WO1989008706A1 - Isoformes de la trophoblastine, nouveaux interferons constitues par lesdites isoformes, leurs procedes d'obtention et leurs applications - Google Patents

Isoformes de la trophoblastine, nouveaux interferons constitues par lesdites isoformes, leurs procedes d'obtention et leurs applications Download PDF

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WO1989008706A1
WO1989008706A1 PCT/FR1989/000116 FR8900116W WO8908706A1 WO 1989008706 A1 WO1989008706 A1 WO 1989008706A1 FR 8900116 W FR8900116 W FR 8900116W WO 8908706 A1 WO8908706 A1 WO 8908706A1
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PCT/FR1989/000116
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English (en)
French (fr)
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Jacques Martal
Gilles Charpigny
Pierre Gaye
Jean-Claude Pernollet
Madia Charlier
Michel Guillomot
Jean-Claude Huet
Pierrette Reinaud
Dominique Hue
Nicole Chene
Claude La Bonnardiere
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Institut National De La Recherche Agronomique (Inr
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Publication of WO1989008706A1 publication Critical patent/WO1989008706A1/fr

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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to trophoblastin isoforms corresponding to Class II interferons endowed, inter alia, with anti-viral properties.
  • WINTENBERGERTORRES described the identification of an anti-antioluteolytic component secreted by the embryo in the sheep, called trophoblast; he also established the protein nature of the trophoblast, which is thermolabile and sensitive to proteases; he further established that the period during which trophoblastin is secreted is very limited since its presence seems critical from day 12 of gestation only and is no longer detectable after days 21-23.
  • the main polypeptides produced in the culture medium of conceptus of day 16 were identified by two-dimensional electrophoresis as having molecular weights of 22 to 26 kDa (by electrophoretic mobility in the presence of SDS) and a migration in a range of isoelectric points of 6.8 to 5.6; minor polypeptides of PM 40-50 and isoelectric points 6.2 to 7.4 have also been identified in the medium.
  • This Publication also reports the hypothesis put forward that the study, after purification, of the proteins produced by the conceptus of bovines, should make it possible to elucidate their respective roles in the process of maintaining CL and to demonstrate that similar proteins produced by the sheep conceptus could be active in cattle.
  • lapinant ⁇ -oTP-1 serum was used for the immunological characterization of proteins secreted by the bovine conceptus.
  • the double immunodiffusion analysis by the Ouchterlony method with anti-oTP-1 serum, produced a double band against the unfractionated proteins secreted by bovine conceptus.
  • One of these two bands has a partial identity both with purified oTP-1 and with the unfractionated proteins secreted by the ovine conceptus.
  • Other experiments carried out in the presence of a radioactive amino acid, confirm the partial homology between the proteins produced by the ovine and bovine concepts and confirm the work of the MARTAL et al. Team. on transfers of inter-specific trophoblastic vesicles in sheep and cows, reported in J.
  • oTP-1 comprises both cysteines in position 1, 29, 99 and 139 and a sequence Cys-Ala-Trp-Gly-Ile-Val-Val-Arg 139 146 that the 'found in most interferons - ⁇ .
  • This Abstract also indicates that the Authors have demonstrated that oTP-1 has the antiviral and antiproliferative properties of an interferon, which leads them to suggest that oTP-1 not only corresponds to a signal of maternal recognition of gestation ( "maternai recognition of pregnancy") in sheep, but could also play the role of local immunomodulator.
  • oTP-1 consists of 3 - 4 isoelectric variants of isoelectric points 5.5 - 5.8, with a molecular mass of 18 kDa and oTP-1 obtained by in vitro translation (pre -oTP-1) from polyadenylated RNA, consists of several polypeptides each of which has a molecular mass of the order of 21 kDa.
  • oTPB or oT-1 or trophoblastin
  • the sequence of 45 amino acids represented in this publication differs from the corresponding sequence represented in the publication of NATURE only by the fact that it comprises a Gln residue in position 5, an Arg residue in position 6, while the sequence of corresponding amino acid published in NATURE comprises in these two successive positions respectively an Arg residue and a Lys residue.
  • the embryonic signal that constitutes trophoblastin is secreted at the time of differentiation of the embryo; its secretion begins on D 9, when the embryo is only an undifferentiated mass of cells and it disappears on the 22nd day of gestation, when the embryo is already formed.
  • the deepening of knowledge relating to the protein designated under the name of trophoblastine would make it possible to lower the threshold of detection of the embryonic signal in question, which is of particular importance in the techniques of transfer and transplantation of embryos, in which it is essential to detect as early as possible if the transferred embryo is alive, and in which it would be even more favorable to be able to detect the viability of the embryos in synthetic media before transplantation.
  • the subject of the present invention is isoforms of trophoblastin or of its embryonic analogs, characterized by a sequence of N-terminal amino acids which corresponds to a common general formula I: Cys-Tyr-Leu-Ser- (X) -Arg-Leu-Met-Leu-Asp-Ala- (U) -Glu-Asn-Leu
  • T 1 isoform is characterized by an N-terminal amino acid sequence which corresponds to formula I 1 below:
  • the T 2 isoform is characterized by an N-terminal amino acid sequence which corresponds to the form I 2 below:
  • the T3 isoform is characterized by an N-terminal amino acid sequence which corresponds to formula I 3 below:
  • the T4 isoform is characterized by an N-terminal amino acid sequence which corresponds to formula 14 below: Cys-Tyr- Leu-Ser-Gln-Arg-Leu-Met-Leu-Asp-Ala-Arg-Glu-Asn-
  • T 5 is characterized by an N-terminal amino acid sequence which corresponds to formula I 5 below: Cys-Tyr-Leu-Ser-Gln-Arg-Leu-Met-Leu-Asp-Ala-Lys- Glu-Asn-Leu
  • the present invention also relates to a process for the isolation of trophoblastin isoforms by high performance liquid phase chromatography (HPLC), characterized in that a culture medium obtained after a concept incubation is subjected of mammals for an appropriate time, to an anion exchange column chromatography balanced by a cationic buffer, then to the action of a salt gradient in a cationic buffer, in the space of an appropriate time interval, followed of an isocratic elution during which the isoforms are successfully collected.
  • HPLC high performance liquid phase chromatography
  • the conceptus collection medium intended to be cultured is constituted by an appropriate buffer devoid of serum albumin and containing polyvinylpyrrolidone at a concentration of approximately 0.05 % to 0.5% weight / volume.
  • the concepts are cultured in a synthetic culture medium devoid of serum albumin and blood serum and containing the polyvinylpyrrolidone at a concentration of about 0.05% to
  • the polyvinylpyrrolidone is preferably chosen from polyvinylpyrrolidones of high molecular weight, in order to promote its elimination by exclusion on a chromatography column or by insolubilization at acid pH.
  • these isoforms each of which has a molecular weight of the order of 20 kDa, have an isoelectric point of approximately 5.4 for the Ti form, of approximately 5.3 for the T 2 form, d '' about 5.2 for the shape
  • the present invention further relates to an agent having antiviral, antiproliferative, antitumor, immunological, immunomodulatory, inhibition of rejection, cell differentiation and inhibition of luteolysis activities at the start of gestation in mammals, as well that of genetic selection, characterized in that it consists of a class II interferon a, which is one of the isoforms of trophoblastin as identified by their N-terminal sequences of formulas I 1 , I 2 , I 3 , I 4 and Is or their mixtures.
  • the present invention further relates to the nucleotide sequences of cDNA of each of the isoforms of trophoblastin, as well as the amino acid sequences. which are deduced from it.
  • the nucleotide sequence of cDNA of the isoform T 2 consists of a polypeptide whose molecular weight is of the order of 23 kDa, corresponding to the precursor of trophoblastin, and which corresponds to formula II below:
  • the present invention further relates to an oligodeoxynucleotide probe for the characterization of clones containing the cDNA of trophoblastin mRNA and its isoforms, which is deduced from the nucleotide sequence of the cDNA of one of the isoforms trophoblastin.
  • such a probe it is characterized in that it corresponds to residues 34 to 43 of the N-terminal sequence of trophoblastin and corresponds to formula III below: 34 35 40 43
  • TTT I which can be replaced by A, G or C.
  • the invention naturally includes any equivalent probe deduced from the sequence of formula II or from the sequences of formulas I 1 , I 2 , I 3 , I 4 , Is suitable for characterize clones containing the trophoblastin mRNA cDNA or the gene thereof.
  • the subject of the present invention is, moreover, a process for obtaining by cloning the cDNA of the mRNA of the isoforms of trophoblastin, characterized in that it comprises the following successive steps:
  • the search for clones containing the cDNA of the trophoblastin mRNA is carried out using monospecific anti-trophoblastin immune serum.
  • the search for said clones is carried out using an oligodeoxynucleotide probe deduced from the nucleotide sequence of the cDNA of one of the isoforms of trophoblastin, and in particular of the oligonucleotide probe of formula III above, by hybridization of said clones with said probe, followed by isolation of inserts of appropriate size and subcloning into an appropriate plasmid (in particular pUC18) to determine that one of the clones s' hybrid with a single 1.1 kb RNA coding for a polypeptide of approximately 23 kDa corresponding to the precursor of trophoblastin and that the other of the clones hybridizes with the mRNA corresponding to the cDNA of one of the isoforms trophoblastin.
  • an oligodeoxynucleotide probe deduced from the nucleotide sequence of the cDNA of one of the isoforms of trophoblastin, and in particular of the
  • the present invention also relates to a kit for detecting an embryonic signal to control the viability of an embryo at an early stage of its development, characterized in that it comprises, as detection agent, at least one trophoblastin isoform of formula I above.
  • the present invention further encompasses the application of the trophoblastin isoforms according to the present invention as agents for protecting embryos during their transfer into the uterus of a recipient mammal, and as agents therapeutic, in particular as antiviral, antiproliferative, antitumour, immunomodulatory agents, in veterinary or human medicine.
  • Trophoblastin is the first isolated class II ⁇ interferon naturally expressed in mammalian tissue. So far, the rare known class II ⁇ interferons (bovine, human, equine, porcine) have only been demonstrated using molecular DNA probes or by expression in bacterial systems. As a result, the properties of class II ⁇ interferons are little known. In addition, the structure of a natural interferon can differ significantly by its glycosylated parts, from interferons obtained by genetic engineering. Trophoblastine has the originality for an interferon not to be secreted following a viral infection. In addition, it is produced in an exceptionally high quantity compared to that of all naturally expressed interferons.
  • the embryos are eliminated and the culture medium is centrifuged at 10,000 g for 20 minutes at 4 ° C., then dialyzed and concentrated against 50 mM TRIS / HCl buffer pH 6.00.
  • Trophoblastin is purified to a high degree of purity by high performance liquid phase chromatography (HPLC) on a DEAE 5 PW column 7.5 mm in diameter and 75 mm in length, eluted at a flow rate of 0.5 ml / min by a gradient from 0 to 0.15 M KCl in a 0.05 M Tris HCl buffer pH 6.00, within 40 min, followed by an isocratic elution at 0.15 M KCl in the same buffer .
  • HPLC high performance liquid phase chromatography
  • the trophoblast is purified to a high degree of purity by high performance liquid chromatography (HPLC) on a column, semi-preparative anion exchange DEAE-5 PW of 21.5 mm in diameter over 150 mm in length, eluted with a flow rate of 4 ml / min, by a gradient from 0 to 0.15 M KCl in a 0.05 M Tris-HCl pH 6.00 buffer, within 60 minutes, followed by isocratic elution at 0.15 M KCl in the same buffer.
  • HPLC high performance liquid chromatography
  • Figure 1 attached shows the isolation of the polypeptides T 1 , T 2 , T 3 in the isocratic part of the gradient close to 0.5 M KCl.
  • Purified trophoblastin has an apparent molecular weight of 20 kDa and an isoelectric point of approximately 5.3. It specifically binds to endometrial membrane receptors, which demonstrates the maintenance of its biological activity after the purification steps.
  • the uterine receptor for this embryonic signal exists at the end of the luteal phase of the oestrian cycle while the embryo is absent.
  • the five trophoblastin isoforms, T 1 , T 2 , T 3 , T 4 , T 5 , isolated in accordance with the present invention, are variants thereof, as demonstrated by the sequencing of the N-terminal end of these five proteins, as shown in Formulas I, I 1 , I 2 and I 3 , I 4 , I 5 above.
  • ovine trophoblastin possesses the antiviral activity of interferons, in the presence of bovine MDBK cells infected with bovine vesicular stomatitis virus (VSV).
  • VSV bovine vesicular stomatitis virus
  • the cells were infected with 100 PFU of VSV (Serotype Indiana) in 0.2 ml of MEM + 2% SVF, then finally covered with 3 ml of MEM + 2% VSF containing 0.6% of agarose, with which they were incubated for another 24 hours at 37 ° C.
  • the viral plaques were counted after staining with crystal violet and the dilution of the protein causing 50% inhibition of the VSV plaques was determined.
  • the titers expressed in international units of human IFN (iu / ml) were deduced from the titer of a standard laboratory IFN included in these tests and calibrated with respect to the human IFN reference Ga23-902-530 (NIH Bethesda, Md, 12,000 iu / ml).
  • the specific activity of isoforms 1, 2, 3 purified by HPLC is respectively 0.6 ⁇ 10 ei / mg of protein, 0.8 ⁇ 10 ei / mg of protein and 0.7 ⁇ 10 s iu / mg of protein in the presence of MDBK and VSV cells (of. Figure 6 attached).
  • the antiviral activity of two pools of culture media for 16-day-old sheep embryos is between 0.25 and 0.68 ui / mg trophoblastin.
  • the differential antiviral activity was measured in cells of different species by inhibition of the effect exerted on VSV carried by plates as described above.
  • the results obtained are collated in Table I below, in which MM6 designates a sheep cell line established from fetal lamb fibroblasts; PD-5 is a pig kidney cell line; WISH is a human cell line and L 929 is a murine cell line.
  • the three purified isoforms were diluted to give the same antiviral titer on the MDBK cells.
  • This Table shows that the three isoforms have the same relative activity pattern, at least on bovine, ovine, porcine and human cells, isoform 2 being more active on murine cells than the other two isoforms, of a factor of about 9 to 27.
  • trophoblastin not only exhibits strong structural homology with interferons, as indicated in the aforementioned FEBS LETTERS Publication, but it also has the antiviral activity characteristic of all families of interferons.
  • seroneutralization the inventors have been able to confirm that the ovine trophoblastin is indeed a class II ⁇ interferon: a class I anti-interferon ⁇ immune serum does not recognize trophoblastin and conversely a monospecific anti-trophoblastin immune serum does not recognize ⁇ interferons class I.
  • Table II illustrates the seroneutralization of viral activity in the presence of interferon (IFN) and anti-IFN immune serum.
  • IFN interferon
  • VSV vesicular stomatitis virus
  • the immuniser used is anti-T rabbit immuniser (mixture of isoforms T 1 , T 2 , T 3 ).
  • EXAMPLE 3 CLONING OF TROPHOBLASTIN.
  • Obtaining clones containing the cDNA for trophoblastin mRNA comprises the following steps. 1. Isolation of total poly (A) + embryonic RNAs, using the guanidine isothiocyanate method from 16-day-old sheep embryos (CATHALA et al., 1983). In order to have a "negative" control corresponding to a late stage during which trophoblastin is no longer secreted, mRNAs were also isolated from embryos 29 days old. After purification on an affinity column (oligo dT cellulose), approximately 50 to 60 ⁇ g of poly (A) + RNA were obtained from around twenty 16-day-old embryos, removed surgically.
  • affinity column oligo dT cellulose
  • trophoblastin precursor and quantification of the corresponding mRNA.
  • the biological activity of embryonic poly (A) + RNA was measured by in vitro translation in two acellular systems (reticulocyte lysate and wheat germ) in the presence of 35 S-methionine. In these two cases, the amount of trophoblastin synthesized was estimated by SDS electrophoresis, followed by immunoprecipitation, respectively using a monospecific antitrophoblastin antiserum prepared by the inventors. Under these conditions, a polypeptide with an apparent molecular weight of ⁇ 23 kDa was detected; it corresponds to the precursor of trophoblastin and represents approximately 1 to 2% of the total mRNAs.
  • the cDNAs were synthesized according to the method of GUBLER and HOFFMAN (1983) which makes it possible to obtain cDNAs of length close to that of the mRNAs. After addition of EcoRI "linkers", these cDNAs were inserted respectively into the EcoRI sites of a plasmid vector (pUC8) and of an expression vector, the bacteriophage Lambda gt 11 ( ⁇ gtll), then cloned into the strains ( DH5 and Y1088) of colibacilli (Esche ⁇ chia coli). The plasmid and phage banks contained respectively 4.10 5 and 1.2.10 6 recombinants per ⁇ g of DNA.
  • Mdp 7 hybridized with a single 1.1 kb mRNA (Northern-blot technique); this size was compatible with that of an mRNA coding for a polypeptide of ⁇ 23 kDa (precursor of the trophoblast). This is the reason why further analysis of these clones was undertaken.
  • IFN ⁇ lI class II ⁇ interferons
  • the amino acid sequence of isoform 2 determined by peptide hydrolysis is strictly identical to cDNA ( Figure 5). No N-glycosylation site (ASN-X-THR or SER) is observed. Furthermore, the region corresponding to the signal peptide has a homology of more than 95% at the nucleotide level and 100% at the level of the primary structure with that of bovine class II interferon ⁇ (CAPON, SHEPARD and GOEDDEL, ( MOL. CELL. BIOL. (1985), 5, 768-779), which suggests that these two molecules could derive from a common ancestral gene.
  • CAPON, SHEPARD and GOEDDEL bovine class II interferon ⁇
  • bovine class I IFN ⁇ 64% with bovine class I IFN ⁇ .
  • bovine class II IFN ⁇ 71% (and 56% with human IFN ⁇ lI), while it is only 48% with bovine class I IFN ⁇ .
  • trophoblastin corresponds to a class II ⁇ embryonic interferon.
  • a quantitative method for detecting the ovine trophoblast has been developed by the inventors. It consists of a radioimmunological assay by competition, using two antibodies, under the following conditions: the ovine trophoblastin used for labeling with Iodine 125 and for the calibration curves, is derived from the purification by HPLC chromatography of culture media sheep embryos. An anti-trophoblastin antiserum after immunization of rabbits is used at a final dilution of 1 / 360,000; it binds 35% radioactive trophoblastin under the dosage conditions established by the Inventors.
  • this method makes it possible to detect 15 pg of trophoblastin in 100 ⁇ l of sample to be tested.
  • ⁇ represent the culture medium for sheep embryos.
  • - Specificity of the assay there is no cross-immunoreactivity with porcine interferons, secretion products from bovine, porcine, rabbit embryos and serum compounds.
  • this assay it is possible to follow the evolution of the secretion of trophoblastin in vivo and in vitro.
  • the sensitivity of the assay makes it possible to detect small amounts of trophoblastin secreted by sheep and goat embryos at very early stages (20ng / embryo in sheep perfusates at 11 days of gestation, 1 ⁇ g / day for an 11-day cultivated embryo in vitro).
  • the cDNA of the trophoblastin isoforms which are the subject of the present invention or of their analogs can, moreover, be used for all the productions of class II ⁇ interferons by genetic engineering methods (bacterial, cellular expression , genetic engineering or through transgenic animals).

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PCT/FR1989/000116 1988-03-18 1989-03-17 Isoformes de la trophoblastine, nouveaux interferons constitues par lesdites isoformes, leurs procedes d'obtention et leurs applications WO1989008706A1 (fr)

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FR8803591A FR2628744B1 (fr) 1988-03-18 1988-03-18 Isoformes de la trophoblastine, nouveaux interferons constitues par lesdites isoformes, leurs procedes d'obtention et leurs applications
FR88/03591 1988-03-18
US30420989A 1989-01-31 1989-01-31
US304,209 1989-01-31

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0367063A1 (de) * 1988-10-26 1990-05-09 The Curators Of The University Of Missouri Isolation und Klonierung von cDNA für das für Rindertrophoblast kodierende Gen
FR2669931A1 (fr) * 1990-11-29 1992-06-05 Transgene Nouveaux variants derives des interferons-alpha et leur procede de production.
FR2669824A1 (fr) * 1990-11-29 1992-06-05 Agronomique Inst Nat Rech Utilisation de variants des interferons alpha pour l'obtention de medicaments.
WO1992009691A1 (fr) * 1990-11-29 1992-06-11 Institut National De La Recherche Agronomique - I.N.R.A. Nouveaux variants derives des interferons de type i, leur procede de production, et leurs applications
WO1993012146A1 (en) * 1991-12-06 1993-06-24 Landsforeningen Til Kraeftens Bekaempelse Trophoblast interferons and their use
WO1994028425A1 (en) * 1993-05-21 1994-12-08 Brigham And Women's Hospital, Inc. Embryotoxic factors
EP0669981A1 (de) * 1992-10-30 1995-09-06 University Of Florida Interferon-tau-zusammensetzungen und verfahren zur verwendung

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0174143A1 (de) * 1984-08-27 1986-03-12 Genentech, Inc. Verschiedenartige Familie von menschlichen Leukozyten-Interferonen, Zusammensetzungen, die diese enthalten, Verfahren zu ihrer Herstellung, sowie DNA und transfektierte Wirte hierfür

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0174143A1 (de) * 1984-08-27 1986-03-12 Genentech, Inc. Verschiedenartige Familie von menschlichen Leukozyten-Interferonen, Zusammensetzungen, die diese enthalten, Verfahren zu ihrer Herstellung, sowie DNA und transfektierte Wirte hierfür

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0367063A1 (de) * 1988-10-26 1990-05-09 The Curators Of The University Of Missouri Isolation und Klonierung von cDNA für das für Rindertrophoblast kodierende Gen
FR2669931A1 (fr) * 1990-11-29 1992-06-05 Transgene Nouveaux variants derives des interferons-alpha et leur procede de production.
FR2669824A1 (fr) * 1990-11-29 1992-06-05 Agronomique Inst Nat Rech Utilisation de variants des interferons alpha pour l'obtention de medicaments.
WO1992009691A1 (fr) * 1990-11-29 1992-06-11 Institut National De La Recherche Agronomique - I.N.R.A. Nouveaux variants derives des interferons de type i, leur procede de production, et leurs applications
US5378823A (en) * 1990-11-29 1995-01-03 Institut National De La Recherche Agronomique-I.N.R.A. Nucleic acids encoding type I interferon variants
WO1993012146A1 (en) * 1991-12-06 1993-06-24 Landsforeningen Til Kraeftens Bekaempelse Trophoblast interferons and their use
EP0669981A1 (de) * 1992-10-30 1995-09-06 University Of Florida Interferon-tau-zusammensetzungen und verfahren zur verwendung
EP1360962A2 (de) * 1992-10-30 2003-11-12 University Of Florida Interferon-tau zusammensetzung und Methode für ihre Verwendung
EP1360962A3 (de) * 1992-10-30 2004-02-04 University Of Florida Interferon-tau zusammensetzung und Methode für ihre Verwendung
WO1994028425A1 (en) * 1993-05-21 1994-12-08 Brigham And Women's Hospital, Inc. Embryotoxic factors

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AU638207B2 (en) 1993-06-24
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