IL86086A - Fusion protein with activities of interleukin-2 and gm-csf and pharmaceutical compositions containing said protein - Google Patents

Fusion protein with activities of interleukin-2 and gm-csf and pharmaceutical compositions containing said protein

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Publication number
IL86086A
IL86086A IL8608688A IL8608688A IL86086A IL 86086 A IL86086 A IL 86086A IL 8608688 A IL8608688 A IL 8608688A IL 8608688 A IL8608688 A IL 8608688A IL 86086 A IL86086 A IL 86086A
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IL
Israel
Prior art keywords
protein
thr
asp
csf
constituent
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Application number
IL8608688A
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Hebrew (he)
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IL86086A0 (en
Original Assignee
Hoechst Ag
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Publication of IL86086A0 publication Critical patent/IL86086A0/en
Publication of IL86086A publication Critical patent/IL86086A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • 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]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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
    • 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

Abstract

Bifunctional proteins obtainable by genetic engineering from an interleukin-2 and a granulocyte-macrophage "colony stimulating" factor fraction have the biological activity of both components, but are distinguished by increased stability. These proteins are thus medicaments which are suitable for the treatment of malignant neoplasms. [EP0288809A1]

Description

86086/2 FUSION PROTEIN WITH ACTIVITIES OF INTERLEUKIN-2 AND GM-CSF AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAID PROTEIN.
GM-CSF -■) 2-,ρρ,>Ί υ Ν n-mtnyon yi >m>N ii-i rv iniN D^ on rnnpn i^w3rri Abstract of the DiscLosure BifunctionaL proteins, obtainable by genetic manipulation, composed of an interleukin-2 and a granulocyte macrophage colony stimulating factor constituent have the biological activity of both components but are distinguished by increased stability. These proteins are thus medicaments which are suitable for the treatment of malignant neoplasms.
InterLeuk in-2, called IL-2 hereinafter, acts as T-cell growth factor. IL-2 potentiates the activity of killer cells such as NK (natural killer) cells, cytotoxic T- cells and LAK (lymphokine-activated killer) cells.
By contrast, granulocyte macrophage colony stimulating factor, called GM-CSF hereinafter, stimulates the formation of granulocytes and macrophages from hemopoietic precursor cells. Combination of the two biological activities is of interest for human treatment with and without administration of cytostatics. However, the stabilities of IL-2 and GM-CSF differ, which may result in problems on direct adm nistration of the two components and thus in a decrease in the therapeutic success.
The problem of the difference in stability can be solved according to the invention by linking these two proteins to a bifunctional protein.
Fusion proteins of the general formula Met - X - Y - Z or Met - Z - Y - X (la) (lb) have already been proposed for the preparation, by genetic manipulation, of optionally modified GM-CSF in which X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, preferably human, IL-2, Y denotes a direct bond if the amino acid or amino acid sequence adjacent to the desired protein allows the desired protein to be cleaved off, or 2 86086/2 otherwise denotes a bridging member which is composed of one or more genetically encodable amino acids and which allows the cleavage off, and Z is a sequence which is composed of genetically encodable amino acids and which represents the desired GM-CSF protein. It is also possible during this to make use - more or less - up to the end of the DNA sequence coding for IL-2, and thus generate biologically active IL-2 - modified where appropriate - as a "by-product" (not prior-published European Patent Application with the publication number (EP-A) 0,228,018 and South African Patent 86/9557).
EP-Al-0 158 198 discloses a hybrid protein consisting of human IFN-^ and human IL-2, GM-CSF is not mentioned.
Schrader et al . (1986) disclose homologies between GM-CSF and IL-2. However, the homolgy is only 25% and Schrader et al. do not teach or suggest any hybrid protein between GM-CSF and IL-2.
The present invention however teaches for the first time that GM-CSF and IL-2 can be combined to result in a bifunctional protein, i.e. without loss of activity.
This was neither taught nor suggested by the above references. Moreover, because of the fact that the stabilities of IL-2 and GM-CSF differ, it was very surprising to obtain a bifunctional protein with greater stability than IL-2 or GM-CSF alone. A further advantage of the present .invention is that because of the greater stability a lower dosage for application is possible . - 2a - 86086/3 The present invention relates to a bifunctional protein composed of a biologically active interleukin-2 (IL-2) constituent and a bilogically active granulocyte macrophage colony stimulating factor (GM-CSF) constituent, wherein the two biologically active protein constituents are linked via a bridge composed of 1 to about 20 genetically encodable amino acids.
Preferably the bridge corresponds to the formula (II) - Asp - (aa)x - Pro - (II) x denoting an integer from 1 to 18, and aa being a genetically encodable amino acid with the exception of Cys. The Bridging member (a.a)^ may denote the amino acid sequence -pro-Met-lle-Thr-Thr-Tyr-Ala-Asp-Asp- or -Pro-Met-lle-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-lle-Asp-Asp- The invention further relates to a pharmaceutical composition comprising a protein as defined above, where appropriate combined with a pharmacologically suitable vehicle. Such compositions can be used for treatment of malignant neoplasms.
- - . In contrast to the earlier proposal, the invention relates not to the use of the proteins as intermediate but to the use in methods for the therapeutic treatment of the human body and to medicaments which contain fusion proteins of this type or which are composed of fusion proteins of this type. A further aspect of the invention relates to the use of these fusion proteins for the preparation of a -medicament for the treatment of malignant neopl asms .
The fusion protein used according to the invention is thus composed of two biologically active components, namely of an IL-2 constituen , which can be modified in a manner known per se, on the one hand, and of a GM-CSF constituent, which can likewise be modified, on the other hand and, where appropriate, of a bridging member corresponding to the definition Y in the formulae given above. The arrangement of the two components preferably corresponds to the formula la. The principle according to the invention can a'lso be used for the preparation of other novel bifunctional proteins.
The figure shows the construe tion of the plasmid pB30 which codes for a bifunctiona I protein according to the invention. - 3 - Modi ications of the IL-2 molecule have been disclosed, reference being made here only to EP-A 0,091,539, 0,109,748, 0,118,617, 0,136,489 and 0,163,249 by way of example .
Furthermore, the not prior-published EP-A 0,219,839 proposes an IL-2 derivative in which the first seven N-terminal amino acids are deleted.
Modifications of the GM-CSF molecule have been proposed in EP-A 0,228,018.
Further alterations to the two active constituents of the molecule can be carried out in a manner known per se, mention being made here only of specific mutagenesis by way of example.
The bridging member Y advantageously has the formula II - Asp - (aa)x - Pro - (II) in .which x denotes an integer up to about 20, and aa denotes any desired genetically encodable amino acid with the exception of cysteine.
It is advantageous in the formula II for the IL-2 con-stituent to be arranged at the left-hand end, and consequently the GM-CSF constituent to be arranged at the right-hand end.
Particularly preferred embodiments of Y have the amino acid sequence -As p-Pro-Met-I I e-Thr-Thr-Tyr-A la-Asp-Asp-Pro or -Asp-Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr- I le-Asp-Asp-Pro-it again being preferable for the IL-2 constituent to be arranged at the left-hand end and the GM-CSF constituent to be arranged at the right-hand end.
The bifunctional proteins according to the invention can be expressed in a manner known per se. It is possible in bacterial expression systems for the route of direct expression to be followed. Suitable for this purpose are all known host-vector systems with hosts such as bacteria of the species Streptomyces, B. subtilis, Salmonella typhimurium or Serratia marcescens, especially E. col .
The DNA sequence which codes for the desired protein is incorporated in a known manner into a vector which ensures satisfactory expression in the chosen expression system.
It is expedient to choose for this purpose the promoter and operator from the group trp, lac, tac, P\_ or PR of phage λ, hsp, omp or a synthetic promoter, as described in, for example, German Of f enLegungsschr ft 3,430,683 and in EP-A 0,173,149. The tac promoter-operator sequence is advantageous and is now commercially available (for example '··'ρ Κ223-3 express ion vec tor , Pharmac ia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, page 63).
On expression of the protein according to the invention, it may prove expedient to modify individual triplets for the first few amino acids after the ATG start codon in order to prevent any base-pairing at the level of the mRNA. Such modifications, such as deletions or additions of individual amino acids, are familiar to the expert, and the invention also relates to them.
For expression in yeasts - preferably S. cerevisiae - it is expedient to use a secretion system, for example heterologous expression via the ct-factor system, which has been described several times.
It is advantageous for the expression of the bifunctional molecule in yeast if dibasic peptide sequences and - 5 - gtycosylat ion sites in the bifunctionaL protein have been destroyed by appropriate exchange of individual amino acid,s. This results in many possible combinations which may also influence the biological action.
The expression of IL-2 in yeast is disclosed in EP-A 0,142,268, and that of GM-CSF in EP-A 0,188,350.
The administration of the bifunctionaL proteins according to the invention corresponds to that of the two components. However, because of the greater stability a lower dosage is possible in many cases, the dosage being in the lower part of the range of those hitherto proposed.
The invention is illustrated in detail in the examples which follow. Unless indicated otherwise, percentage data and ratios relate to weight.
Example 1 The plasmid p159/6 (EP-A2 0,163,249, Figure 5; (1) in the present figure) contains a synthetic gene coding for IL-2 between an EcoRI and a Sail cleavage site. The DNA sequence for this gene is represented in the said EP-A2 as "DNA sequence I". A Taql cleavage site is located in the region of triplets 127 and 128. The IL-2 part-sequence (2) is cut out of this plasmid by cutting with EcoRI and Taql, and is isolated.
The plasmid pHG23 (3) which codes for GM-CSF is disclosed in EP-A2 0,183,350. The GM-CSF cDNA is represented in Figure 2 in this EP-A2. The plasmid pHG23 is obtained when the cDNA sequence is incorporated in the Pstl cleavage site of pBR322, use being made of, on the one hand, the Pstl cleavage site at the 5' end and, on the other hand, a Pstl site introduced at the 3* end by GC tailing. The DNA sequence (4) which contains most of the GM-CSF gene is isolated from this plasmid by cutting with SfaNI and Pstl. - 6 - The following oligonucleotide (5) is synthesized by the phosphite method: 128 ( 133) He lie Se r Thr Leu Asp Pro Met He CG ATC ATC TCT ACC CTG GAC CCG ATG ATC TAG TAG AGA TGG GAC CTG GGC TAC TAG (Taql ) (5) 1 2 Thr Thr Ty r Ala Asp Asp Pro (Ala) (Pro) ACC ACC TAT GCG GAC GAT CCG GC TGG TGG ATA CGC CTG CTA GGC CGT GGG (Sf aNI ) The oligonucleotide (5) extends at the 5' end the DNA sequence of IL-2, there being,, however, Asp in place of Thr in position 133. At the 3' end of this oligonucleo-tide are located the nuc leotides which have been deleted from the cDNA by cutting with SfaNI.
The preparation of the expression pLasmid pEWIOOO (6) is proposed in the (not prior-published) EP-A 0,227,938 (Figure 1). This plasmid is a derivative of the plasmid ptac 11 (Amann et al., Gene 25 (1983) 167 - 178), in which a synthetic sequence which contains a Sail cleavage site has been incorporated in the recognition site for EcoRI. The expression plasmid pKK 177.3 is obtained n this way. Insertion of the lac repressor (Farabaugh, Nature 274 (1978) 765 - 769) results in the plasmid p J F 118. The latter is opened at the unique restriction cleavage site for Aval, and is shortened by about 1000 bp in a known manner by exonuclease treatment and is ligated. The plasmid pEWIOOO (6) is obtained. Opening of this plasmid in the polylinker using the enzymes EcoRI and Pstl results in the linearized expression plasmid (7).
This linearized plasmid DNA (7) is now ligated with the DNA fragment (2) which codes for the IL-2 sequence, with - 7 - the synthetic oL i gonuc L eo t i de (5) and with the cDNA fragment (4). The result is the plasmid pB30 (8) which is transformed into the E. coli strain Mc1061. The plasmid DNA from individual clones is isolated and character zed by restriction analysis.
Example 2 If the following synthetic oligonucleotide 128 (133) He He Se r Thr Leu Asp Pro Met lie Thr Thr Tyr CG ATC ATC TCT ACC CTG GAC CCG ATG ATC ACC ACC TAT TAG TAG AGA TGG GAC CTG GGC TAC TAG TGG TGG ATA (Taql ) 1 2 Leu Glu Glu Leu Thr He Asp Asp Pro (Ala ) (Pro) CTA GAA GAG CTC ACG ATC GAC GAT CCG GC GAT CTT CTC GAG TGC TAG CTG CTA GGC CGT GGG (SfaNI) is used in place of oligonucleotide (5) in the example the result is the plasmid pB31.
Example 3 Competent cells of the E. coli strain W3110 are transformed with the plasmid pB30 or pB31. An overnight culture of the strain is diluted in the ratio of about 1:100 with LB medium (J. H. Miller, Experiments in Molec.
Gen., Cold Spring Harbor Lab., 1972), which contains 50 ug/ml ampicillin, and the growth is followed by measurement of the 00. At OD = 0.5 the culture is adjusted to a concentration of 2 mM in isopropyl-B-D-th iogalactopyra-noside (IPTG) and, after 150 - 180 minutes, the bacteria are spun down. These bacteria are treated in a buffer mixture (7M urea, 0.1% SDS, 0.1M sodium phosphate, pH 7.0) for about 5 minutes, and samples are applied to an SDS polyacrylamide gel electrophoresis plate. This confirms the expression of the bifunctional protein.
The stated conditions appLy to shake cultures; for larger fermentations it is expedient to modify the OD values and nutrient media and vary the IPTG concentrations appro-pr iately .
Example 4 E. coli W3110 cells which contain the plasmid pB30 or pB31 are, after induction, spun down, resuspended in sodium phosphate buffer (pH 7) and again spun down. The bacteria are taken up in the same buffer and then dis- ( R ) rupted (French Press, Dynomill). The disrupted cells are spun down. The supernatant and sediment are analyzed by SDS polyacrylamide gel elec trophorese as described in Example 3. Staining of the protein bands reveals that the bifunctional protein is located in the sediment from the disruption. The sediment is washed several times with chaotropic buffers and finally with water, resulting in further enrichment of the desired protein. The protein concentration is then determined in the aqueous protein suspension. The suspension is now adjusted to a concentration of 5 M in guanidinium hydrochloride and 2 mM in d i t h i o t h re i tol (OTT). The mixture is stirred under nitrogen for about 30 minutes and then diluted with 50 mM tris buffer (pH 8.5) so that the protein concentration s 100 ug/ml. It is now dialyzed against this tris buffer and, after two changes of the buffer, dialyzed against water. The protein treated in this way is sterile filtered and its biological activity is checked. It shows full bio-logical action both in the i n t e r I euk i n-2-dependent CTLL 2 cell proliferation assay and in the human bone marrow assay. Mixed colonies of granulocytes and macrophages are observed in these.
The bifunctional protein can be further purified by i n te r I euk i n-2-spec i f i c affinity chromatography. The protein is still active in both assays. In contrast, an E. coli extract of the untransf ormed strain W3110 which has been treated as described shows no activity. - 9 - Other conditions are expedient for the industrial preparation of the product, for example for the folding of the protein and its purification. Suitable purification processes - which are known per se - are ion exchange, adsorption, gel filtration and preparative HPLC chromatography.

Claims (9)

- 10 86086/2 Patent Claims
1. , A bifunctional protein composed of a biologically actiye interle'ukin-2 (IL-2) constituent and a biologically actiye granulocyte macrophage colony stimulating factor (GM-CSF) constituent, wherein the two biologically active protein constituents are linked via a bridge composed of 1 to about 20 genetically encodable amino acids,
2. · A protein as claimed in Claiml , wherein the bridge corresponds to the formula (II) - Asp - (aa)x - Pro - (II) x denoting an integer from 1 to 18, and aa being a genetically encodable amino acid with the exception of Cys .
3. A protein as claimed in Claim 2* wherein the bridging member (aa)x denotes the amino acid sequence -Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp- or -Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-Ile-Asp-Asp-.
4. A protein as claimed in Claim 2 or 3, wherein the IL-2 constituent is arranged N-terminal and the GM-CSF constituent is arranged C-terminal.
5. A process for the preparation of bifunctional proteins as claimed in any of Claims 1 to 4, which comprises construction, and expression in a host cell, of a gene coding for these proteins.
6. · A pharmaceutical composition comprising a protein as claimed in any of claims 1 to 4, where appropriate combined with a pharmacologically suitable vehicle.
7. The use of a protein as claimed in any of claims 1 to 4 for the preparation of a pharmaceutical composition,for the treatment of malignant 'neoplasms .substantially an hereinbefore described,. 86068/2 - 11 -
8. A pharmaceutical composition containing a bifunctional protein defined in claim 1 as active ingredient.
9. . A pharmaceutical composition for the treatment of malignant neoplasms containing as active ingredient a compound claimed in any of claims 1 to A 10 , Pharmaceutical compositions containing a bifunctional protein defined in claim 1, substantially as hereinbefore described.
IL8608688A 1987-04-16 1988-04-15 Fusion protein with activities of interleukin-2 and gm-csf and pharmaceutical compositions containing said protein IL86086A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
DE19873712985 DE3712985A1 (en) 1987-04-16 1987-04-16 BIFUNCTIONAL PROTEINS

Publications (2)

Publication Number Publication Date
IL86086A0 IL86086A0 (en) 1988-09-30
IL86086A true IL86086A (en) 1995-01-24

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IL8608688A IL86086A (en) 1987-04-16 1988-04-15 Fusion protein with activities of interleukin-2 and gm-csf and pharmaceutical compositions containing said protein

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EP (1) EP0288809B1 (en)
JP (1) JP2667193B2 (en)
KR (1) KR970000187B1 (en)
AR (1) AR242991A1 (en)
AT (1) ATE79135T1 (en)
AU (1) AU613022B2 (en)
CA (1) CA1322157C (en)
DE (2) DE3712985A1 (en)
DK (1) DK170741B1 (en)
ES (1) ES2033981T3 (en)
FI (1) FI98830C (en)
GR (1) GR3006141T3 (en)
HU (1) HU204303B (en)
IE (1) IE61574B1 (en)
IL (1) IL86086A (en)
NO (1) NO176922C (en)
NZ (1) NZ224247A (en)
PH (1) PH25327A (en)
PT (1) PT87237B (en)
ZA (1) ZA882659B (en)

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ES2033981T3 (en) 1993-04-01
NO881658D0 (en) 1988-04-15
PT87237A (en) 1988-05-01
FI98830C (en) 1997-08-25
AR242991A1 (en) 1993-06-30
JPS63301898A (en) 1988-12-08
DE3873397D1 (en) 1992-09-10
NO881658L (en) 1988-10-17
NZ224247A (en) 1990-04-26
KR970000187B1 (en) 1997-01-06
DK170741B1 (en) 1996-01-08
FI881743A0 (en) 1988-04-14
IE881146L (en) 1988-10-16
DK209188D0 (en) 1988-04-15
HU204303B (en) 1991-12-30
NO176922C (en) 1995-06-21
AU1466188A (en) 1988-10-20
PH25327A (en) 1991-04-30
ZA882659B (en) 1988-10-14
DK209188A (en) 1988-10-17
EP0288809A1 (en) 1988-11-02
FI98830B (en) 1997-05-15
ATE79135T1 (en) 1992-08-15
CA1322157C (en) 1993-09-14
GR3006141T3 (en) 1993-06-21
KR880012760A (en) 1988-11-29
NO176922B (en) 1995-03-13
AU613022B2 (en) 1991-07-25
HUT47319A (en) 1989-02-28
DE3712985A1 (en) 1988-11-03
IE61574B1 (en) 1994-11-16
FI881743A (en) 1988-10-17
IL86086A0 (en) 1988-09-30
PT87237B (en) 1992-07-31
EP0288809B1 (en) 1992-08-05
JP2667193B2 (en) 1997-10-27

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