WO2005075648A1 - Interleukin-18 mutant proteins - Google Patents

Abstract

An interleukin-18 mutant protein is provided which is highly stable and retaining biological activities. An interleukin-18 mutant protein may have cysteine residues substituted with other amino acid residues while retaining biological activies. An interleukin-18 mutant protein may also have one or several amino acid residues deleted, substituted or added, and which is more stable than a wild-type interleukin-18 protein.

Description

DESCRIPTION INTERLEUKIN-18 MUT7ΛNT PROTEINS

Background 7Art The present invention relates to interleukin-18 mutant proteins. Human interleukin-18 (hIL-18), initially cloned as an IE-γ-inducing factor secreted by macrophages or Kuppfer cells, strongly augments the production of IFN-γ both in natural killer cells and T cells; hIL-18 plays a key role in many inflammatory diseases including allergy and autoimmune diseases [Okamura H (1995) "Cloning of a new cytokine that induces IFN-γ production by T cells", Nature 378.88-91; Ushio S, M Namaba, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, icallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S, Ikeda M, Okamura H, and Kurimoto , (1996) "Cloning of the coda for human IF-γ-inducing factor, expression in Escherichia coli, and studies on the biologic actives of the protein", J. Immunol. 156:4274-4279; Nakanishi K, "Interleukin-18 is a unique cytokine that stimulates both Thl and Th2 responses depending on its cytokine milieu" (2001) Cytokine Growth Factor Reviews 2:53-72]. The present inventors previously developed a high production system for correctly folded active hIL-18 protein, and it enabled the inventors to determine both the 3D- structure of hIL-18 and the molecular mechanism of its receptor binding mode [Li A, Kato Z, Ohnishi H, Hashimoto K, atsukuma E, Cmoya K, Yamamoto Y, and Kondo N, (2003) "Cptimized gene synthesis and high expression of human interleukin-18", Protein Expression and Purification, 32:110-118; Kato Z, Jee J, S ikano H, Mishima M, Ohki I, Ohnishi H, Li A, Hashimoto K, Matsukuma E, Cmoya K, Yamamoto Y, Yoneda T, Hara T, Kondo N, and Shirakawa M. (2003) "The structure and binding mode of interleukin-18", Nature Structural Biology. 10:966-9714]. This production method and the molecular mechanisms can strongly assist the experimental and medical applications of IL-18; however, one of the most common problems in the application of recombinant proteins to experiments and medicine is the inactivation of the proteins (usually, they form aggregates) [T. /Arakawa, S.J. Prestrelski, W.C. Kenney, and J.F. Carpenter, (1993) "Factors affecting short-term and long-term stabilities of proteins", Advanced Drug Delivery Reviews, 10:1-286]. Recently, therapeutic approaches using recombinant IL-18 have been examined for the purpose of treating cancers including a clinical trial in humans; in the course of such examinations, necessity for IL-18 of a more stable form that will allow therapy to be performed with intertrial reliability has risen [Yamanaka K, Hara I, Nagai H, et al. "Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model", Cancer Immunol Immunother, 1999,48:297- 302; Nagai H, Hara I, Horikawa T, et al. "Antitumor effects on mouse melanoma elicited by local secretion of interleukin-12 and their enhancement by treatment with interleukin-18", Cancer Invest 2000,18:206-13; Herzyk DJ, Bugelski PJ, Hart TK, Wier PJ. "Preclinical safety of recombinant human interleukin-18", Toxicol Pathol. 2003 31:554-61].

Disclosure of Invention It is an object of the present invention to provide interleukin-18 mutant proteins which are highly stable and retaining the biological activities of the wild-type IL-18. The present inventors have succeeded in creating a highly stable hlL- 18 mutant protein based on the 3D-structure of IL-18 and the molecular mechanism of its receptor binding mode. In this mutant protein, the four cysteine residues which were considered not involved in the disulfide bond within the same molecule from the 3D-structure of hIL-18 based on NMR analysis are substituted with other amino acid (i.e. serine residues) . This mutant protein is highly stable under non-reducing conditions, does not form oligomers, and shows no decrease in biological activities. The present invention has been achieved based on these findings. The summary of the present invention is as follows. (1) An interleukin-18 mutant protein comprising: (a) an interleukin-18 mutant protein having an amino acid sequence as shown in SEQ ID NO: 2 but with at least one of the cysteine residues at positions 38, 68, 76 and 127 being substituted with other amino acid residues; (b) an interleukin-18 mutant protein having an amino acid sequence of the mutant protein of (a) above but with one or several amino acid residues other than the amino acid residues at positions 38, 68, 76 and 127 being deleted, substituted or added, and which is more stable than a wild-type interleukin-18 protein having an amino acid sequence as shown in SEQ ID NO: 2 under non-reducing conditions; or (c) an interleukin-18 mutant protein encoded by an DNA that hybridizes under a stringent condition with a complement of SEQ ID NO. 1 or 13 that encodes an interleukin-18 protein, wherein at least amino acid residues of the mutant protein at positions 38, 68, 76 and 127 are other than cysteine residues; or a salt of (a) , (b) or (c) .

(2) The interleukin-18 mutant protein or a salt thereof of (1) above, wherein the other amino acid residues are serine residues.

(3) The interleukin-18 mutant protein or a salt of (1) above, wherein the other amino acid residues are selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, threonine, glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid and glutamic acid residues. (4) The interleukin-18 mutant protein or a salt of (1) above, wherein the interleukin-18 mutant protein is a mutant of a human-derived wild-type interleukin-18 protein. (5) The interleukin-18 mutant protein or a salt of (1) above, wherein the interleukin-18 mutant protein is a mutant of a wild-type interleukin-18 protein derived from an animal other than human. (6) The interleukin-18 mutant protein or a salt of 1) above, having an amino acid sequence as shown in SEQ ID NO: 4. (7) An isolated DNA encoding the protein of (1) above. (8) A recombinant vector comprising the DNA of (7) above. (9) A transformant comprising the recombinant vector of (8) above. (10) A method of producing an interleukin-18 mutant protein, comprising culturing a host transformed with the DNA of (7) above and recovering the interleukin-18 mutant protein from the resultant culture. (11) An agonist or antagonist for an interleukin-18 protein, wherein the agonist or antagonist is the interleukin-18 mutant protein or a salt thereof of (1) above. (12) A pharmaceutical composition comprising the interleukin-18 mutant protein or a salt thereof of (1) above as an active ingredient.

(13) An antibody to the interleukin-18 mutant protein or a salt thereof of (1) above.

Brief Description of Drawings Fig. 1 shows the results of oligomerization assay of wild-type IL-18. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using 10-20% gradient gel. Lane 1: without 2ME, before aeration. Lane 2: without 2ME, after aeration. Lane 3: with 2ME, after aeration. 400 ng of the protein was loaded. The wild-type protein shows a marked oligomerization pattern after aeration, but this pattern disappeared in the presence of 2ME. Fig. 2 shows an alignment of the amino acid sequences of IL-18 proteins. Completely conserved residues among the eight species are boxed with solid line. The cysteine residues are boxed with dotted line. The positions of the cysteine residues in the human sequence are indicated. The secondary structure of human IL-18 previously determined by the inventors is shown above the sequences as open boxes (S; beta-strand) and filled boxes (H; helix) . The intermediate loop regions are indicated as L. C76 and C127 are conserved among all the species, while C38 and C68 were substituted with other residues in some species. Fig. 3a shows the structure of human IL-18. The overall structure is shown using ribbon models, and the four cysteine residues are shown by spacefill representation in dark grey. The top figure and the bottom figure are tilted at 90 degrees to each other. The four cysteine residues do not form any sulfide bonds, and are present on the surface of the structure. Fig. 3b shows the structure of a complex composed of human IL-18 and human IL-18 receptor a (IL-18R ) . The overall structure of hIL-18 is shown using a ribbon model, and the four cysteine residues are shown by spacefill representation in dark grey. hIL-18Rα is shown by spacefill representation in light grey. The four cysteine residues of hIL-18 do not have any direct interaction with hIL-18Rα, which suggests that their influence upon the binding is little. Fig. 4a shows the results of dimerization assay of the IL-18 mutants having one cysteine residue on the surface. SDS-PAGE was carried out on 10- 20% gradient gel. Without 2ME, before aeration. 400 ng of each protein was loaded. Fig. 4b shows the results of dimerization assay of the IL-18 mutants having one cysteine residue on the surface. SDS-PAGE was carried out on 10- 20% gradient gel. Without 2ME, after aeration. 400 ng of each protein was loaded. All the mutants showed dimerization after aeration, indicating that all the cysteine residues of IL-18 are involved in the oligomerization phenomenon. Fig. 5 shows the results of oligomerization assay of IL-18-AS. SDS- PAGE was carried on 10-20% gradient gel. Lane 1: without 2ME, before aeration. Lane 2: without 2ME, after aeration. 400 ng of the protein was loaded. IL-18-AS protein did not show any oligomerization pattern. Fig . 6 shows I FN- γ induction (pg/ml ) by the wild type and the IL-18-AS before and after oxidation . Mean values of triplicate IFN- y induction assays are shown with standard deviation . Solid circle, hIL-18-wild before oxidation; open circle, hIL-18-wild after oxidation; solid triangle, hIL-18-AS before oxidation; and open triangle, hIL-18-AS after oxidation . IL-18-AS showed the same activity as that of the wild type even after oxidation, while wild type protein showed significant reduction in the activity after oxidation .

Best Mode for Carrying Out the Invention The term "mutant protein" used herein means a protein which is different from a standard protein but retains the essential nature of the standard protein. A typical mutant protein has an amino acid sequence which is different from the amino acid sequence of the standard protein. The term "interleukin-18 protein" used herein is a concept encompassing the interleukin-18 protein comprising the amino acid sequence as shown in SEQ ID NO: 2 (wild-type human IL-18) , homologous proteins thereof, and mutant proteins of such homologous proteins. The term "biological activities of interleukin-18 protein" used herein means the metabolic or physiological functions of interleukin-18 protein. These functions include, but are not limited to, IFN-γ production enhancing activity, Fas-mediated cytotoxicity against natural killer cells, immunoregulatory function on pleiotropism including developmental regulation of helper T lymphocyte type 1, activity as antigen, and activity as immunogen. The term "agonist" used herein refers to a substance which has the same affinity and/or biological activity that a certain substance shows to its receptor. The term "antagonist" used herein refers to a substance which may reduce or eliminate the activity of a certain substance having biological activity. The term "antibody" used herein means a protein which is induced in the body by immunological reaction as a result of stimulation with antigen, and has activity of specifically binding to immunogen (antigen) . This term encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibody, humanized antibodies, and Fab or Fab fragments. The term "non-reducing conditions" used herein refers to solution conditions, etc. in the absence of reducing agents where SH groups in the side chains of cysteine residues are capable of being oxidized or capable of forming disulfide bonds with other SH groups. According to the present invention, a novel mutant of interleukin-18 protein has been provided. This mutant is highly stable under non-reducing conditions, does not form oligomers, and is retaining sufficient biological activities. Thus, the mutant protein of the invention is applicable to medicines for human and other animals (such as anti-tumor agents, therapeutics for allergy) and to experiments conveniently. The interleukin- 18 mutant proteins of the invention can be used as agonists or antagonists for interleukin-18 proteins. These agonists and antagonists are stable under non-reducing conditions . 1. Interleukin-18 Mutant Protein The present invention provides an interleukin-18 mutant protein comprising: (a) an interleukin-18 mutant protein having an amino acid sequence as shown in SEQ ID NO: 2 but with at least one of the cysteine residues at positions 38, 68, 76 and 127 being substituted with other amino acid residues; (b) an interleukin-18 mutant protein having an amino acid sequence of the mutant protein of (a) above but with one or several amino acid residues other than the amino acid residues at positions 38, 68, 76 and 127 being deleted, substituted or added, and which is more stable than a wild-type interleukin-18 protein having an amino acid sequence as shown in SEQ ID NO: 2 under non-reducing conditions; or (c) an interleukin-18 mutant protein encoded by an DNA that hybridizes under a stringent condition with a complement of SEQ ID NO. 1 or 13 that encodes an interleukin-18 protein, wherein at least amino acid residues of the mutant protein at positions 38, 68, 76 and 127 are other than cysteine residues; or a salt of (a) , (b) or (c) . Specific examples of the interleukin-18 mutant protein of (a) above include an interleukin-18 mutant protein comprising the amino acid sequence as shown in SEQ ID NO: 2 but with at least one of the cysteine residues at positions 38, 68, 76 and 127 being substituted with other amino acid residues (e.g. serine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, threonine, glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid residue or the like) . Other amino acid residues to be substituted with the cysteine residues at positions 38, 68, 76 and 127 may be those amino acid residues that would not destroy the 3D-structure of interleukin-18. Judging from the structure, it appears that the cysteine residues at position 38 and 68 may be substituted with any amino acid including serine. On the other hand, it appears that, preferably, the cysteine residues at positions 76 and 127 may be substituted with serine, alanine, threonine, glycine or the like. The invention also includes an interleukin-18 mutant protein encoded by a nucleic acid that hybridizes under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequence represented by a complement of SEQ ID NO: 1 or 13. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 95%, or at least 98%, identical to the sequence of a portion or all of a complement of a nucleic acid encoding an interleukin-18 polypeptide. Hybridization of the oligonucleotide to a nucleic acid sample typically is performed under stringent conditions. Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a probe dissociates from a target DNA. This melting temperature is used to define the required stringency conditions. If sequences are to be identified that are related and substantially identical to the probe, rather than identical, then it is useful to first establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt

(e.g., SSC or SSPE) . Then, assuming that 1% mismatching results in a 1 °C decrease in the Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having >95% identity with the probe are sought, the final wash temperature is decreased by 5°C) In practice, the change in Tm can be between 0.5°C and 1.5°C per 1% mismatch. Stringent conditions involve hybridizing at 68°C in 5x SSC/5x Denhardt's solution/1.0% SDS, and washing in 0.2x SSC/0.1%SDS at room temperature. Moderately stringent conditions include washing in 3x SSC at 42°C. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Additional guidance regarding such conditions is readily available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons. N.Y.) at Unit 2.10. In a preferred embodiment, the interleukin-18 mutant protein has an amino acid sequence as shown in SEQ ID NO: 4. The interleukin-18 mutant protein of the invention may be either a mutant of a human-derived wild-type interleukin-18 protein or a mutant of a wild-type interleukin-18 protein derived from an animal other than human. The salt of the interleukin-18 mutant protein of the invention may be a pharmacologically acceptable salt. Particularly preferable are pharmacologically acceptable acid addition salts. Examples of such salts include salts formed with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid) and salts formed with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid) . The interleukin-18 mutant protein or a salt thereof of the invention may be used as an agonist or antagonist for interleukin-18 protein. For example, the interleukin-18 mutant protein or a salt thereof of the invention may be used for preventing and/or treating diseases attributable to interleukin-18 protein (such as autoimmune diseases, allergy, neurological disorders, etc. ) or diseases of which symptoms may be ameliorated by the administration of interleukin-18 protein (such as hepatitis C, tumors, etc. ) . The interleukin-18 mutant protein or a salt thereof of the invention may be used as a reagent for experiments. The interleukin-18 mutant protein or a salt thereof of the invention may be prepared by known methods. For example, the interleukin-18 mutant protein may be produced by obtaining a DNA encoding an interleukin-18 mutant protein of interest as described in sub-section 2 below, integrating the DNA into an appropriate expression vector, introducing the vector into an appropriate host, and allowing the host to produce the mutant protein of interest as a recombinant protein [see, for example, Current Protocols (compact ed. ) : Molecular Biology Experimental Protocols I, II and III, translated by Kaoru Saigo and Yumiko Sano, published by Maruzen Co.; Ausubel, F.M. et al., Short Protocols in Molecular Biology (Third Edition), John Wiley & Sons, Inc. , New York] . Alternatively, the interleukin-18 mutant protein or a salt thereof of the invention may be prepared according to methods of peptide synthesis known in the art. 2. Isolated DNA Encoding Interleukin-18 Mutant Protein An isolated DNA encoding the interleukin-18 mutant protein of the invention may be any DNA as long as it comprises a nucleotide sequence encoding the interleukin-18 mutant protein of the invention. As a specific example of the DNA encoding the interleukin-18 mutant protein of the invention, a DNA comprising the nucleotide sequence as shown in SEQ ID NO: 3 may be given. The invention also includes nucleic acids that hybridize under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequence represented by SEQ ID NO: 1 or 13 or its complement. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding an interleukin-18 polypeptide, or its complement. Hybridization of the oligonucleotide probe to a nucleic acid sample typically is performed under stringent conditions. Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a probe dissociates from a target DNA. This melting temperature is used to define the required stringency conditions. If sequences are to be identified that are related and substantially identical to the probe, rather than identical, then it is useful to first establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt (e.g., SSC or SSPE) . Then, assuming that 1% mismatching results in a 1°C decrease in the Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having >95% identity with the probe are sought, the final wash temperature is decreased by 5°C) . In practice, the change in Tm can be between 0.5°C and 1.5°C per 1% mismatch. Stringent conditions involve hybridizing at 68°C in 5x SSC/5x Denhardt's solution/1.0% SDS, and washing in 0.2x SSC/0.1%SDS at room temperature. Moderately stringent conditions include washing in 3x SSC at 42°C. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid . Additional guidance regarding such conditions is readily available in the art , for example , by Sambrook et al . , 1989, Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press , N . Y . ; and Ausubel et al . (eds . ) , 1995 , Current Protocols in Molecular Biology, ( John Wiley & Sons . N . Y . ) at Unit 2 . 10 . The isolated DNA encoding the interleukin-18 mutant protein of the invention may be prepared, for example, as described below. Briefly, mRNA is extracted from healthy human blood, and cDNA is synthesized from the mRNA using a reverse transcriptase and oligo dT primers. Then, the coding region of mature hIL-18 (157 residues) is amplified by PCR. The resultant PCR product is a DNA encoding the wild- type hIL-18 protein. One example of the amino acid sequence encoding a wild-type hIL-18 protein and one example of the nucleotide sequence of a DNA encoding a wild-type hIL-18 protein are shown in SEQ ID NO: 2 and SEQ ID NO: 1, respectively. The DNA encoding the interleukin-18 mutant protein of the invention may be prepared by mutating the coding region of mature hIL-18 (157 residues) by point mutation mutagenesis. The mutated coding region of mature hIL-18 (157 residues) is amplified by PCR. The resultant PCR product is a DNA encoding the interleukin-18 mutant protein of the invention. Examples of the nucleotide sequence of a DNA encoding an interleukin-28 mutant protein comprising the amino acid sequence as shown in SEQ ID NO: 2 but with at least one of the cysteine residues at positions 38, 68, 76 and 127 being substituted with other amino acid residues are shown in SEQ ID NOS: 3, 5, 7, 9 and 11. The DNA having the nucleotide sequence as shown in SEQ ID NO: 3 encodes an interleukin-18 mutant protein comprising the amino acid sequence as shown in SEQ ID NO: 2 but with all of the cysteine residues at positions 38, 68, 76 and 127 being substituted with serine residues. The DNA having the nucleotide sequence as shown in SEQ ID NO: 5 encodes an interleukin-18 mutant protein comprising the amino acid sequence as shown in SEQ ID NO: 2 but with the cysteine residue at position 38 being substituted with serine residue. The DNA having the nucleotide sequence as shown in SEQ ID NO: 7 encodes an interleukin-18 mutant protein comprising the amino acid sequence as shown in SEQ ID NO: 2 but with the cysteine residue at position 68 being substituted with serine residue. The DNA having the nucleotide sequence as shown in SEQ ID NO: 9 encodes an interleukin-18 mutant protein comprising the amino acid sequence as shown in SEQ ID NO: 2 but with the cysteine residue at position 76 being substituted with serine residue. The DNA having the nucleotide sequence as shown in SEQ ID NO: 11 encodes an interleukin-18 mutant protein comprising the amino acid sequence as shown in SEQ ID NO: 2 but with the cysteine residue at position 127 being substituted with serine residue. The above-described DNA molecules encoding hIL-18 mutant proteins have been prepared using a wild-type hIL-18 gene. It is also possible to prepare a DNA encoding an interleukin-18 mutant protein by designing and synthesizing an optimized cDNA instead of using a wild-type hIL-18 gene. One example of optimized cDNA sequence is shown in SEQ ID NO: 13. It has been confirmed that the expression/production of interleukin-18 protein can be remarkably improved (more than 5-fold) by using this optimized cDNA sequence. 3. Recombinant Vector A recombinant vector comprising a DNA encoding the interleukin-18 mutant protein of the invention may be obtained by inserting a DNA encoding the interleukin-18 mutant protein of the invention into an appropriate expression vector according to known methods (e.g. methods described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Examples of expression vectors useful in the invention include, but are not limited to, plasmids derived from Escherichia coli (e.g. pBR322, pBR325, pUC12, and pUC13) ; plasmids derived from Bacillus subtilis (e.g. pUBllO, pTP5 and pC194) ; plasmids derived from yeast (e.g. pSH19 and pSH15) ; bacteriophages such as L-phage; animal viruses such as retrovirus, vaccinia virus; and insect pathogenic virus such as baculovirus. The expression vector may comprise a promoter, enhancer, splicing signal, polyadenylation signal, selective markers, SV40 replication origin and the like. The expression vector may be a fusion protein expression vector. Various fusion protein expression vectors are commercialized, e.g. pGEX series (Amersham Pharmacia Biotech) , pET CBD Fusion System 34b-38b (Novagen) , pET Dsb Fusion Systems 39b and 40b (Novagen) , and pET GST Fusion System 41 and 42 (Novagen) . 4. Transformant A transformant may be obtained by introducing a recombinant vector comprising a DNA encoding the interleukin-18 mutant protein of the invention into a host. Specific examples of hosts useful in the invention include, but are not limited to, bacterial cells (e.g. bacteria belonging to the genus Escherichia, bacteria belonging to the genus Bacillus such as B. subtilis) , fungal cells (e.g. yeast, fungi belonging to the genus Aspergillus) , insect cells (e.g. S2 cells, Sf cells), animal cells (e.g. CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells) and plant cells. The introduction of a recombinant vector into a host may be performed by methods described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (e.g. the calcium phosphate method, the DEAE-dextran method, transfection, microinjection, lipofection, electroporation, transduction, the scrape loading method, or the shot gun method) or infection. The thus obtained transformant may be cultured in a medium, and an interleukin-18 mutant protein of interest may be recovered from the resultant culture. When the interleukin-18 mutant protein is secreted into the medium, the medium is recovered and then the interleukin-18 mutant protein is separated and purified therefrom. When the interleukin-18 mutant protein is produced within the transformed cell, the cell is lysed and then the interleukin-18 mutant protein is separated and purified from the resultant lysate. When an interleukin-18 mutant protein of interest is expressed in the form of a fusion protein with other protein which will function as a tag, first, the fusion protein is separated and purified. Then, the interleukin- 18 mutant protein of interest can be obtained by cutting the other protein by treating the fusion protein with FactorXa or an appropriate enzyme (enterokinase) . The separation and purification of the interleukin-18 mutant protein may be performed by known methods. For example, methods utilizing the difference in solubility, such as salting out and solvent precipitation; methods utilizing difference in molecular weight, such as dialysis, ultra- filtration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods utilizing difference in electric charge, such as ion exchange chromatography; methods utilizing specific affinity, such as affinity chromatography; methods utilizing difference in hydrophobicity, such as reversed-phase high performance liquid chromatography; methods utilizing difference in isoelectric point, such as isoelectric focusing; and the like may be used. 5. Use of Interleukin-18 Mutant Protein as Agonist The interleukin-18 mutant protein of the invention having equal interferon-γ induction activity and being highly stable under non-reducing conditions, when compared with the wild-type interleukin-18 protein, may be used as an agonist for interleukin-18 protein. The agonist for interleukin- 18 protein may be used for preventing and/or treating diseases of which syrrptoms may be ameliorated by the administration of interleukin-18 protein (such as hepatitis C, tumors, etc.). The agonist for interleukin-18 protein may also be used as a reagent for experiments.

6. Use of Interleukin-18 Mutant Protein as Antagonist The interleukin-18 mutant protein of the invention having lower interferon-γ induction activity and being highly stable under non-reducing conditions, when compared with the wild-type interleukin-18 protein, may be used as an antagonist for interleukin-18 protein. The antagonist for interleukin-18 protein may be used for preventing and/or treating diseases attributable to interleukin-18 protein (such as autoimmune diseases, allergy, neurological disorders, etc.). The antagonist for interleukin-18 protein may also be used as a reagent for experiments.

7. Pharmaceutical Compositions The interleukin-18 mutant protein or a salt thereof of the invention may be used for preventing and/or treating diseases attributable to interleukin-18 protein (such as autoimmune diseases, allergy, neurological disorders, etc.) or diseases of which symptoms may be ameliorated by the administration of interleukin-18 protein (such as hepatitis C, tumors, etc.). The interleukin-18 mutant protein or a salt thereof of the invention may be administered alone or together with carriers, diluents or excipients in an appropriate form of a pharmaceutical composition, orally or parenterally to mammals (e.g. human, rabbit, dog, cat, rat, mouse) . Dose levels may vary depending upon the patient to be treated, the target disease, syrrptoms, administration route, and so on. When the interleukin-18 mutant protein or a salt thereof of the invention is used for preventing and/or treating an excessive immune response of an adult (e.g. pollinosis) , the mutant protein or a salt thereof may be administered by intravenous injection usually at a dose of about 0.1-10.0 μg/kg body weight, preferably 1.0 μg/kg body weight, at a frequency of about 3 times a week, preferably once in two days (preferably, continuous or every-other-day administration) . With respect to other parenteral administration or oral administration, similar dose levels may be used, though the dose may be increased when symptoms are particularly severe. Compositions for oral administration include solid or liquid preparations, such as tablets (including sugar-coated tablets and film- coated tablets) , pills, granules, powders, capsules (including soft capsules), syrups, emulsions and suspensions. These compositions may be prepared according to conventional methods and may contain carriers, diluents or excipients conventionally used in the field of medicine manufacturing. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers or excipients for tablets. Compositions for parenteral administration include, for example, injections and suppositories. Injections may be intravenous injections, subcutaneous injections, intradermal injections, muscle injections, instilment injections, etc. Such injections may be prepared by conventional methods, i.e. by dissolving, suspending or emulsifying the interleukin-18 mutant protein or a salt thereof in an aseptic, aqueous or oily liquid conventionally used in injections. Examples of aqueous liquids for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents. They may be used in combination with a suitable auxiliary solubilizer such as alcohol (e.g. ethanol), polyalcohol (e.g. propylene glycol, polyethylene glycol), nonionic surfactant [e.g. Polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. Examples of oily liquids for injection include sesame oil and soybean oil. They may be used in combination with an auxiliary solubilizer such as benzyl benzoate, benzyl alcohol, etc. Usually, the prepared injections are filled in appropriate ampoules. Suppositories for administration into rectum may be prepared by mixing the interleukin-18 mutant protein or a salt thereof with a conventional suppository base. It is convenient to formulate the above-described pharmaceutical compositions for oral or parenteral administration into unit dosage forms that would give an appropriate dose of the active ingredient. Examples of such unit dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories. Usually, each unit of these dosage forms contains 0.125-1.000 μg of the interleukin-18 mutant protein or a salt thereof. In particular, it is preferred that each unit should contain 0.125- 1.000 μg of the interleukin-18 mutant protein or a salt thereof in injections, and each unit in other dosage forms should contain 0.125-1.000 μg of the interleukin-18 mutant protein or a salt thereof. The above-described pharmaceutical compositions may contain other active ingredients as long as they do not produce undesirable interaction with the interleukin-18 mutant protein or a salt thereof. 8. Antibody An antibody to the interleukin-18 mutant protein or a salt thereof of the invention may be used for detecting and/or quantitatively determining the interleukin-18 mutant protein or a salt thereof of the invention. An antibody to the interleukin-18 mutant protein or a salt thereof of the invention may be obtained by administering to an animal the interleukin- 18 mutant protein or a salt thereof of the invention or a fragment thereof containing the epitope of the protein, according to conventional protocols. The antibody of the invention may be any one of polyclonal antibody, monoclonal antlbody, chimeric antibody, single chain antibody or humanized antibody. Polyclonal antibodies may be prepared according to known methods or modifications thereof. For example, a complex of an immunogen (antigen protein) and a carrier protein is prepared and then administered to animals (immunization) . Fractions containing an antibody to the protein of the invention are harvested from the immunized animals, followed by separation and purification of the antibody. At the time of administration of the complex, complete Freund's adjuvant or incomplete Freund's adjuvant may also be administered to enhance antibody production ability. The administration is carried out generally once in about every 2-6 weeks and about 3-10 times in the total. Polyclonal antibodies can be recovered from the blood, abdominal dropsy or other body fluid, preferably from the blood, of the immunized animals. The separation and purification of polyclonal antibodies may be performed by the same methods for separation and purification of immunoglobulin (e.g. salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, absorption and desorption with ion exchangers, ultracentrifugation, gel filtration, specific purification methods in which an antibody of interest alone is recovered using an antigen-bound solid phase or an active adsorbent such as protein A or protein G, followed by dissociation of the bond) . Monoclonal antibodies may be prepared by the hybridoma method of G. Koehler and C. Milstein described in Nature (1975) 256:495; Science (1980) 208:692. Briefly, after immunization of animals, antibody producing cells are isolated from the spleen of the immunized animals, and then fused to myeloma cells to thereby prepare monoclonal antibody-producing cells. Further, a cell line may be isolated therefrom which reacts specifically with the interleukin-18 mutant protein or a salt thereof but does not cross- react with other antigen proteins substantially. This cell line is cultured, and a monoclonal antibody of interest can be obtained from the resultant culture. Purification of monoclonal antibodies may be performed according to the above-described methods for separation and purification of immunoglobulin. A method for preparing single chain antibodies is disclosed in U.S. Patent No. 4,946,778. A method for preparing humanized antibodies is disclosed in Biotechnology 10:1121- (1992); Biotechnology 10:169- (1992).

Example Hereinbelow, the present invention will be described in more detail with reference to the following Examples. These Exarrples are provided solely for the purpose of illustration and not intended to limit the scope of the present invention.

Materials and Methods

Vector Construction and Protein Expression Construction of the expression vector, and expression and purification of wild-type hIL-18 protein were carried out as described previously [Li A, Kato Z, Ohnishi H, Hashimoto K, Matsukuma E, Cmoya K, Yamamoto Y, and Kondo N (2003) "Optimized gene synthesis and high expression of human interleukin- 18", Protein Expression and Purification, 32:110-1184]. Briefly, the coding region for mature hIL-18 (157 residues) with FactorXa cleavage site immediately before the hIL-18 sequence was amplified by PCR, and the amplified product was cloned into pGEX-4T-l vector (Pharmacia) . BL-21 (DE3) (Novagen) was transformed with this vector, and protein expression was performed as follows: the colony with the highest expression level was cultured overnight in 200 ml of LB medium with 100 μg/ml ampicillin. The culture was transferred into 2 L of LB medium with 100 μg/ml ampicillin and then incubated at 37 °C until OD600 = 0.45; then, it was cooled to 25°C. IPTG (final concentration: 1 mM) was added to the medium when OD600 reached 0.5. The culture was further incubated at 25^°C for five hours. The bacterial cell pellet was resuspended in lysis buffer [50 mM Tris- HC1, pH 8.0, 400 mM KCl, 10 mM 2-mercaptoethanol (hereinafter, sometimes abbreviated to "2ME") and 1 mM EDTA] containing 1 mM PefaBloc (Roche) , lysed by sonication, and centrifuged. The resultant clear lysate was applied to a GST affinity column (Pharmacia) and the column was then washed. The captured fusion protein was eluted with elution buffer (50 mM Tris-HCl, pH 8.0, and 10 mM glutathione) . The protein-containing fractions were concentrated, and then, the protein was cleaved by adding thereto bovine factor-Xa (Funakoshi) at a ratio of 1% (w/w) at 4°C. The mature hIL-18 protein was isolated using Sephacryl S-100 26/60 (Pharmacia). This fraction was stored at 4°C until further experiments.

Oligomerization Assay of the Wild-Type Protein Oligomerization assay of the wild-type protein was carried out in a sealed siliconized Eppendorf tube. A protein solution (400 ng/μl) in phosphate buffered saline (137 mM NaCI, 8.10 mM Na₂HP0₄, 2.68 mM KCl, and 1.47 mM KH₂P0, pH 7.0) was agitated at 150 rpm at 37 °C for 12 hours in an incubator. The sample solution was mixed with 5-fold concentrated SDS-sample buffer [final concentration 2% (w/v) SDS, 10% (v/v) glycerol, 0.002% (w/v) bromophenol blue, 62.5 mM Tris-HCl, pH 6.8, with/without 5% (v/v) 2- mercaptoethanol] and boiled for 5 min. The samples were electrophoresed on SDS-PAGE (10-20% gradient gel) and visualized by Coomassie blue staining. Structural Analyses of hIL-T8-Receptor Complex Multiple sequence alignment of IL-18 proteins was performed using ClustalW with BLOSUM matrix (Rastogi PA, "MacVector. Integrated sequence analysis for the Macintosh", Methods Mol Biol. 2000, 132:47-69). The inventors determined the structure of hIL-18 (PDB code: 1J0S) using nuclear magnetic resonance (NMR) ; herein, the inventors used the modeled structure of hIL-18:hIL18-Rα complex for structural analyses [Kato Z, Jee J, Shikano H, Mishima M, Ohki I, Ohnishi H, Li A, Hashimoto K, Matsukuma E, Cmoya K, Yamamoto ^'Y, Yoneda T, Hara T, Kondo N, and Shirakawa M. (2003) "The structure and binding mode of interleukin-18", Nature Structural Biology 10:966-971]. Structural rendering and analysis were performed with RasMol software [Sayle RA and Milner-White EJ "RASMOL: biomolecular graphics for all", Trends Biochem Sci. 199520:374].

Production and Analyses of Mutant Proteins Site-directed mutagenesis of the hIL-18 gene was performed using GeneEditor in vitro Site-Directed Mutagenesis System (Invitrogen) according to the manufacturer's instructions. Four different primers were designed to introduce cysteine to serine mutations at C38, C68, C76, and C127. The primer sequences were:

C38S: 5'-GACTGATTCTGACTCTAGAGATAATGCACC-3' (SEQ ID NO: 15) C68S: 5'-CTATCTCTGTGAAGTCTGAGAAAATTTCAACTC-3' (SEQ ID NO: 16) C76S: 5^,-GA^AATTTCA^CTCTCTCCTCGAGA^CAA^TATTTCC-3^, (SEQ ID NO: 17) C127S: 5 ' -GATACTTTCTAGCTTCTGAAAAAGΔGΑGΑG-3 ' (SEQ ID NO: 18) All the plasmid sequences harboring respective mutations were confirmed bidirectionally. Expression, purification, and oligomerization assays were performed in the same manner as performed on the wild-type protein. Biological Activity Assay (1) A biological activity assay based on IFN-γ production was carried out as previously described [Shikano H, Kato Z, Kaneko H, Watanabe M, Inoue R, Kasahara K, Takemura M and Kondo N, (2001) "IFN-γ production in response to IL-18 or IL-12 stimulation by peripheral blood mononuclear cells of atopic patients", Clinical and Experimental Allergy, 31: 1263-1270] . Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood of a control donor and suspended at a density of 10⁶ cells /ml in a culture medium. PBMCs were cultured in the presence or absence of recombinant hIL-18 protein for 24 h at 37 °C in a humidified atmosphere containing 5% CC2. The culture supernatant was centrifuged to remove cells and stored at -80 °C until use in assays. IFN-γ concentration was measured using a human IEN-γ enzyme-linked immunosorbent assay kit (JIMRO) .

Biological activity assay (2 ) A biological activity assay based on IFN- y induction was carried out as previously described [K. Konishi, F. Tanabe, M. Taniguchi , H . Yamauchi, T . Tanimoto, M . Ikeda, K. Orita, M . Kurimoto, A simple and sensitive bioassay for the detection of human interleukin-18 /interferon-gamma-inducing factor using human myelomonocytic KG-1 cells , J. Immunol . Methods 209 ( 1997 ) 187 - 191 . ] . Briefly, human myelomonocytic KG-1 cells were grown in the culture medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, L- glutamine (2 mmol/L) , penicillin ( 100 U/mL) and streptomycin ( 100 g/mL) . KG-1 cells ( 3 . 0 x 10⁵ cells ) were cultured in the presence of 0 . 1 - 50 . 0 ng/mL of recombinant hIL-18 for 24 h in a volume of 0.2 ml at 37 °C in a humidified atmosphere containing 5% C0₂. The culture supernatant was centrifuged to remove cells and stored at -80 °C until assay was performed. IFN-γ concentration was measured by fluorometric microvolume assay technology using FMAT 8100HTS system (Applied Biosystems) .

RESULTS

Oligomerization Assay of Wild-Type IL-18 In SDS-PAGE, the wild-type protein showed a marked oligomerization pattern after aeration by agitation (Fig. 1) . However, this oligomerization pattern completely disappeared in the presence of 2ME, which indicates that the oligomerization of the protein is mainly attributable to the intermolecular disulfide bonds in the cysteine residues of hIL-18.

Structural Analyses of hIL-18 and the Molecular Mechanisms of Receptor Binding Alignment of the amino acid sequences of IL-18 proteins showed marked similarities among different species. C76 and C127 are conserved in all the species, while C38 and C68 were replaced by the other residues in several species (Fig. 2) . Conservation among different species usually indicates that the conserved residues are important for the structure or activity; but conservation itself does not show the positions of the residues in the 3D- structure which are involved in intra- or inter-molecular disulfide bonds. The 3D-structure of hIL-18 determined by NMR clearly showed the absence of intramolecular disulfide bonds, and cysteine residues existing on the surface of the structure suggested a possible role of intermolecular disulfide bonds in the oligomerization (Fig. 3a) . The atomic interactions among the cysteine residues and the other residues of hIL-18, and also the positions of the cysteine residues on the complex structure with hIL-18Rα suggested that the replacement of the four cysteine residues with other types of amino acids, especially the conservative residue serine, does not collapse the 3D-structure of hIL-18; the mutated hIL-18 retains the capacity to bind to the receptor (Fig. 3a, 3b) . Based on the structural analyses, five mutant proteins (IL-18-C38, IL-18-C68, IL-18-C76, IL-18-C127 and IL-18- AS) were designed (Table 1) . The DNA sequence and amino acid sequence of wild-type hIL-18 are shown in SEQ ID NOS: 1 and 2, respectively. The DNA sequence and amino acid sequence of mutant IL-18-C38 are shown in SEQ ID NOS: 5 and 6, respectively. The DNA sequence and amino acid sequence of mutant IL-18-C68 are shown in SEQ ID NOS: 7 and 8, respectively. The DNA sequence and amino acid sequence of mutant IL-18-C76 are shown in SEQ ID NOS: 9 and 10, respectively. The DNA sequence and amino acid sequence of mutant IL-18-C127 are shown in SEQ ID NOS: 11 and 12, respectively. The DNA sequence and amino acid sequence of mutant IL-18-AS are shown in SEQ ID NOS: 3 and 4, respectively.

Oligomerization Assay of IL-18 Mutants In SDS-PAGE, mutants having one cysteine residue on the surface showed dimerization after the aeration procedure, but with different degrees (Fig. 4a, 4b) . These findings indicate that all the cysteine residues of hIL-18 are involved in the oligomerization phenomenon but to different extents. On the other hand, IL-18-AS protein did not show any oligomerization pattern even after the aeration procedure (Fig. 5) . This indicates that IL-18-AS protein can exist as a monomer under non-reducing conditions. Biological Activities of the Wild-Type Protein and Mutant Protein IL-18-AS

(1) The biological activities of the wild-type protein and mutant IL-18 -AS protein before aeration were compared. IFN-γ production in the presence of the two IL-18 proteins at four different concentrations showed no significant differences (Table 2) . The biological activities after aeration showed a marked reduction in the wild-type protein, while IL-18-AS did not show any significant reduction in activity even after extensive aeration

(Table 2) . These findings indicate that the newly created mutant, IL-18-AS, is highly stable under non-reducing conditions and yet retaining activities almost equivalent to those of the wild-type protein.

Biological activity of wild and mutant protein, IL-18-AS (2 ) The biological activities of the wild type and IL-18-AS before aeration were compared . IFN- y induction by different concentrations of the two IL-18 proteins showed no significant differences before oxidation ( Fig . 6) . The biological activities after aeration showed a marked reduction in the wild type protein; resulting in about five to ten times lower IFN- γ induction than that before oxidation between 1 and 10 ng/ml . However, IL-18-AS did not show any significant reduction even after extensive aeration ( Fig . 6 ) . These findings indicate that the newly generated mutant , IL-18-AS, is highly stable against non-reductive conditions ; retaining the equivalent biological activity . DISCUSSION The method of the present inventors for the purification of hIL-18 demonstrated that recombinant hIL-18 exists almost as a monomer in solution [Li A, Kato Z, Ohnishi H, Hashimoto K, Matsukuma E, Orioya K, Yamamoto Y, and Kondo N, (2003) "Optimized gene synthesis and high expression of human interleukin-18", Protein Expression and Purification, 32:110-11844]. However, oligomerization and inactivation of IL-18 have been reported [Seya T, Matsumoto M, Shiratori I, Fukumori Y and Toyoshima K, (2001) "Protein polymorphism of human IL-18 identified by monoclonal antibodies", International Journal of Molecular Medicine. 8:585-590; Kikkawa S, Matsumoto M, Shida K, Fukumori Y, Toyoshima K, and Seya T, (2001) "Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18", Biochemical and Biophysical Research Communications 281:461-467; Kikkawa S, Shida K, Okamura H, Begum NA, Matsumoto M, Tsuji S, Nomura M, Suzuki Y, Toyoshima K, and Seya T, "A comparative analysis of the antigenic, structural, and functional properties of three different preparations of recombinant human interleukin-18", J Interferon Cytokine Res. (2000) 20:179-85] and other report of the present inventors has also indicated that a small fraction of the purified protein existed as a dimer or trimer even under intensively reducing conditions. This suggests that the produced IL-18 is partially inactive (data not shown) . However, precise mechanisms of the inactivation have not yet been elucidated. Kikkawa et al. speculated that one of the inactivation mechanisms should be misfolding by the loss of the specific intramolecular covalent bond required for the potent IFN-γ inducing function of IL-18 [Kikkawa S, Matsumoto M, Shida K, Fukumori Y, Toyoshima K, and Seya T (2001) "Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18", Biochemical and Biophysical Research Communications 281:461-467], but the 3D-structure determined by the present inventors revealed that there is no intramolecular disulfide bond in hIL-18 (Fig. 3) . Further, the sidechains of all the cysteine residues exist on the surface of the protein, making it possible for them to be accessible by each other. From these observations, the present inventors speculate that the oligomerization of IL-18 is performed using these free sulfatides on the molecular surface, which leads to the loss of the activity. The present inventors have demonstrated here that wild-type IL-18 forms a large oligomer under non-reducing conditions, losing its IFN-γ inducing activity significantly. The oligomerization mechanism is mediated by the intermolecular disulfide bonds among the four cysteine residues in IL-18 polypeptide, i.e. C38, C68, C76, and C127. To obtain an anti- oxidatively stable hll-18, the present inventors have conservatively mutated all the cysteine residues to serine. As predicted by the 3D-structure analyses (Fig. 3) , it was revealed that the resulting mutant protein hIL-18- AS was highly stable and retained biological activities even after oxidation (Table 2, Fig. 6) . In human fibroblast growth factor (hFGF) , replacement of two of the four cysteine residues with serine residues (C70S and C88S) may increase instability, though the biological activities are retained. However, replacement of the other two cysteine residues with serine residues (C26S and C93S) results in a marked reduction in activity [Seno M, Sasada R, Iwane M, Sudo K, Kurokawa T, Ito K, and Igarashi K, (1988) "Stabilizing basic fibroblast growth factor using protein engineering", Biochemical and Biophysical Research Communications, 151:701-708; G.M. Fox, S.G. Schiffer, M.F. Rohde, L.B. Tsai, A.R. Bank, and T. Arakawa, (1988) "Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine", The Journal of Biological Chemistry, 263:18452-18458]. Biochemical analyses suggested that the two cysteine residues (C26 and C93) form intramolecular disulfide bonds [G.M. Fox, S.G. Schiffer, M.F. Rohde, L.B. Tsai, A.R. Bank, and T. Arakawa, (1988) "Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine", The Journal of Biological Chemistry, 263:18452-18458]. However, the structural determination of hFGF has revealed that C26 and C93 have no intramolecular sulfide bond with their side chains buried inside the molecule. This indicates that while the atomic interactions among the other residues will be important for the activity, the side chains of C70 and C88 are exposed to the solvent without forming any disulfide bonds [Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, Yeh BK, Yayon A, Linhardt RJ, and Mohammad! M, "Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization", Mol Cell. (2000)6:743-5018]. Moreover, the recent structure determination of complexes including hFGF:FGF receptor has also revealed that C26 and C93 exist adjacent to the interface between ligand and receptor, and that C70 and C88 are completely free from the interface of the receptor. Similar observations were made on hIL-18 and its receptor [Kato Z, Jee J, Shikano H, Mishima M, Ohki I, Ohnishi H, Li A, Hashimoto K, Matsukuma E, Qmoya K, Yamamoto Y, Yoneda T, Hara T, Kondo N, and Shirakawa M, (2003) "The structure and binding mode of interleukin-18", Nature Structural Biology, 10:966-971; Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, Yeh BK, Yayon A, Linhardt RJ, and Mohammadi M, "Crystal structure of a ternary FGF-FGFR- heparin complex reveals a dual role for heparin in FGFR binding and dimerization", Mol Cell. (2000) 6:743-50]. In order to use a protein as a therapeutic agent, not only production of the protein in a homogeneous form and in a sufficient quantity, but also a stable formulation suitable for storage and delivery are required. The IL- 18-AS that the present inventors have developed showed considerable stability against oxidation and yet retained almost the same activity as that of the wild-type protein. Although recombinant hIL-18 has been examined in the treatment of cancer [Yamanaka K, Hara I, Nagai H, et al., "Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model", Cancer Immunol Immunother (1999) 48:297-302; Nagai H, Hara I, Horikawa T, et al., "Antitumor effects on mouse melanoma elicited by local secretion of interleukin-12 and their enhancement by treatment with interleukin-18", Cancer Invest (2000) 18:206-13; Herzyk DJ, Bugelski PJ, Hart TK, and Wier PJ, "Preclinical safety of recombinant human interleukin-18", Toxicol Pathol. (2003)31:554-61], the structure-based design of mutants is an effective approach, and a stable form of cytokine can be the first step for development of a more potent cytokine with site-specific modifications including glycosylation or pegylation [Sareneva T, Pirhonen J, Cantell K, and Julkunen I, "N-glycosylation of human interferon-gamma: glycans at Asn- 25 are critical for protease resistance", Biochem J. (1995) 308:9-14; Ohno S, Yokogawa T, Fujii I, Asahara H, Inokuchi H, and Nishikawa K, "Co-expression of yeast amber suppressor tRNATyr and tyrosyl-tRNA synthetase in Escherichia coli: possibility to expand the genetic code", J Biochem. (1998) 124:1065-8; Kiga D, Sakamoto K, Kodama K, Kigawa T, Matsuda T, Yabuki T, Shirouzu M, Harada Y, Nakayama H, Takio K, Hasegawa Y, Endo Y, Hirao I, and Yokoyama S, "An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system", Proc Natl Acad Sci U S A, (2002) 99:9715-20; Wang YS, Youngster S, Grace M, Bausch J, Bordens R, and Wyss DF, "Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications", Adv Drug Deliv Rev. (2002) 54 : 547-70] .

Table 1 Amino acid compositions of wild and mutant proteins

Position 38 68 76 127

Wild- -type Cys Cys Cys Cys

C38 Cys Ser Ser Ser

C68 Ser Cys Ser Ser

C76 Ser Ser Cys Ser

C127 Ser Ser Ser Cys

AS Ser Ser Ser Ser

Table 2 IFN-γ induction (pg/ml ) ^* by wild-type and AS mutant proteins before and after oxidation

hIL-18 Wild-type AS (ng/ l) before after before after

25 120.7±51.1 66.4+29.1 127.9+34.7 145.9+51.3 50 310.5+11.5 148.3+39.1 469.7+88.4 474.2+37.6 100 780.3±123.4 506.0±60.2 800.7+40.5 . 792.5+38.4 400 1063.8+70.0 1148.1+97.1 1157.0+0.9 1056.5±10.8

Mean values of triplicate interferon-γ induction assays are shown with standard deviation. The entire disclosure of Japanese Patent application No. 2004-019567 filed on January 28, 2004 including specification, claims, drawings and summary is incorporated herein by reference in its entirety. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Industrial Applicability According to the present invention, a novel mutant of interleukin-18 protein has been provided. This mutant is highly stable under non-reducing conditions, does not form oligomers, and is retaining sufficient biological activities. Therefore, this mutant is applicable to medicines for human or other animals (such as anti-tumor agents, therapeutics for allergy, etc.) or to experiments conveniently.

Sequence Listing Free Text SEQ ID NO: 1 shows the DNA sequence of wild-type hIL-18. SEQ ID NO: 2 shows the amino acid sequence of wild-type hIL-18. SEQ ID NO: 3 shows the DNA sequence of an IL-18 mutant (IL-18-AS) . SEQ ID NO: 4 shows the amino acid sequence of an IL-18 mutant (IL-18- AS). SEQ ID NO: 5 shows the DNA sequence of an IL-18 mutant (IL-18-C38) . SEQ ID NO: 6 shows the amino acid sequence of an IL-18 mutant (IL-18- C38). SEQ ID NO: 7 shows the DNA sequence of an IL-18 mutant (IL-18-C68) . SEQ ID NO: 8 shows the amino acid sequence of an IL-18 mutant (IL-18- C68). SEQ ID NO: 9 shows the DNA sequence of an IL-18 mutant (IL-18-C76) . SEQ ID NO: 10 shows the amino acid sequence of an IL-18 mutant (IL-18- C76) . SEQ ID NO: 11 shows the nucleotide sequence of an IL-18 mutant (IL-18- C127) . SEQ ID NO: 12 shows the amino acid sequence of an IL-18 mutant (IL-18- C127) . SEQ ID NO: 13 shows the sequence of an optimized cDNA. SEQ ID NO: 14 shows the amino acid sequence encoded by an optimized cDNA. SEQ ID NO: 15 shows the primer sequence which was designed to introduce a cysteine to serine mutation at position 38. SEQ ID NO: 16 shows the primer sequence which was designed to introduce a cysteine to serine mutation at position 68. SEQ ID NO: 17 shows the primer sequence which was designed to introduce a cysteine to serine mutation at position 76. SEQ ID NO: 18 shows the primer sequence which was designed to introduce a cysteine to serine mutation at position 127.

Claims

1. An interleukin-18 mutant protein comprising: (a) an interleukin-18 mutant protein having an amino acid sequence as shown in SEQ ID NO: 2 but with at least one of the cysteine residues at positions 38, 68, 76 and 127 being substituted with other amino acid residues; (b) an interleukin-18 mutant protein having an amino acid sequence of the mutant protein of (a) above but with one or several amino acid residues other than the amino acid residues at positions 38, 68, 76 and 127 being deleted, substituted or added, and which is more stable than a wild-type interleukin-18 protein having an amino acid sequence as shown in SEQ ID NO: 2 under non-reducing conditions; or (c) an interleukin-18 mutant protein encoded by an DNA that hybridizes under a stringent condition with a complement of SEQ ID NO. 1 or 13 that encodes an interleukin-18 protein, wherein at least amino acid residues of the mutant protein at positions 38, 68, 76 and 127 are other than cysteine residues; or a salt of (a) , (b) or (c) .

2. The interleukin-18 mutant protein or a salt thereof according to claim 1, wherein the other amino acid residues are serine residues.

3. The interleukin-18 mutant protein or a salt thereof according to claim 1, wherein the other amino acid residues are selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, threonine, glutamine, asparagine, tyrosine, lysine, arginine, histidine, aspartic acid and glutamic acid residues.

4. The interleukin-18 mutant protein or a salt thereof according to claim 1, wherein said interleukin-18 mutant protein is a mutant of a human- derived wild-type interleukin-18 protein.

5. The interleukin-18 mutant protein or a salt thereof according to claim 1, wherein said interleukin-18 mutant protein is a mutant of a wild- type interleukin-18 protein derived from an animal other than human.

6. The interleukin-18 mutant protein or a salt thereof according to claim 1, having an amino acid sequence as shown in SEQ ID NO: 4.

7. An isolated DNA encoding the protein according to claim 1.

8. A recombinant vector comprising the DNA according to claim 7.

9. A transformant comprising the recombinant vector according to claim

10. A method of producing an interleukin-18 mutant protein, comprising culturing a host transformed with the DNA according to claim 7 and recovering the interleukin-18 mutant protein from the resultant culture.

11. An agonist or antagonist for an interleukin-18 protein, wherein said agonist or antagonist is the interleukin-18 mutant protein or a salt thereof according to claim 1.

12. A pharmaceutical composition comprising the interleukin-18 mutant protein or a salt thereof according to claim 1 as an active ingredient.

13. An antibody to the interleukin-18 mutant protein or a salt thereof according to claim 1.