WO2010112962A1 - Identification, optimization and use of cryptic hla-a24 epitopes for immunotherapy - Google Patents

Identification, optimization and use of cryptic hla-a24 epitopes for immunotherapy Download PDF

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Publication number
WO2010112962A1
WO2010112962A1 PCT/IB2009/005753 IB2009005753W WO2010112962A1 WO 2010112962 A1 WO2010112962 A1 WO 2010112962A1 IB 2009005753 W IB2009005753 W IB 2009005753W WO 2010112962 A1 WO2010112962 A1 WO 2010112962A1
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Prior art keywords
hla
epitope
peptide
restricted
cryptic
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PCT/IB2009/005753
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French (fr)
Inventor
Kostantinos Kosmatopoulos (Kostas)
Jeanne Menez-Jamet
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Vaxon Biotech
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Publication date
Priority to HUE09785926A priority Critical patent/HUE030195T2/en
Priority to CN200980158591.2A priority patent/CN102387813B/en
Priority to CA2756238A priority patent/CA2756238A1/en
Priority to PCT/IB2009/005753 priority patent/WO2010112962A1/en
Priority to US13/258,227 priority patent/US8900600B2/en
Priority to PT97859268T priority patent/PT2413956T/en
Priority to BRPI0924827A priority patent/BRPI0924827A2/en
Priority to DK09785926.8T priority patent/DK2413956T3/en
Application filed by Vaxon Biotech filed Critical Vaxon Biotech
Priority to ES09785926.8T priority patent/ES2608715T3/en
Priority to EP09785926.8A priority patent/EP2413956B1/en
Priority to JP2012502822A priority patent/JP5756792B2/en
Priority to PL09785926T priority patent/PL2413956T3/en
Publication of WO2010112962A1 publication Critical patent/WO2010112962A1/en
Priority to HRP20161710TT priority patent/HRP20161710T1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to the field of peptide immunotherapy.
  • the invention provides novel methods and materials for efficiently treating patients having an HLA- A*2402 phenotype.
  • Peptide vaccination or immunotherapy is a therapeutic approach which is currently the subject of a great number of studies in the context of the treatment of cancer.
  • the principle thereof is based on immunization with peptides which reproduce T cell epitopes of tumor antigens recognized by cytotoxic T lymphocytes (CTLs) 5 which play a major role in the elimination of tumor cells.
  • CTLs cytotoxic T lymphocytes
  • CTLs do not recognize whole protein antigens, but peptide fragments thereof, generally comprising 8 to 10 amino acids, presented by class I major histocompatibility complex (MHC I) molecules expressed on the surface of cells.
  • MHC I major histocompatibility complex
  • the presentation of these peptides is the result of the antigen processing which involves three steps: cytosolic degradation of the antigen by a multienzyme complex called proteasome, translocation of the peptides derived from this degradation in the endoplasmic reticulum (ER) by the TAP transporters, association of these peptides with the MHC I molecules and exportation of the peptide/MHC I complexes to the cell surface.
  • the peptide/MHC I complexes interact with the specific T cell receptor (TCR) on CTL, inducing the stimulation and amplification of these CTL, which become able to attack target cells expressing the antigen from which the peptide is derived.
  • TCR T cell receptor
  • peptide selection takes place, which results in a hierarchy of peptides presentation.
  • Peptides that are preferentially presented by the MHC I molecules are called immunodominant, while peptides which are weakly presented are called cryptic.
  • Immunodominant peptides exhibit a high affinity for the MHC I and are immunogenic while cryptic peptides exhibit a low affinity for MHC I and are non-immunogenic .
  • Tumor antigens are frequently self proteins over-expressed by tumors and expressed at lower levels by normal cells and tissues.
  • the immune system is unable to react against these self antigens because of the self tolerance process.
  • Self-tolerance concerns mainly the immunodominant peptides (Cibotti et al., 1992; Gross et al., 2004), thus explaining the incapacity of these peptides to induce a tumor immunity.
  • Cryptic peptides are much less involved in self tolerance process (Cibotti et al., 1992; Gross et al., 2004; Moudgil et aL, 1999) and can therefore induce an efficient tumor immunity, provided their immunogenicity is enhanced (Engelhorn et al., 2006; Gross et al., 2004).
  • the usual strategy for enhancing the immunogenicity of cryptic peptides which are non-immunogenic because of their low MHC I affinity, consists in increasing their affinity for the MHC I molecules via amino acids substitutions.
  • Peptide affinity for MHC I molecules mainly depends on the presence at well defined positions (primary anchor positions) of residues called "primary anchor residues". These residues are MHC I allele specific.
  • Amino acids substitutions aiming at enhancing affinity for MHC I molecule should preserve the antigenicity of such optimized peptides. Indeed, CTL generated by optimized peptides must cross-react with the corresponding native peptides. Many teams have succeeded in enhancing immunogenicity of already immunogenic peptides by increasing their affinity for HLA-A*0201 (Bakker et al., 1997; Parkhurst et al., 1996; Valmori et al., 1998).
  • HLA-A* 0201 restricted cryptic peptides The inventors have previously described a general strategy to enhance affinity and immunogenicity of HLA-A* 0201 restricted cryptic peptides (Scardino et al., 2002; Tourdot and Gould, 2002) and HLA-B*0702 (WO 2008/010098).
  • HLA-A*2402 is a frequently expressed molecule (27% of the population) and is one of the most common alleles in Japanese and Asian people. Identification and optimization of HLA-A* 2402 restricted tumor cryptic peptides is therefore necessary for developing efficient cancer vaccines for HLA-A* 2402 expressing patients. Several tumor immunogenic peptides presented by HLA-A* 2402 have been described to date (table 1). Antigen Sequence SEQ ID No:
  • the inventors have now found a strategy to identify, in an antigen, cryptic peptides presented by HLA-A*2402 molecule, and to optimize their immunogenicity, preserving the cross-reactivity with the corresponding native cryptic peptides.
  • a first aspect of the present invention is a method for identifying an HLA-A*2402-restricted cryptic epitope in an antigen, comprising a step of selecting, in said antigen, a peptide of 8 to 12 amino acids having a tyrosine (Y) in primary anchor position 2, with the proviso that the peptide does not have, simultaneously, a positively charged amino acid (arginine (R) or lysine (K)) in position 1 and a leucine (L), or a phenylalanine (F) or an isoleucine (I) in C-terminal position.
  • the obtained sequences are those of putative cryptic epitopes.
  • the method for identifying a HLA-A *2402-restricted cryptic epitope in an antigen further comprises step consisting in testing the immunogenicity of each putative cryptic epitope of SEQ ID No: 20, in an appropriate model, and selecting those which are non-immunogenic.
  • an appropriate model is a model which predicts the immunogenicity of the peptide in an individual who expresses HLA-A*2402.
  • An example of such an appropriate model is described in the experimental part and consists of HLA-A*2402 transgenic mice.
  • “native peptide” will be used to designate any peptide of SEQ ID No: 20, whether its non- immunogenicity has been checked or not.
  • the phrase “putative HLA- A*2402-restricted cryptic epitope” will be used to express the fact that the immunogenicity of the peptide has not been tested, and the phrase “confirmed HLA-A*2402 -restricted cryptic epitope” will be used for peptides which have been tested and have proved to be non-immunogenic in an appropriate model.
  • peptide designates not only molecules in which amino acid residues (in L or D configurations) are joined by peptide (-CO-NH-) linkages, but also synthetic pseudopeptides or peptidomimetics in which the peptide bond is modified, especially to become more resistant to proteolysis, and provided their immunogenicity is not impaired by this modification.
  • the selected peptide has 9 to 11 amino acids, more preferably 9 or 10 amino acids and one or more unfavourable amino acids at secondary anchor positions, for example a P (proline) in position 1 and/or a D or E or G or H or P or Q or R or K (glutamic or aspartic acid, glycine, histidine, proline, glutamine, arginine or lysine) at C-terminal position.
  • P proline
  • D or E or G or H or P or Q or R or K glutmic or aspartic acid, glycine, histidine, proline, glutamine, arginine or lysine
  • a second aspect of the present invention is a method for increasing the immunogenicity of a HLA-A*2402-restricted cryptic epitope, comprising a step of substituting the N-terminal residue of said epitope with a positively charged amino acid (R or K), and/or substituting the C-terminal residue of said epitope with an L, F or I.
  • R or K positively charged amino acid
  • the C-terminal modification is the substitution by an L.
  • the peptide can be produced by artificial peptide synthesis or by recombinant expression.
  • the immunogenicity of a HLA-A*2402-restricted cryptic epitope in which the two first residues are RY or KY can be increased by replacing its last amino-acid by an L 5 F or I, preferentially by an L (or by adding a L, I or F at its C- terminus, provided it is not longer than 11 amino acids).
  • sequence .of the selected HLA-A*2402-restricted cryptic epitope is XiYX 2 X 3 X 4 X 5 X 6 X 7 XsXeXiOL (SEQ ID No: 21), wherein Xi is any amino acid but R or K, X 2 to X 6 are any amino acid, and X 7 to X] o are any amino acid or none, the substitution of Xi by R or K is sufficient to increase its immunogenicity.
  • the sequence of the selected HLA-A*2402- restricted cryptic epitope is Xi YX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 XiOXi i (SEQ ID No: 22), wherein Xi is any amino acid but R or K, X 2 to X 6 are any amino acid, and X 7 to Xi 0 are any amino acid or none, and Xn is not an unfavourable amino acids (D or E or G or H or P or Q or R or K ), the substitution of Xi by R or K can be sufficient to increase its immunogenicity.
  • the expression "optimized peptide” or “optimized immunogenic A*2402-restricted epitope” will designate an immunogenic peptide derived from a HLA-A*2402-restricted cryptic epitope (called its "cognate native peptide") by the above method.
  • the optimized peptide can trigger an immune response which cross-recognizes its cognate native peptide.
  • Another aspect of the present invention is hence a method for obtaining a HLA-A*2402-restricted epitope able to trigger an immune response against a HLA-A*2402-restricted cryptic epitope of an antigen, comprising the steps of (i) identifying, in said antigen, one or several native putative HLA-
  • A*2402-restricted cryptic epitopes by the method according to claim 1 ;
  • step (ii) testing the immunogenicity of each native epitope selected in step (i), in an appropriate model, and selecting those which are non-immuno genie;
  • step (iii) for each native epitope selected in step (ii), obtaining an optimized epitope by increasing its immunogenicity, by the method as above-described;
  • step (iv) testing the immunogenicity of each optimized epitope obtained in step (iii), in an appropriate model, and selecting those which are immunogenic;
  • step (v) for each epitope selected in step (iv), testing if the CTLs generated against the optimized epitope also recognize its cognate native epitope, and selecting those for which the test is positive.
  • step (v) the appropriate models which can be used in steps (ii) and (iv) are as described above.
  • step (v) the cross-recognition can be performed by any method known by the skilled artisan, for example as described in the experimental part.
  • the inventors have identified in different tumor associated antigens (hTERT, EphA2, MAGE or Her2/neu), a number of putative HLA-A*2402-restricted cryptic epitopes. When testing the immunogenicity of these epitopes, one of them proved to be immunogenic.
  • the inventors have selected the peptides disclosed in Table 2 below, which are confirmed HLA-A*2402-restricted cryptic epitopes. The peptides are part of the present invention.
  • Her2/neu 300 PYNYLSTDV ID N°9 Table 2 Selected confirmed cryptic HLA-A*2402 restricted peptides
  • the present invention also pertains to optimized peptides derived from the cryptic peptides of SEQ ID Nos: 1 to 9, by a method according to the invention.
  • Preferred examples of optimized peptides are KYGVLLKTL (SEQ ID No: 11), RYMRQFVAL (SEQ ID No: 12), RYVSRLLGI (SEQ ID No: 13), RYGKGWDLL (SEQ ID No: 14), RYLVQVQAL (SEQ ID No: 15), RYWELSNHL (SEQ ID No: 16).
  • SEQ ID No: 13 and SEQ ID No: 15 have been derived from the cryptic HLA-A*2402-restricted epitopes of SEQ ID NOs: 3 and 5, respectively, by substitution of their N-terminal amino-acid with a R.
  • the peptides of SEQ ID Nos: 11, 12, 14 and 16 have been derived from the peptides of SEQ ID Nos: 1, 2, 4 and 6, respectively, by substituting their N-terminal amino-acid with an R or a K and their C-terminal amino-acid with a L.
  • Polyspecific tumor vaccination offers a broader control of tumor cells than monospecific vaccination, thereby reducing the risk of emergence of immune escape variants.
  • Vx-006 is able to induce a polyspecific CD8 cell response both in vivo in HLA-A* 0201 transgenic HHD mice and in vitro in humans, whereas the mixture of TERT 988 Y, HER-2/neu 40 2 ⁇ and MAGE-A 24 8V9 peptides failed to induce a trispecific response.
  • a chimeric polypeptide comprising several epitopes can be more efficient than a mere mixture of the same epitopes to trigger a response against more than one epitope.
  • a chimeric polypeptide comprising a repetition of one single epitope can also trigger a stronger response against said epitope than a peptide consisting of said epitope.
  • a polypeptide organization (either with several different epitopes or with a repetition of one single epitope) can produce new junctional epitopes, especially CD4 restricted epitopes, able to optimize the targeted peptide(s)-specific immune response.
  • peptides bind directly to MHC molecules of every cells present at the site of injection.
  • APC Antigenic Presenting Cells
  • a further aspect of the invention is hence a chimeric polypeptide, comprising one, two, three or more HLA-A*2402-restricted cryptic epitopes or one, two, three or more optimized immunogenic HLA-A*2402-restricted epitopes as described above.
  • the epitopes can be different from each other, and/or the same epitope can be repeated several times.
  • HLA-A*2402- restricted cryptic or optimized immunogenic epitopes described above can hence be advantageously associated to previously described HLA-A* 0201 (WO 02/02716) and/or HLA-B*0702 peptides (WO 2008/010010 and WO 2008/010098), or to immunogenic epitopes derived from previously described tumor associated antigens, comprising CEA, PRAME, Tyrosinase, TRAG-3, NY-Eso-1, P53, Muc-1, PSA/PSMA, survivin, Melan- A/MART-1, TRP-I, TRP-2, WTl, EphAl, EphA2, EphA3, EphA4, G250/MN/CAIX, STEAP, alphafoetoprotein, RAGE-I, PAGE-I.
  • a polyallelic peptides mix comprising at least a peptide according to the present invention and one different HLA- restricted epitope (HLA-A*0201, HLA-A*2402, HLA-B*0702 or others), is also part of the present invention.
  • cryptic epitopes which can advantageously be combined to HLA-A*2402-restricted cryptic epitopes (either in a mix or in a chimeric polypeptide), as well as examples of optimized immunogenic epitopes which can advantageously be combined to optimized immunogenic HLA-A*2402-restricted epitopes, are described in Table 3 below. Of course, these lists are not limitative.
  • Table 3 epitopes which can be combined to HLA-A*2402-restricted epitopes in chimeric polypeptides according to the invention
  • polypeptide can be obtained by chemical synthesis, or by using the technology of genetic engineering (Velders et al., 2001).
  • Another object of the present invention is an isolated nucleic acid molecule designed to cause the expression of a cryptic HLA- A*2402 -restricted epitope, or of an optimized immunogenic HLA-A*2402-restricted epitope, or of a chimeric polypeptide as above-described.
  • designed to cause the expression of a peptide is herein meant that said peptide is expressed as such, isolated from the whole antigen from which its sequence has been selected (and, in appropriate cases, optimized as above- described), when the nucleic acid is introduced in an appropriate cell.
  • the region encoding the epitope or chimeric polypeptide will typically be situated in the polynucleotide under control of a suitable promoter.
  • Bacterial promoters will be preferred for expression in bacteria, which can produce the polypeptide either in vitro, or, in particular circumstances, in vivo.
  • a nucleic acid according to the invention can be administered directly, using an appropriate vector.
  • a tissue-specific, a strong constitutive, or an endogenous promoter can be used to control the peptide expression.
  • Suitable vector systems include naked DNA plasmids, liposomal compositions to enhance delivery, and viral vectors that cause transient expression.
  • viral vectors are adenovirus or vaccinia virus vectors and vectors of the herpes family, especially in a non-replicative form.
  • the present invention also pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising at least, as an active principle, an HLA-A*2402-restricted cryptic epitope as above-described, or an optimized immunogenic epitope polypeptide as mentioned above, or a chimeric polypeptide according to the invention, or a nucleic acid encoding any of these, and/or a vector carrying said nucleic acid.
  • Formulation of pharmaceutical compositions will accord with contemporary standards and techniques. Medicines intended for human administration will be prepared in adequately sterile conditions, in which the active ingredient(s) are combined with an isotonic solution or other pharmaceutical carrier appropriate for the recommended therapeutic use. Suitable formulations and techniques are generally described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton PA).
  • a HLA-A*2402-restricted epitope or a chimeric polypeptide or a nucleic acid according to the invention can be used for the preparation of a composition for preventive or curative immunotherapy, especially, for antiviral or anticancer immunotherapy.
  • a pharmaceutical composition according to the invention is a vaccine.
  • the components described above can be combined with an adjuvant to potentiate the immune response.
  • Classic adjuvants include oil emulsions, like Incomplete Freund's Adjuvant or Montanide, and adherent surfaces such as alum.
  • Adjuvants that recruit and activate dendritic cells particularly via TLR (such as bacterial DNA or bacterial membrane derived proteins) or help elicit cytotoxic T cells are especially useful.
  • Other factors that otherwise boost the immune response or promote apoptosis or elimination of cancer cells can also be included in the composition, such as IL-2 or IL- 12 cytokines or GM-CSF.
  • kits of parts described below can be accompanied with written instructions regarding the use of the composition or combination for eliciting an immune response and/or for the treatment of cancer.
  • WO 2006/120038 the Applicant has described a vaccination protocol which enables the initiation and maintenance of a T cell response targeting sub-dominant/cryptic epitopes.
  • the results reported in WO 2006/120038 demonstrate that injection of a native peptide corresponding to a sub- dominant/cryptic epitope, following vaccination with its cognate optimized peptide, can maintain the immune response initiated by said optimized peptide.
  • a HLA-A* 2402-restricted cryptic epitope can hence be used for the preparation of a medicinal composition for maintaining the CTL immune response initiated by its cognate optimized peptide.
  • An immunogenic peptide having an optimized immunogenic HLA-A*2402-restricted epitope sequence derived from a HLA-A*2402-restricted cryptic epitope can also be used, for the preparation of a medicinal composition for initiating a CTL immune response against said HLA-A*2402- restricted cryptic epitope.
  • the present invention also encompasses a method for vaccinating a patient against a tumoral or viral antigen, wherein said method comprises a first step of vaccination with an optimized immunogenic peptide cognate to a native HLA- A*2402-restricted cryptic epitope of said antigen, followed by a second step of vaccination with said native peptide.
  • the first step and/or the second step can be performed by using a chimeric polypeptide comprising one, two, three or more optimized or cryptic peptides as above-described, instead of single-epitope peptides.
  • the invention also pertains to a kit of parts comprising, in separate formulations or containers (vials, tubes, etc.):
  • peptides which can be part of a kit according to the invention are the peptides of SEQ ID NOs: 1 to 6, which can constitute the first peptide, the second peptide being then derived from said first peptide by a method for increasing its immunogenicity, as described above.
  • kits according to the invention can hence comprise peptides of SEQ ID Nos: 1 and 11 (in separate containers), or peptides of SEQ ID Nos: 2 and 12 (in separate containers), or peptides of SEQ ID Nos: 3 and 13 (in separate containers), or peptides of SEQ ID Nos: 4 and 14 (in separate containers), or peptides of SEQ ID Nos: 5 and 15 (in separate containers), or peptides of SEQ ID Nos: 6 and 16 (in separate containers).
  • kits of parts according to the invention comprise at least one chimeric polypeptide.
  • the kit also comprises at least a peptide cognate to one of the epitopes comprised in the chimeric polypeptide, wherein said cognate peptide is either isolated or included in another chimeric polypeptide.
  • the kit comprises, in separate formulations, a first chimeric polypeptide comprising one, two, three or more HLA-A*2402 -restricted cryptic epitopes, and a second chimeric polypeptide corresponding to its cognate HLA-A*2402-restricted immunogenic chimeric polypeptide (which means that it comprises optimized HLA-A*2402-restricted immunogenic epitopes cognate to the cryptic epitopes comprised in the first chimeric polypeptide).
  • the kit comprises one, two, three or more peptides corresponding to distinct HLA-A*2402-restricted cryptic epitopes, wherein said peptides are either mixed in one single formulation, or separated in several formulations and, in a separate formulation, a chimeric polypeptide comprising the optimized HLA-A* 2402- restricted immunogenic epitopes cognate to said cryptic peptides.
  • kits according to the invention comprise, in separate containers:
  • a polyallelic peptides mix or a polyallelic chimeric polypeptide comprising at least a HLA-A*2402-restricted cryptic epitope as described above and at least one different HLA-restricted cryptic epitope, and
  • a polyallelic peptides mix or a polyallelic chimeric polypeptide comprising at least a HLA-A*2402 -restricted immunogenic epitope cognate to the HLA- A*2402-restricted cryptic epitope recited in (i), and at least another immunogenic epitope cognate to the other cryptic epitope recited in (i).
  • kits according to the invention can comprise, instead of at least part the peptides or chimeric polypeptides, nucleic acid(s) encoding said peptides or chimeric polypeptides.
  • nucleic acid(s) is(are) as above-described.
  • kits According to the invention, mention will be made only of the peptides (native or optimized) included therein; it is understood that chimeric polypeptide(s) (comprising native cryptic epitopes or optimized epitopes) can be enclosed in the kits instead of single-epitope peptides, and that nucleic acid(s) can also be included in addition or instead of at least part of said peptides or chimeric polypeptides.
  • the kit is a vaccination kit, wherein said first (native) and second (cognate optimized) peptides are in separate vaccination doses.
  • the vaccination kit comprises 2 or 3 doses of optimized peptide, and 3, 4, 5 or 6 doses of native peptide.
  • a particular vaccination kit according to the invention is adapted for the first vaccination sequence of 6 injections, and comprises 2 or 3 doses of optimized peptide, and 4 or 3 doses of native peptide.
  • kits comprising at least 2 doses, and up to 40 or 50 doses of native peptide, are also part of the present invention.
  • the vaccination kit can comprise 2 to 3 doses of optimized peptide, and 3 to 40 or up to 50 doses of native peptide.
  • said native and optimized peptides present in the kit are as described above.
  • each dose comprises between 0.1 and 10 mg of peptide, preferably from 1 to 5 mg, or between 1 and 20 mg of polypeptide.
  • each dose is formulated for subcutaneous injection.
  • each dose can be formulated in 0.3 to 1.5 ml of an emulsion of aqueous solution emulsified with Montanide ISA51, used as an adjuvant.
  • Montanide ISA51 used as an adjuvant.
  • the doses are in the form of an aqueous solution.
  • the doses can be in the form of a lyophilized peptide, for extemporaneous preparation of the liquid solution to be injected.
  • Other possible components of said kits are one or several adjuvants, to be added to the peptide compositions before administration, and a notice describing how to use said kits.
  • FIG. 2 Immunogenicity of HLA-A*2402 restricted optimized cryptic peptides.
  • transgenic mice used in the described experiments were obtained by crossing HLA-A24 transgenic mice previously described (Barra et al., 1993) and H2 Kb " H2Db " knock out mice, transgenic for both human ⁇ 2 microglobulin and CD8 ⁇ chain (Perarnau et al., 1999).
  • Peptides were synthesized by Epytop (N ⁇ mes, France).
  • HLA-A*2402 transfected human TAP negative T2-A24 cells were previously described (Miyahara et al., 2005), and were provided by Dr. Lemonnier (Institut Pasteur, Paris, France). AU cell lines were grown in FCS 10% supplemented RPMIl 640 culture medium.
  • mice were injected subcutaneously with 100 ⁇ g of peptide emulsified in Incomplete Freund's Adjuvant (IFA) in the presence of 150 ⁇ g of the I-A b restricted HBVcore ⁇ s T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 112). After 15 days, 5x10 7 spleen cells were stimulated twice in vitro with peptide (10 ⁇ M), at 6 days interval. On day 13 of culture, the bulk responder populations were tested for specific cytotoxicity against target cells expressing HLA-A*2402 and loaded with the same peptide. Cross-recognition assay.
  • IFA Incomplete Freund's Adjuvant
  • mice were injected subcutaneously with 100 ⁇ g of optimized peptide emulsified in Incomplete Freund's Adjuvant (IFA) in the presence of 150 ⁇ g of the I-A b restricted HBVcore 128 T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 112).
  • IFA Incomplete Freund's Adjuvant
  • TPPAYRPPNAPIL I-A b restricted HBVcore 128 T helper epitope
  • 5x10 7 spleen cells were stimulated firstly in vitro with the optimized peptide (10 ⁇ M), and secondly on day 6 of culture with the corresponding native peptide.
  • the bulk responder populations were tested for specific cytotoxicity against targets cells expressing HLA-A*2402 and loaded with the optimized, the native or an irrelevant peptide. Cytotoxic assay.
  • the inventors have selected 10 native peptides according to the selection method described above. First, seven peptides were tested for their capacity to bind HLA- A*2402 molecules. All but two peptides were not or weakly able to bind to the HLA- A*2402.
  • HLA- A24 transgenic mice were then vaccinated with the selected peptides, and fifteen days later, their spleen cells were in vitro stimulated twice at 6 days intervals with the peptide.
  • Peptide-specific CTLs were detected in mice vaccinated with control high affinity peptides selected as having primary Y2 and/or C-terminal anchor motifs (data not shown).
  • Native peptides, which were not able to bind to the HLA-A*2402 were shown to be also non immunogenic (figure 1) and Her2/neu 802, which binds to the HLA-A*2402, was shown to be immunogenic in transgenic mice. This confirms that there is a correlation between binding affinity and immunogenicity for the HLA-A*2402 restricted peptides.
  • Her2/neu 802 PYGCLLDHV ++ 10 Table 5: HLA-A*2402 immunogenicity of selected cryptic peptides. (-) means that none of the mice vaccinated with the corresponding native peptides develops a specific immune response, (+) that less to 50% of vaccinated mice responded, (++) that more that 50% responded. ND: not determined
  • L leucine
  • Table 6 Tumor and HIV derived HLA-A*2402 restricted epitopes Optimized peptides were tested for their immunogenicity (table 7, figure 2), showing that the chosen modification enhances the capacity to induce specific immune response in HLA-A24 transgenic mice for six native peptides.
  • CTLs generated in mice vaccinated with optimized peptides recognized target cells loaded with the corresponding native peptide (figure 2).
  • the inventors describe a method to optimize immunogenicity of HLA-A*2402 restricted cryptic peptides. It consists of a) selecting cryptic peptides with Y2 and unfavourable amino acids in secondary anchor position 1 and/or 9; and b) substituting the unfavourable amino acids at the N-terminal position with a positively charged amino acid (R or K) and the C-terminal residue with a L when this later substitution is necessary.
  • Bennaceur-Griscelli A., Faure, O., criz, P., Firat, H., Chouaib, S., Lemonnier, F.A., Davoust, J., Miconnet, I., Vonderheide, R.H. and Kosmatopoulos, K. (2004) High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy. J Clin Invest,
  • Kitano, S., Okumura, S. Takemitsu, T., Yuta, A., Majima, Y., Lemonnier, F.A., Boon, T. and Shiku, H. (2005) Determination of cellularly processed HLA-A2402-restricted novel
  • H2Kb, H2Db and double H2KbDb knockout mice peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses. Eur J Immunol, 29, 1243- 1252.
  • Velders M.P., Weijzen, S., Eiben, G.L., Elmishad, A.G., Kloetzel, P.M., Higgins, T., Ciccarelli, R.B., Evans, M., Man, S., Smith, L. and Kast, W.M. (2001) Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine. J Immunol, 166, 5366-5373.

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Abstract

The present invention pertains to methods for identifying a HLA-A* 2402- restricted cryptic epitope in an antigen, and for increasing its immunogenicity, in order to obtain HLA-A*2402-restricted epitopes able to trigger an immune response against HLA- A*2402-restricted cryptic epitopes. Isolated peptides consisting of cryptic or optimized HLA- A*2402-restricted epitopes are provided.

Description

IDENTIFICATION, OPTIMIZATION AND USE OF CRYPTIC HLA-A24 EPITOPES FOR IMMUNOTHERAPY
The present invention relates to the field of peptide immunotherapy. In particular, the invention provides novel methods and materials for efficiently treating patients having an HLA- A*2402 phenotype.
Peptide vaccination or immunotherapy is a therapeutic approach which is currently the subject of a great number of studies in the context of the treatment of cancer. The principle thereof is based on immunization with peptides which reproduce T cell epitopes of tumor antigens recognized by cytotoxic T lymphocytes (CTLs)5 which play a major role in the elimination of tumor cells.
It will be recalled that CTLs do not recognize whole protein antigens, but peptide fragments thereof, generally comprising 8 to 10 amino acids, presented by class I major histocompatibility complex (MHC I) molecules expressed on the surface of cells. The presentation of these peptides is the result of the antigen processing which involves three steps: cytosolic degradation of the antigen by a multienzyme complex called proteasome, translocation of the peptides derived from this degradation in the endoplasmic reticulum (ER) by the TAP transporters, association of these peptides with the MHC I molecules and exportation of the peptide/MHC I complexes to the cell surface.
The peptide/MHC I complexes interact with the specific T cell receptor (TCR) on CTL, inducing the stimulation and amplification of these CTL, which become able to attack target cells expressing the antigen from which the peptide is derived.
During the antigen processing, a peptide selection takes place, which results in a hierarchy of peptides presentation. Peptides that are preferentially presented by the MHC I molecules are called immunodominant, while peptides which are weakly presented are called cryptic. Immunodominant peptides exhibit a high affinity for the MHC I and are immunogenic while cryptic peptides exhibit a low affinity for MHC I and are non-immunogenic .
Immunodominant peptides have been widely targeted by tumor vaccines in preclinical and clinical studies with disappointing results (Gross et al., 2004; Rosenberg et al., 2004).
Tumor antigens are frequently self proteins over-expressed by tumors and expressed at lower levels by normal cells and tissues. The immune system is unable to react against these self antigens because of the self tolerance process. Self-tolerance concerns mainly the immunodominant peptides (Cibotti et al., 1992; Gross et al., 2004), thus explaining the incapacity of these peptides to induce a tumor immunity. Cryptic peptides are much less involved in self tolerance process (Cibotti et al., 1992; Gross et al., 2004; Moudgil et aL, 1999) and can therefore induce an efficient tumor immunity, provided their immunogenicity is enhanced (Engelhorn et al., 2006; Gross et al., 2004). The usual strategy for enhancing the immunogenicity of cryptic peptides, which are non-immunogenic because of their low MHC I affinity, consists in increasing their affinity for the MHC I molecules via amino acids substitutions. Peptide affinity for MHC I molecules mainly depends on the presence at well defined positions (primary anchor positions) of residues called "primary anchor residues". These residues are MHC I allele specific. The presence of primary anchor residues, although often necessary, is not sufficient to ensure a high MHC I affinity. It has been shown that residues located outside the primary anchor positions (secondary anchor residues) may exert a favourable or unfavourable effect on the affinity of the peptide for the MHC I. The presence of these secondary anchor residues makes it possible to explain the existence, within peptides having the primary anchor motifs, of a great variability in the binding affinity (Ruppert et al., 1993).
Amino acids substitutions aiming at enhancing affinity for MHC I molecule should preserve the antigenicity of such optimized peptides. Indeed, CTL generated by optimized peptides must cross-react with the corresponding native peptides. Many teams have succeeded in enhancing immunogenicity of already immunogenic peptides by increasing their affinity for HLA-A*0201 (Bakker et al., 1997; Parkhurst et al., 1996; Valmori et al., 1998). The inventors have previously described a general strategy to enhance affinity and immunogenicity of HLA-A* 0201 restricted cryptic peptides (Scardino et al., 2002; Tourdot and Gould, 2002) and HLA-B*0702 (WO 2008/010098).
HLA-A*2402 is a frequently expressed molecule (27% of the population) and is one of the most common alleles in Japanese and Asian people. Identification and optimization of HLA-A* 2402 restricted tumor cryptic peptides is therefore necessary for developing efficient cancer vaccines for HLA-A* 2402 expressing patients. Several tumor immunogenic peptides presented by HLA-A* 2402 have been described to date (table 1). Antigen Sequence SEQ ID No:
Beta-catenin SYLDSGIHF 168
TERT TYVPLLGSL 169
TERT CYGDMENKL 170
TERT AVQVCGPPL 171
KM-HN-1 NYNNFYRFL 172
KM-HN-1 EYSKECLKEF 173
KM-HN-1 EYLSLS DKI 174
MAGE-A2 EYLQLVFGI 175
MAGE-A3 TFPDLESEF 17 6
MAGE-A3 VAELVHFLL 177
MAGE-A4 NYKRCFPVI 178
SAGE LYATVIHDI 179
CEA QYSWFVNGTF 180
CEA TYACFVSNL 181 gp100 / Pmel17 VYFFLPDHL 182
OA1 LYSACFWWL 183 tyrosinase AFLPWHRLF 184
Ep-CAM RYQLDPKFI 185
Her2/neu TYLPTNASL 18 6
PRAME LYVDSLFFL 187
PSMA NYARTEDFF 188
RNF43 NSQPVWLCL 189
WT1 CMTWNQMNL 190
Table 1: Tumor immunogenic HLA- A24 T cell epitopes
As described in the experimental part below, the inventors have now found a strategy to identify, in an antigen, cryptic peptides presented by HLA-A*2402 molecule, and to optimize their immunogenicity, preserving the cross-reactivity with the corresponding native cryptic peptides.
Hence, a first aspect of the present invention is a method for identifying an HLA-A*2402-restricted cryptic epitope in an antigen, comprising a step of selecting, in said antigen, a peptide of 8 to 12 amino acids having a tyrosine (Y) in primary anchor position 2, with the proviso that the peptide does not have, simultaneously, a positively charged amino acid (arginine (R) or lysine (K)) in position 1 and a leucine (L), or a phenylalanine (F) or an isoleucine (I) in C-terminal position. Such an epitope hence has the sequence Xi YX2X3X4X5X6XTXSXPX ioXii (SEQ ID No: 20), wherein Xj to X6 are any amino acid, X7 to Xj0 are any amino acid or none, and X] i ≠ L or F or I if Xj = R or K. When the above selection step is performed alone, the obtained sequences are those of putative cryptic epitopes. Although epitopes responding to the above criteria have a strong probability to be non immunogenic, functional tests are necessary to identify truly cryptic epitopes with certainty. In particular, the inventors have observed that some peptides having a primary sequence as defined above are in fact immunogenic in individuals expressing HLA-A*2402. Hence, in a preferred embodiment, the method for identifying a HLA-A *2402-restricted cryptic epitope in an antigen further comprises step consisting in testing the immunogenicity of each putative cryptic epitope of SEQ ID No: 20, in an appropriate model, and selecting those which are non-immunogenic. For performing this aspect of the invention, an appropriate model is a model which predicts the immunogenicity of the peptide in an individual who expresses HLA-A*2402. An example of such an appropriate model is described in the experimental part and consists of HLA-A*2402 transgenic mice. In this model, the non-immunogenicity of putative cryptic peptides is checked by vaccinating the mice and testing if specific CTL have been generated, by using human cells expressing HLA-A*2402 and loaded with the peptide as target cells. In what follows, the phrases "HLA-A*2402-restricted cryptic epitope" or
"native peptide" will be used to designate any peptide of SEQ ID No: 20, whether its non- immunogenicity has been checked or not. When necessary, the phrase "putative HLA- A*2402-restricted cryptic epitope" will be used to express the fact that the immunogenicity of the peptide has not been tested, and the phrase "confirmed HLA-A*2402 -restricted cryptic epitope" will be used for peptides which have been tested and have proved to be non-immunogenic in an appropriate model.
In the present text, the term "peptide" designates not only molecules in which amino acid residues (in L or D configurations) are joined by peptide (-CO-NH-) linkages, but also synthetic pseudopeptides or peptidomimetics in which the peptide bond is modified, especially to become more resistant to proteolysis, and provided their immunogenicity is not impaired by this modification.
According to a preferred embodiment of the invention, the selected peptide has 9 to 11 amino acids, more preferably 9 or 10 amino acids and one or more unfavourable amino acids at secondary anchor positions, for example a P (proline) in position 1 and/or a D or E or G or H or P or Q or R or K (glutamic or aspartic acid, glycine, histidine, proline, glutamine, arginine or lysine) at C-terminal position.
A second aspect of the present invention is a method for increasing the immunogenicity of a HLA-A*2402-restricted cryptic epitope, comprising a step of substituting the N-terminal residue of said epitope with a positively charged amino acid (R or K), and/or substituting the C-terminal residue of said epitope with an L, F or I.
Preferentially, the C-terminal modification is the substitution by an L.
Of course, in this method, the word "substituting" is to be understood as obtaining a peptide the sequence of which is derived from the sequence of said HLA-
A*2402-restricted cryptic epitope by the mentioned substitution, whatever the technical method used to obtain said peptide. For example, the peptide can be produced by artificial peptide synthesis or by recombinant expression.
In particular, the immunogenicity of a HLA-A*2402-restricted cryptic epitope in which the two first residues are RY or KY can be increased by replacing its last amino-acid by an L5 F or I, preferentially by an L (or by adding a L, I or F at its C- terminus, provided it is not longer than 11 amino acids). When the sequence .of the selected HLA-A*2402-restricted cryptic epitope is XiYX2X3X4X5X6X7XsXeXiOL (SEQ ID No: 21), wherein Xi is any amino acid but R or K, X2 to X6 are any amino acid, and X7 to X] o are any amino acid or none, the substitution of Xi by R or K is sufficient to increase its immunogenicity. More generally, when the sequence of the selected HLA-A*2402- restricted cryptic epitope is Xi YX2X3X4X5X6X7X8X9XiOXi i (SEQ ID No: 22), wherein Xi is any amino acid but R or K, X2 to X6 are any amino acid, and X7 to Xi0 are any amino acid or none, and Xn is not an unfavourable amino acids (D or E or G or H or P or Q or R or K ), the substitution of Xi by R or K can be sufficient to increase its immunogenicity.
In what follows, the expression "optimized peptide" or "optimized immunogenic A*2402-restricted epitope" will designate an immunogenic peptide derived from a HLA-A*2402-restricted cryptic epitope (called its "cognate native peptide") by the above method. In a preferred embodiment of the invention, the optimized peptide can trigger an immune response which cross-recognizes its cognate native peptide. Another aspect of the present invention is hence a method for obtaining a HLA-A*2402-restricted epitope able to trigger an immune response against a HLA-A*2402-restricted cryptic epitope of an antigen, comprising the steps of (i) identifying, in said antigen, one or several native putative HLA-
A*2402-restricted cryptic epitopes, by the method according to claim 1 ;
(ii) testing the immunogenicity of each native epitope selected in step (i), in an appropriate model, and selecting those which are non-immuno genie;
(iii) for each native epitope selected in step (ii), obtaining an optimized epitope by increasing its immunogenicity, by the method as above-described;
(iv) testing the immunogenicity of each optimized epitope obtained in step (iii), in an appropriate model, and selecting those which are immunogenic;
(v) for each epitope selected in step (iv), testing if the CTLs generated against the optimized epitope also recognize its cognate native epitope, and selecting those for which the test is positive.
In this method, the appropriate models which can be used in steps (ii) and (iv) are as described above. In step (v), the cross-recognition can be performed by any method known by the skilled artisan, for example as described in the experimental part.
As disclosed in the experimental part below, the inventors have identified in different tumor associated antigens (hTERT, EphA2, MAGE or Her2/neu), a number of putative HLA-A*2402-restricted cryptic epitopes. When testing the immunogenicity of these epitopes, one of them proved to be immunogenic. The inventors have selected the peptides disclosed in Table 2 below, which are confirmed HLA-A*2402-restricted cryptic epitopes. The peptides are part of the present invention.
Peptide Sequence SEQ ID
TERT 403 PYGVLLKTH ID N°1
TERT 770 PYMRQFVAH ID N°2
HER 780 PYVSRLLGI ID N°3
EphA2 47 PYGKGWDLM ID N°4
EphA2 502 TYLVQVQAL ID N°5
EphA2 817 PYWELSNHE ID N°6
Her2/neu 922 PYDGlPARE ID N°7
MAGE 261 RYEFLWGPR ID N°8
Her2/neu 300 PYNYLSTDV ID N°9 Table 2: Selected confirmed cryptic HLA-A*2402 restricted peptides The present invention also pertains to optimized peptides derived from the cryptic peptides of SEQ ID Nos: 1 to 9, by a method according to the invention. Preferred examples of optimized peptides are KYGVLLKTL (SEQ ID No: 11), RYMRQFVAL (SEQ ID No: 12), RYVSRLLGI (SEQ ID No: 13), RYGKGWDLL (SEQ ID No: 14), RYLVQVQAL (SEQ ID No: 15), RYWELSNHL (SEQ ID No: 16). Among these peptides, SEQ ID No: 13 and SEQ ID No: 15 have been derived from the cryptic HLA-A*2402-restricted epitopes of SEQ ID NOs: 3 and 5, respectively, by substitution of their N-terminal amino-acid with a R. The peptides of SEQ ID Nos: 11, 12, 14 and 16 have been derived from the peptides of SEQ ID Nos: 1, 2, 4 and 6, respectively, by substituting their N-terminal amino-acid with an R or a K and their C-terminal amino-acid with a L. Polyspecific tumor vaccination offers a broader control of tumor cells than monospecific vaccination, thereby reducing the risk of emergence of immune escape variants. In most cases, immunotherapy is then more efficient when targeting several epitopes than when targeting only one epitope, provided the tumour is known to express all targeted antigens. The inventors have previously described a polypeptide composed of HLA-A* 0201 restricted optimized cryptic peptides derived from three different universal tumor antigens (TERT988Y, HER-2/neu402γ and MAGE-A248V9), named Vx-006 (WO 2007/073768). Vx-006 is able to induce a polyspecific CD8 cell response both in vivo in HLA-A* 0201 transgenic HHD mice and in vitro in humans, whereas the mixture of TERT988Y, HER-2/neu402γ and MAGE-A248V9 peptides failed to induce a trispecific response. Hence, a chimeric polypeptide comprising several epitopes can be more efficient than a mere mixture of the same epitopes to trigger a response against more than one epitope. Depending on the context, a chimeric polypeptide comprising a repetition of one single epitope can also trigger a stronger response against said epitope than a peptide consisting of said epitope. Indeed, a polypeptide organization (either with several different epitopes or with a repetition of one single epitope) can produce new junctional epitopes, especially CD4 restricted epitopes, able to optimize the targeted peptide(s)-specific immune response. Moreover, when free peptides are subcutaneously injected, peptides bind directly to MHC molecules of every cells present at the site of injection. As polypeptides need to be processed, vaccination with polypeptides is more efficient to target antigenic peptides to professional Antigenic Presenting Cells (APC) as Dendritic Cells.
A further aspect of the invention is hence a chimeric polypeptide, comprising one, two, three or more HLA-A*2402-restricted cryptic epitopes or one, two, three or more optimized immunogenic HLA-A*2402-restricted epitopes as described above. In a chimeric polypeptide according to the invention, the epitopes can be different from each other, and/or the same epitope can be repeated several times.
It is to be noted that when several epitopes specific for the same HLA molecule are used together, either in a mix or in a chimeric polypeptide, the epitopes are in competition for the binding to the corresponding HLA molecule. Contrarily, by using a mix of different HLA-restricted epitopes (HLA-A*0201, HLA-A*2402, HLA-B*0702 or others), or a chimeric polypeptide comprising the same different HLA-restricted epitopes, there will be no competition for HLA binding, and a polyspecific response will be obtained with certainty, provided all the HLA molecules are expressed in the vaccinated individual.
In a chimeric polypeptide according to the invention, HLA-A*2402- restricted cryptic or optimized immunogenic epitopes described above can hence be advantageously associated to previously described HLA-A* 0201 (WO 02/02716) and/or HLA-B*0702 peptides (WO 2008/010010 and WO 2008/010098), or to immunogenic epitopes derived from previously described tumor associated antigens, comprising CEA, PRAME, Tyrosinase, TRAG-3, NY-Eso-1, P53, Muc-1, PSA/PSMA, survivin, Melan- A/MART-1, TRP-I, TRP-2, WTl, EphAl, EphA2, EphA3, EphA4, G250/MN/CAIX, STEAP, alphafoetoprotein, RAGE-I, PAGE-I. Of course, a polyallelic peptides mix, comprising at least a peptide according to the present invention and one different HLA- restricted epitope (HLA-A*0201, HLA-A*2402, HLA-B*0702 or others), is also part of the present invention.
Examples of cryptic epitopes which can advantageously be combined to HLA-A*2402-restricted cryptic epitopes (either in a mix or in a chimeric polypeptide), as well as examples of optimized immunogenic epitopes which can advantageously be combined to optimized immunogenic HLA-A*2402-restricted epitopes, are described in Table 3 below. Of course, these lists are not limitative.
Figure imgf000009_0001
Figure imgf000010_0001
Table 3: epitopes which can be combined to HLA-A*2402-restricted epitopes in chimeric polypeptides according to the invention
The skilled artisan can chose any known technique to produce such polypeptides. For example, the polypeptide can be obtained by chemical synthesis, or by using the technology of genetic engineering (Velders et al., 2001).
Another object of the present invention is an isolated nucleic acid molecule designed to cause the expression of a cryptic HLA- A*2402 -restricted epitope, or of an optimized immunogenic HLA-A*2402-restricted epitope, or of a chimeric polypeptide as above-described. By "designed to cause the expression of a peptide is herein meant that said peptide is expressed as such, isolated from the whole antigen from which its sequence has been selected (and, in appropriate cases, optimized as above- described), when the nucleic acid is introduced in an appropriate cell. The region encoding the epitope or chimeric polypeptide will typically be situated in the polynucleotide under control of a suitable promoter. Bacterial promoters will be preferred for expression in bacteria, which can produce the polypeptide either in vitro, or, in particular circumstances, in vivo. An example of bacterium that can be used to produce a peptide or polypeptide according to the invention, directly in vivo, is Listeria monocytogenes, which is a facultative intracellular bacterium that enters professional antigen-presenting cells by active phagocytosis (Paterson and Maciag, 2005). Alternatively, a nucleic acid according to the invention can be administered directly, using an appropriate vector. In this case, a tissue-specific, a strong constitutive, or an endogenous promoter can be used to control the peptide expression. Suitable vector systems include naked DNA plasmids, liposomal compositions to enhance delivery, and viral vectors that cause transient expression. Examples of viral vectors are adenovirus or vaccinia virus vectors and vectors of the herpes family, especially in a non-replicative form.
The present invention also pertains to a pharmaceutical composition comprising at least, as an active principle, an HLA-A*2402-restricted cryptic epitope as above-described, or an optimized immunogenic epitope polypeptide as mentioned above, or a chimeric polypeptide according to the invention, or a nucleic acid encoding any of these, and/or a vector carrying said nucleic acid. Formulation of pharmaceutical compositions will accord with contemporary standards and techniques. Medicines intended for human administration will be prepared in adequately sterile conditions, in which the active ingredient(s) are combined with an isotonic solution or other pharmaceutical carrier appropriate for the recommended therapeutic use. Suitable formulations and techniques are generally described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton PA). In particular, a HLA-A*2402-restricted epitope or a chimeric polypeptide or a nucleic acid according to the invention can be used for the preparation of a composition for preventive or curative immunotherapy, especially, for antiviral or anticancer immunotherapy.
In a particular embodiment, a pharmaceutical composition according to the invention is a vaccine. In this latter case, the components described above can be combined with an adjuvant to potentiate the immune response. Classic adjuvants include oil emulsions, like Incomplete Freund's Adjuvant or Montanide, and adherent surfaces such as alum. Adjuvants that recruit and activate dendritic cells particularly via TLR (such as bacterial DNA or bacterial membrane derived proteins) or help elicit cytotoxic T cells are especially useful. Other factors that otherwise boost the immune response or promote apoptosis or elimination of cancer cells can also be included in the composition, such as IL-2 or IL- 12 cytokines or GM-CSF.
Multiple doses and/or different combinations of the immunogenic compositions of this invention can be packaged for distribution separately or together. Each composition or set of compositions, such as the kits of parts described below, can be accompanied with written instructions regarding the use of the composition or combination for eliciting an immune response and/or for the treatment of cancer. In a previous patent application (WO 2006/120038), the Applicant has described a vaccination protocol which enables the initiation and maintenance of a T cell response targeting sub-dominant/cryptic epitopes. The results reported in WO 2006/120038 demonstrate that injection of a native peptide corresponding to a sub- dominant/cryptic epitope, following vaccination with its cognate optimized peptide, can maintain the immune response initiated by said optimized peptide.
According to the invention, a HLA-A* 2402-restricted cryptic epitope can hence be used for the preparation of a medicinal composition for maintaining the CTL immune response initiated by its cognate optimized peptide. An immunogenic peptide having an optimized immunogenic HLA-A*2402-restricted epitope sequence derived from a HLA-A*2402-restricted cryptic epitope can also be used, for the preparation of a medicinal composition for initiating a CTL immune response against said HLA-A*2402- restricted cryptic epitope. The present invention also encompasses a method for vaccinating a patient against a tumoral or viral antigen, wherein said method comprises a first step of vaccination with an optimized immunogenic peptide cognate to a native HLA- A*2402-restricted cryptic epitope of said antigen, followed by a second step of vaccination with said native peptide. In such a method, the first step and/or the second step can be performed by using a chimeric polypeptide comprising one, two, three or more optimized or cryptic peptides as above-described, instead of single-epitope peptides. The invention also pertains to a kit of parts comprising, in separate formulations or containers (vials, tubes, etc.):
(i) a first peptide comprising a sequence of a HLA-A*2402-restricted cryptic epitope, and
(ii) a second peptide comprising a sequence corresponding to an optimized immunogenic epitope cognate to the cryptic epitope recited in (i).
Examples of peptides which can be part of a kit according to the invention are the peptides of SEQ ID NOs: 1 to 6, which can constitute the first peptide, the second peptide being then derived from said first peptide by a method for increasing its immunogenicity, as described above. Preferred kits according to the invention can hence comprise peptides of SEQ ID Nos: 1 and 11 (in separate containers), or peptides of SEQ ID Nos: 2 and 12 (in separate containers), or peptides of SEQ ID Nos: 3 and 13 (in separate containers), or peptides of SEQ ID Nos: 4 and 14 (in separate containers), or peptides of SEQ ID Nos: 5 and 15 (in separate containers), or peptides of SEQ ID Nos: 6 and 16 (in separate containers). Other kits of parts according to the invention comprise at least one chimeric polypeptide. In this embodiment, the kit also comprises at least a peptide cognate to one of the epitopes comprised in the chimeric polypeptide, wherein said cognate peptide is either isolated or included in another chimeric polypeptide. Several preferred variants of such kits are contemplated: in a first embodiment, the kit comprises, in separate formulations, a first chimeric polypeptide comprising one, two, three or more HLA-A*2402 -restricted cryptic epitopes, and a second chimeric polypeptide corresponding to its cognate HLA-A*2402-restricted immunogenic chimeric polypeptide (which means that it comprises optimized HLA-A*2402-restricted immunogenic epitopes cognate to the cryptic epitopes comprised in the first chimeric polypeptide). In a second embodiment, the kit comprises one, two, three or more peptides corresponding to distinct HLA-A*2402-restricted cryptic epitopes, wherein said peptides are either mixed in one single formulation, or separated in several formulations and, in a separate formulation, a chimeric polypeptide comprising the optimized HLA-A* 2402- restricted immunogenic epitopes cognate to said cryptic peptides.
As mentioned above, a poly allelic stimulation (i.e., using epitopes specific for different HLA molecules) can advantageously be performed to obtain a polyspecific response. Accordingly, preferred embodiments of the kits according to the invention comprise, in separate containers:
(i) a polyallelic peptides mix or a polyallelic chimeric polypeptide, comprising at least a HLA-A*2402-restricted cryptic epitope as described above and at least one different HLA-restricted cryptic epitope, and
(ii) a polyallelic peptides mix or a polyallelic chimeric polypeptide, comprising at least a HLA-A*2402 -restricted immunogenic epitope cognate to the HLA- A*2402-restricted cryptic epitope recited in (i), and at least another immunogenic epitope cognate to the other cryptic epitope recited in (i).
Alternatively, the kits according to the invention can comprise, instead of at least part the peptides or chimeric polypeptides, nucleic acid(s) encoding said peptides or chimeric polypeptides. In this case, the nucleic acid(s) is(are) as above-described.
In the following description of some specific kits according to the invention, mention will be made only of the peptides (native or optimized) included therein; it is understood that chimeric polypeptide(s) (comprising native cryptic epitopes or optimized epitopes) can be enclosed in the kits instead of single-epitope peptides, and that nucleic acid(s) can also be included in addition or instead of at least part of said peptides or chimeric polypeptides.
In a particular embodiment of the invention, the kit is a vaccination kit, wherein said first (native) and second (cognate optimized) peptides are in separate vaccination doses. In a preferred embodiment, the vaccination kit comprises 2 or 3 doses of optimized peptide, and 3, 4, 5 or 6 doses of native peptide. A particular vaccination kit according to the invention is adapted for the first vaccination sequence of 6 injections, and comprises 2 or 3 doses of optimized peptide, and 4 or 3 doses of native peptide. In case of long-lasting diseases, it is preferable to maintain the level of immunity obtained after this primo-vaccination, by regular recalls. This can be done, for example, by injections performed every 1 to 6 months. Therefore, complementary kits, comprising at least 2 doses, and up to 40 or 50 doses of native peptide, are also part of the present invention. Alternatively, the vaccination kit can comprise 2 to 3 doses of optimized peptide, and 3 to 40 or up to 50 doses of native peptide. Of course, said native and optimized peptides present in the kit are as described above.
Each dose comprises between 0.1 and 10 mg of peptide, preferably from 1 to 5 mg, or between 1 and 20 mg of polypeptide. In a preferred embodiment, each dose is formulated for subcutaneous injection. For example, each dose can be formulated in 0.3 to 1.5 ml of an emulsion of aqueous solution emulsified with Montanide ISA51, used as an adjuvant. The skilled artisan can choose any other adjuvant(s) in place of (or in addition to) Montanide IS A51. In a particular embodiment, the doses are in the form of an aqueous solution. Alternatively, the doses can be in the form of a lyophilized peptide, for extemporaneous preparation of the liquid solution to be injected. Other possible components of said kits are one or several adjuvants, to be added to the peptide compositions before administration, and a notice describing how to use said kits.
The invention is further illustrated by the following figures and examples. LEGENDS OF FIGURES Figure 1 : Immunogenicity of HLA-A *2402 cryptic peptides. HLA-
A*2402 transgenic mice were vaccinated with the cryptic peptides following the described protocol and generated CTL were tested against T2-A24 targets loaded with peptide as indicated (NR non relevant peptide). Percentage of specific lysis was determined as: Lysis = (Experimental Release - Spontaneous Release) / (Maximal Release - Spontaneous Release) x 100. Four CTL dilutions, corresponding to four CTL/target cells ratio were tested.
Figure 2: Immunogenicity of HLA-A*2402 restricted optimized cryptic peptides. HLA-A*2402 transgenic mice were vaccinated with the optimized peptide following the described protocol and generated CTL were tested against T2-A24 targets loaded with the optimized (immunogenicity), the corresponding native (native peptide cross recognition) or an irrelevant (NR) peptide as indicated. Percentage of specific lysis was determined as: Lysis = (Experimental Release - Spontaneous Release) / (Maximal Release - Spontaneous Release) x 100. Four CTL dilutions, corresponding to four CTL/target cells ratio were tested. EXAMPLES
The examples have been performed using the following materials and methods :esd
Transgenic Mice. The transgenic mice used in the described experiments were obtained by crossing HLA-A24 transgenic mice previously described (Barra et al., 1993) and H2 Kb" H2Db" knock out mice, transgenic for both human β2 microglobulin and CD8α chain (Perarnau et al., 1999).
Peptides. Peptides were synthesized by Epytop (Nϊmes, France).
Cells. HLA-A*2402 transfected human TAP negative T2-A24 cells were previously described (Miyahara et al., 2005), and were provided by Dr. Lemonnier (Institut Pasteur, Paris, France). AU cell lines were grown in FCS 10% supplemented RPMIl 640 culture medium.
Measurement of Peptide Relative Affinity to HLA-A*2402. The protocol used has been described previously (Rohrlich et al., 2003). Briefly, T2-A24 cells were incubated at 37°C for 16 hours with peptides concentrations ranging from 100 μM to 0.1 μM, and then stained with 0041HA monoclonal antibody (mAb)(One Lambda, Inc.) to quantify the surface expression of HLA-A*2402. For each peptide concentration, the HLA-A* 2402 specific staining was calculated as the percentage of staining obtained with 100 μM of the reference peptide standard A24 (AYIDNYNKF, SEQ ID NO: 111). The relative affinity (RA) was determined as: RA = (Concentration of each peptide that induces 30 % of HLA-A*2402-expression / Concentration of the reference peptide that induces 30 % of HLA-A*2402 expression).
CTL Induction in vivo in HLA-A*2402 Transgenic Mice. Mice were injected subcutaneously with 100 μg of peptide emulsified in Incomplete Freund's Adjuvant (IFA) in the presence of 150 μg of the I-Ab restricted HBVcoreπs T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 112). After 15 days, 5x107 spleen cells were stimulated twice in vitro with peptide (10 μM), at 6 days interval. On day 13 of culture, the bulk responder populations were tested for specific cytotoxicity against target cells expressing HLA-A*2402 and loaded with the same peptide. Cross-recognition assay. Mice were injected subcutaneously with 100 μg of optimized peptide emulsified in Incomplete Freund's Adjuvant (IFA) in the presence of 150 μg of the I-Ab restricted HBVcore128 T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 112). After 15 days, 5x107 spleen cells were stimulated firstly in vitro with the optimized peptide (10 μM), and secondly on day 6 of culture with the corresponding native peptide. On day 13, the bulk responder populations were tested for specific cytotoxicity against targets cells expressing HLA-A*2402 and loaded with the optimized, the native or an irrelevant peptide. Cytotoxic assay. Targets were labelled with 100 μCi of Cr51 for 60 min, plated in 96-well V-bottomed plates (3x103 cell/well in 100 μL of RPMI 1640 medium) and, when necessary, pulsed with optimized or native peptides (1 μM) at 37°C for 2 hours. Four dilutions of effector cells were then added in the wells and incubated at 37°C for 4 hours. Percentage of specific lysis was determined as: Lysis = (Experimental Release - Spontaneous Release) / (Maximal Release - Spontaneous Release) x 100.
Example 1: Affinity and Immunogenicity of Selected Cryptic Peptides
The inventors have selected 10 native peptides according to the selection method described above. First, seven peptides were tested for their capacity to bind HLA- A*2402 molecules. All but two peptides were not or weakly able to bind to the HLA- A*2402.
Antigen/position Sequence RA SEQ ID
No
TERT403 PYGVLLKTH - 1
TERT 770 PYMRQFVAH +/- 2
Her2/neu 780 PYVSRLLGI ++ 3
EphA2 47 PYGKGWDLM ND 4
EphA2 502 TYLVQVQAL ND 5
EphA2 817 PYWELSNHE ND 6
Her2/neu 922 PYDGIPARE - 7
MAGE 261 RYEFLWGPR - 8
Her2/neu 300 PYNYLSTDV 9
Her2/neu 802 PYGCLLDHV + 10 Table 4: HLA-A*2402 affinity of cryptic peptides. RA = Relative Affinity =
(Concentration of each peptide that induces 30 % of HLA-A*2402-expression / Concentration of the reference peptide that induces 30 % of HLA-A*2402 expression), (-) means RA>100, (+/-) 10<RA<100, (+) 5<RA<10, (++) RA <5, ND: not determined
HLA- A24 transgenic mice were then vaccinated with the selected peptides, and fifteen days later, their spleen cells were in vitro stimulated twice at 6 days intervals with the peptide. Peptide-specific CTLs were detected in mice vaccinated with control high affinity peptides selected as having primary Y2 and/or C-terminal anchor motifs (data not shown). Native peptides, which were not able to bind to the HLA-A*2402 were shown to be also non immunogenic (figure 1) and Her2/neu 802, which binds to the HLA-A*2402, was shown to be immunogenic in transgenic mice. This confirms that there is a correlation between binding affinity and immunogenicity for the HLA-A*2402 restricted peptides.
Nevertheless, as Her2/neu 780 strongly binds to HLA-A*2402 but is finally non immunogenic, the inventors decided to select native peptides only on their incapacity to induce a specific immune response in HLA- A24 transgenic mice. Finally, only one native peptide selected according to the described selection method was able to generate a specific immune response in HLA-A* 2402 transgenic mice, confirming that the described method allows to efficiently select putative cryptic peptides. Immunogenicity of selected native peptides is shown in table 5.
Antigen/position Sequence Immunogenicity SEQ ID
No
TERT403 PYGVLLKTH 1
TERT770 PYMRQFVAH 2
Her2/neu 780 PYVSRLLGI 3
EphA2 47 PYGKGWDLM 4
EphA2 502 TYLVQVQAL 5
EphA2 817 PYWELSNHE 6
Her2/neu 922 PYDGIPARE ND 7
MAGE 261 RYEFLWGPR 8
Her2/neu 300 PYNYLSTDV 9
Her2/neu 802 PYGCLLDHV ++ 10 Table 5: HLA-A*2402 immunogenicity of selected cryptic peptides. (-) means that none of the mice vaccinated with the corresponding native peptides develops a specific immune response, (+) that less to 50% of vaccinated mice responded, (++) that more that 50% responded. ND: not determined
Example 2: Enhancement of Immunogenicity of the Selected Cryptic Peptides
To enhance HLA-A*2402 affinity and consequently immunogenicity of low affinity peptides with the HLA specific anchor motifs, it was necessary to identify unfavourable secondary anchor motifs and substitute them with favourable motifs. These substitutions must however preserve the conformation of the peptide segment which interacts with the TCR (position 4 to position 8). The interest was, therefore, focused on secondary anchor position 1. Positively charged amino acids (lysine (K) or arginine (R)) are favourable motifs at position 1 whereas a proline (P) is an unfavourable amino acid.
Moreover, as shown in table 6 below, more than 50% of HLA-A*2402 CD8 epitope identified both in tumors and HIV cells, have a leucine (L) in C-terminal position. The inventors hence decided to use L as the C terminal modification to enhance immunogenicity of peptides preferentially having an unfavourable amino acids in this position (aspartic or glutamic acid (D ,E), glycine (G), histidine (H), glutamine (Q), lysine (K), proline (P) or arginine (R)).
Figure imgf000018_0001
Table 6: Tumor and HIV derived HLA-A*2402 restricted epitopes Optimized peptides were tested for their immunogenicity (table 7, figure 2), showing that the chosen modification enhances the capacity to induce specific immune response in HLA-A24 transgenic mice for six native peptides. HLA-A24 transgenic mice vaccinated with the TERT 403KIL9, TERT 770Rl L9, HER 780Rl5 EphA2 47Rl L9, EphA2 502Rl and EphA2 817R1L9 peptides, developed peptide specific CTLs.
Importantly, CTLs generated in mice vaccinated with optimized peptides recognized target cells loaded with the corresponding native peptide (figure 2).
Antigen/posi Modification Sequence Immunogenicity Native peptide Seq ID N0 tion cross recognition
TERT 403 PYGVLLKTH - (0/3) 1 TERT 403 K1L9 KYGVLLKTL + (4/15) + (3/15) 11
TERT 770 PYMRQFVAH - (0/3) 2 TERT 770 R1 L9 RYMRQFVAL ++ (12/18) + (5/18) 12
HER 780 PYVSRLLGI - (0/8) 3 HER 780 R1 RYVSRLLGI + (4/9) + (3/9) 13
EphA2 47 PYGKGWDLM - (0/6) 4 EphA2 47 R1L9 RYGKGWDLL ++ (7/9) ++ (7/9) 14
EphA2 502 TYLVQVQAL - (0/3) 5 EphA2 502 R1 RYLVQVQAL ++ (3/3) ++ (2/3) 15
EphA2 817 PYWELSNHE - (0/3) 6 EphA2 817 R1L9 RYWELSNHL ++ (2/3) ++ (2/3) 16
Her2/neu 922 PYDGIPARE ND 7 Her2/neu 922 R1L9 RYDGIPARL - (0/9) 17
MAGE 261 RYEFLWGPR - (0/3) 8 MAGE 261 L9 RYEFLWGPL - (0/9) 18
Her2/neu 300 PYNYLSTDV - (0/3) 9 Her2/neu 300 R1 L9 RYNYLSTDL - (0/9) 19 Table 7: Native and modified peptides immunogenicity and native peptide cross recognition. (-) means that none of the mice vaccinated with the corresponding native peptides develops a specific immune response, (+) that less to 50% of vaccinated mice responded, (++) that more that 50% responded. (XfY) means that X mice developed a specific response for a total of Y mice vaccinated. ND: not determined
In conclusion, the inventors describe a method to optimize immunogenicity of HLA-A*2402 restricted cryptic peptides. It consists of a) selecting cryptic peptides with Y2 and unfavourable amino acids in secondary anchor position 1 and/or 9; and b) substituting the unfavourable amino acids at the N-terminal position with a positively charged amino acid (R or K) and the C-terminal residue with a L when this later substitution is necessary.
Using these methods of selection/optimization, the inventors also described 6 optimized cryptic peptides that induce specific CTLs in transgenic mice able to recognize cells presenting the corresponding native peptide. REFERENCES
Bakker, A.B., van der Burg, S.H., Huijbens, RJ., Drijfliout, J.W., Melief, C.J., Adema, GJ. and Figdor, CG. (1997) Analogues of CTL epitopes with improved MHC class-I binding capacity elicit anti-melanoma CTL recognizing the wild-type epitope. Int J Cancer, 70, 302-309.
Barra, C, Gournier, H., Garcia, Z., Marche, P.N., Jouvin-Marche, E., Briand, P., Fillipi, P. and Lemonnier, F.A. (1993) Abrogation of H-2-restricted CTL responses and efficient recognition of HLA-A3 molecules in DBA/2 HLA/ A24 responder mice. J Immunol, 150, 3681-3689.
Cibotti, R., Kanellopoulos, J.M., Cabaniols, J.P., Halle-Panenko, O., Kosmatopoulos, K., Sercarz, E. and Kourilsky, P. (1992) Tolerance to a self-protein involves its immunodominant but does not involve its subdominant determinants. Proc Natl Acad Sd USA, 89, 416-420. Engelhorn, M.E., Guevara-Patino, J.A., Noffz, G., Hooper, A.T., Lou, O.,
Gold, J.S., Kappel, BJ. and Houghton, A.N. (2006) Autoimmunity and tumor immunity induced by immune responses to mutations in self. Nat Med, 12, 198-206.
Gross, D.A., Graff-Dubois, S., Opolon, P., Cornet, S., Alves, P.,
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Kitano, S., Okumura, S., Takemitsu, T., Yuta, A., Majima, Y., Lemonnier, F.A., Boon, T. and Shiku, H. (2005) Determination of cellularly processed HLA-A2402-restricted novel
CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE. Clin
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Moudgil, K.D., Southwood, S., Ametani, A., Kim, K., Sette, A. and Sercarz, E.E. (1999) The self-directed T cell repertoire against mouse lysozyme reflects the influence of the hierarchy of its own determinants and can be engaged by a foreign lysozyme. J Immunol, 163, 4232-4237.
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Paterson, Y. and Maciag, P. C. (2005) Listeria-based vaccines for cancer treatment. Curr Opin MoI Ther, 7, 454-460. Perarnau, B., Saron, M.F., San Martin, B.R., Bervas, N., Ong, H.,
Soloski, MJ., Smith, A.G., Ure, J.M., Gairin, J.E. and Lemonnier, F. A. (1999) Single
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Faure, O., Tourdot, S., Chouaib, S., Nadler, L.M., Lemonnier, F.A., Vonderheide, R.H., Cardoso, A.A. and Kosmatopoulos, K. (2002) HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy. J Immunol, 168, 5900-5906.
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Claims

1. A method for identifying a HLA-A*2402-restricted cryptic epitope in an antigen, comprising a step of selecting, in said antigen, a peptide of 8 to 12 amino acids having a tyrosine in position 2, with the proviso that the peptide does not have, simultaneously, a positively charged amino acid (lysine or arginine) in position 1 and a leucine or an isoleucine or a phenylalanine in C-terminal position.
2. The method of claim 1, further comprising a step of testing, in an appropriate model, the immunogenicity of the peptide selected by the method of claim 1, and selecting said peptide if it is non-immunogenic.
3. A method for increasing the immunogenicity of a (putative) HLA-
A*2402-restricted cryptic epitope, comprising a step of substituting the N-terminal residue of said epitope with an arginine or a lysine, and/or a step of substituting the C-terminal residue of said epitope with a leucine or an isoleucine or a phenylalanine, preferentially with a leucine.
4. A method for obtaining a HLA-A*2402-restricted epitope able to trigger an immune response against a HLA-A* 2402-restricted cryptic epitope of an antigen, comprising the steps of
(i) identifying, in said antigen, one or several native (putative) HLA- A* 2402-restricted cryptic epitopes, by the method according to claim 1; (ii) testing the immunogenicity of each native epitope selected in step (i), in an appropriate model, and selecting those which are non-immunogenic;
(iii) for each native epitope selected in step (ii), obtaining an optimized epitope by increasing its immunogenicity, by the method according to claim 3;
(iv) testing the immunogenicity of each optimized epitope obtained in step (iii), in an appropriate model, and selecting those which are immunogenic;
(v) for each epitope selected in step (iv), testing if the CTLs generated against the optimized epitope also recognize its cognate native epitope, and selecting those for which the test is positive.
5. An isolated peptide consisting of a cryptic HLA-A*2402-restricted epitope, wherein said isolated peptide is selected in the group consisting of PYGVLLKTH (SEQ ID NO: 1); PYMRQFVAH (SEQ ID NO: 2); PYVSRLLGI (SEQ ID NO: 3); PYGKGWDLM (SEQ ID NO: 4); TYLVQVQAL (SEQ ID NO: 5); PYWELSNHE (SEQ ID NO: 6); PYDGIPARE (SEQ ID No: 7); RYEFLWGPR (SEQ ID No: 8) and PYNYLSTDV (SEQ ID No: 9).
6. An isolated peptide consisting of an immunogenic HLA-A*2402- restricted epitope derived from a cryptic HLA-A* 2402-restricted epitope according to claim 4 by the method according to claim 2, wherein said isolated peptide is selected in the group consisting of KYGVLLKTL (SEQ ID No: 11); RYMRQFVAL (SEQ ID No: 12); RYVSRLLGI (SEQ ID No: 13); RYGKGWDLL (SEQ ID No: 14); RYLVQVQAL (SEQ ID No: 15); and RYWELSNHL (SEQ ID No: 16).
7. A chimeric polypeptide, comprising one, two, three or more HLA- A*2402-restricted cryptic epitopes according to claim 4.
8. A chimeric polypeptide, comprising one, two, three or more immunogenic HLA-A* 2402-restricted epitopes according to claim 5.
9. An isolated nucleic acid molecule designed to cause the expression of a cryptic HLA-A*2402-restricted epitope according to claim 5, an immunogenic epitope according to claim 6, or a chimeric polypeptide according to claim 7 or claim 8.
10. A pharmaceutical composition comprising at least, as an active principle, an HLA-A*2402-restricted cryptic epitope according to claim 5, or an immunogenic epitope polypeptide according to claim 6, or a chimeric polypeptide according to claim 7 or claim 8, or a nucleic acid according to claim 9.
11. The pharmaceutical composition of claim 10, which is a vaccine.
12. A kit of parts comprising, in separate containers:
(i) a first peptide comprising a sequence of a HLA-A* 2402-restricted cryptic epitope, and
(ii) a second peptide comprising a sequence consisting of a HLA- A* 2402-restricted immunogenic epitope derived from the HLA-A* 2402-restricted cryptic epitope recited in (i) by a method according to claim 3.
13. The kit according to claim 12, wherein said first peptide is an isolated cryptic epitope according to claim 5, and said second peptide is its cognate immunogenic epitope as recited in claim 6.
14. The kit according to claim 12, wherein said first peptide is a chimeric polypeptide comprising one, two, three or more HLA-A* 2402-restricted cryptic epitopes, and/or said second peptide is a chimeric polypeptide comprising one, two, three or more HLA-A* 2402-restricted immunogenic epitopes, wherein at least one immunogenic epitope comprised in the second peptide is cognate to at least one HLA-A*2402-restricted cryptic epitope comprised in the first peptide.
15. The kit according to claim 14, wherein said first peptide is a chimeric polypeptide according to claim 7, and said second peptide is a chimeric polypeptide according claim 8.
16. The kit according to any of claims 12 to 15, which is a vaccination kit, wherein said first and second peptides or chimeric polypeptides are in separate vaccination doses.
17. An isolated peptide according to claim 5 or claim 6, or a chimeric polypeptide according to claim 7 or claim 8, or a nucleic acid according to claim 9, for use as a medicament for preventive or curative immunotherapy.
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