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WO2009106362A1 - Vaccin à adn pour la thérapie et la prophylaxie du cancer du col de l'utérus et son stade préliminaire pré-malin - Google Patents

Vaccin à adn pour la thérapie et la prophylaxie du cancer du col de l'utérus et son stade préliminaire pré-malin Download PDF

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WO2009106362A1
WO2009106362A1 PCT/EP2009/001576 EP2009001576W WO2009106362A1 WO 2009106362 A1 WO2009106362 A1 WO 2009106362A1 EP 2009001576 W EP2009001576 W EP 2009001576W WO 2009106362 A1 WO2009106362 A1 WO 2009106362A1
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nucleic acid
hpv
fragments
vaccine
sequence
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PCT/EP2009/001576
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German (de)
English (en)
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Günter CICHON
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Cichon Guenter
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Publication of WO2009106362A1 publication Critical patent/WO2009106362A1/fr

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • 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
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a nucleic acid which is suitable for one or more
  • HPV Human papilloma virus
  • the nucleic acid upon expression in a mammalian organism, can induce an immune response that results in the destruction of HPV oncoprotein-expressing cells and thus is useful for the prophylaxis and / or treatment of HPV-induced diseases such as cervical cancer and its precursors.
  • the construct according to the invention is distinguished by a safety concept which combines high therapeutic immunogenicity with maximum biological safety and thus creates the prerequisite for a broad and safe clinical application.
  • the prophylactic vaccine (Gardasil® Merck, Cervarix® GlaxoSmithKline), which was also introduced in Germany about a year ago, very reliably prevents first infection with HPV 6, 11, 16 and 18 (in the case of Gardasil) in young people who have not had sexual intercourse yet. However, if a person is already infected with HPV viruses, the vaccine has no effect.
  • its prophylactic efficacy for the prevention of cervical intraepithelial neoplasia (CIN lesions) in 25-year-old women is only 17% regardless of the causative HPV serotype.
  • DNA vaccination has the great advantage over vaccination with peptides or proteins that the patient's antigen processing machinery allows for individual selection of T cell epitopes that arrive at MHC-mediated presentation.
  • the same DNA vaccine can induce very different but nevertheless therapeutically relevant immune responses in immunologically different individuals.
  • a problem in the production and clinical use of oncogene-derived DNA vaccines is the reliable elimination of all potentially transforming properties. It must be assumed that, as part of a DNA vaccination, traces of DNA remain in the patient's body for a lifetime. The DNA entering the body should therefore be completely free of any transforming potential.
  • the object underlying the present invention was to develop a nucleic acid vaccine in which the disadvantages of the prior art are overcome.
  • the nucleic acid vaccine of the invention should combine high therapeutic and prophylactic efficacy with maximum biological safety.
  • the invention therefore provides a method for selecting a nucleic acid which is suitable for forming an immune response in humans, coding for one or more human papilloma virus (HPV) oncoproteins or
  • the method according to the invention is characterized by the following Process steps from:
  • HPV serotypes identify those nucleic acid sequence fragments that are located outside of the transformation-associated peptide motifs, c.
  • a new nucleic acid is designed, wherein one or more of the remaining nucleic acid fragments (F 1 , F 2 , F 3 F n ) are recombinantly ligated while retaining the reading frame (5 '>3') to form a new nucleic acid such that i , the fragment which was previously located at the 3 'end of the respective HPV serotype was now located at the 5' end of the new, synthetic nucleic acid, and ii. optionally further fragments in the reverse order (F n ,
  • An object of the invention are therefore also nucleic acids which have been developed by the method according to the invention.
  • the invention therefore relates to a nucleic acid encoding one or more human papilloma virus (HPV) oncoproteins or fragments thereof, characterized in that partial sequences of the oncoproteins which code for transformation-associated peptide motifs are at least partially eliminated.
  • HPV human papilloma virus
  • the oncoprotein or fragment thereof encoded by the nucleic acid of the invention is preferably derived from one or more HPV serotypes associated with the development of diseases, particularly malignant or premalignant diseases such as cervical cancer or precursors thereof. Examples of these are the HPV serotypes 16, 18, 25, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 or 82. Particularly preferred is the oncoprotein or the Fragment thereof from HPV16 and / or HPV18. More preferably, the nucleic acid encodes oncoproteins or fragments thereof from at least two different serotypes, e.g. from HPV16 and HPV18.
  • sequences coding for transformation-associated peptide motifs may be partially or completely eliminated in the nucleic acids according to the invention. This means that the nucleic acid sequences coding for the corresponding peptide motifs are altered and / or deleted, such that when the nucleic acid is expressed, the resulting polypeptide no longer contains the peptide motif in active form. It may be sufficient for an amino acid present in the transformation-associated peptide motif to be replaced or deleted by another amino acid. On the other hand, 2, 3, 4, 5 or more amino acids may also be replaced or deleted. Most preferably, the amino acids encoding the transformation-associated peptide motif are completely eliminated.
  • the nucleic acid according to the invention codes for one or more HPV oncoproteins or fragments thereof.
  • HPV oncoproteins are the HPV proteins E6, E7 and E5.
  • the HPV oncoproteins are particularly preferably selected from E6 and / or E7, for example from the oncoproteins E6 and / or E7 of the HPV serotypes 16 and / or 18.
  • the nucleic acid of the invention may encode for complete HPV oncoproteins (with transformation-associated peptide motifs eliminated) or fragments of such oncoproteins.
  • the oncoprotein Fragments are chosen so that they contain immunogenic peptide motifs, in particular a T-cell immune response-inducing peptide motifs to produce when used as a nucleic acid vaccine the desired immunizing effect.
  • the fragments are chosen so that they also span transformation-associated peptide motifs, which, however, are eliminated in the context of the present invention.
  • the sequences encoding oncoprotein fragments may each encode 50%, at least 60% each, at least 70% each, at least 80% each, or at least 90% or more of one oncoprotein or more oncoproteins.
  • the nucleic acid encodes a fusion protein containing sequences from multiple oncoproteins or fragments thereof.
  • the individual oncoproteins or fragments may be derived from a single HPV serotype, but preferably from several different HPV serotypes.
  • Oncoproteins are selected from:
  • the MFQ / MARFED motif causes a degradation of the tumor suppressor protein p53 (5, 6).
  • the CxxC motifs cause the formation of zinc finger formations (7, 8), which can cause binding of physiological proteins such as E6AP, p53 or paxillin.
  • the CPEE motif can cause telomerase activation (5).
  • the RRETQL (V) motif in turn, can bind PDZ domain-containing proteins (10-12).
  • the LxCxE motif can mediate the binding of Rb and activate a histone deacetylase, thereby promoting cell proliferation (13).
  • the EDLL / QQLF motif can alter the function of cell cycle proteins such as p21waf-1, p27Kip1, S4, and M2-PK, thus stimulating aerobic glycolysis (14-16).
  • cell cycle proteins such as p21waf-1, p27Kip1, S4, and M2-PK, thus stimulating aerobic glycolysis (14-16).
  • HPV16 E6 oncoprotein GenBank Acc No. AAO 85408, SEQ ID No.
  • transformation-associated peptide motifs which are partially or completely eliminated in the nucleic acid constructs according to the invention: (i) MFQ (position 8-10 (iii) CxxC (position 70-73), (iv) CxxC (position 110-113), (v) CxxC (position 143-146), (vi) CPEE (Position 118-121) and (vii) RRETQL (position 153-158).
  • HPV16 E7 oncoprotein (GenBank Acc No. AAO 85409, SEQ ID No. 3) contains the following transformation-associated peptide motifs which are completely or partially eliminated in the nucleic acid constructs according to the invention: (i) LxCxE (position 22-26 ), (ii) CxxC (position 58-61),
  • transformation-associated peptide motifs are present in particular in the HPV18 E7 oncoprotein (GenBank Acc No. No. Y18491, SEQ ID No. 4), which partially or completely eliminates them in the nucleic acid constructs according to the invention are:
  • the nucleic acid construct according to the invention may have a codon-optimized sequence for expression in the particular target organism provided.
  • the nucleic acid construct preferably has a sequence codon-optimized for human expression. Codons optimized for human expression are listed, for example, in (20-23).
  • a particularly preferred nucleic acid construct encodes sequences from the oncoproteins HPV16-E6, HPV16-E7, HPV18-E6 and HPV18-E7.
  • Such a nucleic acid construct can be used, for example, for the polypeptide p14 having the amino acid sequence according to SEQ ID no. 8 encode and particularly preferably the nucleic acid sequence according to SEQ ID NO. 7 have.
  • the nucleic acid construct is preferably in operative association with an expression control sequence, i. with a sequence enabling expression in a predetermined target cell, such as a predetermined target organism.
  • the expression control sequence allows expression in a human target cell or in a human organism.
  • the expression control sequence preferably contains a suitable promoter, e.g. a viral promoter such as the CMV promoter or another promoter suitable for expression in mammalian cells, particularly in human cells, such as the vaccinia H6 promoter, the RSV promoter or the mouse caveolin-1 (cav-i) Promoter
  • the expression control sequence may comprise a polyadenylation signal such as the CMV polyadenylation signal and optionally enhancers and / or transcription terminators. The use of such expression control sequences in DNA vaccines for human applications is described in (24-27).
  • the nucleic acid construct according to the invention is preferably integrated in a non-viral or viral gene transfer system.
  • non-viral Gene transfer systems are naked DNA, liposomes such as neutral or cationic liposomes, DNA polycation, eg polylysine, DNA complexes optionally adsorbed on particles or coupled to carrier proteins such as transferrin, particle gun (Gene Gun) and electroporation (28-33).
  • viral gene delivery systems are adenoviruses, gutless adenoviruses, adeno-associated viruses, human and murine retroviruses, vaccinia viruses, herpesviruses, baculoviruses, and canary pox viruses Reference is made to (34-39), particularly preferred is the gene transfer system adenoviral gene transfer system.
  • the nucleic acid construct described in the present application is suitable for use in medicine, for example in human medicine, but possibly also in veterinary medicine.
  • the construct is preferably used for the application as a nucleic acid vaccine, in particular for the immunoprophylaxis and / or immunotherapy of papillomavirus-induced diseases, for example cervical cancer or premalignant precursors thereof.
  • a nucleic acid construct is selected which contains sequences from oncoproteins of two or more different HPV serotypes, in particular tumor-associated HPV serotypes such as HPV16 and HPV18. Such a construct can confer immunity to at least two or more different HPV serotypes.
  • nucleic acid constructs according to the invention integrated into suitable gene transfer systems is carried out by known methods.
  • non-viral gene delivery systems such as naked DNA, liposomes, e.g. neutral or cationic liposomes, DNA polycations, e.g. Polylysine complexes optionally coupled to transferrin, or the administration of viral gene delivery systems such as adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, retroviruses, e.g. Murine or human retroviruses, optionally complexed with polycations, can be prepared by known methods, as described, for example, in (24-27).
  • nucleic acid construct according to the invention can be administered alone or in combination with at least one further pharmaceutically active substance, for example a further pharmaceutically active nucleic acid.
  • nucleic acids may be selected from Nucleic acids encoding a cytokine such as TNF- ⁇ , an interleukin, eg, IL-2 or IL-12, or an interferon, eg, interferon- ⁇ or interferon- ⁇ (40-42).
  • the additional pharmaceutically active nucleic acid may be selected from nucleic acids that provide enhanced MHC-mediated presentation of epitopes, such as accelerated ribosomal degradation, eg, by ubiquitinylation (43) or reconstitution of the function of TAPs (transporters associated in antigen presentation in tumor cells (44)).
  • the further pharmaceutically active substance may also be a costimulatory molecule, such as B7, ICAM1, an LFA-3 heat shock protein, CD137-L, CD134-L, GITR-L or CD40, the costimulatory molecule preferably being in a soluble form
  • a costimulatory molecule such as B7, ICAM1, an LFA-3 heat shock protein, CD137-L, CD134-L, GITR-L or CD40
  • the costimulatory molecule preferably being in a soluble form
  • costimulatory molecules are described in (45), (46) and in US 2006/0171949, the disclosure of which is incorporated herein by reference.
  • the molecule is an activator of the GITR signaling pathway, in particular a GITR receptor-binding agonistic antibody or a soluble form of the GITR ligand (GITR-L).
  • GITR-L GITR receptor-binding agonistic antibody
  • GITR-L soluble form of the GITR ligand
  • Examples of corresponding GITR binding molecules are described in US 2007/0098719, the disclosure of which is incorporated herein by reference.
  • the amino acid sequence of the human GITR receptor is in GenBank Acc. No. AA 52387 (SEQ ID No. 5) and the amino acid sequence of the human GITR ligand is in GenBank Acc. No. AAQ 89227 (SEQ ID No. 6).
  • the vaccine according to the invention can also be used together with adjuvants, such as bacterial toxins, e.g. Cholera toxin or thermostable E. coli enterotoxin (47), chemical adjuvants (48) or with cytokines, e.g. with GM-CSF (49).
  • adjuvants such as bacterial toxins, e.g. Cholera toxin or thermostable E. coli enterotoxin (47), chemical adjuvants (48) or with cytokines, e.g. with GM-CSF (49).
  • polypeptide encoded by a nucleic acid of the invention is a polypeptide encoded by a nucleic acid of the invention.
  • the polypeptide according to the invention can be used directly as an immunotherapeutic agent, in medicine, in particular for the immunoprophylaxis and / or immunotherapy of papillomavirus-induced diseases, ie, as a polypeptide vaccine with adjuvants, as previously also for the Nucleic acid construct described.
  • the administration of polypeptide vaccines is described, for example, in WO 93/00436 or WO 96/02677, the disclosure of which is incorporated herein by reference.
  • the polypeptide of the invention is preferably produced by recombinant expression of the nucleic acid construct of the invention in suitable host cells, e.g. in prokaryotic cells such as E. coli, or in eukaryotic cells such as yeast cells, insect cells or mammalian cells, prepared by known methods.
  • suitable host cells e.g. in prokaryotic cells such as E. coli, or in eukaryotic cells such as yeast cells, insect cells or mammalian cells, prepared by known methods.
  • nucleic acid construct with the polypeptide can also be accomplished.
  • Yet another aspect of the invention relates to a combination of agents comprising (i) a nucleic acid vaccine, preferably, a nucleic acid vaccine intended for tumor prophylaxis and / or tumor therapy, and (ii) an activator of the GITR signaling pathway.
  • a nucleic acid vaccine preferably, a nucleic acid vaccine intended for tumor prophylaxis and / or tumor therapy
  • an activator of the GITR signaling pathway GITR activators.
  • Preferred GITR activators are described in US 2006/0171949 and US 2007/0098719, the disclosure of which is incorporated herein by reference.
  • a GITR agonist can be used in all situations where improved reactivity of cytotoxic T cells is required. This applies to all types of chronic persistent infections with pathogens, e.g. chronic hepatitis, AIDS, leprosy or herpes infections, as well as the course of all acute infections with intracellular pathogens.
  • a GITR agonist can help overcome immunological tolerance to any type of malignant tumor alone or in combination with a suitable vaccine, particularly a nucleic acid vaccine.
  • the administration of the individual components of the drug combination can be sequential or simultaneous.
  • Example 1 Cloning of a recombinant therapeutic vaccine gene without transformation-associated peptide motifs
  • An oncogene-encoded protein often mediates its oncogenic effect by binding to the body's own cellular proteins and influencing their physiological action or rapidly degrading them.
  • FIG. 1 shows schematically the protein binding domains present in the oncoprotein HPV18-E6 and their localization
  • Binding studies with synthetic peptides such as LxCxE (AS 22-26 in HPV16-E7) or the PDZ domain-binding RRETQL at the C-terminus of E6 (AS 153-158 in HPV16-E6) document the high affinity of these peptide motifs for cell cycle proteins such as Rb / Histone deacetylase complex or various PDZ domain-containing tumor suppressor proteins such as MAGI-1 or SAP97 / dlg (26,32,37).
  • the therapeutic vaccine gene (p14) 14 DNA fragments from HPV16 and HPV18 E6 and E7 oncogenes were selected and cloned in a mirror image for placement in the wild type genes, taking into account the correct reading frame. Each of the 14 DNA fragments encodes 21-36 amino acids (Table 2). The adenoviral basis vectors and the cloning strategy were used as described in (50).
  • the entire vaccine gene codes for a 415 amino acid (1248 bp).
  • the nucleic acid and amino acid sequence of p14 are shown in Figure 2 (SEQ ID No. 7/8).
  • Expression is under the control of a CMV promoter and a CMV polyadenylation signal.
  • the exclusion of potentially dangerous sequence motifs inevitably leads to the loss of potential T-cell epitopes.
  • the oncogene fragments selected to clone the vaccinating gene were attempted to minimize the loss of therapeutically relevant T-cell epitopes by limiting the inserted deletions to the exact extent of the identified binding motifs.
  • the table shows the presence (1) or absence (0) of the highest affinity T cell epitopes of the p14 vaccine in relation to the four most common MHC class I alleles in the population.
  • the presented p14 vaccine still codes for all 5 of the most affective HLA-A0201 epitopes (about 50% of the population carry this MHC class 1 locus) as well as the 4 most frequent MHC classes 7 of 8 of the highest affinity epitopes.
  • HPV16-E6 HPV18-E6
  • Table 4 The p14 vector no longer shows any transforming activity compared to the starting constructs.
  • FIG. 3 shows that the precipitation of LxCxE-bearing GSTpI 4 fusion constructs after incubation with nuclear extracts leads to a decrease in the Histone deacetylase activity in the supernatant.
  • the effect is independent of the position of the LxCxE motif within the p14 protein ( ⁇ , ⁇ , ⁇ ).
  • Example 2 Animal studies on the prophylactic and therapeutic efficacy of the recombinant HPV vaccine
  • the construct was incorporated into a replication-deficient adenovirus.
  • C57BL6 mice were vaccinated by a single intramuscular administration of 200 .mu.l of an Ad-p14 virus suspension (1 x 10 10 infectious particles).
  • Ad-p14 virus suspension (1 x 10 10 infectious particles).
  • the animals were subcutaneously administered with 1 ⁇ 10 6 HPV16 E6 and E7-expressing C3 or TC1 tumor cells.
  • the animals in the control group had previously been treated instead of Ad p14 with a recombinant adenovirus carrying a reporter gene (bacterial betagalactosidase; lacZ) instead of the p14 gene.
  • Table 5 shows that all animals treated by Ad-p14 single vaccination are protected from an increase in HPV-E6 / E7-expressing C3 and TC1 tumors, respectively, whereas 8 out of 10 or 10 out of 10 animals in the control groups has a fast Show tumor growth.
  • Figure 4a shows that all 10 tumor-bearing mice (C3 tumors) are permanently cured by a single dose of Ad-p14, while the animals treated with control virus (Ad-lacZ) show an unrestrained tumor growth.
  • FIG. 4b shows that, after therapeutic vaccination of tumor-bearing mice (TC1 tumors), tumor volume first decreases. Thereafter, however, the tumors grow out again and only 1 in 10 animals is permanently cured by the vaccine.
  • Figure 5 shows the effect of administering various immunomodulators to increase the therapeutic effect 8 days after therapeutic vaccination with Ad-p14.
  • the combination of vaccination and administration of the DTA-1 antibody leads to complete clearance of the tumors in all tumor-bearing mice (10/10) while the sole administration of the antibody 3 out of 10 animals and the sole vaccination with Ad-p14 only 1 of 10 TC1 - heals tumor-bearing mice.
  • the GITR signaling pathway plays a central role in the attenuation and modulation of immune responses in the murine (16-18) and human immune systems (19).
  • the physiological effect of activating the signaling pathway is strongly cell type dependent. Its activation on murine regulatory T cells leads to a reduction of its immunosuppressive effect on other T lymphocytes, thus weakening the tumor's own immune defense.
  • activation of the GITR signaling pathway on cytotoxic T cells induces an increase in activity.
  • specifically reactive T cells generated as part of a previous vaccination provide improved access to the tumor and, at the same time, enhance it Cytotoxic activity an increase in therapeutic efficacy, which can bring about the decisive therapeutic turn not only in the presented animal model (TC1 tumors), but also in a clinical application.
  • Kanda T Zanma S, Watanabe S et al. Two immunodominant regions of the human papillomavirus type 16 E7 proteins are masked in the nuclei of monkey COS-1 cells. Virology. 1991; 182 (2): 723-731.
  • CD4 + CD25 + regulatory T cells J. Leukocyte Biology. 2007; 82: 93-105. 20. Puigbo et al. Nucleic Acids Research 2007 Vol. 35 W126-W131.
  • CEA transgenic mice Vaccine 21 (2003), pp. 1938-1947. 30. HJ. Mollenkopf, G. Dietrich, J. Novale, L. Grode, KD Diehl and B. Knapp et al., Enhanced protective efficacy of a tuberculosis DNA Vaccine by adsorption on cationic PLG microparticles, Vaccine 22 (2004), pp. 2690-2695.
  • Alimonti et al. TAP expression provides a general method for improving the recognition of malignant cells in vivo Nature Biotechnology vol 18 (2000) pp 515-520).
  • Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines, J Viral 76 (2002), pp. From 4536 to 4546.

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Abstract

La présente invention concerne un acide nucléique, qui code pour une ou plusieurs oncoprotéines du virus du papillome humain (VPH) ou des fragments de celles-ci. L'acide nucléique peut, après expression dans un organisme de mammifère, induire une réponse immunitaire, qui conduit à la destruction de cellules exprimant les oncoprotéines de VPH et, en conséquence, est approprié pour la prophylaxie et/ou le traitement de maladies induites par le VPH, de même que le cancer du col de l'utérus et son stade préliminaire. Vis-à-vis des vaccins à acide nucléique connus, le produit de construction selon l'invention se caractérise par un concept de sécurité, qui allie une immunogénicité thérapeutique élevée avec une sécurité biologique maximale et, par conséquent, crée la condition pour une utilisation clinique large et sans danger.
PCT/EP2009/001576 2008-02-25 2009-02-24 Vaccin à adn pour la thérapie et la prophylaxie du cancer du col de l'utérus et son stade préliminaire pré-malin WO2009106362A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US3105708P 2008-02-25 2008-02-25
DE102008010954.1 2008-02-25
US61/031,057 2008-02-25
DE200810010954 DE102008010954A1 (de) 2008-02-25 2008-02-25 DNA-Vakzine zur Therapie und Prophylaxe von Gebärmutterhalskrebs und seinen prämalignen Vorstufen

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WO2011127924A2 (fr) 2010-04-16 2011-10-20 Charité - Universitätsmedizin Berlin Agent de traitement local de dysplasies cervicales
EP2558116B1 (fr) 2010-04-16 2017-03-22 Charité Universitätsmedizin Berlin Agent de traitement local de dysplasies cervicales
EP2604629A4 (fr) * 2010-08-13 2014-01-15 Genexine Inc Composition contenant un plasmode du papillomavirus humain et un promoteur d'immunité destinée à prévenir ou traiter le cancer du col de l'utérus
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EP2604629A1 (fr) * 2010-08-13 2013-06-19 Genexine Co., Ltd. Composition contenant un plasmode du papillomavirus humain et un promoteur d'immunité destinée à prévenir ou traiter le cancer du col de l'utérus
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WO2016071306A1 (fr) * 2014-11-04 2016-05-12 Crucell Holland B.V. Vaccins thérapeutiques contre le vph16
JP2017534286A (ja) * 2014-11-04 2017-11-24 ヤンセン ファッシンズ アンド プリベンション ベーフェーＪａｎｓｓｅｎ Ｖａｃｃｉｎｅｓ ＆ Ｐｒｅｖｅｎｔｉｏｎ Ｂ．Ｖ． 治療用ｈｐｖ１６ワクチン
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JP2018148890A (ja) * 2014-11-04 2018-09-27 ヤンセン ファッシンズ アンド プリベンション ベーフェーＪａｎｓｓｅｎ Ｖａｃｃｉｎｅｓ ＆ Ｐｒｅｖｅｎｔｉｏｎ Ｂ．Ｖ． 治療用ｈｐｖ１６ワクチン
EP3421046A1 (fr) 2014-11-04 2019-01-02 Janssen Vaccines & Prevention B.V. Vaccins hpv16 thérapeutiques
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EA035461B1 (ru) * 2014-11-04 2020-06-19 Янссен Вэксинс Энд Превеншн Б.В. Терапевтические вакцины против hpv16
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WO2017029360A1 (fr) * 2015-08-20 2017-02-23 Janssen Vaccines & Prevention B.V. Vaccins thérapeutiques contre le vph 18
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