EP0542756A1 - Procede permettant d'introduire un peptide dans le cytosol - Google Patents

Procede permettant d'introduire un peptide dans le cytosol

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Publication number
EP0542756A1
EP0542756A1 EP91911315A EP91911315A EP0542756A1 EP 0542756 A1 EP0542756 A1 EP 0542756A1 EP 91911315 A EP91911315 A EP 91911315A EP 91911315 A EP91911315 A EP 91911315A EP 0542756 A1 EP0542756 A1 EP 0542756A1
Authority
EP
European Patent Office
Prior art keywords
toxin
peptide
cytosol
mutant
cells
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Withdrawn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
EP91911315A
Other languages
German (de)
English (en)
Inventor
Sjur Olsnes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
FORSKNINGSSTIFTELSEN DET NORSKE RADIUMHOSPITAL
Original Assignee
FORSKNINGSSTIFTELSEN DET NORSKE RADIUMHOSPITAL
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by FORSKNINGSSTIFTELSEN DET NORSKE RADIUMHOSPITAL filed Critical FORSKNINGSSTIFTELSEN DET NORSKE RADIUMHOSPITAL
Publication of EP0542756A1 publication Critical patent/EP0542756A1/fr
Withdrawn legal-status Critical Current

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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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
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  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
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  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

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.
EP91911315A 1990-06-27 1991-06-26 Procede permettant d'introduire un peptide dans le cytosol Withdrawn EP0542756A1 (fr)

Applications Claiming Priority (2)

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

Publications (1)

Publication Number Publication Date
EP0542756A1 true EP0542756A1 (fr) 1993-05-26

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ID=19893304

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91911315A Withdrawn EP0542756A1 (fr) 1990-06-27 1991-06-26 Procede permettant d'introduire un peptide dans le cytosol

Country Status (8)

Country Link
EP (1) EP0542756A1 (fr)
JP (1) JPH06503552A (fr)
CA (1) CA2086342A1 (fr)
FI (1) FI925869A (fr)
HU (1) HUT63061A (fr)
LT (1) LTIP835A (fr)
NO (1) NO175188C (fr)
WO (1) WO1992000099A1 (fr)

Families Citing this family (13)

* 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
US5314813A (en) * 1992-02-19 1994-05-24 Scripps Research Institute Drosophila cell lines expressing genes encoding MHC class I antigens and B2-microglobulin and capable of assembling empty complexes and methods of making said cell lines
US5935580A (en) * 1992-04-21 1999-08-10 Institut Pasteur Recombinant mutants for inducing specific immune responses
PT637335E (pt) * 1992-04-21 2007-10-31 Pasteur Institut Mutantes recombinantes para induzir respostas imunitárias específicas
US6455673B1 (en) 1994-06-08 2002-09-24 President And Fellows Of Harvard College Multi-mutant diphtheria toxin vaccines
US20030165543A1 (en) * 2000-01-27 2003-09-04 Langridge William H.R. Transgenic plant-based vaccines
US6777546B2 (en) 1997-10-07 2004-08-17 Loma Linda University Methods and substances for preventing and treating autoimmune disease
US7422747B2 (en) 1997-10-07 2008-09-09 Loma Linda University Transgenic plant-based vaccines
US6004815A (en) * 1998-08-13 1999-12-21 The Regents Of The University Of California Bacteria expressing nonsecreted cytolysin as intracellular microbial delivery vehicles to eukaryotic cells
DE60042687D1 (de) 2000-09-15 2009-09-17 Pasteur Institut Proteinhaltigen Vektoren zur Einführung von Moleküle in CD11b exprimirende Zellen
GB0524408D0 (en) * 2005-11-30 2006-01-11 Glaxosmithkline Biolog Sa Vaccines
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
CA3017143A1 (fr) * 2016-03-10 2017-09-14 The Johns Hopkins University Procedes de production de proteines de fusion de toxine diphterique monomere sans agregat et utilisations therapeutiques

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0108146B1 (fr) * 1982-05-12 1987-01-28 The President And Fellows Of Harvard College Gènes de fusion codant des protéines hybrides, vecteurs de clonage les contenant et leur emploi
FR2532850B1 (fr) * 1982-09-15 1985-12-20 Pasteur Institut Conjugues immunogenes entre un haptene et une molecule porteuse derivee d'une toxine, les vaccins les composant et procede pour leur obtention
US4808700A (en) * 1984-07-09 1989-02-28 Praxis Biologics, Inc. Immunogenic conjugates of non-toxic E. coli LT-B enterotoxin subunit and capsular polymers
IL89504A0 (en) * 1988-03-08 1989-09-10 Univ Wyoming Diphtheria toxin derivative,process for the preparation thereof and pharmaceutical composition containing the same
FR2636842B1 (fr) * 1988-09-27 1994-06-10 Liege Universite Etat Proteine de fusion d'une sequence derivee de la sous-unite b de la toxine cholerique et d'un antigene heterologue doue de proprietes immunogenes, compositions de vaccins les contenant et acides nucleiques recombinants contenant une sequence de nucleotides codant pour ladite proteine de fusion
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 (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9200099A1 *

Also Published As

Publication number Publication date
HU9204125D0 (en) 1993-04-28
LTIP835A (en) 1995-02-27
JPH06503552A (ja) 1994-04-21
FI925869A0 (fi) 1992-12-23
NO902871D0 (no) 1990-06-27
CA2086342A1 (fr) 1991-12-28
NO175188C (no) 1994-09-14
WO1992000099A1 (fr) 1992-01-09
NO902871L (no) 1991-12-30
HUT63061A (en) 1993-07-28
NO175188B (fr) 1994-06-06
FI925869A (fi) 1992-12-23
AU653158B2 (en) 1994-09-22
AU8000191A (en) 1992-01-23

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