AU733126B2 - Polypeptides - Google Patents

Description

AUSTRALIA

Patents Act 1 990 P/00/01 1 28/5/91 Regulation 3.2 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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Name of Applicant: Actual Inventors: Address for Service: Invention Title: KABUSHIKI KAISHA HAYASHIBARA SEIBUTSU KAGAKU KENKYUJO Kakuji TORIGOE; Takanori OKURA; and Masashi KURIMOTO CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia

"POLYPEPTIDES"

The following statement is a full description of this invention, including the best method of performing it known to us:- 23/1 2/97jb9630.cs, 1 Polypeptides Background of the Invention 1. Field of the Invention This invention relates to a novel receptor protein which recognizes a cytokine, more particularly, to a novel polypeptide which recognizes interleukin-18 (hereinafter abbreviated as "IL-18").

2. Description of the Prior Art IL-18 is a type of cytokine or substance which mediates signal transduction in immune system. As seen in e Japanese Patent Kokai Nos.27,189/96 and 193,098/96 and Haruki Okamura et al., Nature, Vol.378, No.6,552, pp.

8 8 9 1 (1995), IL- 18 was provisionally designated as "interferon-gamma inducing factor" immediately after its discovery: This designation was 'changed later into "IL-18" in accordance with the proposal in Shimpei Ushio et al., The Journal of Immunology, Vol.156, pp.4,274-4,279 (1996). IL-18 in mature form consists of 157 o* amino acids and possesses properties of inducing in immunocompetent cells the production of interferon-gamma (hereinafter abbreviated as "IFN-y") which is known as useful biologically-active protein, as well as of inducing and enhancing the generation and cytotoxicity of killer cells.

Energetic studies are now in progress to develop and realize various uses of IL-18 in pharmaceuticals such as antiviral, antimicrobial, antitumor and anti-immunopathic agents which have been in great expectation because of these properties of IL-18.

As described above, in nature, cytokines including IL-

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18 are produced and secreted as substances responsible for signal transduction in immune system. Therefore, excessive amounts of cytokines may disturb the equilibria in immune system when they are produced or administered in the body of mammals.

The surface of usual mammalian cells may bear certain sites or "receptors" which are responsible for recognition of cytokines: Secreted cytokines transduce no signal in cells till they are bound to the receptors. In normal immune system, there would be definite equilibria between respective cytokines and their receptors. Thus, in this field, with the purpose of developing and realizing IL-18 as pharmaceuticals, in addition to the clarification of physiological activities of IL-18, an expedited establishment of mass production and characterization of IL-18 receptor (hereinafter abbreviated as "IL-18R") have been in great expectation.

Summary of the Invention In view of the foregoing, this invention provides a polypeptide as IL-18R which Scan be easily prepared on a large scale.

The second aspect of this invention provides uses of such polypeptide as pharmaceuticals.

The third aspect of this invention provides a DNA which encodes the polypeptide.

The fourth aspect of this invention provides a process to prepare the polypeptide.

The fifth aspect of this invention provides an agent to neutralize IL-18 using the polypeptide.

2 The sixth aspect of this invention provides a method to neutralize IL-18 using the polypeptide.

We energetically and extensively screened various means which might attain these aspects, eventually resulting in the finding that a substance which recognized IL-18 was present in L428 cell, a type of lymphoblastoid cell derived from a patient with Hodgkin's disease. We isolated and characterized this substance, revealing that its nature was proteinaceous, as well as that it well recognized and bound IL-18 even when in isolated form. It was also found that the IL-18R thus identified was efficacious in treatment and prevention of various diseases resulting from excessive immunoreaction, such as autoimmune diseases, because in mammals including human, ILi 18R recognized and neutralized IL-18 which activated immune S system. Further, we have energetically studied L428 cell using as probe some partial amino acid sequences of the IL-18R, S resulting in obtainment of a DNA which did encode IL-18R. We confirmed that a polypeptide obtained by bringing such DNAs into expression in artificial manner well recognized IL-18 and shared some essential physiological activities with the IL-18R separated from L428 cell, as well as that it was preparable in desired amounts by recombinant DNA techniques using such DNA.

Thus we accomplished this invention.

More particularly, this invention attains the first aspect with a polypeptide as IL-18R, which is obtained through gene expression.

This invention attains the second aspect with an agent for IL-18R susceptive diseases, which contains as effective /jiT gredient such polypeptide.

3 This invention attains the third aspect with a DNA which encodes the polypeptide.

This invention attains the fourth aspect with a process to prepare polypeptide, comprising bringing into expression a DNA which encodes the polypeptide, and collecting the resultant polypeptide.

This invention attains the fifth aspect with an agent to neutralize IL-18, which contains as effective ingredient the polypeptide.

This invention attains the sixth aspect with a method to neutralize IL-18, characterized by allowing the polypeptide to act on IL-18.

L428 cell, which is feasible in this invention, have been deposited in the Patent Microorganism Depository, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, 305, Japan, under the accession number of "FERM BP-5777" on and after December 24th, 1996.

Brief Explanation of the Accompanying Drawings FIG. 1 shows that the monoclonal antibody MAb #117-10C a binds to L428 cells and IL-18R while competing with IL-18.

FIG. 2 is an image of intermediate tone given on display, which shows IL-18R on gel electrophoresis visualized by the Western blotting method using the monoclonal antibody MAb #117-10C.

FIG. 3 shows the inhibitory action of the monoclonal T0 antibody MAb #117-10C on the activity of IL-18.

4 FIG. 4 is the chromatogram obtained by applying to IL- 18R an immunoaffinity chromatography using the monoclonal antibody MAb #117-10C.

FIG. 5 is the peptide map of IL-18R.

FIG. 6 shows the structure of the recombinant DNA "pcDNA/HuIL-18R" of this invention.

FIG. 7 shows the structure of the recombinant DNA "pEFHIL18R-14" of this invention.

FIG. 8 shows the structure of the recombinant DNA "pEFHIL18RD1-2-H" of this invention.

FIG. 9 shows the structure of the recombinant DNA "pEFHIL18RD1-H" of this invention.

FIG. 10 shows the structure of the recombinant DNA "pEFMIL18RSHT" of this invention.

Throughout the Figures, the symbol "Pcmv" indicates the cytomegalo virus promotor; "EFlaP", the elongation factor promotor; "IL-18R cDNA", the cDNA encoding the polypeptide of this invention; "EFHIL18R-14 cDNA", the cDNA encoding the soluble polypeptide of human origin according to this invention; "HIL18RD1-2-H cDNA", the cDNA encoding the soluble polypeptide of human origin according to this invention; "HIL18RD1-H cDNA", the cDNA encoding the soluble polypeptide of human origin according to this invention; and "EFMIL18RSHT cDNA", the cDNA encoding the soluble polypeptide of mouse origin according to this invention.

Detailed Description of the Invention This invention relates to a polypeptide as IL-18R, 5 which is obtained through gene expression. The polypeptide of human origin according to this invention usually contains as partial amino acid sequence(s) one or more amino acid sequences of SEQ ID NOs:12 to 19: As a whole, it contains a part or whole of the amino acid sequence of SEQ ID NO:20. While the polypeptide of mouse origin according to this invention usually contains a part or whole of the amino acid sequence of SEQ ID NO:21. Thus, the wording "polypeptide" as referred to in this invention shall include, in addition to those which wholly contain the amino acid sequence of either SEQ ID NO:20 or 21, for example, those which contain the same amino acid sequence but with addition of one or more amino acids, in particular, Sthose which contain one or more amino acids linked to the Cand/or N-termini in SEQ ID NO:20 or 21; those which contain the Ssame amino acid sequence as in SEQ ID NOs:20 and 21 but with deletion of one or more amino acids, in particular, soluble polypeptides which contain the amino acid sequences of SEQ ID NOs:22 to 25; and those which contain either of the amino acid sequences as described above but with a saccharide chain, as far as they are obtainable through gene expression and possess the essential functions of IL-18R. As to IL-18, those of human and mouse origins commonly consisting of 157 amino acids have been *oo a documented: Human IL-18 bears the. amino acid sequence of SEQ ID NO:26 (where the amino acid with symbol "Xaa" represents either isoleucine or threonine), while mouse counterpart, the amino acid sequence of SEQ ID NO:27 (where the amino acid with symbol "Xaa" represents either methionine or threonine).

The polypeptide of this invention is usually prepared Qy'^ 1 by applying recombinant DNA techniques, more particularly, by 6 bringing into expression in artificial manner a DNA which encodes the polypeptide, and collecting the resultant polypeptide. This invention provides, in addition to a DNA which encodes the polypeptide, a process to prepare the polypeptide using recombinant DNA techniques: By practicing such a process according to this invention, desired amounts the polypeptide can be easily obtained.

The DNA which is used in this invention are those which originating natural sources, those which can be obtained by artificially modifying them and those which can be obtained through chemical synthesis, provided that they do encode the polypeptide. Generally, in this field, in case of artificially expressing DNAs which encode polypeptides, one may replace one or more nucleotides in the DNAs with different nucleotides and/or link an appropriate nucleotide sequence to the DNAs, with purpose of improving their expression efficiency and/or the physiological and physicochemical properties of the polypeptides. Such modifications are feasible in the DNA of this invention of course: For example, one can link to the and 3'-termini of the DNA as described above recognition sites for appropriate restriction enzymes, initiation and termination codons, promotors and/or enhancers, as far as the final polypeptide products do retain desired physiological activities.

Thus, the wording "DNA" as referred to in this invention shall mean, in addition to those which encode any polypeptides as described above, those which are complementary thereto, and further those where one or more nucleotides have been replaced with different nucleotides while conserving the amino acid sequence.

7 To obtain such a DNA from natural sources, f or example, mammalian cells including epithelial. cells, endo-thelial cells, interstitial cells, chondracytes, monocytes, granulocytes, lymphocytes, neurocytes and theiLr established -cell lines of human and mouse origins are screened with oligonucleotides as probe which can be prepared with reference to the amino acid sequences of SEQ ID NOs:12 to 25. Examples of preferred cells are cell lines which are obtained by establishing hemopoletic cells including lymphocytes, in particular, JM cells, XDLM'-2 cells, MOLT-16 calls and PEER cells described in Jun Minowada, cancer Review, Vol.10, pp.1-18 :(1988), and lymphoblastoid cells such as L428 cell (FERM BP- 5777), KG-i cell (ATCC CCL-246) and U-937 cells (ATCC CRL- 1593.2). The human and mousea DNAs obtained In this way usually contain a part or whole of respective nuclectide sequences of SEQ ID NOs:). and 2. For example, as shown in SEQ ID NO:7, the DNA obtained from L428 cell, a type of lymphoblastoid cell deri~ved from a patient with Hodgkin's disease, consists of the .:nucleotide sequence of SEQ ID NO:1 encoding the amino acid sequence of SEQ ID NO:-20, and another riucleotide sequence encoding signal peptide which is linked to the 5'-tperminal in :the nucleotide sequence of the SEQ ID. KO:l. Soluble polypeptides with the amino acid sequences of SEQ ID NOs:22 to are usually encoded by respective nucleotide sequences of SEQ ID NOS 3 to 6, which are usually used irx a f orm where, as shown in the nucleotIde sequences of SEQ ID NOs: 8 'to 11, a nucleotide sequence encoding signal peptide is linked to the in the nucleotide sequences of SEQ ID Nos:3 to 6. Such a DNA' can be also obtained through usual chemical synthesis, and in 8any case, DNAs can be amplified to desired levels by PCR method once they become available. By the way, the amino acid sequences of SEQ ID NOs:20 and 21 are described along with the amino acid sequences for signal peptides in P. Parnet et al., The Journal of Biological Chemistry, Vol.271, pp.

3 ,967-3,970 (1996): This paper however makes neither suggestion nor teaching that the polypeptides with the amino acid sequences of SEQ ID NOs:20 and 21 do function as IL-18R.

Such DNA expresses the polypeptide when introduced into an appropriate host of microbe, animal or plant origin.

SThe DNA of this invention is usually prepared into a recombinant DNA prior to introduction into host. Such recombinant DNA, which consists of the DNA of this.invention and an autonomously *o replicable vector, can be easily prepared with usual recombinant DNA techniques, provided that the DNA is available. Examples of vectors which can receive the DNA of this invention are plasmid vectors including pKK223-3, pCDNAI/Amp, BCMGSNeo, pcDL- SRa, pKY4, pCDM8, pCEV4, pME18S and pEF-BOS. Autonomously replicable vectors usually comprises other nucleotide sequences, for example, promotor, enhancer, replication origin, terminator of transcription, splicing sequence and/or selection marker which facilitate the expression of the DNA of this invention in particular hosts. Expression of the DNA becomes artificially regulatable upon external stimuli when it is used in combination with either heat shock protein promotor or interferon-a promotor as disclosed in Japanese Patent Kokai No.163,368/95 by the same applicant.

Conventional methods are feasible in the insertion of the DNA of this invention into such vector. More particularly, 9 a gene with the DNA of this invention and an autonomously replicable vector are first digested with restriction enzyme and/or ultrasonication, then the resultant DNA and vector fragments are ligated. Ligation of DNA and vector fragments become much easier when genes and vectors are digested with restriction enzymes specific to particular nucleotides, for example, AccI, BamHI, BstXI, EcoRI, HindIII, NotI, PstI, SacI, Sall, SmaI, Spel, Xbal and XhoI. To ligate DNA and vector fragments, they are first annealed, if necessary, then exposed to DNA ligase in vivo or in vitro. The recombinant DNA thus e C obtained is unlimitedly replicable in hosts of microbe and animal origins.

Such recombinant DNA is introduced into an appropriate host, prior to use in preparation of the polypeptide. Although conventional hosts of microbe, animal and plant origins are feasible in this invention, it is preferable to choose a host :of yeast or mammalian origin in case that the final use of the polypeptide is pharmaceuticals. Examples of host cells of mammalian origin are epithelial cell, interstitial cell and o o hemopoietic cell of human, monkey, mouse and hamster origins, in particular, 3T3 cell (ATCC CCL-92), C127I cell (ATCC CRL- 1616), CHO-K1 cell (ATCC CCL-61), CV-1 cell (ATCC CCL-70), COS-1 cell (ATCC CRL-1650), HeLa cell (ATCC CCL-2), MOP-8 cell (ATCC CRL-1709) and their mutant strains. To introduce the DNA of this invention into such a host, one can employ conventional methods, for example, DEAE-dextran method, calcium phosphate transfection method, electroporation method, lipofection method, microinjection method and viral infection method using 10

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retrovirus, adenovirus, herpesvirus and vaccinia virus. To select among the resultant transformants a clone which is capable of producing the polypeptide, the transformants are cultivated on culture medium, followed by selecting one or more clones where production of the polypeptide is observed.

Recombinant DNA techniques using host cells of mammalian origin are detailed, for example, Jikken-Igaku-Bessatsu, Saibo-Kogaku Handbook (The handbook for the cell engineering), edited by Toshio KUROKI, Masaru TANIGUCHI and Mitsuo OSHIMURA, published by Yodosha. Co., Ltd., Tokyo, Japan (1992), and Jikken-Igaku- .Bessatsu, Biomanual Series 3, Idenshi-Cloning-Jikken-Ho (The experimental methods for the gene cloning), edited by Takashi YOKOTA and Kenichi ARAI, published by Yodosha Co., Ltd., Tokyo, Japan (1993).

The transformant thus obtained produces and secretes the polypeptide inside and/or outside the host cell when cultivated on culture medium. Such cultivation is feasible with conventional culture media directed to cultivation of transformants, which are usually composed by adding to a bufferized water as base inorganic ions such as sodium ion, potassium ion, calcium ion, phosphoric ion and chloric ion; minor elements, carbon sources, nitrogen sources, amino acids and vitamins which meet to the metabolism of particular hosts; and, if necessary, sera, hormones, cell growth factors and cell adhesion factors. Particular media are, for example, 199 medium, DMEM medium, Ham's F12 medium, IMDM medium, MCDB 104 medium, MCDB 153 medium, MEM medium, RD medium, RITC 80-7 medium, RPMI-1630 medium, RPMI-1640 medium and WAJC 404 medium.

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One can obtain a culture product containing the polypeptide by inoculating on such a culture medium a transformant in an amount of 1x10 4 -1x10 7 cells/ml, preferably, lx0l-1x10l 6 cells/ml, and subjecting the transformant to suspension or monolayer culture at around 37°C for 1 day to 1 week, preferably, 2 to 4 days while replacing the culture medium with a fresh preparation, if necessary. The culture product thus obtained.usually contains about 1 pg/l to 1 mg/l polypeptide, dependently of the type of transformant and cultivation conditions.

The culture product obtained in this way is first subjected to ultrasonication, cell-lytic enzyme and/or detergent **to disrupt cells, if necessary, then polypeptides are separated from the cells or cell debris by filtration and centrifugation, followed by purification. In the purification, a culture product which has been separated from cell or cell debris is subjected to conventional methods common in purification of biologically-active proteins, for example, salting-out, dialysis, filtration, concentration, fractional precipitation, ion-exchange chromatography, gel filtration chromatography,

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adsorption chromatography, isoelectric focusing chromatography, A hydrophobic chromatography, reversed phase chromatography, affinity chromatography, gel electrophoresis and isoelectric focusing gel electrophoresis which are used in combination, if necessary. The purified polypeptide is then concentrated and lyophilized into liquid or solid to meet to its final use. The IL-18 and monoclonal antibody, disclosed in Japanese Patent Kokai No.193,098/96 and Japanese Patent Application No.356,426/96 by the same applicant, are very useful in purification of the polypeptide: Immunoaffinity 12 chromatographies using these do yield a high-purity preparation of the polypeptide with minimized costs and labors.

The polypeptide of this invention exhibits a remarkable efficacy in treatment and prevention of various diseases resulting from excessive immunoreaction because in mammals including human, the polypeptide recognizes and binds IL-18 which may activate immune system. Immune system, which is in nature to defend living bodies from harmful foreign substances, may cause unfavorable results in living bodies because of its nature. When mammals receive a graft of organ, for example, skin, kidney, liver, heart and bone marrow, the 0 0 rejection reaction and immunoreaction against alloantigen may t* activate T-cells, resulting in the occurrence of inflammation Sand proliferation of lymphocytes. Similar phenomena are observed in case that host receives the invasion by heteroantigens, for example, allergens, which are not recognized as self. In autoimmune diseases, allergic reactions are induced 00 by substances which must be recognized as self. The polypeptide of this invention exhibits a remarkable efficacy in treatment and prevention of various diseases resulting from such an immunoreaction because the polypeptide suppresses or regulates the immunoreaction when administered in mammals including human.

Thus, the wording "susceptive diseases" as referred to in this invention shall mean all the diseases resulting from augmented immunoreaction which can be treated and/or prevented by the direct or indirect action of IL-18R: Particular susceptive diseases are, for example, rejection reactions associated with a graft of organ as described above, autoimmune and allergic diseases including pernicious anemia, atrophic gastritis, 13 insulin-resistant diabetes, Wegener granulomatosis, discoid lupus erythematosus, ulcerative colitis, cold agglutininrelating diseases, Goodpasture's syndrome, primary biliary cirrhosis, sympathetic ophtalmitis, hyperthyroidism, juvenile onset type diabetes, Sjogren syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, myasthenia gravis, systemic scleroderma, systemic lupus erythematosus, polyleptic cold hemoglobinuria, polymyositis, periarteritis nodosa, multiple sclerosis, Addison's disease, purpura hemorrhagica, Basedow's disease, leukopenia, Behet's disease, climacterium praecox, rheumatoid arthritis, rheumatopyra, chronic thyroiditis, Hodgkin's disease, HIV-infections, asthma, atopic dermatitis, allergic nasitis, pollinosis and apitoxin-allergy. In addition, the polypeptide of this invention is efficacious in treatment and prevention of septic shock which results from production or administration of excessive IFN-y.

Thus, the agent for susceptive disease, which contains as effective ingredient the polypeptides of this invention, would find a variety of uses as anti-autoimmune-diseases, antiallergies, anti-inflammatories, immunosuppressants, hematopoietics, leukopoietics, thrombopoietics, analgesics and Santipyretics directed to treatment and/or prevention of susceptive diseases as illustrated in the above. The agent according to this invention is usually prepared into liquid, suspension, paste and solid forms which contain the polypeptide :in an amount of 0.00001-100 w/w preferably, 0.0001-20 w/w dependently on the forms of agents as well as on the types and symptoms of susceptive disease.

The agent for susceptive diseases according to this 14 invention includes those which are solely composed of the polypeptide, as well as including those in composition with, for example, one or more physiologically-acceptable carriers, excipients, diluents, adjuvants, stabilizers and, if necessary, other biologically-active substances: Examples of such stabilizer are proteins such as serum albumins and gelatin; saccharides such as glucose, sucrose, lactose, maltose, trehalose, sorbitol, maltitol, mannitol and lactitol; and buffers which are mainly composed of phosphate or succinate.

Examples of the biologically-active substances usable in combination are FK506, glucocorticoid, cyclophosphamide, nitrogen mustard, triethylenethiophosphoramide, busulfan, pheniramine mustard, chlorambucil, azathioprine, 6mercaptopurine, 6-thioguanine, 6-azaguanine, 8-azaguanine, fluorouracil, cytarabine, methotrexate, aminopterin, mitomycin C, daunorubicin hydrochloride, actinomycin D, chromomycin A 3 bleomycin hydrochloride, doxorubicin hydrochloride, cyclosporin A, L-asparaginase, vincristine, vinblastine, hydroxyurea, procarbazine hydrochloride, adrenocortical hormone and auri colloid; receptor antagonists to cytokines other than IL-18, for example, antibodies respectively against interleukin-1 receptor protein, interleukin-2 receptor protein, interleukin-5 receptor protein, interleukin-6 receptor protein, interleukin-8 receptor protein and interleukin-12 receptor protein; and antagonists respectively against TNF-a receptor, TNF-p receptor, interleukin-1 receptor, interleukin-5 receptor and interleukin-8 receptor.

The agent for susceptive diseases according to this invention includes pharmaceuticals in minimal dose unit: The 15 Ct 't wording "pharmaceutical in minimal dose unit" represents those which are prepared into a physically united form suitable for prescription and also allowed to contain the polypeptide in an amount corresponding to its single dose or multiple (up to 4fold) or divisor (up to 1/40) thereof: Examples of such form are injection, liquid, powder, granule, tablet, capsule, sublingual, ophthalmic solution, nasal drop and suppository.

The agent for susceptive diseases according to this invention can be administrated through both oral and parenteral routes to exhibit in each case a remarkable efficacy in treatment and o 9 prevention of susceptive diseases. More particularly, the ee polypeptide is administered through oral route or parenteral :route such as intradermal, subcutaneous, intramuscular or intravenous route at a dose of about 1 pg/time/adult to about Ig/time/adult, preferably, about 10 pg/time/adult to about 100 mg/time/adult 1 to 4 times/day or 1 to 5 times/week over 1 day to 1 year.

The DNA which encodes the polypeptide of this invention is useful in "gene therapies". Particularly, in usual gene therapies, the DNA of this invention is first inserted in a vector derived from virus such as retrovirus, adenovirus or adeno-associated virus and, alternatively, embedded in either cationic- or membrane fusible-liposomes, then the inserted or embedded DNA is directly injected in a patient with an IL-18R susceptive disease and, alternatively, introduced into lymphocytes, which have been collected from the patient, and implanted in the patient. In adoptive immuno gene therapies, by introducing the DNA of this invention into effector cells s "milarly as in the usual gene therapies, the cytotoxicity of

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1 16 N'tt effector cells against tumors and virus-infected cells is enhanced and this would strengthen adoptive immunotherapy. In tumor vaccine gene therapy, tumor cells, which have been extracted from a patient, are introduced with the DNA of this invention similarly as in the usual gene therapies, allowed to proliferate in vitro to a prescribed level and then selftransplanted to the patient: The transplanted tumor cells act as vaccine in the body of the patient, exhibiting a strong and antigen-specific antitumor immunity. Thus, the DNA of this invention exhibits a remarkable efficacy in gene therapies for various diseases including, for example, malignant tumors, vial diseases, infections and autoimmune diseases, as well as in suppression of rejection reaction and excessive immunoreaction associated with grafts of organs and allergic diseases. General procedures for gene therapies are detailed in Jikken-Igaku- Bessatsu, Biomanual UP Series, Idenshichiryo-no-Kisogijutsu (Basic techniques for the gene therapy), edited by.Takashi SHIMADA, Izumi SAITO, and Keiya OZAWA, published by Yodosha Co., Ltd., Tokyo, Japan (1996).

Further, the polypeptide of this invention is useful in affinity chromatography and labelled assay directed to purification and detection of IL-18 because the polypeptide bears properties of recognizing and binding IL-18. In addition, the polypeptide of this invention, in particular, that in soluble form is useful in screening in vivo or in vitro agonists and antagonists to IL-18. Furthermore, the agent to neutralize IL-18 containing as effective ingredient the polypeptide and the method to neutralize IL-18 where IL-18 is exposed to the polypeptide are useful in treatment of various diseases which 17 result from production and administration of excessive IL-18.

The following Examples are to illustrate the way of practicing this invention. The techniques employed in Examples 1 to 9 are common in this field as detailed, for example, Jikken-Igaku-Bessatsu, Saibo-Kogaku Handbook (The handbook for the cell engineering), edited by Toshio KUROKI, Masaru TANIGUCHI and Mitsuo OSHIMURA, published by Yodosha. Co., Ltd., Tokyo, Japan (1992), and Jikken-Igaku-Bessatsu, Biomanual Series 3, Idenshi-Cloning-Jikken-Ho (The experimental methods for the gene cloning), edited by Takashi YOKOTA and Kenichi ARAI, published by Yodosha Co., Ltd., Tokyo, Japan (1993).

Example 1 Preparation and characterization of IL-18R Example 1-1 Preparation of IL-18R Newborn hamsters were intraperitoneally injected with an anti-lymphocyte antibody of rabbit origin to suppress their possible immunoreaction, subcutaneously injected at their dorsal areas with about 5x10 5 cell/animal of L428 cells (FERM BP-5777), a type of lymphoblastoid cell derived from a patient with Hodgkin's disease, and fed in usual manner for 3 weeks. The tumor masses, subcutaneously occurred, about 10g each, were extracted, disaggregated and washed in usual manner in serumfree RPMI-1640 medium (pH thus obtaining proliferated cells.

The proliferated cells were added with a mixture solution (volume ratio of 9:1) of 0.83 w/v NH 4 C1 and 170mM Tris-HCl buffer (pH 7.7) in an amount 10-fold larger than the 18 wet weight of the cells, stirred and collected by centrifugation at 2,000rpm for 10 minutes. The cells were then suspended in an appropriate amount of phosphate buffered saline (hereinafter abbreviated as stirred, collected by centrifugation at 2,000rpm, resuspended to give a cell density of about Ixl0 8 cells/ml in 10mM Tris-HCl buffer (pH 7.2) with ImM MgCl 2 and disrupted with "POLYTRON", a cell disrupter commercialized by Kinematica AG, Littau/Lucerne, Switzerland. The resultant was added with 10mM Tris-HCl buffer (pH 7.2) containing both ImM MgCl 2 and 1M sucrose to give a final sucrose concentration of 0.2M, and centrifuged at 1,000rpm to collect the supernatant which was then centrifuged at 25,000rpm for 60 minutes, followed by collecting the precipitate. The precipitate was added with adequate amounts of 12mM 3- (3-cholamidopropyl)dimethylammonio] 1-propanesulfonic acid (hereinafter abbreviated as "CHAPS"), ethylenediaminetetraacetatic acid (hereinafter abbreviated as "EDTA") and ImM phenylmethylsulfonylfluoride, stirred at for 16 hours, and centrifuged at 25,000rpm for 60 min, followed by collecting the supernatant.

The supernatant was charged to a column of "WHEAT GERM LECTIN SEPHAROSE 6B", a gel product for affinity chromatography commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, pre-equilibrated in PBS with 12mM CHAPS, and the column was washed with PBS containing 12mM CHAPS, and then charged with PBS containing both 0.5 M N-acetyl-D-glucosamine and 12mM CHAPS while monitoring the protein content in the eluate with the absorbance of ultraviolet at a wave length of 280nm. The fractions with an absorbance of 0.16-0.20 were collected and pooled, thus obtaining about 25 liters of aqueous solution with 19 a protein content of about 1 mg/ml per 1012 starting cells.

A small portion of the solution was sampled, added with 4ng human IL-18 which had been 125 I-labelled in usual manner, incubated at 40C for 1 hour, added with appropriate amounts of "POLYETHYLENE GLYCOL 6000", a polyethylene glycol preparation with an averaged molecular weight of 6,000 daltons, commercialized by E. Merck, Postfach, Germany, and allowed to stand under ice-chilling conditions for 30 minutes to effect binding reaction. The reaction product was centrifuged at 6,000rpm for 5 minutes and the resultant precipitate was collected to determine the level of radioactivity. In parallel, there was provided another sections as control in which 3pg nonlabelled human IL-18 was used along with 'I 25 -labelled human IL- 18 and treated similarly as above. Comparison with control revealed that the radioactivity of the precipitate from the sample solution was significantly higher. This indicated that the aqueous solution obtained in the above did contain IL-18R and the I-18R recognized and bound IL-18R when exposed to IL-18R Example 1-2 Binding ability to monoclonal antibody L428 cells (FERM BP-5777) were suspended in RPMI-1640 medium (pH7.4), supplemented with 0.1 v/v bovine serum albumin and also containing 0.1 v/v NaN 3 to give a cell density of 4x10 7 cells/ml, while monoclonal antibody MAb#117-10C specific to human IL-18R, obtained by the method described in Japanese Patent Application No.356,426/96 by the same applicant, was dissolved in another preparation of RPMI-1640 medium supplemented ,with 0.1 w/v% bovine serum albumin to give Sdifferent concentrations of 0.019 pg/ml, 0.209 pg/ml, 2.3 pg/ml, iij 20 25.3 pg/ml and 139.5 pg/ml.

Fifty microliter aliquots of the cell suspension prepared in the above were mixed with 50pl of either solution with different monoclonal antibody concentrations, agitated at for 2 hours, added with 50pl of RPMI-1640 medium supplemented with 0.1 v/v bovine serum albumin and also containing 4ng 1 25 I-labelled human IL-18 prepared in usual manner, and agitated at the same temperature for an additional minutes. Subsequently, each cell suspension was added with 200pl mixture solution (volume ratio 1:1) of dibutylphthalate and diocthylphtalate and centrifuged at 10,000rpm and 200C for minutes, followed by collecting the resultant precipitates containing the cells which were then determined for radioactivity using "MODEL ARC-300", a gamma-ray counter commercialized by Aloka Co., Ltd, Tokyo, Japan.

In parallel, there were provided additional two sections where the monoclonal antibody was neglected, while 4ng 12 5I-labelled human IL-18 was treated similarly as in the sample testing section with or without 4 micrograms of non-labelled human IL-18 (hereinafter referred to as "non-specific binding section" and "whole binding section" respectively). The levels of radioactivity found in "non-specific binding section" and "whole binding section" were put in Formula 1 together with that found in the sample testing section to calculate percent inhibition. The results were as shown in FIG. 1.

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I,

Formula 1 (Whole binding) (Testing) Percent Inhibition x 100 (Whole binding) (Non-specific binding) Fifty microliter aliquots of an IL-18R in aqueous solution obtained by the method in Example 1-1 were added with 501i1 solution with different concentrations for monoclonal antibody MAb #117-10C prepared similarly as above, agitated at for 2 hours, added with 4ng 12 5 1-labelled human IL-18, and agitated at 40C for an additional 30 minutes. Subsequently, each mixture was added with 50pl of 4 mg/ml y-globulin, allowed to stand under ice-chilling conditions for 30 minutes, added with 250p1 of PBS with 20 w/v polyethylene glycol, allowed to stand under ice-chilling conditions for an additional minutes, and centrifuged at 6,000rpm at 40C for 5 minutes, followed by collecting the resultant precipitates which were then determined for radioactivity similarly as above.

At the same time, there were provided additional two sections where the monoclonal antibody was neglected, while 4ng of 1 2 5 I-labelled human IL-18 were treated similarly as in the sample testing section with or without 4pg of non-labelled human IL-18 (hereinafter referred to as "whole binding section" and "non-specific binding section" respectively). The levels of radioactivity found in these two section were put in Formula 1 together in that found in the sample testing section to calculate percent inhibition. The results were as shown in FIG.l.

22 As seen in FIG. 1, in both cases of using L428 cell and IL-18R in solution, the binding of IL-18 to L428 cell and IL-18R were inhibited much more as the concentration of monoclonal antibody MAb #117-10C elevated. This indicated that the monoclonal antibody MAb #117-10C was bound to the possible IL-18R on the surface of L428 cell in a fashion competing with IL-18, as well as that the aqueous solution obtained by the method in Examplel-ldid contain a protein capable of recognizing IL-18 or IL-18R and the monoclonal antibody MAb #117-10C specifically reacted with the IL-18R.

Example 1-3 Western blotting A portion of the IL-18R in aqueous solution obtained 'by the method in Example -1was sampled, added with 2/3 volume of a mixture solution of 2.5 w/v sodium dodecyl sulfate and 50 v/v glycerol, incubated at 370C for 1 hour, and separated into respective proteinaceous components on conventional SDS- PAGE using 10-20% gradient gel but using no reducing agent. The proteinaceous components on the gel were transferred in usual manner to a nitrocellulose membrane which was then soaked for 1 hour in an appropriate amount of 50mM Tris-HC1 buffer with lO1g/ml of monoclonal antibody MAb #117-10C obtained by the methods described in Japanese Patent Application No.356,426/96 by the same applicant, 10 v/v "BLOCK ACE", an immobilizing agent commercialized by Dainippon Seiyaku Co., Ltd., Osaka, Japan, and 0.05 v/v "TWEEN 20", a detergent commercialized by City Chemical Corp., New York, and washed in 50mM Tris- HCl buffer (pH7.5) with 0.05 v/v TWEEN20" to remove the r maining antibody. The membrane was then soaked in Tris-HCl i 9-23 1 buffer (pH 7.5) with an appropriate amount of an anti-mouse immunoglobulin antibody of rabbit origin prelabelled with horse radish peroxidase, 10 v/v "BLOCK ACE" and 0.05 v/v "TWEEN for 1 hour to effect reaction, washed in 50mM Tris-HCl buffer (pH 7.5) with 0.05 v/v "TWEEN 20" and developed using "ECL kit", a kit for development commercialized by Amersham Corp., Arlington Heights, U.S.A.

At the same time, there was provided another section without the monoclonal antibody MAb #117-10C as control and it was treated similarly as above. The molecular weight markers were bovine serum albumin (67,000 daltons), ovalbumin (45,000 daltons), carbonic anhydrase (30,000 daltons), trypsin inhibitor (20,100 daltons) and a-lactoalbumin (14,000 daltons). The results were as shown in FIG. 2.

In the gel electrophoresis in FIG. 2, Lane 2 (with monoclonal antibody) bore a distinct band of IL-18R which was never found in Lane 3 (without monoclonal antibody).

Example 1-4 Inhibition of IL-18 activity KG-1 cells (ATCC CCL246), an established cell line derived from a patient with acute myelogenous leukemia, were suspended in RPMI-1640 medium (pH supplemented with 10 v/v fetal bovine serum and also containing 100g/ml kanamycin and 18.8mM Na 2

HPO

4 to give a cell density of 1x10 7 cells/ml, added with monoclonal antibody MAb #117-10C, obtained by the method described in Japanese Patent Application No.356,426/96 by the same applicant, to give a concentration of 10pg/ml and incubated at 370C for 30 minutes.

The KG-1 cells in suspension were distributed on 96- 24 well microplate to give respective amounts of 50pl/well, added with 50pl of human IL-18 which had been dissolved in a fresh preparation of the same medium to give respective concentrations of Ong/ml, 1.56ng/ml, 3.12ng/ml, 6.25ng/ml, 12.5ng/ml and further added with 50pl/well of lipopolysaccharide in a fresh preparation of the above medium, and incubated at 370C for 24 hours, after which each supernatant was collected and determined for IFN-y content by conventional enzyme immunoassay. In parallel, there were provided additional sections without the monoclonal antibody MAb #117-10C for respective IL-18 concentrations as control and they were treated similarly as above. The results were as shown in FIG. 3. The IFN-y contents in FIG. 3 were calibrated with reference to the standardized IFN-y preparation Gg23-901-530 available from the International Institute of Health, USA, and expressed in the International Unit(IU).

The results in FIG. 3 indicated that the presence of a.

monoclonal antibody MAb #117-10C inhibited the induction of IFNy by IL-18 in KG-1 cell as immunocompetent cell. This also indicated that monoclonal antibody MAb #117-10C blocked the IL- 18R on the surface of KG-1 cell in a fashion competing with Il- 18, thus preventing the signal transduction of IL-18 to KG-1 cell.

Example ,o Purification of IL-18R Seventy-eight milligrams of a monoclonal antibody MAb #117-10C, obtained by the method described in Japanese Patent Application No.356,426/96 by the same applicant, was dissolved in an appropriate amount of distilled water and the solution was 25 dialyzed against borate buffer (pH 8.5) with 0.5M NaCI at for 16 hours. Thereafter, in usual manner, an appropriate amount of "CNBr-ACTIVATED SEPHAROSE 4B", a CNBr-activated gel, commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, was added to the dialyzed solution and allowed to react at 40C for 18 hours under gentle stirring conditions to immobilize the monoclonal antibody MAb #117-10C on the gel.

The gel was packed into column in a plastic cylinder, equilibrated with 2mM CHAPS, charged with an IL-18R in aqueous solution obtained by the method in Example 1-1, and applied with PBS with 12mM CHAPS to remove non-adsorbed components. The column was then applied with 35mM ethylamine containing 2mM CHAPS (pH 10.8) while collecting the eluate in every 8ml fractions which were then checked for presence of IL-18R by the method in Example 1-1 using 1 25 I-labelled human IL-18. The chromatogram obtained in this operation was as shown in FIG.4.

As seen in FIG. 4, IL-18R was eluted in a single sharp "peak when immunoaffinity chromatography using monoclonal antibody MAb #117-10C was applied to a mixture of IL-18R and e contaminants such as the aqueous solution of IL-18R in Example 1-1. The fractions corresponding to this single peak were collected, pooled and lyophilized, thus obtaining a purified IL- 18R in solid form.

[o Thereafter, a portion of the purified IL-18R was sampled, incubated in PBS at 1000C for 5 minutes, and determined for residual activity by the method in Example 1-2, resulting in no binding to IL-18 which proved that IL-18R was inactivated by heating. This would support that the nature of this receptor is proteinaceous.

26 Further, a portion of the purified IL-18R obtained in the above was dissolved in an appropriate amount of PBS, dialyzed against PBS at ambient temperature overnight, added with an appropriate amount of 12 5I-labelled human IL-18 prepared by the method in Example 1-1 and ImM "BS 3 a polymerizing agent commercialized by Pierce, Rockford, and allowed to stand at OoC for 2 hours to form a conjugate of IL-18R and 1251labelled human IL-18. The reaction mixture was added with Tris- HC1 buffer (pH7.5), allowed to stand at OOC for an additional 1 hour to suspend the conjugation reaction, separated into respective proteinaceous components on SDS-PAGE using a set of molecular weight markers and dithiothreitol as reducing agent, and subjected to autoradiogram analysis.

The apparent molecular weight for this conjugate of IL-18R and 125 I-labelled human IL-18 was about 50,000 to 200,000 daltons when estimated with reference to the mobility of molecular weight markers on the autoradiogram. Since the molecular weight of IL-18 is about 20,000 daltons, the molecular weight of IL-18R can be estimated about 30,000-180,000 daltons on the assumption that IL-18R binds one human IL-18 molecule.

Example 1-6 Peptide mapping of IL-18R A purified IL-18R obtained by the method in Example 1-5 was electrophoresed on SDS-PAGE using 7.5 w/v gel with 2 w/v dithiothreitol as reducing agent, and the gel was then Ssoaked for 5 minutes in a mixture solution of 40 v/v aqueous methanol and 1 v/v acetic acid with 0.1 w/v Coomassie Brilliant Blue for development, and soaked for an additional 2 hours for destaining in the same solution but without Coomassie 27 Brilliant Blue, after which the stained part in the gel, molecular weight of 80,000-110,000 daltons, was cut off, added with 50 v/v aqueous acetonitrile containing 0.2 M (NH 4 2

CO

3 and repeatedly agitated at ambient temperature. Thereafter, the gel slices were lyophilized, added with 0.2M (NH 4 2

CO

3 (pH allowed to stand for 5 minutes to effect swelling, added with appropriate amounts of ImM hydrochloric acid with O.lpg/pl "SEQUENCING GRADE MODIFIED TRYPSIN", a reagent of trypsin commercialized by Promega Corp., Madison, and 0.2 M

(NH

4 2

CO

3 (pH and allowed to react at 370C overnight.

After suspending with 10 v/v aqueous acetic acid solution, the reaction mixture was added with a mixture solution of 0.1 v/v trifluoroacetic acid and 60 v/v aqueous acetonitrile and agitated at ambient temperature, after which the resultant supernatant was collected, concentrated in vacuo and centrifugally filtered, thus obtaining a concentrate with peptide fragments.

The concentrate was charged to "pRPC C2/C18 SC2.1/10", column for high-performance liquid chromatography commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, pre-equilibrated with 0.065 v/v trifluoroacetic acid, and then applied at a flow rate of 100pl/min with 0.055 v/v trifluoroacetic acid containing 80 v/v aqueous acetonitrile under liner gradient of acetonitrile increasing from 0 to 80 v/v over 160 minutes immediately after application of the eluent.

While monitoring the absorbance at a wavelength of 240nm, the eluate was fractioned to separately collect respective peptide fragments which eluted about 45, 50, 55, 58, 62, 72, 75 and 77 minutes after application of the eluent. The peptide fragments 28 (hereinafter referred to as "peptide fragment "peptide fragment "peptide fragment "peptide fragment 4", "peptide fragment "peptide fragment "peptide fragment 7" and "peptide fragment 8" in the order of elution) were analyzed in usual manner for amino acid sequence using "MODEL 473A", a protein sequencer commercialized by Perkin-Elmer Corp., Norwalk, U.S.A, revealing that the peptide fragments 1 to 8 bore the amino acid sequences of SEQ ID NOs:12 to 19 respectively.

The peptide map obtained by this operation was as shown in Example 2 Preparation of DNA Example 2-1 Preparation of total RNA In usual manner, L428 cells (FERM BP-5777) were suspended in RPMI-1640 medium (pH7.2) supplemented with 10 v/v fetal bovine serum, and proliferated at 370C while scaling up i the cultivation. When the cell density reached a prescribed level, the proliferated cells were collected, suspended in sodium citrate (pH7.0) containing both 6M guanidine isothiocyanate and 0.5 w/v% sodium N-laurylsarcosinate, and then disrupted with a homogenizer.

Aliquots of 0.1M EDTA (pH 7.5) containing 5.7M CsCl 2 were placed in 35ml-reaction tubes, poured with the cell disruptant obtained in the above in layer over the EDTA in each tube, and subjected to ultracentrifugation at 200C at 25,000rpm for 20 hours to collect the RNA fraction. The RNA fraction was distributed in 15ml-centrifugation tubes, added with an equivolume each of a mixture solution of chloroform/1-butanol 29 (volume ratio agitated for 5 minutes and centrifuged at 4oC at 10,000rpm for 10 minutes, after which the aqueous layer was collected, added with 2.5-fold volume of ethanol and allowed to stand at -200C for 2 hours to precipitate the total RNA. The precipitate was collected, washed with 75 v/v aqueous ethanol, and then dissolved in 0.5ml of sterilized distilled water to obtain a solution of the total RNA originating from L428 cell.

Example 2-2 Preparation of mRNA An aqueous solution containing total RNA solution obtained by the method in Example 2-1 was added with 0.5ml of Tris-HCl buffer (pH containing both ImM EDTA and 0.1 w/v sodium N-laurylsarcosinate, to bring the total volume to 1 ml. The mixture solution was added with 1 ml of "OLIGOTEXM- <SUPER>", a latex with an oligonucleotide of (dT) 30 commercialized by Japan Roche K. Tokyo, Japan, reacted at 650C for 5 minutes and rapidly cooled in an ice-chlling bath.

:Thereafter, the reaction mixture was added with 0.2ml of NaCl, incubated at 370C for 10 minutes, centrifuged at 10,000rpm for 10 minutes to collect the resultant precipitate in pellet form which was then suspended in 0.5ml of sterilized distilled S"water and incubated at 650C for 5 minutes to desorb the mRNA from the latex. The obtained solution was added with an appropriate amount of ethanol, and the resultant precipitate was collected and lyophilized to obtain a solid of mRNA.

Example 2-3 Preparation of DNA fragment encoding polypeptide Four microliters of 25mM MgCl 2 2pl of 100mM Tris-HCl buffer (pH 8.3) containing 500mM KC1, lpl of 25mM dNTP mix, 30 pl of 40units/pl ribonuclease inhibitor and ipl of 200units/pl reverse transcriptase were placed in a 0.5ml-reaction tube, added with 10 ng of an mRNA, obtained by the method in Example 2-2, along with an appropriate amount of random hexanucleotides, and added with sterilized distilled water to bring the total volume of 20pl. The obtained mixture was incubated first at 420C for 20 minutes, then at 990C for 5 minutes to suspend the reaction, thus obtaining a reaction mixture containing a first strand cDNA.

Twenty microliters of the reaction mixture was added with lpl of 2.5 units/pl "CLONED Pfu POLYMERASE", a DNA polymerase commercialized by Stratagene Cloning Systems, California, 10pl of the reaction buffer and lpl of dNTP mix, both commercialized by Stratagene Cloning Systems, added with 0.lpg each of oligonucleotides as sense and antisense primers having respective nucleotide sequences as shown with TCAGTCGACGCCACCATGAATTGTAGAGAA-3 and 5 GAAGCGGCCGCATCATTAAGACTCGGAAAGAAC-3 which had been prepared on the basis of the amino acid sequence described in P. Parnet et al., The Journal of Biological Chemistry, Vol.271, pp.

3 9 6 7 3 9 7 0 (1996), added with sterile distilled water to bring the total *o volume to 1001l. The resultant mixture was subjected first to S* 3-time cycles of incubating at 950C for 1 minute, 420C for 2 minutes and 720C for 3 minutes in the given order, then to time cycles of incubating at 950C for 1 minute, 600C for 2 minutes and 720C for 3 minutes in the given order to effect PCR *reaction.

Fifty nanograms of the obtained PCR product was added with 1 ng of "pCR-Script Cam a plasmid vector 31 commercialized by Stratagene Cloning Systems, California, and then subjected to ligation reaction at 160C for 2 hours using "DNA LIGATION KIT VERSION a DNA ligation kit commercialized by Takara Syuzo, Co., Ltd., Otsu, Shiga, Japan, to insert the DNA fragment of the PCR product in the plasmid vector. A portion of the reaction product was sampled and used in usual manner to transform "XL1-BLUE MRF' KAN", an Escherichia coli strain commercialized by Stratagene Cloning Systems, California, U.S.A.

Example 3 Preparation of recombinant DNA A transformant obtained by the method in Example 2-3 was inoculated in LB medium containing 30pg/ml chloramphenicol and cultivated at 370C for 18 hours, after which the cells were collected from the culture and treated in usual manner to obtain the plasmid DNA. After confirming by the dideoxy method that the plasmid DNA contained the nucleotide sequence of SEQ ID NO:7, the plasmid DNA was exposed to both restriction enzymes NotI and SalI, and 100 ng of the obtained DNA fragment was added with 10ng of "pcDNAI/Amp", a plasmid vector with a modified multiple cloning site, commercialized by Invitrogen Corporation, San Diego, which had been predigested with both restriction enzymes NotI and XhoI, and subjected to ligation reaction at 160C for 2 hours using "LIGATION KIT VERSION a ligation kit commercialized by Takara Syuzo Co., Ltd., Otsu, *Shiga, Japan. A portion of the reaction product was sampled and .9 introduced in usual manner into "XL1-BLUE MRF' KAN", a strain of Escherichia coli commercialized by Stratagene Cloning 32 Systems, California, to obtain a transformant "cDNA/HuIL-18R" which contained a recombinant DNA "pcDNA/HuIL- 18R" of this invention. The recombinant DNA "pcDNA/HuIL-18R" was analyzed in usual manner, revealing that in the recombinant DNA, a DNA "IL-18R cDNA", which contained the nucleotide sequence of SEQ ID NO:1 encoding the polypeptide of this invention, was linked downstream the cytomegalo virus promotor Pcmv, as shown in FIG. 6.

Example 4 Preparation of transformant A transformant "cDNA/HuIL-18R" obtained by the method in Example 3 was inoculated in LB medium (pH 7.5) containing lOOpg/ml ampicillin and cultured at 370C for 18 hours, after which the cells were collected from the culture and treated in usual manner to obtain the plasmid DNA. Separately, COS-1 cell (ATCC CRL-1650), a fibroblastic cell line derived from a kidney o of African green monkey was proliferated in usual manner, and 20 micrograms of the plasmid DNA obtained in the above was S* introduced by conventional electroporation method into 1 x 10 7 COS-1 cells to obtain transformant cells which contained the DNA 99 4 of this invention.

9999 Example Preparation of polypeptide DMEM medium (pH 7.2) supplemented with 10 v/v fetal bovine serum was distributed in flat-bottomed culture bottles, inoculated with transformant cells, obtained by the method in Example 4, to give a cell density of 1 x 10 5 cells/ml, and cultured at 370C in 5 v/v CO 2 incubator for 3 days. After removing the supernatant from the culture, PBS containing both 33 EDTA and 0.02 w/v NaN 3 was placed in the culture bottles to desorb the proliferated cells.

After washing in PBS, the proliferated cells were rinsed in a buffer containing 20mM HEPES, 10mM KC1, 1.5mM MgCl 2 and O.imM EDTA (hereinafter referred to as "hypotonic buffer"), and suspended in a fresh preparation of the hypotonic buffer to give a cell density of 2 x 10 7 cells/ml. The cell suspension was homogenized with a Dounce-type homogenizer under icechilling conditions, and the resultant homogenate was centrifuged at 15,000rpm at 5 minutes to remove both cell nuclei i and intact cells, and dialyzed overnight against PBS containing 2mM CHAPS.

The dialyzed product was charged to a column of immobilized monoclonal antibody MAb #117-10C, prepared by the method in Example 1-5, which was then applied with PBS containing 12mM CHAPS to remove non-adsorbed components.

Thereafter, the column was applied with 35 mM ethylamine (pHlO.8) containing 2 mM CHAPS while collecting and fractionating the eluate.

Each fraction was then checked for presence of the polypeptide of human origin by the method in Example 1-1 using 12 5 I-labelled human IL-18, selected and pooled to obtain per 108 starting cells about 2 ml of an aqueous solution which contained a polypeptide with the amino acid sequence of SEQ ID NO:20. The protein content in the solution was about The polypeptide thus obtained was studied for nphysicochemical properties by the methods in Example 1. As the result, the polypeptide obtained in this Example contained each 34 amino acid sequence in SEQ ID NOs:12 to 19 as partial amino acid sequences, as well as exhibiting physiological activities which were similar to those of the IL-18R from L428 cell.

Example 6 Soluble polypeptide from human origin Example 6-1 Preparation of recombinant DNA One nanogram of a recombinant DNA "pcDNA/HuIL-18R" obtained by the method in Example 3, 10pl of 10xPCR buffer and 1pl of 25mM dNTP mix were placed in O.5ml-reaction tube, added with 1 microliter of 2.5 -units/microliter Pfu DNA polymerase, added with appropriate amounts of oligonucleotides as sense and .antisense primers having respective nucleotide sequences as shown with 5'-TCAGTCGACGCCACCATGAATTGTAGAGAATTA-3' and GAAGCGGCCGCATCATTATCTTGTGAAGACGTG-3 and with sterile distilled water to bring the total volume to 100 pl. The resultant mixture was subjected first to 3-time cycles of incubating at 940C for 1 minute, 420C for 2 minutes and 720C for 3 minutes in the given order, then to 35-time cycles of incubating at 940C for 1 minute, 600C for 2 minutes and 720C for 3 minutes in the given order to effect PCR reaction.

Fifty nanograms of the obtained PCR product was added with Ing of "pCR-SCRIPT a plasmid vector commercialized by Takara Syuzo Co. Ltd., Otsu, Shiga, Japan, and reacted using "DNA LIGATION KIT VERSION a DNA ligation kit commercialized by Takara Shuzo Co. Ltd., Otsu, Shiga, Japan, at 160C for 2 hours to insert the DNA fragment as the PCR product into the plasmid vector. A portion of the reaction product was sampled A and "XL1-BLUE MRF' KAN", a strain of Escherichia coli 35 commercialized by Stratagene Cloning Systems, California, was transformed therewith in usual manner.

The transformant obtained in the above was inoculated in LB medium (pH 7.5) containing 100g/ml ampicillin and cultivated at 370C for 18 hours, after which the cells were collected from the culture and treated in usual manner to obtain the plasmid DNA. After confirming by the dideoxy method that the plasmid DNA contained the nucleotide sequence of SEQ ID the plasmid DNA was exposed to both restriction enzymes NotI and SalI, and 100 ng of the resultant DNA fragment was added with 10ng of "pEF-BOS", a plasmid vector prepared in accordance with the method described in S. Mizushima, Nucleic Acid Research, Vol.18, No.17, pp.

5 3 3 2 (1990) with slight modification and also predigested with both restriction enzymes NotI and XhoI, and subjected to ligation reaction using "LIGATION KIT VERSION a DNA ligation kit commercialized by Takara Shuzo Co., Ltd., Otsu, Shiga, Japan, at 160C for 2 hours.

A portion of the reaction product was sampled and introduced in usual manner into "XL1-BLUE MRF' KAN", a strain of Escherichia coli commercialized by Stratagene Cloning Systems, California, thus obtaining a transformant "EFHIL18R-14" which contained a recombinant DNA "pEFHIL18R-14" of this invention.

The recombinant DNA "pEFHIL18R-14" was analyzed in usual manner, revealing that in the recombinant DNA, a cDNA "EFHIL18R-14 cDNA", which contained the nucleotide sequence of SEQ ID NO:6 encoding the polypeptide of this invention, was located downstream the elongation factor 1 promotor EFlaP as shown in FIG. 7.

36 Example 6-2 Preparation of transformant A transformant "EFHIL18R-14" obtained by the method in Example 6-1 was inoculated in LB medium (pH 7.5) containing lOOg/ml ampicillin and cultivated at 370C for 18 hours, after which the cells were collected from the culture and treated in usual manner to obtain the plasmid DNA. Separately, COS-1 cell (ATCC CRL-1650), a fibroblastoid cell line derived from a kidney of African green monkey, was.proliferated in usual manner, and micrograms of the plasmid DNA obtained in the above was introduced by conventional electroporation method into 1 x 10 7 COS-1 cells to obtain transformant cells which contained the DNA of this invention.

Example 6-3 Preparation of soluble polypeptide "ASF104", a serum-free nutrient culture medium c6mmercialized by Ajinomoto Co., Inc., Tokyo, Japan, was distributed in flat-bottomed culture bottles, inoculated with ransformant cells, obtained by the method in Example 6-2, to givee a cell density of 1 x 10 5 cells/ml, and cultured in usual manner at 370C in 5 v/v CO 2 incubator for 3 days. The supernatant was collected from the culture and charged to a column of an immobilized monoclonal antibody #117-10C prepared by the method in Example 1-5, after which the column was applied first with PBS containing 12mM CHAPS to remove non-adsorbed components, then with 35mM ethylamine (pH 10.8) containing 2 mM CHAPS while collecting and fractionating the eluate. Each fraction was checked for presence of human soluble polypeptide by the method in Example 1-1 using 12 5I-labelled human IL-18, 37 selected and pooled to obtain per 108 starting cells about 2 ml of an aqueous solution which contained a polypeptide with the amino acid sequence of SEQ ID NO:22. The protein content in the solution was about The soluble polypeptide thus obtained was studied for physicochemical properties by the method in Example 1. As the result, the soluble polypeptide obtained in this Example contained each amino acid sequences in SEQ ID NOs:12 to 17 and 19 as partial sequences, as well as exhibiting physiological activities which were similar to the IL-18R from L428 cell.

Example 7 Soluble polypeptide of human origin One nanogram of an recombinant DNA "pEFHIL18R-14" obtained by the method in Example 6-1, 10pl of 10xPCR buffer and lpl of 25mM dNTP mix were placed in 0.5ml-reaction tube, added with 1 il of 2.5units/pl Pfu DNA polymerase, further added with appropriate amounts of oligonucleotides as sense and antisense primers having respective nucleotide sequences as shown with TCAGTCGACGCCACCATGAATTGTAGAG-3' and 5 GAAGCGGCCGCTCATTAGTGATGGTGATGGTGATGTGCAACATGGTTAAGCTT-3', and filled up to 100l with sterile distilled water. The resultant *0 mixture was subjected first to 3-time cycles of incubating at 940C for 1 minute, 420C for 2 minutes and 720C for 1 minute in the given order, then to 35-time cycles of incubating at 940C for 1 minute, 640C for 1 minute and 720C for 1 minute in the given order to effect PCR reaction, thus obtaining a DNA fragment which consisted of the nucleotide sequence of SEQ ID a digestion site for restriction enzyme SalI and a Kozak's sequence both linked to the 5'-terminal of the 38 nucleotide sequence of SEQ ID NO:5, and a digestion site for restriction enzyme NotI and a nucleotide sequence encoding (His) 6 tag both linked to the 3'-terminal of the nucleotide sequence of SEQ ID NO:5. This DNA fragment was introduced similarly as in Example 6-1 in "XL1-Blue MRF'Kan", a strain of Escherichia coli commercialized by Stratagene Cloning Systems, California, to obtain a transformant which contained a recombinant DNA "pEFHIL18RDl-2-H" according to this invention.

Analysis of the recombinant DNA in usual manner confirmed that in this recombinant DNA a cDNA "HIL18RD1-2-H", which contained the nucleotide sequence of SEQ ID NO:5 encoding the polypeptide of this invention, was located downstream the elongation factor promotor EFlaP as shown in FIG. 8.

The recombinant DNA "pEFHIL18RDl-2-H" was introduced in COS-1 cells similarly as in Example 6-2 using the transformant thus obtained, and the COS-1 cells were then cultivated similarly as in Example 6-3. The supernatant of the resultant culture was concentrated with membrane filtration, and charged on a column of "Ni-NTA Spin Kit", a gel product for affinity chromatography commercialized by QIAGEN GmbH, Hilden, Germany, which was then applied with PBS containing imidazole to remove the non-adsorbed fractions. Thereafter, the column was applied with PBS containing 250mM imidazole, and the eluate was collected in fractions while checking the presence of human soluble polypeptide in each fraction by the method in Example 1-1 using 125I-labelled human IL-18, after which the fractions with the polypeptide were collected and pooled, thus obtaining about 2ml of an aqueous solution containing the polypeptide with the amino acid sequence of SEQ ID NO:23 per 39 starting 108 cells. The protein content in the solution was about The soluble polypeptide thus obtained was studied for physicochemical properties by the method in Example 1. As the result, the soluble polypeptide obtained in this Example contained a part or whole of each amino acid sequences in SEQ ID NOs:14 to 16 and 19 as partial amino acid sequences, as well as exhibiting physiological activities which were similar to those of IL-18R from L428 cell.

Example 8 Soluble polypeptide of human origin A transformant containing a recombinant DNA "pEFHIL18RDl-H" according to this invention was prepared similarly as in Example 7, except that sense and antisense primers were replaced with oligonucleotides having respective nucleotide sequences as shown with TCAGTCGACGCCACCATGAATTGTAGAG- 3 and 5 GAAGCGGCCGCTCATTAGTGATGGTGATGGTGATGTCTTTCAGTGAAACAGCT-3' Analysis of the recombinant DNA in usual manner confirmed that in the recombinant DNA a cDNA "HIL18RD1-H", which contained the nucleotide sequence of SEQ ID NO:3 encoding the polypeptide of this invention, was located downstream the elongation factor promotor EFlaP as shown in FIG. 9. Thereafter, similarly as in Example 7, the recombinant DNA was introduced in COS-1 cells and brought into expression, thus obtaining about 2ml of an aqueous solution containing a polypeptide with the amino acid sequence of SEQ ID NO:24 per 108 starting cells. The protein content in the solution was about The polypeptide of this invention thus obtained were 40 studied for physicochemical properties by the method in Example 1. As the result, the soluble polypeptide obtained in this Example contained each amino acid sequences of SEQ ID NOs:14 and as partial amino acid sequences, as well as exhibiting physiological activities which were similar to those of the IL- 18R from L428 cell.

Example 9 Soluble polypeptide of mouse origin Example 9-1 Preparation of recombinant DNA A reaction product containing a first strand cDNA was obtained by subjecting an mRNA, prepared in usual manner from mouse liver, in place with that from L428 cell to the same reaction to synthesize first strand cDNA as in Example 2-3. The reaction product was treated by the same PCR method as in Example 2-3, except that the sense and antisense primers were replaced with oligonucleotides having respective nucleotide sequence as shown with 5'-TCAGTCGACGCCACCATGCATCATGAAGAA-3' and -GAAGCGGCCGCATCATTAGTGATGGTGATGGTGATGTGTAAAGACATGGCC-3', which had been prepared on the basis of the amino acid sequence described in P. Parnet et al., The Journal of Biological Chemistry, Vol.271, pp.3,967-3,970 (1996) and also the S. nucleotide sequence of SEQ ID NO:1: This operation gave a DNA fragment which comprised the nucleotide sequence of SEQ ID NO:11, a digestion site for restriction enzyme SalI linked to the 5'-terminal in the nucleotide sequence of the SEQ ID NO:11, and a cleavage site for restriction enzyme NotI and a nucleotide sequence encoding (His) 6 tag both linked to the 3'-terminal in 41 the nucleotide sequence of the SEQ ID NO:11.

According to the method in Example 6-1, this DNA fragment was introduced into "XL1-BLUE MRF' KAN", a strain of Escherichia coli commercialized by Stratagene Cloning Systems, California, to transform. After a plasmid DNA was collected from the transformant and confirmed to contain the nucleotide sequence of SEQ ID NO:11, the plasmid DNA was introduced into "XL1-BLUE MRF' KAN", a strain of Escherichia coli strain commercialized by Stratagene Cloning Systems, California, to obtain a transformant "EFMIL18RSHT" which contains a recombinant DNA "pEFMIL18RSHT" according to this invention. Analysis in usual manner confirmed that in the recombinant DNA "pEFMIL18RSHT" a cDNA "EFMIL18RSHT cDNA", which contained the nucleotide sequence of SEQ ID NO:4 encoding the polypeptide of this invention, was linked to downstream of the elongation factor 1 promotor EFlaP, as shown in FIG. Example 9-2 Preparation of transformant and soluble polypeptide According to the method in Example 6-2, a plasmid DNA was collected from a transformant "EFMIL18RSHT" obtained by the method in Example 9-1, and introduced into COS-1 cells to obtain transformant cells which contained a DNA encoding a soluble polypeptide of mouse origin.

"ASF104", a serum-free nutrient culture medium commercialized by Ajinomoto Co., Inc., Tokyo, Japan, was distributed in flat-bottomed culture bottles, inoculated with the transformed COS-1 cells to give a cell density of 1 x lO 5 cells/ml, and cultivated in usual manner at 370C in 5 v/v 42

CO

2 incubator for 3 days. The supernatant was collected from the resultant culture and charged to a column of "Ni-NTA", a gel product for affinity chromatography, commercialized by QIAGEN GmbH, Hilden, Germany, after which the column was applied first with PBS containing 20mM imidazole to remove non-adsorbed components, then with PBS containing 250mM imidazole while collecting and fractionating the eluate. The fractions were checked for presence of mouse soluble polypeptide by the method in Example 1-1 using 125 I-labelled mouse IL-18, selected and pooled, thus obtaining per 108 starting cells about 2 ml of an aqueous solution which contained a polypeptide with the amino acid sequence of SEQ ID NO:25. The protein content in the solution was about lOOg/ml. The soluble polypeptide thus obtained was studied in accordance with the method in Example 1, revealing that it efficiently neutralized mouse IL-18.

Example Liquid agent Either polypeptide obtained by the method in Examples to 8 was separately dissolved in aliquots of physiological saline containing as stabilizer 1 w/v "TREHAOSE", a powdered crystalline trehalose commercialized by Hayashibara Co., Ltd., Okayama, Japan, to give respective concentration of 1 mg/ml, and the resultant mixtures were separately and sterilely filtered with membrane in usual manner to obtain four distinct liquid agents.

The products, which are excellent in stability, are useful as injection, ophthalmic solution and collunarium in treatment and prevention of susceptive diseases including autoimmune diseases.

43 Example 11 Dried injection One hundred milligrams of either polypeptide obtained by the methods in Example 5 to 8 was separately dissolved in aliquots of physiological saline containing 1 w/v sucrose as stabilizer, the resultant solutions were separately and sterilely filtered with membrane, distributed in vials in every 1 ml aliquot, lyophilized and sealed in usual manner to obtain four distinct pulverized agents.

The products, which are excellent in stability, are useful as dried injection in treatment and prevention of susceptive diseases including autoimmune diseases.

Example 12 Ointment "HI-BIS-WAKO 104", a carboxyvinylpolymer Scommercialized by Wako Pure Chemicals, Tokyo, Japan, and "TREHAOSE", a powdered crystalline trehalose commercialized by Hayashibara Co., Ltd., Okayama, Japan, were dissolved in sterilized distilled water to give respective concentrations of 1.4 w/w and 2.0 w/w and either polypeptide obtained by the methods in Examples 5 to 8 was separately mixed with aliquots **of the resultant solution to homogeneity, and adjusted to pH7.2 to obtain four distinct paste agents containing about 1 mg/g of the polypeptide of this invention each.

The products, which are excellent in both spreadablity and stability, are useful as ointment in treatment and prevention of susceptive diseases including autoimmune diseases.

Example 13 Tablet 44 Aliquots of "FINETOSE", a pulverized anhydrous crystalline alpha-maltose commercialized by Hayashibara Co., Ltd., Okayama, Japan, were separately admixed with either polypeptide, obtained by the methods in Examples 5 to 8, and aliquots of "LUMIN" as cell activator, [bis-4-(lethylquinoline)][y-4'-(l-ethylquinoline)] pentamethionine cyanine, to homogeneity, and the resultant mixtures were separately tableted in usual manner to obtain four distinct types of tablets, about 200 mg each, containing about Img/tablet of the polypeptide of this invention and also Img/tablet of LUMIN each.

The products, which are excellent in swallowability and stability and also bears an cell activating property, are useful as tablet in treatment and prevention of susceptive diseases including autoimmune diseases.

Experiment.

Acute toxicity test In usual manner, a variety of agents, obtained by the methods in Examples 10 to 13 were percutaneously or orally administrated or intraperitoneally injected to 8 week-old mice.

As the result, the LD 50 of each sample was proved about 1 mg or higher per body weight of mouse in terms of the amount of the polypeptide, regardless of administration route. This does support that the polypeptide of this invention is safe when incorporated in pharmaceuticals directed to use in mammals including human.

As explained above, this invention is based on the discovery of a novel receptor protein which recognizes IL-18.

/s The polypeptide of this invention exhibits a remarkable efficacy 45 in relief of rejection reaction associated with grafts of organs and also in treatment and prevention of various disease resulting from excessive immunoreaction because the polypeptide bears properties of suppressing and regulating immunoreaction in mammals including human. Further, the polypeptide of this invention is useful in clarification of physiological activities of IL-18, establishment of hybridoma cells which are capable of producing monoclonal antibodies specific to IL-18R, and also affinity chromatography and labelled assay to purify and detect IL-18. In addition, the polypeptide of this invention, in particular, that in soluble form is useful in screening in vivo and in vitro agonists and antagonists to IL-18. The polypeptide of this invention, which bears these outstanding usefulness, can be easily prepared in desired amounts by the process according to this invention using recombinant DNA techniques.

This invention, which exhibits these remarkable effects, would be very significant and contributive to the art.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be oo interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not *a to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

feature, integer, step, component or group thereof.

46 SEQUENCE LISTING (1)INFORMATION FOR SEQ ID NO:1: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:1563 base pairs (B)TYPE:nucleic acid (C)strandedness:double (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:1..1563 (C)IDENTIFICATION METHOD:E (xi)SEQUENCE DESCRIPTION:SEQ ID NO:1:

GAA

Glu 1

TTC

Phe

ACC

Thr TCT TGT Ser Cys TAT CTG Tyr Leu ACC AAA Thr Ls a. a a a 0* a a a. a. S a a CTG AAC Leu Asn 50 35

CCA

Pro

GAG

Glu 65

ATG

Met TTT TGG Phe Trp AAA AAT Lys Asn AAA CAC AGC Lys His Ser ACT TCA CGT CCC CAC ATT Thr Ser Arg Pro His Ile 5 AAA CAT TGC TCG TGT TCA Lys His Cys Ser Cys Ser 25 AGC TGG TAC AAA AGC AGT Ser Trp Tyr Lys Ser Ser 40 AGG AGT TCC TCG AGA ATT Arg Ser Ser Ser Arg Ile 55 CCA GTT GAG TTG AAT GAC Pro Val Glu Leu Asn Asp 70 TAT ACT CAG AAA TGG AAA Tyr Thr Gin Lys Trp Lys TGT TTC ACT GAA AGA CAA Cys Phe Thr Glu Arg Gin 100 105 TTT TTT CAG ATA ACC TGT Phe Phe Gin Ile Thr Cys 120 AGC ACA TCA TTG TAT AAG Ser Thr Ser Leu Tyr Lys 135 AAA AAC CCA ACG ATA AAG Lys Asn Pro Thr Ile Lys 150 TAC TCC TGC GTG CAT TTC Tyr Ser Cys Val His Phe 165 ACC AAA ACC TTC AAT ATA Thr Lys Thr Phe Asn Ile 180 185 CCG GTT CTT CTT GGA CCA Pro Val Leu Leu Gly Pro 200 AAA AAC GTA AGG CTC AAC Lys Asn Val Arg Leu Asn ACT GTG GTT GAA GGG GAA CCT Thr Val Val Glu Gly Glu Pro 10 1 CTT GCA CAT GAG ATT GAA ACA Leu Ala His Glu Ile Glu Thr GGA TCA CAG GAA CAT GTG GAG Gly Ser Gln Glu His Val Glu GCT TTG CAT GAT TGT GTT TTG Ala Leu His Asp Cys Val Leu ACA GGA TCT TAC TTT TTC CAA Thr Gly Ser Tyr Phe Phe Gin 75 TTA AAT GTC ATC AGA AGA AAT Leu Asn Val Ile Arg Arg Asn 90 GTA ACT AGT AAA ATT GTG GAA Val Thr Ser Lys Ile Val Glu 110 GAA AAC AGT TAC TAT CAA ACA Glu Asn Ser Tyr Tyr Gin Thr 125 AAC TGT AAA AAG CTA CTA CTG Asn Cys Lys Lys Leu Leu Leu 140 AAG AAC GCC GAG TTT GAA GAT Lys Asn Ala Glu Phe Glu Asp 155 160 CTT CAT CAT AAT GGA AAA CTA Leu His His Asn Gly Lys Leu 170 175 ACA ATA GTG GAA GAT CGC AGT Thr Ile Val Glu Asp Arg Ser 190 AAG CTT AAC CAT GTT GCA GTG Lys Leu Asn His Val Ala Val 205 TGC TCT GCT TTG CTG AAT GAA Cys Ser Ala Leu Leu Asn Glu 48 96 144 192 240 288 336 384 432 480 528 576 624 672 GTT AAA Val Lys CTG GTC Leu Val 130 GAG AAC Glu Asn

AAA

Lys 115

AAC

Asn

AAT

Asn 145

CAG

Gin GGG TAT Gly Tyr TTT AAT ATC Phe Asn Ile AAT ATA Asn Ile GAA TTA Glu Leu

GTT

Val 195

GGA

Gly 47 a a* a a 0@ a a .a 210 215 GAG GAT GTA ATT TAT TGG ATG Glu Asp Val Ile Tyr Trp Met 225 230 AAT ATA CAT GAA GAG AAA GAA Asn Ile His Glu Glu Lys Glu 245 TGG CAT GCT TCA AAA GTA TTG Trp His Ala Ser Lys Val Leu 260 CTA AAT GTT TTA TAT AAT TGC Leu Asn Val Leu Tyr Asn Cys 275 ACC AAA AGC TTC ATC TTG GTG Thr Lys Ser Phe Ile Leu Val 290 295 CAC GTC TTC ACA AGA GGA ATG His Val Phe Thr Arg Gly Met 305 310 GTA GTG TGC CTA GTG ACT GTG Val Val Cys Leu Val Thr Val 325 CTA TTT TAT AGA CAT TTA ACG Leu Phe Tyr Arg His Leu Thr 340 AAA ACA TAT GAT GCT TTT GTG Lys Thr Tyr Asp Ala Phe Val 355 AAT GGA GAG GAG CAC ACC TTT Asn Gly Glu Glu His Thr Phe 370 375 GAG AAA CAT TTT GGG TAT AAG Glu ys His Phe Gly Tyr Lys 385 390 CCT GGA GGA GCT GTT GTT GAT Pro Gly Gly Ala Val Val Asp 405 CGA AGA CTA ATC ATT GTC CTA Arg Arg Leu Ile Ile Val Leu 420 AGG TAT GAA CTT GAA AGT GGA Arg Tyr Glu Leu Glu Ser Gly 435 ATT AAA ATA ATC TTA ATT GAA Ile Lys Ile Ile Leu Ile Glu 450 455 TTG CCC CAA TCA CTA AAG CTT Leu Pro Gin Ser Leu Lys Leu 465 470 AAG GCC GAT AAA TCT CTT TCT Lys Ala Asp Lys Ser Leu Ser 485 CTT TAC TTA ATG CCT GCA AAA Leu Tyr Leu Met Pro Ala Lys 500 GAA GTC TTG CCT GTT CTT TCC Glu Val Leu Pro Val Leu Ser 220 TTC GGG GAA GAA AAT Phe Gly Glu Glu Asn 235 ATG AGA ATT ATG ACT Met Arg Ile Met Thr 250 AGA ATT GAA AAT ATT Arg Ile Glu Asn Ile 265 ACT GTG GCC AGC ACG Thr Val Ala Ser Thr 280 AGA AAA GAC ATG GCT Arg Lys Asp Met Ala 300 ATC ATA GCT GTT TTG Ile Ile Ala Val Leu 315 TGT GTC ATT TAT AGA Cys Val Ile Tyr Arg 330 AGA AGA GAT GAA ACA Arg Arg Asp Glu Thr GGA TCG GAT Gly Ser Asp CCA GAA GGC Pro Glu Gly 255 GGT GAA AGC Glyv Glu Ser

CCT

Pro 240

AAA

Lys

AAT

Asn 270 GGA GGC Gly Gly ACA GAC 864 Thr Asp 285

GAT

Asp

ATC

Ile

GTT

Val

TTA

Leu

TGC

Cys 365 ATC CCA GGC Ile Pro Gly TTG GTG GCA 960 Leu Val Ala 320 GAC TTG GTT 1008 Asp Leu Val 335 ACA GAT GGA 1056 Thr Asp Gly 350 CGA CCT GAA 1104 Arg Pro Glu AGG GTG TTG 1152 Arg Val Leu 912

TCT

Ser 360

GCT

Ala

TTA

Leu

GAA

Glu

AGT

Ser

CTC

Leu 440

TTT

Phe

TTG

Leu

TAT

Tyr

ACA

Thr

GAG

Glu 720 768 816 345

TAC

Tyr CTA AAA GAA Leu Lys Glu GTG GAG ATT TTG CCC Val Glu Ile Leu Pro 380 TGC ATA TTT GAA AGG Cys Ile Phe Glu Arg 395 ATC CAC TCA CTG ATA Ile His Ser Leu Ile 410 AAA AGT TAT ATG TCT Lys Ser Tyr Met Ser GAT GTA Asp Val GTG 1200 Val 400 GAG AAA AGC 1248 Glu Lys Ser 415 AAT GAG GTC 1296 Asn Glu Val 425

CAT

His GAA GCA TTG Glu Ala Leu

GTG

Val 445 430

GAA

Glu AGA AAA 1344 Arg Lys ACA TTC 1392 Thr Phe ACA CCT GTT ACT GAC TTC Thr Pro Val Thr Asp Phe 460 AAA TCT CAC AGA GTT CTG Lys Ser His Arg Val Leu 475 AAC TCA AGG TTC TGG AAG Asn Ser Arg Phe Trp Lys 490 GTC AAG CCA GGT AGA GAC Val Lys Pro Gly Arg Asp

AAG

Lys TGG 1440 Trp 480 AAC CTT 1488 Asn Leu 495 GAA CCG 1536 Glu Pro 1563 505

TCT

Ser 510 48 515 520 (2)INFORMATION FOR SEQ ID NO:2: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:1557 base pairs (B)TYPE:nucieic acid (C)strandedness double (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:1..1557 (C)IDENTIFICATION METHOD:S (xi)SEQUENCE DESCRIPTION:SEQ ID NO:2:

G

S

S

0 *5

TCA

Ser 1

CCT

Pro

GAA

Glu

AGA

Arg

ACC

Thr

TCT

Ser

AGA

Arg

GTT

Val AAA AGT TGT ATT CAC CGA Lys Ser Cys Ile His Arg 5 TTT TAT CTG AAG CCA TGT Phe Tyr Leu Lys Pro Cys ACA GCC ACC ATG AGA TGG Thr Ala Thr Met Arg Trp GAG CTG AAC AAC AGA AGC Glu Leu Asn Asn Arg Ser 55 TTG GAA TTC TGG CCA GTT Leu Giu Phe Trp Pro Val 70 CAA GTC GGA AAT GAT CGT Gin Val Gly Asn Asp Arg AAC AAA CAC AGC TGT TTC Asn Lys His Ser Cys Phe 100 GAA GTT AAC AAA TCT CTG Glu Vai Asn Lys Ser Leu TCA CAA ATT Ser Gin Ile 10 GGC ATA TCT Gly Ile Ser 25 TTC AAA GGC Phe Lys Gly 40 TCG CCC AGA Ser Pro Arg GAG ATG GAG Glu Met Glu CGC AAT TGG Arg Asn Trp 90 TCT GAC AAG Ser Asp Lys 105 CAT ATC ACT His Ile Thr 120 TGG CTG TAT Trp Leu Tyr AAG GAT GCC Lys Asp Ala GTC CAC CAT Val His His 170 ACA GTT ATT Thr Val lie 185 AAG TGT GAG Lys Cys Glu 200 TGC AGT GCT Cys Ser Ala CAT GTG GTA GAG His Val Val Glu GCA CCA GTG CAC Ala Pro Val His AGT GCT TCA CAT Ser Ala Ser His GTC ACT TTT CAT Val Thr Phe His GAT GAG GGA ACG Asp Giu Gly Thr 75 ACC TTA AAT GTC Thr Leu Asn Val CTC GTG ACA AGC Leu Val Thr Ser 110 TGT AAG AAT CCT Cys Lys Asn Pro 125 AAG AAC TGT AAG Lys Asn Cys Lys 140 GAG TTT GGA GAT Glu Phe Gly Asp 155 AAT GGG ACA CGG Asn Gly Thr Arg GAA GGA AGG AGT Glu Gly Arg Ser 190 AAG GTT GGT GTA Lys Val Gly Val 205 TCA TTG AAT AAA Ser Leu Asn Lys 220 GGA GAA Gly Glu AGG AAT Arg Asn GAG TAT Glu Tyr GAT CAC Asp His TAC ATT Tyr Ile ACC AAA Thr Lys AGA GAT Arg Asp AAC TAT Asn Tyr GAA ATA Glu Ile GAG GGC Glu Gly 160 TAC AAC Tyr Asn 175 AAA GTA Lys Val GAA CTA Glu Leu GAC GAT Asp Asp 48 96 144 192 240 288 336 384 432 480 528 576 624 672

GAA

Glu

TCC

Ser 145

TAC

Tyr

ATC

Ile

ACT

Thr 115 GAG CTG ATC CAG GAC ACA Glu Leu Ile Gin Asp Thr 130 135 AAA ACC CCA AGG ATC CTG Lys Thr Pro Arg Ile Leu 150 TAC TCC TGC GTG TTT TCT Tyr Ser Cys Val Phe Ser 165 ACC AAG ACT GTC AAT ATA Thr Lys Thr Val Asn Ile 180 CCA GCT ATT TTA GGA CCA Pro Ala Ile Leu Gly Pro 195 GGA AAG GAT Gly Lys Asp 210 GTG GAG TTG AAC Val Giu Leu Asn 215 49 a a.

a. a a a. a a 0 CTG TTT TAT TGG AGC ATC AGG AAA GAG Leu Phe Tyr Trp Ser Ile Arg Lys Glu 225 230 CAA GAA GAC AGG AAG GAG ACG ACA ACA Gin Glu Asp Arg Lys Glu Thr Thr Thr 245 CAT GCT TCA AAA ATA CTG AGA TTT CAG His Ala Ser Lys Ile Leu Arg Phe Gin 260 265 AAT GTT TTA TAT AAT TGC ACC GTG GCC Asn Val Leu Tyr Asn Cys Thr Val Ala 275 280 AAG AGC TTC GTC TTG GTG AGA AAA GAA Lys Ser Phe Val Leu Val Arg Lys Glu 290 295 GTC TTT ACA GGA GGA GTA ACT GTG CTT Val Phe Thr Gly Gly Val Thr Val Leu 305 310 GTG TGT ATA GTG ATT TTG TGT GTC ATT Val Cys Ile Val Ile Leu Cys Val Ile 325 TTC TAT AGG CGC ATA GCG GAA AGA GAC Phe Tyr Arg Arg Ile Ala Glu Arg Asp 340 345 ACA TAT GAT GCC TTT GTG TCT TAC CTG Thr Tyr Asp Ala Phe Val Ser Tyr Leu 355 360 AAA GAA GAG TAT ACT TTT GCT GTG GAG Lys Glu Glu Tyr Thr Phe Ala Val Glu 370 375 AAA CAG TTT GGG TAT AAG TTA TGC ATA Lys Gin Phe Gly Tyr Lys Leu Cys Ile 385 "390 GGC GGA GCT GTT GTC GAG GAG ATC CAT Gly Gly Ala Val Val Glu Glu Ile His 405 AGG CTA ATC ATC GTT CTC AGC CAG AGT Arg Leu Ile Ile Val Leu Ser Gin Ser 420 425 CGT GAG CTC GAG AGT GGA CTC CAC GAA Arg Glu Leu Glu Ser Gly Leu His Glu 435 440 AAG ATC ATC TTA ATT GAG TTT ACT CCA Lys Ile Ile Leu Ile Glu Phe Thr Pro 450 455 CCC CCG TCG CTG AAA CTC CTG AAG TCC Pro Pro Ser Leu Lys Leu Leu Lys Ser 465 470 GCT GAC AGT CCC TCC ATG AAC TCA AGG Ala Asp Ser Pro Ser Met Asn Ser Arg 485 CTG ATG CCC GCA AAA GCC GTC AAG CCA Leu Met Pro Ala Lys Ala Val Lys Pro 500 505 CGG TCT GTT CTC TCA GCA CCT Arg Ser Val Leu Ser Ala Pro 515 GAC AGC TCA GAC Asp Ser Ser Asp 235 TGG ATT TCT GAA Trp Ile Ser Glu 250 AAA ATT ACT GAA Lys Ile Thr Glu AAC GAA GAA GCC Asn Glu Glu Ala 285 ATA CCT GAT ATC Ile Pro Asp Ile 300 GTT CTC GCC TCT Val Leu Ala Ser 315 TAT AAA GTT GAC Tyr Lys Val Asp 330 GAG ACA CTA ACA Glu Thr Leu Thr AAA GAG TGT CAT Lys Glu Cys His CCT AAT Pro Asn GGC AAA Gly Lys 255 AAC TAT Asn Tyr 270 ATA GAC Ile Asp CCA GGC Pro Gly

CTC

Leu

ACC

Thr

CAT

His 816 864 912 GTG GCA GCA 960 Val Ala Ala 320 TTG GTT CTG 1008 Leu Val Leu 335 GAT GGT AAA 1056 Asp Gly Lys 350 CCT GAG AAT 1104 Pro Glu Asn GTC CTG GAG 1152 Val Leu Glu GTG 720 Val 240 CTG 768 Leu 365 ACG TTA CCC AGG Thr Leu Pro Arg 380 TTT GAA AGA GAT GTG Phe Glu Arg Asp Val 395 TCA CTG ATA GAG AAA Ser Leu Ile Glu Lys 410 TAC CTG ACT AAC GGA Tyr Leu Thr Asn Gly GTG CCT 1200 Val Pro 400 AGC CGG 1248 Ser Arg 415 GCC AGG 1296 Ala Arg AAG ATT 1344 Lys Ile GCA CTG GTA Ala Leu Val

GAG

Glu 445 430

AGG

Arg GCC AGC AAC ATC ACC Ala Ser Asn Ile Thr 460 TAC AGA GTT CTA AAA Tyr Arg Val Leu Lys 475 TTC TGG AAG AAT CTT Phe Trp Lys Asn Leu 490 TGG AGA GAG GAG TCG Trp Arg Glu Glu Ser TTT CTC 1392 Phe Leu

TGG

Trp AGG 1440 Arg 480 GTT TAC 1488 Val Tyr 495 GAG GCG 1536 Glu Ala 1557 510 50 (3)INFORMATION FOR SEQ ID NO:3: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH:312 base pairs TYPE: nucleic acid (C )strandedness :double (D )TOPOLOGY: linear (ii )MOLECULE TYPE: cDNA (ix )FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:l. .312 (C)IDENTIFICATION METHOD:S (xi)SEQUENCE DESCRIPTION:SEQ ID NO:3: GAA TCT TGT ACT TCA CGT CCC CAC ATT Glu Ser Cys Thr Ser Arg Pro His Ile 1 5 TTC TAT CTG AAA CAT TGC TCG TGT TCA Phe Tyr Leu Lys His Cys Ser Cys Ser 25 ACC ACC AAA AGC TGG TAC AAA AGC AGT Thr Thr Lys Ser Trp Tyr Lys Ser Ser 40 CTG AAC CCA AGG AGT TCC TCG AGA ATT Leu Asn Pro Arg Ser Ser Ser Arg Ile 50 55 GAG TTT TGG CCA GTT GAG TTG AAT GAC Giu Phe Trp Pro Val Giu Leu Asn Asp 65 70 ATG AAA AAT TAT ACT CAG AAA TGG AAA Met Lys Asn Tyr Thr Gln Lys Trp Lys AAA CAC AGC TGT TTC ACT GAA AGA Lys His Ser Cys Phe Thr Glu Arg 100 ACT GTG GTT GAA Thr Val Val Glu 10 CTT GCA CAT GAG Leu Ala His Glu GGA TCA CAG GAA Gly Ser Gin Glu GCT TTG CAT GAT Aia Leu His Asp ACA GGA TCT TAC Thr Gly Ser Tyr 75 TTA AAT GTC ATC Leu Asn Val Ile 90 GGG GAA CCT Gly Glu Pro ATT GAA ACA Ile Glu Thr

CAT

His GTG GAG Val Glu TGT GTT TTG Cys Vai Leu 48 96 144 192 240 288 312 V9609

S.

.0 5 00.' '90:0 00 0 TTT TTC Phe Phe

CAA

Gin

AAT

Asn

AGA

Arg

AGA

Arg (4)INFORMATION FOR SEQ ID NO:4: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH:921 base pairs (B)TYPE:nucleic acid (C )strandedness :double (D )TOPOLOGY: linear (ii )MOLECULE TYPE: cDNA (ix )FEATURE: NAME/KEY: mat peptide (B)LOCATION:1. .921 IDENTIFICATION METHOD :S (xi)SEQUENCE DESCRIPTION:SEQ ID NO:4: TCA AAA AGT TGT ATT CAC CGA TCA CAA ATT CAT GTG GTA GAG GGA GAA Ser Lys Ser Cys Ile His Arg Ser Gin Ile His Val Val Giu Gly Giu 1 5 10 CCT TTT TAT CTG AAG CCA TGT GGC ATA TCT GCA CCA GTG CAC AGG AAT Pro Phe Tyr Leu Lys Pro Cys Gly Ile Ser Ala Pro Val His Arg Asn 25 GAA ACA GCC ACC ATG AGA TGG TTC AAA GGC AGT GCTTCA CAT GAG TAT Glu Thr Ala Thr Met Arg Trp Phe Lys Giy Ser Ala Ser His Glu Tyr 40 48 96 144 51 AGA GAG Arg Glu ACC TTG Thr Leu TCT CAA Ser Gin AGA AAC Arg Asn CTG AAC AAC AGA AGC Leu Asn Asn Arg Ser 55 GAA TTC TGG CCA GTT Glu Phe Trp Pro Val 70 GTC GGA AAT GAT CGT Val Gly Asn Asp Arg AAA CAC AGC TGT TTC Lys His Ser Cys Phe 100

GTT

Val

GAA

Glu

TCC

Ser 145

TAC

Tyr GAA GTT Glu Val 115 GAG CTG Glu Leu 130 AAA ACC Lys Thr TAC TCC Tyr Ser

AAC

Asn AAA TCT CTG Lys Ser Leu ATC CAG GAC ACA Ile Gin Asp Thr 135 CCA AGG ATC CTG Pro Arg Ile Leu 150 TGC GTG TTT TCT Cys Val Phe Ser 165 ACT GTC AAT ATA Thr Val Asn Ile 180 TCG CCC AGA GTC Ser Pro Arg Val GAG ATG GAG GAT Glu Met Glu Asp 75 CGC AAT TGG ACC Arg Asn Trp Thr 90 TCT GAC AAG CTC Ser Asp Lys Leu 105 CAT ATC ACT TGT His Ile Thr Cys 120 TGG CTG TAT AAG Trp Leu Tyr Lys AAG GAT GCC GAG Lys Asp Ala Glu 155 GTC CAC CAT AAT Val His His Asn 170 ACA GTT ATT GAA Thr Val Ile Glu 185 AAG TGT GAG AAG Lys Cys Glu Lys 200 TGC AGT GCT TCA Cys Ser Ala Ser AAA GAG GAC AGC Lys Glu Asp Ser 235 ACA ACA TGG ATT Thr Thr Trp Ile 250 TTT CAG AAA ATT Phe Gin Lys Ile 265 GTG GCC AAC GAA Val Ala Asn Glu ACT TTT CAT GAT CAC Thr Phe His Asp His GAG GGA ACG TAC ATT Glu Gly Thr Tyr Ile TTA AAT GTC ACC AAA Leu Asn Val Thr Lys GTG ACA AGC AGA GAT Val Thr Ser Arg Asp 110 AAG AAT CCT AAC TAT Lys Asn Pro Asn Tyr 125 AAC TGT AAG GAA ATA Asn Cys Lys Glu Ile 140 TTT GGA GAT GAG GGC Phe Gly Asp Glu Gly 160 GGG ACA CGG TAC AAC Gly Thr Arg Tyr Asn 175 GGA AGG AGT AAA GTA Gly Arg Ser Lys Val 190 GTT GGT GTA GAA CTA Val Gly Val Glu Leu 205 TTG AAT AAA GAC GAT Leu Asn Lys Asp Asp 220 TCA GAC CCT AAT GTG Ser Asp Pro Asn Val 0S OS

S

5* S 0 5* 00 S 5 0* Oe OS 0 0 *005 0000 @5 .500 a 55 0* S 0e 3 0

S

ATC ACC AAG Ile Thr Lys 192 240 288 336 384 432 480 528 576 624 672 720 768 816 864 912 921 ACT CCA GCT ATT Thr Pro Ala Ile 195 TTA GGA CCA Leu Gly Pro GGA AAG Gly Lys 210 CTG TTT Leu Phe 225

CAA

Gin GAT GTG GAG TTG AAC Asp Val Glu Leu Asn 215 TAT TGG AGC ATC AGG Tyr Trp Ser Ile Arg 230 GAC AGG AAG GAG ACG Asp Arg Lys Glu Thr 245 TCA AAA ATA CTG AGA Ser Lys Ile Leu Arg

GAA

Glu CAT GCT His Ala AAT GTT TTA Asn Val Leu 275 AAG AGC TTC Lys Ser Phe 290 GTC TTT ACA Val Phe Thr 305 260

TAT

Tyr TCT GAA GGC AAA Ser Glu Gly Lys 255 ACT GAA AAC TAT Thr Glu Asn Tyr 270 GAA GCC ATA GAC Glu Ala Ile Asp 285 GAT ATC CCA GGC Asp Ile Pro Gly 300 240

CTG

Leu

CTC

Leu

ACC

Thr

CAT

His AAT TGC ACC Asn Cys Thr GTC TTG GTG AGA Val Leu Val Arg 295 280

AAA

Lys GAA ATA CCT Glu Ile Pro FOR SEQ ID (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:621 base pairs (B)TYPE:nucleic acid (C)strandedness:double 52 (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:1..621 (C)IDENTIFICATION METHOD:S (xi)SEQUENCE DESCRIPTION:SEQ ID

GAA

Glu 1

TTC

Phe

TCT

Ser

TAT

Tyr ACC ACC Thr Thr CTG AAC Leu Asn GAG TTT Glu Phe 65 TGT ACT TCA Cys Thr Ser 5 CTG AAA CAT Leu Lys His AAA AGC TGG Lys Ser Trp CCA AGG AGT Pro Arg Ser TGG CCA GTT Trp Pro Val CGT CCC CAC Arg Pro His TGC TCG TGT Cys Ser Cys TAC AAA AGC Tyr Lys Ser 40 TCC TCG AGA Ser Ser Arg 55 GAG TTG AAT Glu Leu Asn ATT ACT GTG GTT Ile Thr Val Val 10 TCA CTT GCA CAT Ser Leu Ala His 25 AGT GGA TCA CAG Ser Gly Ser Gin ATT GCT TTG CAT Ile Ala Leu His GAC ACA GGA TCT Asp Thr Gly Ser 75 AAA TTA AAT GTC Lys Leu Asn Val 90 CAA GTA ACT AGT Gin Vai Thr Ser 105 TGT GAA AAC AGT Cys Glu Asn Ser GAA GGG GAA CCT Glu Gly Giu Pro GAG ATT GAA ACA Glu Ile Glu Thr GAA CAT GTG GAG Glu His Val Glu GAT TGT GTT TTG Asp Cys Val Leu TAC TTT TTC Tyr Phe Phe it o *0

J

0*

CAA

Gin ATG AAA AAT TAT ACT Met Lys Asn Tyr Thr 85 AAA CAC AGC TGT TTC Lys His Ser Cys Phe 100 GTT AAA AAA TTT TTT Val Lys Lys Phe Phe 115 CTG GTC AAC AGC ACA Leu Val Asn Ser Thr 130 GAG AAC AAT AAA AAC Glu Asn Asn Lys Asn 145 GAG GGG TAT TAC TCC Gin Gly Tyr Tyr Ser 165 TTT AAT ATC ACC AAA Phe Asn Ile Thr Lys 180 AAT ATA GTT CCG GTT Asn Ile Val Pro Val 195 70

CAG

Gin AAA TGG Lys Trp ACT GAA AGA Thr Giu Arg CAG ATA ACC Gin Ile Thr 120 TCA TTG TAT Ser Leu Tyr 135 CCA ACG ATA Pro Thr Ile

ATC

Ile

AAA

Lys

TAC

Tyr 125 AGA AGA AAT Arg Arg Asn ATT GTG GAA Ile Val Glu 110 TAT CAA ACA Tyr Gin Thr CTA CTA CTG Leu Leu Leu 48 96 144 192 240 288 336 384 432 480 528 576 621 150

TGC

Cys AAG AAC TGT AAA AAG Lys Asn Cys Lys Lys 140 AAG AAG AAC GCC GAG Lys Lys Asn Ala Glu 155 TTC CTT CAT CAT AAT Phe Leu His His Asn 170 ATA ACA ATA GTG GAA Ile Thr Ile Val Glu TTT GAA Phe Glu

GAT

Asp 160 GTG CAT Val His ACC TTC AAT Thr Phe Asn CTT CTT GGA Leu Leu Gly 200

GGA

Gly

GAT

Asp 190

GTT

Val AAA CTA Lys Leu 175 CGC AGT Arg Ser

GCA

Ala 185

CCA

Pro AAG CTT AAC Lys Leu Asn

CAT

His 205 (6)INFORMATION FOR SEQ ID NO:6: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:927 base pairs (B)TYPE:nucleic acid (C)strandedness:double (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:mat peptide 53 (B)LOCATION:1..927 (C)IDENTIFICATION METHOD:E (xi)SEQUENCE DESCRIPTION:SEQ ID NO:6: GAA TCT TGT ACT TCA CGT CCC Glu Ser Cys Thr Ser Arg Pro 1 5 TTC TAT CTG AAA CAT TGC TCG Phe Tyr Leu Lys His Cys Ser ACC ACC AAA AGC TGG TAC AAA Thr Thr Lys Ser Trp Tyr Lys CTG AAC CCA AGG AGT TCC TCG Leu Asn Pro Arg Ser Ser Ser 55 GAG TTT TGG CCA GTT GAG TTG Glu Phe Trp Pro Val Glu Leu 70 ATG AAA AAT TAT ACT CAG AAA Met Lys Asn Tyr Thr Gin Lys 85 AAA CAC AGC TGT TTC ACT GAA Lys His Ser Cys Phe Thr Glu 100 GTT AAA AAA TTT TTT CAG ATA Val Lys Lys Phe Phe Gin Ile 115 CTG GTC AAC AGC ACA TCA TTG Leu Val Asn Ser Thr Ser Leu 130 -135 GAG AAC AAT AAA AAC CCA ACG Glu Asn Asn Lys Asn Pro Thr 145 150 CAG GGG TAT TAC TCC TGC GTG Gin Gly Tyr Tyr Ser Cys Val 165 TTT AAT ATC ACC AAA ACC TTC Phe Asn Ile Thr Lys Thr Phe 180 AAT ATA GTT CCG GTT CTT CTT Asn Ile Val Pro Val Leu Leu 195 GAA TTA GGA AAA AAC GTA AGG Glu Leu Gly Lys Asn Val Arg 210 215 GAG GAT GTA ATT TAT TGG ATG Glu Asp Val Ile Tyr Trp Met 225 230 AAT ATA CAT GAA GAG AAA GAA Asn Ile His Glu Glu Lys Glu 245 TGG CAT GCT TCA AAA GTA TTG Trp His Ala Ser Lys Val Leu 260 CTA AAT GTT TTA TAT AAT TGC Leu Asn Val Leu Tyr Asn Cys CAC ATT His Ile TGT TCA Cys Ser 25 AGC AGT Ser Ser 40 AGA ATT Arg Ile AAT GAC Asn Asp TGG AAA Trp Lys AGA CAA Arg Gin 105 ACC TGT Thr Cys 120 TAT AAG Tyr Lys ATA AAG Ile Lys CAT TTC His Phe AAT ATA Asn Ile 185 GGA CCA Gly Pro 200 CTC AAC Leu Asn TTC GGG Phe Gly ATG AGA Met Arg AGA ATT Arg Ile 265 ACT GTG Thr Val ACT GTG GTT GAA GGG GAA CCT Thr Val Val Glu Gly Glu Pro 10 CTT GCA CAT GAG ATT GAA ACA Leu Ala His Glu Ile Glu Thr GGA TCA CAG GAA CAT GTG GAG Gly Ser Gin Glu His Val Glu GCT TTG CAT GAT TGT GTT TTG Ala Leu His Asp Cys Val Leu ACA GGA TCT TAC TTT TTC CAA Thr Gly Ser Tyr Phe Phe Gin 75 TTA AAT GTC ATC AGA AGA AAT Leu Asn Val Ile Arg Arg Asn 90 GTA ACT AGT AAA ATT GTG GAA Val Thr Ser Lys Ile Val Glu 110 GAA AAC AGT TAC TAT CAA ACA Glu Asn Ser Tyr Tyr Gin Thr 125 AAC TGT AAA AAG CTA CTA CTG Asn Cys Lys Lys Leu Leu Leu 140 AAG AAC GCC GAG TTT GAA GAT Lys Asn Ala Glu Phe Glu Asp 155 160 CTT CAT CAT AAT GGA AAA CTA Leu His His Asn Gly Lys Leu 170 175 ACA ATA GTG GAA GAT CGC AGT Thr Ile Val Glu Asp Arg Ser 190 AAG CTT AAC CAT GTT GCA GTG Lys Leu Asn His Val Ala Val 205 TGC TCT GCT TTG CTG AAT GAA Cys Ser Ala Leu Leu Asn Glu 220 GAA GAA AAT GGA TCG GAT CCT Glu Glu Asn Gly Ser Asp Pro 235 240 ATT ATG ACT CCA GAA GGC AAA Ile Met Thr Pro Glu Gly Lys 250 255 GAA AAT ATT GGT GAA AGC AAT Glu Asn Ile Gly Glu Ser Asn 270 GCC AGC ACG GGA GGC ACA GAC Ala Ser Thr Gly Gly Thr Asp 48 96 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816 864 54 275 280 285 ACC AAA AGC TTC ATC TTG GTG AGA AAA GAC ATG GCT GAT ATC CCA GGC 912 Thr Lys Ser Phe Ile Leu Val Arg Lys Asp Met Ala Asp Ile Pro Gly 290 295 300 CAC GTC TTC ACA AGA 927 His Val Phe Thr Arg 305 (7)INFORMATION FOR SEQ ID NO:7: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:1620 base pairs (B)TYPE:nucieic acid (C)strandedness:double (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (vi)ORIGINAL SOURCE: (A)ORGANISM:lymphobiastoid cell derived from a patient with Hodgkin's disease (B)INDIVIDUAL ISOLATE:L428 (FERM BP-5777) (ix)FEATURE: (A)NAME/KEY:sig peptide (B)LOCATION:1..57 (C)IDENTIFICATION METHOD:E .ix)FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:58..1620 (C)IDENTIFICATION METHOD:E (xi)SEQUENCE DESCRIPTION:SEQ ID NO:7: ATG AAT TGT AGA GAA TTA CCC TTG ACC CTT TGG GTG CTT ATA TCT GTA 48 Met Asn Cys Arg Glu Leu Pro Leu Thr Leu Trp Val Leu Ile Ser Val -10 AGC ACT GCA GAA TCT TGT ACT TCA CGT CCC CAC ATT ACT GTG GTT GAA 96 Ser Thr Ala Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val Glu 1 5 GGG GAA CCT TTC TAT CTG AAA CAT TGC TCG TGT TCA CTT GCA CAT GAG 144 Gly Glu Pro Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His Glu 20 ATT GAA ACA ACC ACC AAA AGC TGG TAC AAA AGC AGT GGA TCA CAG GAA 192 Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gln Glu 35 40 CAT GTG GAG CTG AAC CCA AGG AGT TCC TCG AGA ATT GCT TTG CAT GAT 240 His Val Glu Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His Asp 55 TGT GTT TTG GAG TTT TGG CCA GTT GAG TTG AAT GAC ACA GGA TCT TAC 288 Cys Val Leu Glu Phe Trp Pro Val Giu Leu Asn Asp Thr Gly Ser Tyr 70 TTT TTC CAA ATG AAA AAT TAT ACT CAG AAA TGG AAA TTA AAT GTC ATC 336 Phe Phe Gin Met Lys Asn Tyr Thr Gin Lys Trp Lys Leu Asn Val Ile 85 AGA AGA AAT AAA CAC AGC TGT TTC ACT GAA AGA CAA GTA ACT AGT AAA 384 Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg Gin Val Thr Ser Lys 100 105 ATT GTG GAA GTT AAA AAA TTT TTT CAG ATA ACC TGT GAA AAC AGT TAC 432 Ile Val Glu Val Lys Lys Phe Phe Gin Ile Thr Cys Giu Asn Ser Tyr 110 115 120 125 55 TAT CAA ACA CTG GTC AAC AGC ACA Tyr Gin Thr Leu Val Asn Ser Thr 130 CTA CTA CTG GAG AAC AAT AAA AAC Leu Leu Leu Glu Asn Asn Lys Asn 145 TTT GAA GAT CAG GGG TAT TAC TCC Phe Glu Asp Gin Gly Tyr Tyr Ser 160 165 GGA AAA CTA TTT AAT ATC ACC AAA Gly Lys Leu Phe Asn Ile Thr Lys 175 180 GAT CGC AGT AAT ATA GTT CCG GTT Asp Arg Ser Asn Ile Val Pro Val 190 195 GTT GCA GTG GAA TTA GGA AAA AAC Val Ala Val Glu Leu Gly Lys Asn 210 CTG AAT GAA GAG GAT GTA ATT TAT Leu Asn Glu Glu Asp Val Ile Tyr 225 TCG GAT CCT AAT ATA CAT GAA GAG Ser Asp Pro Asn Ile His Glu Glu 240 245 GAA GGC AAA TGG CAT GCT TCA AAA Glu Gly Lys Trp His Ala Ser Lys 255 260 GAA AGC AAT CTA AAT GTT TTA TAT Glu Ser Asn Leu Asn Val Leu Tyr 270 275 GGC ACA GAC ACC AAA AGC TTC ATC Gly Thr Asp Thr Lys Ser Phe Ile 290 ATC CCA GGC CAC GTC TTC ACA AGA Ile Pro Gly His Val Phe Thr Arg 305 TTG GTG GCA GTA GTG TGC CTA GTG Leu Val Ala Val Val Cys Leu Val 320 325 GAC TTG GTT CTA TTT TAT AGA CAT Asp Leu Val Leu Phe Tyr Arg His 335 340 ACA GAT GGA AAA ACA TAT GAT GCT Thr Asp Gly Lys Thr Tyr Asp Ala 350 355 CGA CCT GAA AAT GGA GAG GAG CAC Arg Pro Glu Asn Gly Glu Glu His 370 AGG GTG TTG GAG AAA CAT TTT GGG Arg Val Leu Glu Lys His Phe Gly 385 GAT GTA GTG CCT GGA GGA GCT GTT Asp Val Val Pro Gly Gly Ala Val 400 405 GAG AAA AGC CGA AGA CTA ATC ATT Glu Lys Ser Arg Arg Leu Ile Ile 415 420

TCA

Ser

CCA

Pro 150 TTG TAT AAG AAC Leu Tyr Lys Asn 135 ACG ATA AAG AAG Thr Ile Lys Lys TGC GTG CAT TTC CTT Cys Val His Phe Leu 170 ACC TTC AAT ATA ACA Thr Phe Asn Ile Thr 185 CTT CTT GGA CCA AAG Leu Leu Gly Pro Lys 200 GTA AGG CTC AAC TGC Val Arg Leu Asn Cys 215 TGG ATG TTC GGG GAA Trp Met Phe Gly Glu 230 155

CAT

His CAT AAT His Asn ATA GTG GAA Ile Val Glu CTT AAC Leu Asn TCT GCT Ser Ala 220 GAA AAT Glu Asn 235

CAT

His 205

TTG

Leu

GGA

Gly TGT AAA AAG Cys Lys Lys 140 AAC GCC GAG Asn Ala Glu AAA GAA ATG AGA ATT ATG Lys Glu Met Arg Ile Met 250 GTA TTG AGA ATT GAA AAT Val Leu Arg Ile Glu Asn 265 AAT TGC ACT GTG GCC AGC Asn Cys Thr Val Ala Ser 280 TTG GTG AGA AAA GAC ATG Leu Val Arg Lys Asp Met 295 GGA ATG ATC ATA GCT GTT Glv Met Ile Ile Ala Val 480 528 576 624 672 720 768 816 864 912 960

ACG

Thr

GCT

Ala 300

GGA

Gly 285

GAT

Asp ACT CCA Thr Pro ATT GGT Ile Gly 310 ACT GTG TGT GTC ATT Thr Val Cys Val Ile 330 TTA ACG AGA AGA GAT Leu Thr Arg Arg Asp 345 TTT GTG TCT TAC CTA Phe Val Ser Tyr Leu 360 ACC TTT GCT GTG GAG Thr Phe Ala Val Glu 375 TAT AAG TTA TGC ATA Tyr Lys Leu Cys Ile 390 GTT GAT GAA ATC CAC Val Asp Glu Ile His 410 GTC CTA AGT AAA AGT Val Leu Ser Lys Ser 425 315

TAT

Tyr GAA ACA TTA 1104 Glu Thr Leu AAA GAA TGC 1152 Lys Glu Cys 365 ATT TTG CCC 1200 Ile Leu Pro 380 TTT GAA AGG 1248 Phe Glu Arg 395 TCA CTG ATA 1296 Ser Leu Ile TAT ATG TCT 1344 Tyr Met Ser TTG ATC 1008 Leu Ile AGA GTT 1056 Arg Val 56 AAT GAG GTC AGG TAT GAA CTT GAA AGT Asn Glu Val Arg Tyr Glu Leu Glu Ser 430 435 GAA AGA AAA ATT AAA ATA ATC TTA ATT Glu Arg Lys Ile Lys Ile Ile Leu Ile 450 TTC ACA TTC TTG CCC CAA TCA CTA AAG Phe Thr Phe Leu Pro Gin Ser Leu Lys 465 470 CTG AAG TGG AAG GCC GAT AAA TCT CTT Leu Lys Trp Lys Ala Asp Lys Ser Leu 480 485 AAG AAC CTT CTT TAG TTA ATG CCT GCA Lys Asn Leu Leu Tyr Leu Met Pro Ala 495 500 GAG GAA CCG GAA GTG TTG CCT GTT CTT Asp Glu Pro Glu Val Leu Pro Val Leu 510 515 GGA GTC GAT GAA GGA TTG GTG Gly Leu His Giu Ala Leu Val 440 445 GAA TTT AGA CCT GTT AGT GAG Glu Phe Thr Pro Val Thr Asp 455 460 GTT TTG AAA TGT CAC AGA GTT Leu Leu Lys Ser His Arg Val 475 TCT TAT AAG TGA AGG TTC TGG Ser Tyr Asn Ser Arg Phe Trp 490 AAA AGA GTC AAG CCA GGT AGA Lys Thr Val Lys Pro Gly Arg 505 TCC GAG TCT Ser Glu Ser 520 1392 1440 1488 1536 1584 1620 (8)INFORMATION FOR SEQ ID NO:8: (i)SEQUENGE GHARAGTERISTIGS: (A)LENGTH:369 base pairs (B)TYPE:nucleic acid (C)strandedness:double (D)TOPOLOGY:linear (ii)MOLEGULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:sig peptide (B)LOGATION:1..57 (C)IDENTIFIGATION METHOD:S (ix)FEATURE: .(A)NAME/KEY:mat peptide (B)LOCATION:58..369 (C)IDENTIFICATION METHOD:S (xi)SEQUENCE DESGRIPTION:SEQ ID NO:8:

ATG

Met

AGC

Ser

GGG

Gly

ATT

Ile

CAT

His

TGT

Gys

TTT

Phe AAT TGT AGA GAA TTA CCC TTG Asn Cys Arg Glu Leu Pro Leu -15 ACT GGA GAA TGT TGT ACT TCA Thr Ala Glu Ser Gys Thr Ser 1 5 GAA CCT TTC TAT GTG AAA CAT Glu Pro Phe Tyr Leu Lys His 20 GAA ACA ACC ACC AAA AGG TGG Glu Thr Thr Thr Lys Ser Trp 35 GTG GAG CTG AAG CCA AGG AGT Val Giu Leu Asn Pro Arg Ser GTT TTG GAG TTT TGG CCA GTT Val Leu Glu Phe Trp Pro Val TTC CAA ATG AAA AAT TAT ACT Phe Gin Met Lys Asn Tyr Thr 85 ACC CTT Thr Leu -10 CGT CCG Arg Pro TGC TCG Cys Ser TAG AAA Tyr Lys TCC TCG Ser Ser 55 GAG TTG Glu Leu 70 CAG AAA Gin Lys TGG GTG GTT ATA Trp Val Leu Ile CAC ATT ACT GTG His Ile Thr Val TGT TCA CTT GCA Cvs Ser Leu Ala

TCT

Ser

GTT

Val

GTA

Val

GAA

Glu CAT GAG His Glu AGG AGT Ser Ser 40 AGA ATT Arg Ile GGA TGA GAG Gly Ser Gin

GAA

Glu 48 96 144 192 240 288 336 GCT TTG Ala Leu AAT GAG ACA Asn Asp Thr TGG AAA TTA Trp Lys Leu

GGA

Gly

AAT

Asn GAT GAT His Asp TCT TAG Ser Tyr GTC ATC Val Ile 57 AGA AGA AAT AAA CAC AGC TGT TTC ACT GAA AGA Arg Arg Asn Lys His Ser Cys Phe Thr Glu Arg 100 (9)INFORMATION FOR SEQ ID NO:9: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:678 base pairs (B)TYPE:nucleic acid (C)strandedness:double (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:sig peptide (B)LOCATION:1..57 (C)IDENTIFICATION METHOD:S (ix)FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:58..678 (C)IDENTIFICATION METHOD:S (xi)SEQUENCE DESCRIPTION:SEQ ID NO:9: 369 0* ATG AAT TGT Met Asn Cys AGC ACT GCA Ser Thr Ala GGG GAA CCT Gly Glu Pro ATT GAA ACA Ile Glu Thr AGA GAA Arg Glu -15 GAA TCT Glu Ser 1 TTC TAT Phe Tyr ACC ACC Thr Thr TTA CCC TTG ACC Leu Pro Leu Thr

CTT

Leu -10

CCC

Pro TGT ACT Cys Thr CTG AAA Leu Lys 20 AAA AGC Lvs Ser TCA CGT Ser Arg 5 CAT TGC His Cys TGG GTG CTT ATA Trp Val Leu Ile CAC ATT ACT GTG His Ile Thr Val TGT TCA CTT GCA Cvs Ser Leu Ala TCT GTA Ser Val GTT GAA Val Glu CAT GAG His Glu

TCG

Ser TGG TAC AAA Trp Tyr Lys 30

CAT

His GTG GAG CTG Val Glu Leu

AAC

Asn 50

TTT

Phe 35

CCA

Pro

AGC

Ser 40

AGA

Arg

AGT

Ser GGA TCA CAG Gly Ser Gin AGG AGT TCC Arg Ser Ser

TCG

Ser 55

TTG

Leu ATT GCT TTG Ile Ala Leu

CAT

His

TCT

Ser

GAA

Glu

GAT

Asp

TAC

Tyr TGT GTT TTG Cys Val Leu

GAG

Glu

ATG

Met TGG CCA GTT Trp Pro Val

GAG

Glu 70

CAG

Gin AAT GAC ACA Asn Asp Thr

GGA

Gly

AAT

Asn 48 96 144 192 240 288 336 384 432 480 528 576 TTT TTC Phe Phe AGA AGA Arg Arg ATT GTG Ile Val

CAA

Gln

AAT

Asn AAA AAT TAT Lys Asn Tyr

ACT

Thr 85

TTC

Phe AAA TGG AAA Lys Trp Lys

TTA

Leu

GTA

Val GTC ATC Val Ile AAA CAC AGC Lys His Ser

TGT

Cys 100

TTT

Phe ACT GAA AGA Thr Glu Arg

CAA

Gin 105

TGT

Cys ACT AGT AAA Thr Ser Lys GAA GTT AAA Glu Val Lys 110

TAT

Tyr

AAA

Lys 115

AAC

Asn TTT CAG ATA Phe Gin Ile

ACC

Thr 120

TAT

Tyr GAA AAC AGT Glu Asn Ser

TAC

Tyr 125 CAA ACA CTG Gln Thr Leu

GTC

Val 130

AAC

Asn AGC ACA TCA Ser Thr Ser

TTG

Leu 135

ACG

Thr AAG AAC Lys Asn CTA CTA CTG Leu Leu Leu TTT GAA GAT Phe Glu*Asp 160

GAG

Glu 145

CAG

Gin AAT AAA AAC Asn Lys Asn GGG TAT TAC Gly Tyr Tyr

TCC

Ser 165

CCA

Pro 150

TGC

Cys 58 GTG CAT TTC Val His Phe

CTT

Leu 170 ATA AAG AAG Ile Lys Lys TGT AAA AAG Cys Lys Lys 140 AAC GCC GAG Asn Ala Glu 155 CAT CAT AAT His His Asn GGA AAA CTA TTT AAT ATC ACC AAA ACC TTC Gly Lys Leu Phe Asn Ile Thr Lys Thr Phe 175 180 GAT CGC AGT AAT ATA GTT CCG GTT GTT CTT Asp Arg Ser Asn Ile Val Pro Val Leu Leu 190 195 GTT GCA Val Ala AAT ATA Asn Ile 185 GGA CCA Gly Pro 200 ACA ATA GTG GAA Thr Ile Val Glu AAG CTT AAC CAT Lys Leu Asn His 205 624 672 678 FOR SEQ ID NO:l0: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:984 base pairs (B)TYPE:nucleic acid (C)strandedness:double (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:sig peptide (B)LOCATION:1..57 (C)IDENTIFICATION METHOD:E (ix)FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:58..984 (C)IDENTIFICATION METHOD:E (xi)SEQUENCE DESCRIPTION:SEQ ID N0:10:

S

*5 ATG AAT TGT AGA GAA TTA CCC TTG ACC Met Asn Cys Arg Glu Leu Pro Leu Thr AGC ACT GCA GAA TCT TGT ACT TCA CGT Ser Thr Ala Glu Ser Cys Thr Ser Arg 1 5 GGG GAA CCT TTC TAT CTG AAA CAT TGC Gly Glu Pro Phe Tyr Leu Lys His Cys 20 ATT GAA ACA ACC ACC AAA AGC TGG TAC Ile Glu Thr Thr Thr Lys Ser Trp Tyr 35 CAT GTG GAG CTG AAC CCA AGG AGT TCC His Val Giu Leu Asn Pro Arg Ser Ser TGT GTT TTG GAG TTT TGG CCA GTT GAG Cys Val Leu Glu Phe Trp Pro Val Glu 70 TTT TTC CAA ATG AAA AAT TAT ACT GAG Phe Phe Gin Met Lys Asn Tyr Thr Gin 85 AGA AGA AAT AAA CAC AGC TGT TTC ACT Arg Arg Asn Lys His Ser Cys Phe Thr 100 ATT GTG GAA GTT AAA AAA TTT TTT GAG Ile Val Giu Val Lys Lys Phe Phe Gin 110 115 TAT CAA ACA CTG GTC AAC AGC ACA TCA Tyr Gin Thr Leu Val Asn Ser Thr Ser 130 CTA CTA CTG GAG AAC AAT AAA AAC CCA CTT TGG GTG CTT ATA TCT GTA Leu Trp Val Leu le Ser Val -10 CCC CAC ATT ACT GTG GTT GAA Pro His Ile Thr Val Val Glu TCG TGT TCA CTT GCA CAT GAG Ser Cys Ser Leu Ala His Glu AAA AGC AGT GGA TCA CAG GAA Lys Ser Ser Gly Ser Gin Glu 40 TCG AGA ATT GCT TTG CAT GAT Ser Arg Ile Ala Leu His Asp 55 TTG AAT GAG ACA GGA TCT TAC Leu Asn Asp Thr Gly Ser Tyr AAA TGG AAA TTA AAT GTC ATC Lys Trp Lys Leu Asn Val Ile GAA AGA CAA GTA ACT AGT AAA Glu Arg Gin Val Thr Ser Lys 105 ATA ACC TGT GAA AAC AGT TAC Ile Thr Cys Giu Asn Ser Tyr 120 TTG TAT AAG AAC TGT AAA AAG Leu Tyr Lys Asn Cys Lys Lys 135 140 ACG ATA AAG AAG AAC GCC GAG 48 96 144 192 240 288 336 384 432 480 528 59 Leu Leu Leu TTT GAA GAT Phe Glu Asp 160 GGA AAA CTA Gly Lys Leu 175 GAT CGC AGT Asp Arg Ser 190

GTT

Val GCA GTG Ala Val CTG AAT GAA Leu Asn Glu TCG GAT CCT Ser Asp Pro 240 GAA GGC AAA Glu Gly Lys 255 GAA AGC AAT Glu Ser Asn Glu Asn Asn Lys Asn Pro 145 150 CAG GGG TAT TAC TCC TGC Gin Gly Tyr Tyr Ser Cys 165 TTT AAT ATC ACC AAA ACC Phe Asn Ile Thr Lys Thr 180 AAT ATA GTT CCG GTT CTT Asn Ile Val Pro Val Leu 195 GAA TTA GGA AAA AAC GTA Glu Leu Gly Lys Asn Val 210 GAG GAT GTA ATT TAT TGG Glu Asp Val Ile Tyr Trp 225 230 AAT ATA CAT GAA GAG AAA Asn Ile His Glu Glu Lys 245 TGG CAT GCT TCA AAA GTA Trp His Ala Ser Lys Val 260 CTA AAT GTT TTA TAT AAT Leu Asn Val Leu Tyr Asn 275 ACC AAA AGC TTC ATC TTG Thr Lys Ser Phe Ile Leu 290 CAC GTC TTC ACA AGA His Val Phe Thr Arg 305 Thr Ile Lys Lys Asn Ala Glu 155 GTG CAT TTC CTT CAT CAT AAT Val His Phe Leu His His Asn 170 TTC AAT ATA ACA ATA GTG GAA Phe Asn Ile Thr Ile Val Glu 185 CTT GGA CCA AAG CTT AAC CAT Leu Gly Pro Lys Leu Asn His 200 205 AGG CTC AAC TGC TCT GCT TTG Arg Leu Asn Cys Ser Ala Leu 215 220 ATG TTC GGG GAA GAA AAT GGA Met Phe Gly Glu Glu Asn Gly 235 GAA ATG AGA ATT ATG ACT CCA Glu Met Arg Ile Met Thr Pro 250 TTG AGA ATT GAA AAT ATT GGT Leu Arg Ile Glu Asn Ile Gly 265 TGC ACT GTG GCC AGC ACG GGA Cys Thr Val Ala Ser Thr Gly 280 285 GTG AGA AAA GAC ATG GCT GAT Val Arg Lys Asp Met Ala Asp 295 300 576 624 672 720 768 816 864 912 960 984 270

GGC

Gly ACA GAC Thr Asp ATC CCA GGC Ile Pro Gly (11)INFORMATION FOR SEQ ID NO:11: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH:975 base pairs (B)TYPE:nucleic acid (C)strandedness:double (D)TOPOLOGY:linear (ii)MOLECULE TYPE:cDNA (ix)FEATURE: (A)NAME/KEY:sig peptide (B)LOCATION:1..54 (C)IDENTIFICATION METHOD:S (ix)FEATURE: (A)NAME/KEY:mat peptide (B)LOCATION:55..975 (C)IDENTIFICATION METHOD:S (xi)SEQUENCE DESCRIPTION:SEQ ID NO:11: ATG CAT CAT GAA GAA TTA ATC TTG ACA CTC TGC ATT Met His His Glu Glu Leu Ile Leu Thr Leu Cys Ile -10 AGT GCC TCA AAA AGT TGT ATT CAC CGA TCA CAA ATT Ser Ala Ser Lys Ser Cys Ile His Arg Ser Gin Ile 1 5 GGA GAA CCT TTT TAT CTG AAG CCA TGT GGC ATA TCT CTC ATT GTT AAA Leu Ile Val Lys CAT GTG GTA GAG His Val Val Glu GCA CCA GTG CAC 48 96 144 60 Gly

AGG

Arg

GAG

Glu

GAT

Asp

TAC

Tyr

ACC

Thr

AGA

Arg

AAC

Asn

GAA

Glu

GAG

Glu S. 55 *5 5* S 5** 555.55 S.

S.

Glu Pro Phe Tyr Leu Lys Pro 20 AAT GAA ACA GCC ACC ATG AGA Asn Glu Thr Ala Thr Met Arg TAT AGA GAG CTG AAC AAC AGA Tyr Arg Glu Leu Asn Asn Arg CAC ACC TTG GAA TTC TGG CCA His Thr Leu Glu Phe Trp Pro 70 ATT TCT CAA GTC GGA AAT GAT Ile Ser Gin Val Gly Asn Asp 85 AAA AGA AAC AAA CAC AGC TGT Lys Arg Asn Lys His Ser Cys 100 GAT GTT GAA GTT AAC AAA TCT Asp Val Glu Val Asn Lys Ser 115 TAT GAA GAG CTG ATC CAG GAC Tyr Glu Glu Leu Ile Gin Asp 130 ATA TCC AAA ACC CCA AGG ATC Ile Ser Lys Thr Pro Arg Ile 145 150 GGC TAC TAC TCC TGC GTG TTT Gly Tyr Tyr Ser Cys Val Phe 160 165 AAC ATC ACC AAG ACT GTC AAT Asn Ile Thr Lys Thr Val Asn 180 GTA ACT CCA GCT ATT TTA GGA Val Thr Pro Ala Ile Leu Gly 195 CTA GGA AAG GAT GTG GAG TTG Leu Gly Lys Asp Val Glu Leu 210 GAT CTG TTT TAT TGG AGC ATC Asp Leu Phe Tyr Trp Ser Ile 225 230 GTG CAA GAA GAC AGG AAG GAG Val Gin Glu Asp Arg Lys Glu 240 245 CTG CAT GCT TCA AAA ATA CTG Leu His Ala Ser Lys Ile Leu 260 CTC AAT GTT TTA TAT AAT TGC Leu Asn Val Leu Tyr Asn Cys 275 ACC AAG AGC TTC GTC TTG GTG Thr Lys Ser Phe Val Leu Val 290 CAT GTC TTT ACA His Val Phe Thr 305 Cys Gly Ile Ser Ala 25 TGG TTC AAA GGC AGT Trp Phe Lys Gly Ser 40 AGC TCG CCC AGA GTC Ser Ser Pro Arg Val 55 GTT GAG ATG GAG GAT Val Glu Met Glu Asp CGT CGC AAT TGG ACC Arg Arg Asn Trp Thr TTC TCT GAC AAG CTC Phe Ser Asp Lys Leu 105 CTG CAT ATC ACT TGT Leu His Ile Thr Cys 120 ACA TGG CTG TAT AAG Thr Trp Leu Tyr Lys 135 CTG AAG GAT GCC GAG Leu Lys Asp Ala Glu 155 TCT GTC CAC CAT AAT Ser Val His His Asn 170 ATA ACA GTT ATT GAA Ile Thr Val Ile Glu 185 CCA AAG TGT GAG AAG Pro Lys Cys Glu Lys 200 AAC TGC AGT GCT TCA Asn Cys Ser Ala Ser 215 AGG AAA GAG GAC AGC Arg Lys Glu Asp Ser 235 ACG ACA ACA TGG ATT Thr Thr Thr Trp Ile 250 AGA TTT CAG AAA ATT Arg Phe Gin Lys Ile 265 ACC GTG GCC AAC GAA Thr Val Ala Asn Glu 280 AGA AAA GAA ATA CCT Arg Lys Glu Ile Pro 295 Pro Val His GCT TCA CAT Ala Ser His ACT TTT CAT Thr Phe His GAG GGA ACG Glu Gly Thr TTA AAT GTC Leu Asn Val GTG ACA AGC Val Thr Ser 110 AAG AAT CCT Lys Asn Pro 125 AAC TGT AAG Asn Cys Lys 140 TTT GGA GAT Phe Gly Asp GGG ACA CGG Gly Thr Arg GGA AGG AGT Gly Arg Ser 190 GTT GGT GTA Val Gly Val 205 TTG AAT AAA Leu Asn Lys 220 TCA GAC CCT Ser Asp Pro TCT GAA GGC Ser Glu Gly ACT GAA AAC Thr Glu Asn 270 GAA GCC ATA Glu Ala Ile 285 GAT ATC CCA Asp Ile Pro 300 192 240 288 336 384 432 480 528 576 624 672 720 768 816 864 912 960 975

TAC

Tyr 175

AAA

Lys

GAA

Glu

GAC

Asp

AAT

Asn

AAA

Lys 255

TAT

Tyr

GAC

Asp

GGC

Gly 61 (12)INFORMATION FOR SEQ ID NO:12: (i )SEQUENCE CHARACTERISTICS: (B)TYPE:amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (v )FRAGMENT TYPE: internal fragment (xi)SEQUENCE DESCRIPTION:SEQ ID NO: 12: Trp His Ala Ser Lys, 1 (13)INFORMATION FOR SEQ ID NO:13: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH:7 (B )TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (v)FRAGMENT TYPE:internal fragment (xi )SEQUENCE DESCRIPTION:SEQ ID NO:13: Ile Met Thr Pro Glu Gly Lys :1 (14)INFORMATION FOR SEQ ID NO:14: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH: 13 (B )TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (v )FRAGMENT TYPE: internal fragment (xi)SEQUENCE DESCRIPTION:SEQ ID NO:14: Ser Ser Gly Ser Gln Glu His Val Glu Leu Asn Pro Arg 1 5 FOR SEQ ID (i )SEQUENCE CHARACTERISTICS: (A)LENGTH:4 (B )TYPE: amino acid D )TOPOLOGY: linear ii )MOLECULE TYPE: peptide (v)FRAGMENT TYPE:internal fragment (xi )SEQUENCE DESCRIPTION:SEQ ID Ser Trp Tyr Lys 1 (16)INFORMATION FOR SEQ ID NO:16: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH: (B )TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (v )FRAGMENT TYPE: internal fragment (xi)SEQUENCE DESCRIPTION:SEQ ID NO:16: 62 Leu Asn His Val Ala Val Glu Leu Gly Lys 1 5 (17)INFORMATION FOR SEQ ID NO:17: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH: 6 (B )TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (v)FRAGMENT TYPE:internal fragment (xi)SEQUENCE DESCRIPTION:SEQ ID NO: 17: Ser Phe Ile Leu Val Arg 1 (i8)INFORMATION FOR SEQ ID NO:18: (i )SEQUENCE CHARACTERISTICS: LENGTH (B )TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE:peptide (v )FRAGMENT TYPE: internal fragment (xi)SEQUENCE DESCRIPTION:SEQ ID NO:18: Thr Val Lys Pro Gly Arg Asp Giu Pro Giu Val Leu Pro Val Leu 1 5 10 (19)INFORMATION FOR SEQ ID NO:19: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH: 11 *(B)TYPE: amino acid (D )TOPOLOGY: linear (ii)MOLECULE TYPE:peptide (v )FRAGMENT TYPE: internal fragment (xi )SEQUENCE DESCRIPTION:SEQ ID NO: 19: Ser Asn Ile Val Pro Val Leu Leu Gly Pro Lys (20)INFORMATION FOR SEQ ID (i )SEQUENCE CHARACTERISTICS: (A)LENGTH: 521 (B )TYPE: amino acid (D )TOPOLOGY: linear (ii)MOLECULE TYPE:peptide (xi)SEQUENCE DESCRIPTION:SEQ ID Glu Ser Cys Thr Ser Arg Pro His Ilie Thr Val Val 1 5 10 Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His 25 Thr Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser.Gin 40 Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His 55 Glu Phe Trp Pro Val Giu Leu Asn Asp Thr Gly Ser 70 75 Glu Gly Glu Pro Glu Ile Glu Thr Glu His Val Glu Asp Cys Val Leu Tyr Phe Phe Gin 63 Met Lys Asn Tyr Thr Gin Lys Trp Lys Leu Asn Val Ile Arg Arg Asn 90 Lys His Ser Cys Phe Thr Glu Arg Gin Val Thr Ser Lys Ile Val Glu 100 105 110 Val Lys Lys Phe Phe Gin Ile Thr Cys Glu Asn Ser Tyr Tyr Gin Thr 115 120 125 Leu Val Asn Ser Thr Ser Leu Tyr Lys Asn Cys Lys Lys Leu Leu Leu 130 135 140 Glu Asn Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu Phe Glu Asp 145 150 155 160 Gin Gly Tyr Tyr Ser Cys Val His Phe Leu His His Asn Gly Lys Leu 165 170 175 Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu Asp Arg Ser 180 185 190 Asn Ile Val Pro Val Leu Leu Gly Pro Lys Leu Asn His Val Ala Val 195 200 205 Glu Leu Gly Lys Asn Val Arg Leu Asn Cys Ser Ala Leu Leu Asn Glu 210 215 .220 Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu Asn Gly Ser Asp Pro 225 230 235 240 Asn Ile His Glu Glu Lys Glu Met Arg Ile Met Thr Pro Glu Gly Lys 245 250 255 Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn Ile Gly Glu Ser Asn 260 265 270 S Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly Gly Thr Asp S275 280 285 Thr Lys Ser Phe Ile Leu Val Arg Lys Asp Met Ala Asp Ile Pro Gly 290 295 300 His Val Phe Thr Arg Gly Met Ile Ile Ala Val Leu Ile Leu Val Ala 305 310 315 320 Val Val Cys Leu Val Thr Val Cys Val Ile Tyr Arg Val Asp Leu Val 325 330 335 Leu Phe Tyr Arg His Leu Thr Arg Arg Asp Glu Thr Leu Thr Asp Gly 340 345 350 Lys Thr Tyr Asp Ala Phe Val Ser Tyr Leu Lys Glu Cys Arg Pro Glu "355 360 365 Asn Gly Glu Glu His Thr Phe Ala Val Glu Ile Leu Pro Arg Val Leu 370 375 380 Glu Lys His Phe Gly Tyr Lys Leu Cys Ile Phe Glu Arg Asp Val Val 385 390 395 400 Pro Gly Gly Ala Val Val Asp Glu Ile His Ser Leu Ile Glu Lys Ser S405 410 415 Arg Arg Leu Ile Ile Val Leu Ser Lys Ser Tyr Met Ser Asn Glu Val 420 425 430 Arg Tyr Glu Leu Glu Ser Gly Leu His Glu Ala Leu Val Glu Arg Lys 435 440 445 Ile Lys Ile Ile Leu Ile Glu Phe Thr Pro Val Thr Asp Phe Thr Phe 450 455 460 Leu Pro Gin Ser Leu Lys Leu Leu Lys Ser His Arg Val Leu Lys Trp 465 470 475 480 Lys Ala Asp Lys Ser Leu Ser Tyr Asn Ser Arg Phe Trp Lys Asn Leu 485 490 495 Leu Tyr Leu Met Pro Ala Lys Thr Val Lys Pro Gly Arg Asp Glu Pro 500 505 510 Glu Val Leu Pro Val Leu Ser Glu Ser 515 520 64 (21)INFORMATION FOR SEQ ID NO:21: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH: 519 (B )TYPE: amino acid TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION:SEQ ID NO:21: Ser Lys Ser Cys Ile His Arg Ser Gin Ile His Vai Val Giu Gly Glu 0e I. 0 0 *0 0 '0 0 *0 0 0e *0 Pro Giu Arg Thr Ser Arg Val Giu Ser 145 Tyr Ile Thr Gly Leu 225 Gin His Asn Lys Vai 305 Vai Phe Thr Lys Lys Phe Thr Giu Leu Gin Asn Giu Giu 130 Lys Tyr Thr Pro Lys 210 Phe Giu Ala Vai Ser 290 Phe Cys Tyr Tyr Giu 370 Gin Tyr Ala Leu Giu Vai Lys Vai 115 Leu Thr Ser Lys Ala 195 Asp Tyr Asp Ser leu 275 Phe Thr Ile Arg Asp 355 Giu Phe Leu Thr Asn Phe Gly His 100 Asn Ile Pro Cys Thr 180 Ile Val Trp Arg Lys 260 Tyr Val Gly Vai Arg 340 Ala Tyr Gly Lys Pro Met Arg Asn Arg Trp Pro 70 Asn Asp Ser Cys Lys Ser Gin Asp Arg Ile 150 Val Phe 165 Val Asn Leu Gly Giu Leu Ser Ile 230 Lys Giu 245 Ile Leu Asn Cys Leu Val Gly Val 310 Ile Leu 325 Ile Ala Phe Val Thr Phe Tyr Lys Cys Gly Ile 25 Trp Phe Lys 40 Ser Ser Pro 55 Val Giu Met Arg Arg Asn Phe Ser Asp 105 Leu His Ile 120 Thr Trp Leu 135 Leu Lys Asp Ser Val His Ile Thr Vai 185 Pro Lys Cys 200 Asn Cys Ser 215 Arg Lys Giu Thr Thr Thr Arg Phe Gin 265 Thr Val Ala 280 Arg Lys Giu 295 Thr Val Leu Cys Val Ile Glu Arg Asp 345 Ser Tyr Leu 360 Ala Val Giu 375 Leu Cys Ile Ser Ala Gly Ser Arg Val Glu Asp 75 Trp Thr 90 Lys Leu Thr Cys Tyr Lys Ala Glu 155 His Asn 170 Ile Giu Glu Lys Ala Ser Asp Ser 235 Trp Ile 250 Lys Ile Asn Giu Ile Pro Val Leu 315 Tyr Lys 330 Giu Thr Lys Giu Thr Leu Phe Giu Pro Val His Arg Asn Ala Ser His Giu Tyr Thr Phe His Asp His Glu Gly Thr Tyr Ile Leu Asn Vai Thr Lys Val Thr Ser Arg Asp 110 Lys Asn Pro Asn Tyr 125 Asn Cys Lys Giu Ile 140 Phe Giy Asp Giu Gly 160 Gly Thr Arg Tyr Asn 175 Gly Arg Ser Lys Val 190 Val Gly Val Giu Leu 205 Leu Asn Lys Asp Asp 220 Ser Asp Pro Asn Vai 240 Ser Giu Gly Lys Leu 255 Thr Giu Asn Tyr Leu 270 Glu Ala Ile Asp Thr 285 Asp Ile Pro Gly His 300 Ala Ser Val Ala Ala 320 Val Asp Leu Val Leu 335 Leu Thr Asp Gly Lys 350 Cys His Pro Giu Asn 365 Pro Arg Val Leu Giu 380 Arg Asp Val Val Pro 65 385 390 395 400 Gly Gly Ala Val Val Glu Giu Ile His Ser Leu Ile Giu Lys Ser Arg 405 410 415 Arg Leu Ile Ile Val Leu Ser Gin Ser Tyr Leu Thr Asn Gly Ala Arg 420 425 430 Arg Glu Leu Giu Ser Gly Leu His Giu Ala Leu Val Giu Arg Lys Ile 435 440 445 Lys Ile Ile Leu Ile Glu Phe Thr Pro Ala Ser Asn Ile Thr Phe Leu 450 455 460 Pro Pro Ser Leu Lys Leu Leu Lys Ser Tyr Arg Val Leu Lys Trp Arg 465 470 475 480 Ala Asp Ser Pro Ser Met Asn Ser Arg Phe Trp Lys Asn Leu Val Tyr 485 490 495 Leu Met Pro Ala Lys Ala Val Lys Pro Trp Arg Glu Glu Ser Giu Ala 500 505 510 Arg Ser Val Leu Ser Aia Pro 515 (22)INFORMATION FOR SEQ ID NO:22: (i )SEQUENCE CHARACTERISTICS: LENGTH:309 (B )TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE:peptide (xi)SEQUENCE DESCRIPTION:SEQ ID NO:22: .s a C. 4* a 0 *e *000 a a. '*0

C.

(9 '9(9 *0 9* 9 (9 9 9 0* Glu Ser Cys Thr Ser 1 5 Phe Tyr Leu Lys His 20 Thr Thr Lys Ser Trp 35 Leu Asn Pro Arg Ser Glu Phe Trp Pro Val 65 Met Lys Asn Tyr Thr Lys His Ser Cys Phe 100 Val Lys Lys Phe Phe 115 Leu Val Asn Ser Thr 130 Glu Asn Asn Lys Asn 145 Gin Gly Tyr Tyr Ser 165 Phe Asn Ile Thr Lys 180 Asn Ile Val Pro Val 195 Giu Leu Gly Lys Asn 210 Giu Asp Val Ile Tyr 225 Asn Ile His Glu Glu Arg Pro His Cys Tyr Ser Giu 70 Gin Thr Gin Ser Pro 150 Cys Thr Leu Val Trp 230 Lys Ser Cys Lys Ser 40 Ser Arg 55 Leu Asn Lys Trp Glu Arg Ile Thr 120 Leu Tyr 135 Thr Ile Val His Phe Asn Leu Gly 200 Arg Leu 215 Met Phe Giu Met Ile Thr Val 10 Ser Leu Ala 25 Ser Gly Ser Ile Ala Leu Asp Thr Gly 75 Lys Leu Asn 90 Gin Val Thr 105 Cys Giu Asn Lys Asn Cys Lys Lys Asn 155 Phe Leu His 170 Ile Thr Ile 185 Pro Lys Leu Asn Cys Ser Gly Giu Glu 235 Arg Ile Met Val His Gin His Ser Val Ser Ser Lys 140 Ala His Val Asn Ala 220 Asn Thr Giu Giu Glu Asp Tyr Ile Lys Tyr 125 Lys Glu Asn Glu His 205 Leu Gly Pro Gly Ile His Cys Phe Arg Ile 110 Tyr Leu Phe Gly Asp 190 Val Leu Ser Glu Giu Giu Val Val Phe Arg Val Gin Leu Giu Lys 175 Arg Ala Asn Asp Gly Pro Thr Giu Leu Gin Asn Glu Thr Leu Asp 160 Leu Ser Val Giu Pro 240 Lys 66 245 250 255 Trp His Ala Ser Lys Val Leu Arg Ile Glu Asn Ile Gly Glu Ser Asn 260 265 270 Leu Asn Val Leu Tyr Asn Cys Thr Val Ala Ser Thr Gly Gly Thr Asp 275 280 285 Thr Lys Ser Phe Ile Leu Val Arg Lys Asp Met Ala Asp Ile Pro Gly 290 295 300 His Val Phe Thr Arg 305 (23)INFORMATION FOR SEQ ID NO:23: (i )SEQUENCE CHARACTERISTICS: LENGTH :207 (B)TYPE:amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (xi )SEQUENCE DESCRIPTION:SEQ ID NO:23: Giu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val Giu Gly Giu Pro @5

S

S

9* 5 S S @9 S. S .5 5

S.

*9 5 .5

S

9 59** ~S *S (S S. @9 5 (9 9 6 9* 595 95* .5

C

9 .5 '9* Phe Thr Leu Giu Met Lys Val Leu Giu 145 Gin Phe Asn Tyr Thr Asn 50 Phe Lys His tys Val 130 Asn Gly Asn Ile Leu Lys 20 Lys Ser 35 Pro Arg Trp Pro Asn Tyr Ser Cys 100 Lys Phe 115 A sn Ser Asn Lys Tyr Tyr Ile Thr 180 Val Pro 195 His Trp Ser Vai Thr Phe Phe Thr Asn Ser 165 Lys Val Cys Tyr Ser Giu 70 Gin Thr Gin Ser Pro 150 Cys Thr Leu Ser Lys Ser 55 Leu Lys Glu Ile Leu 135 Thr Vai Phe Leu Cys Ser 25 Ser Ser 40 Arg Ilie Asn Asp Trp Lys Arg Gin 105 Thr Cys 120 Tyr Lys Ile Lys His Phe Asn Ile 185 Gly Pro 200 Leu Giy Ala Thr Leu 90 Val Giu Asn Lys Leu 170 Thr Lys Ala Ser Leu Gly 75 Asn Thr Asn Cys Asn 155 His Ile Leu His Gin His Ser Val Ser Ser Lys 140 Ala His Val Asn Giu Giu Asp Tyr Ile Lys Tyr 125 Lys Giu Asn Giu His 205 Ile His Cys Phe Arg Ile 110 Tyr Leu Phe Gly Asp 190 Val Giu Val Val Phe Arg Val Gin Leu Glu Lys 175 Arg Ala Thr Giu Leu Gin Asn Giu Thr Leu Asp 160 Leu Ser (24)INFORMATION FOR SEQ ID NO:24: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH: 104 (B )TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION: SEQ ID NO:24: Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val Val Giu Gly Giu Pro 1 5 10 Phe Tyr Leu Lys His Cys Ser Cys Ser Leu Ala His Glu Ile Giu Thr 25 67 The Thr Lys Ser Trp Tyr Lys Ser Ser Gly Ser Gin Giu His Val Giu 40 Leu Asn Pro Arg Ser Ser Ser Arg Ile Ala Leu His Asp Cys Val Leu 55 Giu Phe Trp Pro Vai Glu Leu Asn Asp Thr Gly Ser Tyr Phe Phe Gin 70 75 Met Lys Asn Tyr Thr Gin Lys Trp Lys Leu Asn Vai Ile Arg Arg Asn 90 Lys His Ser Cys Phe Thr Giu Arg 100 FOR SEQ ID (i )SEQUENCE CHARACTERISTICS: A) LENGTH: 307 (B )TYPE :amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION:SEQ ID Ser Lys Ser Cys Ile His Arg Ser Gin Ile His Val Val Giu Giy Glu I. a a. a (a 0 a. I. a a a. 0* a a 'a I.

a. a a 0* Pro Giu Arg Thr Ser Arg Val Giu Ser 145 Tyr Ile Thr Gly Leu 225 Gin His Asn Lys Phe Thr Giu 50 Leu Gin Asn Giu Giu 130 Lys Tyr Thr Pro Lys 210 Phe Giu Ala Vai Ser 290 Tyr Ala 35 Leu Giu Val Lys Vai 115 Leu Thr Ser Lys Ala 195 Asp Tyr Asp Ser Leu 275 Phe Leu 20 Thr Asn Phe Gly His 100 Asn Ile Pro Cys Thr 180 Ile.

Val Trp Arg Lys 260 Tyr Val Lys Met Asn Trp Asn 85 Ser Lys Gin Arg Val 165 Val Leu Giu Ser Lys 245 Ile Asn Leu Pro Arg Arg Pro 70 Asp Cys Ser Asp Ile 150 Phe Asn Gly Leu Ile 230 Giu Leu Cys Val Cys Trp Ser 55 Val Arg Phe Leu Thr 135 Leu Ser Ile Pro Asn 215 Arg Thr Arg Thr Arg 2'95 Gly Ile 25 Phe Lys 40 Ser Pro Giu Met Arg Asn Ser Asp 105 His Ile 120 Trp Leu Lys Asp Vai His Thr Vai 185 Lys Cys 200 Cys Ser Lys Giu Thr Thr Phe Gin 265 Val Ala 280 Lys Giu Ser Ala Pro Giy Ser Ala Arg Val Thr Giu Asp Giu 75 Trp Thr Leu 90 Lys Leu Val Thr Cys Lys Tyr Lys Asn 140 Ala Giu Phe 155 His Asn Giy 170 Ile Giu Gly Giu Lys Val Ala Ser Leu 220 Asp Ser Ser 235 Trp Ile Ser 250 Lys Ile Thr Asn Giu Giu Ile Pro Asp 300 Val Ser Phe Gly Asn Thr Asn 125 Cys Gly Thr Arg Gly 205 Asn Asp Giu Giu Ala 285 His His His Thr Val Ser 110 Pro Lys Asp Arg Ser 190 Vai Lys Pro Gly Asn 270 Ile Arg Giu Asp Tyr Thr Arg Asn Giu Glu Tyr 175 Lys Giu Asp Asn Lys 255 Tyr Asp Asn Tyr His Ile Lys Asp Tyr Ile Gly 160 Asn Vai Leu Asp Vai 240 Leu Leu Thr Ile Pro Gly His 68 Val Phe Thr 305 (26)INFORMATION FOR SEQ ID NO:26: (i )SEQUENCE CHARACTERISTICS: (A LENGTH: 15 7 (B )TYPE: amino acid TOPOLOGY: linear (ii )MOLECULE TYPE peptide (xi )SEQUENCE DESCRIPTION:SEQ ID NO:26: Tyr Phe Giy Lys Leu Giu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn 1 5 10 Asp Gin Vai Leu Phe Ile Asp Gin Giy Asn Arg Pro Leu Phe Giu Asp 25 Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile 40 Ile Ser Met Tyr Lys Asp Ser Gin Pro Arg Gly Met Ala Val Thr Ile 55 Ser Val Lys Cys Glu Lys Ile Ser Xaa Leu Ser Cys Giu Asn Lys Ile 70 75 Ile Ser Phe Lys Giu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys 90 Se Asp Ile Ile Phe Phe Gin Arg Ser Val Pro Giy His Asp Asn Lys 105 110 Met Gin Phe Giu Ser Ser Ser Tyr Giu Giy Tyr Phe Leu Aia Cys Giu 115 120 125 Lys Giu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Giu Asp Giu Leu 130 135 140 *Giy Asp Arg Ser Ile Met Phe Thr Vai Gin Asn Giu Asp 145 150 (27)fNFORMATION FOR SEQ ID NO:27: (i )SEQUENCE CHARACTERISTICS: (A)LENGTH:157 TYPE: amino acid (D )TOPOLOGY: linear (ii )MOLECULE TYPE :peptide (xi)SEQUENCE DESCRIPTION:SEQ ID NO:27: *:Asn Phe Giy Arg Leu His Cys Thr Thr Aia Vai Ile Arg Asn Ile Asn 1 5 10 Asp Gin Val Leu Phe Vai Asp Lys Arg Gin Pro Vai Phe Giu Asp Met 25 Thr Asp Ile Asp Gin Ser Aia Ser Giu Pro Gin Thr Arg Leu Ile Ile 40 Tyr Met Tyr Lys Asp Ser Giu Vai Arg Giy Leu Aia Vai Thr Leu Ser 55 Vai Lys Asp Ser Lys Xaa Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile 70 75 Ser Phe Giu Giu Met Asp Pro Pro Giu Asn Ile Asp Asp Ile Gin Ser 90 Asp Leu Ile Phe Phe Gin Lys Arg Vai Pro Gly His Asn Lys Met Giu 100 105 110 Phe Giu Ser Ser Leu Tyr Giu Giy His Phe Leu Ala Cys Gin Lys Giu 115 120 125 Asp Asp Aia Phe Lys Leu Ile Leu Lys Lys Lys Asp Giu Asn Giy Asp 69 130 135 140 Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gin Ser 145 150 155 e* 9* C. 70

Claims (24)

1. A polypeptide having the function as an interleukin-18 receptor of recognising interleukin-18, which is obtained through artificial gene expression.

2. The polypeptide of claim 1, which is obtainable by bringing into expression a human or mouse gene in a host of animal, plant or microbe origin.

3. The polypeptide of claim 1, wherein said gene contains a part or whole of the nucleotide sequence of SEQ ID No: 1 or 2.

4. The polypeptide of claim 1, wherein said gene contains the nucleotide sequence of SEQ ID No: 3 or 4. The polypeptide of claim 1, wherein said gene contains a nucleotide sequence of any one of SEQ ID Nos: 5 to 11.

6. The polypeptide of claim 1, which contains one or more amino acid sequences of SEQ ID Nos: 12 to 19. S: 7. The polypeptide of claim 1, which contains a part or whole of the amino acid sequence of SEQ ID No: 20 or 21. S 8. The polypeptide of claim 1, which contains an amino acid sequence of any one of SEQ ID Nos: 22 to

9. An agent for interleukin-18 receptor susceptive diseases, which contains as effective ingredient the polypeptide of claim 1.

10. The agent of claim 9, which contains as stabilizer a protein, saccharide and/or buffer.

11. An anti-autoimmune disease agent in accordance with claim 9. .12. An immunosuppressant in accordance with claim 9.

13. An isolated DNA capable of expressing the polypeptide of claim 1 in a host cell.

14. The DNA of claim 13 containing a nucleotide sequence originating from human or mouse. The DNA of claim 13, which contains a part or whole of either the nucleotide sequence of SEQ ID No: 1, SEQ ID No: 2 or their complementary sequences.

16. The DNA if claim 13, which contains the nucleotide sequence of SEQ ID No: 3, SEQ ID No:4 or their complementary sequences.

17. The DNA of claim 13, which contains the nucleotide sequence of SEQ ID SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ ID No:9, SEQ ID SEQ ID No:11 or their complementary sequences.

18. The DNA sequence of claim 13, wherein, based on the degeneracy of the genetic code, one or more nucleotides are replaced with different nucleotides while conserving the amino acid sequence.

19. The DNA of claim 13, which is placed in an autonomously relicable vector. The DNA of claim 13, which is placed in a host of animal, plant or microbe origin.

21. A process for preparing a polypeptide, comprising bringing into expression an isolated DNA capable of expressing the polypeptide of claim 1 in a host cell; and collecting the resultant polypeptide.

22. The process of claim 21, wherein the resultant polypeptide is collected through a step which includes salting out, dialysis, filtration, concentration, fractional precipitation, ion-exchange chromatography, gel filtration chromatography, adsorption chromatography, isoelectric focusing chromatography, hydrophobic chromatography, reversed phase chromatography, affinity chromatography, gel electrophoresis, and/or isoelectric focusing gel electrophoresis. S 23. The process of claim 21, wherein the resultant polypeptide is collected through a step which includes immunoaffinity chromatography using a monoclonal antibody.

24. An agent to neutralize interleukin-18, which contains as effective ingredient the polypeptide of claim 1. A method to neutralize interleukin-18, characterized by allowing the polypeptide of claim 1 to act on interleukin-18.

26. A polypeptide having the function as an interleukin-18 receptor of recognising interleukin-18 obtained through artificial gene expression, substantially as herein described with reference to any one of the Examples and/or accompanying Figures but excluding any comparative examples.

27. An agent as defined in claim 9 for interleukin-18 receptor susceptive diseases substantially as herein described with reference to any one of the Examples and/or accompanying Figures.

28. A DNA as defined in claim 13, substantially as herein described with reference to any one of the Examples, and/or accompanying Figures.

29. A process to prepare a polypeptide of claim 1 which process is substantially as herein described with reference to any one of the Examples, and/or accompanying Figures. An agent of claim 24 to neutralise interleukin-18, substantially as herein described with reference to any one of the Examples and/or 4*i accompanying Figures.

31. A method of claim 25 which method is substantially as herein described Swith reference to any one of the Examples and/or accompanying Figures.

32. A method for the treatment or prophylaxis of interleukin-18-related diseases in a patient requiring said treatment or prophylaxis which method comprises administering to said patient an effective amount of the polypeptide of claim 1.

33. Use of the polypeptide of claim 1 in the preparation of a medicament for the treatment of prophylaxis of interleukin-18-related diseases. Dated this 15 th day of January 2001 KABUSHIKI KAISHA HAYASHIBARA SEIBUTSU KAGAKU KENKYUJO By their patent attorneys CALLINAN LAWRIE