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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
  • C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/708—Specific hybridization probes for papilloma
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
  • G01N33/56988—HIV or HTLV
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57411—Specifically defined cancers of cervix
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/01—DNA viruses
  • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

• the invention relates to a papillomavirus DNA (HPV63) or variants of this papillomavirus, and more particularly to probes derived from this papillomavirus. It also relates to products genetically and im unologically linked to this papillomavirus, as well as to methods using these various products for the in vitro diagnosis of papillomavirus infections and, for some of them, for vaccination against these same papillomaviruses. or papillomavirus variants.
• product genetically or immunologically linked to a papillomavirus means the various products derived from its initial DNA, whether they are corresponding RNAs or recombinant DNAs containing all or part of this initial DNA, expression products of these DNAs, where appropriate recombinant, in competent cell hosts and antibodies directed against these products. They are therefore polypeptides resulting from the transcription and translation of all or part of the different open reading phases of the initial DNA. They are also antibodies induced in vivo by said polypeptides.
• papillomavirus covers a large number of viruses having in common to be held responsible for several forms of viral infections spreading between skin warts or relatively benign mucous membranes and hyperplasias liable to degenerate into intra- neoplasias. epithelial and in skin or mucosal cancers.
• papillomavirus infections mention will also be made more particularly of skin warts (in particular, common and plantar warts), wart-shaped epidermodysplasia, flat or intermediate skin or mucous warts, intraepithelial neoplasia and skin cancer, epidermodysplasia wart cancer, intraepithelial neoplasia and cancer of the cervix and genitals external, condylomas and papiHomes.
• skin warts in particular, common and plantar warts
• wart-shaped epidermodysplasia flat or intermediate skin or mucous warts
• intraepithelial neoplasia and skin cancer epidermodysplasia wart cancer
• intraepithelial neoplasia and cancer of the cervix and genitals external condylomas and papiHomes.
• papillomaviruses although very different from each other, have sizes of the order of 7000-8000 base pairs.
• their genomes may have certain degrees of homology, evaluated in hybridization tests carried out under conditions known as “non-stringent” or “not strict” or, on the contrary, under conditions of “stringent hybridization” or " strict ".
• Hybridization tests under non-strict or non-stringent conditions involve the mutual contacting of DNAs originating from two virus isolates under the following conditions described by HEILMAN CA. et al, 1980, J. Virol., 3, 395-407, and CROISSANT et al, 1982, CR Acad. Se. Paris, 294, 581-586 (heteroduplex molecules). It will be recalled that these non-strict or non-stringent conditions imply the performance of hybridization tests under the following conditions, in particular of medium and temperature: Non-strict conditions (Tm-40 , C);
• Hybridization tests under strict or stringent conditions involve the mutual contacting of DNAs originating from two virus isolates under the conditions described by KREMSDORF, D. ET AL. ((1982), J. Virol. 43_: 436-447 and 1983, J. Virol. 4_8: 340-351) and Davis R.. et al., 1971, Methods Enzy ol., 21, 413-418 (heteroduplex molecules).
• papillomaviruses which exhibit percentages of cross-hybridization of less than 50% under strict or stringent conditions belong to different types. Viruses for which we observe, under these strict or stringent conditions, percentages (or degrees) of hybridization greater than 50% are considered to belong to the same type. They can form different subtypes within this same type.
• two HPVs belong to different types if their genomes have less than 50% cross hybridization under strict hybridization conditions, that is to say after hybridization in saturated liquid medium and treatment of SI nuclease hybrids.
• Two HPVs represent subtypes of the same type if their genomes have incomplete homology, but greater than 50%. Differences in only a small number of cleavage sites by restriction enzymes define variants.
• DNA-HPVs DNAs derived from these papillomaviruses, in particular genetic recombinants comprising all or part of the genomes (called DNA-HPVs) of these papillomaviruses, as hybridization probes.
• DNA-HPVs genetic recombinants comprising all or part of the genomes
• hybridization probes The use of mixtures or "cocktails" of DNA-HPVs, for the preparation of mixtures of hybridization probes, has also been described.
• These mixtures of probes are then capable of being used, sometimes more effectively than isolated probes, for the in vitro diagnosis of various categories of infections, or even of the risk levels which accompany the discovery in a patient of certain papillomaviruses.
• These categories of infections include those which are likely to be accompanied by genital neoplasia and, in particular, uterine cancer: see for example the cocktails indicated below (if we disregard DNA-HPV which is the object of the present invention).
• the invention relates to a newly isolated papillomavirus, the genomic DNA which can be obtained from this new papillomavirus, fragments of this genomic DNA, as well as the new hybridization probes which can be formed from this DNA-HPV. or these DNA-HPV fragments.
• HPV63 The DNA of this new HPV (hereinafter called HPV63), which has been isolated several times, from lesions having the clinical characteristics of genital intraepithelial neoplasias of the cervix, is characterized by the map of restriction shown in Figure 3 attached.
• the physical map gives the position of sites of cleavages by various restriction endonucleases.
• the origin of each card is constituted by a single cut-off site, in this case a BamHI site.
• the distances of the other sites from the origin are expressed as a percentage of genome length.
• the invention also relates to all variants of these papillomaviruses, belonging respectively to the same subtypes, therefore capable of hybridizing with 1 ⁇ DN-HPV63 under stringent conditions.
• the invention also relates to "cocktails" of probes already described in the prior patents, but however supplemented with a probe derived from the papillomavirus according to the present application and, consequently, even more advanced diagnostic kits or “kits”.
• Hybridization probes constructed from the genome of HPV63 are particularly useful, for diagnosis and / or screening in vitro, under the conditions which have been described in the prior patents and which will be recalled below, of the types of papillomavirus which constitute a risk, in particular of genital neoplasias and cancers of the cervix.
• the invention also relates to fragments of each of 1 ⁇ DN-HPV63 or to DNA fragments, in particular of equivalent length, capable of hybridizing with the preceding one, in particular under strict conditions.
• it relates to the recombinant DNAs containing all or part of the DNA-HPV mentioned above, and more particularly to the recombinant DNAs containing, respectively, fragments corresponding to the El, E2, E4, E6-E7, L1, L2 genes and to the intergenic region, NC, or alternatively fragments containing sequences corresponding to the intergenic regions of each of said DNA-HPVs.
• probes which can be formed from these DNA-HPV or to starting from any fragment contained in this DNA-HPV, since it is capable of hybridizing with the previous one under strict conditions and under non-strict conditions with the DNA-HPV corresponding to the types previously described HPV16, HPV18 and HPV33 and in vitro diagnostic methods involving said probes or the mixtures containing them.
• HPV63 Molecular cloning and characterization of a new type of HPV associated with genital neoplasias
• HPV16 The new type of HPV has been demonstrated in DNA extracted from mild dysplasia (grade I intraepithelial neoplasia), by hybridization under non-strict conditions (Tm-40%), using a mixture of specific probes for HPV16, 18 and 33.
• Tm-40% non-strict conditions
• the study of the sensitivity of this DNA to various restriction endonucleases has shown that the BamHI enzyme once cuts the viral sequences, generating molecules of about 8
• the recombinant plasmids were detected by hybridization of replicas of the infected bacterial cultures, with a mixture of the probes corresponding to types 16, 18 and 33, under non-strict conditions. Several recombinant plasmids, containing all of the viral sequences, have been isolated. The cleavage of the recombinant plamides by the BamHI insertion enzyme generates an 8 Kb fragment which hybridizes with probes 16, 18 and 33 under non-strict conditions. Cleavage of recombinant plasmids and DNA of the original lesion by mixing enzymes
• the invention therefore also relates to any recombinant DNA containing the above-mentioned DNA-HPV or fragments of this DNA-HPV, in particular hybridization probes formed from these recombinant DNAs and specially adapted for the detection of '' an infection by the papillomavirus according to the invention or of a variant or subtype of this papillomavirus.
• hybridization probes formed from these recombinant DNAs and specially adapted for the detection of '' an infection by the papillomavirus according to the invention or of a variant or subtype of this papillomavirus.
• These probes can either be labeled themselves or be modified at the level of certain nucleotides, in particular with a view to their coupling, direct or indirect, with a separate marker.
• nucleic acids possibly contained in the test sample for its possible DNA content of the corresponding papillomavirus or 1 • one of its variants.
• the method according to the invention for the in vitro diagnosis on a biological sample to be tested is therefore characterized by bringing a probe as defined above into contact with the nucleic acids of this sample, if necessary previously made accessible to the probe, preferably under stringent hybridization conditions, and by detecting the hybrid formed between the desired viral DNA, possibly present in the sample and said probe.
• HPV63 radioactive probes prepared from the DNA of the purified HPV63 made it possible to determine the pathogenic power of this virus.
• HPV63 is therefore a type of genital tropism HPV with oncogenic potential. It is very desirable to incorporate it into everything mixture of HPV DNA intended for the preparation of molecular probes, for the diagnosis or screening of the types of HPV constituting a risk for the development of genital neoplasias and, in particular, cervical cancers.
• the probe is therefore associated with probes derived from other papillomaviruses, in particular from those designated below:
• HPV16 18, 33, 39, 54 or alternatively still, with HPV6, 11, 42, 54, for the in vitro diagnosis of genital neoplasms and cancers of the cervix, condylomas and papillomas,
• each of the probes according to the invention or the mixtures containing the above-mentioned probe can in particular be used as follows, it being understood of course that the diagnostic tests described cannot be considered as limiting the conditions of use of the probes or mixtures of probes according to the invention.
• this involves, for example, identifying an HPV in a biopsy, in cells obtained by scraping lesions, or in biopsy sections fixed by the Carnoy mixture (ethanol, chloroform, acetic acid 6 : 3: 1) and included in the paraffin.
• the examination requires the prior extraction of DNA from the samples according to methods of which the principle is known and involves the analysis of this DNA by molecular hybridization experiments, carried out under strict or less strict conditions, to using radioactive probes (labeled with 32 p or 35 S) prepared from the HPV according to the invention or mixtures of DNAs or HPVs containing it.
• radioactive probes labeled with 32 p or 35 S
• This method comprises, after denaturation of the DNA, the deposit of an aliquot of DNA on membranes (nitrocellulose or "Genescreenplus”), the hybridization of each membrane, under the usual conditions, with a mixture of probes and the detection of radioactive hybrids, by exposure of the membranes to contact with an x-ray film.
• a replica hybridization method can also be used. This method comprises the electrophoretic separation in agarose gel of the DNA fragments generated after treatment of the DNA by restriction enzymes, the transfer of the fragments, after alkaline denaturation, on membranes (nitrocellulose, "Genescreenplus”) and their hybridization, under usual conditions, with the appropriate mixture of probes. The formation of radioactive hybrids is detected after exposure of the membranes in contact with an X-ray film.
• the radioactive probes consist either of DNAs from HPVs labeled by the nick-translation method, or by RNAs prepared by transcription of viral DNAs inserted into a vector, for example of the SP6 type.
• the use of radioactive probes has the advantage of great sensitivity, but this does not exclude the use of non-radioactive probes, for example biotinylated probes which are capable of being recognized by antibodies or labeled themselves, either themselves recognized by antibodies carrying an enzymatic, fluorescent marker, etc.
• the invention also relates to competent cell cultures transformed with recombinant DNAs of the above-mentioned type, in particular those in which the nucleotide sequence corresponding to the papillomavirus DNA is placed under the control of elements for transcription and regulation of this nucleotide sequence in said cell culture.
• the invention relates to the polypeptides resulting in particular from the respective expressions of the El, E2, E4, E6, E7, Ll, L2 genes of 1 ⁇ DN-HPV63.
• the process according to the invention for the production of these polypeptides therefore comprises the transformation of competent cell cultures with the recombinant DNAs containing the corresponding nucleotide sequences originating from HPV63, so that the nucleotide sequence corresponding to one of said proteins can be expressed in this cell host, the recovery of these polypeptides from the products synthesized by the competent cell host and the purification (for example by contacting the expression products previously extracted from the cell cultures or from the medium in which these have been developed, with antibodies previously formed against such polypeptides).
• the expression products of the L2 sequences of each of the papillomavirus genomes according to the invention are however of very particular interest, in that they can themselves be used for the in vivo production of antibodies capable of recognizing the products of expression of the L2 gene in biological samples affected by a papillomavirus of the HPV63 type or of a variant thereof, and this more particularly when the preparations of the genus in question have been previously fixed.
• the invention also relates to hybrid polypeptides containing the above-mentioned polypeptides and respectively derived from HPV63, for example the L2 protein fused to other polypeptide sequences, insofar as these do not essentially modify the immunogenic properties of the protein. L2.
• these other polypeptide fragments can in particular result from the mode of production used for these hybrid polypeptides, for example by genetic engineering.
• these hybrid polypeptides contain a sequence derived from ⁇ -galactosidase.
• Such products can in particular be obtained by transformation of E. coli with appropriate vectors (phages or plasmids) modified by all or part of the lactose operon and comprising, in addition, inserted downstream of the promoter of the lactose operon (or any other suitable promoter, for example phage ⁇ ), the nucleotide sequence derived from an L2 gene derived from the papillomavirus concerned according to the invention.
• plasmids or phages of this type comprising at least part of the gene for the ⁇ -galactosidase of the lacton operon.
• the polypeptides according to the invention can also, when they have been purified, be used in techniques for purifying the antibodies which correspond to them, in particular from sera from animals which had been immunized with these polypeptides.
• these polypeptides can be fixed on affinity columns.
• the antibody purification operations then consist in passing the serum containing them in contact with affinity columns carrying the above-mentioned polypeptides.
• the antibodies selectively attached to these columns can then be recovered by dissociation of the antigen-antibody complexes, using an appropriate buffer, having an adequate ionic strength, for example an aqueous solution of a salt such than ammonium acetate. Acidified solutions can also be used.
• the invention also relates to a method for producing antibodies against said polypeptides, in particular against the expression products of the E6, E7 or, preferably L2 genes of HPV63, this method comprising the immunization of a suitable living host with said polypeptides and the recovery of the antibodies formed from a serum of the immunized host, in particular by bringing these sera into contact with the polypeptides corresponding to the purified state and by the recovery of these antibodies from the antigen- antibodies formed.
• the invention relates to the compositions involving the antibodies according to the invention (or groups containing these antibodies in association with antibodies from other papillomaviruses), in particular the compositions containing the antibodies derived from the compositions or "cocktails" of DNA-HPVs listed above and to which it is again referred to in the following (or groups of corresponding antibodies).
• the invention relates to the above-mentioned antibodies or mixtures of antibodies, previously purified, in association with an appropriate pharmaceutical vehicle.
• This composition is then capable of being used for the treatment of the given condition, as soon as it has been diagnosed clinically, after an in vitro diagnostic test on a histological or cytological sample taken of the patient.
• This composition (in particular in the form of a serum) can be administered, preferably parenterally. This serum is then capable of causing a regression of the infections induced by papillomaviruses of the HPV63 type.
• These antibodies can more particularly be used in tests for the diagnosis of an infection with respect to one of the papillomaviruses according to the invention or of variants of these papillomaviruses, insofar as histological sections coming from infected persons may also contain expression products of certain of the structural genes, in particular L2, of papillomaviruses of the same type.
• the invention therefore also relates to a method for in vitro diagnosis, in particular of general neoplasias and cancers of the cervix, comprising bringing into contact histopathological sections originating from lesions induced in the persons concerned under conditions allowing production of an antigen-antibody complex and the detection of this antigen-antibody complex.
• the detection is done on preparations fixed beforehand under dissociating conditions, for example with the medium or mixture of CARNOY already mentioned above (also described in the work of L. LISON, entitled "Histochemistry and cytochemistry of animals”) .
• the anti-L2 antibodies which may be attached can be recognized by other antibodies formed against the former, these other antibodies carrying appropriate markers, preferably non-radioactive. These markers are for example of an enzymatic or fluorescent nature.
• the antibodies thus selected can therefore be used to diagnose in vitro the types of corresponding conditions.
• the invention finally relates to the corresponding vaccine compositions containing one or preferably several other L2 proteins, in combination with a pharmaceutically acceptable vehicle suitable for the chosen mode of administration, in particular by parenteral.
• a pharmaceutically acceptable vehicle suitable for the chosen mode of administration in particular by parenteral.
• the strain containing 1 ⁇ DN-HPV63 was deposited at the CNCM on December 21, 1989 under the deposit number 1-918.
• the strain deposited consists of a culture of E. coli K12 strain NM522 harboring a recombinant plasmid BamHI itself containing the DNA of HPV63.

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