Osteoclastgenic inhibitory agent comprising interleukin-18

[Technical Field to which the Invention Affiliates] The present invention relates to an osteoclast formation inhibitor comprising interleukin-18 (hereinafter abbreviated as "IL-18") or a functional derivative thereof.

[0002] In a healthy living body, bone formation by osteoblasts and bone resorption by osteoclasts are maintained in a well-balanced manner, and as a result, bone tissue always becomes new bone without changing its basic shape. It has been replaced and remains healthy. In addition, this balance plays an important role in maintaining homeostasis of the living body, such as keeping the calcium concentration in the blood constant. On the other hand, when this balance is lost, especially when the amount of bone resorption exceeds the amount of bone formation, not only bone-related diseases but also various other diseases are caused. Therefore, elucidation of a series of bone resorption mechanisms in living organisms, especially elucidation of the formation mechanism of osteoclasts, is not only scientifically significant but also clinically extremely significant, and has received a great deal of attention. I'm collecting.

[0003] However, it can be said that the mechanism of osteoclast formation has not been completely elucidated even though, for example, interleukin-1 as a promoter and interleukin-4 as an inhibitor have been confirmed. Not in. It is considered that this is because the formation of osteoclasts in the living body is also controlled in a complicated and closely related manner by many promoters and inhibitors, like various phenomena in the living body. There is. From these facts, it is desired to establish an effective osteoclast formation inhibitor not only from a scientific point of view but also from a clinical point of view.

[0004] In view of such circumstances, an object of the present invention is to provide an effective and novel osteoclast formation inhibitor.

[0005] [Means for Solving Problems] [0006] IL-18 is a type of cytokine that is a signal transmitter in the immune system. At the time of its discovery, this cytokine was first discovered in JP-A-8-27189, JP-A-8-193098, and Haruki Okamura et al., "Nature," Vol. 378, No. 6,552, pp. 88-91 (1995). It was described as an interferon-γ inducer, as seen in, but subsequently according to the proposal in Simpei Ushio et al., The Journal of Immunology, Vol. 156, pp. 4,274-4,279 (1996). , IL-18 came to be called. IL-18 is abbreviated as interferon-γ (hereinafter abbreviated as IFN-γ) and granulocyte macrophage colony stimulating factor (hereinafter abbreviated as GM-CSF), which are useful as physiologically active substances in immunocompetent cells. ), In addition, it has the property of enhancing the cytotoxicity of killer cells and inducing the production of killer cells.

[0007] On the other hand, in the process of advancing research to solve the above problems, the present inventors have the ability to inhibit the formation of osteoclasts from precursor osteoclasts in-vitro, which is derived from mouse bone marrow. It was found that a specific gene is specifically expressed in a large amount in a species of osteoclast cell line. Further detailed analysis revealed that the gene encodes IL-18 having the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing. Based on these findings, further diligent research revealed that IL-18 having the amino acid sequence and its functional derivative significantly inhibited the formation of osteoclasts, and that this inhibition was mainly induced and produced by the IL-18. It was found that it was due to the action of GM-CSF. The present invention has been completed based on the above-mentioned original knowledge.

[0008] That is, the present invention solves the above-mentioned problems with an osteoclast formation inhibitor comprising IL-18 or a functional derivative thereof.

[Embodiments of the Invention] The osteoclast formation inhibitor of the present invention comprises IL-18 or a functional derivative thereof. The IL-18 here includes all the polypeptides having the above-mentioned properties as IL-18, and the source and origin thereof do not matter. The IL-18 used in the present invention has, for example, SEQ ID NOs: 1, 2 and 3 in the sequence listing as a partial amino acid sequence in the middle portion, and further the amino acid sequences shown in SEQ ID NOs: 4 and 5, and has a sequence number as a whole. IL-18 having the amino acid sequence shown in 6 or 7 can be mentioned. Further, the functional derivative referred to in the present specification is one of the amino acids in the amino acid sequence as long as it does not substantially lose the property of inhibiting the formation of osteoclastic cells among the properties of IL-18 as described above. One or two or more substituted with other amino acids, one or two or more amino acids added to the N-terminal and / or C-terminal of these amino acid sequences, one or one in the middle part of these amino acid sequences. Those with two or more amino acids inserted, those with one or two or more deleted N-terminal and / or C-terminal amino acids in these amino acid sequences, and one or 2 amino acids in the middle part of these amino acid sequences. It means that more than one is deleted. As such a functional derivative, for example, in the specification of Japanese Patent Application No. 9-20906 (priority claim in Japanese Patent Application No. 9-329715) by the same patent applicant, the property as IL-18 is substantially exhibited. The amino acid sequence of the functional derivative whose stability has been improved while retaining it is described in detail. Furthermore, the functional derivative referred to in the present specification also includes the above-mentioned IL-18 or a polypeptide in which a sugar chain is added to the functional derivative thereof. The above IL-18 or its functional derivative (hereinafter, abbreviated as "the IL-18" including both IL-18 and its functional derivative) is abbreviated by a cell culture method or the like. It may be isolated from a natural source or artificially synthesized by recombinant DNA technology or peptide synthesis.

[0010] From an economical point of view, the method using recombinant DNA technology is advantageous, and in such a method, usually, a DNA encoding the IL-18 is introduced into an appropriate host derived from a microorganism or an animal or plant to form a trait. After culturing this as a transformant by a conventional method, the culture is purified by a conventional method for purifying cytokines to obtain the IL-18. The DNA used here may be any DNA as long as it contains the DNA encoding the IL-18, and a DNA having an appropriate sequence may be used depending on the purpose of use of the osteoclast formation inhibitor and the method of application. You can choose. For example, in Japanese Patent Application Laid-Open No. 8-193098, Japanese Patent Application Laid-Open No. 8-231598, and Japanese Patent Application Laid-Open No. 8-27189 by the same patent applicant, DNA containing a DNA encoding IL-18 derived from mice and humans was introduced. The method for producing IL-18 obtained by culturing the transformed transformant microorganism is also described in the specification of Japanese Patent Application No. 8-185305 (claimed priority in Japanese Patent Application No. 9-187418) by the same patent applicant. Describes in detail a method for producing IL-18 obtained by culturing a transformant animal cell into which DNA containing chromosomal DNA, which encodes human-derived IL-18, has been introduced. Furthermore, DNA containing DNA encoding a human IL-18 functional derivative was introduced into the specification of Japanese Patent Application No. 9-20906 (priority claim in Japanese Patent Application No. 9-329715) by the same patent applicant. The method for producing IL-18 obtained by culturing transformant animal cells is described in detail.

[0011] While the recombinant DNA technology as described above is economically advantageous as described above, the IL-18 obtained by such a method is originally in vivo depending on the host used and the DNA sequence. There may be slight differences in physicochemical properties from IL-18, which is produced and functions. However, in the specification of Japanese Patent Application No. 8-67434 (claimed priority in Japanese Patent Application Laid-Open No. 8-269105 (Japanese Patent Application Laid-Open No. 9-289896)) by the same patent applicant, it was cultivated as a natural source. The method for producing IL-18 using human cells is also described in the specification of Japanese Patent Application No. 8-213267 (priority claim in Japanese Patent Application No. 9-213885) by the same patent applicant. The method for producing IL-18 using 1β-converting enzyme is described in detail, and is the IL-18 obtained by these methods substantially the same in physicochemical properties as the IL-18 produced and functioning in vivo? Or, since it is considered to be extremely close to the same, the production amount is slightly low, but it is advantageous in terms of low side effects when used as a drug premised on administration to warm-blooded animals including humans, for example. is there. When applying the purification method using a monoclonal antibody specific to the IL-18 disclosed in JP-A-8-231598 by the same patent applicant, the high-purity IL-18 is minimized. It can be obtained with limited cost and effort.

[0012] The osteoclast formation inhibitor of the present invention comprises the IL-18 thus obtained, and inhibits the formation of osteoclasts regardless of in-vitro or in-vivo. Includes all forms used for. For example, a kit for screening a bone-related disease agent as a medium component for cell culture of animal cells and the like, which inhibits the formation of osteoclasts and improves the maintenance, proliferation and / or differentiation of desired cells. It can be advantageously used as a constituent, as a bone resorption regulator, and as an osteoclast-related disease agent. The bone resorption regulator referred to here is a drug that regulates bone resorption to a normal range by inhibiting the formation of osteoclasts in the living body and improves poor physical condition such as relatively minor joint pain. Includes health foods, etc. In addition, the osteoclast-related disease agent referred to here includes an agent for preventing and / or treating all diseases associated with the excessive formation and / or function of osteoclasts in vivo. Target diseases include, for example, hypercalcemia, osteoclastoma, Paget's disease, osteosarcoma, arthritis, rheumatoid arthritis, osteoarthritis, primary hyperthyroidism, osteopenia, osteoporosis, etc. Can be mentioned. Depending on the dosage form and the subject of use, the osteoclast formation inhibitor of the present invention is usually prepared in the form of a liquid, paste or solid, and the IL-18 is preferably 0.000002 to 100% (w / w). Contains 0.0002 to 0.5% (w / w).

[0013] The osteoclast formation inhibitor of the present invention is not only in the form of IL-18 alone, but also in other forms such as, for example, a carrier, an excipient, a diluent, an immunostimulatory agent, an antibiotic, and a serum glucose as a stabilizer. And proteins such as gelatin, glucose, maltose, maltotriose, malttetraose, trehalose, sucrose, isomaltose, lactose, panose, sucrose, palatinose, lactosucrose, raffinose, fructo-oligo, galactooligosaccharide, lentinan, dextrin and purulan Sugars such as sugars such as sorbitol, maltose, sucrose such as lactitol and maltotriitol, sugars such as maltotriitol, buffers mainly composed of phosphates or citrates, 2-mercaptoethanol, dithiotreitol and reduced forms. Reducing agents such as glutathione, and if necessary, interferon-α, interferon-β, interferon-γ, interleokin 2, interleokin 3, interleokin 6, interleoclast 12, TNF-α, TNF-β, GM-CSF, estrogen, progesterone, chlormaginone acetate, calcitonin, somatokine, somatomedin, insulin-like growth factor, ipriflavon, paracyloid hormone, noruethisterone, sucrose, ancitabine, citarabin, fluorouracil, tetrahydrofurylfluorouracil, methotrexate, vitamin D₂, Active vitamin D, crestin, L-aspartinase and other physiologically active substances such as pisibanil, and one or 2 calcium salts such as calcium lactate, calcium chloride, calcium hydrogen phosphate, calcium L-aspartate. It also includes the form as a composition with more than a species. When the osteoclast formation inhibitor of the present invention is administered to a warm-blooded animal including humans, for example, as an osteoclast-related disease agent, a physiologically acceptable substance from the above is appropriately selected. It is desirable to select the composition as the composition.

[0014] Further, the osteoclast formation inhibitor of the present invention also includes a drug in the form of a dosage unit when administered to a warm-blooded animal including humans, and the drug in the form of a dosage unit is defined as the drug in the form of a dosage unit. The IL-18 is contained, for example, in an amount corresponding to a single dose or an integral multiple (up to 4 times) or a divisor (up to 1/40) thereof, and is physically separated and suitable for administration. Means a drug in the dosage form of. Examples of the drug in such a dosage form include injections, liquids, powders, granules, tablets, capsules, sublinguals, eye drops, nasal drops, suppositories and the like.

[0015] The osteoclast-related disease according to the present invention, whether orally or parenterally, the osteoclast-related inhibitor as an osteoclast-forming inhibitor. It is effective in the treatment and prevention of. Although it depends on the type and symptoms of the disease, specifically, while observing the patient's symptoms and the course after administration, the IL is about 0.5 μg to 100 mg / dose per adult, preferably about 2 μg to 10 mg / dose. -18 may be orally administered at doses of 2 to 6 times / day or 2 to 10 times / week for 1 day to 1 year, or parenterally, intradermally, subcutaneously, intramuscularly or intravenously. ..

[0016] Next, an experimental example will be shown, and the preparation of the IL-18, the physicochemical properties of the IL-18, and the biological action will be described.

[Experimental Example 1] <Preparation of human IL-18> Autonomous replication in which cDNA encoding human IL-18 is ligated according to the method described in JP-A-8-231598 by the same patent applicant. A possible recombinant DNA "pKGFHH2" was prepared. As a result of analysis by the dideoxy method, as shown in FIG. 1, in this recombinant DNA, the cDNA "KGFHH2 cDNA" containing the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing is linked downstream of the Tac promoter "Ptac". It was. In addition, this recombinant DNA "pKGFHH2" contained all the base sequences encoding the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 and 5 in the sequence listing. That is, these amino acid sequences are based on the 46th to 63rd, 88th to 105th, 400th to 420th, 151st to 165th and 214th to 228th bases in the base sequence shown in SEQ ID NO: 8, respectively. It was coded.

[0018] Further, according to the method described in JP-A-8-231598, this recombinant DNA "pKGFHH2" was subjected to Escherichia coli Y1090 strain (ATCC). 37197) was introduced, cultured, and the produced polypeptide was purified by immunoaffinity chromatography. As a result, purified human IL-18 having a purity of 95% or more was obtained in a yield of about 25 mg per liter of the culture solution. This purified human IL-18 was analyzed as shown below according to the method described in JP-A-8-193098 by the same patent applicant, and its biological action and physicochemical properties were confirmed. That is, when human lymphocytes collected from healthy subjects by a conventional method are cultured in the presence of various concentrations of purified human IL-18, IFN-γ production depending on the concentration of purified human IL-18 present is observed. It was confirmed that this purified IL-18 has a biological action that induces the production of IFN-γ in lymphocytes, which are immunocompetent cells. SDS-polyacrylamide gel electrophoresis under non-reducing conditions according to the method reported by UK Remli in Nature, Vol. 227, pp. 680-685 (1970), corresponds to a molecular weight of 18,500 ± 3,000 daltons. While the main band capable of inducing IFN-γ was shown at the position where it was subjected to, the isoelectric point was shown at 4.9 ± 1.0 when chromatographic focusing was performed according to a conventional method. In addition, when the N-terminal was analyzed using a protein sequencer "473A type" manufactured by Perkin Elmer according to a conventional method, the sequence number in which methionine was bound to the N-terminal in the amino acid sequence shown in SEQ ID NO: 8 of the sequence listing. It was confirmed that it had the amino acid sequence shown in 9.

[Experimental Example 2] <Preparation of Human IL-18> Japanese Patent Application No. 8-67434 by the same patent applicant (Priority claim in Japanese Patent Application No. 8-269105 (Japanese Unexamined Patent Publication No. 9-289896)) THP-1 cells (ATCC TIB202), a type of myelomonocytic cell line derived from human acute monocytic leukemia, are transplanted subcutaneously into the back of a newborn newborn hamster according to the method described in the above specification. , Raised for 3 weeks. Then, the tumor mass (about 15 g / animal) generated under the skin is removed and dispersed, the cells are disrupted, the produced polypeptide is purified by immunoaffinity chromatography, and the purified human IL-18 is hamstered. It was obtained with a yield of about 50 ng per animal.

[0020] This purified human IL-18 was analyzed as shown below according to the method also described in Japanese Patent Application No. 8-67434 to confirm its biological action and physicochemical properties. That is, when human lymphocytes collected from healthy subjects by a conventional method are cultured in the presence of various concentrations of purified human IL-18, IFN-γ production depending on the concentration of purified human IL-18 present is observed. It was confirmed that this purified IL-18 has a biological action that induces the production of IFN-γ in lymphocytes, which are immunocompetent cells. SDS-polyacrylamide in the presence of 2% (w / v) dithiothreitol as a reducing agent, according to the method reported by UK Remli in Nature, Vol. 272, pp. 680-685 (1970). Gel electrophoresis showed a major band capable of inducing IFN-γ at positions corresponding to molecular weights of 18,000 to 19,500 daltons. Furthermore, according to the peptide map whose method is also described in the specification of Japanese Patent Application No. 8-67434, a polypeptide fragment obtained by treating this purified human IL-18 with a Sigma cross-tripine agent is prepared by Toso. When the amino acid sequences of individual peptide fragments were analyzed from the N-terminal side by subjecting them to high performance liquid chromatography using the column of "ODS-120T" manufactured by ODS-120T, SEQ ID NOs: 10, 11, 12 and 13 in the sequence listing were analyzed. The amino acid sequence shown in is obtained. Each of these amino acid sequences is in the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing.<u style="single">No.</u>It was in perfect agreement with the 148th to 157th, 1st to 13th, 45th to 58th and 80th to 96th sequences. The above results show that IL-18 obtained by the method of Experimental Example 2 has the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing, and the portions shown in SEQ ID NOs: 1, 2, 3, 4 and 5 thereof. It shows that it has all the amino acid sequences.

[Experimental Example 3] &lt;Preparation of Functional Derivatives&gt; According to the method described in the specification of Japanese Patent Application No. 9-20906 (priority claim in Japanese Patent Application No. 9-329715) by the same patent applicant. DNA encoding a human IL-18 functional derivative in which the 38th cysteine in the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing is replaced with serine, the 68th cysteine is replaced with serine, and the 76th cysteine is replaced with alanine. An autonomously replicable recombinant DNA "pCSHIGIF / MUT35" was prepared. Analysis by the dideoxy method revealed that, as shown in Fig. 2, in this recombinant DNA, Karsten Henko et al. In "Journal of Molecular Biology", Vol. 185, pp. 227-260 (1985). The DNA "IGIF / MUT35" having the nucleotide sequence shown in SEQ ID NO: 14 in the sequence listing is linked to the downstream of the nucleotide sequence encoding the signal peptide of subtype α2b in the reported human interferon-α in the same reading frame. And further downstream, there was a stop codon for protein synthesis. The amino acid sequence encoded by this DNA is as shown in SEQ ID NO: 14 in the sequence listing, with the 38th cysteine in the amino acid sequence of SEQ ID NO: 6 in the sequence listing being serine, the 68th cysteine being serine, and the 76th cysteine. It consisted of replacing the second cysteine with alanine. In addition, this DNA is an amino acid sequence in which all the amino acid sequences shown in SEQ ID NOs: 1, 2, 3 and 4 in the sequence listing and the cysteine of the fifth amino acid of the amino acid sequence shown in SEQ ID NO: 5 are replaced with alanine. It contained a base sequence encoding. That is, these amino acid sequences are based on the 46th to 63rd, 88th to 105th, 400th to 420th, 151st to 165th and 214th to 228th bases in the base sequence shown in SEQ ID NO: 14, respectively. It was coded.

[0022] Further, according to the method described in the specification of Japanese Patent Application No. 9-20906 (claimed priority in Japanese Patent Application No. 9-329715) by the same patent applicant, this recombinant DNA "pCSHIGIF / MUT35" is applied. It is introduced into COS-1 cells (ATCC CRL1650), which is a type of cell line derived from the kidney of African green monkey, cultured, and the produced polypeptide is purified by immunoaffinity chromatography to purify human IL-18 function. The sex derivative was obtained with a yield of about 40 ng per 1 ml of culture medium. Further, according to the method described in the specification of Japanese Patent Application No. 9-20906 (priority claim in Japanese Patent Application No. 9-329715) by the same patent applicant, this purified human IL-18 functional derivative is prepared as follows. The biological action and physicochemical properties were confirmed by analysis as shown in. That is, KG-1 cells (ATCC), which is a type of cell lineage derived from human acute myeloid leukemia in the presence of various concentrations of this purified human IL-18 functional derivative. When CCL246) was cultured, IFN-γ production was observed depending on the concentration of the purified human IL-18 functional derivative present, and this purified IL-18 functional derivative was used as an immunocompetent cell for KG-1 cells. It was confirmed that it has a biological action that induces the production of IFN-γ in. SDS-polyacrylamide in the presence of 2% (w / v) dithiothreitol as a reducing agent, according to the method reported by UK Remuri in Nature, Vol. 227, pp. 680-685 (1970). Gel electrophoresis showed a major band capable of inducing IFN-γ at positions corresponding to molecular weights of 18,000 to 19,500 daltons. In addition, when the N-terminal was analyzed using a protein sequencer "473A type" manufactured by Perkin Elmer according to a conventional method, it was completely the same as the amino acid sequence of the N-terminal part in the amino acid sequence shown in SEQ ID NO: 14 of the sequence listing. It was confirmed that it had a matching amino acid sequence shown in SEQ ID NO: 15 in the sequence listing.

[Experimental Example 4] &lt;Preparation of Functional Derivatives&gt; According to the method described in the specification of Japanese Patent Application No. 9-20906 (claimed priority in Japanese Patent Application No. 9-329715) by the same patent applicant. Human IL in which the 38th cysteine in the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing is replaced with serine, the 68th cysteine is replaced with serine, the 76th cysteine is replaced with alanine, and the 127th cysteine is replaced with serine. -18 An autonomously replicable recombinant DNA "pCSHIGIF / MUT42" was prepared, in which a DNA encoding a functional derivative was ligated. Analysis by the dideoxy method revealed that in this recombinant DNA, Karsten Henko et al., "Journal of Molecular Biology," Vol. 185, pp. 227-260 (1985). The DNA "IGIF / MUT42" having the nucleotide sequence shown in SEQ ID NO: 16 in the sequence listing is linked to the downstream of the nucleotide sequence encoding the signal peptide of subtype α2b in the reported human interferon-α in the same reading frame. And further downstream, there was a stop codon for protein synthesis. The amino acid sequence encoded by this DNA is as shown in SEQ ID NO: 16 in the sequence listing, with the 38th cysteine in the amino acid sequence in the SEQ ID NO: 6 being serine, the 68th cysteine being serine, and the 76th cysteine. It consisted of substituting cysteine with alanine and the 127th cysteine with serine. In addition, in this DNA, all the amino acid sequences shown in SEQ ID NOs: 1, 2, 3 and 4 in the sequence listing and the cysteine of the fifth amino acid in the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing are replaced with alanine. It contained a base sequence encoding an amino acid sequence. That is, these amino acid sequences are the 46th to 63rd, 88th to 105th, 400th to 420th, 151st to 165th and 214th to 228th positions in the base sequence shown in SEQ ID NO: 16 in the sequence listing, respectively. It was encoded by a base.

[0024] Further, according to the method described in the specification of Japanese Patent Application No. 9-20906 (priority claim in Japanese Patent Application No. 9-329715) by the same patent applicant, this recombinant DNA "pCSHIGIF / MUT42" is subjected to. The polypeptide was introduced into COS-1 cells, cultured, and the produced polypeptide was purified by immunoaffinity chromatography to obtain a purified human IL-18 functional derivative in a yield of about 20 ng per 1 ml of culture medium. Further, according to the method described in the specification of Japanese Patent Application No. 9-20906 (priority claim in Japanese Patent Application No. 9-329715) by the same patent applicant, this purified human IL-18 functional derivative is prepared as follows. The biological action and physicochemical properties were confirmed by analysis as shown in. That is, when KG-1 cells were cultured in the presence of various concentrations of this purified human IL-18 functional derivative, IFN-γ production was observed depending on the concentration of the purified human IL-18 functional derivative present. It was confirmed that this purified IL-18 functional derivative has a biological action that induces the production of IFN-γ in KG-1 cells as immunocompetent cells. SDS-polyacrylamide in the presence of 2% (w / v) dithiothreitol as a reducing agent, according to the method reported by UK Remuri in Nature, Vol. 227, pp. 680-685 (1970). Gel electrophoresis showed a major band capable of inducing IFN-γ at positions corresponding to molecular weights of 18,000 to 19,500 daltons. In addition, when the N-terminal was analyzed using a protein sequencer "473A type" manufactured by Perkin Elmer according to a conventional method, the amino acid sequence of the N-terminal part in the amino acid sequence shown in SEQ ID NO: 16 of the sequence listing was completely matched. It was confirmed that it had a matching amino acid sequence shown in SEQ ID NO: 15 in the sequence listing.

[Experimental Example 5] &lt;Preparation of human IL-18&gt; According to the method described in the specification of Japanese Patent Application No. 8-185305 (priority claim in Japanese Patent Application No. 9-187418) by the same patent applicant. , A recombinant DNA "pBGHuGF" capable of autonomous replication was prepared by ligating the chromosomal DNA encoding human IL-18. As a result of analysis by the dideoxy method, as shown in FIG. 4, in this recombinant DNA, the DNA "HuIGIF" having the base sequence shown in SEQ ID NO: 17 in the sequence listing, which is a chromosomal DNA encoding human IL-18, is restricted. Enzyme Hind It was connected downstream of the site cut by III. As shown in SEQ ID NO: 17 in the sequence listing, this chromosomal DNA "HuIGIF" consists of 11,464 bp, and is 83 to 1,453, 1,466 to 4,848, 4,984 to 6,317, and 6,452 from the 5'end of this sequence. The exons were divided by four introns located at the 11th and 224th positions. In the sequence excluding these introns, the 3rd to 11443th bases from the 5'end are the parts encoding the human IL-18 precursor, and the 4866th to 4983th bases are active human ILs. It was the part that coded -18. In addition, this DNA contained all the base sequences encoding the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 and 5 in the sequence listing. That is, these amino acid sequences are encoded by the bases 4,911 to 4,928, 4,953 to 4,970, 11,372 to 11,392, 6,350 to 6,364 and 6,413 to 6,427, respectively, in the base sequence shown in SEQ ID NO: 17. It had been.

[0026] Further, according to the method described in the specification of Japanese Patent Application No. 8-185305 (priority claim in Japanese Patent Application No. 9-187418) by the same patent applicant, this recombinant DNA "pBGHuGF" is applied to Chinese. CHO-K1 cells (ATCC), which are cell lines derived from hamster ovary cells It was introduced into CCL61), cultured, and the supernatant of the cell disruption obtained by culturing THP-1 cells was allowed to act on the culture supernatant, and the produced polypeptide was purified by immunoaffinity chromatography to purify humans. IL-18 was obtained with a yield of about 15 mg per liter of culture. Further, this purified human IL-18 is subjected to the following according to the method described in the specification of Japanese Patent Application No. 8-185305 (priority claim in Japanese Patent Application No. 9-187418) by the same patent applicant. The biological action and physicochemical properties were confirmed. That is, when human lymphocytes collected from healthy subjects by a conventional method are cultured in the presence of various concentrations of purified human IL-18, IFN-γ production depending on the concentration of purified human IL-18 present is observed. It was confirmed that this purified IL-18 has a biological action that induces the production of IFN-γ in lymphocytes, which are immunocompetent cells. SDS-polyacrylamide in the presence of 2% (w / v) dithiothreitol as a reducing agent, according to the method reported by UK Remuri in Nature, Vol. 227, pp. 680-685 (1970). Gel electrophoresis showed a major band capable of inducing IFN-γ at positions corresponding to molecular weights of 18,000 to 19,500 daltons. In addition, the N-terminal has the amino acid sequence shown in SEQ ID NO: 15 in the sequence listing that completely matches the amino acid sequence of the N-terminal portion of active IL-18 among the amino acid sequences shown in SEQ ID NO: 17 in the sequence listing. Was.

[Experimental Example 6] &lt;Preparation of mouse IL-18&gt; 8 μl of 25 mM magnesium chloride, 10 μl of 10 × PCR buffer, 1 μl of 25 mM dNTP mix, 2.5 units / μl amplitac DNA polymerase in a 0.5 ml reaction tube. 1 μl, prepared from a phage DNA clone according to the method described in JP-A-8-27189, has the nucleotide sequence shown in SEQ ID NO: 18 in the sequence listing, and has the amino acid sequence shown in SEQ ID NO: 7, mouse IL-18. 5 ́-ATAGAATTCAAATGAACTTTGGCCGACTTCACTG-3 ́ and 5 ́-ATAAAGCTTCTAACTTTGATGTAAGTT-3 Appropriate amounts of sense primer and antisense primer of the base sequence represented by ́ were added to make 100 μl with sterile distilled water. By conventional methods, the mixture is incubated at 94 ° C for 1 minute, 43 ° C for 1 minute, 72 ° C for 1 minute, and in this order for 3 cycles, followed by an additional 1 minute at 94 ° C. The PCR reaction was repeated 40 times in this order for 1 minute at 60 ° C and 1 minute at 72 ° C.

[0028] This PCR product and the Stratagene plasmid vector "pCR-Script SK (+)" are ligated with DNA ligase according to a conventional method to form recombinant DNA, which is then combined with the Stratagene Escherichia coli strain "pCR-Script SK (+)" by the competent cell method. It was introduced into XL-1 Blue MRF ́Kan and transformed. The transformant was inoculated into L-broth medium (pH 7.2) containing 50 μg / ml ampicillin, cultured with shaking at 37 ° C for 18 hours, and then the culture was centrifuged to collect the transformant, which was usual. Recombinant DNA was isolated by applying the alkaline-SDS method. When a part of this recombinant DNA was taken and analyzed by the dideoxy method, the Eco RI cleavage site and Hind were located at the 5 ́ and 3 ́ ends of the nucleotide sequence shown in SEQ ID NO: 18 in the sequence listing, respectively. III Cleavage site, and methionine codon for starting polypeptide synthesis and methionine codon for terminating polypeptide synthesis at the sites corresponding immediately before and after the N-terminal and C-terminal, respectively, in the amino acid sequence shown in SEQ ID NO: 18. It contained DNA with a TAG codon. In addition, this recombinant DNA contained all the base sequences encoding the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 and 5 in the sequence listing. That is, these amino acid sequences are based on the 46th to 63rd, 85th to 102nd, 394th to 414th, 148th to 162nd and 211th to 225th bases in the base sequence shown in SEQ ID NO: 18, respectively. It was coded.

[0029] Therefore, after cleaving the remaining recombinant DNA with restriction enzymes Eco RI and Hind III according to a conventional method, the obtained Eco RI was obtained using the DNA ligation kit "DNA ligation kit version 2" manufactured by Takara Shuzo. -Recombinant DNA "pKGFMH2" that can be replicated by reacting 10 ng of Pharmacia plasmid vector "pKK223-3", which has been previously cut with 0.1 μg of Hind III DNA fragment with the same restriction enzyme, at 16 ° C for 30 minutes and ligating them. Got Escherichia coli Y1090 strain (ATCC37197) was transformed with this recombinant DNA pKGFMH2 by the competent cell method, and the obtained transformant "KGFMH2" was inoculated into L-broth medium (pH 7.2) containing 50 μg / ml ampicillin. Then, the cells were shake-cultured at 37 ° C for 18 hours. The culture was centrifuged to collect a transformant, and a recombinant DNA "pKGFMH2" was extracted by applying a normal SDS-alkali method to a part of the transformant. As a result of analysis by the dideoxy method, as shown in FIG. 5, in the recombinant DNA pKGFMH2, the cDNA KGFMH2 containing the nucleotide sequence shown in SEQ ID NO: 18 in the sequence listing is included. The cDNA was linked downstream of the Tac promoter Ptac.

[0030] Ampicillin was added to L-broth medium (pH 7.2) sterilized by autoclave to a concentration of 50 μg / ml, cooled to 37 ° C, inoculated with transformant KGFMH2, and shaken to 37. Seed culture was performed at ° C for 18 hours. Take 18 liters of the same fresh medium in 20 liters of jar fermenta, sterilize in the same manner, add ampicillin, cool to 37 ° C, inoculate 1% (v / v) of the seed culture obtained above, and 37 The cells were aerated and stirred at ° C for 8 hours. The culture is centrifuged to collect cells, suspended in a mixed solution (pH 7.3) containing 150 mM sodium chloride, 16 mM disodium hydrogen phosphate and 4 mM sodium dihydrogen phosphate, ultrasonically crushed, and then centrifuged. The crushed material was removed, and about 2 liters of the supernatant was collected.

[0031] To about 2 liters of the obtained supernatant, 10 mM phosphate buffer (pH 7.3) containing ammonium sulfate was added so that ammonium sulfate was 40% saturated, the precipitate was removed by centrifugation, and then ammonium sulfate was further added to the supernatant. Was added until it was 85% saturated, left at 4 ° C for 18 hours, and then centrifuged at about 8,000 rpm for 30 minutes to collect the precipitate. Next, this precipitate was dissolved in 10 mM phosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate to make about 1,300 ml, and after filtration, it was preliminarily equilibrated with the same fresh buffer. CL-4B load a column of about 800 ml, wash the column with the same fresh buffer, and then apply 10 mM phosphate buffer (pH 6.6) at SV1.5 under a concentration gradient of ammonium sulfate that drops from 1.5 M to 0 M. The liquid was passed. The fraction eluted when the ammonium sulfate concentration is around 1 M is collected, membrane-concentrated, dialyzed against 10 mM phosphate buffer (pH 6.5) at 4 ° C for 18 hours, and then 10 mM phosphate buffer (pH 6. A column of about 55 ml of Pharmacia "DEAE-5PW" equilibrated in 5) was loaded. After washing the column with the same fresh buffer solution, a 10 mM phosphate buffer solution (pH 6.5) was passed through the column at SV5.5 under a concentration gradient of sodium chloride increasing from 0 M to 0.5 M, and the sodium chloride concentration was 0.2. Fractions eluted near M were collected. After that, the collected eluate was concentrated in the same manner as above to make about 9 ml, dialyzed against PBS at 4 ° C for 18 hours, and then equilibrated with fresh PBS in advance. The column of "75" was loaded. Further, fresh PBS was passed through the column to collect an IFN-γ-inducible fraction, and the membrane was concentrated to obtain purified mouse IL-18. The yield of this purified mouse IL-18 was about 350 μg per 1 liter of the culture solution.

[0032] This purified mouse IL-18 was analyzed as shown below according to the method described in JP-A-8-27189 to confirm its biological action and physicochemical properties. That is, when mouse splenocytes collected by a conventional method were cultured in the presence of various concentrations of the purified mouse IL-18, IFN-γ production was observed depending on the concentration of the purified mouse IL-18 present. It was confirmed that purified IL-18 has a biological action that induces the production of IFN-γ in Tile cells, which are immunocompetent cells. SDS-polyacrylamide gel electrophoresis under non-reducing conditions results in a molecular weight of 19,000 ± 5,000 daltons, according to the method reported by UK Remli in Nature, Vol. 227, pp. 680-685 (1970). The main band capable of inducing IFN-γ was shown at the corresponding position. Further, the N-terminal had the amino acid sequence shown in SEQ ID NO: 19, which corresponds to the N-terminal portion in the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing.

Next, Experimental Example 7 will be shown to explain the biological action of IL-18 used in the present invention in more detail, and Experimental Example 8 will be shown to explain its toxicity.

[Experimental Example 7] &lt;Biological Action&gt; [0035] [Experimental Example 7-1] &lt;Induction of GM-CSF Production&gt; Blood was collected from a healthy subject using a heparin-added syringe, and serum-free RPMI1640 medium ( It was diluted 2-fold with pH 7.4). Blood was layered on Ficoll, and lymphocytes collected by centrifugation were washed with RPMI1640 medium (pH 7.4) supplemented with 10% (v / v) fetal bovine serum, and then placed in the same fresh medium with a cell density of 1. × 10⁶Floating to individual / ml, 2 ml / well was dispensed into a 12-well plate.

[0036] Separately, IL-18 obtained by the method of Experimental Example 1 was prepared in RPMI1640 medium (pH 7.4) supplemented with 10% (v / v) fetal bovine serum to a concentration of 1 μg / ml, and placed on the above plate. Dispense 20-200 μl / well, add 10 μl / well of fresh same medium containing 500 μg / ml concanavalin A, then 5% CO₂Incubator was cultured at 37 ° C for 48 hours. After culturing, 0.1 ml of the culture supernatant was collected from each well, and the GM-CSF content was measured by a usual enzyme immunoassay method. At the same time, a system in which only IL-18 was omitted was provided and treated in the same manner as above to serve as a control. The results are shown in Table 1.

[0037] [Table 1]<img file="JP3955352B2_D0001.tif" />[0038] The results in Table 1 show that when IL-18 was allowed to act in the presence of concanavalin A as a cofactor, lymphocytes as immunocompetent cells produced GM-CSF depending on the concentration of IL-18. Is shown. It was also confirmed that IL-18 and its functional derivatives obtained by the methods of Experimental Examples 2 to 5 were also subjected to the same operation separately, and all of them also induced the production of GM-CSF. It was. On the other hand, for IL-18 obtained by the method of Experimental Example 6, splenocytes collected from mice by a conventional method were used instead of lymphocytes collected from human blood used in Experimental Example 7-1. When tested according to Experimental Example 7-1, it was confirmed that it also induces the production of GM-CSF.

[0039] [Experimental Example 7-2] &lt;Inhibition of osteoclast formation&gt; [0040] [Experimental Example 7-2 (a)] TJ Martin et al., "Journal of Cellular Biochemistry", No. As described in Volume 56 (1994), pp. 357 to 366, etc., in general, in order for osteoclast progenitor cells to differentiate and osteoclasts to be formed, a precursor derived from bone marrow hematopoietic stem cells. Contact with osteoclasts and bone marrow stromal cells is a prerequisite. Also, G.D. Ludman, Endocrin Review, Volume 17 (1996).<u style="single">、3</u>As described on pages 08 to 332, the characteristics of osteoclasts are that they are polynuclear, have tartrate-resistant acid phosphatase (hereinafter abbreviated as "TRAP") activity, and have a calcium receptor. It is generally recognized that it is something to have. On the other hand, in the co-culture system of osteoblasts and bone marrow cells described in N. Udagawa et al., "Journal of Experimental Medicine", Vol. 182 (1995), pp. 1461 to 1468, for example, 1α , 25-Dihydroxyvitamin D₃, Prostaglandin E₂, Osteoclast-like cells (hereinafter sometimes abbreviated as "OCL") in response to any factor of corticosteroid, interleukin 1, interleukin 6 or interleukin 11. Is recognized. The OCL formed here has the characteristics of osteoclasts in vivo. Therefore, this co-culture system reproduces the process of osteoclast formation in vivo well in vitro, and by using this system, experiments on osteoclast formation and inhibitors against it should be performed. Can be done.

[0041] Using this co-culture system, the inhibitory effect of IL-18 on osteoclast formation was investigated. Osteoblasts are treated with 0.1% (w / v) collagenase (Australia, Worthington Biochemical Company) and 0.2% (w / v) dispase (joint liquor purification) on the calvaria of newborn mice according to a conventional method. Prepared. Bone marrow cells were prepared from mature mice according to a conventional method. 0.4 ml of α-MEM medium supplemented with 10% (v / v) fetal bovine serum per well of a 48-well plate (hereinafter, simply referred to as medium throughout Experimental Example 4-2), 2 × Ten⁴1 primary osteoblast and 5 × 10⁵5% CO of individual bone marrow cells₂ Co-cultured in an incubator at 37 ° C for 7 days. This was used as a negative control. On the other hand, in another well, 1α, 25-dihydroxyvitamin D₃(Made by Wako Junyaku) and Prostaglandin E₂(American, made by Sigma), concentration 10 each^-8M and 10^-7All were cultured in the same manner as the negative control except that the medium added so as to become M was used. This was used as a positive control. In yet another well, the same concentration of 1α, 25-dihydroxyvitamin D as the positive control₃And prostaglandin E₂In addition, all the cells were cultured in the same manner as the positive control except that the medium prepared by the method of Experimental Example 6 was added so as to have a concentration of 0.01 to 10 ng / ml. In each case, on the 3rd day of culture, each well was replaced with fresh medium having the same composition as previously used. After culturing for 6 days, the cells were fixed according to the method described in N. Udagawa et al., "Journal of Experimental Medicine", Vol. 182 (1995), pp. 1461 to 1468, and became TRAP activity. Based on this, the number of stained cells (hereinafter referred to as "TRAP positive cells") was counted per well. Through this Experimental Example 4-2, the same culture system was provided for each of 4 wells, and the average value of the number of TRAP-positive cells per well was calculated. The results are shown in Table 2.

[0042] [Table 2]<img file="JP3955352B2_D0002.tif" />As shown in Table 2, the formation of TRAP-positive cells was hardly observed in the negative control, whereas the formation of TRAP-positive cells was remarkable in the positive control. On the other hand, in the system in which IL-18 was further added to the positive control, the formation of TRAP-positive cells was inhibited depending on the concentration, and when the IL-18 concentration was 8 ng / ml or more, the inhibition was maximum, and the TRAP-positive cells were inhibited. The number was equivalent to that of the negative control. The above indicates that the IL-18 does have an effect of inhibiting OCL formation in in vitro, and further strongly suggests that the IL-18 inhibits osteoclast formation. ing.

[Experimental Example 7-2 (b)] As described above, there are various factors that promote the formation of osteoclast-like cells in the co-culture system used through this Experimental Example 7-2. It has been confirmed that there is. Therefore, in Experimental Example 7-2 (b), whether or not the inhibitory effect of IL-18 on osteoclast formation shown in Experimental Example 7-2 (a) is specific to a certain factor. I examined. That is, 1α, 25-dihydroxyvitamin D₃, Prostaglandin E₂, Parathyroid hormone, either interleukin 1 or interleukin 11 at a concentration of 10 respectively^-8M, 10^-7All were cultured in the same manner as the negative control of Experimental Example 7-2 (a) except that the medium added to M, 200 ng / ml, 100 ng / ml or 20 ng / ml was used. These were used as positive controls. On the other hand, in another well, all except using a medium in which IL-18 obtained by the method of Experimental Example 6 was added so as to be 10 ng / ml in addition to one of the factors having the same concentration as this positive control. The cells were cultured in the same manner as the positive control. The number of TRAP-positive cells was compared as in Experimental Example 7-2 (a) after culturing. The results are shown in Table 3.

[0045] [Table 3]<img file="JP3955352B2_D0003.tif" />[0046] As shown in Table 3, remarkable formation of TRAP-positive cells was observed in all positive controls. On the other hand, when IL-18 was added to the positive control, the formation of TRAP-positive cells was almost completely inhibited. This strongly suggests that IL-18 has an action of inhibiting osteoclast formation in general regardless of the cause of osteoclast formation.

[Experimental Example 7-2 (c)] Next, the formation of osteoclasts by the IL-18 confirmed by Experimental Example 7-2 (a) and Experimental Example 7-2 (b). It was investigated whether the inhibition was due to the action of GM-CSF whose production was induced by the IL-18. For the negative control and the positive control, the same system as shown in Experimental Example 7-2 (a) was used, respectively. Another u<u style="single">D</u>In Le, the same concentration of 1α, 25-dihydroxyvitamin D as this positive control₃And prostaglandin E₂(I) Anti-mouse GM-CSF polyclonal antibody (manufactured by R &amp; D Systems, USA) is added so as to have a concentration of 10 μg / ml, or (ii) Method of Experimental Example 6 Add IL-18 to a concentration of 10 ng / ml, or add anti-mouse GM-CSF polyclonal antibody to a concentration of 10 μg / ml in addition to (iii) ii, or (iv) Mouse GM- Add CSF (manufactured by R &amp; D Systems, USA) to a concentration of 0.1 ng / ml, or add anti-mouse GM-CSF polyclonal antibody to a concentration of 10 μg / ml in addition to (v) iv. All were cultured in the same manner as the positive control, except that one of the media added as described above was used. The number of TRAP-positive cells was compared as in Experimental Example 7-2 (a) after culturing. The results are shown in Table 4. The codes of i to v shown in Table 4 are the same as the codes of the culture systems other than the control system described here.

[0048] [Table 4]<img file="JP3955352B2_D0004.tif" />As shown in Table 4, the formation of TRAP-positive cells was almost completely inhibited by IL-18 (culture system ii), but this inhibition was almost completely inhibited by the addition of anti-mouse GM-CSF polyclonal antibody. It was released (culture system iii). On the other hand, mouse GM-CSF was also found to inhibit the formation of TRAP-positive cells like IL-18 (culture system iv), but this inhibition was almost completely achieved by the addition of anti-mouse GM-CSF polyclonal antibody. Was released (culture system v). In addition, the antibody alone had no effect on the formation of TRAP-positive cells (culture system i). The above results strongly suggest that the inhibition of IL-18 on osteoclast formation is mainly due to the action of GM-CSF induced and produced by the IL-18.

[Experimental Example 8] &lt;Acute toxicity test&gt; According to a conventional method, any of the IL-18 obtained by the methods of Experimental Examples 1 to 6 was separately percutaneously, orally or orally obtained in 8-week-old mice. It was administered by injection intraperitoneally. As a result, the LD50 of these IL-18s was about 1 mg / kg mouse body weight or more by any administration route. This confirms that IL-18 is safe to combine with drugs that are intended for administration to warm-blooded animals such as humans.

[0051] Further, the GM-CSF induced and produced by the IL-18 is described in "Nikkei Bio Yearbook 96" (1995), pp. 498 to 499, published by Nikkei BP, in Japan. Although it has not yet been clinically applied, it has already been clinically applied in the United States and Europe, and it can be said that there is no problem with its safety. The above is a disease in which the osteoclast formation inhibitor of the present invention can be continuously used for a long period of time in warm-blooded animals including humans without causing serious side effects, and osteoclast formation and / or function is involved. It has been shown to be effective in the treatment and prevention of.

[0052] Examples are shown below, and the osteoclast formation inhibitor of the present invention will be described.

[Example 1] &lt;Liquid&gt; 2 mg / mg of any of the IL-18 obtained by the methods of Experimental Examples 1 to 6 in physiological saline containing 1% (w / v) human serum albumin as a stabilizer. It was dissolved to ml and sterilized by microfiltration according to a conventional method to obtain a liquid preparation.

[0054] All of these products have excellent stability and are useful as a medium component for cell culture, and also for treating / preventing bone resorption regulators, hypercalcemia, osteoclastoma, osteoporosis, etc. It is useful as an injection, an eye drop, and a nasal drop as an osteoclast-related disease agent.

[Example 2] &lt;Drying agent&gt; 50 mg of any of the IL-18 obtained by the methods of Experimental Examples 1 to 6 in 100 ml of physiological saline containing 1% (w / v) purified gelatin as a stabilizer. It was dissolved, sterilized by microfiltration according to a conventional method, dispensed into vials in 1 ml increments, freeze-dried, and then sealed.

[0056] All of these products have excellent stability and are useful as a medium component for cell culture, and also for treating / preventing bone resorption regulators, hypercalcemia, osteoclastoma, osteoporosis, etc. It is useful as a dry injection as an agent for osteoclast-related diseases.

[Example 3] &lt;Drying agent&gt; Dissolve 50 mg of any of the IL-18 obtained by the methods of Experimental Examples 1 to 6 in 100 ml of physiological saline containing 1% (w / v) trehalose as a stabilizer. Then, it was sterilized by microfiltration according to a conventional method, 1 ml each was dispensed into a vial, freeze-dried, and then sealed.

[0058] All of these products have excellent stability and are useful as a medium component for cell culture, and also for treating / preventing bone resorption regulators, hypercalcemia, osteoclastoma, osteoporosis, and the like. It is useful as a dry injection as an agent for osteoclast-related diseases.

[Example 4] &lt;Ointment&gt; Concentrations of carboxyvinyl polymer "Hibiswaco 104" manufactured by Wako Pure Chemical Industries, Ltd. and high-purity trehalose in sterile distilled water 1.4% (w / w) and 2.0% (w / w, respectively). ), And one of the IL-18s obtained by the methods of Experimental Examples 1 to 6 is uniformly mixed, adjusted to pH 7.2, and a paste containing about 1 mg of the IL-18 per 1 g. I got a paste.

[0060] This product is excellent in spreadability and stability, and is an ointment as a bone resorption regulator and an osteoclast-related disease agent for treating / preventing hypercalcemia, osteoclastoma, osteoporosis, etc. It is useful as an agent.

[Example 5] &lt;Tablet&gt; Hayashibara's anhydrous crystal α-maltose powder "Fine Tooth" is uniformly mixed with any of the IL-18 obtained by the methods of Experimental Examples 1 to 6 and lumin as a cell activator. The resulting mixture was tableted by a conventional method to obtain tablets containing about 2 mg each of the IL-18 and lumin per tablet (about 200 mg) of the product.

[0062] All of these products have excellent ingestibility and stability, and also have a cell activating effect, and are used for treating / preventing bone resorption regulators, hypercalcemia, osteoclastoma, osteoporosis, etc. It is useful as a tablet as an agent for osteoclast-related diseases.

[Effect of the Invention] As described above, the osteoclast formation inhibitor of the present invention remarkably inhibits the formation of osteoclasts regardless of in-vitro or in-vivo, and thus cell culture. It is useful as a medium component for bone resorption, and also as a disease agent for treating and preventing osteoclast-related diseases such as hypercalcemia, osteoclastoma and osteoporosis. Demonstrate.

[0064] This invention exhibits such remarkable effects and effects, and can be said to be an invention of great significance to contribute to the field.

[0065] [Sequence list]<img file="JP3955352B2_D0005.tif" /> 【0066】<img file="JP3955352B2_D0006.tif" /> 【0067】<img file="JP3955352B2_D0007.tif" /> 【0068】<img file="JP3955352B2_D0008.tif" /> 【0069】<img file="JP3955352B2_D0009.tif" /> 【0070】<img file="JP3955352B2_D0010.tif" /> 【0071】<img file="JP3955352B2_D0011.tif" /> 【0072】<img file="JP3955352B2_D0012.tif" /><img file="JP3955352B2_D0013.tif" /> 【0073】<img file="JP3955352B2_D0014.tif" /> 【0074】<img file="JP3955352B2_D0015.tif" /> 【0075】<img file="JP3955352B2_D0016.tif" /> 【0076】<img file="JP3955352B2_D0017.tif" /> 【0077】<img file="JP3955352B2_D0018.tif" /> 【0078】<img file="JP3955352B2_D0019.tif" /><img file="JP3955352B2_D0020.tif" /> 【0079】<img file="JP3955352B2_D0021.tif" /> 【0080】<img file="JP3955352B2_D0022.tif" /><img file="JP3955352B2_D0023.tif" /> 【0081】<img file="JP3955352B2_D0024.tif" /><img file="JP3955352B2_D0025.tif" /><img file="JP3955352B2_D0026.tif" /><img file="JP3955352B2_D0027.tif" /><img file="JP3955352B2_D0028.tif" /><img file="JP3955352B2_D0029.tif" /><img file="JP3955352B2_D0030.tif" /><img file="JP3955352B2_D0031.tif" /><img file="JP3955352B2_D0032.tif" /><img file="JP3955352B2_D0033.tif" /> 【0082】<img file="JP3955352B2_D0034.tif" /><img file="JP3955352B2_D0035.tif" /> 【0083】<img file="JP3955352B2_D0036.tif" />BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the structure of recombinant DNA pKGFHH2.

FIG. 2 is a diagram showing the structure of recombinant DNA pCSHIGIF / MUT35.

FIG. 3 is a diagram showing the structure of recombinant DNA pCSHIGIF / MUT42.

FIG. 4 is a diagram showing the structure of recombinant DNA pBGHuGF.

FIG. 5 is a diagram showing the structure of recombinant DNA pKGFMH2.

[Code description] KGFHH2 cDNA cDNA IGIF / MUT35 encoding the IL-18 DNA IGIF / MUT42 encoding the IL-18 DNA HuIGIF encoding the IL-18 DNA KGFMH2 cDNA encoding the IL-18 Encoding cDNA 5S 5S Ribosome RNA Gene Ptac tac Promoter rrnBT1T2 Ribosome RNA Operon Transcription Termination Region AmpR Ampicillin Resistance Gene pBR322ori Replication Initiator in Escherichia coli CMV Cytomegalovirus Promoter IFNss Sequence encoding signal peptide of subtype α2b in human interferon-α