EP0510018A1 - Peptide a signal lipoproteique fusionne avec des polypeptides antigeniques - Google Patents

Peptide a signal lipoproteique fusionne avec des polypeptides antigeniques

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Publication number
EP0510018A1
EP0510018A1 EP19910901263 EP91901263A EP0510018A1 EP 0510018 A1 EP0510018 A1 EP 0510018A1 EP 19910901263 EP19910901263 EP 19910901263 EP 91901263 A EP91901263 A EP 91901263A EP 0510018 A1 EP0510018 A1 EP 0510018A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
plasmid
fusion
lipoprotein
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19910901263
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German (de)
English (en)
Inventor
Peter Lau
Clément Rioux
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National Research Council of Canada
Original Assignee
National Research Council of Canada
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Filing date
Publication date
Application filed by National Research Council of Canada filed Critical National Research Council of Canada
Publication of EP0510018A1 publication Critical patent/EP0510018A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • Lipoprotein signal peptide fused to antigenic polypeptides Lipoprotein signal peptide fused to antigenic polypeptides
  • the present invention relates to a fusion plasmid suitable for the expression of polypeptides of any composition or size modified in vivo by the addition of a lipid moiety.
  • synthetic peptides are commonly rendered immunogenic by being coupled to a high-molecular weight carrier protein, such as keyhole limpet hae ocyanin (KLH) , which has been shown to induce a helper T-cell response.
  • KLH keyhole limpet hae ocyanin
  • FCA complete (FCA) or the incomplete (FIA) Freund's adjuvants.
  • FCA is a water-in-oil emulsion containing killed mycobacteria, whereas FIA is devoid of bacteria.
  • the active ingredient in FIA that can substitute for the mycobacteria in FCA is muramyl dipeptide (MDP; N-acetyl- muramyl-L—alanyl-D- isoglutamine) .
  • MDP muramyl dipeptide
  • MDP muramyl dipeptide
  • MDP N-acetyl- muramyl-L—alanyl-D- isoglutamine
  • the chemical nature of the synthetic analog of the bacterial lipoprotein is N-palmitoyl-S-(2RS)-2,3- bis-(palmitoyloxy)propyl) )-cysteinyl-serine. Its abbreviation is Pam, Cys-Ser.
  • this analog as well as the native lipoprotein of the outer membrane of E. coli, were found to be polyclonal activators for antibody production by B-lymphocytes as shown by Bessler et al. in (1985) The J. of Immunol. 135,1900-1905. In another study, Hopp demonstrated in (1984) Mol. Immunol.
  • This fusion plasmid comprises a DNA sequence encoding a lipoprotein signal peptide, and at least the first amino acid of a mature lipoprotein. Also, it may optionally comprise a short amino acid segment with a /3-turn structure or an exogenous protease recognition sequence.
  • a DNA sequence encoding the desired polypeptide can then be inserted into the fusion plasmid downstream from the above- mentioned DNA sequence to produce a lipid modified polypeptide.
  • the fusion plasmid of the present invention is preferably derived from a prokaryotic expression vector with signals for the strong transcription of the fusion genes and effective translation.
  • the lipoprotein signal peptide contained in the fusion plasmid will preferably be a bacterial lipoprotein signal peptide.
  • the first amino acid of the mature lipoprotein will more preferably be cysteine.
  • the preferred cysteine codon there may preferably be a DNA sequence coding for a short amino acid sequence with a 3-turn structure or an exogenous protease recognition cleavage sequence.
  • This fusion plasmid therefore allows for synthesis of polypeptides as part of recombinant lipopeptides secreted across the cytoplasmic membrane and recovered from the cells.
  • the lipid moiety of the lipopeptides synthesized through the fusion plasmid of the present invention can also act as an intrinsic carrier/adjuvant for anti-peptide antibody production, making the lipopeptides useful in the development of vaccines.
  • the presence of a hydrophobic region in the lipopeptide derivatives may facilitate their passage across biological membranes.
  • the lipid portion can render a peptide more fat-soluble and can therefore be useful in enhancing the delivery of polypeptide drugs.
  • a recombinant fusion protein comprising a polypeptide and at least the first amino acid of a mature lipoprotein, said amino acid having attached thereto at least one fatty acid.
  • the first amino acid is a cysteine residue bearing fatty acids at its sulfhydryl and ⁇ -NH 2 groups.
  • the fusion protein will further comprise a short amino acid sequence comprising a /3-turn or the recognition sequence of an exogenous protease.
  • Figures la and lb represent a schematic representation of the construction of the fusion plasmid pKLY3 from the commercially available prokaryotic expression vector pKK233-2.
  • Figure lc represents the insertion of the coding sequence for the P2 peptide of the epidermal growth factor receptor (EGF-R) into plasmid pKLY3 to produce plasmid pKLY4.
  • EGF-R epidermal growth factor receptor
  • Figure 2 represents the nucleotide sequence of the Ncol-Hindlll DNA fragment inserted into plasmid pKLY2.
  • Figure 3 represents the nucleotide sequence of the Sphl-BamHI DNA fragment inserted into plasmid pKLY3.
  • the present invention relates to fusion plasmids for the expression and in vivo lipid modification of polypeptides.
  • the DNA sequences coding for the polypeptides are inserted in frame with a DNA region of the plasmid corresponding to the 5' end of a lipoprotein gene.
  • the lipoprotein amino terminus gene includes codons for the lipoprotein signal peptide, and at least the first amino acid of the mature lipoprotein, preferably a cysteine residue.
  • a cysteine codon there may be added codons for a short amino acid segment capable of forming a ⁇ - turn. This amino acid segment may also comprise a protease recognition cleavage site.
  • the fusion plasmid is under the control of transcription and translation elements allowing for inducible expression in transformed bacteria.
  • the nature of the expression vector, lipoprotein amino terminus, 0-turn peptide and polypeptide is as follows.
  • the fusion plasmids can be derived from prokaryotic expression vectors replicated at a moderate or high number of copies per cell. Suitable levels of expression of the fusion genes can be obtained through the use of a strong inducible promoter giving no or very low transcription under non-induced conditions in order to prevent inhibition of bacterial growth by toxic fusion products. The presence of strong terminators of transcription downstream of the fusion gene prevents readthrough transcription that could make the plasmid unstable.
  • the translationa ⁇ signals of the fusion gene consist of a ribosome binding site and an AUG initiation codon, possibly provided by the plasmid vector, which codes for the first amino acid of the lipoprotein signal peptide.
  • the presence in the fusion plasmid of the origin of replication of a single-stranded DNA phage allows, upon phage infection, the synthesis of high yields of single stranded DNA during replication.
  • the single stranded DNA can be used for site directed mutagenesis on the plasmid. Lipoprotein amino terminus
  • the lipoprotein signal peptide to be used in the context of the present invention is a bacterial lipoprotein signal peptide having between 16 and 29 amino acids in length such as that described in Klein et al. , 1988, Protein Eng., 2:15-20.
  • This signal peptide will contain a positively charged amino terminal region, a central hydrophobic region and a carboxyl-ter inal region which has a recognition sequence for signal peptidase II (SPasell).
  • SPasell recognition sequence which is sometimes referred to as a "lipoprotein box” consists of Leu-X-Y- Cys (the position of cys is numbered +1 and leu -3). In this sequence, the cysteine is an invariant residue, leu at the -3 position can be replaced by a chemically similar residue, and X and Y are small amino acids.
  • Cleavage of a lipoprotein signal peptide in vivo through the action of the appropriate cell protease requires prior lipid modification of the first amino acid of the mature lipoprotein.
  • this lipid modification mainly consists in palmitoylation at the sulfhydryl group of the cysteine residue at position +1.
  • the site of cleavage is on the amino terminal side of the modified cysteine of the mature protein (viz. Leu- X-Y-Cys).
  • mature lipoproteins contain a tetrapeptide such as Gln-Ala-Asn- Tyr (QANY) forming a ⁇ -turn structure which may favor the cleavage action of SPasell.
  • a tetrapeptide such as Gln-Ala-Asn- Tyr (QANY) forming a ⁇ -turn structure which may favor the cleavage action of SPasell.
  • These natural tetrapeptides which may be part of the plasmid of the present invention, can be replaced by Ile-Glu-Gly-Arg ( EGR) which is a specific recognition cleavage site for factor Xa protease as described by Nagar & Thogersen in 1984, Nature, 309:810-812. This protease cleaves on the ⁇ arboxy terminal side of the arginine residue.
  • EGR Ile-Glu-Gly-Arg
  • protease recognition sequences such as Asp-Asp-Asp-Asp-Lys for enterokinase and Pro-X Gly- Pro-Y for collagenase (the arrows indicate the cleavage sites) also fall within the scope of the present invention.
  • protease recognition sequences possessing the appropriate characteristics may be used in the context of the present invention.
  • any sequence coding for a desired polypeptide, once fused to the gene portion coding for the desired lipoprotein amino terminus and preferably to those amino acids constituting a / S-turn is expected to produce, after expression in a plasmid and in vivo processing, a polypeptide with an N-terminal lipid modified cysteine.
  • the DNA sequence coding for the desired polypeptide will be either synthetic oligonucleotides or an appropriate restriction endonuclease fragment.
  • the nature of the polypeptide that can be expressed in vivo is basically independent of its amino acid composition or size contrary to experience in chemical synthesis of peptides.
  • the lipopeptides thus synthesized can be used for many applications. Hence, they can serve to raise specific anti-peptide antibodies useful to identify particular clones in a recombinant DNA expression library. Antibodies recognizing peptide fragments in a native protein will help to study the structure and function of the protein and to develop synthetic peptide vaccines. Also, because the lipid portion of these derivatives could facilitate the passage across biological membranes, there is potential for the creation of efficient delivery systems for certain peptide drugs.
  • fusion genes are constructed are well known to those skilled in the art.
  • a DNA sequence coding for the desired lipoprotein amino terminus will be generally fused to the 5'-phosphate end of oligonucleotides or restriction endonuclease fragments coding for the desired polypeptide sequence.
  • the following description of a preferred embodiment of the present invention can be used as a basis for the construction of other fusion genes and is not to be interpreted as limiting the scope of the present invention. Description of a preferred embodiment
  • the gene coding for both the lipoprotein signal sequence of the ColE2- lasmid coded lysis protein also known as bacteriocin release protein and the N-terminal 5 amino acid residues of the mature lysis protein, is fused to the synthetic oligonucleotides coding for the P2 peptide corresponding to the C-terminal phosphorylation site of EGF-R described by Hayden et, al. in (1986) Proc. West. Pharmacol. Sec. 29. 459-461 using the following procedure.
  • the fusion vector was constructed using plasmid pKK233-2 shown in Figure la.
  • This plasmid derivative of pBR322 is expressed in a moderate number of copies per cell. It provides a strong regulated trp-lac fusion promoter (pTac) inducible by IPTG, an analog of lactose. Downstream of this promoter are a ribosome-binding site followed by an ATG translation initiation codon, a multiple cloning site and the rrn B transcription terminators.
  • the origin of replication of phage Fl (obtained as a 514 bp Rsal fragment from plasmid pEMBL8) was inserted into the PvuII site of pKK233-2 to obtain pKLYl shown in Figure la.
  • Plasmid pKLY2 was digested at the unique restriction sites Ncol and Hindlll for insertion of the synthetic oligonucleotide of Figure 2 with the N- terminal portion of the colicin E, lysis gene. Digestion at Ncol exposes the ATG start codon. After gene assembly, the ATG is fused in frame with the signal sequence of the lysis gene followed by five codons for the amino acids Cys-Gln-Ala-Asn-Tyr (CQANY) of the mature protein, a GTA codon for valine creating a unique Sna BI restriction site, a TAA stop codon and a BamHI restriction site close to the Hindlll ligation site.
  • CQANY Cys-Gln-Ala-Asn-Tyr
  • Tetrapeptide QANY can contribute to the formation of a ⁇ -turn.
  • the cysteine codon TGT of the colicin E 2 lysis gene was replaced by TGC. This silent change introduced a unique Sphl restriction site in the recombinant plasmid pKLY3 shown in Figure lb.
  • Restriction digests of pKLY3 at the restriction sites Sphl and BamHI allow for directional insertion of annealed oligonucleotides or restriction fragments with sequences coding for the desired polypeptide followed by a termination codon (Method I).
  • the fusion can be created by loop-in mutagenesis on pKLY3 single stranded DNA using oligonucleotides with complementary sequences for annealing (Method II). In both these methods, the nature of the coding sequences between the cysteine codon and the coding sequences of the polypeptides can be varied.
  • the unique restriction site SnaBI in pKLY3 allows for blunt-end ligation of restriction fragments or annealed oligonucleotides with the desired polypeptide coding capacity immediately downstream of the tyrosine residue of the mature colicin E 2 lysis protein (Method III).
  • the fusion of the N-terminal portion of the colicin E 2 lysis protein to the peptide P2 which consists of 12 amino acids from the C-terminal phosphorylation site of the epidermal growth factor receptor (EGF-R amino acids 1137-1148) was made according to method I.
  • the synthetic oligonucleotides contained the sequence for the tetrapeptide QANY and the peptide P2 followed by the TAA stop codon and the diagnostic restriction site Xbal as seen in Figure 3.
  • the cells were incubated at 37°C with agitation (250 rpm).
  • the processing of the fusion proteins into mature lipopeptides was determined by jj vivo labelling experiments with tritiated palmitate ( [3H]-palmitate) .
  • Total cell proteins were then precipitated with 10% (w/v) trichloroacetic acid (TCA) for 30 min. at 4° C.
  • TCA trichloroacetic acid
  • the protein pellets obtained by centrifugation at 18,000 g for 15 minutes were washed twice in methanol to remove lipids.
  • the dried pellets were resuspended in sample buffer and analyzed by electrophoresis on discontinuous Tricine-Sodiu Dodecyl Sulfate (SDS) polyacrylamide gels for the separation of low molecular weight proteins following the method described by Schagger and Von Jagow in (1987) Anal. Bioche 166. 368-379.
  • the labelled protein bands were detected by fluorography.
  • transf- ormants with the fusion plasmid pKLY3 produced, upon induction, a palmitate-containing compound of less than 2,300 daltons. These two compounds were not found in cells containing the vector pKLY2 independently of induction.
  • the inducible products of pKLY3 and pKLY4 would be translocated across the cytoplasmic membrane as indicated by the loss of mature products in cells treated with globomycin (an inhibitor of signal peptidase II) for 5 min before adding [ 3 H]-palmitate in in vivo labelling experiments.
  • the results showed the presence of a precursor form of the induced product from PKLY3.
  • alkaline phosphatase provides a simple way to monitor the level of expression and secretion of fusion products since it is active in cells only when present in the periplasm.
  • DH ⁇ F'IQ transformants containing plasmid pKLY5 produced an enzymatically active pal itate-containing derivative of CQANY-alkaline phosphatase. This product had an apparent molecular weight of approximately 50,000 daltons on a discontinuous Tricine-SDS polyacrylamide gel and was absent from transformants treated with globomycin.
  • the fractionation of IPTG induced transformants containing PKLY4 or PKLY5 into periplasmic and intracellular, outer membrane, and cytoplasmic membrane components was performed by standard procedures.
  • the radiolabelled cells were harvested and suspended in 10 mM sodium phosphate (pH 7.0). The cells were disrupted by two passages through a French pressure cell at 20,000 lb/in .
  • the cell lysate was centrifuged at 4,500 X g for 10 min at 4°C, and the cell envelope fraction was obtained by centrifugation at 100,000 X g for 40 min at
  • the cytoplasmic membrane was solubilized differentially with the detergent sodium lauryl sarcosinate.
  • the outer membrane was sedimented by centrifugation at 100,000 X g for 40 min at 4°C and solubilized in water.
  • Analysis of the TCA precipitated and methanol washed components of the various fractions by electrophoresi ⁇ on discontinuous Tricine-SDS polyacrylamide gels showed that the lipid derivatives of CQANY-P2 and of CQANY-alkaline phosphatase were mostly associated with the outer membrane.
  • the lipid modified polypeptides synthesized in E. coli were secreted across the cytoplasmic membrane.
  • the change in the nature of the ⁇ -turn forming tetrapeptide had no effect on the in vivo production of a stable lipid modified derivative of peptide P2.
  • Cleavage of the IEGR-containing lipopeptide by factor Xa protease should allow the release of the free P2 peptide or any other peptide of interest.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Plasmide de fusion destiné à la synthèse in vivo d'un polypeptide à modification lipidique. Le plasmide comporte une première séquence ADN codant un peptide à signal lipoprotéique, de préférence un peptide bactérien à signal lipoprotéique, ainsi qu'au moins le premier aminoacide d'une lipoprotéine mûre, de préférence la cystéine. Dans la séquence ADN, le codon cystéinique préféré peut être suivi de codons pour quelques aminoacides qui forment une structure à tour-beta. Ces aminoacides peuvent également comprendre un site de clivage à reconnaissance de protéase exogène et spécifique. Une deuxième séquence ADN codant le polypeptide souhaité peut s'introduire dans le plasmide de fusion afin de produire le polypeptide à modification lipidique souhaité.
EP19910901263 1989-12-26 1990-12-27 Peptide a signal lipoproteique fusionne avec des polypeptides antigeniques Withdrawn EP0510018A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US45647389A 1989-12-26 1989-12-26
US456473 1989-12-26

Publications (1)

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EP0510018A1 true EP0510018A1 (fr) 1992-10-28

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EP19910901263 Withdrawn EP0510018A1 (fr) 1989-12-26 1990-12-27 Peptide a signal lipoproteique fusionne avec des polypeptides antigeniques

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EP (1) EP0510018A1 (fr)
AU (1) AU7034691A (fr)
CA (1) CA2032914A1 (fr)
WO (1) WO1991009952A1 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5777095A (en) * 1988-10-24 1998-07-07 Symbicom Aktiebolag Osp A and B Sequence of Borrelia burgdonferi strains ACA1 and IP90
US6143872A (en) * 1988-10-24 2000-11-07 Symbicom Aktiebolag Borrelia burdorferi Osp A and B proteins and immunogenic peptides
AU681572B2 (en) * 1991-10-21 1997-09-04 Med Immune, Inc. Bacterial expression vectors containing DNA encoding secretion signals of lipoproteins
FR2708622B1 (fr) * 1993-08-02 1997-04-18 Raymond Hamers Vecteur recombinant contenant une séquence d'un gène de lipoprotéine de structure pour l'expression de séquences de nucléotides.
US6335018B1 (en) 1995-05-01 2002-01-01 Aventis Pasteur Limited High molecular weight major outer membrane protein of moraxella
US6440425B1 (en) * 1995-05-01 2002-08-27 Aventis Pasteur Limited High molecular weight major outer membrane protein of moraxella
ZA964896B (en) 1995-06-07 1997-01-08 Connaught Lab Expression of lipoproteins
EP1741718A3 (fr) * 1995-06-07 2007-03-28 Sanofi Pasteur Inc. Expression de lipoproteins
US6251405B1 (en) * 1995-06-07 2001-06-26 Connaught Laboratories, Inc. Immunological combination compositions and methods
CN108135181A (zh) * 2015-09-04 2018-06-08 斯克利普斯研究院 定新型抗生素及相关组合物的方法
US11446398B2 (en) 2016-04-11 2022-09-20 Obsidian Therapeutics, Inc. Regulated biocircuit systems
WO2020086742A1 (fr) 2018-10-24 2020-04-30 Obsidian Therapeutics, Inc. Régulation de protéine accordable par er
EP3935159A1 (fr) 2019-03-08 2022-01-12 Obsidian Therapeutics, Inc. Compositions d'anhydrase carbonique humaine 2 et procédés de régulation accordable
US20220267398A1 (en) 2019-06-12 2022-08-25 Obsidian Therapeutics, Inc. Ca2 compositions and methods for tunable regulation
US20220259284A1 (en) 2019-06-12 2022-08-18 Obsidian Therapeutics, Inc. Ca2 compositions and methods for tunable regulation
US20220348937A1 (en) 2019-09-06 2022-11-03 Obsidian Therapeutics, Inc. Compositions and methods for dhfr tunable protein regulation

Non-Patent Citations (1)

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Title
See references of WO9109952A1 *

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WO1991009952A1 (fr) 1991-07-11
CA2032914A1 (fr) 1991-06-27
AU7034691A (en) 1991-07-24

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