WO2001093804A2 - Conjugues du virus de l'hepatite c - Google Patents

Conjugues du virus de l'hepatite c Download PDF

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WO2001093804A2
WO2001093804A2 PCT/US2001/017302 US0117302W WO0193804A2 WO 2001093804 A2 WO2001093804 A2 WO 2001093804A2 US 0117302 W US0117302 W US 0117302W WO 0193804 A2 WO0193804 A2 WO 0193804A2
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seq
conjugate
hcv
immunogenic
ompc
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PCT/US2001/017302
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WO2001093804A3 (fr
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Anthony J. Conley
Philip M. Mckenna
Craig T. Przysiecki
Paul M. Keller
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Merck & Co., Inc.
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Priority to EP01939609A priority Critical patent/EP1290230A4/fr
Priority to JP2002501378A priority patent/JP2004509846A/ja
Priority to CA002409287A priority patent/CA2409287A1/fr
Priority to US10/296,353 priority patent/US20030224011A1/en
Publication of WO2001093804A2 publication Critical patent/WO2001093804A2/fr
Publication of WO2001093804A3 publication Critical patent/WO2001093804A3/fr

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    • 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
    • A61K39/29—Hepatitis virus
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • HCV hepatitis C virus
  • HCV protein and artificial peptides Recombinant HCV protein used to elicit antibodies recognizing HCV include structural proteins El and E2. (Choo, et al, Proc. Natl. Acad. Sci. USA, 97:1294-1298, 1994.) Artificial peptides having amino acid sequences different from that found in HCV and yet able to generate antibodies recognizing HCV protein have been selected using phage display libraries. (Prezzi, et al., The Journal of Immunology .756-4504-4513, 1996; and Puntoriero, et al. EMBO Journal 77:3521-3533, 1998.) Such artificial peptides are referred to as HCV mimotopes.
  • the present invention features HCV conjugates able to induce an immune response recognizing different strains and variants of HCV.
  • the conjugates contain a polypeptide or protein complex carrier and one or more HCV mimotopes.
  • Preferred HCV mimotopes provide antigens able to generate antibodies recognizing the hypervariable region of the HCV E2 protein.
  • a first aspect of the present invention describes an HCV conjugate.
  • the conjugate comprises a polypeptide or protein complex carrier and an immunogenic HCV peptide, or a pharmaceutically acceptable salt thereof.
  • the HCV peptide is covalently joined to the carrier. Additional groups such as other types of HCV peptides, reactive site capping groups, and anions may also be attached to the carrier.
  • Covalently joined refers to the presence of a covalent linker that is stable to hydrolysis under physiological conditions.
  • the covalent linker is stable to other reactions that may occur under physiological conditions including adduct formation, oxidation, and reduction.
  • HCV peptide comprises one or more antigenic determinants that can elicit an immune response recognizing HCV.
  • Preferred antigenic determinants are HCV mimotopes that can elicit antibodies recognizing different strains and variants of HCV.
  • Examples of HCV peptides are those comprising the HCV mimotope sequence of SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6, or SEQ. ID. NO. 7.
  • the HCV conjugate comprises: a polypeptide or protein complex carrier, immunogenic HCV peptide PEPi, and immunogenic HCV peptide PEP2, wherein PEPi and PEP2 are each covalently joined to the carrier though an independently selected covalent linker and comprises a different sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6, and SEQ. ID. NO. 7; or a pharmaceutically acceptable salt thereof.
  • SEQ. ID. NO. 1 amino acid
  • SEQ. ID. NO. 2 amino acid
  • SEQ. ID. NO. 3 amino acid peptide
  • SEQ. ID. NO. 4 amino acid peptide
  • the HCV conjugate may contain additional peptides including, but not limited, to peptides of SEQ. ID. NOs. 1-7.
  • Another aspect of the present invention describes an HCV conjugate produced by a process comprising: (a) joining a plurality of linkers to reactive sites on a polypeptide or protein complex carrier; (b) joining two or more different HCV immunogenic peptides to the plurality of linkers; and (c) capping the product of step (b). Capping is achieved by attaching a capping group to linkers not reacted with immunogenic peptides.
  • Each of the two or more different HCV immunogenic peptides comprises either SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6, or SEQ. ID. NO. 7.
  • Reactive sites are groups capable of conjugation.
  • Preferred reactive sites are nucleophilic sites, such as the epsilon a ino group of lysine.
  • a plurality of linkers refers to more than one linker.
  • the process can contain additional steps such as joining one or more anions and joining additional HCV immunogenic peptides other than those comprising SEQ. ID. NOs. 1-7. Such additional HCV immunogenic peptides are in addition to the two or more different HCV immunogenic peptides of SEQ. ID. NOs. 1-7.
  • the first HCV conjugate comprises a polypeptide or protein complex carrier covalently joined to a first immunogenic HCV peptide comprising SEQ. ID. NO. 1, to a second immunogenic HCV peptide comprising SEQ. ID. NO. 2 and to a third immunogenic HCV peptide comprising SEQ. ID. NO. 4, or a pharmaceutically acceptable salt thereof; and the second conjugate comprises an immunogenic HCV peptide comprising SEQ. ID. NO. 7, or a pharmaceutically acceptable salt thereof.
  • the first and second HCV conjugates may contain additional groups attached to the respective carriers such as additional types of HCV peptides, reactive site capping groups, and anions.
  • Another aspect of the present invention describes a method of inducing an immune response in a subject.
  • the method comprises the step of inoculating the subject with an effective amount of an HCV conjugate described herein.
  • Another aspect of the present invention describes a method of making an HCV conjugate containing multiple immunogenic HCV peptides. The method comprises the step of simultaneously conjugating the peptides to a carrier.
  • Another aspect of the present invention describes an antisera containing antibodies recognizing HCV.
  • the antisera is made by a process comprising the steps of: (a) inoculating a subject with an effective amount of an HCV conjugate described herein to produce antibodies; and (b) removing the antibodies from the subject.
  • the present invention features HCV conjugates containing a polypeptide or protein complex carrier and one or more HCV mimotopes.
  • the conjugates are able to elicit antibodies recognizing the HCV mimotope used to induce the immune response and also recognizing different strains and variants of HCV.
  • Different techniques can be employed to use an antigen to elicit an immune response.
  • the examples provided below include data illustrating benefits to using an immunogenic protein carrier complex and to using particular antigens or antigen combinations.
  • HCV mimotope delivered to a subject as part of a conjugate containing immunogenic polypeptide or protein complexes have advantages in eliciting an immune response compared to the same HCV mimotope peptide delivered to the subject using other techniques such as chimeric mimotope-E2 proteins and DNA vectors of chimeric mimotope-E2 proteins.
  • antigen combinations were found that were more effective in eliciting cross-reacting antibodies when present on a single conjugate than when present in a cocktail containing individual conjugates. Such antigen combinations are preferably produced using simultaneous conjugation techniques.
  • HCV conjugates are useful for eliciting an immune response in a subject.
  • the immune response results in the production of antibodies recognizing HCV.
  • the production of the immune response and the resulting antibodies can be employed in therapeutic or diagnostic applications.
  • a “subject” refers to a mammal capable of producing antibodies. Examples of subjects include humans, chimpanzees, mice and horses. Preferred subjects are humans. Other subjects, even those that do not become infected with HCV, can be used to generate anti-HCV antibodies.
  • Diagnostic applications include using HCV antibodies for detecting the presence of HCV in a test subject. Such antibodies can be obtained from subjects that have been induced to generate an immune response using an HCV conjugate described herein.
  • Therapeutic applications include treating a subject infected with HCV and prophylactically treating a subject. Such applications involve the use or production of HCV neutralizing antibodies.
  • the ability of HCV conjugates described herein to produce antibodies reacting with different strains and variants of HCV is useful for treating or hindering initial infections of HCV, and for guarding against mutated forms of HCV produced in vivo.
  • An immunogenic HCV peptide comprises one or more HCV mimotopes and may also contain additional groups.
  • Preferred additional groups are those enhancing an immune response and those involved in conjugation to a carrier. Additional groups that may be useful in enhancing an immune response include immunogenic peptides such as those containing an immunostimulatory invasin domain or a helper T cell epitope. (E.g., see, Wang, International Publication Number
  • HCV mimotopes can be produced and incorporated into an HCV conjugate using standard conjugation techniques.
  • HCV peptides can be made up of common amino acids found in proteins and unnatural amino acids not normally found in proteins. Common amino acids found in proteins are the L-enantiomer (except for glycine which does not have a chiral center). Standard nomenclature used to reference common amino acids found in proteins is as follows:
  • HCV conjugates may contain one or more different immunogenic HCV peptides.
  • at least one immunogenic HCV peptide comprises, consists essentially of, or consists of, an HCV mimotope sequence that is either SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6 or SEQ. ID. NO. 7.
  • Reference to "consists essentially” indicates that additional groups may be present that do not substantially effect the ability of the mimotope to elicit an immune response. Examples of additional groups encompassed by reference to consists essentially of include reporter amino acids and groups involved in conjugation.
  • Hl- SEQ. ID. NO. 4 XTHTTGGVVGHATSGLTSLFSPGPSQK
  • immunogenic HCV peptides comprise, consist essentially of, or consist of, the following sequences: SEQ. ID. NO. 8 XTHTTGGQAGHQAHSLTGLFSPGAKQNXl ⁇ 2;
  • SEQ. ID. NO. 10 XTTVVGGSQSHTVRGLTSLFSPGASQNXl ⁇ 2; SEQ. ID. NO. il XTHTTGGVVGHATSGLTSLFSPGPSQKXl ⁇ 2; SEQ. ID. NO. 12 TTTTTGGQVGHQTSGLTGLFSPGAQQNXl ⁇ 2; SEQ. ID. NO. 13 TTTTTGGVQGHTTRGLVRLFSLGSKQNXl ⁇ 2; and SEQ. ID. NO. 14 XTHTTGGVVSHQTRSLVGLFSPGPQQNXl ⁇ 2; SEQ. ID. NOs. 8-14 corresponds to SEQ. ID. NOs.
  • Xl ⁇ 2 where ⁇ l is a reporter amino acid and ⁇ 2 is a sulfur containing amino acid.
  • Each X for SEQ. ID. NOs. 8-14 is either glutamine or pyroglutamate, preferably glutamine.
  • a reporter amino acid is an amino acid other than one of the twenty common amino acids listed above. Examples of reporter amino acids are as follows: Nva - norvaline; Nle - norleucine; Abu - 2-aminobutyric acid; Dbu - 2,4- diaminobutyric acid; Dpr - 2,3 diaminopropionic acid; and Chg - cyclohexylglycine.
  • a sulfur containing amino acid contains a reactive sulfur group.
  • sulfur containing amino acids include cysteine and unnatural amino acids such as homocysteine. Additionally, the reactive sulfur may exist in a disulfide form prior to activation and reaction with carrier.
  • immunogenic HCV peptides comprise, consist essentially of, or consist of, the following sequences: SEQ. ID. NO. 15 XTHTTGGQAGHQAHSLTGLFSPGAKQNX3C; SEQ. ID. NO. 16 XTTTTGGQVSHATHGLTGLFSLGPQQKX3C; SEQ. ID. NO. 17 XTTVVGGSQSHTVRGLTSLFSPGASQNX3C; SEQ. ID. NO. 18 XTHTTGGVVGHATSGLTSLFSPGPSQKX3C; SEQ. ID. NO. 19 TTTTTGGQVGHQTSGLTGLFSPGAQQNX3C; SEQ. ID. NO. 20 TTTTTGGVQGHTTRGLVRLFSLGSKQNX3C; and SEQ. ID. NO. 21 XTHTTGGWSHQTRSLVGLFSPGPQQNX3C.
  • SEQ. ID. NOs. 15-21 correspond to SEQ. ID. NOs. 8-14 were the ⁇ l reporter group (shown in SEQ. ID. NOs. 15-21 as ⁇ 3) is either norvaline, norleucine, 2-aminobutyric acid, 2,4-diaminobutyric acid, 2,3 diaminopropionic acid, or cyclohexylglycine; and the X sulfur containing amino acid is cysteine.
  • Each X for SEQ. ID. NOs. 15-21 is either glutamine or pyroglutamate, preferably glutamine.
  • SEQ. ID. NOs. 22 and 23 examples of an immunogenic HCV peptide containing more than one mimotope is provided by SEQ. ID. NOs. 22 and 23.
  • SEQ. ID. NOs. 22 and 23 which contains mimotopes of both SEQ. ID. NOs. 1 and 7 are as follows: SEQ. ID. NO. 22 XT-HTTGGQAGHQAHSLTGLFSPGAKQN QTHTTGGVVSHQTRSLVGLFSPGPQQNXl ⁇ 2;
  • Peptides can be produced using techniques well known in the art. Such techniques include chemical and biochemical synthesis. Examples of techniques for chemical synthesis of polypeptides are provided in Vincent, in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990. Examples of techniques for biochemical synthesis involving the introduction of a nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989.
  • HCV mimotopes are covalently joined to a polypeptide or protein complex carrier.
  • Carriers can be used to provide a scaffold for producing conjugates containing multiple antigens and to enhance the immune response.
  • Preferred polypeptide and protein complex carriers are immunogenic carriers able to enhance an immune response. Enhancement of the immune response can come from, for example, providing T helper cell epitopes or providing adjuvant activity.
  • immunogenic carriers include the Outer Membrane Protein Complex of Neisseria meningitidis (OMPC), human serum albumin, tetanus toxoid, MMP-derived from OMPC, diptheria toxoid, hepatitis B virus surface antigen, hepatitis B virus core antigen, and human rotavirus VP6 capsid protein.
  • a preferred carrier is OMPC.
  • OMPC contains numerous groups available for conjugation. The availability of a group for conjugation includes the type of group present and the position of the group in OMPC. Nucleophilic functionalities available for conjugation can be determined using techniques well know in the art. (See Emini, et al. U.S. Patent No. 5,606,030.) One type of group that can be used for conjugation is primary amino groups present on amino acids such as the epsilon amino group of lysine and the alpha amino group of protein N-terminal amino acids. OMPC can be obtained using techniques well known in the art such as those described by Fu, U.S. Patent No. 5,494,808.
  • the present application identifies preferred combinations of HCV mimotopes, and a preferred mimotope, that can be used to elicit antibodies cross reacting to different strains and variants of HCV. While combinations of mimotopes can be used as cocktails of different conjugates, it appears that greater efficacy can be achieved by producing a multiple conjugate containing a combination of different mimotopes.
  • the conjugate comprises, consists essentially or, consists of, two or more different types of immunogenic HCV peptides joined to the carrier, one or more capping groups joined to the carrier, and may contain one or more anions joined to the carrier.
  • at least three different types of immunogenic HCV peptides are present, three different types of immunogenic HCV are present, four different types of immunogenic HCV peptides are present, five different types of immunogenic HCV peptides are present, or six different types of immunogenic HCV peptides are present.
  • Multiple conjugates preferably contain two or more immunogenic
  • HCV peptides that comprise or consist essentially of a different HCV mimotope sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2,
  • SEQ. ID. NO. 3 SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6, and SEQ. ID.
  • Each of the immunogenic HCV peptides can be joined to the carrier through an independently selected covalent linker.
  • such peptides are conjugated to
  • immunogenic HCV peptide combinations include those comprising the following sequence: (1) SEQ. ID. NOs. 1, 2, and 4; (2) SEQ. ID. NOs.
  • OMPC represents the Outer Membrane Protein Complex of Neisseria meningitidis
  • anion is a low molecular weight moiety having an anionic character at physiological pH; preferably, the anion is an ionized form of carboxylic, sulfonic, propionic or phosphonic acid;
  • Li is a covalent linker joining PEPi to OMPC, wherein each Li that is present may be the same or different;
  • L2 is a covalent linker joining PEP2 to OMPC, wherein each L2 that is present may be the same or different;
  • L3 is a covalent linker joining PEP3 to OMPC, wherein each L3 that is present may be the same or different;
  • L4 is a covalent linker joining PEP4 to OMPC, wherein each L4 that is present may be the same or different;
  • L5 is a covalent linker joining PEP5 to OMPC, wherein each L5 that is present may be the same or different;
  • L ⁇ is a covalent linker joining PEP6 to OMPC, wherein each L ⁇ that is present may be the same or different;
  • L7 is a covalent linker joining anion to OMPC, wherein each L7 and each anion that is present may be the same or different;
  • L8 is a covalent linker joining capping group to OMPC, wherein each L8 and each capping group that is present may be the same or different;
  • PEPi, PEP2, PEP3, PEP4, PEP5, and PEP6, are immunogenic peptides, provided that at least two of PEPi, PEP2, PEP3, PEP4, PEP5, and PEP6 comprise a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, and SEQ. ID. NO. 6; a, b, c, d, e, f, g, and h is each an individually selected coupling load, wherein a, b, c, and h are each greater than 0; or a pharmaceutically acceptable salt thereof.
  • a capping group is a chemical moiety that can react with a linker reactive group and inhibits the ability of the group to which it is attached to undergo further reaction.
  • Suitable capping groups can readily be obtained for different linkers.
  • suitable capping groups for maleimide activated OMPC include thiol containing compounds such as N-acetylhomocysteine, n-acetylcysteine, and mercaptoethanol.
  • thiol containing compounds such as N-acetylhomocysteine, n-acetylcysteine, and mercaptoethanol.
  • Different types of capping groups can be employed including those that are, or not, anionic in character.
  • the capping groups present in a conjugate have the same chemical structure.
  • the "coupling load” is equal to the moles of a particular group divided by moles of lysine times 100.
  • the coupling load of a + b + c + d + e + f + g + h is about 25.
  • the coupling load of a + b + c + d + e + f is preferably about 5 to about 20, wherein the coupling load of individual members that are present is preferably at least about 0.2.
  • the overall coupling load a + b + c + d + e + f is at least 6, 7, or 8 and is no greater than 18, 19, or 20; and the coupling load of a, b, c, d, e, and f, when present (i.e., greater than zero), is individually at least about 0.4, preferably at least about 1.
  • each of Li, L2, L3, L4, L5, L6, L7, and Lg that are present independently have the structure:
  • R is selected from the group consisting of alkylene, substituted alkylene, and phenyl; one of Rl and R2 is either hydrogen, alkyl, substituted alkyl or -SO3H, and the other of Ri and R2 is the position to which PEPi, PEP2, PEP3, PEP4, PEP5, PEP6, anion or capping group is joined; preferably one of Rl and R2 is hydrogen; and each anion is either carboxylic, sulfonic, propionic, or phosphonic acid.
  • Alkylene refers to a hydrocarbon group containing only carbon- carbon single bonds between the carbon atoms.
  • the alkylene hydrocarbon group contains 1 to 12 carbon atoms and may be straight-chain or contain one or more branches or cyclic groups.
  • the alkylene group is attached at two locations to other functional groups or structural moieties.
  • the alkylene group is either -(CH2)0-3-C6Hl0-(CH2)0-3--- or -(CH2) ⁇ -3-C5H8-(CH2) ⁇ -3". More preferably, the alkylene is either -C ⁇ Hio-, or -C6H10-CH2-.
  • Substituted alkylene refers to an alkylene where one or more of the hydrogens is replaced with a moiety that is either -NH2, -NHCOCH3, alkyl amino, carboxy, carboxy alkyl, sulphono, phosphono, halogen, -OH, -CN, -SH, -NO , -C ⁇ - 2 alkyl substituted with 1 to 5 halogens, -CF 3 , -OCH 3 , or -OCF 3 .
  • Alkyl refers to a hydrocarbon group consisting of 1 to 8 carbon atoms joined by carbon-carbon single bonds.
  • the alkyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups.
  • the alkyl is one to four carbon atoms.
  • Substituted alkyl refers to an alkyl where one or more of the hydrogens is replaced with a moiety that is either -NH2, -NHCOCH3, alkyl amino, carboxy, carboxy alkyl, sulphono, phosphono, halogen, -OH, -CN, -SH, -NO 2 , -C 1 . 2 alkyl substituted with 1 to 5 halogens, -CF 3 , -OCH 3 , or -OCF 3 .
  • each of PEPi, PEP2, PEP3, PEP4, PEP5, and PEP6 consists of a different sequence selected from the group consisting of SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, SEQ. ID. NO. 11, SEQ. ID. NO. 12, SEQ. ID. NO. 13, SEQ. ID. NO. 14 and SEQ. ID. NO. 22, or from the group consisting of SEQ. ID. NO. 15, SEQ. ID. NO. 16, SEQ. ID. NO. 17, SEQ. ID. NO. 18, SEQ. ID. NO. 19, SEQ. ID. NO. 20, SEQ. ID. NO. 21 and SEQ. ID. NO. 23; and is joined to OMPC though the sulfur on its carboxy terminus amino acid.
  • each of Li, L2, L3, L4, L5, L ⁇ , L7, and L ⁇ that are present independently have the following structure: wherein OMPC is joined at position "z", and Ri and R2 are as described above.
  • g is zero (i.e., the anion and L7 are not present).
  • the HCV conjugate contains a polypeptide or protein complex carrier and comprises, consists essentially of, or consists of, (1) an immunogenic HCV peptide that comprises, consists essentially of, or consists of, the HCV mimotope sequence of SEQ. ID. NO. 7; (2) one or more capping groups joined to the carrier: and (3) may contain one or more anions joined to the carrier.
  • OMPC represents the Outer Membrane Protein Complex of Neisseria meningitidis; each anion is a low molecular weight moiety having an anionic character at physiological pH; preferably, the anion is an ionized form of carboxylic, sulfonic, propionic or phosphonic acid;
  • Li is a covalent linker joining the immunogenic HCV peptide to OMPC, wherein each Li may be the same or different;
  • L2 is a covalent linker joining the anion to OMPC, wherein each L2 and each anion that is present may be the same or different;
  • L3 is a covalent linker joining the capping group to OMPC, wherein each L3 and each capping group that is present may be the same or different; n, m, and p is each an individually selected coupling load, wherein n and p are each greater than 0; or a pharmaceutically acceptable salt thereof.
  • the coupling load of n + m + p is about 25.
  • n is at least about 0.4, at least about 1, at least about 3, at least 5, or at least about 8 and is no greater than about 20 or about 18.
  • each of Li, L2 and L3 independently contains the following structure:
  • R is selected from the group consisting of alkylene, substituted alkylene, and phenyl; and one of R3 and R4 is either hydrogen, alkyl, substituted alkyl or -SO3H, and the other of R3 and R4 is the position to which the immunogenic peptide, anion, or capping group binds; preferably one of R3 and R4 is hydrogen.
  • the immunogenic HCV peptide consists of SEQ. ID. NO. 14 or SEQ. ID. NO. 20 and is joined to OMPC though the sulfur group of its carboxy terminus amino acid.
  • Li, L2 and L3 independently have the following structure:
  • each anion that is present is either carboxylic, sulfonic, or phosphoric acid, and no anions are present (m is zero and L 2 is not present).
  • Immunogenic HCV peptides containing HCV mimotopes can be conjugated to polypeptide and protein complex carriers through a covalent linker using conjugation techniques well known in the art. (See, Tolman, et al, U.S. Patent No. 5,274,122, Emini, et al, U.S. Patent No. 5,606,030, and Conley, et al., U.S. Patent No. 5,763,574.)
  • the conjugates can also be modified, for example, to contain one or more anionic groups.
  • a covalent linker joining an immunogenic HCV peptide or anion to a carrier is stable under physiological conditions.
  • linkers are nonspecific cross-linking agents, monogeneric spacers and bigeneric spacers.
  • Non-specific cross-linking agents and their use are well known in the art.
  • Examples of such reagents and their use include reaction with glutaraldehyde; reaction with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide, with or without admixture of a succinylated carrier; periodate oxidation of glycosylated substituents followed by coupling to free amino groups of a protein carrier in the presence of sodium borohydride or sodium cyanoborohydride; diazotization of aromatic amino groups followed by coupling on tyrosine side chain residues of the protein; reaction with isocyanates; or reaction of mixed anhydrides. See, generally, Briand, et al, J. Mm. Meth. 78:59, 1985.
  • Monogeneric spacers and their use are well known in the art. Monogeneric spacers are bifunctional and require functionalization of only one of the partners of the reaction pair before conjugation takes place.
  • An example of a monogeneric spacer and its use involves coupling an immunogenic HCV peptide to one end of the bifunctional molecule adipic acid dihydrazide in the presence of carbodiimide. A diacylated hydrazine presumably forms with pendant glutamic or aspartic carboxyl groups of the carrier. Conjugation then is performed by a second coupling reaction with carrier protein in the presence of carbodiimide.
  • Bigeneric spacers and their use are well known in the art. Bigeneric spacers are formed after each partner of the reaction pair is functionalized. Conjugation occurs when each functionalized partner is reacted with its opposite partner to form a stable covalent bond or bonds. (See, for example, Marburg, et al. , J. Am. Chem. Soc. 705:5282-5287, 1986; and Marburg, et al., U.S. Patent No. 4,695,624.)
  • Process 1 la React a fraction of the protein carrier nucleophilic groups with a reagent to generate a thiol group.
  • a reagent is N-acetyl homocysteine thiolactone
  • lb React the product of step la with a reagent comprising an electrophile and an anion having a negative charge at physiological pH.
  • An example of such a reagent is maleimidoalkanoic acid
  • lc React the product of step lb with immunogenic peptides previously derivatized so as to append an electrophilic group.
  • a bifunctional electrophilic reagent is maleimidoalkanoic acid hydroxysuccinimide ester;
  • step 2b React the product from step 2a with a reagent comprising both a nucleophile and an anion.
  • a reagent comprising both a nucleophile and an anion.
  • An example of such a reagent is ⁇ -mercaptoacetic acid.
  • step 2c React the product of step 2b with immunogenic peptides containing a nucleophile, such as a thiol group.
  • reagents include N-(bromoacetyl)-6-amino caprole acid, and succinic anhydride;
  • step 3b React the residual fraction of nucleophilic groups on the product of step 3a with a reagent, for example with N-acetyl homocysteine thiolactone, which generates thiol groups on the protein carrier; and
  • Step 3c React the product of step 3b with immunogenic peptides previously derivatized so as to append an electrophilic group, for example, a group comprising maleimide.
  • Process 4 4a React a fraction of the protein carrier nucleophilic groups with a reagent comprising both an electrophile and an anion or incipient anion, such as N-(bromoacetyl)-6-amino caproic acid or succinic anhydride; 4b. React the residual protein nucleophilic groups on the product of step 4a with a bifunctional electrophilic reagent to append electrophilic sites onto the protein. Examples of such reagents are maleimidoalkanoic acid hydroxysuccinimide ester; and
  • step 4c React the product of step 4b with immunogenic peptides containing a nucleophilic group, such as a thiol.
  • Process 5 5a React a fraction of the protein carrier amino groups with a crosslinker having the structure:
  • R is selected from the group consisting of alkylene, substituted alkylene, and phenyl; and Ri is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and -SO3H; where alkylene, substituted alkylene, alkyl, and substituted alkyl are as described above, including preferred groups;
  • step 5b React the product of step 5a with immunogenic peptides containing a nucleophilic group, such as a thiol.
  • Processes 1-5 will generally involve additional steps such as removal of excess reagent from one step prior to commencing a second step and the capping of unreacted functionalities.
  • the Reaction Scheme provided below illustrates the use of process 5 along with additional steps to produce a conjugate.
  • the components listed in the Reaction Scheme such as the carrier, crosslinker, capping groups, and mimotope are for illustration purposes. Based on the present disclosure, one of ordinary skill in the art can replace the components in the Reaction Scheme with components well known in the art and components described herein.
  • N-acetylcystiene NAC
  • HCV conjugates can be formulated and administered to a subject using the guidance provided herein along with techniques well known in the art. Guidelines for pharmaceutical administration in general are provided in, for example, Modern Vaccinology, Ed. Kurstak, Plenum Med. Co. 1994; Remington's Pharmaceutical Sciences 18 th Edition, Ed. Gennaro, Mack Publishing, 1990; and Modern Pharmaceutics 2 nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990, each of which are hereby incorporated by reference herein.
  • HCV conjugates can be prepared as acidic or basic salts.
  • Pharmaceutically acceptable salts in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary -mimonium salts that are formed, e.g., from inorganic or organic acids or bases.
  • salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyan
  • Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the subject; the route of administration; the desired effect; and the particular compound employed.
  • the conjugates can be used in multi-dose vaccine formats. It is expected that a dose would consist of the range of 1 ⁇ g to 1.0 mg total protein, in an embodiment of the present invention the range is 0.1 mg to 1.0 mg.
  • the HCV conjugate is preferably formulated with an adjuvant.
  • adjuvants are alum, AIPO4, alhydrogel, Lipid-A and derivatives or variants thereof, Freund's complete or incomplete adjuvant, neutral liposomes, liposomes containing vaccine and cytokines or chemokines.
  • booster doses may subsequently be administered to maintain antibody titers.
  • An example of a dosing regime would be day 1, 1 month, a third dose at either 4, 6 or 12 months, and additional booster doses at distant times as needed.
  • each peptide consisted of 29 residues, including a 27-mer mimotope, a carboxy penultimate unnatural residue, and a carboxy terminal cysteine present as a carboxamide. Examples of such peptides include the following:
  • Each X for SEQ. ID. NOs. 24-31 is either glutamine or pyroglutamate, preferably glutamine.
  • the peptides were synthesized with X being glutamine.
  • peptides were synthesized as a 30-mer including an N-terminal glycine, cysteine, an unnatural residue, and a 27-mer mimotope sequence with the terminal residue present as a carboxamide.
  • a cyclized mimotope peptide For examining a cyclized mimotope peptide, the synthesis and cyclization followed a strategy published previously (Conley, et al, Vaccine 72:445- 451, 1994). Briefly, the peptide was synthesized with an N-terminal glycine, cysteine, lysine, followed by the 27-mer mimotope sequence, an additional penultimate carboxy terminal aspartic acid, and the terminal unusual amino acid. Condensation between the ⁇ -amino of lysine and the ⁇ -carboxyl of the aspartic acid yielded the cyclic product.
  • Custom synthesized hepatitis C virus region derived peptides were used as ELISA antigens. These were prepared either without (Table 1 A) or with (Table IB) a biotin group for standard peptide adsorption or strepavidin biotin capture ELISA formats, respectively.
  • N. meningitidis OMPC was activated at pH 8.0 (10 mM HEPPS buffer) by reaction with an excess of the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC,
  • the percent coupling load of mimotope peptide to total OMPC lysine was calculated from the ratio of the moles of unnatural amino acid in the mimotope over the moles of lysine multiplied by 100. Quantitation was based on amino acid analysis of the acid hydrolysed conjugate, 6 N HC1 for 20 hours at 110°C. For lysine- containing mimotopes a correction was made to the observed lysine value to obtain the OMPC lysine contribution. Protein concentrations were determined by Lowry assay using BSA as a standard. SDS-PAGE analysis was performed to provide evidence of covalent linkage of the mimotope peptides to protein components of OMPC. Coupling loads were in the range of 8 to 19%.
  • SCPN simultaneously conjugated peptide vaccines
  • Nva - norvaline Nle - norleucine
  • Abu 2-aminobutyric acid
  • Dbu 2,4-diaminobutyric acid
  • Dpr 2,3 diaminopropionic acid
  • Chg - cyclohexylglycine
  • Example 3 DNA Vector Immunogens Six individual expression plasmids were constructed to elicit immune responses by DNA delivery to primates. The six mimotope sequences, SEQ. ID. NOs. 1, 3, 4, 5, 6, and 7 were individually appended to the ectodomain sequences of the HCV E2 of strain N (Hayashi, et al, J. Hepatol. 77:S94-S107, 1993), to generate a chimeric protein. The HVR encoding sequences were removed from this E2 sequence and a (his) 6 tag was added to the carboxyl terminus. These constructs were cloned in the plasmid Vljnstpa in frame with the tissue plasminogen activator (tPA) leader sequences. (Montgomery, et al, Pharmacol. Ther. 74:195-205, 1997.)
  • tPA tissue plasminogen activator
  • chimeric mimotope-containing glycoproteins were produced as secreted products by transient expression in 293T cell cultures.
  • 293T cells were cultured in poly D-lysine-coated 175 cm flasks (BioCoat, Becton-Dickinson) and were transfected at 60 to 70% confluence.
  • the DNA at l ⁇ g/ ⁇ l in sterile 10 mM Tris, 1 mM EDTA, pH 8.0, was mixed with the Lipofectamine Plus component of the Lipofectamine Plus kit (G BCO), according to instructions, using approximately 25 ⁇ g DNA/flask.
  • Opti-MEM based lipofectamine-DNA medium was removed and replaced with 24 ml of a protein-free medium supplemented with L-glutamine and penicillin/streptomycin (CELLGRO Free - complete serum, protein, hormone, growth factor free medium, CellGro, Herndon, VA).
  • This medium was harvested every 24 hours post-transfection for 3 harvests, clarified by low speed centrifugation, and stored at 4°C until all materials were collected, pooled, and passed through a 0.22 ⁇ filter. The medium was then dialyzed against 10 mM imidazole, 0.5 M NaCl, 20 mM Na phosphate, pH 7.8 (starting buffer) and filtered again using a 0.22 ⁇ filter. Approximately 2.0 L of material was loaded on a 10 ml volume Ni-
  • NTA column (Qiagen, Valencia, CA) at a flow rate of approximately 3 ml/minute at 4°C.
  • the column was washed with starting buffer, then eluted stepwise with: (a) 40 ml of buffer with 20 mM imidazole; (b) 10 ml of buffer with 40 mM imidazole; (c) 30 ml of buffer with 100 mM imidazole; and (d) 15 ml of buffer with 250 mM imidazole. Essentially all the chimeric glycoprotein eluded with the 100 mM imidazole buffer.
  • each glycoprotein' s major eluded fraction was dialyzed into water for N-terminal protein sequence analysis, phosphate buffered saline for ELISA studies, or 5 mM triethanolamine, 150 mM NaCl, pH 7.8 for subsequent vaccine formulation. Purity was assessed by SDS-PAGE followed by western blot using either an anti-HCV gpE2 specific murine monoclonal antibody or an anti-tetra-his antibody (Qiagen, Valencia, CA). Identity and completion of leader processing was confirmed by N-terminal amino acid sequence analysis.
  • Example 5 Vaccine Formulation The first studies in rabbits were carried out using peptide conjugate vaccines formulated in Freunds complete and incomplete adjuvants. Peptide OMPC conjugate vaccines were prepared in 500 ⁇ g total protein doses in 10 mM HEPPS, pH 8.0. Further studies then were performed with each dose adsorbed onto 1.4 mg/ml AIPO4 in 6 mM triethanolamine saline pH 7.0-8.0 to which monophosphoryl lipid-A (MPL-A) was already adsorbed. The weight ratio of AIPO4 to MPL-A was 3.5 to 1. The purified chimeric proteins were also prepared in 500 ⁇ g total protein doses in 5 mM triethanolamine, 150 mM NaCl, pH 7.8.
  • DNA vector vaccines were resuspended in sterile PBS, pH 7.5 at 5.0 mg/ml.
  • New Zealand white rabbits (2 to 2.5 Kg) were injected at the start of each study and at weeks 4 and 8. The animals were bled to prepare serum biweekly through week 12.
  • Rhesus monkeys, Maccca mulatta (>2.0 Kg) were immunized with the mimotope OMPC conjugate vaccines and the chimeric mimotope glycoprotein vaccines at day 1 and weeks 4, 8, and 26.
  • Each dose was inoculated intramuscularly and equally distributed between 2 sites in each deltoid muscle.
  • the monkeys were immunized with the DNA vector vaccines prepared in sterile PBS.
  • Each animal was inoculated intramuscularly using a Biojector (Bioject, Inc. Portland, OR) needle-less device fitted with a number 3 syringe.
  • Each dose was equally distributed between two sites in each quadracep muscle.
  • the animals were bled and sera were prepared biweekly through week 12, monthly through week 24, then biweekly again through week 32.
  • HRP-conjugated antisera were used as secondary antibodies.
  • mouse monoclonal antibody a goat anti-mouse IgG, Fc fragment specific antibody was used (Jackson Immuno. Res., West Grove, PA).
  • rabbit antibodies a goat anti rabbit IgG-Fc was used (Bethyl Labs, Montgomery, TX).
  • monkey antibodies a goat anti-monkey IgG H+L was used (Bethyl Labs). All these secondary antibodies were used at a 1:4000 dilution. All blots were developed on Hyperfilm using ECL or ECL + reagents (Amersham, Piscataway, NJ).
  • Example 8 ELISA of Immune Sera to Mimotopes and HVR Peptides
  • Antibody responses were determined against the homologous immunizing peptide, heterologous mimotopes, or HVR peptides by an enzyme-linked immunoadsorbent assay (ELISA) essentially as described (Tolman, et al., 1993. Int. J. Peptide Protein Res. 41:455-466, 1993 and Conley, et al, Vaccine 72:445-451, 1994) or by use of streptavidin capture of biotinylated peptide ELISA.
  • ELISA enzyme-linked immunoadsorbent assay
  • microtiter plates were coated with 0.25 ⁇ g of peptide in PBS in a humid atmosphere, overnight at 4°C. The plates were washed extensively, blocked with IX blocker BSA (Reacti-Bind, Pierce, Rockford, II), washed extensively, then reacted with dilutions of test and control sera for 1 hour at 37°C. The plates then were washed extensively and reacted to the conjugated secondary antibody. The plate was processed for reaction by standard methods, and absorbance was measured at 450 nm.
  • IX blocker BSA Reacti-Bind, Pierce, Rockford, II
  • HCV E2 glycoprotein capture ELISA was used to measure anti- mimotope responses to recombinant glycoproteins.
  • ELISA plates were coated with 50 pg of either of two murine anti-HCV E2 monoclonal antibodies overnight at 4°C in a humid environment. These Mabs were prepared from hybridoma cell cultures using serum-free medium and purified using HiTrap Protein G (Amersham Pharmacia, Piscataway, NJ). These two antibodies appeared to bind to conserved determinants, located at a distance from the N-terminus of E2, and by analysis did not interfere with anti-mimotope or anti-HVR sera binding to E2 in ELISA or BIACore.
  • Anti-mimotope R9 antibodies recognized 4 of 4 HVR peptides, while anti-mimotope R6 and F78 antibodies recognized 3 of 3 HVR peptides.
  • anti-mimotope D6 antibodies only recognized the three variant sequences and were of low titer.
  • the anti-mimotope HI antibodies recognized none of the H strain variants.
  • the ELISA titers for the HCV H strain HVR variant peptides showed a response to variant #1 and #3 only from one of the rabbits. The remainder of the sera did not recognize any of the HCV H strain HVR sequences.
  • the three SCPVs consisted of one vaccine containing three mimotopes, one containing five mimotopes and a third containing six mimotopes.
  • the peptide ELISA studies again showed that SCPVs could elicit high titer anti-mimotope responses (Table 5).
  • the R9 peptide was the highest coupled peptide in both SCPV number 2 and number 4 (Table 2).
  • Rhesus monkeys were used in primate immunogenicity studies and to compare three ways to deliver mimotope based vaccines with the goal of eliciting broadly HNR-reactive antibodies.
  • Peptide conjugates were prepared for five singular mimotopes, one SCPN consisting of five mimotopes, and a conjugate consisting of the HVR sequence of HCV strain BK. ⁇ 5 conjugation occurred at a low efficiency.
  • the two most broadly reactive sera sets in these experiments were from the M63-conjugate and the SCPV consisting of five mimotope peptides.
  • the anti-M63 sera recognized 5 of 10 peptides in the standard ELISA and 11 of 11 biotinylated peptides in the capture ELISA.
  • the anti-five mimotope sera from SCPV animals recognized 6 of 10 peptides in the standard ELISA and 8 of 11 biotinylated peptides in the capture ELISA. Between these two sets all peptides are recognized with the exception of the 147-07 sequence.
  • the reaction to the HVC H strain variant #2 is of low titer and should be grouped as a rare reactor. Thus, between a singular mimotope conjugate using M63 sequence and the SCPV, 19 of 21 HVR sequences were recognized.
  • each set of immune sera strongly recognized E2 displaying its cognate mimotope (Table 6C) plus additional chimeric E2.

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Abstract

La présente invention concerne des conjugués du VHC, capables d'induire une réponse immunitaire reconnaissant différentes souches et variantes du VHC. Les conjugués contiennent un support de complexe polypeptidique ou protéique et un ou plusieurs mimotopes VHC. Les mimotopes VHC préférés sont à l'origine d'antigènes capables de produire des anticorps reconnaissant des domaines hypervariables de la protéine VHC E2.
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WO2005058968A1 (fr) * 2003-12-18 2005-06-30 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Procede de production d'un conjugue porteur de peptides a antigenicite elevee
EP1885395A2 (fr) * 2005-05-16 2008-02-13 Merck & Co., Inc. Procédé d'augmentation de l'immunigénicité des antigènes de plasmodium
US20080145373A1 (en) * 2003-12-17 2008-06-19 Elan Pharmaceuticals, Inc. A-beta immunogenic peptide carrier conjugates and methods of producing same

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US5274122A (en) * 1992-10-15 1993-12-28 Merck & Co., Inc. Acidic derivatives of homocysteine thiolactone
WO1999058561A1 (fr) * 1998-05-14 1999-11-18 Pasteur Merieux Serums & Vaccins Mimotopes du virus de l'hepatite c
GB9810756D0 (en) * 1998-05-19 1998-07-15 Angeletti P Ist Richerche Bio Mimotopes of hypervariable region 1 of the e2 glycoprotein of hcv and uses thereof

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Cited By (10)

* Cited by examiner, † Cited by third party
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US20080145373A1 (en) * 2003-12-17 2008-06-19 Elan Pharmaceuticals, Inc. A-beta immunogenic peptide carrier conjugates and methods of producing same
US8227403B2 (en) 2003-12-17 2012-07-24 Wyeth Llc A-β immunogenic peptide carrier conjugates and methods of producing same
EP2479184A3 (fr) * 2003-12-17 2013-09-04 Janssen Alzheimer Immunotherapy Conjugués transporteurs de peptides bêta immunogènes et leurs procédés de production
US9089510B2 (en) 2003-12-17 2015-07-28 Janssen Sciences Ireland Uc A-β immunogenic peptide carrier conjugates and methods of producing same
US9095536B2 (en) 2003-12-17 2015-08-04 Janssen Sciences Ireland Uc Aβ immunogenic peptide carrier conjugates and methods of producing same
US9125847B2 (en) 2003-12-17 2015-09-08 Janssen Sciences Ireland Uc A-β immunogenic peptide carrier conjugates and methods of producing same
WO2005058968A1 (fr) * 2003-12-18 2005-06-30 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Procede de production d'un conjugue porteur de peptides a antigenicite elevee
US20090104220A1 (en) * 2003-12-18 2009-04-23 Elisabetta Bianchi Method to Make a Peptide-Carrier Conjugate with a High Immunogenicity
EP1885395A2 (fr) * 2005-05-16 2008-02-13 Merck & Co., Inc. Procédé d'augmentation de l'immunigénicité des antigènes de plasmodium
EP1885395A4 (fr) * 2005-05-16 2009-12-02 Merck & Co Inc Procédé d'augmentation de l'immunigénicité des antigènes de plasmodium

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