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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
  • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/545—IL-1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/61—Growth hormone [GH], i.e. somatotropin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/034—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

• the present invention concerns a process for preparing a protein or polypeptide which contains an amino-terminal extension, preferably with at least two amino acids, in addition to the desired polypeptide chain.
• proteins or polypeptides may be produced biosynthe ically by cultivating a host organism which contains a vector with the DNA sequence, such as a plasmid, which codes for the desired polypeptide.
• the resulting polypeptide is isolated from the cultivation medium and purified, e.g. chromato- graphically.
• polypeptides that are produced biosynthetically in this way are so atostatin , insulin and human growth hormone.
• Another advantag . e of transport of the desired protein out into the periplasma is that the protein here folds sub ⁇ stantially correctly to provide the native protein with the correct folding for the active protein. This involves essential advantages in the biosynthetic production of proteins on an industrial scale because then it is not necessary to effect subsequent folding, which is very difficult to perform in vitro.
• the signal sequence is cut by an enzyme contained in the bacterium during or after the transport of the protein through the inner membrane. This cut, however, is not completely specific since partially non-specific cutting of the signal sequence takes place depending upon the cultivation methods, strain of bacterium, etc., or sub- sequent enzymatic reactions occur, whereby the N-terminus of the desired protein is digested by peptidases in the periplas a to an uncontrolled degree.
• a desired or mature protein by means of a microorganism coding for a desired protein with an amino terminal extension which comprises at least two amino acids, cf. the DK Patent Application 3448/84.
• the desired protein with the extension is collected in the cytoplasma, from which it is to be isolated and purified.
• the extension may be digested specifically to obtain the desired protein with a correct N-terminal in a pure state, using e.g. the enzyme DAP 1 and/or DAP 4.
• S is a signal sequence
• E is an amino extension with at least two amino acids, at least one of which is charged
• P is the desired mature protein.
• the desired polypeptide is synthetized with associated extension.
• the signal sequence will cause the amino-terminal-extended protein to be transported through the membrane enclosing the cytoplasma
• amino-terminal- extended polypeptide will occur in correctly folded form. If a microorganism with a periplasmatic space is used, the desired amino-extended polypeptide will normally be collected in it. It happens that under certain circum- ces, which are not fully understood, the protein also passes the outer membrane to the cultivation medium it ⁇ self.
• the isolated amino-terminal-extended polypeptide can then be used as a substrate for e.g. the enzyme DAP 1 to produce the desired polypeptide, P.
• the amino-terminal- extended polypeptide for production of the desired mature protein in a high yield and in a pure state by utilizing at the final purification the difference in charge between the mature protein and the amino-terminal- extended protein which might be left after the enzymatic cleavage.
• the process of the invention can be used generally for preparation of an arbitrary protein with an amino terminal extension which can be subsequently removed enzymatically .
• proteins which can be produced by the process of the invention are amino-terminal-extended IGF 1, amino- terminal-extended 22K hGH, amino-terminal-extended 20K hGH, amino-terminal-extended IL 1 and amino-ter inal- ex ended insulin.
• the amino terminal extension, E. preferably comprises 2 amino acids, as mentioned, and in particular preferably 4 to 16 amino acids.
• the amino terminal extension contains at least one charged amino acid.
• the presence of one or more charged amino acids permits effective purification and isolation of the desired protein by chromatography .
• the invention also concerns a DNA sequence, a plasmid containing this DNA sequence, and microorganisms in which such a plasmid has been inserted.
• any microorganism into which a plasmid may be inserted can be used in the process of the invention.
• E. coli lends itself particularly well for the purpose, but also other microorganisms may be used.
• An example of this is B. subtilis.
• This microorganism does not, as is the case with E. coli, have an outer membrane and accordingly a periplasmatic space.
• the resulting polypeptide is transported through the cell membrane and out into the cultivation medium with simultaneous cutting of the signal sequence.
• the polypeptide will be correctly folded and can be isolated from the cultivation medium in a manner known per se, cf. by chromatography on an ion-exchange resin.
• the polypeptide produced by the process of the invention which comprises the amino-terminal extension, is suitable as a starting material in the preparation of the mature protein, P.
• the amino terminal extension in a suitable manner, it is possible to obtain specific enzymatic digestion of the extension.
• one of the enzymes DAP 1 or DAP 4 may be used for this pur ⁇ pose, which causes stepwise cleavage of the extension, preferably by removal of two amino acids at a time, followed by isolation of the resulting mature protein from possibly non-reacted or partially reacted, extended polypeptide in a known manner, such as by chromatography.
• the invention moreover concerns an amino-terminal-extended interleukin 1 (IL-1) as well as a preparation containing the biologically active polypeptide.
• IL-1 interleukin 1
• Interleukin 1 is a polypeptide which is produced in various animal cells, such as monocytes.
• the full amino acid sequence of IL-1 and the precursor of IL-1 are known.
• IL-1 has various biological effects. Thus, it was shown recently that IL-1 has a cytotoxic effect on the Langer- hans's islets, i.e. /.-cells in the pancreas, and may thus cause insulin dependent diabetes ellitus (IDDM). The effect occurs in connection with the binding of IL-1 to specific receptors of the /3-cells.
• IDDM insulin dependent diabetes ellitus
• IL-1 The production of excess of IL-1 can take place by various biological influences.
• This embodiment of the invention is based on the finding that amino-terminal-extended IL-1 counteracts the cyto ⁇ toxic effect of IL-1, such as against .-cells in the pancreas.
• Amino-terminal-extended IL—1 is believed to bind to the same receptors as IL-1 on the - c e l l s and thus block these receptors. This would prevent the cytotoxic effect of IL-1.
• the present invention also concerns amino-terminal- extended IL-1. Further, the invention concerns prepa ⁇ rations containing amino-terminal-extended IL-1, which may e.g. be used for therapeutic or prophylactic treat ⁇ ment of IDDM.
• the invention moreover concerns a DNA-strueture coding for amino-terminal-extended IL-1 or amino-terminal- extended IL-1 derivatives.
• the extension of the IL-1 and IL-1 derivatives may have different lengths and e.g. compris * e most of the pre- sequence of the naturally occurring IL-1. Preferably, however, a considerably shorter amino terminal extension is used, e.g. comprising 2 to 6 amino acids.
• polypeptides of the present type are amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids
• the polypeptide may be modified in a manner known per se or be amended, e.g. by replacing one or more amino acids by other amino acids or by removal of one or more amino acids in the C-terminus.
• the polypeptide of the invention can be used therapeutic- ally for combatting or preventing i.a. diabetes and autoimmune diseases. It is also proposed to use the poly ⁇ peptide for treatment of patients who are subjected to septic shock under the action of endotoxiantibodies.
• pharmaceutical preparations in particular injectable preparations are used, containing the poly ⁇ peptide in a pharmaceutically acceptable carrier medium.
• the present polypeptide is produced biosynthetically by cultivation of a microorganism or a cell line which contains a gene coding for the polypeptide, which is then recovered, purified and processed to the desired preparation.
• the restriction sites EcoRI/BamHI have inserted between them a DNA segment containing a promotor, a Shine Dalgarno sequence and the signal sequence from outer membrane protein A (2), followed by a synthetic DNA linker containing the recognition sequence for the restriction enzymes Narl, Bglll, PvuII and Ba HI.
• the plasmid pHD167 is likewise a derivative of the plasmid pAT153.
• This plasmid contains a promotor, a Shine Dalgarno sequence, a synthetic gene coding for IGF1, and a transcription terminator.
• the above-mentioned sequences are inserted between the EcoRI and BamHI restriction sites in pAT153.
• the plasmid pHD176 contains the plasmid region, the promotor region and the signal sequence from the plasmid pHD165 as well as the transcription terminator and the gene for IGF1 from the plasmid pHD167, isolated as an Ace III/BamHI DNA fragment, and a small synthetic DNA linker coding for amino terminal excusion.
• the plasmid pHD176 contains the coding region for the outer membrane protein A signal sequence, followed by the amino terminal extension Ala-Glu-Ala-Glu, and the coding sequence for IGF1.
• the plasmid pHD141 shown in fig. 2 is a derivative of the plasmid pHD167 in which the coding sequence for IGF1 has been replaced by the coding sequence for amino-terminal- extended 20K-hGH.
• the plasmid pHD223 contains the sequences contained in the plasmid pHD141 between Clal/EcoRI, a Clal/Narl DNA fragment coding for the signal sequence from pHD165 as well as a synthetic DNA linker coding for an amino terminal excusion.
• the plasmid pHD223 thus contains the coding region for the outher membrane protein A signal sequence, followed by the amino terminal extension Ala-Glu-Ala-Glu , followed by the coding sequence for 20K-hGH.
• the plasmid pHD106-9 shown in fig. 3 is a derivative of the vector pUC18 into which the coding region for the signal sequence from E. coli fimbria protein K88 (3) has been inserted between the restriction sites BamHI/Pstl.
• the plasmid pHD117-4 SP13 is a derivative of the plasmid pHD167, in which the coding region for IGF1 has been replaced by the coding region for amino-terminal-extended 22K-hGH.
• the plasmid pHD148-22K-hGH contains the signal sequence from the fimbria protein K88, followed by the amino terminal extension Ala-Glu-Ala-Glu-Ala-Glu, followed by the coding region for 22K-hGH.
• the plasmid pHD163 shown in fig. 4 is a derivative of the plasmid pHD167 in which the coding region for IGF1 has been replaced by the coding region for amino-terminal- extended IL-1.
• the plasmid pHD185 is a derivative of the plasmid pAT153, into which a synthetic DNA fragment containing the coding region for the outer membrane protein A signal sequence as well- as the recognition sites for the restriction enzymes Narl, Bglll, PuvII and BamHI have been inserted between the restriction sites EcoRI/BamHI.
• the plasmid pHD163/185 thus contains the coding region for the signal sequence from the outer membrane protein A, followed by the amino terminal extension Met-Glu-Ala-Glu- Phe-Asp and the coding region for interleukin 1.
• the plasmid pHD165 which contains a promotor and the above-mentioned signal sequence was cut with the enzymes Narl/Ba HI, and then the plasmid fragment was purified.
• the plasmid pHD167 containing e.g. the coding sequence for human IGF1 followed by transcription terminator, was cut with the enzymes AccI I I/BamHI , and then a fragment of about 600 base pairs was purified. These two fragments as well as a synthetic DNA linker, containing a restriction overhang for the enzymes Narl/AccIII (coding for amino terminal extension) were then ligated together to form the plasmid pHD176.
• the ligation mixture was subsequently transformed into the bacterium E. coli MC1061, plated on ampicillin containing aga plates and incubated overnight at 37 °C.
• a plurality of bacteria colonies was selected and cultivated for further characterization by restriction enzyme analysis.
• the clones pHD176-D, -F were then selected.
• the clone pHD176-F was cultivated on a large scale, and then the bacteria were harvested by centri- fugation.
• the bacteria were then partly exploded by means of osmotic shock, so that the proteins present in the periplasma were released from the bacteria.
• the amino-terminal- extended IGF1 was purified. This material was then used as a substrate for DAP 1, whereby the N-terminal extension was specifically removed.
• the expression level in the above-mentioned cultivation of the bacterium HD176-F was about 5 mg of amino-terminal- extended IGF1 per litre of bacteria culture with an 0D600 of about 1.
• the plasmid pHD141 containing the gene for 20K human growth hormone, was cut with the enzymes Clal/EcoRI, and then the plasmid fragment was purified.
• the plasmid pHD165 was cut with the enzymes Clal/Narl, and then a fragment of about 120 base pairs was purified. These fragments were subsequently ligated together in the presence" of a synthetic DNA linker with an overhang for the restriction enzymes Narl/EcoRI to form the plasmid pHD223.
• the ligation mixture was transformed into E. coli MC1.061 and plated on ampicillin containing growth plates
• the expression level was of the same order as mentioned in example 1.
• the plasmid pHD106-9 which contains another signal sequence described above, was cut with the restriction enzymes BamHI/Hindlll . A fragment of 190 base pairs was then purified. This fragment was subsequently cut with the enzyme BstNI, treated with SI nuclease to produce a blunt end, phenol-extract ⁇ d, ethanol-precipitated, and then cut with the enzyme Clal. A fragment of 60 base pairs was then purified.
• the plasmid pHD117-4 SP13 was cut with the enzymes Clal/EcoRIj and then the plasmid fragment was purified. The two mentioned purified fragments were ligated together in the presence of a BstNI/EcoRI linker to form the plasmid pHD148-22K-hGH.
• the ligation mixture was transformed into the bacterium E. coli MC1061 and plated on ampicillin containing growth plates. After incubation overnight, a plurality of colonies was selected for further character- ization by restriction enzyme analysis and DNA sequence determination. Cultivation and isolation of the resulting amino-terminal- extended 22K hGH were performed in the same manner as stated in example 1.
• the plasmid pHD185 containing the coding sequence for a signal sequence, was cut with the enzymes Clal/Narl, and then a DNA fragment of about 90 base pairs was purified. These two fragments were then ligated together to form the plasmid pHD163/185.
• the obtained plasmid was inserted into E. coli, which was cultivated in the usual manner.
• the resulting amino- terminal-extended human IL-1 was purified and converted into human IL-1 by reaction with DPA 1.
• a gene coding for the amino acids 117-269 in the IL-1 precursor was produced. It has been found by comparative studies of messenger RNA sequences of high-level- expressed and low-level-expressed genes in bacteria, respectively, that there is a strong correlation between codon use and expression efficiency. Accordingly, it was decided to use the codons which are most frequently used in high-level-expressed E. coli genes. Useful cloning sites were inserted inside the gene as well as at both ends. The gene was extended with a DNA sequence which codes for the amino terminal extension Met-Glu-Ala-Glu (MEAE). The gene was produced by stepwise cloning between restriction sites of oligonucleotides having an average length of 80 to 100 nucleotides. The sequence of the gene for MEAE-IL-1 is shown in fig. 5.
• E. coli was selected as the host organism since glyco- sylation or other modifications of IL-1 have not been reported.
• the expression was controlled by a synthetic promotor (SP13) and an .optimum Shine Dalgarno sequence.
• SP13 synthetic promotor
• the used promotor was selected because it is known that it is effective for expression of e.g. bhGH in E. coli (up to 20 ⁇ of total bacteria protein) (Bio Technology,
• IL-1 Extraction of IL-1 was performed as described in Bio Technology, Vol. 4, Dec. 1986, p. 1078. Purification of the N-terminal-extended IL-l- derivatives was performed on FF-S sepharose, FF-Q sepharose and gel filtration colonies, respec i ely. The derivatives in question were characterized by amino acid analyses, N-terminal sequence analysis, molecular weight determination by the firm atomic bombardment method (Barber M. et al.; (1981) J. Chem. Soc., Chem. Comm. Vol. 1981, p. 325-327), SDS and native electrophoresis.
• a DNA segment with the coding region for pheny1-alanine-aspartic acid was inserted into the clone pHD230. It was inserted in such a manner that IL-1 was extended with the sequence Met- Glu-Ala-Glu-Phe-Asp- .
• the gene for amino-terminal- extended IL-1 was expressed in E. coli and purified as described above.
• the clone was prepared by in vitro mutagenesis on the plasmid pHD230.
• the amino-terminal-extended IL-1 was expressed and purified as described above.
• the N-terminal methionine group was cleaved in vivo by an E. coli enzyme Example 10
• the resulting clone pHD228 was then cut with the enzymes Narl/Clal, and then a fragment coding for the below amino acid sequence was inserted.
• the gene was expressed in E. coli and purified as described above.
• the inserted amino acid sequence had the following composition:
• the specific bioactivity (units/ng IL-1-3) is determined by simultaneous measurement of the biological response in the comitogenic mouse thymocyte profileration assay (LAF) and by quantization by means of IL-l-/_ Elisa of the corresponding amount of IL-1-3 protein.
• LAF assay is performed as described above (Scand. 3. Immunol. 26:611, 1987, Scand. J. Immunol. : Endotoxin stimulated human onocyte secretion of interleukin 1, tumor necrosis factor alpha and prostaglandin E show stable in erindi idual difference, in press).
• Thymocytes are isolated from 5 to 7 weeks old male mice of the C3H/he mice strain (Charles River, Federal Republic of Germany). After washing of the cells, these are plated in shallow microtiter plates (Nunc, Roskilde) in a medium consisting of a mixture of 90? ⁇ RpMI-164 and 10? ⁇ ac ⁇ cumulated heat inactivated human serum, the medium having been added to the sample in a dilution series beginning with 20 vol? ⁇ and diluted 1:2 in up to 8 steps. The test series is repeated 3 times. PHA (Difco) 5 ,ug/ml is added to each well. After 48 hours' incubation at 37 °C 1 ,uCi tritiated thy idin is added per well. The cultures are harvested on glass fibre filters (Skatron, Norway) after another 18 hours' incubation by means of a semi-automatic cell culture harvester. Then the filters are counted in a beta counter.
• the results are calculated as follows: The average of triplicates is calculated. Linear regression is performed for each dilution series, comprising a count above 3 x background. The highest counts in the dilution series are included only if they are more than 10? ⁇ higher than the count in the next dilution step. Linear regression is performed either semi-logarithmlcly or double-logarith- micly. This results in parallel linear curves which directly allow quantization of the biological response with respect to a standard consisting of recombl ⁇ ant I -l- 100 ng/ml (WHO standard). The results can then be stated in Units/ml, the WHO standard containing 100 U/ng.
• IL-1-5 Elisa is performed according a standardized Elisa method (Cistro ⁇ ).
• Results appear from the enclosed table. It is noted that the specific activity of MEAE-IL-1-/3 is found to be 50 to 100 times weaker than recombinant full length IL-1-3. The results are obtained from 3 independent experiments.
• the pancreas is removed from 5 to 7 days old rats of the Wistar strain, and the islets are isolated by collagenase degradation.
• the islets are cultivated in RPMI-1640 + 10? ⁇ NCS at 37 °C in a moist atmosphere. After 1 week's incubation the islets are washed in RPMI-1640 + 0.5? ⁇ HS and incubated further for 6 days with and without IL-1.
• the insulin secretion from the islets Is determined in Radioim unoassay (RIA).
• RIA Radioim unoassay
• Fig. 3 shows a standard curve for rIL-1. It will be seen that the insulin secretion is stimulated at very low rIL-1 concentrations (0.001 ng/ml) (about 130? ⁇ ), while the insulin secretion is inhibited to about 50? ⁇ at a somewhat higher concentration (0.1 ng/ml). References:

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• 1988
  • 1988-06-23 WO PCT/DK1988/000101 patent/WO1988010299A1/en not_active Application Discontinuation
  • 1988-06-23 AU AU19870/88A patent/AU1987088A/en not_active Abandoned
  • 1988-06-23 EP EP88905733A patent/EP0371041A1/en not_active Withdrawn
  • 1988-06-23 JP JP63505494A patent/JPH03503596A/ja active Pending
• 1989
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