WO1999058561A1 - Hepatitis c virus mimotopes - Google Patents
Hepatitis c virus mimotopes Download PDFInfo
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- WO1999058561A1 WO1999058561A1 PCT/FR1999/001155 FR9901155W WO9958561A1 WO 1999058561 A1 WO1999058561 A1 WO 1999058561A1 FR 9901155 W FR9901155 W FR 9901155W WO 9958561 A1 WO9958561 A1 WO 9958561A1
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- peptide
- ser
- hepatitis
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Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24041—Use of virus, viral particle or viral elements as a vector
- C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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 the treatment and prevention of hepatitis C and in particular to any peptide mimicking conformational epitopes of structural antigens of the hepatitis C virus (HCV) and to any poly nucleotide integrated in a vector allowing the expression of said peptides and their use for therapeutic, prophylactic, in particular vaccine, and / or diagnostic purposes.
- HCV hepatitis C virus
- HCV is an enveloped virus with positive strand RNA and represents the major etiological agent of non-A, non-B hepatitis. This virus is involved in chronic liver infections and in late complications leading to cirrhosis of the liver and hepatocellular carcinoma (HCC). The virus is mainly transmitted by blood.
- Immune reactions in response to infection appear limited or at least inadequate, with chronicity appearing in 80% of cases after primary infection.
- Chronically infected patients have abnormally high blood levels of enzymes from liver cell necrosis, especially alanine aminotransferase (ALAT), a high level of antibodies specific for viral antigens, and have viremia detectable by RT-PCR.
- AZA alanine aminotransferase
- Prospective studies in blood donors have shown the coexistence of significant anti-HCV antibody levels, normal ALT levels, and transient or fluctuating viremia suggesting the existence of healthy carriers of infection.
- the main target of HCV diagnosis is the structural protein of the nucleocapsid. This protein is well conserved among the various HCV genotypes and anti-capsid antibodies are present in the majority of chronically infected patients. (Manzini, P. et al., 1993. Liver 13: 222-226; Bukh, J .. et al. 1994. PNAS 91: 8239-8243; Harada, S., et al. 1991. J. Virol. 65 : 3015-3021).
- the recombinant protein C22-3 covering amino acids 2 to 120 of the nucleocapsid, is the major component of commercial anti-HCV tests called "second generation" (Hosein, B., et al. 1991.
- the hepatitis C virus genome consists of 2 non-coding portions located respectively at the 5 'and 3' ends of the genome framing a single open reading segment (ORF) which codes for 3 structural proteins (the nucleocapsid protein or core ( C), and the two envelope proteins E1 and E2) and 6 non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a and NS5b) (Bukh, J., et al. 1995 Seminars in liver disease 15: 41- 63).
- ORF open reading segment
- a unique poly-protein precursor (Grakoui, A., et al. 1993. J. Virol 67: 1385-1395; Grakoui, A., et al. 1993. PNAS 90: 10583-10587; Grakoui, A., et al. 1993. J. Virol. 67: 2832-2843; Tanji, Y., et al. 1994. J. Virol 68: 8418-8422; Manabe, S., et al. 1994. Virology 198: 636-644) is directly translated from viral RNA (Choo, QL et al. 1989.
- HCV nucleocapsid contains several highly immunogenic linear epitopes (Chiba, J., H. et al. 1991. PNAS 88: 4641-4645; Yoshikawa, A. , K. et al. 1992. J. Immunol. Meth. 148: 143-150; Parmley, SF, et al. 1988. Gene 73: 305-318; Winter, G., et al. Annu Rev Immunol 12: 433 -455; Kohler, G., et al. 1975. Nature: 495-497; Huse, WD, et al. 1989.
- the present invention aims to overcome these needs by identifying peptide structures for the therapeutic or prophylactic treatment of hepatitis C capable of reacting with an antibody specific for an antigen of HCV structure, comprising an amino acid sequence which mimics an epitope conformational of an antigen of the structure of said virus without however corresponding to a continuous amino acid sequence of this antigen, characterized in that this peptide notably comprises, as desired, the following sequences 1 to 7
- the present invention also relates to any recombinant vector comprising a functional expression cassette allowing the expression of a polynucleotide coding for a peptide meeting the criteria defined above.
- the present invention also relates to a therapeutic or prophylactic composition of hepatitis C, in particular intended for vaccine use, the active principle of which comprises a peptide meeting the criteria defined above and / or a recombinant vector coding for said peptide.
- the present invention also relates to the use of a peptide meeting the criteria defined above as a reagent for the diagnosis of hepatitis C and / or of susceptibility to chronicity in the event of infection established by the virus of hepatitis C, said diagnosis comprising the evaluation, from a blood sample, of the humoral and / or cell-mediated response specific for this peptide.
- a peptide meeting the criteria defined above as a reagent for the diagnosis of hepatitis C and / or of susceptibility to chronicity in the event of infection established by the hepatitis C virus, said diagnosis including evaluation of the delayed hypersensitivity response following intradermal or subcutaneous administration of this peptide.
- Peptide means a sequence of at least 6 amino acids linked together by a peptide bond obtained by chemical synthesis or by genetic recombination techniques, preferably between 6 and 50 amino acids and in particular between 20 and 40 amino acids .
- Structural antigen means a molecular entity which binds at least to a specific antibody while also participating in the spatial configuration of the object from which it originates, in this case HCV.
- “By conformal epitope” means a molecule which constitutes the specific binding site to an antibody and which is represented by an amino acid sequence which does not correspond to a continuous amino acid sequence of the natural protein against which this is directed. antibody.
- this amino acid sequence of the conformational epitope is not homologous to a continuous amino acid sequence of the natural protein, the homology being defined by the combination of the two criteria
- amino acid identity criterion determined by the ratio between the number of amino acids of a peptide according to the invention which are identical to those of a sequence of the same size carried by the natural protein, and the total number amino acids of said peptide.
- identity in amino acids will preferably not exceed 50%, even 60% or 70% or 80% or even 90%.
- sequence identity criterion determined by the ratio between the number of amino acids of a peptide according to the invention which are both identical and are in the same chain position as those of a sequence of the same size carried by the natural protein, and the total number of amino acids of said peptide.
- the chain identity will not exceed 70 to 80%.
- “By mimotope” means an epitope which mimics the three-dimensional structure of another epitope by binding to the specific binding site of the same antico ⁇ s
- “By CDR3” means the hyper-variable amino acid chain region of the heavy and light chain of immunoglobulins and which is located at the site of specific interaction with the epitope.
- Conjugate means the association of the peptide as defined in the invention with any other molecule, by physical or chemical methods, intended to induce or strengthen the immunogenicity of the starting peptide.
- Immunogenicity means the capacity of a molecular entity, after inoculation into a mammal, to induce a production of antico ⁇ s specifically directed against this entity.
- “By poly nucleotide” means either an RNA sequence, or a DNA sequence, or a cDNA sequence resulting from the reverse transcription of a sequence of natural or synthetic origin, with or without bases modified.
- “Mucosal route” means a mode of administration which brings the pharmaceutical composition directly into contact with the different types of mucous membranes in the body.
- Parenter route means a mode of administration which puts the pharmaceutical composition directly in contact with the internal tissues or organs of the organism.
- the invention therefore relates to any peptide which mimics a conformational epitope of a structural antigen of the hepatitis C virus and which is recognized by a specific antico ⁇ s of this antigen.
- a peptide according to the invention can be advantageously represented by one of the 7 sequences as follows SEQ ID NO: 1 Gln-Leu-Ile-Thr-Lys-Pro-Leu
- rFabC14 is mainly characterized in heavy VH3 chains by a CDR3 (Complementary-Determining Region) of sequence DLYYDDMSYE, either in 3-letter code Asp-Leu-Tyr-Tyr-Asp-Asp-Met-Ser-Tyr -Glu (SEQ ID NO: 8), and in the Vi amb dal light chains of a CDR3 of sequence GTWDNSLSA, ie Gly-Thr-T ⁇ -Asp-Asn-Ser-Leu-Ser-Ala (SEQ ID NO: 9 ).
- CDR3 Complementary-Determining Region
- the second, designated rFabC3, is mainly characterized in heavy V H 3 chains by a CDR3 of sequence DPLEYFDTSDYDFVDF, namely Asp-Pro-Leu-Glu-Tyr-Phe-Asp-Thr- Ser-Asp-Tyr-Asp-Phe- Val-Asp-Phe (SEQ ID NO: 10), and in the light chains V kappa 4 of a CDR3 of sequence QQYYSTP, ie Gln-Gln-Tyr-Tyr-Ser-Thr-Pro (SEQ ID NO: 11) .
- chimeric antico ⁇ s composed of human and non-human amino acid sequences can be formed as described by Winter et al. (1991) Nature 349: 293; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86: 4220; Shaw et al. (1987) J Immunol. 138: 4534; and Brown et al. (1987) Cancer Res. 47: 3577; Riechmann et al. (1988) Nature 332: 323: Verhoeyen et al. (1988) Science 239: 1534; Jones et al. (1986) Nature 321: 522; EP-A-519,596,; GB-A-2,276,169.
- the present invention also relates to peptides comprising a repetition (2 or more) of the peptide according to the invention or a combination of different peptides according to l invention, as well as peptides comprising both repeats and combinations.
- the peptides can be joined by covalent bonds or non-covalent bonds, for example, one can advantageously cite the method developed by Posnett et al (J. Biol. Chem. (1988) 263: 1719) which n does not alter the epitope or epitopes carried by the peptides and results in the formation of octamers of the same peptide or of different peptides.
- the peptides according to the invention can thus be conjugated to known immunogenic proteins such as serum albumin, thyroglobulin, ovalbumin, gelatin, hemocyanin (e.g. Keyhole Limpet Haemocyanin KLH), seroglobulins, tetanus toxoid, etc. or to T helper epitopes among which there may be mentioned the theratope.
- immunogenic proteins such as serum albumin, thyroglobulin, ovalbumin, gelatin, hemocyanin (e.g. Keyhole Limpet Haemocyanin KLH), seroglobulins, tetanus toxoid, etc. or to T helper epitopes among which there may be mentioned the theratope.
- Heterobifunctional agents such as SPDP, carbodiimide, glutaraldehyde, biotin / avidin system, etc. can be used, for example.
- the peptides can also be coupled to lipopolysaccharides, polysaccharides, 1 glycopeptides, muramyl peptide analogs, fatty acids, etc.
- a peptide is coupled with a fatty acid of the palmitoyl-lysine type as described in EP 491628 (Biovector) or (Pam) 3 Cys-Ser as described in EP547681 (Merck), for example, the technical content of said patents being inco ⁇ orated by reference in the subject of the invention.
- the peptides can also be formulated with alum, monophosphoryl Lipid A, pluronics, SAF1, Ribi trehalose-6,6-dimycolate or other immunostimulatory compounds known to those skilled in the art to increase the immunogenicity of the peptide to which these compounds are linked.
- the present invention also relates to the isolated DNA fragments coding for the peptides according to the invention and which can be used to produce the peptides by expression of the DNA sequence in an appropriate expression system. Taking into account the degeneracy of the code, those skilled in the art are perfectly capable of determining the different DNA sequences capable of coding for the different peptides in accordance with the invention.
- the expression system is an in vitro expression system for the production of the peptides for their subsequent use, e. g. as a diagnostic reagent, as an antigenic component or as a vaccine component.
- in vitro expression systems or vectors are perfectly known to those skilled in the art and mention may be made, for example, of bacteria such as E. coli, eukaryotic cells such as yeasts, in particular S. cerevisiae. , baculovirus, in particular spread on insect cells, etc.
- the invention therefore also relates to an expression cassette comprising such a DNA fragment and regulatory sequences allowing the expression of this DNA fragment in an appropriate in vitro expression system.
- the expression system is an in vivo expression system for generating an immune reaction, preferably protective, in the patient treated.
- the expression system which can be replicative or not replicative, will express the peptide in vivo. Those skilled in the art have at their disposal such systems.
- plasmids in particular naked plasmids, eg according to WO-A-90 11092, WO-A-93 19813, WO-A-94 21797 and WO-A-95 20660, poxviruses, such as vaccinia virus and avian pox (fowlpox, pigeonpox, canarypox, etc.), adenoviruses, etc.
- poxviruses such as vaccinia virus and avian pox (fowlpox, pigeonpox, canarypox, etc.), adenoviruses, etc.
- the invention therefore also relates to expression cassettes comprising such a DNA fragment and the means for regulating expression in the chosen expression system. It also relates to the expression system or expression vector, comprising such an expression cassette, in particular plasmid, poxvirus, adenovirus, as seen above.
- the invention also relates to the use of at least one peptide in accordance with the invention in combination or not with at least one recombinant vector in accordance with the invention for the preparation of a pharmaceutical composition intended for preventing or curing of a condition linked to hepatitis C.
- a composition according to the invention may comprise preparations which may be in the form of creams, lyophilized or non-lyophilized powders, solutions, suspensions, for administration by mucous route such as oral , nasal, rectal, genital, cutaneous, for example.
- sterile injectable preparations may be in the form of solutions, suspensions or emulsions .
- the preparations may contain excipients and / or stabilizing agents suitable for the mode of administration.
- Preparations intended for vaccine use may also contain adjuvants or be incorporated into delivery systems compatible with use in human medicine.
- adjuvants such as Alum (Aluminum phosphate or aluminum hydroxide) inco ⁇ or in a conventional manner in vaccines, the incomplete Freund's adjuvant, Monophosphorylated Lipid A (MPL), QS21, Polyphosphazene, muramyl dipeptide (MDP) or its derivatives, the use of antigen delivery systems such as emulsions (MF59, SAF1, RIBI, SB 62), ISCOMS, liposomes, microspheres composed of PLGA polymers of well-calibrated diameter , or possibly pseudo virions.
- Alum Alphazene
- MDP muramyl dipeptide
- antigen delivery systems such as emulsions (MF59, SAF1, RIBI, SB 62), ISCOMS, liposomes, microspheres composed of PLGA polymers of well-calibrated diameter , or possibly pseudo
- the doses and routes of administration of these pharmaceutical compositions will be determined by taking into account the nature of the composition, the level of expression of the peptide of interest by the recombinant vector, if included in the preparation, of the age, sex and weight of the individual receiving the preparation.
- the relative importance of the carrier molecule in the conjugate will also be taken into account if it is included in the composition.
- the doses of peptides administered may reach 1 to 5 mg but more generally will be between 5 ⁇ g and 1 mg per injection, preferably 50 to 500 ⁇ g.
- the recombinant vector coding for the peptide of interest may be administered or used to transfect or infect the cells of interest at a minimum dose of 10 3 ′ 5 infecting units (pfu).
- the recombinant vector will be used in a dose scale ranging from 10 4 to 10 10 pfu depending on the expression efficiency of the peptide by this vector and in particular in a dose scale ranging from 10 6 to 10 9 pfu, for example.
- the pharmaceutical composition comprises several recombinant vectors coding for peptides of different interest
- these same dose scales can be applied to these combinations.
- Those skilled in the art will be able to refer to the protocols and clinical trials using preparations based on recombinant vectors, in particular recombinant pox viruses, recombinant adenoviruses, already produced in humans to agree on the appropriate number of pfu that must contain pharmaceutical composition.
- the dosage should be sufficient to induce a response at least equivalent to that of the product in the form of a modified or unmodified peptide and / or induce a level of expression of the peptide equivalent to that obtained using the recombinant vectors already mentioned.
- the quantities of plasmids contained in the pharmaceutical compositions may be in scales ranging from 1 ⁇ g to 100 mg, preferably between 0.1 mg to 10 mg. Those skilled in the art will be able to refer to the protocols and clinical trials already carried out in humans, using plasmid DNA preparations to agree on the dose of plasmid which the pharmaceutical composition must contain.
- the pharmaceutical composition may be administered all at once or several times to achieve the desired level of response, in particular the level and quality of the desired antico ⁇ s and / or specific cell-mediated response, identified as that guaranteeing protection against accidental contamination.
- the chosen administrative voice to respect the deadlines allocated between each injection, which may preferably be 1 month, 2 months or 6 months and / or make use of a combined or serial manner during the duration of the medical protocol, in particular the vaccine protocol, of different pharmaceutical compositions relating to the peptide, to the recombinant vector or to the plasmid of interest that a person skilled in the art is capable of controlling.
- the level of protection it may also be necessary to give booster injections at regular intervals.
- the pharmaceutical composition for the treatment of hepatitis C, can be administered at once or several times and in a manner which can be very closely spaced, in particular within periods of less than a week, in order to reach the desired level of response. , in particular that which makes it possible to note the absence of hepatitis C virus in the blood by the PCR test.
- those skilled in the art may refer to the protocol used by Pol S., et al. 1998 Acta Gastroenterol. Belg. 61: 228, for the treatment of chronic hepatitis B using a vaccine antigen of the hepatitis B envelope.
- the pharmaceutical composition comprising the peptide, the vector or the plasmid of interest may be combined or used alternating with conventional treatment of this condition, including interferon ⁇ .
- the pharmaceutical composition comprising one or more peptides of interest, the corresponding recombinant vector (s) of interest as well as the plasmid (s) interest in stimulating cells of the patient's immune system in vitro or ex vivo and then reinjecting them into the body of the individual.
- This methodology was notably developed in the immunotherapeutic treatment of cancer.
- the subject of the invention is finally the use of the peptides of interest as a reagent for the diagnosis of hepatitis C and / or of susceptibility to chronicity in the event of established infection.
- conformational epitopes of HCV structural antigens have been defined.
- HCV protective antico ants which very often recognize conformational epitopes and thus make it possible to distinguish individuals who are healthy or who have contracted an old infection from which they are cured (having protective antico ⁇ s) of those who are chronically infected (not having protective antico ⁇ s).
- the present invention therefore also relates to a method of diagnosing hepatitis C and / or of susceptibility to chronicity in the event of established infection, the said method being based on the analysis of the humoral response, the cell-mediated response and / or the delayed hypersensitivity response.
- immuno-enzymatic, radio-immunological or western blotting methods well known to those skilled in the art can be used, such as, for example, the ELISA, RIA, RIPA or IRMA methods.
- the peptides according to the invention can be brought into contact with human cells of the immune system and the specific lymphoproliferative response can be evaluated as is well known to those skilled in the art.
- the delayed hypersensitivity response can be evaluated by administering the peptide of interest by the intra-dermal or subcutaneous route and by measuring the intensity of the late local inflammatory response which is generally observed within at least 6 hours. after the injection and preferably between 18 and 48 hours after the injection.
- FIG. 1 represents the characteristics of recognition of the 12 "positive” isolates with respect to different fragments of the HCV nucleocapsid (M48, S18D, V22G and P42Y antigens) by ELISA.
- Figures 2-4 show the binding inhibition curves, respectively, of the
- sequences coding for the heavy and light chain parts of the selected Fab molecules can be isolated and synthesized, and cloned into any vector or replicon allowing their expression.
- Any suitable expression system can be used, for example bacteria, yeasts, insect, amphibian and mammalian cells.
- Expression systems in the bacteria include those described in Chang et al. (1978) Nature 275: 615, Goeddel et al. (1979) Nature 281: 544, Goeddel et al. (1980) Nucleic Acids Res. 8: 4057, EP-A-36,776, US-A-4,551,433, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80: 21-25, and Siebenlist et al. (1980) Cell 20: 269.
- Expression systems in yeasts include those described in Hinnen et al. (1978) Proc. Natl. Acad. Sci.
- lymphocytes from a healthy HCV carrier were used as starting material to produce the combinatorial library of antico ⁇ s.
- the lymphocyte cDNA was obtained from RNA using a methodology developed by Sodoyer R. et al. (1997) Human Antibodies 8: 37.
- the heavy and light chain library was constructed using the phagemids pVH (pM 831) and pVL (pM452). The two libraries were then associated in a "Random" fashion by subcloning VL genes in the heavy chain library.
- the phagemid library obtained is then infected with the phage helper Ml 3 VCS thus allowing the expression of Fab on the surface of the phages.
- rFabC3 recognizes the S18D antigen which corresponds to the N terminal portion of the capsid going from amino acid 2 to 21.
- r FabC14 does not recognize S18D. The complete nucleotide sequence of these 2 recombinant antico ⁇ s has been produced.
- rFab C3 contains a VH3 heavy chain associated with
- Kappa 4 whose CDR3 have the respective sequence DPLEYFDTSDYDFVDF, i.e. Asp-Pro-Leu-Glu-Tyr-Phe-Asp-Thr-Ser-Asp-Tyr-Asp-Phe-Val-Asp-Phe (SEQ ID NO: 10) , and QQYYSTP, ie Gln-Gln-Tyr-Tyr-Ser-Thr-Pro (SEQ ID NO: 11).
- RFabC14 contains a VH3 heavy chain associated with a lambdal light chain whose CDR3s have the respective sequence DLYYDDMSYE, ie in 3-letter code Asp-Leu-Tyr-
- the clone producing the monoclonal antibody was deposited on February 9, 1996 under the deposit number CNCM 1-1673 with the National Collection of Cultures of microorganisms of the Institut Pasteur (CNCM) and is therefore accessible to the public.
- CNCM 1-1673 National Collection of Cultures of microorganisms of the Institut Pasteur (CNCM) and is therefore accessible to the public.
- a person skilled in the art can therefore easily reproduce the invention, and in particular select the mimotopes of the HCV envelope using this monoclonal antico ⁇ s or reproduce an equivalent of this antico ⁇ s using the conventional tools of molecular biology.
- the DNA which codes for immunoglobulins and in particular the CDR3 fragments of the heavy chain of immunoglobulins is sequenced.
- the peptides among which those comprising the sequences 1 to 7 (see the sequence list below) can be obtained from a library of peptides or prepared by conventional chemical synthesis. Such a synthesis can be carried out in a homogeneous solution or in the solid phase. In homogeneous solution, one proceeds according to the method described by Houbenweyl in the work “Method der organishen Chemie” edited by E. Wunsh, vol. 15-1 and II, THEME, Stuttgart, 1974. In solid phase, one proceeds according to the method described by Atherton and Shepart in their work “Solid phase peptide synthesis", IRL Press, Oxford, 1989.
- phage bank expressing random peptides at their commercially available surface was used to characterize the structure of the epitopes recognized by the soluble Fabs corresponding to the Fabs expressed on the surface of the 12 positive isolates.
- Soluble Fabs are derived by excision of gene III from positive isolates. These sFab are then used to sensitize immunotubes which are used for selection, by panning, within the phage library expressing the peptides random those which interact specifically with the Fabs of interest. After several amplification and selection cycles according to standard procedures (Maniatis et al.), The specific phages selected are amplified in E. Coli.
- Mini phage DNA preparations are carried out according to the procedures described in the works of Maniatis, the DNA is sequenced using an automatic sequencer from which the sequence of the peptide expressed by the phage.
- the peptides containing the sequences Gln-Leu-Ile-Thr-Lys-Pro-Leu, His-Ala-Phe-Pro-His-Leu-His and Ser-Ala-Pro- Ser-Ser-Lys- Asn were isolated using the recombinant antico ⁇ s FabC3 and FabC14.
- peptides are capable of completely inhibiting the interaction of the antico ⁇ s FabC3 and FabC14 with the M48 antigen whereas they do not have this inhibitory effect with respect to a monoclonal antico ⁇ s 2C6F6 which recognizes a linear epitope located between amino acids 18 and 24 of the M48 antigen (figs 2 to 4).
- the peptides containing the sequences Gly-Glu-Thr-Arg-Ala-Pro-Leu, Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro- Arg-Pro, T ⁇ -Gln- Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-Thr and Met-Leu-Pro-Ser-Val-Leu-Asp were isolated using the monoclonal antibody H2. These same sequences are also recognized by other monoclonal antico ⁇ s specific for the HCV envelope.
- a peptide according to Example 1 is produced by chemical synthesis ("Method der organishen Chemie” edited by E. Wunsh, vol. 15-1 and II, THIEME, Stuttgart, 1974; Atherton and Shepart in their book “Solid phase peptide synthesis ", IRL Press, Oxford, 1989) or obtained from the expression product of a recombinant vector and in particular of a recombinant baculovirus using the techniques developed by Smith et al USA 4,745,051. Water-in-oil emulsions are then prepared using squalene as a constituent of the organic phase, Tween 80 or a mixture of Tween 80 and SPAN as a surfactant, the aqueous phase containing the peptide solution.
- a vaccine formulation based on liposomes containing a peptide according to Example 1 is prepared by referring to works such as "Liposomes as Drug Carriers" edited by G. Gregoriadis, 1988, or in volumes 1 to 3 of "Liposome Technology edited by G. Gregoriadis, 1984.
- a vaccine formulation based on ISCOMs containing a peptide according to Example 1 is prepared by referring to the reference articles of B Morein et al, 1984, Nature 308: 457 or Immunology to Day, 1987, 8 (11): 333.
- a vaccine formulation is prepared based on microparticles comprising a peptide according to Example 1 mimicking a conformational epitope of HCV structural antigen.
- many synthetic or natural polymers are used such as the methyl methacrylate polymer (Troster SD et al, 1992, J. Micro-encaps. 9:19) but often poly (d, 1 -lactide- co-glycolide) also called PLGA is the referent because of its biodegradability, its safety and its applications already old in the medical field.
- the PLGA micro particles loaded with peptides are prepared in particular by double water-in-oil-in-water emulsion.
- the peptide is dissolved in the aqueous phase and then emulsified in a solution of PLGA in the organic phase such as dichloromethane.
- a solution of PLGA in the organic phase such as dichloromethane.
- the water in oil emulsion is obtained by high speed stirring of the peptide solution in the organic solution of PLGA.
- a second aqueous phase containing an appropriate concentration of surfactant such as polyvinyl alcohol is added to the first emulsion to thereby produce the double emulsion.
- surfactants are also used such as bile salts or poly (oxyethylene glycerol monoleate) to stabilize the double emulsion (Rafati H et al., 1997, Vaccine 15: 1888).
- the microparticles of PLGA are washed several times in distilled water and then lyophilized and kept at 5 ° C.
- Example 6 Vaccine composition
- Peptides with fewer than 20 amino acids can be weakly immunogenic.
- a vaccine formulation is prepared based on polymers of the same peptide or different peptides, in the form of octamers comprising a branched poly-lysine structure with 8 lateral arms on which the same are fixed.
- peptide or different peptides according to Example 1 using the method developed by Posnett DN et al. (1988) J. Biol. Chem. 263: 1719.
- Example 7 Vaccine composition
- a vaccine formulation is prepared comprising a peptide according to Example 1 coupled to a carrier molecule such as tetanus or diphtheria toxoid using methods well known to those skilled in the art. Nevertheless, to best preserve the conformation of the antigenic site carried by the peptide the use of "spacer arms" is recommended, as well as the use of glutaraldehyde as a conjugating agent should be avoided as much as possible.
- Example 8 Vaccine composition comprising a lipopeptide
- a vaccine formulation comprising a peptide according to Example 1 coupled to one or more chains derived from fatty acids, including N ⁇ palmitoyl-lysine, N, N-dipalmitoyl-lysine, pimelautide, trimexautide or to a group.
- steroid including N ⁇ [(cholest-5-enyl-3-oxy) -acetyl)] - lysine or (cholest-5-enyl-3-oxy) acetic acid according to the process described in patent EP0491628 (INSERM) so as to obtain a lipopeptide.
- a vaccine composition comprising a recombinant poxvirus encoding a peptide according to Example 1 mimicking a conformational epitope of HCV structural antigen.
- Recombinant poxviruses are obtained by homologous recombination, for example, using expression cassettes (plasmid) containing the poly nucleotide which codes for the peptide of interest under the dependence of promoters of poxviruses (H6, I3L) according to the methods described. in US 5,863,542.
- a vaccine composition comprising several peptides according to example 1 using the same modes of preparations developed in examples 1 to 8, peptides which may or may not be in the form of conjugates, comprising or not the sequences 1 to 7 cited in the list of sequences.
- a vaccine composition is prepared in particular comprising both one or more peptides mimicking one or more conformational epitopes of the nucleocapsid and one or more peptides mimicking one or more conformational epitopes of the HCV envelope
- Example 11 Expression of Several Peptides by a Poxvirus
- a vaccine composition comprising a recombinant poxvirus encoding several peptides according to Example 1.
- the use and preparation of recombinant vectors encoding several epitopes is well known to those skilled in the art (Toes RE et al. (1997) Proc Natl Acad Acad Sci USA 94: 14660, Thomson SA et al. (1996) J. Immunol 157: 822) and is also applicable to the preparation of recombinant poxviruses encoding multiple mimotopes.
- a vaccine composition is prepared comprising a recombinant canaripox (recombinant ALVAC) encoding multiple mimotopes of the nucleocapsid and of the HCV envelope.
- Detection of specific HCV antibodies is carried out by ELISA using one or more peptides according to Example 1 for the diagnosis of hepatitis C from a biological sample. For this, a sample of physiological fluid (blood, plasma, serum) is taken, a sample which is then reacted in the presence of a peptide according to the invention. The peptide itself is used as a diagnostic reagent.
- an indirect diagnostic test of the ELISA type in which the peptide fixed on a support (well) is placed in the presence of the sample to be tested, while the revelation of the antigen-antico ⁇ s fixation is ensured by a labeled anti-Ig.
- a test by competition or displacement in which a peptide according to Example 1 is used, and a labeled antico ⁇ s specific for the peptide.
- the peptide is again attached to a solid support.
- the competition test the peptide attached to its support is put simultaneously in the presence of the sample (antico ⁇ s of the sample) and of a labeled antico ⁇ s specific for the peptide.
- antico ⁇ s marked antico ⁇ s coupled to peroxidase are used.
- monoclonal and polyclonal antico ⁇ s or recombinant antico ⁇ s specific for the peptide according to Example 1 are also used, which are sometimes in the form of Fab or F (ab ′) 2 fragments.
- Detection of specific HCV antibodies is carried out by immunochromatography using one or more peptides according to the invention for the diagnosis of hepatitis C from a biological sample.
- the peptide according to Example 1 is fixed on a strip-type support and reference is made to the article by Robert FN Zurk et al., Clin. Chem.
- a study of the specific lymphoproliferative response to one or more peptides according to Example 1 is implemented for the diagnosis of hepatitis C from a biological sample.
- the patient's blood is collected on a heparin tube.
- the lymphocytes are then separated by centrifugation on hypaque Ficoll and then distributed in 96-well sterile microplates at the rate of 2 10 5 cells per round-bottom well under a final volume of 200 ⁇ l of complete culture medium (RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 50 U / ml of penicillin, 50 ⁇ g / ml of streptomycin and 5% of AB complemented serum) and placed in the presence of variable concentrations of the peptide in accordance with the invention (concentrations ranging from lng / ml to 50 ⁇ g / ml) .
- Each concentration of peptide is tested in a trip liquid to overcome biological variations as much as possible. Multiple combinations of peptides can also be tested in the indicated concentration range, for example a combination resulting from the association of an envelope mimotope with a nucleocapsid mimotope in the indicated concentration range.
- 0.5 ⁇ ci of tritiated thymidine is added to each well.
- the cellular DNA is collected from each culture well on filters after precipitation with ethanol and the degree of tritiated thymidine incorporation is measured using a liquid scintillation counter. which reflects the intensity of the lymphoproliferative response.
- the results are expressed in the form of stimulation index (average of the cpm of the lymphocyte culture wells containing a given concentration of peptide / average of the cpm of the lymphocyte culture wells without peptide).
- the lymphoproliferative response is considered positive when the stimulation index is greater than 3.
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Abstract
The invention concerns a peptide for the therapeutic or prophylactic treatment of hepatitis C, capable of reacting with a hepatitis C virus structure antigen specific antibody, comprising an amino acid sequence which imitates a conformational epitope of an antigen structure of said virus without, however, corresponding to a continuous amino acid sequence of said antigen, characterised in that said peptide comprises in particular assorted sequences 1 to 7. The invention also concerns a recombinant vector comprising a functional expression cassette for expressing a polynucleotide coding for said peptide. The invention further concerns a or composition for treating or preventing hepatitis C, in particular to be used as vaccine, whereof the active principle comprises said peptide, if required conjugate, and/or a recombinant vector coding for said peptide. The invention finally concerns the use of said peptide as reagent for diagnosing hepatitis C and/or susceptibility to chronicity in the event of hepatitis C infection.
Description
Mimotopes du Virus de l'hépatite CHepatitis C virus mimotopes
La présente invention se rapporte au traitement et à la prévention de l'hépatite C et notamment à tout peptide mimant des épitopes conformationels d'antigènes de structure du virus de l'hépatite C (VHC) et à tout poly nucléotide intégré dans un vecteur permettant l'expression desdits peptides et leur utilisation à des fins thérapeutiques, prophylactiques, notamment vaccinales, et/ou de diagnostiques.The present invention relates to the treatment and prevention of hepatitis C and in particular to any peptide mimicking conformational epitopes of structural antigens of the hepatitis C virus (HCV) and to any poly nucleotide integrated in a vector allowing the expression of said peptides and their use for therapeutic, prophylactic, in particular vaccine, and / or diagnostic purposes.
Domaine de l'inventionField of the invention
Le VHC est un virus enveloppé à ARN à brin positif et représente l'agent étiologique majeur de l'hépatite non- A, non-B. Ce virus est impliqué dans les infections chroniques du foie et dans les complications tardives conduisant à une cirrhose du foie et à un carcinome hépato-cellulaire (HCC). Le virus est principalement transmis par le sang.HCV is an enveloped virus with positive strand RNA and represents the major etiological agent of non-A, non-B hepatitis. This virus is involved in chronic liver infections and in late complications leading to cirrhosis of the liver and hepatocellular carcinoma (HCC). The virus is mainly transmitted by blood.
Les réactions immunitaires en réponse à l'infection semblent limitées ou tout au moins inadéquates, la chronicité apparaissant dans 80 % des cas après infection primaire. Les patients chroniquement infectés ont des taux sanguins anormalement élevés en enzymes provenant de la nécrose des cellules hépatiques, notamment en alanine aminotransférase (ALAT), un taux élevé d'anticorps spécifiques des antigènes viraux et présentent une virémie détectable par RT-PCR. Des études prospectives menées sur des donneurs de sang ont montré la coexistence de taux significatifs d'anticorps anti-HCV, de taux normaux en ALAT, et d'une virémie transitoire ou fluctuante ce qui suggère l'existence de porteurs sains de rinfection.Immune reactions in response to infection appear limited or at least inadequate, with chronicity appearing in 80% of cases after primary infection. Chronically infected patients have abnormally high blood levels of enzymes from liver cell necrosis, especially alanine aminotransferase (ALAT), a high level of antibodies specific for viral antigens, and have viremia detectable by RT-PCR. Prospective studies in blood donors have shown the coexistence of significant anti-HCV antibody levels, normal ALT levels, and transient or fluctuating viremia suggesting the existence of healthy carriers of infection.
Il n'existe pas aujourd'hui de test commercialement disponible qui permet d'analyser la réponse humorale en distinguant les malades porteurs chroniques d'HCV, des patients ayant contractés une infection ancienne dont ils sont guéris, ou des porteurs sains.Today, there is no commercially available test which makes it possible to analyze the humoral response by distinguishing patients suffering from chronic HCV, patients having contracted an old infection from which they are cured, or healthy carriers.
La cible principale du diagnostic HCV est la protéine structurale de la nucléocapside. Cette protéine est bien conservée parmi les différents génotypes d'HCV et les anticorps anti-capside sont présents chez la majorité des patients chroniquement infectés. (Manzini, P. et al., 1993. Liver 13:222-226 ; Bukh, J.. et al. 1994. P.N.A.S. 91:8239-8243 ; Harada, S., et al. 1991. J. Virol. 65:3015-3021). Par exemple, la protéine recombinante C22-3, couvrant les acides aminés de 2 à 120 de la nucléocapside est le composant majeur des tests commerciaux anti-HCV dit "de seconde génération" (Hosein, B., étal. 1991. P.N.A.S. 88:3647-3651 ; Grakoui, A., et al. 1993. J. Virol. 67:2832-2843).
Le génome du virus de l'hépatite C consiste en 2 portions non codantes situées respectivement aux extrémités 5' et 3' du génome encadrant un seul segment ouvert de lecture (ORF) qui code pour 3 protéines de structure ( la protéine nucléocapsidique ou core (C), et les deux protéines d'enveloppe El et E2) et 6 protéines non structurelles (NS2, NS3, NS4a, NS4b, NS5a et NS5b) (Bukh, J., et al. 1995 Seminars in liver disease 15 : 41-63). Un précurseur poly protéique unique (Grakoui, A., et al. 1993. J. Virol 67:1385-1395 ; Grakoui, A., et al. 1993. P.N.A.S. 90:10583-10587 ; Grakoui, A., et al. 1993. J. Virol. 67:2832-2843 ; Tanji, Y., et al. 1994. J. Virol 68:8418-8422 ; Manabe, S., et al. 1994. Virology 198:636-644) est directement traduit à partir de l'ARN viral (Choo, Q.L. et al. 1989. Science 244:359-352) et subit des modifications post-traductionnelles à la fois par les protéases de l'hôte et par des protéases virales (Manabe, S., et al. 1994. Virology 198:636- 644 ; Hijikata, M., et al. 1993. J. Virol 67:4665-4675). Les protéines structurales, qui consistent en la capside C et les deux protéines d'enveloppe El et E2, sont générées par coupures séquentielles dans le premier tiers de la poly protéine, tandis que les protéines non structurales (NS2, NS3, NS4 et NS5) sont générées à partir des deux tiers restant.The main target of HCV diagnosis is the structural protein of the nucleocapsid. This protein is well conserved among the various HCV genotypes and anti-capsid antibodies are present in the majority of chronically infected patients. (Manzini, P. et al., 1993. Liver 13: 222-226; Bukh, J .. et al. 1994. PNAS 91: 8239-8243; Harada, S., et al. 1991. J. Virol. 65 : 3015-3021). For example, the recombinant protein C22-3, covering amino acids 2 to 120 of the nucleocapsid, is the major component of commercial anti-HCV tests called "second generation" (Hosein, B., et al. 1991. PNAS 88: 3647-3651; Grakoui, A., et al. 1993. J. Virol. 67: 2832-2843). The hepatitis C virus genome consists of 2 non-coding portions located respectively at the 5 'and 3' ends of the genome framing a single open reading segment (ORF) which codes for 3 structural proteins (the nucleocapsid protein or core ( C), and the two envelope proteins E1 and E2) and 6 non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a and NS5b) (Bukh, J., et al. 1995 Seminars in liver disease 15: 41- 63). A unique poly-protein precursor (Grakoui, A., et al. 1993. J. Virol 67: 1385-1395; Grakoui, A., et al. 1993. PNAS 90: 10583-10587; Grakoui, A., et al. 1993. J. Virol. 67: 2832-2843; Tanji, Y., et al. 1994. J. Virol 68: 8418-8422; Manabe, S., et al. 1994. Virology 198: 636-644) is directly translated from viral RNA (Choo, QL et al. 1989. Science 244: 359-352) and undergoes post-translational modifications both by host proteases and by viral proteases (Manabe, S ., et al. 1994. Virology 198: 636-644; Hijikata, M., et al. 1993. J. Virol 67: 4665-4675). The structural proteins, which consist of the capsid C and the two envelope proteins El and E2, are generated by sequential cleavages in the first third of the poly protein, while the non-structural proteins (NS2, NS3, NS4 and NS5) are generated from the remaining two-thirds.
Plusieurs études basées sur la modélisation de la protéine avec des peptides synthétiques ont indiqué que la nucléocapside du HCV contient plusieurs épitopes linéaires fortement immunogènes (Chiba, J., H. et al. 1991. P.N.A.S. 88:4641-4645 ; Yoshikawa, A., K. et al. 1992. J. Immunol. Meth. 148:143-150 ; Parmley, S.F., et al. 1988. Gène 73:305- 318 ; Winter, G., et al. Annu Rev Immunol 12:433-455 ; Kohler, G., et al. 1975. Nature : 495-497 ; Huse, W.D., et al. 1989. Science 246:1275-1281 ; Hoogenboom, H.R., et al. 1992. Immunol Rev 130:41-68 ; Burton, D.R., et al. 1994. In Advances in Immunology, Vol 57, vol. 57.F.J. Dixon, éd. Académie Press Inc, 525 B Street Suite 1900, San Diego, CA 92101-4495, p. 191-280 ; Marks, J.D., et al. 1992. Bio/Technology 10:779-783 ; Waterhouse, P., et al. 1993. Nucleic Acids Res 21:2265-2266 ; Me Cafferty, J., et al. 1990. Nature 348:552-554). Inversement, on sait très peu de chose à propos des épitopes naturels, conformationnels de la nucléocapside. Cela est principalement dû au fait que très peu d'anticorps ont été produits à partir d'individus infectés par HCV (Siemoneit, K., et al. 1994. Hybridoma 13:9-13 ; Cerino, A., et al. 1993. J. Immunol. 151:7005-7015 ; Akastuka, T., et al. 1993. Hepatology 18:503-510)).Several studies based on protein modeling with synthetic peptides have indicated that the HCV nucleocapsid contains several highly immunogenic linear epitopes (Chiba, J., H. et al. 1991. PNAS 88: 4641-4645; Yoshikawa, A. , K. et al. 1992. J. Immunol. Meth. 148: 143-150; Parmley, SF, et al. 1988. Gene 73: 305-318; Winter, G., et al. Annu Rev Immunol 12: 433 -455; Kohler, G., et al. 1975. Nature: 495-497; Huse, WD, et al. 1989. Science 246: 1275-1281; Hoogenboom, HR, et al. 1992. Immunol Rev 130: 41- 68; Burton, DR, et al. 1994. In Advances in Immunology, Vol 57, vol. 57. FJ Dixon, éd. Académie Press Inc, 525 B Street Suite 1900, San Diego, CA 92101-4495, p. 191- 280; Marks, JD, et al. 1992. Bio / Technology 10: 779-783; Waterhouse, P., et al. 1993. Nucleic Acids Res 21: 2265-2266; Me Cafferty, J., et al. 1990. Nature 348: 552-554). Conversely, very little is known about natural, conformational epitopes of the nucleocapsid. This is mainly due to the fact that very few antibodies have been produced from individuals infected with HCV (Siemoneit, K., et al. 1994. Hybridoma 13: 9-13; Cerino, A., et al. 1993 J. Immunol. 151: 7005-7015; Akastuka, T., et al. 1993. Hepatology 18: 503-510)).
Résumé de l'inventionSummary of the invention
Face à la problématique liée au risque important de développement d'hépato carcinome ou de cirrhose consécutif à l'infection par le VHC le besoin existe d'identifier
une composition pharmaceutique permettant de traiter efficacement ou de prévenir cette affection virale.Faced with the problem linked to the significant risk of development of hepatocellular carcinoma or cirrhosis following infection with HCV, there is a need to identify a pharmaceutical composition for effectively treating or preventing this viral condition.
Il existe aussi un besoin de développer des réactifs rentrant notamment dans la composition de kits immunologiques qui permettent de distinguer entre les porteurs sains, les malades chroniquement infectés et les sujets guéris qui ont fait une infection ancienne.There is also a need to develop reagents used in particular in the composition of immunological kits which make it possible to distinguish between healthy carriers, chronically infected patients and cured subjects who have had an old infection.
La présente invention vise à pallier ces besoins en identifiant des structures peptidiques pour le traitement thérapeutique ou prophylactique de l'hépatite C capable de réagir avec un anticorps spécifique d'un antigène de structure du VHC , comprenant une séquence en acides aminés qui mime un épitope conformationnel d'un antigène de structure dudit virus sans toutefois correspondre à une séquence continue d'acides aminés de cet antigène, caractérisé en ce que ce peptide comprend notamment au choix les séquences 1 à 7 suivantesThe present invention aims to overcome these needs by identifying peptide structures for the therapeutic or prophylactic treatment of hepatitis C capable of reacting with an antibody specific for an antigen of HCV structure, comprising an amino acid sequence which mimics an epitope conformational of an antigen of the structure of said virus without however corresponding to a continuous amino acid sequence of this antigen, characterized in that this peptide notably comprises, as desired, the following sequences 1 to 7
SEQ ID NO : 1 Gln-Leu-Ile-Thr-Lys-Pro-LeuSEQ ID NO: 1 Gln-Leu-Ile-Thr-Lys-Pro-Leu
SEQ ID NO : 2 His-Ala-Phe-Pro-His-Leu-HisSEQ ID NO: 2 His-Ala-Phe-Pro-His-Leu-His
SEQ ID NO : 3 Ser-Ala-Pro-Ser-Ser-Lys-AsnSEQ ID NO: 3 Ser-Ala-Pro-Ser-Ser-Lys-Asn
SEQ ID NO : 4 Gly-Glu-Thr-Arg-Ala-Pro-Leu SEQ ID NO : 5 Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro-Arg-ProSEQ ID NO: 4 Gly-Glu-Thr-Arg-Ala-Pro-Leu SEQ ID NO: 5 Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro-Arg-Pro
SEQ ID NO : 6 T -Gln-Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-ThrSEQ ID NO: 6 T -Gln-Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-Thr
SEQ ID NO : 7 Met-Leu-Pro-Ser-Val-Leu-Asp.SEQ ID NO: 7 Met-Leu-Pro-Ser-Val-Leu-Asp.
La présente invention concerne également tout vecteur recombinant comprenant une cassette d'expression fonctionnelle permettant l'expression d'un polynucleotide codant pour un peptide répondant aux critères définis ci dessus.The present invention also relates to any recombinant vector comprising a functional expression cassette allowing the expression of a polynucleotide coding for a peptide meeting the criteria defined above.
La présente invention concerne aussi une composition thérapeutique ou prophylactique de l'hépatite C, notamment destiné à un usage vaccinal, dont le principe actif comprend un peptide répondant aux critères définis ci dessus et/ou un vecteur recombinant codant pour ledit peptide.The present invention also relates to a therapeutic or prophylactic composition of hepatitis C, in particular intended for vaccine use, the active principle of which comprises a peptide meeting the criteria defined above and / or a recombinant vector coding for said peptide.
Enfin la présente invention concerne aussi l'utilisation d'un peptide répondant aux critères définis ci dessus en tant que réactif pour le diagnostic de l'hépatite C et/ou de la susceptibilité à la chronicité en cas d'infection établie par le virus de l'hépatite C, ledit diagnostic comprenant l'évaluation, à partir d'un échantillon de sang, de la réponse humorale et/ou à médiation cellulaire spécifique de ce peptide.
L'utilisation d'un peptide répondant aux critères définis ci dessus en tant que réactif pour le diagnostic de l'hépatite C et/ou de la susceptibilité à la chronicité en cas d'infection établie par le virus de l'hépatite C, ledit diagnostic comprenant l'évaluation de la réponse d' hypersensibilté retardée consécutive à l'administration intradermique ou sous cutanée de ce peptide.Finally, the present invention also relates to the use of a peptide meeting the criteria defined above as a reagent for the diagnosis of hepatitis C and / or of susceptibility to chronicity in the event of infection established by the virus of hepatitis C, said diagnosis comprising the evaluation, from a blood sample, of the humoral and / or cell-mediated response specific for this peptide. The use of a peptide meeting the criteria defined above as a reagent for the diagnosis of hepatitis C and / or of susceptibility to chronicity in the event of infection established by the hepatitis C virus, said diagnosis including evaluation of the delayed hypersensitivity response following intradermal or subcutaneous administration of this peptide.
L'utilisation d'un peptide répondant aux critères définis ci dessus et/ou d'un vecteur recombinant codant pour ledit peptide pour la préparation d'une composition thérapeutique ou prophylactique destinée au traitement ou a la prévention de l'hépatite C.The use of a peptide meeting the criteria defined above and / or of a recombinant vector coding for said peptide for the preparation of a therapeutic or prophylactic composition intended for the treatment or prevention of hepatitis C.
Description de l'inventionDescription of the invention
Dans le contexte de la présente invention, différents termes employés sont ci-après définis:In the context of the present invention, various terms used are defined below:
"Par peptide" on entend une séquence d'au moins 6 acides aminés liés entre eux par une liaison peptidique obtenu par synthèse chimique ou par des techniques de recombinaison génétique, de préférence entre 6 et 50 acides aminés et notamment entre 20 et 40 acides aminés."Peptide" means a sequence of at least 6 amino acids linked together by a peptide bond obtained by chemical synthesis or by genetic recombination techniques, preferably between 6 and 50 amino acids and in particular between 20 and 40 amino acids .
"Par antigène de structure" on entend une entité moléculaire qui se lie au moins à un anticorps spécifique tout en participant également à la configuration spatiale de l'objet dont il est issu, en l'occurrence ici le VHC."Structural antigen" means a molecular entity which binds at least to a specific antibody while also participating in the spatial configuration of the object from which it originates, in this case HCV.
"Par épitope conformationel" on entend une molécule qui constitue le site de liaison spécifique à un anticorps et qui est représentée par une séquence d'acides aminés qui ne correspond pas à une séquence continue en acides aminées de la protéine naturelle contre laquelle est dirigé cet anticorps. De préférence, cette séquence en acides aminés de l'épitope conformationnel n'est pas homologue à une séquence continue en acides aminés de la protéine naturelle, l'homologie étant définie par la combinaison des deux critères"By conformal epitope" means a molecule which constitutes the specific binding site to an antibody and which is represented by an amino acid sequence which does not correspond to a continuous amino acid sequence of the natural protein against which this is directed. antibody. Preferably, this amino acid sequence of the conformational epitope is not homologous to a continuous amino acid sequence of the natural protein, the homology being defined by the combination of the two criteria
- le critère d'identité des acides aminés déterminé par le rapport entre le nombre d'acides aminés d'un peptide selon l'invention qui sont identiques à ceux d'une séquence de même taille portée par la protéine naturelle, et le nombre total d'acides aminés dudit peptide. De préférence l'identité en acides aminés ne dépassera pas de préférence 50%, voire 60% ou 70% ou 80% ou même 90%.
- Le critère d'identité d'enchainement déterminé par le rapport entre le nombre d'acides aminés d'un peptide selon l'invention qui sont à la fois identiques et se trouvent à la même position d'enchainement que ceux d'une séquence de même taille portée par la protéine naturelle, et le nombre total d'acides aminés dudit peptide. De préférence l'identité d'enchainement ne dépassera pas 70 à 80%.- the amino acid identity criterion determined by the ratio between the number of amino acids of a peptide according to the invention which are identical to those of a sequence of the same size carried by the natural protein, and the total number amino acids of said peptide. Preferably the identity in amino acids will preferably not exceed 50%, even 60% or 70% or 80% or even 90%. - The sequence identity criterion determined by the ratio between the number of amino acids of a peptide according to the invention which are both identical and are in the same chain position as those of a sequence of the same size carried by the natural protein, and the total number of amino acids of said peptide. Preferably the chain identity will not exceed 70 to 80%.
"Par mimotope" on entend un épitope qui mime la structure tridimensionnelle d'un autre épitope en se fixant sur le site de liaison spécifique du même anticoφs"By mimotope" means an epitope which mimics the three-dimensional structure of another epitope by binding to the specific binding site of the same anticoφs
"Par CDR3" on entend la région hyper variable d'enchaînement en acides aminés de la chaîne lourde et légère des immunoglobulines et qui se situe au niveau du site d'interaction spécifique avec l'épitope."By CDR3" means the hyper-variable amino acid chain region of the heavy and light chain of immunoglobulins and which is located at the site of specific interaction with the epitope.
"Par conjugué" on entend l'association du peptide tel que défini dans l'invention à toute autre molécule, par des procédés physiques ou chimiques, ayant pour vocation d'induire ou de renforcer l'immunogénicité du peptide de départ."Conjugate" means the association of the peptide as defined in the invention with any other molecule, by physical or chemical methods, intended to induce or strengthen the immunogenicity of the starting peptide.
"Par immunogénicité" on entend la capacité d'une entité moléculaire, après inoculation à un mammifère, à induire une production d'anticoφs spécifiquement dirigé contre cette entité."Immunogenicity" means the capacity of a molecular entity, after inoculation into a mammal, to induce a production of anticoφs specifically directed against this entity.
"Par poly nucléotide" on entend soit une séquence d'ARN, soit une séquence d'ADN, soit d'une séquence d'ADNc résultant de la transcription inverse d'une séquence d'origine naturelle ou de synthèse, avec ou sans bases modifiées."By poly nucleotide" means either an RNA sequence, or a DNA sequence, or a cDNA sequence resulting from the reverse transcription of a sequence of natural or synthetic origin, with or without bases modified.
"Par voie muqueuse", on entend un mode d'administration qui met en contact directement la composition pharmaceutique avec les différents types de muqueuses de l'organisme."Mucosal route" means a mode of administration which brings the pharmaceutical composition directly into contact with the different types of mucous membranes in the body.
"Par voie parentérale", on entend un mode d'administration qui met directement en contact la composition pharmaceutique avec les tissus ou organes interne de l'organisme."Parenteral route" means a mode of administration which puts the pharmaceutical composition directly in contact with the internal tissues or organs of the organism.
L'invention vise donc tout peptide qui mime un épitope conformationnel d'un antigène de structure du virus de l'hépatite C et qui est reconnu par un anticoφs spécifique de cet antigène. Un peptide selon l'invention peut être représenté avantageusement par l'une des 7 séquences telle que suit
SEQ ID NO : 1 Gln-Leu-Ile-Thr-Lys-Pro-LeuThe invention therefore relates to any peptide which mimics a conformational epitope of a structural antigen of the hepatitis C virus and which is recognized by a specific anticoφs of this antigen. A peptide according to the invention can be advantageously represented by one of the 7 sequences as follows SEQ ID NO: 1 Gln-Leu-Ile-Thr-Lys-Pro-Leu
SEQ ID NO : 2 His-Ala-Phe-Pro-His-Leu-HisSEQ ID NO: 2 His-Ala-Phe-Pro-His-Leu-His
SEQ ID NO : 3 Ser-Ala-Pro-Ser-Ser-Lys-AsnSEQ ID NO: 3 Ser-Ala-Pro-Ser-Ser-Lys-Asn
SEQ ID NO : 4 Gly-Glu-Thr-Arg-Ala-Pro-Leu SEQ ID NO : 5 Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro-Arg-ProSEQ ID NO: 4 Gly-Glu-Thr-Arg-Ala-Pro-Leu SEQ ID NO: 5 Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro-Arg-Pro
SEQ ID NO : 6 Tφ-Gln-Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-ThrSEQ ID NO: 6 Tφ-Gln-Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-Thr
SEQ ID NO : 7 Met-Leu-Pro-Ser-Val-Leu-Asp.SEQ ID NO: 7 Met-Leu-Pro-Ser-Val-Leu-Asp.
Des peptides synthétiques, représentant des épitopes linéaires et très immunogéniques de la nucléocapside ont déjà été identifiés par l'homme du métierSynthetic peptides, representing linear and very immunogenic epitopes of the nucleocapsid have already been identified by a person skilled in the art
(Siemoneit, K., et al. 1994. Hybridoma 13:9-13 ; Cerino, A., et al. 1993. J. Immunol.(Siemoneit, K., et al. 1994. Hybridoma 13: 9-13; Cerino, A., et al. 1993. J. Immunol.
151:7005-7015 ; Akastuka, T., et al. 1993. Hepatology 18:503-510) sans toutefois être capable d'isoler des épitopes conformationnels.151: 7005-7015; Akastuka, T., et al. 1993. Hepatology 18: 503-510) without however being able to isolate conformational epitopes.
A partir d'une banque combinatoire d'anticoφs obtenue à partir du sang périphérique d'un porteur sain asymptomatique du virus de l'hépatite C, défini en ce que la sérologie vis à vis du VHC est positive, que le taux d'ALAT est normal et qu'il n'y a pas de virémie détectable par PCR, et d'une librairie synthétique de peptides, on peut identifier, par le biais de mimotopes, de nouveaux épitopes conformationnels présents sur les antigènes de structure du VHC, notamment des épitopes qui peuvent se situer dans la région comprise entre les acides aminés 1 à 72 de la nucléocapside et en particulier dans la région comprise entre les acides aminés 1 à 20 de la nucléocapside, de même que des épitopes qui se situent dans la région comprise entre les acides aminés 1 à 191 et la région comprise entre les acides aminés 235 à 242 de la protéine d'enveloppe El ainsi que les épitopes qui se situent dans la région comprise entre les acides aminés 1 à 396 et la région comprise entre les acides aminés 429 et 570 de la protéine d'enveloppe E2. L'identification de ces mimotopes a nécessité pour leur mise en œuvre d'un processus technologique sophistiqué, à savoir :From a combinatorial bank of anticoφs obtained from the peripheral blood of a healthy asymptomatic carrier of the hepatitis C virus, defined in that the serology with respect to HCV is positive, that the rate of ALT is normal and that there is no viremia detectable by PCR, and from a synthetic library of peptides, it is possible to identify, by means of mimotopes, new conformational epitopes present on the antigens of structure of the HCV, in particular epitopes which may lie in the region between amino acids 1 to 72 of the nucleocapsid and in particular in the region between amino acids 1 to 20 of the nucleocapsid, as well as epitopes which are situated in the region between amino acids 1 to 191 and the region between amino acids 235 to 242 of the envelope protein E1 as well as the epitopes which are located in the region between amino acids 1 to 396 and the region between amino acids 429 and 570 of the envelope protein E2. The identification of these mimotopes required for their implementation a sophisticated technological process, namely:
-la constitution d'une librairie combinatoire d'anticoφs suffisamment complexe pour refléter au mieux le répertoire naturel en anticoφs d'un individu, et de façon avantageuse le répertoire en anticoφs d'un individu porteur sain du VHC ; la sélection au sein de cette banque, d'anticoφs recombinants spécifiques d'antigènes de structure du VHC ; -la sélection de peptides spécifiques des anticoφs recombinants, à partir d'une librairie synthétique aléatoire de peptides obtenue par recombinaison moléculaire, la dite sélection pouvant se faire par ELISA ;
-la caractérisation de ces peptides comme étant des mimotopes d'épitopes conformationnels du VHC en ce qu'il n'y a pas de correspondance entre la séquence en acides aminés dudit peptide avec une quelconque séquence continue en acides aminés retrouvés dans les antigènes de structure du VHC d'une part et d'autre part en ce que ce peptide est capable d'inhiber l'interaction de l'anticoφs recombinant avec l'antigène de structure qui à servi à la sélection du dit anticoφs recombinant.-the constitution of a combinatorial library of anticoφs sufficiently complex to best reflect the natural repertoire in anticoφs of an individual, and advantageously the repertoire in anticoφs of an individual carrying a healthy HCV; the selection from this bank of recombinant anticoφs specific for antigens of HCV structure; the selection of peptides specific for recombinant anticoφs, from a random synthetic library of peptides obtained by molecular recombination, said selection being able to be made by ELISA; the characterization of these peptides as being mimotopes of conformational epitopes of HCV in that there is no correspondence between the amino acid sequence of said peptide with any continuous sequence of amino acids found in the structural antigens HCV on the one hand and on the other hand in that this peptide is capable of inhibiting the interaction of the recombinant anticoφs with the structural antigen which served for the selection of the said recombinant anticoφs.
Pour ce criblage on peut isoler deux anticoφs recombinants spécifiques, par exemple. Le premier, désigné rFabC14, se caractérise principalement dans les chaînes VH3 lourdes par un CDR3 (Complementary-Determining Région) de séquence DLYYDDMSYE, soit en code à 3 lettres Asp-Leu-Tyr-Tyr-Asp-Asp-Met-Ser-Tyr-Glu (SEQ ID NO : 8), et dans les chaînes légères Viambdal d'un CDR3 de séquence GTWDNSLSA, soit Gly-Thr-Tφ-Asp-Asn-Ser-Leu-Ser-Ala (SEQ ID NO : 9). Le deuxième, désigné rFabC3, se caractérise principalement dans les chaînes VH3 lourdes par un CDR3 de séquence DPLEYFDTSDYDFVDF, soit Asp-Pro-Leu-Glu-Tyr-Phe-Asp-Thr- Ser-Asp-Tyr-Asp-Phe-Val-Asp-Phe (SEQ ID NO : 10), et dans les chaînes légères Vkappa4 d'un CDR3 de séquence QQYYSTP, soit Gln-Gln-Tyr-Tyr-Ser-Thr-Pro (SEQ ID NO : 11).For this screening, two specific recombinant anticoφs can be isolated, for example. The first, designated rFabC14, is mainly characterized in heavy VH3 chains by a CDR3 (Complementary-Determining Region) of sequence DLYYDDMSYE, either in 3-letter code Asp-Leu-Tyr-Tyr-Asp-Asp-Met-Ser-Tyr -Glu (SEQ ID NO: 8), and in the Vi amb dal light chains of a CDR3 of sequence GTWDNSLSA, ie Gly-Thr-Tφ-Asp-Asn-Ser-Leu-Ser-Ala (SEQ ID NO: 9 ). The second, designated rFabC3, is mainly characterized in heavy V H 3 chains by a CDR3 of sequence DPLEYFDTSDYDFVDF, namely Asp-Pro-Leu-Glu-Tyr-Phe-Asp-Thr- Ser-Asp-Tyr-Asp-Phe- Val-Asp-Phe (SEQ ID NO: 10), and in the light chains V kappa 4 of a CDR3 of sequence QQYYSTP, ie Gln-Gln-Tyr-Tyr-Ser-Thr-Pro (SEQ ID NO: 11) .
L'homme du métier peut utiliser tout anticoφs ayant un squelette de structure VH3, Viambdal pour produire rFabC14 et tout anticoφs de structure VH3, VI^^^ pour produire rFabC3. Il peut facilement reproduire ces anticoφs, ayant les mêmes caractéristiques de reconnaissance, la production d'anticoφs recombinants par mutagénèse ou clonage à partir de CDR d'intérêt faisant partie des capacités normales de l'homme du métier.Those skilled in the art can use any anticoφs having a backbone of structure VH3, Viambdal to produce rFabC14 and any anticoφs of structure V H 3, VI ^^^ to produce rFabC3. It can easily reproduce these anticoφs, having the same recognition characteristics, the production of recombinant anticoφs by mutagenesis or cloning from CDRs of interest forming part of the normal capacities of those skilled in the art.
Ainsi des anticoφs chimères composés de séquences d'acides aminés humaines et non humaines peuvent être formés comme décrit par Winter et al. (1991) Nature 349 : 293 ; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86 : 4220 ; Shaw et al. (1987) J Immunol. 138 : 4534 ; and Brown et al. (1987) Cancer Res. 47 : 3577 ; Riechmann et al. (1988) Nature 332 : 323 : Verhoeyen et al. (1988) Science 239 : 1534 ; Jones et al. (1986) Nature 321 : 522 ; EP-A-519,596, ; GB-A-2,276,169.Thus chimeric anticoφs composed of human and non-human amino acid sequences can be formed as described by Winter et al. (1991) Nature 349: 293; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86: 4220; Shaw et al. (1987) J Immunol. 138: 4534; and Brown et al. (1987) Cancer Res. 47: 3577; Riechmann et al. (1988) Nature 332: 323: Verhoeyen et al. (1988) Science 239: 1534; Jones et al. (1986) Nature 321: 522; EP-A-519,596,; GB-A-2,276,169.
Pour induire ou plutôt renforcer l'immunogénicité du peptide mimant un épitope conformationnel du VHC, la présente invention a également pour objet des peptides comprenant une répétition (2 ou plus) du peptide conforme à l'invention ou une combinaison de différents peptides conformes à l'invention, ainsi qu'à des peptides comprenant à la fois des répétitions et des combinaisons.
Dans de tels cas, les peptides peuvent être joints par des liaisons covalentes ou des liaisons non covalentes, par exemple, on peut citer avantageusement la méthode développée par Posnett et al ( J. Biol. Chem. (1988) 263: 1719) qui n'altère pas le ou les épitopes porté par les peptides et aboutit à la formation d'octamères du même peptide ou de peptides différents.To induce or rather reinforce the immunogenicity of the peptide mimicking a conformational epitope of HCV, the present invention also relates to peptides comprising a repetition (2 or more) of the peptide according to the invention or a combination of different peptides according to l invention, as well as peptides comprising both repeats and combinations. In such cases, the peptides can be joined by covalent bonds or non-covalent bonds, for example, one can advantageously cite the method developed by Posnett et al (J. Biol. Chem. (1988) 263: 1719) which n does not alter the epitope or epitopes carried by the peptides and results in the formation of octamers of the same peptide or of different peptides.
Dans le cadre notamment des préparations antigéniques et des formulations vaccinales qui sont décrites ci-après, on peut aussi préférer conjuguer par liaison covalente les peptides de l'invention à des molécules immunogènes usuellement utilisées pour rendre immunogène les peptides de petite taille.In the context in particular of the antigenic preparations and vaccine formulations which are described below, it is also possible to prefer to conjugate, by covalent bonding, the peptides of the invention to immunogenic molecules usually used to make small peptides immunogenic.
Les peptides selon l'invention peuvent ainsi être conjugués aux protéines immunogènes connues telles que les sérum albumines, thyroglobuline, ovalbumine, gélatine, hémocyanine (e.g. Keyhole Limpet Haemocyanin KLH), séroglobulines, anatoxine tétanique, etc. ou à des épitopes T helper parmi lesquelles on peut citer le thératope.The peptides according to the invention can thus be conjugated to known immunogenic proteins such as serum albumin, thyroglobulin, ovalbumin, gelatin, hemocyanin (e.g. Keyhole Limpet Haemocyanin KLH), seroglobulins, tetanus toxoid, etc. or to T helper epitopes among which there may be mentioned the theratope.
Les techniques de conjugaison sont aussi parfaitement connues de l'homme de l'art. On peut recourir par exemple aux agents hétérobifonctionnels tels que SPDP, carbodiimide, glutaraldéhyde, système biotine/avidine, etc.Conjugation techniques are also perfectly known to those skilled in the art. Heterobifunctional agents such as SPDP, carbodiimide, glutaraldehyde, biotin / avidin system, etc. can be used, for example.
On peut aussi coupler les peptides à des lipopolysaccharides, polysaccharides, 1 glycopeptides, analogues du muramyl peptide, acides gras, etc. De préférence, on effectue le couplage d'un peptide avec un acide gras du type palmitoyl-lysine tel que décrit dans EP 491628 (Biovector) ou (Pam)3 Cys-Ser tel que décrit dans EP547681 (Merck), par exemple, le contenu technique desdits brevets étant incoφoré par référence dans l'objet de l'invention.The peptides can also be coupled to lipopolysaccharides, polysaccharides, 1 glycopeptides, muramyl peptide analogs, fatty acids, etc. Preferably, a peptide is coupled with a fatty acid of the palmitoyl-lysine type as described in EP 491628 (Biovector) or (Pam) 3 Cys-Ser as described in EP547681 (Merck), for example, the technical content of said patents being incoφorated by reference in the subject of the invention.
Les méthodes pour lier de manière opérationnelle des peptides individuels par des chaînes latérales portant des résidus d'acide aminé, afin de former un conjugué immunogène, par exemple un polymère polypeptidique ramifié, sont aussi bien connues de l'homme de l'art. Par ces méthodes, on cherche à établir des liaisons sur différentes chaînes latérales par un ou plusieurs types de groupes fonctionnels afin d'obtenir une structure dans laquelle les structures peptidiques sont liées par covalence tout en étant séparées par au moins une chaîne latérale. Comme groupes fonctionnels, on peut citer les groupes aminés epsilon, les groupes bêta- ou gamma carboxylique, les groupes thiol (-SH) et les cycles aromatiques (par exemple tyrosine et histidine). Des méthodes pour lier des polypeptides à l'aide de ces groupes fonctionnels sont décrites dans Erlanger (1980 Method of
Enzymology, 70 : 85), Aurameas et al. , (1978 Scand. J. Immunol. , Vol.8, suppl. 7, 7-23) et US-A-4 193 795. En outre, il est également possible de mettre en œuvre une réaction de couplage dirigée telle que décrite dans Rodwell et al. , (1985 Biotech 3, 889-894). Les peptides peuvent également être modifiés pour incoφorer des bras d'espacement tels que hexaméthylène diamine ou d'autres molécules bi-fonctionnelles de tailles similaires.Methods for operably linking individual peptides through side chains carrying amino acid residues to form an immunogenic conjugate, for example a branched polypeptide polymer, are also well known to those of skill in the art. By these methods, it is sought to establish links on different side chains by one or more types of functional groups in order to obtain a structure in which the peptide structures are covalently linked while being separated by at least one side chain. As functional groups, there may be mentioned the epsilon amino groups, the beta- or gamma carboxylic groups, the thiol groups (-SH) and the aromatic rings (for example tyrosine and histidine). Methods for linking polypeptides using these functional groups are described in Erlanger (1980 Method of Enzymology, 70: 85), Aurameas et al. , (1978 Scand. J. Immunol., Vol.8, suppl. 7, 7-23) and US-A-4 193 795. In addition, it is also possible to carry out a directed coupling reaction as described in Rodwell et al. , (1985 Biotech 3, 889-894). Peptides can also be modified to incorporate spacer arms such as hexamethylene diamine or other bi-functional molecules of similar sizes.
Les peptides peuvent être également formulés avec de l'alum, du monophosphoryl Lipid A, pluronics, SAF1, Ribi trehalose-6,6-dimycolate ou autres composés immunostimulants connus de l'homme de l'art pour accroître l'immunogénicité du peptide auquel ces composés sont liés.The peptides can also be formulated with alum, monophosphoryl Lipid A, pluronics, SAF1, Ribi trehalose-6,6-dimycolate or other immunostimulatory compounds known to those skilled in the art to increase the immunogenicity of the peptide to which these compounds are linked.
Néanmoins toutes ces méthodes de conjugaison, de modification, de répétition ou de combinaison de peptides conformes à l'invention doivent respecter au mieux la conformation originelle du peptide.However, all these methods of conjugation, modification, repetition or combination of peptides in accordance with the invention must best respect the original conformation of the peptide.
La présente invention a aussi pour objet les fragments d'ADN isolés codant pour les peptides selon l'invention et pouvant être utilisés pour produire les peptides par expression de la séquence d'ADN dans un système d'expression approprié. En tenant compte de la dégénérescence du code, l'homme de l'art est parfaitement à même de déterminer les différentes séquences d'ADN aptes à coder pour les différents peptides conformes à l'invention.The present invention also relates to the isolated DNA fragments coding for the peptides according to the invention and which can be used to produce the peptides by expression of the DNA sequence in an appropriate expression system. Taking into account the degeneracy of the code, those skilled in the art are perfectly capable of determining the different DNA sequences capable of coding for the different peptides in accordance with the invention.
Selon un premier aspect de l'invention, le système d'expression est un système d'expression in vitro pour la production des peptides en vue de leur utilisation ultérieure, e. g. comme réactif de diagnostic, comme composant antigénique ou comme composant vaccinal. De tels systèmes ou vecteurs d'expression in vitro sont parfaitement connus de l'homme du métier et l'on peut citer à titre d'exemple les bactéries telles que E. Coli, les cellules eucaryotes telles que les levures, notamment S. cerevisiae, le baculovirus, notamment propagé sur cellules d'insectes, etc.According to a first aspect of the invention, the expression system is an in vitro expression system for the production of the peptides for their subsequent use, e. g. as a diagnostic reagent, as an antigenic component or as a vaccine component. Such in vitro expression systems or vectors are perfectly known to those skilled in the art and mention may be made, for example, of bacteria such as E. coli, eukaryotic cells such as yeasts, in particular S. cerevisiae. , baculovirus, in particular spread on insect cells, etc.
L'invention a donc aussi pour objet une cassette d'expression comprenant un tel fragment d'ADN et des séquences régulatrices permettant l'expression de ce fragment d'ADN dans un système d'expression in vitro approprié.The invention therefore also relates to an expression cassette comprising such a DNA fragment and regulatory sequences allowing the expression of this DNA fragment in an appropriate in vitro expression system.
Selon un deuxième aspect de l'invention le système d'expression est un système d'expression in vivo pour générer chez le patient traité une réaction immunitaire, de préférence protectrice. En d'autres termes, le système d'expression, qui peut être réplicatif
ou non réplicatif, va exprimer le peptide in vivo. L'homme du métier a à sa disposition de tels systèmes. A titre d'exemples préférés, on peut citer les plasmides, notamment plasmides nus, e. g. selon WO-A-90 11092, WO-A-93 19813, WO-A-94 21797 et WO-A- 95 20660, les poxvirus , tels que le virus de la vaccine et les pox aviaires (fowlpox, pigeonpox, canarypox, etc.), les adénovirus, etc.According to a second aspect of the invention, the expression system is an in vivo expression system for generating an immune reaction, preferably protective, in the patient treated. In other words, the expression system, which can be replicative or not replicative, will express the peptide in vivo. Those skilled in the art have at their disposal such systems. By way of preferred examples, mention may be made of plasmids, in particular naked plasmids, eg according to WO-A-90 11092, WO-A-93 19813, WO-A-94 21797 and WO-A-95 20660, poxviruses, such as vaccinia virus and avian pox (fowlpox, pigeonpox, canarypox, etc.), adenoviruses, etc.
L'invention a donc aussi pour objet des cassettes d'expression comprenant un tel fragment d'ADN et les moyens de régulation de l'expression dans le système d'expression choisi. Elle a aussi pour objet le système d'expression ou vecteur d'expression, comprenant une telle cassette d'expression, en particulier plasmide, poxvirus, adénovirus, comme vu ci- dessus.The invention therefore also relates to expression cassettes comprising such a DNA fragment and the means for regulating expression in the chosen expression system. It also relates to the expression system or expression vector, comprising such an expression cassette, in particular plasmid, poxvirus, adenovirus, as seen above.
L'invention se rapporte également à l'utilisation d'au moins un peptide conforme à l'invention en association ou non avec au moins un vecteur recombinant conforme à l'invention pour la préparation d'une composition pharmaceutique destinée à prévenir ou à guérir d'une affection liée à l'hépatite C. Une composition selon l'invention peut comprendre des préparations pouvant être sous forme de crèmes, de poudres lyophilisées ou non, de solutions, de suspensions, pour des administrations par voie muqueuse telle qu'oral, nasal, rectal, génital, cutanée, par exemple. Pour des administrations parentérales telle que intra dermique, sous cutanée, intra musculaire, intra veineuse, intra artériel, intra lymphatique ou intra péritonéale, par exemple, les préparations injectables stériles pourront être selon les cas sous forme de solutions, de suspensions ou d'émulsions. Outre le ou les principes actifs, conforme à l'objet de l'invention, les préparations pourront contenir des excipients et/ou des agents stabilisants adaptés au mode d'administration.The invention also relates to the use of at least one peptide in accordance with the invention in combination or not with at least one recombinant vector in accordance with the invention for the preparation of a pharmaceutical composition intended for preventing or curing of a condition linked to hepatitis C. A composition according to the invention may comprise preparations which may be in the form of creams, lyophilized or non-lyophilized powders, solutions, suspensions, for administration by mucous route such as oral , nasal, rectal, genital, cutaneous, for example. For parenteral administration such as intra dermal, subcutaneous, intramuscular, intravenous, intra arterial, intra lymphatic or intra peritoneal, for example, sterile injectable preparations may be in the form of solutions, suspensions or emulsions . In addition to the active ingredient (s), in accordance with the subject of the invention, the preparations may contain excipients and / or stabilizing agents suitable for the mode of administration.
Les préparations destinées à un usage vaccinal pourront également contenir des adjuvants ou être incoφorées dans des systèmes de délivrance compatibles avec un usage en médecine humaine. On peut rapporter notamment l'usage des adjuvants comme l'Alum (Aluminium phosphate ou hydroxyde d'aluminium) incoφoré de façon classique dans les vaccins, l'adjuvant incomplet de Freund, Lipide A monophosphorylé (MPL), QS21, Polyphosphazène, muramyl dipeptide (MDP) ou ses dérivés, l'usage de système de délivrance de l'antigène comme les émulsions (MF59, SAF1,RIBI,SB 62), les ISCOMS, les liposomes, les micro sphères composées de polymères de PLGA de diamètre bien calibré, ou éventuellement les pseudo virions.Preparations intended for vaccine use may also contain adjuvants or be incorporated into delivery systems compatible with use in human medicine. We can report in particular the use of adjuvants such as Alum (Aluminum phosphate or aluminum hydroxide) incoφor in a conventional manner in vaccines, the incomplete Freund's adjuvant, Monophosphorylated Lipid A (MPL), QS21, Polyphosphazene, muramyl dipeptide (MDP) or its derivatives, the use of antigen delivery systems such as emulsions (MF59, SAF1, RIBI, SB 62), ISCOMS, liposomes, microspheres composed of PLGA polymers of well-calibrated diameter , or possibly pseudo virions.
Les doses et voies d'administration de ces compositions pharmaceutiques seront déterminées en prenant en compte la nature de la composition, le niveau d'expression du
peptide d'intérêt par le vecteur recombinant s'il est inclus dans la préparation, de l'âge, du sexe et du poids de l'individu recevant la préparation. Il sera également tenu compte de l'importance relative de la molécule porteuse dans le conjugué s'il est inclus dans la composition.The doses and routes of administration of these pharmaceutical compositions will be determined by taking into account the nature of the composition, the level of expression of the peptide of interest by the recombinant vector, if included in the preparation, of the age, sex and weight of the individual receiving the preparation. The relative importance of the carrier molecule in the conjugate will also be taken into account if it is included in the composition.
Compte tenu de tous ces facteurs qui sont connus et appréciés par l'homme du métier, les doses de peptides administrées pourront atteindre 1 à 5 mg mais plus généralement se situeront entre 5μg et 1 mg par injection, de préférence 50 à 500μg. Le vecteur recombinant codant pour le peptide d'intérêt pourra être administré ou utilisé pour transfecter ou infecter les cellules d'intérêt à une dose minimale de 103'5 unités infectantes (pfu). De façon préférentielle, le vecteur recombinant sera utilisé dans une échelle de dose allant de 104 à 1010 pfu en fonction de l'efficacité d'expression du peptide par ce vecteur et notamment dans une échelle de dose allant de 106 à 109 pfu, par exemple. Lorsque la composition pharmaceutique comprend plusieurs vecteurs recombinant codant pour des peptides d'intérêt différent, il est bien entendu que ces mêmes échelles de doses pourront être appliquées à ces combinaisons. L'homme de l'art pourra se référer aux protocoles et essais cliniques utilisant des préparations à base de vecteurs recombinants, notamment les pox virus recombinants, les adénovirus recombinants, déjà réalisés chez l'homme pour convenir du nombre approprié de pfu que doit renfermer la composition pharmaceutique.Given all of these factors which are known and appreciated by those skilled in the art, the doses of peptides administered may reach 1 to 5 mg but more generally will be between 5 μg and 1 mg per injection, preferably 50 to 500 μg. The recombinant vector coding for the peptide of interest may be administered or used to transfect or infect the cells of interest at a minimum dose of 10 3 ′ 5 infecting units (pfu). Preferably, the recombinant vector will be used in a dose scale ranging from 10 4 to 10 10 pfu depending on the expression efficiency of the peptide by this vector and in particular in a dose scale ranging from 10 6 to 10 9 pfu, for example. When the pharmaceutical composition comprises several recombinant vectors coding for peptides of different interest, it is understood that these same dose scales can be applied to these combinations. Those skilled in the art will be able to refer to the protocols and clinical trials using preparations based on recombinant vectors, in particular recombinant pox viruses, recombinant adenoviruses, already produced in humans to agree on the appropriate number of pfu that must contain pharmaceutical composition.
Lorsque la composition pharmaceutique comprend un plasmide contenant le système d'expression du peptide d'intérêt, le dosage devra être suffisant pour induire une réponse au moins équivalente à celle du produit sous forme de peptide modifié ou non et/ou induire un niveau d'expression du peptide équivalent à celui obtenu à l'aide des vecteurs recombinants déjà cités. Par exemple, les quantités de plasmides contenus dans les compositions pharmaceutiques pourront se situer dans des échelles allant de lμg à lOOmg, de façon préférentielle entre 0,1 mg à lOmg. L'homme de l'art pourra se référer aux protocoles et essais cliniques déjà réalisés chez l'homme, utilisant des préparations d'ADN plasmidique pour convenir de la dose de plasmide que doit renfermer la composition pharmaceutique.When the pharmaceutical composition comprises a plasmid containing the expression system of the peptide of interest, the dosage should be sufficient to induce a response at least equivalent to that of the product in the form of a modified or unmodified peptide and / or induce a level of expression of the peptide equivalent to that obtained using the recombinant vectors already mentioned. For example, the quantities of plasmids contained in the pharmaceutical compositions may be in scales ranging from 1 μg to 100 mg, preferably between 0.1 mg to 10 mg. Those skilled in the art will be able to refer to the protocols and clinical trials already carried out in humans, using plasmid DNA preparations to agree on the dose of plasmid which the pharmaceutical composition must contain.
Pour la prévention de l'hépatite C, la composition pharmaceutique pourra être administrée en une seule fois ou à plusieurs reprises pour atteindre le niveau de réponse désirée, notamment le niveau et la qualité de la réponse anticoφs et/ou à médiation cellulaire spécifique désirée, identifiée comme celle garantissant la protection vis à vis d'une contamination accidentelle. Pour atteindre cet objectif il pourra être nécessaire, outre la composition de la préparation, la voix d'administration choisie, de respecter les délais
impartis entre chaque injection, qui peuvent être de façon préférentielle de 1 mois, 2 mois ou 6 mois et/ou de faire usage de façon combinée ou sériée pendant la durée du protocole médical, notamment vaccinal, de compositions pharmaceutiques différentes se rapportant au peptide, au vecteur recombinant ou au plasmide d'intérêt que l'homme de l'art est capable de maîtriser. Il pourra être également nécessaire pour maintenir le niveau de protection de pratiquer des injections de rappel à intervalles réguliers.For the prevention of hepatitis C, the pharmaceutical composition may be administered all at once or several times to achieve the desired level of response, in particular the level and quality of the desired anticoφs and / or specific cell-mediated response, identified as that guaranteeing protection against accidental contamination. To achieve this objective it may be necessary, in addition to the composition of the preparation, the chosen administrative voice, to respect the deadlines allocated between each injection, which may preferably be 1 month, 2 months or 6 months and / or make use of a combined or serial manner during the duration of the medical protocol, in particular the vaccine protocol, of different pharmaceutical compositions relating to the peptide, to the recombinant vector or to the plasmid of interest that a person skilled in the art is capable of controlling. To maintain the level of protection, it may also be necessary to give booster injections at regular intervals.
Pour le traitement de l'hépatite C la composition pharmaceutique, notamment vaccinale, pourra être administrée en une seule fois ou à plusieurs reprises et de façon pouvant être très rapprochée, notamment dans des délais inférieurs à une semaine, pour atteindre le niveau de réponse désirée, notamment celui qui permet de constater l'absence de virus de l'hépatite C dans le sang par le test PCR. A titre d'exemple, l'homme de l'art pourra se référer au protocole utilisé par Pol S., et al. 1998 Acta Gastroenterol. Belg. 61:228, pour le traitement de l'hépatite B chronique utilisant un antigène vaccinal de l'enveloppe de l'hépatite B. Au besoin, la composition pharmaceutique comprenant le peptide, le vecteur ou le plasmide d'intérêt pourra être associé ou utilisé en alternance avec un traitement conventionnel de cette affection, notamment l'interféron α.For the treatment of hepatitis C, the pharmaceutical composition, in particular the vaccine composition, can be administered at once or several times and in a manner which can be very closely spaced, in particular within periods of less than a week, in order to reach the desired level of response. , in particular that which makes it possible to note the absence of hepatitis C virus in the blood by the PCR test. By way of example, those skilled in the art may refer to the protocol used by Pol S., et al. 1998 Acta Gastroenterol. Belg. 61: 228, for the treatment of chronic hepatitis B using a vaccine antigen of the hepatitis B envelope. If necessary, the pharmaceutical composition comprising the peptide, the vector or the plasmid of interest may be combined or used alternating with conventional treatment of this condition, including interferon α.
Que ce soit pour la prévention ou le traitement de l'hépatite C il pourra être également utile d'utiliser la composition pharmaceutique comprenant un ou plusieurs peptides d'intérêt, le ou les vecteurs recombinants d'intérêt correspondants ainsi que le ou les plasmides d'intérêt pour stimuler les cellules du système immunitaire du patient in vitro ou ex vivo et de les réinjecter ensuite dans l'organisme de l'individu. Cette méthodologie a notamment été développée dans le traitement immunothérapeutique du cancer.Whether for the prevention or treatment of hepatitis C, it may also be useful to use the pharmaceutical composition comprising one or more peptides of interest, the corresponding recombinant vector (s) of interest as well as the plasmid (s) interest in stimulating cells of the patient's immune system in vitro or ex vivo and then reinjecting them into the body of the individual. This methodology was notably developed in the immunotherapeutic treatment of cancer.
L'invention a pour objet enfin l'utilisation des peptides d'intérêt en tant que réactif pour le diagnostic de l'hépatite C et/ou de la susceptibilité à la chronicité en cas d'infection établie. Pour la première fois des épitopes conformationnels d'antigènes de structure du VHC ont été définis. On peut donc utiliser ces peptides, à des fins diagnostiques, pour rechercher préférentiellement des anticoφs protecteurs du VHC, qui, très souvent reconnaissent des épitopes conformationnels et permettre ainsi de distinguer les individus porteurs sains ou ayant contracté une infection ancienne dont ils sont guéris ( possédant des anticoφs protecteurs) de ceux qui sont chroniquement infectés ( ne possédant pas d'anticoφs protecteurs).The subject of the invention is finally the use of the peptides of interest as a reagent for the diagnosis of hepatitis C and / or of susceptibility to chronicity in the event of established infection. For the first time conformational epitopes of HCV structural antigens have been defined. We can therefore use these peptides, for diagnostic purposes, to preferentially seek HCV protective antico ants, which very often recognize conformational epitopes and thus make it possible to distinguish individuals who are healthy or who have contracted an old infection from which they are cured (having protective anticoφs) of those who are chronically infected (not having protective anticoφs).
La présente invention a donc aussi pour objet une méthode de diagnostique de l'hépatite C et/ou de susceptibilité à la chronicité en cas d'infection établie, la dite méthode
étant basée sur l'analyse de la réponse humorale, de la réponse à médiation cellulaire et/ou de la réponse d'hypersensibilité retardée.The present invention therefore also relates to a method of diagnosing hepatitis C and / or of susceptibility to chronicity in the event of established infection, the said method being based on the analysis of the humoral response, the cell-mediated response and / or the delayed hypersensitivity response.
Pour l'analyse de la réponse humorale on pourra utiliser des méthodes immuno- enzymatiques, radio- nmunologies ou de western blotting bien connues de l'homme du métier, comme par exemple les méthodes ELISA, RIA, RIPA ou IRMA.For the analysis of the humoral response, immuno-enzymatic, radio-immunological or western blotting methods well known to those skilled in the art can be used, such as, for example, the ELISA, RIA, RIPA or IRMA methods.
Pour l'analyse de l'ύrimunité à médiation cellulaire on pourra mettre en contact les peptides conforme à l'invention avec des cellules humaines du système immunitaire et évaluer la réponse lymphoproliférative spécifique comme cela est bien connu de l'homme du métier. On peut enfin évaluer la réponse d'hypersensibilité retardée en administrant le peptide d'intérêt par voie intra dermique ou sous cutanée et en mesurant l'intensité de la réponse inflammatoire locale tardive qui s'observe généralement dans un délai d'au moins 6 heures après l'injection et de façon préférentielle entre 18 et 48 heures après l'injection.For the analysis of cell-mediated immunity, the peptides according to the invention can be brought into contact with human cells of the immune system and the specific lymphoproliferative response can be evaluated as is well known to those skilled in the art. Finally, the delayed hypersensitivity response can be evaluated by administering the peptide of interest by the intra-dermal or subcutaneous route and by measuring the intensity of the late local inflammatory response which is generally observed within at least 6 hours. after the injection and preferably between 18 and 48 hours after the injection.
Description des figuresDescription of the figures
La figure 1 représente les caractéristiques de reconnaissance des 12 isolats "positifs" vis à vis de différents fragments de la nucléocapside du VHC (antigènes M48, S18D, V22G et P42Y) par ELISA.FIG. 1 represents the characteristics of recognition of the 12 "positive" isolates with respect to different fragments of the HCV nucleocapsid (M48, S18D, V22G and P42Y antigens) by ELISA.
Les figures 2-4 représentent les courbes d'inhibition de la liaison, respectivement, duFigures 2-4 show the binding inhibition curves, respectively, of the
FabC3 soluble, du FabC14 so lubie et de l'anticoφs monoclonal 2C6F6 à l'antigène de la nucléocapside M48 en présence de concentrations croissantes (de raison 10) en peptides ayant pour séquences QLITKPL, HAFPHLH ou SAPSSKN, obtenues par compétition enSoluble FabC3, fabC14 and the monoclonal antibody 2C6F6 to the M48 nucleocapsid antigen in the presence of increasing concentrations (of reason 10) in peptides having sequences QLITKPL, HAFPHLH or SAPSSKN, obtained by competition in
ELISA.ELISA.
La présente invention est décrite plus en détail ci-après à l'aide du complément de description qui va suivre, qui se réfère à des exemples de sélection d'anticoφs dirigés contre les antigènes de structure du VHC, de sélection de peptides selon l'invention, de compositions vaccinales selon l'invention et d'utilisation des peptides selon l'invention pour le diagnostic de l'hépatite C. Il va de soi, toutefois que ces exemples sont donnés à titre d'illustration de l'objet de l'invention dont ils ne constituent en aucune manière une limitation.
Exemple 1 : Sélection de peptidesThe present invention is described in more detail below with the aid of the additional description which follows, which refers to examples of selection of antico contres directed against structural antigens of HCV, selection of peptides according to invention, vaccine compositions according to the invention and use of the peptides according to the invention for the diagnosis of hepatitis C. It goes without saying, however, that these examples are given by way of illustration of the subject of the invention. invention of which they do not in any way constitute a limitation. EXAMPLE 1 Selection of Peptides
1) La fabrication d'anticoφs recombinants utilisant des méthodes de biologie moléculaire se sont largement développées depuis les dix dernières années et sont maintenant bien connues de l'homme du métier. Il est aussi connu que la spécificité d'un anticoφs recombinant est portée essentiellement par les CDR3 des chaînes légères et lourdes. La connaissance de l'enchaînement en acides aminés qui représente le CDR3 et de la structure du squelette des chaînes lourdes et légères d'un anticoφs donné est suffisante pour que l'homme de métier puisse reproduire et reconstituer un anticoφs recombinant équivalent ayant les mêmes caractéristiques de reconnaissance dudit anticoφs.1) The manufacture of recombinant anticoφs using molecular biology methods have been widely developed over the past ten years and are now well known to those skilled in the art. It is also known that the specificity of a recombinant anticoφs is carried essentially by the CDR3 of the light and heavy chains. Knowledge of the amino acid chain which represents CDR3 and of the skeleton structure of the heavy and light chains of a given anticoφs is sufficient for those skilled in the art to be able to reproduce and reconstitute an equivalent recombinant anticoφs having the same characteristics. of recognition of said anticoφs.
Les séquences codant pour les parties de chaînes lourdes et légères des molécules Fab sélectionnées peuvent être isolées et synthétisées, et clonées dans tout vecteur ou réplicon permettant leur expression.The sequences coding for the heavy and light chain parts of the selected Fab molecules can be isolated and synthesized, and cloned into any vector or replicon allowing their expression.
Tout système d'expression approprié peut être utilisé, par exemple bactéries, levures, cellules d'insecte, d'amphibien et de mammifère. Les systèmes d'expression dans la bactérie incluent ceux décrits dans Chang et al. (1978) Nature 275 : 615, Goeddel et al. (1979) Nature 281 : 544, Goeddel et al. (1980) Nucleic Acids Res. 8 : 4057, EP-A- 36,776, US-A-4,551,433, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80 : 21-25, and Siebenlist et al. (1980) Cell 20 : 269. Les systèmes d'expression dans les levures incluent ceux décrit dans Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75 : 1929, Ito et al. (1983) J. Bacteriol. 153 : 163, Kurtz et al. (1986) Mol. Cell. Biol. 6 : 142, Kunze et al. (1985) J. Basic Microbiol. 25 : 141, Gleeson et al. (1986) J. Gen. Microbiol. 132 : 3459, Roggenkamp et al. (1986) Mol. Gen. Genêt. 202 : 302, Das et al. (1984) J. Bacteriol. 158 : 1165, De Louvencourt et al. (1983) J. Bacteriol. 154 : 737, Van denAny suitable expression system can be used, for example bacteria, yeasts, insect, amphibian and mammalian cells. Expression systems in the bacteria include those described in Chang et al. (1978) Nature 275: 615, Goeddel et al. (1979) Nature 281: 544, Goeddel et al. (1980) Nucleic Acids Res. 8: 4057, EP-A-36,776, US-A-4,551,433, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80: 21-25, and Siebenlist et al. (1980) Cell 20: 269. Expression systems in yeasts include those described in Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75: 1929, Ito et al. (1983) J. Bacteriol. 153: 163, Kurtz et al. (1986) Mol. Cell. Biol. 6: 142, Kunze et al. (1985) J. Basic Microbiol. 25: 141, Gleeson et al. (1986) J. Gen. Microbiol. 132: 3459, Roggenkamp et al. (1986) Mol. Gen. Broom. 202: 302, Das et al. (1984) J. Bacteriol. 158: 1165, De Louvencourt et al. (1983) J. Bacteriol. 154: 737, Van den
Berg et al. (1990) Bio/Technology 8 : 135, Kunze et al. (1985) J. Basic Microbiol. 25 : 141, Cregg et al. (1985) Mol. Cell. Biol. 5 : 3376, US-A- 4,837,148 et 4,929,555, Beach et al. (1981) Nature 300 : 706, Davidow et al. (1985) Curr. Genêt. 10 : 380, Gaillardin et al. (1985) Curr. Genêt. 10 : 49, Ballance et al. (1983) Biochem. Biophys. Res. Commun. 112 : 284-289, Tilburn et al. (1983) Gène 26 : 205-221, Yelton et al. (1984)Berg et al. (1990) Bio / Technology 8: 135, Kunze et al. (1985) J. Basic Microbiol. 25: 141, Cregg et al. (1985) Mol. Cell. Biol. 5: 3376, US-A- 4,837,148 and 4,929,555, Beach et al. (1981) Nature 300: 706, Davidow et al. (1985) Curr. Broom. 10: 380, Gaillardin et al. (1985) Curr. Broom. 10:49, Ballance et al. (1983) Biochem. Biophys. Res. Common. 112: 284-289, Tilburn et al. (1983) Gene 26: 205-221, Yelton et al. (1984)
Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Kelly et al. (1985) EMBO J. 4 : 475479 ; EP-A-244,234 et WO-A-91/00357. L'expression de gènes hétérologues dans les insectes peut être réalisée comme décrit dans US-A-4,745,051, EP-A- 127,839 et EP-A- 155,476, Vlak et al. (1988) J. Gen. Virol. 69 : 765-776, Miller et al. (1988) Ann. Rev. Microbiol. 42 : 177, Carbonell et al. (1988) Gène 73 : 409, Maeda et al. (1985) NatureProc. Natl. Acad. Sci. USA 81: 1470-1474, Kelly et al. (1985) EMBO J. 4: 475479; EP-A-244,234 and WO-A-91/00357. The expression of heterologous genes in insects can be carried out as described in US-A-4,745,051, EP-A-127,839 and EP-A-155,476, Vlak et al. (1988) J. Gen. Virol. 69: 765-776, Miller et al. (1988) Ann. Rev. Microbiol. 42: 177, Carbonell et al. (1988) Gene 73: 409, Maeda et al. (1985) Nature
315 : 592-594, Lebacq-Verheyden et al. (1988) Mol. Cell. Biol. 8 : 3129, Smith et al. (1985) Proc. Natl. Acad. Sci. USA 82 : 8404, Miyajima et al. (1987) Gène 58 : 273, et
Martin et al. (1988) DNA 7 : 99. De nombreux souches et variants de baculovirus et cellules d'insectes permissives sont décrits dans Luckow et al. (1988) Bio/Technology 6 : 47-55, Miller et al. (1986) GENERIC ENGINEERING , Setlow, J.K. et al. Eds. Vol. 8, Plénum Publishing, pp. 277-279, and Maeda et al. (1985) Nature 315 : 592-594. L'expression en cellules de mammifère peut être réalisée comme décrit dans Dijkema et al. (1985) EMBO J. 4 : 761, Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79 : 6777, Boshart et al. (1985) Cell 41 : 521, et US-A-4,399,216. On peut aussi se reporter à Ham et al. (1979) Meth. Enz. 58 : 44, Barnes et al. (1980) Anal. Biochem. 102 : 255, US-A-4,767,704, 4,657,866 ; 4,927,762 ; 4,560,655 ; brevet US RE 30,985, WO-A- 90/103430 et WO-A-87/00195.315: 592-594, Lebacq-Verheyden et al. (1988) Mol. Cell. Biol. 8: 3129, Smith et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8404, Miyajima et al. (1987) Gene 58: 273, and Martin et al. (1988) DNA 7: 99. Many strains and variants of baculovirus and permissive insect cells are described in Luckow et al. (1988) Bio / Technology 6: 47-55, Miller et al. (1986) GENERIC ENGINEERING, Setlow, JK et al. Eds. Flight. 8, Plénum Publishing, pp. 277-279, and Maeda et al. (1985) Nature 315: 592-594. Expression in mammalian cells can be achieved as described in Dijkema et al. (1985) EMBO J. 4: 761, Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79: 6777, Boshart et al. (1985) Cell 41: 521, and US-A-4,399,216. We can also refer to Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980) Anal. Biochem. 102: 255, US-A-4,767,704, 4,657,866; 4,927,762; 4,560,655; US patent RE 30,985, WO-A- 90/103430 and WO-A-87/00195.
Nous avons ainsi utilisé les lymphocytes du sang périphérique provenant d'un porteur sain du VHC comme matériel de départ pour produire la librairie combinatoire d'anticoφs. L'ADNc des lymphocytes a été obtenu à partir de l'ARN en utilisant une méthodologie développée par Sodoyer R. et al. (1997) Human Antibodies 8: 37. La librairie de chaînes lourdes et légères a été construite en utilisant les phagemides pVH (pM 831) et pVL (pM452). Les deux librairies ont été ensuite associées de façon "Random" par sous clonage des gènes VL dans la librairie de chaines lourdes. La librairie de phagemides obtenue est ensuite infectée par le phage helper Ml 3 VCS permettant ainsi l'expression des Fab à la surface des phages. Après une première sélection des phages exprimant les Fab à leur surface par "panning" contre une portion de l'antigène de la capside ( Cl 19) qui correspond à la portion N terminale de la capside allant de l'acide aminé 1 à 119, ils sont soumis à un deuxième cycle de sélection contre l'antigène M48 qui correspond à la portion N terminale de la capside allant de l'acide aminé 1 à 48. 12 isolats ont été caractérisés comme reconnaissant 1We used peripheral blood lymphocytes from a healthy HCV carrier as starting material to produce the combinatorial library of anticoφs. The lymphocyte cDNA was obtained from RNA using a methodology developed by Sodoyer R. et al. (1997) Human Antibodies 8: 37. The heavy and light chain library was constructed using the phagemids pVH (pM 831) and pVL (pM452). The two libraries were then associated in a "Random" fashion by subcloning VL genes in the heavy chain library. The phagemid library obtained is then infected with the phage helper Ml 3 VCS thus allowing the expression of Fab on the surface of the phages. After a first selection of the phages expressing the Fabs on their surface by "panning" against a portion of the capsid antigen (Cl 19) which corresponds to the N terminal portion of the capsid ranging from amino acid 1 to 119, they are subjected to a second selection cycle against the M48 antigen which corresponds to the N terminal portion of the capsid ranging from amino acid 1 to 48. 12 isolates were characterized as recognizing 1
'antigène M48 parmi lesquels ceux exprimant l'anticoφs r FabC3 et r FabC14 ( voir figure 1). De plus, r FabC3 reconnaît bien l'antigène S18D qui correspond à la portion N terminale de la capside allant de l'acide aminé 2 à 21. Par contre, r FabC14 ne reconnaît pas S18D. La séquence nucléotidique complète de ces 2 anticoφs recombinants a été réalisée. rFab C3 contient une chaîne lourde VH3 associée à'M48 antigen including those expressing the anticoφs r FabC3 and r FabC14 (see Figure 1). In addition, r FabC3 recognizes the S18D antigen which corresponds to the N terminal portion of the capsid going from amino acid 2 to 21. On the other hand, r FabC14 does not recognize S18D. The complete nucleotide sequence of these 2 recombinant anticoφs has been produced. rFab C3 contains a VH3 heavy chain associated with
Kappa 4 dont les CDR3 ont pour séquence respective DPLEYFDTSDYDFVDF, soit Asp-Pro-Leu-Glu-Tyr-Phe-Asp-Thr-Ser-Asp-Tyr-Asp-Phe-Val-Asp-Phe (SEQ ID NO : 10), et QQYYSTP, soit Gln-Gln-Tyr-Tyr-Ser-Thr-Pro (SEQ ID NO : 11). RFabC14 contient une chaîne lourde VH3 associée à une chaîne légère lambdal dont les CDR3 ont pour séquence respective DLYYDDMSYE, soit en code à 3 lettres Asp-Leu-Tyr-Kappa 4 whose CDR3 have the respective sequence DPLEYFDTSDYDFVDF, i.e. Asp-Pro-Leu-Glu-Tyr-Phe-Asp-Thr-Ser-Asp-Tyr-Asp-Phe-Val-Asp-Phe (SEQ ID NO: 10) , and QQYYSTP, ie Gln-Gln-Tyr-Tyr-Ser-Thr-Pro (SEQ ID NO: 11). RFabC14 contains a VH3 heavy chain associated with a lambdal light chain whose CDR3s have the respective sequence DLYYDDMSYE, ie in 3-letter code Asp-Leu-Tyr-
Tyr-Asp-Asp-Met-Ser-Tyr-Glu (SEQ ID NO : 8), et GTWDNSLSA, soit Gly-Thr-Tφ- Asp-Asn-Ser-Leu-Ser-Ala (SEQ ID NO : 9).
2) L'anticoφs monoclonal H2, dirigé contre les protéines d'enveloppe El et E2 du VHC a été utilisé pour l'identification de mimotopes de l'enveloppe du VHC. Le procédé d'obtention et les caractéristiques de cet anticoφs sont décrits de façon précise dans WO9729129 (Institut Pasteur). Le clone produisant l'anticoφs monoclonal a été déposé le 9 février 1996 sous le numéro de dépôt CNCM 1-1673 auprès de la Collection Nationale de Cultures de microorganismes de l'Institut Pasteur (CNCM) et est donc accessible au public. L'homme du métier pourra donc aisément reproduire l'invention, et notamment sélectionner les mimotopes de l'enveloppe du VHC en utilisant cet anticoφs monoclonal ou reproduire un équivalent de cet anticoφs en utilisant les outils classiques de la biologie moléculaire. A partir du clone, on séquence l'ADN qui code pour les immunoglobulines et notamment les fragments CDR3 de la chaîne lourdes des irnmunoglobulines. Connaissant la structure du squelette des chaînes lourdes et légères de l'immunoglobuline monoclonale d'une part et du CDR3 d'autre part, il est alors facile de reproduire un équivalent de l'anticoφs monoclonal H2 par substitution du CDR3 d'une autre immunoglobuline par le CDR3 porté par l'anticoφs monoclonal H2.Tyr-Asp-Asp-Met-Ser-Tyr-Glu (SEQ ID NO: 8), and GTWDNSLSA, i.e. Gly-Thr-Tφ- Asp-Asn-Ser-Leu-Ser-Ala (SEQ ID NO: 9). 2) The monoclonal anticoφs H2, directed against the envelope proteins El and E2 of the HCV was used for the identification of mimotopes of the envelope of the HCV. The process for obtaining and the characteristics of this anticoφs are described precisely in WO9729129 (Institut Pasteur). The clone producing the monoclonal antibody was deposited on February 9, 1996 under the deposit number CNCM 1-1673 with the National Collection of Cultures of microorganisms of the Institut Pasteur (CNCM) and is therefore accessible to the public. A person skilled in the art can therefore easily reproduce the invention, and in particular select the mimotopes of the HCV envelope using this monoclonal anticoφs or reproduce an equivalent of this anticoφs using the conventional tools of molecular biology. From the clone, the DNA which codes for immunoglobulins and in particular the CDR3 fragments of the heavy chain of immunoglobulins is sequenced. Knowing the skeleton structure of the heavy and light chains of the monoclonal immunoglobulin on the one hand and of the CDR3 on the other hand, it is then easy to reproduce an equivalent of the monoclonal anticoφs H2 by substitution of the CDR3 for another immunoglobulin by the CDR3 carried by the monoclonal anticoφs H2.
3) Sélection des peptides d'intérêt: les peptides, parmi lesquels ceux comprenant les séquences 1 à 7 (voir la liste de séquence ci-après) peuvent être obtenus à partir d'une librairie de peptides ou préparée par synthèse chimique classique. Une telle synthèse peut être réalisée en solution homogène ou en phase solide. En solution homogène, on procède selon la méthode décrite par Houbenweyl dans l'ouvrage "Méthode der organishen Chemie" édité par E. Wunsh, vol. 15-1 et II, THEME, Stuttgart, 1974. En phase solide, on procède selon la méthode décrite par Atherton et Shepart dans leur ouvrage "Solid phase peptide synthesis", IRL Press, Oxford, 1989.3) Selection of the peptides of interest: the peptides, among which those comprising the sequences 1 to 7 (see the sequence list below) can be obtained from a library of peptides or prepared by conventional chemical synthesis. Such a synthesis can be carried out in a homogeneous solution or in the solid phase. In homogeneous solution, one proceeds according to the method described by Houbenweyl in the work "Method der organishen Chemie" edited by E. Wunsh, vol. 15-1 and II, THEME, Stuttgart, 1974. In solid phase, one proceeds according to the method described by Atherton and Shepart in their work "Solid phase peptide synthesis", IRL Press, Oxford, 1989.
Une banque de phages exprimant des peptides random à leur surface commercialement disponible (pHD7, NEB) a été utilisée pour caractériser la structure des épitopes reconnus par les Fab solubles correspondant aux Fab exprimés à la surface des 12 isolats positifs. Les Fab solubles (sFab) sont dérivés par excision du gène III des isolats positifs. Ces sFab sont ensuite utilisés pour sensibiliser des immunotubes qui servent à la sélection, par panning, au sein de la librairie de phages exprimant les peptides random ceux qui interagissent spécifiquement avec les Fab d'intérêt. Après plusieurs cycles d'amplification et de sélection selon des procédures standard (Maniatis et al.), les phages spécifiques sélectionnés sont amplifiés dans E. Coli. Des mini préparations d'ADN phagiques sont réalisées selon les procédures décrites dans les ouvrages de Maniatis, l'ADN est séquence à l'aide d'un séquenceur automatique à partir duquel on en déduit la
séquence du peptide exprimé par le phage. A titre d'exemple les peptides contenant les séquences Gln-Leu-Ile-Thr-Lys-Pro-Leu, His-Ala-Phe-Pro-His-Leu-His et Ser-Ala-Pro- Ser-Ser-Lys-Asn ont été isolés en utilisant les anticoφs recombinants FabC3 et FabC14. Ces peptides sont capables d'inhiber totalement l'interaction de l'anticoφs FabC3 et FabC14 avec l'antigène M48 alors qu'ils n'ont pas cet effet inhibiteur vis à vis d'un anticoφs monoclonal 2C6F6 qui reconnaît un épitope linéaire situé entre les acides aminés 18 et 24 de l'antigène M48 (figs 2 à 4). De même, les peptides contenant les séquences Gly-Glu-Thr-Arg-Ala-Pro-Leu, Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro- Arg-Pro, Tφ-Gln-Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-Thr et Met-Leu-Pro-Ser-Val- Leu-Asp ont été isolés en utilisant l'anticoφs monoclonal H2. Ces mêmes séquences sont reconnues également par d'autres anticoφs monoclonaux spécifiques de l'enveloppe du VHC.A phage bank expressing random peptides at their commercially available surface (pHD7, NEB) was used to characterize the structure of the epitopes recognized by the soluble Fabs corresponding to the Fabs expressed on the surface of the 12 positive isolates. Soluble Fabs (sFab) are derived by excision of gene III from positive isolates. These sFab are then used to sensitize immunotubes which are used for selection, by panning, within the phage library expressing the peptides random those which interact specifically with the Fabs of interest. After several amplification and selection cycles according to standard procedures (Maniatis et al.), The specific phages selected are amplified in E. Coli. Mini phage DNA preparations are carried out according to the procedures described in the works of Maniatis, the DNA is sequenced using an automatic sequencer from which the sequence of the peptide expressed by the phage. By way of example, the peptides containing the sequences Gln-Leu-Ile-Thr-Lys-Pro-Leu, His-Ala-Phe-Pro-His-Leu-His and Ser-Ala-Pro- Ser-Ser-Lys- Asn were isolated using the recombinant anticoφs FabC3 and FabC14. These peptides are capable of completely inhibiting the interaction of the anticoφs FabC3 and FabC14 with the M48 antigen whereas they do not have this inhibitory effect with respect to a monoclonal anticoφs 2C6F6 which recognizes a linear epitope located between amino acids 18 and 24 of the M48 antigen (figs 2 to 4). Similarly, the peptides containing the sequences Gly-Glu-Thr-Arg-Ala-Pro-Leu, Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro- Arg-Pro, Tφ-Gln- Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-Thr and Met-Leu-Pro-Ser-Val-Leu-Asp were isolated using the monoclonal antibody H2. These same sequences are also recognized by other monoclonal anticoφs specific for the HCV envelope.
Exemple 2 : Formulations vaccinalesExample 2 Vaccine Formulations
Un peptide selon l'exemple 1 est produit par synthèse chimique ("Méthode der organishen Chemie" édité par E. Wunsh, vol. 15-1 et II, THIEME, Stuttgart, 1974 ; Atherton et Shepart dans leur ouvrage "Solid phase peptide synthesis", IRL Press, Oxford, 1989 ) ou obtenu à partir du produit d'expression d'un vecteur recombinant et notamment d'un baculovirus recombinant en utilisant les techniques développées par Smith et al USA 4,745,051. Des émulsions eau dans huile sont ensuite préparées en utilisant le squalène comme constituant de la phase organique, le Tween 80 ou un mélange de Tween 80 et de SPAN comme surfactant, la phase aqueuse contenant la solution de peptide. Lorsque l'hydophobicité du peptide est très importante, on procède à la réalisation d' émulsions huile dans eau dans lesquelles le peptide sera associé à la phase organique. Au besoin des immunostimulants comme le QS 21, des dérivés du MPL ou tout autre adjuvant sont incoφorés dans la préparation de ces émulsions.A peptide according to Example 1 is produced by chemical synthesis ("Method der organishen Chemie" edited by E. Wunsh, vol. 15-1 and II, THIEME, Stuttgart, 1974; Atherton and Shepart in their book "Solid phase peptide synthesis ", IRL Press, Oxford, 1989) or obtained from the expression product of a recombinant vector and in particular of a recombinant baculovirus using the techniques developed by Smith et al USA 4,745,051. Water-in-oil emulsions are then prepared using squalene as a constituent of the organic phase, Tween 80 or a mixture of Tween 80 and SPAN as a surfactant, the aqueous phase containing the peptide solution. When the hydophobicity of the peptide is very high, oil-in-water emulsions are produced in which the peptide will be associated with the organic phase. If necessary, immunostimulants such as QS 21, derivatives of MPL or any other adjuvant are incorporated into the preparation of these emulsions.
Exemple 3 : Formulations vaccinalesEXAMPLE 3 Vaccine Formulations
On prépare une formulation vaccinale à base de liposomes contenant un peptide selon l'exemple 1 en se référant aux ouvrages tels que " Liposomes as Drug Carriers " édité par G. Gregoriadis, 1988, ou aux volumes 1 à 3 de "Liposome Technology édité par G. Gregoriadis, 1984.
Exemple 4 : Formulations vaccinalesA vaccine formulation based on liposomes containing a peptide according to Example 1 is prepared by referring to works such as "Liposomes as Drug Carriers" edited by G. Gregoriadis, 1988, or in volumes 1 to 3 of "Liposome Technology edited by G. Gregoriadis, 1984. EXAMPLE 4 Vaccine Formulations
On prépare une formulation vaccinale à base d'ISCOMs contenant un peptide selon l'exemple 1 en se référant aux articles de référence de B Morein et al, 1984, Nature 308:457 ou Immunology toDay, 1987, 8(11) : 333.A vaccine formulation based on ISCOMs containing a peptide according to Example 1 is prepared by referring to the reference articles of B Morein et al, 1984, Nature 308: 457 or Immunology to Day, 1987, 8 (11): 333.
Exemple 5 : Formulations vaccinalesEXAMPLE 5 Vaccine Formulations
On prépare une formulation vaccinale à base de micro particules comprenant un peptide selon l'exemple 1 mimant un épitope conformationnel d'antigène de structure du VHC. Pour la préparation de micro particules ou de nanoparticules, de nombreux polymères synthétiques ou naturels sont utilisés comme le polymère de methyl métacrylate (Troster S.D. et al, 1992, J. Micro-encaps. 9:19) mais souvent le poly (d, 1-lactide- co-glycolide) encore appelé PLGA est le réfèrent du fait de sa biodégrabilité, de son innocuité et de ses applications déjà anciennes dans le domaine médical. Les micro particules de PLGA chargées en peptides sont préparées notamment par double émulsion eau dans huile dans eau. Le peptide est solubilisé en phase aqueuse puis émulsionné dans une solution de PLGA en phase organique comme le dichlorométhane. L'émulsion eau dans huile est obtenue par agitation à haute vitesse de la solution de peptide dans la solution organique de PLGA. Puis une seconde phase aqueuse contenant une concentration appropriée de surfactant tel que l'alcool polyvinylique est ajoutée à la première émulsion pour réaliser ainsi la double émulsion. D'autres surfactants sont également utilisés comme les sels biliaires ou le poly (oxyéthylène glycérol monoleate ) pour stabiliser la double émulsion (Rafati H et al., 1997, Vaccine 15 : 1888). Après agitation pendant une nuit pour permettre l'évaporation du solvant, les micro particules de PLGA sont lavées plusieurs fois dans l'eau distillée puis lyophilisée et gardées à 5°C.A vaccine formulation is prepared based on microparticles comprising a peptide according to Example 1 mimicking a conformational epitope of HCV structural antigen. For the preparation of micro particles or nanoparticles, many synthetic or natural polymers are used such as the methyl methacrylate polymer (Troster SD et al, 1992, J. Micro-encaps. 9:19) but often poly (d, 1 -lactide- co-glycolide) also called PLGA is the referent because of its biodegradability, its safety and its applications already old in the medical field. The PLGA micro particles loaded with peptides are prepared in particular by double water-in-oil-in-water emulsion. The peptide is dissolved in the aqueous phase and then emulsified in a solution of PLGA in the organic phase such as dichloromethane. The water in oil emulsion is obtained by high speed stirring of the peptide solution in the organic solution of PLGA. Then a second aqueous phase containing an appropriate concentration of surfactant such as polyvinyl alcohol is added to the first emulsion to thereby produce the double emulsion. Other surfactants are also used such as bile salts or poly (oxyethylene glycerol monoleate) to stabilize the double emulsion (Rafati H et al., 1997, Vaccine 15: 1888). After stirring overnight to allow the solvent to evaporate, the microparticles of PLGA are washed several times in distilled water and then lyophilized and kept at 5 ° C.
Exemple 6 : Composition vaccinaleExample 6: Vaccine composition
Les peptides qui comportent moins de 20 acides aminés peuvent être faiblement immunogéniques. Pour augmenter immunogénicité des peptides selon l'exemple 1, on prépare une formulation vaccinale à base de polymères du même peptide ou de peptides différents, sous forme d'octamères comprenant une structure poly-lysine ramifiée à 8 bras latéraux sur lesquels sont fixés le même peptide ou des peptides différents selon l'exemple 1 en mettant en œuvre le procédé développé par Posnett D.N et al. (1988) J. Biol. Chem. 263 : 1719.
Exemple 7 : Composition vaccinalePeptides with fewer than 20 amino acids can be weakly immunogenic. To increase the immunogenicity of the peptides according to Example 1, a vaccine formulation is prepared based on polymers of the same peptide or different peptides, in the form of octamers comprising a branched poly-lysine structure with 8 lateral arms on which the same are fixed. peptide or different peptides according to Example 1 using the method developed by Posnett DN et al. (1988) J. Biol. Chem. 263: 1719. Example 7: Vaccine composition
On prépare une formulation vaccinale comprenant un peptide selon l'exemple 1 couplé à une molécule porteuse comme l'anatoxine tétanique ou diphtérique en utilisant des procédés bien connus de l'homme de métier. Néanmoins, pour conserver au mieux la conformation du site antigénique porté par le peptide l'usage de "bras espaceurs" est recommandé de même qu'il faut éviter autant que possible le recours au glutaraldehyde comme agent de conjugaison.A vaccine formulation is prepared comprising a peptide according to Example 1 coupled to a carrier molecule such as tetanus or diphtheria toxoid using methods well known to those skilled in the art. Nevertheless, to best preserve the conformation of the antigenic site carried by the peptide the use of "spacer arms" is recommended, as well as the use of glutaraldehyde as a conjugating agent should be avoided as much as possible.
Exemple 8 : Composition vaccinale comprenant un lipopeptideExample 8: Vaccine composition comprising a lipopeptide
On prépare une formulation vaccinale comprenant un peptide selon l'exemple 1 couplé à une ou plusieurs chaînes dérivés d'acides gras parmi lesquels la Nε palmitoyl-lysine, la N,N-dipalmitoyl-lysine, le pimélautide, le trimexautide ou à un groupement stéroïdique parmi lesquels le Nε[(cholest-5-ényl-3-oxy)-acétyl)]-lysine ou l'acide (cholest-5-ényl-3-oxy) acétique selon le procédé décrit dans le brevet EP0491628 (INSERM) de façon à obtenir un lipopeptide.A vaccine formulation is prepared comprising a peptide according to Example 1 coupled to one or more chains derived from fatty acids, including Nε palmitoyl-lysine, N, N-dipalmitoyl-lysine, pimelautide, trimexautide or to a group. steroid including Nε [(cholest-5-enyl-3-oxy) -acetyl)] - lysine or (cholest-5-enyl-3-oxy) acetic acid according to the process described in patent EP0491628 (INSERM) so as to obtain a lipopeptide.
Exemple 9 : Expression des peptides par des poxvirusEXAMPLE 9 Expression of the Peptides by Poxviruses
On prépare une composition vaccinale comprenant un poxvirus recombinant codant pour un peptide selon l'exemple 1 mimant un épitope conformationnel d'antigène de structure du VHC. Les poxvirus recombinants sont obtenus par recombinaison homologue, par exemple, en utilisant des cassettes d'expression (plasmide) contenant le poly nucléotide qui code pour le peptide d'intérêt sous la dépendance de promoteurs des poxvirus ( H6, I3L) selon les procédés décrits dans US 5,863,542.A vaccine composition is prepared comprising a recombinant poxvirus encoding a peptide according to Example 1 mimicking a conformational epitope of HCV structural antigen. Recombinant poxviruses are obtained by homologous recombination, for example, using expression cassettes (plasmid) containing the poly nucleotide which codes for the peptide of interest under the dependence of promoters of poxviruses (H6, I3L) according to the methods described. in US 5,863,542.
Exemple 10 : Combinaison de peptidesExample 10 Combination of Peptides
On prépare une composition vaccinale comprenant plusieurs peptides selon l'exemple 1 en utilisant les mêmes modes de préparations développés dans les exemples 1 à 8, peptides qui peuvent être sous forme de conjugués ou non, comprenant ou non les séquences 1 à 7 citées dans la liste des séquences. On prépare notamment une composition vaccinale comprenant à la fois un ou des peptides mimant un ou des épitopes conformationnels de la nucléocapside et un ou des peptides mimant un ou des épitopes conformationnels de l'enveloppe du VHC
Exemple 11 : Expression de plusieurs peptides par un poxvirusA vaccine composition is prepared comprising several peptides according to example 1 using the same modes of preparations developed in examples 1 to 8, peptides which may or may not be in the form of conjugates, comprising or not the sequences 1 to 7 cited in the list of sequences. A vaccine composition is prepared in particular comprising both one or more peptides mimicking one or more conformational epitopes of the nucleocapsid and one or more peptides mimicking one or more conformational epitopes of the HCV envelope Example 11 Expression of Several Peptides by a Poxvirus
On prépare une composition vaccinale comprenant un poxvirus recombinant codant pour plusieurs peptides selon l'exemple 1. L'utilisation et la préparation de vecteurs recombinants codant pour plusieurs épitopes est bien connu de l'homme de métier ( Toes RE et al. (1997) Proc. Natl. Acad. Sci. USA 94: 14660 , Thomson SA et al. (1996) J. Immunol. 157: 822) et est applicable aussi à la préparation de poxvirus recombinants codant pour des mimotopes multiples. On prépare notamment une composition vaccinale comprenant un canaripox recombinant ( ALVAC recombinant) codant pour des mimotopes multiples de la nucléocapside et de l'enveloppe du VHC.A vaccine composition is prepared comprising a recombinant poxvirus encoding several peptides according to Example 1. The use and preparation of recombinant vectors encoding several epitopes is well known to those skilled in the art (Toes RE et al. (1997) Proc Natl Acad Acad Sci USA 94: 14660, Thomson SA et al. (1996) J. Immunol 157: 822) and is also applicable to the preparation of recombinant poxviruses encoding multiple mimotopes. In particular, a vaccine composition is prepared comprising a recombinant canaripox (recombinant ALVAC) encoding multiple mimotopes of the nucleocapsid and of the HCV envelope.
Exemple 13 : DiagnosticExample 13: Diagnosis
On met en œuvre une détection d'anticoφs spécifiques du VHC par ELISA en utilisant un ou plusieurs peptides selon l'exemple 1 pour le diagnostic de l'hépatite C à partir d'un échantillon biologique. Pour cela, on prélève un échantillon de fluide physiologique (sang, plasma, sérum), échantillon que l'on fait ensuite réagir en présence d'un peptide selon l'invention. On utilise le peptide lui-même comme réactif de diagnostic. On recourt généralement -soit à un test de diagnostic indirect, de type ELISA dans lequel le peptide fixé sur un support ( puits) est mis en présence de l'échantillon à tester, tandis que la révélation de la fixation antigène-anticoφs est assurée par un anti-Ig marqué.Detection of specific HCV antibodies is carried out by ELISA using one or more peptides according to Example 1 for the diagnosis of hepatitis C from a biological sample. For this, a sample of physiological fluid (blood, plasma, serum) is taken, a sample which is then reacted in the presence of a peptide according to the invention. The peptide itself is used as a diagnostic reagent. We generally use either an indirect diagnostic test, of the ELISA type in which the peptide fixed on a support (well) is placed in the presence of the sample to be tested, while the revelation of the antigen-anticoφs fixation is ensured by a labeled anti-Ig.
-soit à un test par compétition ou de déplacement, dans lequel on utilise un peptide selon l'exemple 1 , et un anticoφs marqué spécifique du peptide. Le peptide est là aussi fixé à un support solide. Dans le test de compétition, on met le peptide fixé à son support simultanément en présence de l'échantillon (anticoφs de l'échantillon) et d'un anticoφs marqué spécifique du peptide. On utilise comme anticoφs marqués des anticoφs couplés à la peroxydase. Dans le test de compétition ou de déplacement on utilise également des anticoφs monoclonaux et polyclonaux ou anticoφs recombinants spécifiques du peptide selon l'exemple 1 , qui sont parfois sous forme de fragments Fab ou F(ab')2 or a test by competition or displacement, in which a peptide according to Example 1 is used, and a labeled anticoφs specific for the peptide. The peptide is again attached to a solid support. In the competition test, the peptide attached to its support is put simultaneously in the presence of the sample (anticoφs of the sample) and of a labeled anticoφs specific for the peptide. As anticoφs marked, anticoφs coupled to peroxidase are used. In the competition or displacement test, monoclonal and polyclonal anticoφs or recombinant anticoφs specific for the peptide according to Example 1 are also used, which are sometimes in the form of Fab or F (ab ′) 2 fragments.
Exemple 13 : DiagnosticExample 13: Diagnosis
On met en œuvre une détection d'anticoφs spécifiques du VHC par immunochromatographie en utilisant un ou plusieurs peptides selon l'invention pour le diagnostic de l'hépatite C à partir d'un échantillon biologique
Pour cela, le peptide selon l'exemple 1 est fixé sur un support de type bandelette et on se réfère à l'article de Robert F.N Zurk et al., Clin. Chem. 31/7, 1144-1150 (1985) ainsi qu'aux brevets ou demandes de brevet WO-A-88/08 534, WO-A-91/12528, EP-A-291 176, EP-A-299428, EP-A-291 194, EP-A-284 232, US-A-5 120 643, US-A-5 030 558, US-A-5 266 497, US-A-4 740 468, US-A-5 266 497, US-A-4 855 240, US-A-5 451 504, US-A-5 141 850, US-A-5 232 835 et US-A-5 238 652 pour mettre en œuvre le procédé.Detection of specific HCV antibodies is carried out by immunochromatography using one or more peptides according to the invention for the diagnosis of hepatitis C from a biological sample. For this, the peptide according to Example 1 is fixed on a strip-type support and reference is made to the article by Robert FN Zurk et al., Clin. Chem. 7/31, 1144-1150 (1985) and in patents or patent applications WO-A-88/08 534, WO-A-91/12528, EP-A-291 176, EP-A-299428, EP -A-291 194, EP-A-284 232, US-A-5 120 643, US-A-5 030 558, US-A-5 266 497, US-A-4 740 468, US-A-5 266 497, US-A-4 855 240, US-A-5 451 504, US-A-5 141 850, US-A-5 232 835 and US-A-5 238 652 for implementing the method.
Exemple 14 : DiagnosticExample 14: Diagnosis
On met en œuvre une étude de la réponse lymphoproliférative spécifique à un ou plusieurs peptides selon l'exemple 1 pour le diagnostic de l'hépatite C à partir d'un échantillon biologique. Le sang du patient est recueilli sur tube héparine. Les lymphocytes sont ensuite séparés par centrifugation sur Ficoll hypaque puis distribués en micro plaques 96 puits stériles à raison de 2 105 cellules par puits à fond rond sous un volume final de 200μl de milieu de culture complet ( RPMI 1640 supplémenté par 25mM HEPES, 2mM L- glutamine , 50U/ml de pénicilline, 50μg/ml de streptomycine et 5% de sérum AB dé complémenté) et mis en présence de concentrations variables du peptide conforme à l'invention (concentrations allant de lng/ml à 50μg/ml). Chaque concentration de peptide est testée en trip liquette pour s'affranchir au mieux des variations biologiques. Des combinaisons multiples de peptides peuvent être également testées dans la gamme de concentration indiquée, par exemple une combinaison résultant de l'association d'un mimotope de l'enveloppe avec un mimotope de la nucléocapside dans la gamme de concentration indiquée. Après 5 jours de culture à 37 °c sous 5% CO2, 0,5μci de thymidine tritiée est ajouté à chaque puits. Après une nouvelle incubation de 16 heures, on recueille l'ADN cellulaire de chaque puits de culture sur des filtres après précipitation à l'éthanol et on mesure le taux d'incoφoration de thymidine tritiée à l'aide d'un compteur à scintillation liquide qui reflète l'intensité de la réponse lymphoproliférative. Les résultats sont exprimés sous la forme d'index de stimulation (moyenne des cpm des puits de culture lymphocytaire contenant une concentration donnée en peptide/ moyenne des cpm des puits de culture lymphocytaire sans peptide). La réponse lymphoproliférative est considérée comme positive lorsque l'index de stimulation est supérieur à 3.
A study of the specific lymphoproliferative response to one or more peptides according to Example 1 is implemented for the diagnosis of hepatitis C from a biological sample. The patient's blood is collected on a heparin tube. The lymphocytes are then separated by centrifugation on hypaque Ficoll and then distributed in 96-well sterile microplates at the rate of 2 10 5 cells per round-bottom well under a final volume of 200 μl of complete culture medium (RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 50 U / ml of penicillin, 50 μg / ml of streptomycin and 5% of AB complemented serum) and placed in the presence of variable concentrations of the peptide in accordance with the invention (concentrations ranging from lng / ml to 50 μg / ml) . Each concentration of peptide is tested in a trip liquid to overcome biological variations as much as possible. Multiple combinations of peptides can also be tested in the indicated concentration range, for example a combination resulting from the association of an envelope mimotope with a nucleocapsid mimotope in the indicated concentration range. After 5 days of culture at 37 ° C. under 5% CO2, 0.5 μci of tritiated thymidine is added to each well. After a further 16 hour incubation, the cellular DNA is collected from each culture well on filters after precipitation with ethanol and the degree of tritiated thymidine incorporation is measured using a liquid scintillation counter. which reflects the intensity of the lymphoproliferative response. The results are expressed in the form of stimulation index (average of the cpm of the lymphocyte culture wells containing a given concentration of peptide / average of the cpm of the lymphocyte culture wells without peptide). The lymphoproliferative response is considered positive when the stimulation index is greater than 3.
Claims
RevendicationsClaims
1) Peptide pour le traitement thérapeutique ou prophylactique de l'hépatite C, capable de réagir avec un anticoφs spécifique d'un antigène de structure du virus de l'hépatite C, comprenant une séquence en acides aminés qui mime un épitope conformationnel d'un antigène de structure dudit virus sans toutefois correspondre à une séquence continue d'acides aminés de cet antigène, caractérisé en ce que ce peptide comprend notamment au choix les séquences là 71) Peptide for the therapeutic or prophylactic treatment of hepatitis C, capable of reacting with an anticoφs specific for a structural antigen of the hepatitis C virus, comprising an amino acid sequence which mimics a conformational epitope of a structural antigen of said virus without however corresponding to a continuous amino acid sequence of this antigen, characterized in that this peptide comprises in particular the sequences there 7
SEQ ID NO : l Gln-Leu-Ile-Thr-Lys-Pro-LeuSEQ ID NO: l Gln-Leu-Ile-Thr-Lys-Pro-Leu
SEQ ID NO : 2 His-Ala-Phe-Pro-His-Leu-HisSEQ ID NO: 2 His-Ala-Phe-Pro-His-Leu-His
SEQ ID NO : 3 Ser-Ala-Pro-Ser-Ser-Lys-AsnSEQ ID NO: 3 Ser-Ala-Pro-Ser-Ser-Lys-Asn
SEQ ID NO : 4 Gly-Glu-Thr-Arg-Ala-Pro-LeuSEQ ID NO: 4 Gly-Glu-Thr-Arg-Ala-Pro-Leu
SEQ ID NO : 5 Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro-Arg-Pro SEQ ID NO : 6 Tφ-Gln-Ser-Tyr-Pro-Met-Phe-Asn-Asn-Thr-Leu-ThrSEQ ID NO: 5 Ser-Val-Ser-Val-Gly-Met-Lys-Pro-Ser-Pro-Arg-Pro SEQ ID NO: 6 Tφ-Gln-Ser-Tyr-Pro-Met-Phe-Asn-Asn -Thr-Leu-Thr
SEQ ID NO : 7 Met-Leu-Pro-Ser-Val-Leu-Asp.SEQ ID NO: 7 Met-Leu-Pro-Ser-Val-Leu-Asp.
2) Peptide selon la revendication 1, pour laquelle l'antigène de structure est représenté par la capside ou l'enveloppe du virus de l'hépatite C.2) Peptide according to claim 1, for which the structural antigen is represented by the capsid or the envelope of the hepatitis C virus.
3) Peptide selon la revendication 2, pour laquelle l'anticoφs spécifique est dirigé contre la capside et comprend au niveau de la région hyper variable CDR3 de la chaîne lourde VH3 la séquence polypeptidique Asp-leu-Tyr-Tyr-Asp-Asp-Met- Ser-Tyr-Glu et au niveau de la région hyper variable CDR3 de la chaîne légère3) Peptide according to claim 2, for which the specific anticoφs is directed against the capsid and comprises at the level of the CDR3 hyper variable region of the VH3 heavy chain the polypeptide sequence Asp-leu-Tyr-Tyr-Asp-Asp-Met - Ser-Tyr-Glu and at the CDR3 hyper variable region of the light chain
Viambdal la séquence Gly-Thr-Tφ-Asp-Asn-Ser-Leu-Ser-AlaViambdal the sequence Gly-Thr-Tφ-Asp-Asn-Ser-Leu-Ser-Ala
4) Peptide selon la revendication 2, pour laquelle l'anticoφs spécifique est dirigé contre la capside et comprend au niveau de la région hyper variable CDR3 de la chaîne lourde VH3 la séquence polypeptidique Asp-Pro-Leu-Glu-Tyr-Phe-Asp-4) Peptide according to claim 2, for which the specific anticoφs is directed against the capsid and comprises at the CDR3 hyper variable region of the VH3 heavy chain the polypeptide sequence Asp-Pro-Leu-Glu-Tyr-Phe-Asp -
Thr-Ser-Asp-Tyr-Asp-Phe-Val-Asp-Phe et au niveau de la région hyper variableThr-Ser-Asp-Tyr-Asp-Phe-Val-Asp-Phe and at the level of the hyper variable region
CDR3 de la chaîne légère Vkappa4 la séquence Gln-Gln-Tyr-Tyr-Ser-Thr-ProCDR3 of the Vkappa4 light chain the sequence Gln-Gln-Tyr-Tyr-Ser-Thr-Pro
5) Peptide comprenant l'enchaînement et/ou la répétition d'un ou plusieurs peptides selon l'une des revendications 1 à 4.
5) Peptide comprising the sequence and/or repetition of one or more peptides according to one of claims 1 to 4.
6) Conjugué comprenant au moins un peptide selon l'une des revendications 1 à 4 lié à une molécule pour induire ou renforcer l'immunogénicité dudit peptide.6) Conjugate comprising at least one peptide according to one of claims 1 to 4 linked to a molecule to induce or reinforce the immunogenicity of said peptide.
7) Vecteur recombinant comprenant une cassette d'expression fonctionnelle permettant l'expression d'un poly nucléotide codant pour un peptide selon l'une des revendications 1 à 5.7) Recombinant vector comprising a functional expression cassette allowing the expression of a polynucleotide encoding a peptide according to one of claims 1 to 5.
8) Vecteur recombinant selon la revendication 9, caractérisé en ce qu'il est un adénovirus, un poxvirus, un baculovirus, un phage ou un plasmide.8) Recombinant vector according to claim 9, characterized in that it is an adenovirus, a poxvirus, a baculovirus, a phage or a plasmid.
9) Composition thérapeutique ou prophylactique de l'hépatite C, notamment destiné à un usage vaccinal, dont le principe actif comprend un peptide selon l'une des revendications 1 à 5, le cas échéant conjugué selon la revendication 6, et /ou un vecteur recombinant codant pour ledit peptide selon l'une des revendications 7 et9) Therapeutic or prophylactic composition for hepatitis C, in particular intended for vaccination use, the active principle of which comprises a peptide according to one of claims 1 to 5, where appropriate conjugated according to claim 6, and/or a vector recombinant encoding said peptide according to one of claims 7 and
8.8.
10) Composition selon la revendication 9 dont le principe actif est sous la forme d'une formulation associée à un adjuvant compatible permettant l'administration d'une dose efficace par voie muqueuse ou parentérale.10) Composition according to claim 9, the active principle of which is in the form of a formulation associated with a compatible adjuvant allowing the administration of an effective dose by mucosal or parenteral route.
11) Utilisation d'un peptide selon l'une des revendications 1 à 5 en tant que réactif pour le diagnostic de l'hépatite C et/ou de la susceptibilité à la chronicité en cas d'infection établie par le virus de l'hépatite C, ledit diagnostic comprenant l'évaluation, à partir d'un échantillon de sang, de la réponse humorale et/ou à médiation cellulaire spécifique de ce peptide.11) Use of a peptide according to one of claims 1 to 5 as a reagent for the diagnosis of hepatitis C and/or susceptibility to chronicity in the event of established infection with the hepatitis virus C, said diagnosis comprising the evaluation, from a blood sample, of the humoral and/or cell-mediated response specific to this peptide.
12) Utilisation d'un peptide selon l'une des revendications 1 à 5 en tant que réactif pour le diagnostic de l'hépatite C et/ou de la susceptibilité à la chronicité en cas d'infection établie par le virus de l'hépatite C, ledit diagnostic comprenant l'évaluation de la réponse d'hypersensibilité retardée consécutive à l'administration intradermique ou sous cutanée de ce peptide.12) Use of a peptide according to one of claims 1 to 5 as a reagent for the diagnosis of hepatitis C and/or susceptibility to chronicity in the event of established infection with the hepatitis virus C, said diagnosis comprising the evaluation of the delayed hypersensitivity response following intradermal or subcutaneous administration of this peptide.
13) Utilisation d'un peptide selon l'une des revendications 1 à 5,1e cas échéant d'un conjugué selon la revendication 6 et/ou d'un vecteur recombinant selon l'une des
revendications 7 et 8 pour la préparation d'une composition thérapeutique ou prophylactique telle que celle revendiquée à la revendication 9, destinée au traitement ou a la prévention de l'hépatite C.
13) Use of a peptide according to one of claims 1 to 5, 1e where appropriate a conjugate according to claim 6 and/or a recombinant vector according to one of claims 7 and 8 for the preparation of a therapeutic or prophylactic composition such as that claimed in claim 9, intended for the treatment or prevention of hepatitis C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU37144/99A AU3714499A (en) | 1998-05-14 | 1999-05-14 | Hepatitis c virus mimotopes |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9806335 | 1998-05-14 | ||
| FR98/06335 | 1998-05-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1999058561A1 true WO1999058561A1 (en) | 1999-11-18 |
Family
ID=9526521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/FR1999/001155 WO1999058561A1 (en) | 1998-05-14 | 1999-05-14 | Hepatitis c virus mimotopes |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU3714499A (en) |
| WO (1) | WO1999058561A1 (en) |
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| EP1290230A2 (en) * | 2000-06-02 | 2003-03-12 | Merck & Co., Inc. | Hepatitis c virus conjugates |
| FR2848566A1 (en) * | 2002-12-11 | 2004-06-18 | Centre Nat Rech Scient | New peptides recognized by autoantibodies against human thyroperoxidase, useful for diagnosis and treatment of autoimmune diseases and thyoid cancer, also antibodies specific for them |
| EP1832653A1 (en) * | 1998-10-23 | 2007-09-12 | Institute Of Virology | MN gene and protein |
| EP1970070A2 (en) * | 2002-06-07 | 2008-09-17 | Xigen S.A. | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
| US7635681B2 (en) | 2002-01-09 | 2009-12-22 | Xigen Sa | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
| US8080517B2 (en) | 2005-09-12 | 2011-12-20 | Xigen Sa | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
| US8183339B1 (en) | 1999-10-12 | 2012-05-22 | Xigen S.A. | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
| US8236924B2 (en) | 1999-10-12 | 2012-08-07 | Xigen Sa | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
| US8748395B2 (en) | 2005-09-12 | 2014-06-10 | Xigen Inflammation Ltd. | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
| US8981052B2 (en) | 2010-06-21 | 2015-03-17 | Xigen Inflammation Ltd. | JNK inhibitor molecules |
| US9006185B2 (en) | 2008-05-30 | 2015-04-14 | Xigen Inflammation Ltd. | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases |
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| US9610330B2 (en) | 2008-05-30 | 2017-04-04 | Xigen Inflammation Ltd. | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases |
| US10023615B2 (en) | 2008-12-22 | 2018-07-17 | Xigen Inflammation Ltd. | Efficient transport into white blood cells |
| US9915658B2 (en) | 2009-05-20 | 2018-03-13 | Ortho-Clinical Diagnostics, Inc. | Reagents for HCV antigen-antibody combination assays |
| US10041946B2 (en) | 2009-05-20 | 2018-08-07 | Ortho-Clinical Diagnostics Inc. | Reagents for HCV antigen-antibody combination assays |
| US8981052B2 (en) | 2010-06-21 | 2015-03-17 | Xigen Inflammation Ltd. | JNK inhibitor molecules |
| US9624267B2 (en) | 2010-06-21 | 2017-04-18 | Xigen Inflammation Ltd. | JNK inhibitor molecules |
| US9150618B2 (en) | 2010-10-14 | 2015-10-06 | Xigen Inflammation Ltd. | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory eye diseases |
| US10596223B2 (en) | 2011-12-21 | 2020-03-24 | Xigen Inflammation Ltd. | JNK inhibitor molecules for treatment of various diseases |
| US10624948B2 (en) | 2013-06-26 | 2020-04-21 | Xigen Inflammation Ltd. | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases |
| US11779628B2 (en) | 2013-06-26 | 2023-10-10 | Xigen Inflammation Ltd. | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases |
| US11331364B2 (en) | 2014-06-26 | 2022-05-17 | Xigen Inflammation Ltd. | Use for JNK inhibitor molecules for treatment of various diseases |
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