc 240

ctggtcagag ccacacctgt ctcgcagacc accacagctg ccactgcctc agttagaagc 300

acaaaggacc cctgcccctc ccagccccca gtgttcccag cagctaagca gtgtccagca 360

ttggaagtga cctggccaga ggtggaagtg ccactgaatg gaacgctgag cttatcctgt 420

gtggcctgca gccgcttccc caacttcagc atcctctact ggctgggcaa tggttccttc 480

attgagcacc tcccaggccg actgtgggag gggagcacca gccgggaacg tgggagcaca 540

ggctgggctg agggcaacct tgccccccac ccaagaagcc ctgccctcca gccacagcag 600

tccacagcag cagggttaag actcagcaca gggccagcag cagcacaacc ttgaccagag 660

cttgggtcct acctgtctac ctggagtgaa cagtccctga ctgcctgtag gctgcgtgga 720

tgcgcaacac accccctcct tctctgcttt gggtcccttc tctcaccaaa ttcaaactcc 780

attcccacct acctagaaaa tcacagcctc cttataatgc ctcctcctcc tgccattctc 840

tctccaccta tccattagcc ttcctaacgt cctactcctc acactgctct actgctcaga 900

aaccaccaag actgttgatg ccttagcctt gcactccagg gccctacctg catttcccac 960

atgactttct ggaagcctcc caactattct tgcttttccc agacagctcc cactcccatg 1020

tctctgctca tttagtcccg tcttcctcac cgccccagca ggggaacgct caagcctggt 1080

tgaaatgctg cctcttcagt gaagtcatcc tctttcagct ctggccgcat tctgcagact 1140

tcctatcttc gtgctgtatg tttttttttt cccccttcac tctaatggac tgttccaggg 1200

aagggatggg ggcagcagct gcttcggatc cacactgtat ctgtgtcatc cccacatggg 1260

tcctcataaa ggattattca atggaggcat cctgacatct gtccatttag gcttcagttc 1320

cactcccagg aactttgcct gtcccacgag ggagtatggg 1360

<210>8

<211>161

<212>PRT

<213>Homo sapiens

<220>

<221> Signal

<222>(1)..(28)

<400>8

Met Arg His Asn Trp Thr Pro Asp Leu Ser Pro Leu Trp Val Leu Leu

1 5 10 15

Leu Cys Ala His Val Val Thr Leu Leu Val Arg Ala Thr Pro Val Ser

20 25 30

Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr Lys Asp Pro

35 40 45

Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys Gln Cys Pro Ala

50 55 60

Leu Glu Val Thr Trp Pro Glu Val Glu Val Pro Leu Asn Gly Thr Leu

65 70 75 80

Ser Leu Ser Cys Val Ala Cys Ser Arg Phe Pro Asn Phe Ser Ile Leu

85 90 95

Tyr Trp Leu Gly Asn Gly Ser Phe Ile Glu His Leu Pro Gly Arg Leu

100 105 110

Trp Glu Gly Ser Thr Ser Arg Glu Arg Gly Ser Thr Gly Trp Ala Glu

115 l20 125

Gly Asn Leu Ala pro His pro Arg Ser Pro Ala Leu Gln Pro Gln Gln

130 135 140

Ser Thr Ala Ala Gly Leu Arg Leu Ser Thr Gly Pro Ala Ala Ala Gln

145 150 155 160

Pro

<210>9

<211>7812

<212>DNA

<213>Homo sapiens

<400>9

gtcgacggta cccccgggaa agatttaata cgactcacta tagggcggga cagaattgat 60

ctgtgagaga ctcatctagt tcatacccta ggtgaccctg ggggtggcat gggggtagat 120

tagagatccc agtctggtat cctctggaga gtaggagtcc caggagctga aggtttctgg 180

ccactgaact ttggctaaag cagaggtgtc acagctgctc aagattccct ggttaaaaag 240

tgaaagtgaa atagagggtc ggggcagtgc tttcccagaa ggattgctcg gcatcctgcc 300

cttcccagaa gcagctctgg tgctgaagag agcactgcct ccctgtgtga ctgggtgagt 360

ccatattctc tctttgggtc tcaattttgc cttccctaat gaaggggtaa gattggacta 420

ggtaagcatc ttacaaccat ttgtggtcat gagagctggg gtggggaagg attgtcactt 480

gaccccccca gctctgtttc taagtgctga aagagctcca ggctatgcta cgggaggaga 540

agccagctac tgaggaaaag ccagctactg agaaaaagcg ggagtggttt accattctcc 600

tcccccacct ttcaccagag aagaggacgt tgtcacacat aaagagccag gctcaccagc 660

tcctgacgca tgcatcatga ccatgagaca caactggaca ccaggtaggc cttggggcta 720

cgcatgggca ggcggggtag ggtgaggtct atgaacagaa tggagcaatg ggctaacccg 780

gagccttcac tccaaggcaa accacccagc gcacctggtg ctgttgcttt aagaacctgg 840

gcagatattg tagctctggc tccagtctaa agcttctctg tactctgttc aataaagggc 900

taaggggtgg gtgctgaggg gtccctcttc ccgctctgat tccctggcta gaacccagac 960

atctctgggc tggagttaca tccttacccg ggcagcccac tctgtctcca gagccgctga 1020

cctgtaactg tcctttcctc agacctcagc cctttgtggg tcctgctcct gtgtgcccac 1080

gtcgtcactc tcctggtcag agccacacct gtctcgcaga ccaccacagc tgccactgcc 1140

tcagttagaa gcacaaagga cccctgcccc tcccagcccc cagtgttccc agcagctaag 1200

cagtgtccag cattggaagt gacctggcca gaggtggaag tgccactgag taagaagcac 1260

agtggtggag ggtgggctat gggcacagag gttcccaggg tcgggttgac tcctgagcgc 1320

cagtcccctt ctgcccatgt accaccagct gagccagctg ggctgagcac gcaccattct 1380

ccctccccaa cccagtgtca tgggtgcagg cttggcgcag ctcccaagat gctccctatc 1440

aaataggaca gagaactcaa gacataagta atggtcacag gacctcccag agccttggtt 1500

gcagtggacc ccaaggccag cccctccacc cagagcctgc tggcctctgg ccatctcaga 1560

ggagcagcag ccatccagca ctgcctctgt cacctgggct cccaagtcac cgaggctggg 1620

cactagaaaa ggtcatcctg aggagacagg ttcagaagag gattcatcac gtgaaccaag 1680

gaccattcct cacattcccc gtgtttaggg ctagggcctc tcggagacaa ctgcacttct 1740

gtaacggacg ttcccaccta ggtggtgtgc agagcagttc tctaggttcc agatgcatgg 1800

ggactggggg gagctggcag agagggcaca gcagagcagg gtaggggaag ggcctgctct 1860

tctgaagagc taactgctgc ctgtgtccct agatggaacg ctgagcttat cctgtgtggc 1920

ctgcagccgc ttccccaact tcagcatcct ctactggctg ggcaatggtt ccttcattga 1980

gcacctccca ggccgactgt gggaggggag caccaggtga gggtcgcagc agccaggtgg 2040

gtgggaagga agccttctgc ggccttctca tgacctttcc ttcccttccg ctccagccgg 2100

gaacgtggga gcacaggtac gcagctgtgc aaggccttgg tgctggagca gctgacccct 2160

gccctgcaca gcaccaactt ctcctgtgtg ctcgtggacc ctgaacaggt tgtccagcgt 2220

cacgtcgtcc tggcccagct ctgggtgagg agcccaagga gaggcctcca ggaacaggag 2280

gagctctgct tccatatgtg gggaggaaag ggtgggctct gccagagcag cctgtgaact 2340

aatgcccagc attcctcaag gtcagccaga caaaaaggaa cttaggtctt gggcagagga 2400

ggtgtagcct ggggcaaagt gatgagatgt ccctcctttc cttggcctga tccttgtctg 2460

ccttcacttc cctaggctgg gctgagggca accttgcccc ccacccaaga agccctgccc 2520

tccagccaca gcagtccaca gcagcagggt taagactcag cacagggcca gcagcagcac 2580

aaccttgacc agagcttggg tcctacctgt ctacctggag tgaacagtcc ctgactgcct 2640

gtaggctgcg tggatgcgca acacaccccc tccttctctg ctttgggtcc cttctctcac 2700

caaattcaaa ctccattccc acctacctag aaaatcacag cctccttata atgcctcctc 2760

ctcctgccat tctctctcca cctatccatt agccttccta acgtcctact cctcacactg 2820

ctctactgct cagaaaccac caagactgtt gatgccttag ccttgcactc cagggcccta 2880

cctgcatttc ccacatgact ttctggaagc ctcccaacta ttcttgcttt tcccagacag 2940

ctcccactcc catgtctctg ctcatttagt cccgtcttcc tcaccgcccc agcaggggaa 3000

cgctcaagcc tggttgaaat gctgcctctt cagtgaagtc atcctctttc agctctggcc 3060

gcattctgca gacttcctat cttcgtgctg tatgtttttt ttttccccct tcactctaat 3120

ggactgttcc agggaaggga tgggggcagc agctgcttcg gatccacact gtatctgtgt 3180

catccccaca tgggtcctca taaaggatta ttcaatggag gcatcctgac atctgttcat 3240

ttaggcttca gttccactcc caggaacttt gcctgtccca cgagggagta tgggagagat 3300

ggactgccac acagaagctg aagacaacac ctgcttcagg ggaacacagg cgcttgaaaa 3360

agaaaagaga gaacagccca taatgctccc cgggagcaga ggccactaat ggagagtggg 3420

aagagcctgg aaagatgtgg cctcaggaaa agggatgaga gaaaggaggt ggtatggaag 3480

actcagcagg aacaaggtag gcttcaaaga gcctatattc ctctttttcc cacaccgatc 3540

aagtcaactc agtactcacg ggagaaaaat agactttatt tacaagtaat aacatttaga 3600

aaagatccat ccccggccct taaaaacctt cccatcactc caaatcccac cccagtgcaa 3660

gtctggggaa ggtagggtgt gagctgctgc tgaaggctgt cccccaaccc cactcctgag 3720

acacagggcc catccgtcct gggaaagagc atcctctggc aggtgctccc accaggtcag 3780

acccagtcct ggacttcaag agtgagggcc cctgctgggc ccagccacca ggacagcagg 3840

aaccagggcc tactcctctt atggtccctt ctagatccag aggctaagag gaagactggc 3900

caggcccaag gacccagcca tcaaaaccag cctcaaatct ggttgtgatg gagaagtgac 3960

tttgctttaa gaaaaaagga ggcaaggtag ggagagcgcc cacactgtcc atgctccagg 4020

ccccctgggc cagctccgag aaggcgccag tgaaggacca gggaccaggc cagggtgcgg 4080

gcaggcatca ctgtctctag gggtttggct actgttggcc tgggagctga gagaaggcac 4140

tgagagggac agtaggcgga ggaccaggtg acggcagcat cggggacaca ggtggggcca 4200

ctcactggta ctggcccttt agtgctttgc ctgaaagaga cacagtcaca tggccagatg 4260

agaacttgcg atactagcct gcacccactg gctgggaaga tctcttcctg ctcccacgcc 4320

cctgtctgga tcccctccct tgtgagcccc agggttatca gttgctggct gtgcctgagc 4380

agctctgggt gctctccatg agaatggggc catctgtctt ctctccttgg agaggagcta 4440

ccaggacagg gacacctctt accccacacc ctccagcagc ctggcgtggc cccatcttgg 4500

atgctacttg gtggggcggt ctggggggtg cccatgctct catcgggttt ccctccccca 4560

tcctgccagt gcctctacct tgcccttggc tcgaggggtg gcaccaatgg cggcagcagt 4620

ggcggcgctg gctgtggtgg tggcaatgcg cggagaacgg cgggttccac tgcgagtgtt 4680

gggggaagcc ttggacaggg ccttctttga ggctccccgc cgcagaaggc tgttccctag 4740

cttcttgggt gtgttgagga tgctgaaggc catcgactgg cgccggtcag cctgcaagga 4800

agggctgtca gaccgggaga cccaatgctg ccttcccagg ccagcgtgct gtgccacgct 4860

gtaccagcaa ggtcccgcca gggcgtcgct tcatccccct tcagccccag cctcacctgt 4920

ttagtagaag ctggagctgc tttcttctgg gcctcagtag tgctctgttt gcgcccttca 4980

tgtcggtctc ggggagtcat ggggcgtggg aaacagctgg tggccttctt agactatgga 5040

gaagaggaca gttaggcaga cagtagcaag aggagtcaca tctgaagcca ggtgtcttgt 5100

cctctcagag ctgagtggac cttgtaagtc aacgtgcaac ctgctcccct tcccaactct 5160

gggccagatc cttcccttcc caacagttcc catccatggg tcaggccctt ggagagaggg 5220

aaagagaggg ggaagtgagg gaaggagaga gaaggctccc tttagtcctt ggtgagctgg 5280

gcctgacctg agcacagtgc tggagtaaca cccaggagcc accgcgccta cctcaggagt 5340

tccagggccc tggtggggct ctagggagac ccgtttgcgc tgctgccggg tggtgatgcc 5400

agtgccctcg gctatctgga ttggctgcat gctggctcgg cgcagggtct cttgggggtc 5460

tccagttttc atctcctcat ctgtgatggt gcccaggctc agggaaggct gcatgggtgg 5520

aagaggtggt cagtggacca tagctgtatg gagatggagg aggacctggg gctgttccag 5580

aactctacac tcgcccgaca cttatggtcg ggacccttcc tgcctacgag gtagaaagac 5640

acaagcctcc tttcctgttc tgctttctac ctaagccctg ggcaaatggc acaagcagtg 5700

cagtcctgac cagattcctc tctgagctcc tgcctacccc cagggacttc acccctgagt 5760

gccctccagc tgtctgttcc acctggaaca tgagaaggtc accccttccc ctcttcggcc 5820

agtcagtgat ccagggccct agtgctcagg ctagatcagc aggtgggatt ccaaggaagg 5880

gcagggatgg gaggccctgc acagtgaccc caggcctcac cctggactcc agggatagca 5940

ggtcttcaga tgtggggggc acactcgatt gcgctgctgc agctctgcaa tgcggttcca 6000

gtcatccagc tgctcaggct catcctggca agtgcccatg tagaagctgt tccttcctgt 6060

ggaaggcagg gaagtgggaa caaatgagcc tggagtcggc aggtcacctc ctggccctgg 6120

catcttgcca gcctttgctg ccacctaccc cataaacttg aagcccggca caccagtctg 6180

attcagtgcc gcaggtgcag gagtacggca cacagactat ttctatccta ggggcttgct 6240

caccaccttc tccctggaga gggcagaaga ggtcacacgc agagactgct actacatctt 6300

attcacctgc caaggcttgg tggccaacac ccagaggaac aaattaagga ccgggaatta 6360

attcccaggg gctccctggt gcccaaagga caagagcttc caagaagagt ctggccagcc 6420

tggcctttcc agcagcccat caccgcctga gaagggcatg gaggactccc cacagctaag 6480

tgtcacaatt gtgctgggaa tcccgggccc ttaactctgg ctaagagtgc ccccaacaca 6540

gccagcccct agatgggcag gtaaggaagg ccctgaggct gcaggaagga ggggcaggtg 6600

gagctggatg gtagcaagga ggccagcctt ggatttttaa aaagctttcc tcttttccct 6660

gtgccacgat ccaccttcca gtctaatttt ggggtatagt aagtccctgt agtcccctca 6720

cctggagggg ccccactgga caccccggcc tgggaacgac gagcagaact gcgagtggtg 6780

gggcggtagc caggcaagct gagcagggct gagttgccat aatcgggaga acccaggcga 6840

gctagagact gagtagagga ggtggctcgc aggctagcct gggaagcagg agcagaccgc 6900

gtgctgtaga acgatgagtt ggcgctgtct ggctcttcca catctagctt ctggaagaca 6960

gagtgaatct gttgcagtgt acagtccctg gcactgtaca gaagcttccc attcccttcc 7020

gaagccctca gatcccacgg cacatccatg tattcccaac tgctttgcaa aggtccttaa 7080

agtgtgtgtc tgcaagaaat gggccttgtc gacagaagcc ctcacaaggt ggtgctgatg 7140

ttgtcaagac tcttctacgc atttttttca tggagtctat tcataatgct ttgaggtagg 7200

gaatgcagag tgtttatcgg cccattttgg agatgaagtg caaagaaata aagtgactag 7260

ccccaaatca cactgctagg aagtatcaga gctggggcta ggccccatgt ctcctgacta 7320

gtcaggctca tcccacagcc tctgctgtcc ctcagtccaa acttccaggg cccttaccat 7380

gttccagaac ttcccccaac ttcttggtag cagggggcac cctaaacaca caggcccccc 7440

ctgctgtacc aggggccccc tctcccctcc tcccaaacct ccccttcaag atgtggaaac 7500

aaaggcaagg gcctgcagcc tgtcaggcag tccactgggc agcaacaatg cctctcagct 7560

gcatggggca tgctgggagg cacaggatgg gctgcagctt cgccacgttc tctcccttca 7620

ccctgcacag gctcagtgct acgcatggag agaatgctag ccttagtcag gaggcaggga 7680

tctaatccta gccctgcctt tttcttcaga agtgccctta accaagtcac tccccttttt 7740

aagacctctc agctttccca ctgtaacatg gactggctgc tcatccctcc ctgctcctga 7800

ctgagtgccc ag 7812

<210>10

<211>40

<212>PRT

<213>Homo sapiens

<400>10

Thr Pro Val Ser Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser

1 5 10 15

Thr Lys Asp Pro Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys

20 25 30

Gln Cys pro Ala Leu Glu Val Thr

35 40

Claims (42)

1.An isolated IL-18 binding protein IL-18BP, wherein the IL-18BP comprises the amino acid sequence of SEQ ID NO: 10 and is selected from:

(a) has the amino acid sequence of SEQ ID NO: 2; or

(b) A polypeptide as defined in (a) which is free of a leader sequence.

2. The isolated IL-18BP of claim 1, wherein said polypeptide is a non-viral protein.

3. The isolated IL-18BP of claim 1, wherein said polypeptide is a human protein.

4. The isolated IL-18BP of claim 1, wherein said polypeptide has a molecular weight of 40 kilodaltons, as determined by SDS-PAGE under non-reducing conditions.

5. The isolated IL-18BP of claim 1, wherein said polypeptide is a fusion protein.

6. The isolated IL-18BP of claim 1, wherein said polypeptide is in soluble form.

7. The isolated IL-18BP according to any one of claims 1 to 6, wherein the polypeptide is a non-glycosylated IL-18 BP.

8. An isolated IL-18BP selected from the group consisting of:

(a) has the amino acid sequence of SEQ ID NO: 2; or

(b) A polypeptide as defined in (a) which is free of a leader sequence.

9. The isolated IL-18BP of claim 8, wherein said polypeptide is a non-viral protein.

10. The isolated IL-18BP of claim 8, wherein said polypeptide is a human protein.

11. The isolated IL-18BP of claim 8, wherein said polypeptide has a molecular weight of 40 kilodaltons, as determined by SDS-PAGE under non-reducing conditions.

12. The isolated IL-18BP of claim 8, wherein said polypeptide is a fusion protein.

13. The isolated IL-18BP of claim 8, wherein said polypeptide is in soluble form.

14. The isolated IL-18BP according to any one of claims 8 to 13, wherein the polypeptide is a non-glycosylated IL-18 BP.

15. A pharmaceutical composition comprising the isolated IL-18BP of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising DNA encoding the IL-18BP of any one of claims 1 to 14.

17. A DNA capable of hybridizing under stringent conditions, or capable of hybridizing under stringent conditions but which, due to the degeneracy of the genetic code, hybridizes with SEQ ID NO: 1, which DNA is capable of encoding an isolated IL-18BP according to any one of claims 1 to 14.

18. DNA encoding the isolated IL-18BP of any one of claims 1 to 14, said isolated IL-18BP comprising the amino acid sequence SEQ ID NO: 10.

19. a DNA encoding the isolated IL-18BP of any one of claims 1 to 14, said isolated IL-18BP comprising the amino acid sequence SEQ ID NO: 10.

20. a DNA which hybridizes under stringent conditions to the DNA of claim 18 or which is capable of hybridizing under stringent conditions due to the degeneracy of the genetic code and which is capable of encoding the isolated IL-18BP of any one of claims 1 to 14.

21. The DNA of any one of claims 17 to 20 operably linked to other DNA sequences to facilitate expression.

22. The DNA according to any one of claims 17 to 21 which is genomic DNA.

23. The DNA according to any one of claims 17 to 22 which is cDNA.

24. The DNA of claim 23, wherein the cDNA is seq id NO: 1, or a cDNA sequence of the DNA sequence of 1.

25. The DNA of claim 23 or 24, wherein the cDNA is suitable for expression in a bacterial host.

26. A replicable expression vector comprising the DNA of any one of claims 17 to 25.

27. A transformed host cell comprising the expression vector of claim 26.

28. The transformed host cell of claim 27, which is a eukaryotic cell.

29. The transformed host cell of claim 27, which is a prokaryotic cell.

30. A method of producing the isolated IL-18BP of any one of claims 1 to 14, comprising culturing the host cell of any one of claims 27 to 29 under conditions suitable for expression of IL-18BP, and isolating the IL-18 BP.

31. An antibody to an isolated IL-18BP according to any one of claims 1 to 14.

32. The antibody of claim 31, which is a polyclonal antibody.

33. The antibody of claim 31, which is a monoclonal antibody.

34. The antibody of claim 31, which is an anti-idiotype antibody.

35. The antibody of claim 31, which is a chimeric antibody.

36. The antibody of claim 31, which is a humanized antibody.

37. A method of isolating the isolated IL-18BP of any one of claims 1 to 14, comprising:

(a) the human fluid is passed through a column coupled to IL-18,

(b) eluting a protein capable of binding IL-18, and

(c) purifying said protein.

38. Use of an isolated IL-18BP according to any one of claims 1 to 14 for the preparation of a pharmaceutical composition for the treatment of an autoimmune disease, diabetes, rheumatoid arthritis, transplant rejection, inflammatory bowel disease, sepsis, multiple sclerosis, ischemic heart disease, ischemic brain injury, chronic hepatitis, psoriasis, chronic pancreatitis or acute pancreatitis.

39. Use of the isolated IL-18BP of any one of claims 1 to 14 for the purification of IL-18.

40. Use of an antibody according to any one of claims 31 to 36 in an assay for detection of IL-18 BP.

41. Use of a DNA encoding the isolated IL-18BP of any one of claims 1 to 14 in the preparation of a gene therapy vector.

42. Use of the DNA of any one of claims 17 to 25 in the manufacture of the isolated IL-18BP of any one of claims 1 to 14.