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WO1992000099A1 - Procede permettant d'introduire un peptide dans le cytosol - Google Patents

Procede permettant d'introduire un peptide dans le cytosol Download PDF

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Publication number
WO1992000099A1
WO1992000099A1 PCT/NO1991/000093 NO9100093W WO9200099A1 WO 1992000099 A1 WO1992000099 A1 WO 1992000099A1 NO 9100093 W NO9100093 W NO 9100093W WO 9200099 A1 WO9200099 A1 WO 9200099A1
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WIPO (PCT)
Prior art keywords
toxin
peptide
cytosol
mutant
cells
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PCT/NO1991/000093
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English (en)
Inventor
Sjur Olsnes
Original Assignee
Forskningsstiftelsen Det Norske Radiumhospital
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Filing date
Publication date
Application filed by Forskningsstiftelsen Det Norske Radiumhospital filed Critical Forskningsstiftelsen Det Norske Radiumhospital
Priority to JP3510777A priority Critical patent/JPH06503552A/ja
Priority to AU80001/91A priority patent/AU653158C/en
Publication of WO1992000099A1 publication Critical patent/WO1992000099A1/fr
Priority to FI925869A priority patent/FI925869A0/fi

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Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • 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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—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
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention is directed to a method of in- troducing a peptide into the cytosol, and more specifically to a novel principle in vaccine production against viruses, intracellular parasites and bacteria and against malignant cells.
  • MHC histocompati- bility antigens
  • MHC Class I MHC Class I
  • the common way today to immunize against such structures is to use attenuated live viruses that are able to enter cells and replicate such that the peptides in question are formed in the cells and can be presented at the cell surface. In this way the population of the relevant cytotoxic CD8 + cells is expanded and upon later exposure to the corres ⁇ ponding virulant virus strain, the organism has an immune protection.
  • toxin molecules that are of very low toxicity (Barbieri, J.T. & Collier, R.J. Infect. Immun. 55, 1647-1651 (1987)). If the toxins were able to carry s into cells additional peptide material, such non-toxic mutants could be useful for vaccine purposes to carry into the cytosol antigenic peptides (Cerundolo et al. Nature 345, 449 (1990)) that can be presented by Class I MHC antigens. Such antigenic sequences can be obtained from a number of viruses, bacteria o and parasites, and it is also possible to derive such struc ⁇ tures from certain malignant cells.
  • the present invention relates to a method of introducing a peptide into the cytosol by linking the peptide to a bacterial or plant toxin, or a mutant thereof. Further, the present invention relates to a method of preparing a vaccine by linking a peptide to a bacterial or plant toxin, or a mutant thereof to translocate the peptide into the cytosol for subsequent presentation at the cell surface by Class I MHC antigens to elicit a Class I restricted immune response and to expand the relevant population of CD8 + T-lymphocytes. Also, the present invention relates to vaccines which have been produced by the above-mentioned method, as well as the use of such vaccines against viruses, intracellular bacteria and para ⁇ sites, and against molecules associated with malignancies.
  • FIG. 1 N-terminal extensions of diphtheria toxin.
  • pBD-lS The coding region of the diphtheria toxin gene carrying a triple mutation changing Glu 148 to Ser, and where Gly 1 was replaced by initiator Met placed behind a T3 promotor to give pBD-lS (McGill, S., Stenmark, H., Sandvig, K. & Olsnes, S. : EMBO J. 8, 2843-2848 (1989)).
  • pBD-1 was cleaved with Ncol, and an oligonucleotide encoding the oligopeptide MGVDEYNEMPMPVN (referred to as B3) was inserted.
  • pGD-2 encodes diphtheria toxin with its natural signal sequence, MSRKLFASILIGALLGIGAPPSAHA (referred to as ss), after an SP6 promotor.
  • the plasmid was obtained by digesting pGD-1 (McGill, S., Stenmark, H., Sandvig, K. & Olsnes, S.: EMBO J. 8, 2843-2848 (1989)) with Hindlll and Pstl, removing the overhangs with S x -nuclease and religating to form pGD-2.
  • FIG. 2 Translocation to the cytosol of A-fragment with N-terminally added B3 oligopeptide.
  • pBD-1 and pB-B3-Dl were transcribed and translated in vitro.
  • the corresponding trans ⁇ lation products (DT and B3-DT) were added to Vero cells grow ⁇ ing as monolayers in 24-well microtiter plates and kept at 24°C for 20 min in the presence of 10 ⁇ M monensin (McGill, S., Stenmark, H., Sandvig, K. & Olsnes, S.: EMBO J. 8, 2843-2848 (1989)).
  • the cells were washed twice with Hepes medium and subsequently treated with 0.4 ⁇ g/ml TPCK (N-tosyl-L-phenyl- alanine chloromethyl ketone)-treated trypsin in Hepes medium containing 10 ⁇ M monensin for 5 min at 20°C.
  • the cells were washed and exposed to Hepes medium, pH 4.43, containing 10 mM Na-gluconate to increase the buffering capacity at the low pH. After 2 min at 37°C, the cells were washed with Hepes medium, pH 7.4, and then treated with 3 mg/ml pronase in Hepes medium, pH 7.4, containing 10 ⁇ M monensin for 5 min at 37°C.
  • TPCK N-tosyl-L-phenyl- alanine chloromethyl ketone
  • the cells which were detached from the plastic by the treatment, were recovered by centrifugation and washed once with Hepes medium containing 1 mM NEM (N-ethyl maleimide) and 1 mM PMSF (phenylmethylsulfonyl fluoride).
  • NEM N-ethyl maleimide
  • PMSF phenylmethylsulfonyl fluoride
  • the cells were lysed with Triton X-100 in phosphate buffered saline containing 1 mM PMSF and 1 mM NEM, nuclei were removed by centrifugation and the protein in the supernatant fraction was precipitated with 10% (w/v) trichloroacetic acid or immunoprecipitated with anti-B3 antibodies adsorbed to protein A-Sepharose.
  • FIG.3 Translocation to the cytosol of diphtheria toxin with signal sequence.
  • lane 3 the cells were treated as in lane 2, except that 6 times more translation product was used and the cells were then exposed to pH 4.8 and pronase as in Fig. 2.
  • the cells were lysed with Triton X-100 and the nuclei were removed.
  • the lysed cells were either analyzed with non-reducing SDS-PAGE (15% gel) directly (lanes 5-8) or they were treated with saponin and the membrane pellets (lanes 9 and 10) and the supernatant fractions (lanes 11 and 12) were analyzed separately.
  • Diphtheria toxin is synthesized by pathogenic strains of Corynebacterium diphtheriae as a single chain polypeptide.
  • the protein is easily split ( “nicked” ) at a trypsin-sensitive site to yield two disulfide-linked fragments, A and B (Pappen- heimer, A.M., Jr. Annu. Rev. Biochem. 46, 69-94 (1977)).
  • the B-fragment (37 kD) binds to cell surface receptors
  • the A-fragment (21 kD) is an enzyme that is trans ⁇ located to the cytosol where it inactivates elongation factor 2 by ADP-ribosylation and thus blocks protein synthesis (Van Ness, B.G., Hovard, J.B. & Bodley, J.W. J. Biol. Chem. 255, 10710-10716 (1980)).
  • the translocation which normally occurs across the limiting membrane of endosomes, is triggered by the low pH in the acidic vesicles (Draper, R.K. & Simon, M.I. J. Cell Biol.
  • a mutant toxin which contains a triple mutation changing Glu 148 , which is located in the enzymatically active site of the toxin, to Ser (Barbieri, J. T. & Collier, R.J. Infect. Immun. 55, 1647-1651 (1987)).
  • the modified toxin has strongly reduced toxicity.
  • toxin with B3 was selectively precipitated with anti-B3 (lane 4), but not with a control serum (lane 5). Toxin without B3 was not precipitated with anti-B3 (lane 3).
  • the dialyzed translation products were bound to Vero cells, nicked on the cells with low concentrations of trypsin, and then the cells were exposed to pH 4.8. Under these conditions part of the bound toxin was translocated to the cytosol and thereby became shielded against pronase added to s the medium (Moskaug, 3.0. , Sandvig, K. & Olsnes, S. J. Biol. Chem. 263, 2518-2525 (1988)). In the case of diphtheria toxin as such, two fragments (MW 21 kD and 25 kD) were protected under these conditions (Fig.
  • toxin carrying its normal signal sequence (25 amino acids). As shown in Fig. 3, lane 2, this protein was nicked by trypsin into a 23.5 kD A-fragment and a 37 kD B- fragment. (In this experiment the toxin was only partially nicked. Partially nicked 125 I-labelled natural toxin is shown for comparison in lane 1). When the toxin with signal sequence was bound to cells, nicked, and then exposed to pH 4.8, two fragments (23.5 kD and 25 kD) were protected against pronase (lane 8).
  • Protected A-fragment with uncleaved signal sequence is also shown in lane 3, where the material was precipitated with an anti-diphtheria toxin serum which binds the whole toxin, the A-fragment, as well as whole B-fragment (see lanes 1 and 2), but not the 25 kD-fragment.
  • the pronase-treated cells were treated with saponin, the extended A-fragment was released to the medium (lanes 4 and 12), whereas the 25 kD fragment remained in the membrane fraction (lane 10).

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Abstract

Procédé permettant d'introduire un peptide dans le cytosol par liaison du peptide à une toxine bactérienne ou végétale, ou un mutant de celle-ci. Procédé de préparation d'un vaccin par liaison d'un peptide à une toxine bactérienne ou végétale, ou à un mutant de celle-ci de façon à effectuer la translocation dudit peptide dans le cytosol pour présentation ultérieure à la surface de la cellule par des antigènes de complexe majeur d'histocompatabilité (CMH) de classe I, afin de provoquer une réponse immunitaire limitée et d'augmenter la population de lymphocytes T CD8+. Vaccins obtenus grâce à ladite méthode et utilisation de ceux-ci contre les virus, les bactéries et les parasites intracellulaires ainsi que les molécules associées aux malignités.
PCT/NO1991/000093 1990-06-27 1991-06-26 Procede permettant d'introduire un peptide dans le cytosol WO1992000099A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP3510777A JPH06503552A (ja) 1990-06-27 1991-06-26 細胞質ゾル内にペプチドを導入する方法
AU80001/91A AU653158C (en) 1990-06-27 1991-06-26 Method of introducing a peptide into the cytosol
FI925869A FI925869A0 (fi) 1990-06-27 1992-12-23 Foerfarande foer infoerande av en peptid i cytosol

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NO902871 1990-06-27
NO902871A NO175188C (no) 1990-06-27 1990-06-27 Fremgangsmåte for fremstilling av et peptidkonjugat med evne til å trenge inn i cellecytosol

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WO1992000099A1 true WO1992000099A1 (fr) 1992-01-09

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EP (1) EP0542756A1 (fr)
JP (1) JPH06503552A (fr)
CA (1) CA2086342A1 (fr)
FI (1) FI925869A0 (fr)
HU (1) HUT63061A (fr)
LT (1) LTIP835A (fr)
NO (1) NO175188C (fr)
WO (1) WO1992000099A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5503829A (en) * 1992-04-21 1996-04-02 Institut Pasteur Recombinant mutants for inducing specific immune responses
US5935580A (en) * 1992-04-21 1999-08-10 Institut Pasteur Recombinant mutants for inducing specific immune responses
WO2000009733A1 (fr) * 1998-08-13 2000-02-24 The Regents Of The University Of California Vecteurs d'apport intracellulaire
US6043057A (en) * 1988-09-16 2000-03-28 Vitec Aktiebolag Recombinant systems for expression of the cholera B-sub-unit with the aid of foreign promoters and/or leader peptides
WO2000048638A3 (fr) * 1999-02-16 2001-03-01 Harvard College Vaccins à base de toxine diphtérique à mutations multiples
EP1254156A1 (fr) * 2000-01-27 2002-11-06 Loma Linda University Vaccins a base de plantes transgeniques
US6777546B2 (en) 1997-10-07 2004-08-17 Loma Linda University Methods and substances for preventing and treating autoimmune disease
JP2006022111A (ja) * 1992-02-19 2006-01-26 Scripps Res Inst:The ｉｎｖｉｔｒｏにおける細胞障害性Ｔ細胞の活性化
WO2007062832A3 (fr) * 2005-11-30 2007-09-07 Glaxosmithkline Biolog Sa Vaccins
US7422747B2 (en) 1997-10-07 2008-09-09 Loma Linda University Transgenic plant-based vaccines
US9370564B2 (en) 2000-09-15 2016-06-21 Institut Pasteur Vectors for molecule delivery to CD11b expressing cells
CN109790545A (zh) * 2016-03-10 2019-05-21 约翰·霍普金斯大学 产生不含聚集物的单体白喉毒素融合蛋白的方法和治疗用途
US11965009B2 (en) 2016-03-10 2024-04-23 The Johns Hopkins University Methods of producing aggregate-free monomeric diphtheria toxin fusion proteins and therapeutic uses

Citations (6)

* Cited by examiner, † Cited by third party
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FR2532850A1 (fr) * 1982-09-15 1984-03-16 Pasteur Institut Conjugues immunogenes entre un haptene et une molecule porteuse derivee d'une toxine, les vaccins les composant et procede pour leur obtention
EP0172107A1 (fr) * 1984-08-10 1986-02-19 Praxis Biologics, Inc. Conjugués immunogènes d'unité secondaire d'entérotoxine LT-B d'E. coli et polymères capsulaires
US4675382A (en) * 1982-05-12 1987-06-23 President And Fellows Of Harvard College Hybrid protein
EP0332174A2 (fr) * 1988-03-08 1989-09-13 The University Of Wyoming Agents cellulaires cytotoxiques spécifiques
WO1990003437A1 (fr) * 1988-09-27 1990-04-05 L'universite De L'etat A Liege Proteines de fusion de la sous-unite b de la toxine cholerique et d'un antigene heterologue et acides nucleiques les encodant
WO1991009871A1 (fr) * 1989-12-22 1991-07-11 Seragen Incorporated Molecules hybrides presentant une region de translocation et une region de liaison cellulaire

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4675382A (en) * 1982-05-12 1987-06-23 President And Fellows Of Harvard College Hybrid protein
FR2532850A1 (fr) * 1982-09-15 1984-03-16 Pasteur Institut Conjugues immunogenes entre un haptene et une molecule porteuse derivee d'une toxine, les vaccins les composant et procede pour leur obtention
EP0172107A1 (fr) * 1984-08-10 1986-02-19 Praxis Biologics, Inc. Conjugués immunogènes d'unité secondaire d'entérotoxine LT-B d'E. coli et polymères capsulaires
EP0332174A2 (fr) * 1988-03-08 1989-09-13 The University Of Wyoming Agents cellulaires cytotoxiques spécifiques
WO1990003437A1 (fr) * 1988-09-27 1990-04-05 L'universite De L'etat A Liege Proteines de fusion de la sous-unite b de la toxine cholerique et d'un antigene heterologue et acides nucleiques les encodant
WO1991009871A1 (fr) * 1989-12-22 1991-07-11 Seragen Incorporated Molecules hybrides presentant une region de translocation et une region de liaison cellulaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Dialog Information Services, File 155, Medline 67-91, Dialog Accession No. 07152866, S. McGILL et al.: "Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants", & EMBO J Oct 1989, 8 (10), p2843-8. *
Dialog Information Services, File 155, Medline 67-91, Dialog Accession No. 07731463, H. STENMARK et al.: "Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol", & J CELL BIOL (US) Jun 1991, 113 (5), p1025-32. *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6043057A (en) * 1988-09-16 2000-03-28 Vitec Aktiebolag Recombinant systems for expression of the cholera B-sub-unit with the aid of foreign promoters and/or leader peptides
JP4584794B2 (ja) * 1992-02-19 2010-11-24 ザ スクリプス リサーチ インスティチュート ｉｎｖｉｔｒｏにおける細胞障害性Ｔ細胞の活性化
JP2006022111A (ja) * 1992-02-19 2006-01-26 Scripps Res Inst:The ｉｎｖｉｔｒｏにおける細胞障害性Ｔ細胞の活性化
US5679784A (en) * 1992-04-21 1997-10-21 Institut Pasteur Recombinant mutants for inducing specific immune responses
US5935580A (en) * 1992-04-21 1999-08-10 Institut Pasteur Recombinant mutants for inducing specific immune responses
US5503829A (en) * 1992-04-21 1996-04-02 Institut Pasteur Recombinant mutants for inducing specific immune responses
US6455673B1 (en) 1994-06-08 2002-09-24 President And Fellows Of Harvard College Multi-mutant diphtheria toxin vaccines
US7115725B2 (en) 1994-06-08 2006-10-03 President And Fellows Of Harvard College Multi-mutant diphtheria toxin vaccines
US7422747B2 (en) 1997-10-07 2008-09-09 Loma Linda University Transgenic plant-based vaccines
US6777546B2 (en) 1997-10-07 2004-08-17 Loma Linda University Methods and substances for preventing and treating autoimmune disease
WO2000009733A1 (fr) * 1998-08-13 2000-02-24 The Regents Of The University Of California Vecteurs d'apport intracellulaire
WO2000048638A3 (fr) * 1999-02-16 2001-03-01 Harvard College Vaccins à base de toxine diphtérique à mutations multiples
EP1254156A4 (fr) * 2000-01-27 2003-05-21 Univ Loma Linda Vaccins a base de plantes transgeniques
EP1254156A1 (fr) * 2000-01-27 2002-11-06 Loma Linda University Vaccins a base de plantes transgeniques
US9370564B2 (en) 2000-09-15 2016-06-21 Institut Pasteur Vectors for molecule delivery to CD11b expressing cells
US10004794B2 (en) 2000-09-15 2018-06-26 Institut Pasteur Vectors for molecule delivery to CD11b expressing cells
WO2007062832A3 (fr) * 2005-11-30 2007-09-07 Glaxosmithkline Biolog Sa Vaccins
CN109790545A (zh) * 2016-03-10 2019-05-21 约翰·霍普金斯大学 产生不含聚集物的单体白喉毒素融合蛋白的方法和治疗用途
US11965009B2 (en) 2016-03-10 2024-04-23 The Johns Hopkins University Methods of producing aggregate-free monomeric diphtheria toxin fusion proteins and therapeutic uses

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CA2086342A1 (fr) 1991-12-28
NO175188B (fr) 1994-06-06
NO902871L (no) 1991-12-30
HUT63061A (en) 1993-07-28
LTIP835A (en) 1995-02-27
FI925869L (fi) 1992-12-23
NO902871D0 (no) 1990-06-27
JPH06503552A (ja) 1994-04-21
HU9204125D0 (en) 1993-04-28
NO175188C (no) 1994-09-14
FI925869A0 (fi) 1992-12-23
AU653158B2 (en) 1994-09-22
EP0542756A1 (fr) 1993-05-26
AU8000191A (en) 1992-01-23

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