Human Pancreatic Elastase
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curopaiscnes Katentamt European Patent Office (jj) Publication number: O 1 98 645 B1 Office europeen des brevets cUHOPeAN PATENT SPECIFICATION © intci.*: C12N 15/57, C12N 9/66, 22.11.90 C07H 21/04, C12N1/20, Application number: 86302557.3 A61K 37/54 Date of filing: 07.04.86 jy nuinan pancreauc eiasiase. rropnetor: sankyo company LIMITED, 27.04.85 JP 91986/85 No. 1-6, 3-chome Nihonbashi Honcho Chuo-ku, 26.07.85 JP 163964/85 Tokyo(JP) 23.10.85 JP 236686/85 inventor: Takiguchi, Yo C/o Bio-Science Research Laboratory, Sankyo Company Limited 22.10.86 Bulletin 86/43 Mo. 2-58, 1-chome, Hiromachi Shinagawa-ku rokyo140(JP) Inventor: Tani, Tokyo C/o Bio-Science Research ui^nv/aLiui i ui ii ic yi ai u li ic Ljctlcl II. Laboratory, Sankyo Company Limited 22.11.90 Bulletin 90/47 Mo. 2-58, 1-chome, Hiromachi Shinagawa-ku rokyo 140(JP) nventor: Kawashima, Ichiro C/o Bio-Science Research -ab., Sankyo Company Limited No. 2-58, 1-chome, \T BE CH DE FR GB IT LI LU NL SE Hiromachi Shinagawa-ku Tokyo 140(JP) nventor: Eurukawa, Hidehiko C/o Bio-Science Research Lab., Sankyo Company Limited Ao. 2-58, 1-chome, Hiromachi IWIVI VI VSI IbU . Shinagawa-ku =P-A- 0157 604 rokyo 140(JP) nventor: Ohmine, Toshinori C/o Bio-Science Research -ab., Sankyo Company Limited No. 3I0CHIMICA ET BIOPHYSICA ACTA, vol. 623, no. 1, 2-58, 1-chome, u 1980 -iiromachi Shinagawa-ku Tokyo 140(JP) i/lay 29, (Amsterdam); C. LARGMAN ET nventor: Ohsumi, Jun C/o Bio-Science U.::"Human Pancreatic Proelastase 2 Sequence of the Research Lab., Vctivation Peptide", 208-212 Sankyo Company Limited No. 2-58, 1-chome, Hiromachi pp. Shinagawa-ku Tokyo 140(JP) 0 representative: Gibson, Christian John Robert et al, A ARKS & CLERK 57/60 Lincoln's Inn Fields, London VC2A3LS(GB) c iiiuiiuio iiuiii ii le puuuuaiion or me mention ot tne grant ot tne turopean patent, any person may give notice to le European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned tatement. It shall not be deemed to have been filed until the opposition fee has been paid (Art 99(1) European patent * c anvention). II- ACTORUM AG EP 0198 645 B1 uescription Background of the Invention 5 This invention relates to human pancreatic elastase. Elastase is a serine protease, capable of hydrolyzing the fibrous insoluble protein known as elastin. Elastin is a scleroprotein forming connective tissues, tendons, aortic integuments and cervical bundles of higher animals. Elastin is only slightly degraded by pepsin and trypsin. In the course of their study on arteriosclerosis, Balo' et ai observed degradation of the elastin fibers 10 of blood vessel walls, and postulated the presence of a degrading enzyme [Balo', J and Banga, I: Schweiz Z Pathol Bacteriol, 12, 350 (1949)]. Subsequently, Banga discovered an enzyme in the pancreas which specifically degrades elastin. The enzyme was isolated in the form of crystals and named "elastase" [Banga, I: Acta Physiol Acad Sci Hung, 3, 317 (1952)]. Elastase has been confirmed to exist in the pancreas of most animals, including humans, monkeys, 15 cats, rabbits, etc. The level is about 3.1 mg/g-pancreas in human beings, about 2.2 mg/g in bovine animals and about 10.2 mg/g in rats. A correlation is recognized between elastase activity and the age of a human being: a marked lowering in elastase activity is observed in the pancreas and plasma of males over 40 and females over 60 years [Loeven, W A and Baldwin, Maureen M: Gerontologia, 17, 170 (1971)]. In the case of patients with arteriosclerosis, the elastase activity in the pancreas was found to be 20 markedly lower than that of healthy people, and in some cases it had completely disappeared [Balo', J, and Banga, I: Nature, 178, 310 (1956)]. Subsequent studies have also demonstrated that elastase not on- ly degrades elastin but also promotes elastin biosynthesis. Studies on the pharmacological action of elastase have been carried out in rats and rabbits, and have revealed the following effects: 25 1) inhibition of the deposition of lipids and calcium on arterial walls; 2) elimination of cholesterol and calcium from arterial walls; 3) selective degradation of denatured elastin; 4) promotion of synthesis of elastin fibers in the arterial walls; 30 5) lowering of serum lipids; 6) improvement of lipoprotein metabolism. In clinical studies conducted on the basis of the studies mentioned above, the following effects have become apparent after oral administration of elastase: 35 1) restoration of elasticity and expandability of arterial walls; 2) improvement of serum lipid abnormality; 3) improvement of serum lipoprotein metabolism. 40 Elastase extracted and purified from porcine pancreas was used for the above studies. However, with administration of porcine elastase to human beings, there is the risk of antibody formation due to the foreign protein, and the danger of anaphylaxis with repeated administration (Japanese Patent Applica- tion Laid-open 11180/1984). Accordingly, human elastase is preferable for human use. However it has been extremely difficult to procure human elastase in sufficient quantities from traditional sources. 45 At present, two kinds of human pancreatic elastase are known, elastase 1 and elastase 2. They have not been fully characterized. For example, for pancreatic elastase 2, a partial sequence of 16 amino acid residues representing 12 amino acid residues in the activation peptide and 4 amino acid residues at the N-terminal end of the elastase have been sequenced [Largman, C et al; Biochim Biophys Acta, 623, 208 (1 980)]. This partial sequence is Cys Gly Asp Pro Thr Tyr Pro Pro Tyr Val Thr Arg Val Val Gly Gly. Objects of the Present Invention It is an object of this invention to make human pancreatic elastase more readily available, with the pos- sibility of obtaining the elastase in substantially pure form. It is a further object of this invention to elimi- 55 nate the continuing dependency on human pancreas for adequate supplies of human pancreatic elastase. It is a yet further object to produce new elastase compounds and elastase-like compounds. Summary of the Present Invention 30 This invention embraces the use of genetic engineering for the production of human pancreatic elastase. Recombinant DNA technology is employed, in order to provide a base sequence coding for a human pancreatic elastase or a functionally similar compound. Correspondingly, human pancreatic elastase and functionally similar compounds now become readily available for the first time. Indeed, nov- el elastases have been discovered and are part of this invention. 35 More specifically, this invention provides a compound which includes an amino acid sequence for a EP0 198 645 B1 human pancreatic elastase selected from the group of human pancreatic elastase IIB, human pancreatic elastase IMA, human pancreatic elastase NIB, and any compounds corresponding to human pancreatic elastase IIB, IIIA or NIB with one or more deleted, inserted, replaced or altered amino acids. The se- quences for human pancreatic elastases IIB, IIIA, and IIB are given below. The present invention also 5 embraces DNA which codes for the compounds provided by this invention. Thus, through the use of genetic engineering, it is now possible in accordance with the present inven- tion to provide a DNA which codes for a compound capable of functioning as a human pancreatic elastase. Novel human pancreatic elastases are provided, as well as derivative compounds which func- tion as human pancreatic elastases. Such compounds, which include compounds arising from silent muta- 10 tions, fusion proteins and other compounds functionally effective as a human pancreatic elastase, are included. Thus, substantially similar protein effective as a human pancreatic elastase, that is, com- pounds corresponding to human pancreatic elastases with one or more deleted, replaced or altered ami- no acids, and compounds corresponding to human pancreatic elastases with one or more extra amino ac- ids, are also included. More especially, precursor compounds such as proelastases and preproelastas- 15 es, especially when expressed by a recombinant DNA sequence, as well as elastases obtained by activation of such precursor compounds, are part of this invention. Further examples of precursor com- pounds within this invention include fusion proteins comprising an elastase (optionally in the form of a proelastase or preproelastase) and an amino acid sequence derived from another protein. In particular, such fusion proteins can be obtained when the DNA coding for an elastase is inserted into an expres- 20 sion gene downstream of a promoter. Elastase IIB This invention provides human pancreatic elastase IIB having the following amino acid sequence of 25 formula (III): (N)-Cys Gly Val Ser Thr Tyr Ala Pro Asp Met Ser Arg Met Leu Gly Gly Glu Glu Ala Arg Pro Asn Ser Trp Pro Trp Gin Val Ser Leu Gin Tyr Ser Ser Asn Gly Gin Trp Tyr His 30 Thr Cys Gly Gly Ser Leu lie Ala Asn Ser Trp Val Leu Thr Ala Ala His Cys He Ser Ser Ser Arg lie Tyr Arg Val Met Leu Gly Gin His Asn Leu Tyr Val Ala Glu Ser Gly Ser Leu Ala Val Ser Val Ser Lys lie Val 35 Val His Lys Asp Trp Asn Ser Asn Gin Val Ser Lys Gly Asn Asp He Ala Leu Leu Lys Leu Ala Asn Pro Val Ser Leu Thr Asp Lys He Gin Leu Ala Cys Leu Pro Pro Ala Gly Thr He Leu Pro Asn Asn Tyr Pro Cys Tyr 40 Val Thr Gly Trp Gly Arg Leu Gin Thr Asn Gly Ala Leu Pro Asp Asp Leu Lys Gin Gly Arg Leu Leu Val Val Asp Tyr Ala Thr Cys Ser Ser Ser Gly Trp Trp Gly Ser Thr Val Lys Thr Asn Met He Cys Ala Gly Gly Asp 45 Gly Val lie Cys Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Asn Cys Gin Ala Ser Asp Gly Arg Trp Glu Val His Gly lie Gly Ser Leu Thr Ser Val Leu Gly Cys Asn Tyr Tyr Tyr Lys Pro Ser He Phe Thr Arg Val Ser 50 Asn Tyr Asn Asp Trp He Asn Ser Val lie Ala Asn Asn - (C) For the human pancreatic elastase IIB itself, the N-terminal end simply terminates with a hydrogen at- om.