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WO2002034770A1 - Identification d"epitopes du vhc restreints par hla-dr - Google Patents

Identification d"epitopes du vhc restreints par hla-dr Download PDF

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Publication number
WO2002034770A1
WO2002034770A1 PCT/GB2001/004636 GB0104636W WO0234770A1 WO 2002034770 A1 WO2002034770 A1 WO 2002034770A1 GB 0104636 W GB0104636 W GB 0104636W WO 0234770 A1 WO0234770 A1 WO 0234770A1
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Prior art keywords
polypeptide
amino acids
patient
epitope
hcv
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PCT/GB2001/004636
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English (en)
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Andrew James Godkin
Howard Thomas
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Imperial College Innovations Limited
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Priority to AU2002210683A priority Critical patent/AU2002210683A1/en
Publication of WO2002034770A1 publication Critical patent/WO2002034770A1/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
    • A61K38/00—Medicinal preparations containing 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to methods relating to hepatitis C infection and to compounds and compositions that may be useful in such methods.
  • the hepatitis C virus is a single stranded RNA virus, encoded by a genome of approximately 9,500 nucleotides. It is a member of the Flaviviridae family, being most closely related to GBN-A, GBN-B and GBN-C, the other Hepaciviruses (1).
  • the virion which has been visualized with electron microscopy in lymphoblastoid cell cultures (2), consists of the positive strand R ⁇ A molecule, surrounded by the core (nucleocapsid) and two envelope proteins (E1 ,E2).
  • the HCV genome contains a long translational open reading frame (OR ), which encodes a single polypeptide that is cleaved co- and post-translationally by cellular and viral proteinases to yield a number of structural and non-structural proteins (3)
  • the structural proteins comprise of core, E l and E2.
  • the non-structural proteins which are not expected to be constituents of the virion, are ⁇ S2, NS3, NS4a, NS4b, NS5a, and NS5b. These have a variety of functions, necessary for viral replication, including proteinase (NS2, NS3), NTPase/helicase (NS3) and RNA-dependent RNA polymerase (RdRp) (NS5b) activities.
  • Translation of the HCV ORF is directed via a 340 nucleotide long 5' non-coding region (5'NCR), functioning as an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • HCV Hepatitis C virus
  • helper CD4+ T cell response may directly influences the outcome after acute viral infections.
  • One of their key functions is to help an adequate cytotoxic T cell (CTL) response to develop.
  • CTL cytotoxic T cell
  • Identification of antigen-specific CTLs with MHC-peptide tetramers has revealed non-cytopathic viral infections such as EBV (2) or LCMN (3) lead to a massive expansion of CTL which is associated with control or elimination of the viral infection.
  • Successful clearance of HCN also appears to be associated with a broader, more powerful CTL response during the acute infection (4, 5).
  • persistent viral infection in HIV (6) and HCN (7) are associated with progressive loss of CTL or low magnitude responses.
  • CD4+ T cells are critical for maintaining effective cytotoxic T lymphocyte responses and hence control of viraemia [reviewed in (8)]. Many of the data are derived from the mouse model of LCMV infection. In CD4-/- knockout mice high dose LCMN- WE infection or low dose LCMV-DOCLLE (a more virulent strain) induces an initial CTL response which later disappears and chronic viraemia ensues. Remarkably, even transient CD4+ depletion during infection with certain virulent LCMV strains is enough to cause long term loss of functional CTLs (9).
  • HLA-DR1 1 Al *0101 , Bl* l 101
  • HLA class II molecules bind short pathogen derived peptide epitopes, 12 - 20 amino acids in length, and are instrumental in activating CD4+ T lymphocytes. Hence this HLA association offers a powerful argument for the central role of CD4+ T cells.
  • PBMCs peripheral blood mononuclear cells
  • the HCV genome of approximately 9000 bases encodes a series of proteins including the structural proteins core and envelope (El , E2), and the non-structural proteins NS2-5 (1 1).
  • Identifying specific T cell epitopes allows new questions to be addressed [reviewed in (20)], such as epitope- specific T cell frequencies, their role in memory, hierarchies of response, and cytokine production (e.g. Thl IFN ⁇ -producing, Th2 IL4-producing or ThO IFN ⁇ / IL4).
  • the difficulty in identifying CD4+ epitopes is in part due to the increased degeneracy and promiscuity of peptide binding to HLA class II compared to class I molecules (21) and the poorly understood rules of antigen processing.
  • HLA-DR 11 -restricted T cell epitopes derived from several viral proteins. We describe methods of treatment and diagnosis of patients with or at risk of HCN infection and compounds and compositions that may be useful in such methods.
  • a first aspect of the invention provides the use of a polypeptide having one or more copies of one or more epitopes selected from epitopes present in the regions between amino acids 31-45, 141-155, 1207-1221, 2268-2284 and/or 2941-2955 of a native HCN polypeptide, in the manufacture of a medicament for the treatment of a patient with or at risk of hepatitis C infection, wherein the patient is of HLA type DR1 1 or DR12 and wherein the polypeptide (1) does not comprise a sequence of more than 30 contiguous amino acids from a native HCV polypeptide and/or (2) the polypeptide is of about 8 to about 100 amino acids in length.
  • a further aspect of the invention provides a method of treating a patient with or at risk of hepatitis C infection wherein a polypeptide having one or more copies of one or more epitopes selected from epitopes present in the regions between amino acids 31-45, 141-155, 1207- 1221 , 2268-2284 and or 2941 -2955 of a native HCV polypeptide, is administered to the patient, wherein the patient is of HLA type DRl 1 or DR12 and wherein the polypeptide (1) does not comprise a sequence of more than 30 contiguous amino acids from a native HCN polypeptide and/or (2) the polypeptide is of about 8 to about 100 amino acids in length.
  • epitopes present in the said regions are T cell-stimulating epitopes. It is preferred that a selected epitope is a T cell-stimulating epitope.
  • a further aspect of the invention provides the use of a polypeptide comprising or consisting of at least eight contiguous amino acids selected from the regions between amino acids 31 -45, 141-155, 1207- 1221 , 2268-2284 and/or 2941-2955 of a native HCV polypeptide, in the manufacture of a medicament for the treatment of a patient with or at risk of hepatitis C infection, wherein the patient is of HLA type DRl 1 or DR12 and wherein the polypeptide (1) does not comprise a sequence of more than 30 contiguous amino acids from a native HCV polypeptide and/or (2) the polypeptide is of about 8 to about 100 amino acids in length.
  • a further aspect of the invention provides a method of treating a patient with or at risk of hepatitis C infection wherein a polypeptide comprising or consisting of at least eight contiguous amino acids selected from the regions between amino acids 31-45, 141 -155, 1207-1221, 2268-2284 and or 2941-2955 of a native HCV polypeptide, is administered to the patient, wherein the patient is of HLA type DRl 1 or DRl 2 and wherein the polypeptide ( 1) does not comprise a sequence of more than 30 contiguous amino acids from a native HCV polypeptide and or (2) the polypeptide is of about 8 to about 100 amino acids in length.
  • the said regions contain T cell-stimulating epitopes. It is preferred that the said contiguous amino acids contain a T cell- stimulating epitope.
  • the patient is a human patient.
  • Methods of determining the HLA type of a patient will be well known to those skilled in the art.
  • a method involving polymerase chain reaction (PCR) amplification may be used, as described, for example, in Kimura & Sasazuki (1992) 11 International Histocompatibility Workshop reference protocol for the HLA DNA typing technique, p397-419, in K Tsuji, M Aizawa and T Sasazuki (ed), HLA 1991 , Oxford University Press, Oxford, United Kingdom or Nevinny-Sticke et al (1991) Nonradiactive HLA class II typing using polymerase chain reaction and digoxigenin-l l-2'-3'-dideoxy- uridine-triphosphate labeled oligonucleotide probes.
  • PCR polymerase chain reaction
  • the patient is of HLA type DRl 1.
  • the polypeptide may have one or more copies of epitope(s) from one of the said regions, ie from the region between amino acids 31-45 or 141 -155 or 1207-1221 or 2268-2284 or 2941-2955 of a native HCV polypeptide.
  • the polypeptide may have one or more copies of epitope(s) from one such region and additionally one or more copies of epitope(s) from one or more different such regions.
  • the polypeptide may have or comprise one or more copies of an amino acid sequence from the region between amino acids 31-45 or 141-155 or 1207-1221 or 2268-2284 or 2941-2955 of a native HCV polypeptide, or one or more copies of amino acid sequences from one or more different such regions.
  • native HCV polypeptide any naturally occurring HCV polypeptide. It is preferred that the HCV polypeptide is that encoded by the viral genotype la, based on Simmond's classification, as known to those skilled in the art.
  • HCV-J isolate refers to that of the unprocessed HCN polypeptide, as used for the HCV-J isolate according to Kato et al (1990) PNAS 81, 9524-9528, as well known to those skilled in the art.
  • Other HCV isolates may be aligned with this sequence in order to identify the equivalent amino acid.
  • type 2 isolates may contain 4 extra codons/amino acids in the E2 sequence, whilst type 3 sequences have an insertion of 2 amino acids compared to type 1 sequences.
  • EP 0 979, 867, for example, and references therein discusses types and subtypes of HCV polypeptide.
  • the term native HCV polypeptide includes any variation already observed within any of the described regions of HCV.
  • mutations may be introduced, provided that the mutated sequence is immunologically cross-reactive with the unmutated sequence (ie retains the same epitope).
  • an antibody and/or T cell response elicited by the mutated sequence should also recognise the unmutated sequence.
  • a mutation is a conservative substitution, as well known to those skilled in the art.
  • conservative substitutions is intended combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Mutations may be made using the methods of protein engineering and site-directed mutagenesis as well known to those skilled in the art.
  • the three-letter and one-letter amino acid code of the IUPAC-IUB Biochemical Nomenclature Commission is used herein.
  • the sequence of polypeptides are given N-terminal to C-terminal as is conventional.
  • Xaa represents any amino acid.
  • the amino acids are L-amino acids, in particular it is preferred that the amino acid residues immediately flanking (such as those within 10 to 20 residues) of the epitope-forming amino acid sequence consist of L-amino acid residues but they may be D-amino acid residues.
  • the polypeptide does not comprise a sequence of more than 30 contiguous (ie consecutive) amino acids from a native HCV polypeptide, preferably that encoded by viral genotype la. It will be appreciated that the polypeptide may comprise more than one sequence of up to 30 contiguous amino acids from a native HCN polypeptide, for example a sequence of up to 30 contiguous amino acids including the amino acid sequence of region 31-45 from a native HCV polypeptide and additionally a sequence of up to 30 contiguous amino acids including the amino acid sequence of region 141-155 from a native HCV polypeptide.
  • the polypeptide does not comprise a sequence of more than 28, 25, 22, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9 or 8 contiguous amino acids from the native HCV polypeptide. It is preferred that the polypeptide comprises or consists of a sequence or sequences of between about 8 and 18 or 20 contiguous amino acids from the native HCV polypeptide, preferably about 15 contiguous amino acids from the native HCV polypeptide.
  • the polypeptide may be between about 8 and 100 amino acids in length. It is further preferred that the polypeptide is between about 8 and 50, 40, 30, 20, 18, 16 or 15 amino acids in length. This may have advantages in terms of ease of preparation and/or administration/formulation of the polypeptide when compared with longer polypeptides.
  • the polypeptide may alternatively be longer than 100 amino acids in length.
  • the polypeptide may comprise a "carrier" portion, as known to those skilled in the art.
  • the carrier portion may serve to aid handling, delivery or immunogenic presentation of the HCN epitope(s) of interest.
  • the polypeptide may comprise a non-HCV viral polypeptide or fragment thereof, for example a hepatitis B vims (HBV) polypeptide or fragment thereof, as discussed further below.
  • HBV hepatitis B vims
  • the polypeptide may be a antiidiotypic antibody that has an epitope as defined above, as known to those skilled in the art.
  • Polypeptides in which one or more of the amino acid residues are chemically modified, before or after the polypeptide is synthesised may be used providing that the function of the polypeptide, namely the production of a specific immune response in vivo, remains substantially unchanged.
  • modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as ⁇ -t-butoxycarbonyl. Such modifications may protect the polypeptide from in vivo metabolism.
  • the epitope(s) may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier. It may be advantageous for the polypeptide to be formed as a loop, with the ⁇ -terminal and C-terminal ends joined together, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed. If the epitope, for example epitope-forming amino acid sequence, is covalently linked to a carrier, preferably a polypeptide, then the arrangement is preferably such that the epitope-forming amino acid sequence forms a loop.
  • a carrier preferably a polypeptide
  • a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system.
  • the epitope(s) as defined above in relation to the preceding aspects of the invention may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
  • More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr- Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-1.
  • the latter compound may variously be regarded as a carrier or as an adjuvant or as both.
  • cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate, the latter agent exploiting the -SH group on the C-terminal cysteine residue (if present).
  • Any of the conventional ways of cross-linking polypeptides may be used, such as those generally described in O'Sullivan et al Anal. Biochem. (1979) 100, 100- 108.
  • the first portion may be enriched with thiol groups and the second portion reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP), a heterobifunctional cross- linking agent which incorporates a disulphide bridge between the conjugated species.
  • NHS iodoacetic acid
  • SPDP N- succinimidyl-3-(2-pyridyldithio)propionate
  • Amide and thioether bonds for example achieved with m-maleimidobenzoyl-N- hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.
  • cross-linking agents include S-acetylthioglycolic acid N- hydroxysuccinimide ester (SAT A) which is a thiolating reagent for primary amines which allows deprotection of the sulphydryl group under mild conditions (Julian et al (1983) Anal. Biochem. 132, 68), dimethylsuberimidate dihydrochloride and N-N'-o- phenylenedimaleimide.
  • SAT A S-acetylthioglycolic acid N- hydroxysuccinimide ester
  • polypeptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express the polypeptide as a fusion product with a peptide sequence which acts as a carrier.
  • Kabigen's "Ecosec" system is an example of such an arrangement.
  • the epitope(s) of the invention may be linked to other antigens to provide a dual effect.
  • a further aspect of the invention provides the use of (1) a peptidomimetic compound conesponding to a polypeptide (therapeutic polypeptide) as defined in relation to any of the preceding aspects of the invention, and/or (2) a polynucleotide capable of expressing a said polypeptide and/or (3) an antibody reactive with an epitope as defined in relation to a preceding aspect of the invention, in the manufacture of a medicament for the treatment of a patient with or at risk of hepatitis C infection, wherein the patient is of HLA type DRl 1 or DRl 2.
  • the said peptidomimetic compound, polypeptide, polynucleotide or antibody may also be useful in the manufacture of a diagnostic reagent for use in diagnosis of a patient with or at risk of hepatitis C infection, preferably wherein the patient is of HLA type DRl 1 or DRl 2.
  • a further aspect of the invention provides a method of treating a patient with or at risk of hepatitis C infection, wherein the patient is administered (1) a peptidomimetic compound conesponding to a polypeptide (therapeutic polypeptide) as defined in relation to any of the preceding aspects of the invention, and/or (2) a polynucleotide capable of expressing a said polypeptide and/or (3) an antibody reactive with an epitope as defined in relation to a preceding aspect of the invention, wherein the patient is of HLA type DRl 1 or DR12.
  • peptidomimetic refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent, but that avoids the undesirable features.
  • morphine is a compound which can be orally administered, and which is a peptidomimetic of the peptide endorphin.
  • Therapeutic applications involving peptides are limited, due to lack of oral bioavailability and to proteolytic degradation.
  • peptides are rapidly degraded in vivo by exo- and endopeptidases, resulting in generally very short biological half-lives.
  • Another deficiency of peptides as potential therapeutic agents is their lack of bioavailability via oral administration.
  • Degradation of the peptides by proteolytic enzymes in the gastrointestinal tract is likely to be an important contributing factor. The problem is, however, more complicated because it has been recognised that even small, cyclic peptides which are not subject to rapid metabolite inactivation nevertheless exhibit poor oral bioavailability.
  • Retro-inverso peptidomimetics in which the peptide bonds are reversed, can be synthesised by methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159 3230-3237. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
  • a common theme among many of the synthetic strategies has been the introduction of some cyclic moiety into a peptide-based framework.
  • the cyclic moiety restricts the conformational space of the peptide structure and this frequently results in an increased affinity of the peptide for a particular biological receptor.
  • An added advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.
  • RCM ring closing metathesis
  • an antibody is included an antibody or other immunoglobulin, or a fragment or derivative thereof, as discussed further below.
  • variable heavy (V H ) and variable light (V L ) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
  • variable domains that antigenic specificity is confened by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • variable domains include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the N H and N L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci.
  • ScFv molecules we mean molecules wherein the N H and N L partner domains are linked via a flexible oligopeptide.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • Whole antibodies, and F(ab') 2 fragments are "bivalent". By “bivalent” we mean that the said antibodies and F(ab') 2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.
  • the antibody has an affinity for the epitope of between about 10 5 .M “ ' to about 10 12 .M “ ', more preferably at least 10 8 .M".
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in 'Monoclonal Antibodies: A manual of techniques “, H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hunell (CRC Press, 1982). Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799). Suitably prepared non-human antibodies can be "humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • the polynucleotide capable of expressing the said polypeptide may be prepared using techniques well known to those skilled in the art and as described below.
  • the polynucleotide is capable of expressing the said polypeptide in the patient.
  • the said polypeptide may be expressed from any suitable polynucleotide (genetic construct) as is described below and delivered to the patient.
  • the genetic construct which expresses the polypeptide comprises the said polypeptide coding sequence operatively linked to a promoter which can express the transcribed polynucleotide (eg mRNA) molecule in a cell of the patient, which may be translated to synthesise the said polypeptide.
  • Suitable promoters will be known to those skilled in the art, and may include promoters for ubiquitously expressed, for example housekeeping genes or for tissue-specific genes, depending upon where it is desired to express the said polypeptide, as discussed further below.
  • the genetic construct can be DNA or RNA it is prefened if it is DNA.
  • the genetic constmct is adapted for delivery to a human cell.
  • the constructs of the invention may be introduced into the cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the (dividing) cell.
  • Targeted retroviruses are available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre-existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).
  • Retroviral methods such as those described below, may only be suitable when the cell is a dividing cell.
  • Retroviral DNA constructs which encode the said polypeptide may be made using methods well known in the art.
  • To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 ⁇ m pore-size filter and stored at -70°.
  • the retrovirus it is convenient to inject directly retroviral supernatant to which 10 ⁇ g/ml Polybrene has been added. The injection may be made into the area in which the target cells are present. It may be desirable to express the antigenic polypeptides in antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • Other methods involve simple delivery of the construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time.
  • An example of the latter approach includes liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).
  • Other methods of delivery include adenoviruses carrying external DNA via an antibody-polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18) and transferrin-polycation conjugates as carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414).
  • the DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle.
  • naked DNA and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the patient to be treated.
  • Non-viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1 144.
  • Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle.
  • Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell- selective moiety into a fibre protein.
  • Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells are also useful for delivering the genetic construct to a cell.
  • Other suitable viruses or virus-like particles include HSV, AAV, vaccinia and parvovirus.
  • Immunoliposomes are especially useful in targeting to cell types which over-express a cell surface protein for which antibodies are available.
  • MPB-PE N-[4-(p- maleimidophenyl)butyryl]-phosphatidylethanolamine
  • MPB-PE is incorporated into the Hposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the hposomal surface.
  • the liposome is conveniently loaded with the genetic construct for delivery to the target cells, for example, by forming the said liposomes in a solution of the genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 ⁇ m and 0.2 ⁇ m pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifiigation at 80 000 x g for 45 min. Freshly prepared MPB-PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight.
  • the immunoliposomes are separated from unconjugated antibodies by ultracentrifiigation at 80 000 x g for 45 min.
  • Immunoliposomes may be injected, for example intraperitoneally or directly into a site where the target cells are present.
  • adenoviruses carrying external DNA via an antibody-polylysine bridge see Curiel Prog. Med. Virol. 40, 1- 18
  • transferrin- polycation conjugates as carriers
  • a polycation-antibody complex is formed with the genetic construct, wherein the antibody is specific for either wild- type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody.
  • the polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone.
  • the adenovirus because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is polylysine.
  • DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.
  • a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells is employed. This is accomplished by conjugating the iron-transport protein transfemn to polycafions that bind nucleic acids.
  • Human transfemn, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage.
  • These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell.
  • the transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs).
  • complexes of transferrin-polycation and the DNA constructs or other genetic constructs are supplied to the target cells, a high level of expression from the construct in the cells is expected.
  • High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used.
  • adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the genetic construct, the construct is taken up by the cell by the same route as the adenovirus particle.
  • This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types.
  • the suitable delivery vehicle comprising the genetic construct may be desirable to locally perfuse an area comprising target cells with the suitable delivery vehicle comprising the genetic construct for a period of time; additionally or alternatively the delivery vehicle or genetic construct can be injected directly into accessible areas comprising target cells. It may be beneficial to deliver the delivery vehicle or genetic construct systemically.
  • the genetic constructs can be prepared using methods well known in the art.
  • expression of the said polypeptide is directly or indirectly (see below) under the control of a promoter that may be regulated, for example by the concentration of a small molecule that may be administered to the patient when it is desired to activate or repress (depending upon whether the small molecule effects activation or repression of the said promoter) expression of the said polypeptide.
  • the expression construct is stable ie capable of expressing the said polypeptide (in the presence of any necessary regulatory molecules) in the said cell for a period of at least one week, one, two, three, four, five, six, eight months or one or more years.
  • a preferred construct may comprise a regulatable promoter.
  • regulatable promoters include those refened to in the following papers: Rivera et al ( 1999) Proc Natl Acad Sci USA 96(15), 8657-62 (control by rapamycin, an orally bioavailable dnig, using two separate adenovirus or adeno-associated virus (AAV) vectors, one encoding an inducible human growth hormone (hGH) target gene, and the other a bipartite rapamycin-regulated transcription factor); Magari et al (1997) J Clin Invest 100(11), 2865-72 (control by rapamycin); Bueler (1999) Biol Chem 380(6), 613-22 (review of adeno-associated viral vectors); Bohl et al (1998) Blood 92(5), 1512-7 (control by doxycycline in adeno-associated vector); Abruzzese et al (1996) J Mol Med 74(7), 379-92 (reviews induction factors e.g., hormones, growth factors
  • Tetracycline - inducible vectors may also be used. These are activated by a relatively non-toxic antibiotic that has been shown to be useful for regulating expression in mammalian cell cultures. Also, steroid- based inducers may be useful especially since the steroid receptor complex enters the nucleus where the DNA vector must be segregated prior to transcription.
  • This system may be further improved by regulating the expression at two levels, for example by using a tissue-specific promoter and a promoter controlled by an exogenous inducer/repressor, for example a small molecule inducer, as discussed above and known to those skilled in the art.
  • one level of regulation may involve linking the appropriate sequence encoding the polypeptide to an inducible promoter whilst a further level of regulation entails using a tissue-specific promoter to drive the gene encoding the requisite inducible transcription factor (which controls expression of the polypeptide from the inducible promoter).
  • Control may further be improved by cell-type-specific targeting of the genetic construct.
  • a further aspect of the invention provides a chimaenc polypeptide comprising 1) a first epitope selected from epitopes present in a region between amino acids 31-45, 141- 155, 1207-1221, 1245- 1259, 2268-2284 and/or 2941-2955 of a native HCV polypeptide and 2) a second epitope (and optionally further epitopes) selected from said epitopes, wherein the first and second epitope are present in different said regions, and wherein the chimaeric polypeptide is not a native HCN polypeptide or fragment thereof.
  • the chimaeric polypeptide of the invention includes appropriate preferences as indicated above in relation to the polypeptide as defined in relation to preceding aspects of the invention. It is preferred that a said selected epitope is a T cell-stimulating epitope.
  • the chimaeric polypeptide comprises amino acid sequences corresponding to amino acids 31-45, 141 -155, 1207-1221, 1245- 1259, 2268-2284 and/or 2941-2955 of the HCV polypeptide.
  • a further aspect of the invention provides a peptidomimetic compound conesponding to the chimaeric polypeptide of the invention. It will be appreciated that a chimaeric polypeptide of the invention may be used in a method or use of a previous aspect of the invention.
  • the chimaeric polypeptide may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein.
  • Temporary N-amino group protection is afforded by the 9-fJuorenylmefhyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide.
  • Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6- trimethylbenzenesulphonyl derivative (in the case of arginine).
  • glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities.
  • the solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent).
  • the peptide-to- resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/l-hydroxybenzotriazole mediated coupling procedure.
  • Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers.
  • Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
  • FAB fast atom bombardment
  • a further aspect of the invention provides a polynucleotide encoding or capable of expressing a chimaeric polypeptide of the invention.
  • the polynucleotide of the invention may be administered to a patient, as discussed above.
  • the polynucleotide may be expressed in a suitable host to produce a polypeptide of the invention.
  • the DNA encoding the chimaeric polypeptide of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the chimaeric polypeptide of the invention.
  • Such techniques include those disclosed in US Patent Nos.
  • the DNA encoding the chimaeric polypeptide of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and conect reading frame for expression.
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
  • the DNA insert may be operatively linked to an appropriate promoter.
  • Bacterial promoters include the E.coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the phage ⁇ PR and PL promoters, the phoA promoter and the trp promoter.
  • Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters and the promoters of retroviral LTRs. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will desirably also contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation. (Hastings et al, International Patent No. WO 98/16643, published 23 April 1998)
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector and it will therefore be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence marker, with any necessary control elements, that codes for a selectable trait in the transformed cell.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracyclin, kanamycin or ampicillin resistance genes for culturing in E.coli and other bacteria.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • polypeptide of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (for example using a tag fused to the polypeptide), hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • bacteria eg. E.coli and Bacillus subtil is
  • yeasts eg.
  • Saccaromyces cerevisiae transformed with, for example, yeast expression vectors; insect cell systems transformed with, for example, viral expression vectors (eg. baculovirus) ; plant cell systems transfected with, for example viral or bacterial expression vectors; animal cell systems transfected with, for example, adenovirus expression vectors.
  • yeast expression vectors for example, yeast expression vectors
  • viral expression vectors eg. baculovirus
  • the vectors include a prokaryotic replicon, such as the Col E 1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic cell types.
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E.coli, transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical prokaryotic vector plasmids are: pUC18, pLfC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA, USA); p7) ⁇ 99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).
  • a typical mammalian cell vector plasmid is pSNL available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, NJ, USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YTps) and incorporate the yeast selectable markers HIS 3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls.
  • One such method involves ligation via homopolymer tails.
  • Another method involves ligation via cohesive ends.
  • a further method uses synthetic molecules called linkers and adaptors. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.
  • a desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
  • the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • the techniques described above may also be used in the production of a polypeptide (therapeutic polypeptide) as defined in relation to earlier aspects of the invention.
  • a further aspect of the invention provides the use of a chimaeric polypeptide, polynucleotide or peptidomimetic compound of the invention in the manufacture of a medicament for the treatment of a patient with or at risk of hepatitis C infection.
  • a further aspect of the invention provides a method of treating a patient with or at risk of hepatitis C infection, wherein the patient is administered a chimaeric polypeptide, polynucleotide or peptidomimetic compound of the invention. It is prefened that the patient is of HLA type DRl 1 or DRl 2, preferably DR1 1.
  • the relevant agent may be administered as a prophylactic vaccine (or the medicament may be prepared as a prophylactic vaccine).
  • the agent/medicament may be administered to a patient at risk of HCV infection, with the intention of immunising the patient against HCV infection.
  • healthcare workers may be at a greater risk of HCV infection (for example as a consequence of needle stick injuries) than other occupational groups.
  • Intravenous drug users may also be at greater risk.
  • the relevant agent/medicament may be administered as a therapeutic vaccine.
  • the agent/medicament may be administered to a patient with HCV infection.
  • the patient may have acute or chronic HCV infection, preferably acute
  • HCV infection Methods of detecting HCV infection are well known to those skilled in the art, and are mentioned in Example 1.
  • PCR may be used to detect HCV nucleic acid.
  • the patient may also be administered other antiviral treatment, for example is, has been, or will be administered ⁇ -interferon, as known to those skilled in the art.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a chimaeric polypeptide, polynucleotide, or peptidomimetic compound of the invention and a physiologically acceptable excipient.
  • the composition may further comprise an component for increasing the antigenicity and/or immieuxicity of the composition, for example an adjuvant and/or a cytokine.
  • Suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms” of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Pluronic" is a Registered Trade Mark.
  • the aforementioned compounds or a formulation thereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • a compound Whilst it is possible for a compound to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the compound and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Prefened unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • the following delivery systems may be particularly suitable for use with polypeptide or polynucleotide active ingredients.
  • Polypeptides may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections.
  • An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
  • the polypeptide can be administered by a surgically implanted device that releases the drug directly to the required site.
  • Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis.
  • the direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.
  • Electroporation therapy (EPT) systems can also be employed for the administration of polypeptides.
  • EPT Electroporation therapy
  • a device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
  • Polypeptides can be delivered by electroincorporation (El).
  • El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin.
  • the particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
  • ReGel injectable system that is thermo-sensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.
  • Polypeptides may also be delivered orally.
  • the process may employ a natural process for oral uptake of vitamin Bj 2 in the body to co-deliver proteins and peptides.
  • the protein or peptide can move through the intestinal wall.
  • Complexes are synthesised between vitamin B 12 analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B 12 portion of the complex and significant bioacfivity of the drug portion of the complex.
  • IF intrinsic factor
  • Polypeptides can also be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targetting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al ( 1998), Trends Cell Biol 8, 84-87.
  • a further aspect of the invention provides a polypeptide (1207- 1221 polypeptide) comprising or consisting of at least eight contiguous amino acids selected from the region comprised between amino 1207- 1221 of a native HCV polypeptide, and/or having an epitope selected from epitopes present in the said region, wherein the polypeptide is of about 8 to about 100 amino acids and/or does not comprise a sequence of more than 30 contiguous amino acids from a native HCV polypeptide.
  • Relevant preferences indicated in relation to earlier aspects of the invention for example concerning the length of the polypeptide or sequences therein derived from a HCV polypeptide, also apply to this aspect of the invention.
  • the polypeptide may be useful in relation to previous aspects of the invention.
  • a further aspect of the invention provides a polynucleotide capable of expressing a polypeptide (1207- 1221 polypeptide) of the previous aspect of the invention.
  • a still further aspect of the invention provides a peptidomimetic compound corresponding to the polypeptide (1207-1221 polypeptide) of the previous aspect of the invention.
  • a further aspect of the invention provides the said polypeptide, peptidomimetic compound or polynucleotide for use in medicine.
  • a further aspect of the invention provides the use of the said polypeptide ( 1207-1221 polypeptide), peptidomimetic compound or polynucleotide in the manufacture of a medicament for the treatment of a patient with or at risk of HCV infection.
  • a further aspect of the invention provides a pharmaceutical composition comprising a said polypeptide, peptidomimetic compound or polynucleotide and a pysiologically acceptable excipient, preferences for which are as discussed above.
  • the composition may comprise a suitable adjuvant.
  • the polypeptide may be modified to increase its antigenicity and/or combined with a compound for increasing its antigenicity and/or immunogenicity, as indicated above.
  • the polypeptide may be mannosylated.
  • the polypeptide may comprise a non-HCV viral polypeptide, for example a HBV polypeptide, for example a HBV core protein, as described in, for example, Clarke et al (1987) Nature 330(6146), 381 -384 or Chambers et al (1996) J Virol 70(6), 4045-4052.
  • a HBV polypeptide for example a HBV core protein, as described in, for example, Clarke et al (1987) Nature 330(6146), 381 -384 or Chambers et al (1996) J Virol 70(6), 4045-4052.
  • a further aspect of the invention provides a method of determining the relative prospects of a particular outcome for a subject of exposure to HCV and/or HCV infection, comprising the step of determining whether the subject has an immune response and/or determining the nature of the immune response, to one or more epitopes present in a region between amino acids 31-45, 141-155, 1207- 1221 , 2268-2284 and or 2941-2955 of a native HCV polypeptide. It is preferred that the patient is of HLA type DRl l or DRl 2, preferably DRl l .
  • the method may be used to determine the relative prospect of the patient developing chronic HCV infection.
  • the method may comprise the step of determining whether the patient has a T cell response to the epitope(s).
  • the method may comprise the step of determining whether the T cells produce IFN ⁇ and/or IL10. Production of IFN ⁇ may indicate that the patient is predisposed to clear the HCV infection, whereas production of IL10 with only low or non-detectable levels of IFN ⁇ may indicate that the patient is predisposed to develop chronic HCV infection.
  • the method may comprise the step of comparing responses to different epitopes. Suitable methods for investigating the immune response of the patient are described, for example, in Example 1.
  • a further aspect of the invention provides a method of selecting a method of treatment for a subject with or at risk of a HCV infection wherein a method according to the previous aspect of the invention is used.
  • the method may be used to prioritise treatment with ⁇ -interferon or other agents, for example treatment according to previous aspects of the invention.
  • the method may be used to determine which epitope(s) to administer to the patient; if a patient has a strong response to a particular epitope then it may not be necessary to administer that epitope to the patient.
  • Example 1 Characterisation of novel HLA-DR11 restricted hepatitis C virus (HCV) epitopes reveals both qualitative and quantitative differences in HCN-specific CD4+ T cell responses in chronically infected and non-viraemic patients.
  • HCV hepatitis C virus
  • the CD4+ T cells response appears to be critical in deciding the fate of many viral infections. Clearance of HCN, which often causes persistent infection, has a strong association with the MHC class II antigen HLA-DR1 1 suggesting a key role for CD4+ T cells.
  • HLA-DR 1 1 -restricted T cell epitopes derived from several viral proteins, using a CD4+ T cell epitope prediction program based on complete HLA class ll-eluted pooled peptide sequence data [ (22, 23) and additional data] and highly sensitive ELISPOT assays. This enabled us to compare specific CD4+ T cell populations in HLA-DR1 1 patients who have either successfully eliminated the virus or who are chronically infected, revealing fundamental differences in the qualitative and quantitative characteristics of T cell responses.
  • the frequency and phenotype of memory cells is likely to reflect the magnitude of the initial immune response, and suggests a high frequency of IFN ⁇ -secreting CD4+ T cells to multiple epitopes are important in clearance of HCN.
  • Study population Patients were diagnosed with HCN exposure by the clinical history and the presence of anti-HCV antibodies. Chronic infection was diagnosed by a positive viral specific nested PCR reaction +/- abnormal serum alanine transaminase levels. A cleared infection was diagnosed by at least three negative HCV PCRs separated by 6- 12 months and normal liver function tests. The HLA typing was performed using a PCR-based method to obtain the genotype. These tests were performed by the hospital reference laboratories. Ethical committee approval (St Mary's ⁇ HS Trust) was granted for obtaining blood samples from patients.
  • the peptides were made according to the viral genotype la based on Simmond's classification.
  • the candidate peptides 15 or 16 amino acids in length were synthesized commercially (ECHAZ micro collections, Tubingen, Germany).
  • Each peptide was dissolved in DMSO, the majority entering solution at 50 mg / ml, and frozen at -20 °C until further use.
  • HLA-DR11 pool sequencing data was obtained as previously described for other alleles [(23)]. This HLA-DR11 matrix was used to scan the viral polypeptide and the top 4% of predicted ligands synthesised commercially.
  • ELISPOT enzyme-linked immunospot
  • T cells obtained from TCLs grown as described below
  • R-5 RPMI with 5% heat treated fetal calf serum
  • the wells were pre-coated with monoclonal antibody to the cytokine of interest.
  • the peptides were tested in duplicate wells, having been added to a final concentration of 10 ⁇ g / ml, and compared to control wells with no peptide.
  • the plate was incubated at 37°C, 5% CO : for 18 hours for IFN ⁇ and 1L10, and 40 hours for IL4.
  • the plate was then developed and specific cytokine-producing T cells were enumerated at the single cell level by counting the number of spots per well minus the background. The plates were assessed by an independent observer blinded to the well contents.
  • 2xl0 5 PBMCs at a concentration of 2 m cells / ml were grown with 10 ⁇ g/ml of peptide, using R-10 (RPMI plus 10% human AB serum plus antibiotics/L- glutamine supplement) and incubated in a humidified atmosphere containing 5% C0 2 at 37 °C.
  • R-10 RPMI plus 10% human AB serum plus antibiotics/L- glutamine supplement
  • the medium was supplemented with IL2 (10% Lymphocult T, Biotest) and fresh R-10 on days 6 and 9.
  • IL2 10% Lymphocult T, Biotest
  • the lines were washed three times in RPMI and assayed as described above.
  • the anti- HLA-DR monoclonal antibody L243 and anti-DQ la3 were added 1 hour prior to peptide at 20 ⁇ g / ml.
  • BCLs EBV transformed B cell lines
  • HLA-DRl l pool sequence data was tested on a series of known HLA-DRl l ligands (24) using the previously described epitope- prediction program (22, 23). Table 1 demonstrates this approach was highly effective in the accurate identification of peptides from a parent protein that could be presented by HLA-DR1 1.
  • the candidate peptides were given scores that ranked all of them to within the top 4% of predicted ligands, with three out of eight of the tested ligands receiving scores ranking them ⁇ 1 %.
  • HCV polypeptide of 301 1 amino acids was scanned with the HLA- DR1 1 matrix and the top 4% of predicted epitopes were synthesised. For initial screening these peptides were made up into a series of pools containing five or six peptides. Fifteen HLA-DR1 1 positive patients were screened using an ex vivo IFN ⁇ ELISPOT assay with 24 pools. Pools 1, 2, 11, 17 and 19 had a frequency of positive responses (ie >2x background) in more than 25% of individuals. The responses were dominated by individuals who had cleared the virus (data not shown).
  • Epitopes derived from three different viral proteins are identified by specific IFN ⁇ -producing CD4+ T cells in all non-viraemic HLA-DRll patients.
  • IL10 production from epitope-specific T cells reveals differential responses between chronically infected and non-viraemic patients.
  • IL10 is a cytokine produced by T cells which is thought to play an immunomodulatory role by down regulating antigen specific immune responses. IL 10 may favour viral persistence by inhibiting a Thl -type cell mediated immune response (25, 26).
  • Figure 2b summarizes the production of IL10 from TCLs generated to the above four epitopes. The magnitude of the measured responses is reduced when compared to IFN ⁇ both in the proportion of positive lines, and the frequencies of responding T cells within the line. The patients in group A had stronger IL10 responses compared to group B to epitope 2.2 and 19.3, with an equal magnitude for 1.2.
  • RNA viruses like HCV lack the genomic capacity to produce immunomodulatory cytokines, but there is some evidence that epitope variants can favour IL I O production in certain cases (28).
  • the prevalence of ILIO may reflect the response of the immune system to a chronic antigenic load, switching off IFN ⁇ production and favouring ILI O (29). This strategy might minimize immunopathology in these chronically infected individuals. Chronically infected patients produced no ILIO to 2.2, which is the only epitope to which they produced some IFN ⁇ .
  • IL4 production was also measured but revealed very low frequency responses in both groups. There were slightly stronger responses in group A to peptides 2.2, 11.5, and 19.3 (% positive lines producing >200 cells / million group A vs. group B 9.7 vs. 0, 22.7 vs. 14, 6.45 vs. 0) and in group B to 1.2 ( 0 vs. 5).
  • the patients in group A who produced IL4 in particular lines tended to also produce IFN ⁇ / IL10 from the same lines giving a ThO phenotype.
  • the IL4-producing lines in group B either produced this cytokine alone, or with 1L10, suggesting a Th2 phenotype.
  • FIG. 3 compares the ex vivo epitope-specific T cell responses to the frequencies of specific cells measured in the short term T cell lines. There does not appear to be a direct correlation in most cases. Although in certain instances high frequency cultured responses reflect an apparently high precursor frequency measured ex vivo, there are many instances of very high cultured frequencies being grown out from PBMCs demonstrating very low ex vivo measured frequencies. This raises a number of important points. Firstly, the methods used to measure responding cells may give differing results.
  • ex vivo measured frequencies shown in Figure 1 are greater than published precursor frequencies in HCV patients measured by T cell proliferation in limited dilution assays, even though these use whole protein as antigen and might theoretically contain multiple epitopes (14). Indeed the size of such frequencies is emphasized by comparison to other ex vivo measured responses e.g. in Figure 1 , influenza haemagglutinin universal epitope (HA 305-320 sequence
  • ACPKYVKQNTLKLATG 25 cells / million and tetanus toxoid 22 cells / million in patient 1.
  • the epitope-specific cells will not divide in standard proliferation assays without the addition of IL2 (data not shown), suggesting a degree of T cell anergy or suppression.
  • CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

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Abstract

L"invention concerne l"utilisation d"un polypeptide comprenant une ou plusieurs copies d"un ou de plusieurs épitopes choisis parmi des épitopes présents dans les régions situées entre les acides aminés 31-45, 141-155, 1207-1221, 2268-2284 et/ou 2941-2955 d"un polypeptide VHC natif. Ce polypeptide est utilisé dans la préparation d"un médicament destiné au traitement d"un patient présentant un risque d"infection par le VHC, ce patient possédant un antigène HLA de type DR11 ou DR12. Ledit polypeptide (a) ne comprend aucune séquence de plus de 30 acides aminés contigus provenant d"un polypeptide VHC natif, et/ou (b) présente une longueur d"environ 8 à environ 100 acides aminés. L"invention concerne également une méthode destinée à pronostiquer une issue particulière pour un sujet exposé au VHC et/ou à une infection par le VHC. Cette méthode consiste à déterminer si le sujet présente une réponse immunitaire à un ou plusieurs épitopes présents dans une région située entre les acides aminés 31-45, 141-155, 1207-1221, 2268-2284 et/ou 2941-2955 d"un polypeptide VHC natif. Les épitopes présents dans les régions susmentionnées sont considérés comme des épitopes stimulant les lymphocytes T restreints par un antigène HLA.
PCT/GB2001/004636 2000-10-25 2001-10-18 Identification d"epitopes du vhc restreints par hla-dr WO2002034770A1 (fr)

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Publication number Priority date Publication date Assignee Title
FR2843115A1 (fr) * 2002-08-02 2004-02-06 Commissariat Energie Atomique Melange de peptides issus des proteines c et ns3 du virus de l'hepatite c et leurs applications
WO2004069864A1 (fr) * 2003-01-07 2004-08-19 Biomerieux Polypeptides f’ du virus de l’hepatite c, epitopes et leurs applications diagnostiques et therapeutiques
WO2012072088A1 (fr) 2010-12-02 2012-06-07 Bionor Immuno As Conception d'échafaudage peptidique
WO2013182661A1 (fr) 2012-06-06 2013-12-12 Bionor Immuno As Peptides issus de protéines virales pour l'utilisation en tant qu'immunogènes et réactifs de dosage
EP3338798A1 (fr) 2011-01-06 2018-06-27 Bionor Immuno AS Peptide multimère

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WO2001021189A1 (fr) * 1999-07-19 2001-03-29 Epimmune Inc. Induction de reponses immunitaires cellulaires au virus de l'hepatite c mettant en oeuvre des compositions de peptides et d'acide nucleique

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WO1995027733A1 (fr) * 1994-04-08 1995-10-19 The Government Of The United States Of America, Represented By The Department Of Health & Human Services Peptide de noyau du virus de l'hepatite c pour la stimulation des lymphocytes t cytotoxiques et le diagnostic de l'exposition au vhc
WO2001021189A1 (fr) * 1999-07-19 2001-03-29 Epimmune Inc. Induction de reponses immunitaires cellulaires au virus de l'hepatite c mettant en oeuvre des compositions de peptides et d'acide nucleique

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GODKIN ET AL.: "Use of complete eluted peptide sequence data from HLA-DR and -DQ molecules to predict T-cell epitopes, and the influence of the non-binding terminal regions of ligands in epitope selection", JOURNAL OF IMMUNOLOGY, vol. 161, 1998, pages 850 - 858, XP002188420 *
LAMONACA VINCENZO ET AL: "Conserved hepatitis C virus sequences are highly immunogenic for CD4+ T cells: Implications for vaccine development.", HEPATOLOGY, vol. 30, no. 4, October 1999 (1999-10-01), pages 1088 - 1098, XP002188421, ISSN: 0270-9139 *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2843115A1 (fr) * 2002-08-02 2004-02-06 Commissariat Energie Atomique Melange de peptides issus des proteines c et ns3 du virus de l'hepatite c et leurs applications
WO2004014936A3 (fr) * 2002-08-02 2004-07-22 Commissariat Energie Atomique Melange de peptides issus des proteines c et ns3 du virus de l'hepatite c et leurs applications.
WO2004069864A1 (fr) * 2003-01-07 2004-08-19 Biomerieux Polypeptides f’ du virus de l’hepatite c, epitopes et leurs applications diagnostiques et therapeutiques
WO2012072088A1 (fr) 2010-12-02 2012-06-07 Bionor Immuno As Conception d'échafaudage peptidique
EP3338798A1 (fr) 2011-01-06 2018-06-27 Bionor Immuno AS Peptide multimère
WO2013182661A1 (fr) 2012-06-06 2013-12-12 Bionor Immuno As Peptides issus de protéines virales pour l'utilisation en tant qu'immunogènes et réactifs de dosage
US10501499B2 (en) 2012-06-06 2019-12-10 Bionor Immuno As Peptides derived from viral proteins for use as immunogens and dosage reactants

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