Source: http://www.patentsencyclopedia.com/app/20120082692
Timestamp: 2018-04-24 16:45:03
Document Index: 791925200

Matched Legal Cases: ['ART-1', 'art-127', 'art-127', 'art126', 'art126', 'ART-1']

Identification, Optimization and Use of Cryptic HLA-A24 Epitopes for Immunotherapy - Patent application
Patent application title: Identification, Optimization and Use of Cryptic HLA-A24 Epitopes for Immunotherapy
Inventors: Kostantinos(kostas) Kosmatopoulos (Paris, FR) Jeanne Menez-Jamet (Montrouge, FR)
Assignees: VAXON BIOTECH
Patent application number: 20120082692
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.
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 5.
8. A chimeric polypeptide, comprising one, two, three or more immunogenic HLA-A*2402-restricted epitopes according to claim 6.
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 claim 12, which is a vaccination kit, wherein said first and second peptides or chimeric polypeptides are in separate vaccination doses.
[0001] 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.
[0002] 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), which play a major role in the elimination of tumor cells.
[0003] 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: [0004] cytosolic degradation of the antigen by a multienzyme complex called proteasome, [0005] translocation of the peptides derived from this degradation in the endoplasmic reticulum (ER) by the TAP transporters, [0006] association of these peptides with the MHC I molecules and exportation of the peptide/MHC I complexes to the cell surface.
[0007] 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.
[0008] 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.
[0009] 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).
[0010] 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.
[0011] 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).
[0012] 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).
[0013] 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.
[0014] 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).
[0015] 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.
[0016] Several tumor immunogenic peptides presented by HLA-A*2402 have been described to date (table 1).
TABLE-US-00001 TABLE 1 Tumor immunogenic HLA-A24 T cell epitopes 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 EYLSLSDKI 174 MAGE-A2 EYLQLVFGI 175 MAGE-A3 TFPDLESEF 176 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 186 PRAME LYVDSLFFL 187 PSMA NYARTEDFF 188 RNF43 NSQPVWLCL 189 WT1 CMTWNQMNL 190
[0017] 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.
[0018] 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 X1YX2X3X4X5X6X7X8X.sub.9X10X- 11 (SEQ ID No: 20), wherein X1 to X6 are any amino acid, X7 to X10 are any amino acid or none, and X11≠L or F or I if X1=R or K.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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 L, 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 X1YX2X3X4X5X6X7X8X.sub.9X10L (SEQ ID No: 21), wherein X1 is any amino acid but R or K, X2 to X6 are any amino acid, and X7 to X10 are any amino acid or none, the substitution of X1 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 X1YX2X3X4X5X6X7X8X.sub.9X10X- 11 (SEQ ID No: 22), wherein X1 is any amino acid but R or K, X2 to X6 are any amino acid, and X7 to X10 are any amino acid or none, and X11 is not an unfavourable amino acids (D or E or G or H or P or Q or R or K), the substitution of X1 by R or K can be sufficient to increase its immunogenicity.
[0027] 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.
[0028] 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
[0029] (i) identifying, in said antigen, one or several native putative HLA-A*2402-restricted cryptic epitopes, by the method according to claim 1;
[0030] (ii) testing the immunogenicity of each native epitope selected in step (i), in an appropriate model, and selecting those which are non-immunogenic;
[0031] (iii) for each native epitope selected in step (ii), obtaining an optimized epitope by increasing its immunogenicity, by the method as above-described;
[0032] (iv) testing the immunogenicity of each optimized epitope obtained in step (iii), in an appropriate model, and selecting those which are immunogenic;
[0033] (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.
[0034] 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.
[0035] 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.
TABLE-US-00002 TABLE 2 Selected confirmed cryptic HLA-A*2402 restricted peptides Peptide Sequence SEQ ID TERT 403 PYGVLLKTH ID No 1 TERT 770 PYMRQFVAH ID No 2 HER 780 PYVSRLLGI ID No 3 EphA2 47 PYGKGWDLM ID No 4 EphA2 502 TYLVQVQAL ID No 5 EphA2 817 PYWELSNHE ID No 6 Her2/neu 922 PYDGIPARE ID No 7 MAGE 261 RYEFLWGPR ID No 8 Her2/neu 300 PYNYLSTDV ID No 9
[0036] 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.
[0037] 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/neu402Y 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/neu402Y 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.
[0038] 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.
[0039] 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.
[0040] 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-1, TRP-2, WT1, EphA1, EphA2, EphA3, EphA4, G250/MN/CAIX, STEAP, alphafoetoprotein, RAGE-1, PAGE-1. 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.
[0041] 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.
TABLE-US-00003 TABLE 3 epitopes which can be combined to HLA-A*2402- restricted epitopes in chimeric polypeptides according to the invention HLA-A*0201 Native peptide Optimized peptide Antigen Sequence No Name Sequence No Mart-127 AAGIGILTV 23 Mart-127Y1 YAGIGILTV 24 Mart126 EAAGIGILTV 25 Mart126L27 ELAGIGILTV 26 Gp100177 AMLGTHTMEV 27 Gp100177Y1 YMLGTHTMEV 28 Gp100178 MLGTHTMEV 29 Gp100178Y1 YLGTHTMEV 30 Gp100154 KTWGQYWQV 31 Gp100154Y1 YTWGQYWQV 32 Gp100154M155 KMWGQYWQV 33 Gp100570 SLADTNSLAV 34 Gp100570Y1 YLADTNSLAV 35 Gp100209 TDQVPFSV 36 Gp100209Y1 YDQVPFSV 37 Gp100209M210 YMQVPFSV 38 Gp100476 VLYRYGSFSV 39 Gp100476Y1 YLYRYGSFSV 40 Gp100457 LLDGTATLRL 41 Gp100457Y1 YLDGTATLRL 42 HER-2/neu799 QLMPYGCLL 43 HER-2/neu799Y1 YLMPYGCLL 44 HER-2/neu369 KIFGSLAFL 45 HER-2/neu369Y1 YIFGSLAFL 46 HER-2/neu789 CLTSTVQLV 47 HER-2/neu789Y1 YLTSTVQLV 48 HER-2/neu48 HLYQGCQW 49 HER-2/neu48Y1 YLYQGCQW 50 HER-2/neu773 VMAGVGSPYV 51 HER-2/neu773Y1 YMAGVGSPYV 52 HER-2/neu5 ALCRWGLL 53 HER-2/neu5Y1 YLCRWGLL 54 HER-2/neu851 VLVKSPNHV 55 HER-2/neu851Y1 YLVKSPNHV 56 HER-2/neu661 ILLVVVLGV 57 HER-2/neu661Y1 YLLVVVLGV 58 HER-2/neu650 PLTSIISAV 59 HER-2/neu650Y1 YLTSIISAV 60 HER-2/neu466 ALIHHNTHL 61 HER-2/neu466Y1 YLIHHNTHL 62 HER-2/neu402 TLEEITGYL 63 HER-2/neu402Y1 YLEEITGYL 64 HER-2/neu391 PLQPEQLQV 65 HER-2/neu391Y1 YLQPEQLQV 66 HER-2/neu971 ELVSEFSRM 67 HER-2/neu971Y1 YLVSEFSRM 68 EphA261 DMPIYMYSV 69 EphA261Y1 YMPIYMYSV 70 HER2911 TVWELMTFGA 71 HER911Y1V10 YVWELMTFGV 74 HER4911 TIWELMTFGG 72 HER1911 TVWELMTFGS 73 HER2722 KVKVLGSGA 75 HER722Y1V9 YVKVLGSGV 79 HER3722 KLKVLGSGV 76 HER4722 RVKVLGSGA 77 HER1722 KIKVLGSGA 78 HER2845 DLAARNVLV 80 HER845Y1 YLAARNVLV 82 HER3845 NLAARNVLL 81 HER2904 DVWSYGVTV 83 HER904Y1 YVWSYGVTV 85 HER4904 DVWSYGVTI 84 HER2933 DLLEKGERL 86 HER933Y1 YLLEKGERL 88 HER1933 SILELKGERL 87 HER2945 PICTIDVYMI 89 HER945Y1 YICTIDVYMV 93 HER3945 QICTIDVYMV 90 HER4945 PICTIDVYMV 91 HER1945 PICTIDVYKI 92 MAGE-A248G9 YLEYRQVPG 94 MAGE-A248V9 YLEYRQVPV 96 MAGE-A248D9 YLEYRQVPD 95 TERT988 DLQVNSLQTV 97 TERT988Y1 YLQVNSLQTV 98 TERT572 RLFFYRKSV 99 TERT572Y1 YLFFYRKSV 100 HLA-B*0702 Native peptide Optimized peptide Name Sequence No Name Sequence No TERT444 FPRRLVQLL 101 TERT444A1 APRRLVQLL 102 CEA188/554 SPRLQLSNG 103 CEA188/554L9 SPRLQLSNL 104 HER-2/neu1069 APRSPLAPS 105 HER-2/neu1069L9 APRSPLAPL 106 HER-2/neu870 SPKANKEIL 107 HER-2/neu760A1 APKANKEIL 108 HER-2/neu246 GPKHSDCLA 109 HER-2/neu246A1 APKHSDCLA 110
[0042] 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).
[0043] 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.
[0044] 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.).
[0045] 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 anti-cancer immunotherapy.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] The invention also pertains to a kit of parts comprising, in separate formulations or containers (vials, tubes, etc.):
[0051] (i) a first peptide comprising a sequence of a HLA-A*2402-restricted cryptic epitope, and
[0052] (ii) a second peptide comprising a sequence corresponding to an optimized immunogenic epitope cognate to the cryptic epitope recited in (i).
[0053] 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).
[0054] 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.
[0055] 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.
[0056] As mentioned above, a polyallelic 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:
[0057] (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
[0058] (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).
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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 ISA51. 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.
[0063] The invention is further illustrated by the following figures and examples.
[0064] FIG. 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)×100. Four CTL dilutions, corresponding to four CTL/target cells ratio were tested.
[0065] FIG. 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)×100. Four CTL dilutions, corresponding to four CTL/target cells ratio were tested.
[0066] The examples have been performed using the following materials and methods:esd
[0067] 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.sup.- H2 Db.sup.- knock out mice, transgenic for both human β2 microglobulin and CD8α chain (Perarnau et al., 1999).
[0068] Peptides. Peptides were synthesized by Epytop (N mes, France).
[0069] 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). All cell lines were grown in FCS 10% supplemented RPMI1640 culture medium.
[0070] 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).
[0071] 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 HBVcore128 T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 112). After 15 days, 5×107 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.
[0072] 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, 5×107 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.
[0073] Cytotoxic assay. Targets were labelled with 100 μCi of Cr51 for 60 min, plated in 96-well V-bottomed plates (3×103 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)×100.
Affinity and Immunogenicity of Selected Cryptic Peptides
[0074] 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.
TABLE-US-00004 TABLE 4 HLA-A*2402 affinity of cryptic peptides. Antigen/position Sequence RA SEQ ID No TERT 403 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 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
[0075] 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 (FIG. 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.
[0076] 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.
TABLE-US-00005 TABLE 5 HLA-A*2402 immunogenicity of selected cryptic peptides. 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 (-) means that none of the mice vaccinated with the corresponding native peptides developes a specific immune response, (+) that less to 50% of vaccinated mice responded, (++) that more that 50% responded. ND: not determined
Enhancement of Immunogenicity of the Selected Cryptic Peptides
[0077] 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.
[0078] 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)).
TABLE-US-00006 TABLE 6 Tumor and HIV derived HLA-A*2402 restricted epitopes Antigen Sequence No reference Beta-catenin SYLDSGIHF 113 http://www.cancerimmunity.org/peptidedatabase/mutation.htm HM-HN-1 NYNNFYRFL 114 http://www.cancerimmunity.org/peptidedatabase/tumorspecific.htm KM-HN-1 EYSKECLKEF 115 http://www.cancerimmunity.org/peptidedatabase/differentiation.htm KM-HN-1 EYLSLSDKI 116 http.//www.cancerimmunity.org/peptidedatabase/overexpressed.htm MAGE-A2 EYLQLVFGI 117 MAGE-A3 VAELVHFLL 119 MAGE-A4 NYKRVFPVI 120 SAGE LYATVIHDI 121 CEA QYSWFVNGTF 122 CEA TYACFVSNL 123 gp100/Pmel17 VYFFLPDHL 124 OA1 LYSACFWWL 125 tyrosinase AFLPWHRLF 126 Ep-CAM RYQLDPKFI 127 Her2/neu TYLPTNASL 128 PRAME LYVDSLFFL 129 PSMA NYARTEDFF 130 RNF43 NSQPVWLCL 131 TERT TYVPLLGSL 132 Ref peptides TERT TERT CYGDMENKL 133 TERT AVQVCGPPL 134 WT1 CMTWNQMNL 135 p17 HYMLKHLVW 136 http://hiv-web.lanl.gov/content/immunology/tables/ctl_summary.html p17 KYKLKHIVW 137 p17 LYNTVATL 138 p17 LYCVHQKI 139 p17-p24 NYPIVQNL 140 p24 EIYKRWIIL 141 p24 IYKRWIIL 142 p24 IYKRWIILGL 143 p2p7p1p6 LYPLASLRSL 144 RT DAYFSVPL 145 RT VYYDPSKDL 146 RT IYQEPFKNL 147 Integrase GYIEAEVI 148 gp160 LFCASDAKAY 149 gp160 RYLRDQQL 150 gp160 RYLKDQQLL 151 gp160 RYLRDQQLL 152 gp160 RYLRDQQLLGI 153 gp160 YLKDQQLL 154 gp160 YLRDQQLL 155 gp160 WYIKIFIMI 156 gp160 SYRRLRDLL 157 Nef TYKAAVDL 158 Nef HSQRRQDIL 159 Nef RQDILDLWI 160 Nef GYFPDWQNY 161 Nef NYTPGPGVRY 162 Nef RYPLTFGW 163 Nef RYPLTFGWCF 164 Nef FYPLTFGWCY 165 Nef DSRLAFHHM 166 Nef AFHHVAREL 167
[0079] Optimized peptides were tested for their immunogenicity (table 7, FIG. 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 770R1L9, HER 780R1, EphA2 47R1L9, EphA2 502R1 and EphA2 817R1L9 peptides, developed peptide specific CTLs.
[0080] Importantly, CTLs generated in mice vaccinated with optimized peptides recognized target cells loaded with the corresponding native peptide (FIG. 2).
TABLE-US-00007 TABLE 7 Native and modified peptides immunogenicity and native peptide cross recognition. Native peptide cross Antigen/position Modification Sequence Immunogenicity recognition SEQ ID No TERT 403 K1L9 PYGVLLKTH -(0/3) +(3/15) 1 TERT 403 KYGVLLKTL +(4/15) 11 TERT 770 R1L9 PYMRQFVAH -(0/3) +(5/18) 2 TERT 770 RYMRQFVAL ++(12/18) 12 HER 780 R1 PYVSRLLGI -(0/8) +(3/9) 3 HER 780 RYVSRLLGI +(4/9) 13 EphA2 47 R1L9 PYGKGWDLM -(0/6) ++(7/9) 4 EphA2 47 RYGKGWDLL ++(7/9) 14 EphA2 502 R1 TYLVQVQAL -(0/3) ++(2/3) 5 EphA2 502 RYLVQVQAL ++(3/3) 15 EphA2 817 R1L9 PYSELSNHE -(0/3) ++(2/3) 6 EphA2 817 RYWELSNHL ++(2/3) 16 Her2/neu 922 R1L9 PYDGIPARE ND 7 Her2/neu 922 RYDGIPARL -(0/9) 17 MAGE 261 L9 RYEFLWGPR -(0/3) 8 MAGE 261 RYEFLWGPL -(0/9) 18 Her2/neu 300 R1L9 PYNYLSTDV -(0/3) 9 Her2/neu 300 RYNYLSTDL -(0/9) 19 (-) 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. (X/Y) means that X mice develop a specific response for a total of Y mice vaccinated. ND: not determined.
[0081] 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.
[0082] 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.
[0083] Bakker, A. B., van der Burg, S. H., Huijbens, R. J., Drijfhout, J. W., Melief, C. J., Adema, G. J. and Figdor, C. G. (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. [0084] 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. [0085] 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 Nad Acad Sci USA, 89, 416-420. [0086] Engelhorn, M. E., Guevara-Patino, J. A., Noffz, G., Hooper, A. T., Lou, O., Gold, J. S., Kappel, B. J. and Houghton, A. N. (2006) Autoimmunity and tumor immunity induced by immune responses to mutations in self. Nat Med, 12, 198-206. [0087] Gross, D. A., Graff-Dubois, S., Opolon, P., Cornet, S., Alves, P., Bennaceur-Griscelli, A., Faure, O, Guillaume, 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, 113, 425-433. [0088] Miyahara, Y., Naota, H., Wang, L., Hiasa, A., Goto, M., Watanabe, M., 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 Cancer Res, 11, 5581-5589. [0089] 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. [0090] Parkhurst, M. R., Salgaller, M. L., Southwood, S., Robbins, P. F., Sette, A., Rosenberg, S. A. and Kawakami, Y. (1996) Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues. J Immunol, 157, 2539-2548. [0091] Paterson, Y. and Maciag, P. C. (2005) Listeria-based vaccines for cancer treatment. Curr Opin Mol Ther, 7, 454-460. [0092] Perarnau, B., Saron, M. F., San Martin, B. R., Bervas, N., Ong, H., Soloski, M. J., Smith, A. G., Ure, J. M., Gairin, J. E. and Lemonnier, F. A. (1999) Single H2 Kb, H2 Db and double H2 KbDb knockout mice: peripheral CD8+ T cell repertoire and anti-lymphocytic choriomeningitis virus cytolytic responses. Eur J Immunol, 29, 1243-1252. [0093] Rohrlich, P. S., Cardinaud, S., Firat, H., Lamari, M., Briand, P., Escriou, N. and Lemonnier, F. A. (2003) HLA-B*0702 transgenic, H-2 KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus. Int Immunol, 15, 765-772. [0094] Rosenberg, S. A., Yang, J. C. and Restifo, N. P. (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med, 10, 909-915. [0095] Ruppert, J., Sidney, J., Celis, E., Kubo, R. T., Grey, H. M. and Sette, A. (1993) Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell, 74, 929-937. [0096] Scardino, A., Gross, D. A., Alves, P., Schultze, J. L., Graff-Dubois, S., 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. [0097] Tourdot, S. and Gould, K. G. (2002) Competition between MHC class I alleles for cell surface expression alters CTL responses to influenza A virus. J Immunol, 169, 5615-5621. [0098] Valmori, D., Gervois, N., Rimoldi, D., Fonteneau, J. F., Bonelo, A., Lienard, D., Rivoltini, L., Jotereau, F., Cerottini, J. C. and Romero, P. (1998) Diversity of the fine specificity displayed by HLA-A*0201-restricted CTL specific for the immunodominant Melan-AIMART-1 antigenic peptide. J Immunol, 161, 6956-6962. [0099] 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.
19019PRTArtificialCryptic HLA-A2402 restricted peptide 1Pro Tyr Gly Val Leu Leu Lys Thr His1 529PRTArtificialCryptic HLA-A2402 restricted peptide 2Pro Tyr Met Arg Gln Phe Val Ala His1 539PRTArtificialCryptic HLA-A2402 restricted peptide 3Pro Tyr Val Ser Arg Leu Leu Gly Ile1 549PRTArtificialCryptic HLA-A2402 restricted peptide 4Pro Tyr Gly Lys Gly Trp Asp Leu Met1 559PRTArtificialCryptic HLA-A2402 restricted peptide 5Thr Tyr Leu Val Gln Val Gln Ala Leu1 569PRTArtificialCryptic HLA-A2402 restricted peptide 6Pro Tyr Trp Glu Leu Ser Asn His Glu1 579PRTArtificialCryptic HLA-A2402 restricted peptide 7Pro Tyr Asp Gly Ile Pro Ala Arg Glu1 589PRTArtificialCryptic HLA-A2402 restricted peptide 8Arg Tyr Glu Phe Leu Trp Gly Pro Arg1 599PRTArtificialCryptic HLA-A2402 restricted peptide 9Pro Tyr Asn Tyr Leu Ser Thr Asp Val1 5109PRTArtificialCryptic HLA-A2402 restricted peptide 10Pro Tyr Gly Cys Leu Leu Asp His Val1 5119PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 11Lys Tyr Gly Val Leu Leu Lys Thr Leu1 5129PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 12Arg Tyr Met Arg Gln Phe Val Ala Leu1 5139PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 13Arg Tyr Val Ser Arg Leu Leu Gly Ile1 5149PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 14Arg Tyr Gly Lys Gly Trp Asp Leu Leu1 5159PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 15Arg Tyr Leu Val Gln Val Gln Ala Leu1 5169PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 16Arg Tyr Trp Glu Leu Ser Asn His Leu1 5179PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 17Arg Tyr Asp Gly Ile Pro Ala Arg Leu1 5189PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 18Arg Tyr Glu Phe Leu Trp Gly Pro Leu1 5199PRTArtificialOptimized peptide derived from cryptic peptides of SEQ ID Nos 1 to 9 19Arg Tyr Asn Tyr Leu Ser Thr Asp Leu1 52012PRTArtificialCryptic HLA-A2402 restricted peptide 20Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 102112PRTArtificialCryptic HLA-A2402 restricted peptide 21Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu1 5 102212PRTArtificialCryptic HLA-A2402 restricted peptide 22Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10239PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 23Ala Ala Gly Ile Gly Ile Leu Thr Val1 5249PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 24Tyr Ala Gly Ile Gly Ile Leu Thr Val1 52510PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 25Glu Ala Ala Gly Ile Gly Ile Leu Thr Val1 5 102610PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 26Glu Leu Ala Gly Ile Gly Ile Leu Thr Val1 5 102710PRTArtificialpaptide 27Ala Met Leu Gly Thr His Thr Met Glu Val1 5 102810PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 28Tyr Met Leu Gly Thr His Thr Met Glu Val1 5 10299PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 29Met Leu Gly Thr His Thr Met Glu Val1 5309PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 30Tyr Leu Gly Thr His Thr Met Glu Val1 5319PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 31Lys Thr Trp Gly Gln Tyr Trp Gln Val1 5329PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 32Tyr Thr Trp Gly Gln Tyr Trp Gln Val1 5339PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 33Lys Met Trp Gly Gln Tyr Trp Gln Val1 53410PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 34Ser Leu Ala Asp Thr Asn Ser Leu Ala Val1 5 103510PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 35Tyr Leu Ala Asp Thr Asn Ser Leu Ala Val1 5 10368PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 36Thr Asp Gln Val Pro Phe Ser Val1 5378PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 37Tyr Asp Gln Val Pro Phe Ser Val1 5388PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 38Tyr Met Gln Val Pro Phe Ser Val1 53910PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 39Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val1 5 104010PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 40Tyr Leu Tyr Arg Tyr Gly Ser Phe Ser Val1 5 104110PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 41Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu1 5 104210PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 42Tyr Leu Asp Gly Thr Ala Thr Leu Arg Leu1 5 10439PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 43Gln Leu Met Pro Tyr Gly Cys Leu Leu1 5449PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 44Tyr Leu Met Pro Tyr Gly Cys Leu Leu1 5459PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 45Lys Ile Phe Gly Ser Leu Ala Phe Leu1 5469PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 46Tyr Ile Phe Gly Ser Leu Ala Phe Leu1 5479PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 47Cys Leu Thr Ser Thr Val Gln Leu Val1 5489PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 48Tyr Leu Thr Ser Thr Val Gln Leu Val1 5498PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 49His Leu Tyr Gln Gly Cys Gln Trp1 5508PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 50Tyr Leu Tyr Gln Gly Cys Gln Trp1 55110PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 51Val Met Ala Gly Val Gly Ser Pro Tyr Val1 5 105210PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 52Tyr Met Ala Gly Val Gly Ser Pro Tyr Val1 5 10538PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 53Ala Leu Cys Arg Trp Gly Leu Leu1 5548PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 54Tyr Leu Cys Arg Trp Gly Leu Leu1 5559PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 55Val Leu Val Lys Ser Pro Asn His Val1 5569PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 56Tyr Leu Val Lys Ser Pro Asn His Val1 5579PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 57Ile Leu Leu Val Val Val Leu Gly Val1 5589PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 58Tyr Leu Leu Val Val Val Leu Gly Val1 5599PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 59Pro Leu Thr Ser Ile Ile Ser Ala Val1 5609PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 60Tyr Leu Thr Ser Ile Ile Ser Ala Val1 5619PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 61Ala Leu Ile His His Asn Thr His Leu1 5629PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 62Tyr Leu Ile His His Asn Thr His Leu1 5639PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 63Thr Leu Glu Glu Ile Thr Gly Tyr Leu1 5649PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 64Tyr Leu Glu Glu Ile Thr Gly Tyr Leu1 5659PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 65Pro Leu Gln Pro Glu Gln Leu Gln Val1 5669PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 66Tyr Leu Gln Pro Glu Gln Leu Gln Val1 5679PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 67Glu Leu Val Ser Glu Phe Ser Arg Met1 5689PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 68Tyr Leu Val Ser Glu Phe Ser Arg Met1 5699PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 69Asp Met Pro Ile Tyr Met Tyr Ser Val1 5709PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 70Tyr Met Pro Ile Tyr Met Tyr Ser Val1 57110PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 71Thr Val Trp Glu Leu Met Thr Phe Gly Ala1 5 107210PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 72Thr Ile Trp Glu Leu Met Thr Phe Gly Gly1 5 107310PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 73Thr Val Trp Glu Leu Met Thr Phe Gly Ser1 5 107410PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 74Tyr Val Trp Glu Leu Met Thr Phe Gly Val1 5 10759PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 75Lys Val Lys Val Leu Gly Ser Gly Ala1 5769PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 76Lys Leu Lys Val Leu Gly Ser Gly Val1 5779PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 77Arg Val Lys Val Leu Gly Ser Gly Ala1 5789PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 78Lys Ile Lys Val Leu Gly Ser Gly Ala1 5799PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 79Tyr Val Lys Val Leu Gly Ser Gly Val1 5809PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 80Asp Leu Ala Ala Arg Asn Val Leu Val1 5819PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 81Asn Leu Ala Ala Arg Asn Val Leu Leu1 5829PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 82Tyr Leu Ala Ala Arg Asn Val Leu Val1 5839PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 83Asp Val Trp Ser Tyr Gly Val Thr Val1 5849PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 84Asp Val Trp Ser Tyr Gly Val Thr Ile1 5859PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 85Tyr Val Trp Ser Tyr Gly Val Thr Val1 5869PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 86Asp Leu Leu Glu Lys Gly Glu Arg Leu1 58710PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 87Ser Ile Leu Glu Leu Lys Gly Glu Arg Leu1 5 10889PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 88Tyr Leu Leu Glu Lys Gly Glu Arg Leu1 58910PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 89Pro Ile Cys Thr Ile Asp Val Tyr Met Ile1 5 109010PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 90Gln Ile Cys Thr Ile Asp Val Tyr Met Val1 5 109110PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 91Pro Ile Cys Thr Ile Asp Val Tyr Met Val1 5 109210PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 92Pro Ile Cys Thr Ile Asp Val Tyr Lys Ile1 5 109310PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 93Tyr Ile Cys Thr Ile Asp Val Tyr Met Val1 5 10949PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 94Tyr Leu Glu Tyr Arg Gln Val Pro Gly1 5959PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 95Tyr Leu Glu Tyr Arg Gln Val Pro Asp1 5969PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 96Tyr Leu Glu Tyr Arg Gln Val Pro Val1 59710PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 97Asp Leu Gln Val Asn Ser Leu Gln Thr Val1 5 109810PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 98Tyr Leu Gln Val Asn Ser Leu Gln Thr Val1 5 10999PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 99Arg Leu Phe Phe Tyr Arg Lys Ser Val1 51009PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 100Tyr Leu Phe Phe Tyr Arg Lys Ser Val1 51019PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 101Asp Pro Arg Arg Leu Val Gln Leu Leu1 51029PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 102Ala Pro Arg Arg Leu Val Gln Leu Leu1 51039PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 103Ser Pro Arg Leu Gln Leu Ser Asn Gly1 51049PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 104Ser Pro Arg Leu Gln Leu Ser Asn Leu1 51059PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 105Ala Pro Arg Ser Pro Leu Ala Pro Ser1 51069PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 106Ala Pro Arg Ser Pro Leu Ala Pro Leu1 51079PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 107Ser Pro Lys Ala Asn Lys Glu Ile Leu1 51089PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 108Ala Pro Lys Ala Asn Lys Glu Ile Leu1 51099PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 109Gly Pro Lys His Ser Asp Cys Leu Ala1 51109PRTArtificialEpitope which can be combined to HLA-A2402-restricted epitope 110Ala Pro Lys His Ser Asp Cys Leu Ala1 51119PRTArtificialReference peptide standard A24 111Ala Tyr Ile Asp Asn Tyr Asn Lys Phe1 511213PRTArtificialI-Ab restricted HBVcore128 T helper epitope 112Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu1 5 101139PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 113Ser Tyr Leu Asp Ser Gly Ile His Phe1 51149PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 114Asn Tyr Asn Asn Phe Tyr Arg Phe Leu1 511510PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 115Glu Tyr Ser Lys Glu Cys Leu Lys Glu Phe1 5 101169PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 116Glu Tyr Leu Ser Leu Ser Asp Lys Ile1 51179PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 117Glu Tyr Leu Gln Leu Val Phe Gly Ile1 51189PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 118Thr Phe Pro Asp Leu Glu Ser Glu Phe1 51199PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 119Val Ala Glu Leu Val His Phe Leu Leu1 51209PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 120Asn Tyr Lys Arg Cys Phe Pro Val Ile1 51219PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 121Leu Tyr Ala Thr Val Ile His Asp Ile1 512210PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 122Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe1 5 101239PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 123Thr Tyr Ala Cys Phe Val Ser Asn Leu1
51249PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 124Val Tyr Phe Phe Leu Pro Asp His Leu1 51259PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 125Leu Tyr Ser Ala Cys Phe Trp Trp Leu1 51269PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 126Ala Phe Leu Pro Trp His Arg Leu Phe1 51279PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 127Arg Tyr Gln Leu Asp Pro Lys Phe Ile1 51289PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 128Thr Tyr Leu Pro Thr Asn Ala Ser Leu1 51299PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 129Leu Tyr Val Asp Ser Leu Phe Phe Leu1 51309PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 130Asn Tyr Ala Arg Thr Glu Asp Phe Phe1 51319PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 131Asn Ser Gln Pro Val Trp Leu Cys Leu1 51329PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 132Thr Tyr Val Pro Leu Leu Gly Ser Leu1 51339PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 133Cys Tyr Gly Asp Met Glu Asn Lys Leu1 51349PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 134Ala Val Gln Val Cys Gly Pro Pro Leu1 51359PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 135Cys Met Thr Trp Asn Gln Met Asn Leu1 51369PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 136His Tyr Met Leu Lys His Leu Val Trp1 51379PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 137Lys Tyr Lys Leu Lys His Ile Val Trp1 51388PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 138Leu Tyr Asn Thr Val Ala Thr Leu1 51398PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 139Leu Tyr Cys Val His Gln Lys Ile1 51408PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 140Asn Tyr Pro Ile Val Gln Asn Leu1 51419PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 141Glu Ile Tyr Lys Arg Trp Ile Ile Leu1 51428PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 142Ile Tyr Lys Arg Trp Ile Ile Leu1 514310PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 143Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu1 5 1014410PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 144Leu Tyr Pro Leu Ala Ser Leu Arg Ser Leu1 5 101458PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 145Asp Ala Tyr Phe Ser Val Pro Leu1 51469PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 146Val Tyr Tyr Asp Pro Ser Lys Asp Leu1 51479PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 147Ile Tyr Gln Glu Pro Phe Lys Asn Leu1 51488PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 148Gly Tyr Ile Glu Ala Glu Val Ile1 514910PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 149Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr1 5 101508PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 150Arg Tyr Leu Arg Asp Gln Gln Leu1 51519PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 151Arg Tyr Leu Lys Asp Gln Gln Leu Leu1 51529PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 152Arg Tyr Leu Arg Asp Gln Gln Leu Leu1 515311PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 153Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile1 5 101548PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 154Tyr Leu Lys Asp Gln Gln Leu Leu1 51558PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 155Tyr Leu Arg Asp Gln Gln Leu Leu1 51569PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 156Trp Tyr Ile Lys Ile Phe Ile Met Ile1 51579PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 157Ser Tyr Arg Arg Leu Arg Asp Leu Leu1 51588PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 158Thr Tyr Lys Ala Ala Val Asp Leu1 51599PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 159His Ser Gln Arg Arg Gln Asp Ile Leu1 51609PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 160Arg Gln Asp Ile Leu Asp Leu Trp Ile1 51619PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 161Gly Tyr Phe Pro Asp Trp Gln Asn Tyr1 516210PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 162Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr1 5 101638PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 163Arg Tyr Pro Leu Thr Phe Gly Trp1 516410PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 164Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe1 5 1016510PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 165Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr1 5 101669PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 166Asp Ser Arg Leu Ala Phe His His Met1 51679PRTArtificialTumor and HIV derived HLA-A2402 restricted epitope 167Ala Phe His His Val Ala Arg Glu Leu1 51689PRTArtificialTumor immunogenic HLA- A24 T cell epitope 168Ser Tyr Leu Asp Ser Gly Ile His Phe1 51699PRTArtificialTumor immunogenic HLA- A24 T cell epitope 169Thr Tyr Val Pro Leu Leu Gly Ser Leu1 51709PRTArtificialTumor immunogenic HLA- A24 T cell epitope 170Cys Tyr Gly Asp Met Glu Asn Lys Leu1 51719PRTArtificialTumor immunogenic HLA- A24 T cell epitope 171Ala Val Gln Val Cys Gly Pro Pro Leu1 51729PRTArtificialTumor immunogenic HLA- A24 T cell epitope 172Asn Tyr Asn Asn Phe Tyr Arg Phe Leu1 517310PRTArtificialTumor immunogenic HLA- A24 T cell epitope 173Glu Tyr Ser Lys Glu Cys Leu Lys Glu Phe1 5 101749PRTArtificialTumor immunogenic HLA- A24 T cell epitope 174Glu Tyr Leu Ser Leu Ser Asp Lys Ile1 51759PRTArtificialTumor immunogenic HLA- A24 T cell epitope 175Glu Tyr Leu Gln Leu Val Phe Gly Ile1 51769PRTArtificialTumor immunogenic HLA- A24 T cell epitope 176Thr Phe Pro Asp Leu Glu Ser Glu Phe1 51779PRTArtificialTumor immunogenic HLA- A24 T cell epitope 177Val Ala Glu Leu Val His Phe Leu Leu1 51789PRTArtificialTumor immunogenic HLA- A24 T cell epitope 178Asn Tyr Lys Arg Cys Phe Pro Val Ile1 51799PRTArtificialTumor immunogenic HLA- A24 T cell epitope 179Leu Tyr Ala Thr Val Ile His Asp Ile1 518010PRTArtificialTumor immunogenic HLA- A24 T cell epitope 180Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe1 5 101819PRTArtificialTumor immunogenic HLA- A24 T cell epitope 181Thr Tyr Ala Cys Phe Val Ser Asn Leu1 51829PRTArtificialTumor immunogenic HLA- A24 T cell epitope 182Val Tyr Phe Phe Leu Pro Asp His Leu1 51839PRTArtificialTumor immunogenic HLA- A24 T cell epitope 183Leu Tyr Ser Ala Cys Phe Trp Trp Leu1 51849PRTArtificialTumor immunogenic HLA- A24 T cell epitope 184Ala Phe Leu Pro Trp His Arg Leu Phe1 51859PRTArtificialTumor immunogenic HLA- A24 T cell epitope 185Arg Tyr Gln Leu Asp Pro Lys Phe Ile1 51869PRTArtificialTumor immunogenic HLA- A24 T cell epitope 186Thr Tyr Leu Pro Thr Asn Ala Ser Leu1 51879PRTArtificialTumor immunogenic HLA- A24 T cell epitope 187Leu Tyr Val Asp Ser Leu Phe Phe Leu1 51889PRTArtificialTumor immunogenic HLA- A24 T cell epitope 188Asn Tyr Ala Arg Thr Glu Asp Phe Phe1 51899PRTArtificialTumor immunogenic HLA- A24 T cell epitope 189Asn Ser Gln Pro Val Trp Leu Cys Leu1 51909PRTArtificialTumor immunogenic HLA- A24 T cell epitope 190Cys Met Thr Trp Asn Gln Met Asn Leu1 5
Patent applications by Jeanne Menez-Jamet, Montrouge FR
20140296182 METHOD AND COMPOSITION FOR TREATING LICE INFESTATION
20140296181 METHODS OF MODULATING ONCOGENIC FUSION PROTEINS
20140296180 CYCLIC OLIGOSACCHARIDES FOR USE IN THE TREATMENT AND PREVENTION OF BACTERIAL INFECTION
20140296179 NUTRITIONAL COMPOSITION COMPRISING IMMUNOGLOBULINS AND OLIGOSACCHARIDES
20140296178 Combination Therapy with a Gallium Complex
2010-11-04 Production and use of epitope-tagged hepatitis c virus particle
2010-05-06 System and method for modulating and optimizing immunotherapy
2010-11-04 Flt4 (vegfr-3) as a target for tumor imaging and anti-tumor therapy
2010-11-04 Codon-optimized dna molecules encoding the receptor binding domains of clostridium difficile toxins a and b, and methods of use thereof
2010-10-21 Chitosan/carbon nanotube composite scaffolds for drug delivery
2013-10-10 Identification, optimization and use of cryptic hla-b7 epitopes for immunotherapy
2012-06-07 Identification, optimization and use of shared hla-b*0702 epitopes for immunotherapy
2011-10-20 Identification, optimization and use of cryptic hla-b7 epitopes for immunotherapy
2009-07-09 Polynucleotides encoding mhc class i-restricted htert epitopes, analogues thereof or polyepitopes