Immunogenic peptides of the cyclin B1 tumor antigen

Abstract

The present invention relates to peptides comprising at least one CD4.sup.+ T epitope, which is immunodominant in vitro, of the cyclin B1 tumor antigen, said peptides being capable of stimulating a specific human CD4.sup.+ T lymphocyte response in subjects who have varied HLA II molecules, and to the use of these peptides as a cancer vaccine and as a reagent for the diagnosis of cancer or the immunomonitoring of the cellular response against cyclin B1 during cancer or during an anticancer treatment.

Claims

1. A method of inducing a cyclin B1-specific human CD4.sup.+ T lymphocyte response in a subject, the method comprising administering a peptide to the subject, the said peptide consisting of a sequence of from 11 to 30 amino acids which is derived from the human cyclin B1 sequence of SEQ ID NO: 1 and which comprises at least one human cyclin B1 CD4.sup.+ T epitope which is immunodominant in vitro, said peptide being selected from the group consisting of: a) the sequences of 11 to 30 consecutive amino acids of the human cyclin B1 sequence of SEQ ID NO: 1 comprising at least residues 3 to 11, 19 to 27, 376 to 384, 27 to 35, 120 to 128, 282 to 290, 287 to 295, 305 to 313, 317 to 325, 322 to 330, 325 to 333, 379 to 387, 412 to 420, 418 to 426 or 422 to 430 of said sequence SEQ ID NO: 1, and b) the sequences of 11 to 30 amino acids having at least 70% identity with a sequence in a), with the exclusion of the sequence of 15 amino acids made up of residues 279 to 293 of said sequence SEQ ID NO: 1, said peptide being capable of stimulating a cyclin B1-specific human CD4.sup.+ T lymphocyte response.

2. The method as claimed in claim 1, characterized in that the average strength of the in vitro response of human CD4.sup.+ T lymphocytes specific for said peptide is at least 2.5% in a group of subjects expressing varied HLA II molecules including at least the HLA-DR1, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR11, HLA-DR13 and HLA-DR15 molecules, and the frequency of responders in vitro to said peptide is at least 55% in said group of subjects.

3. The method as claimed in claim 1, characterized in that the said peptide is selected from the group consisting of the sequences SEQ ID NO: 10, 12, 13, 26, 57, 58, 61, 63, 64, 65, 76, 77, 82, 83, 84, 87, 88, 89 and 91.

4. The method as claimed in claim 1, characterized in that the said peptide is a mixture of peptides comprising: (i) at least one first peptide comprising a cyclin B1 CD4.sup.+ T epitope which is immunodominant in vitro, as defined in claim 1, and (ii) at least one second peptide comprising at least one CD4.sup.+ T epitope other than said epitope which is immunodominant in vitro, and/or one CD8.sup.+ T epitope, and/or one B epitope.

5. The method as claimed in claim 4, characterized in that the said second peptide is a peptide comprising a cyclin B1 CD4.sup.+ T epitope, capable of binding to at least 6 different predominant HLA II molecules chosen from the HLA-DR1, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR11, HLA-DR13, HLA-DR15, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DP401 and HLA-DP402 molecules, selected from the group made up of: a) the sequences of 11 to 30 consecutive amino acids of the human cyclin B1 sequence of SEQ ID NO: 1 comprising at least residues 170-178, 201-209, 204-212, 209-217, 214-222, 218-226, 223-231, 227-235, 243-251, 246-254, 252-260, 269-277, 301-309, 344-352, 365-373, 368-376 or 371-379 of said sequence SEQ ID NO: 1, and b) the sequences of 11 to 30 amino acids having at least 70% identity with a sequence in a).

6. The method as claimed in claim 5, characterized in that the said second peptide is selected from the group consisting of the sequences SEQ ID NO: 33, 39, 40, 41, 42, 43, 44, 45, 48, 49, 50, 54, 60, 69, 73, 74, 75, 85, 86 and 90.

7. The method as claimed in claim 4, characterized in that the said second peptide is a peptide comprising a cyclin B1 CD4.sup.+ T epitope, capable of binding to 3 to 5 different predominant HLA II molecules chosen from the HLA-DR1, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR11, HLA-DR13, HLA-DR15, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DP401 and HLA-DP402 molecules, selected from the group consisting of: a) the sequences of 11 to 30 consecutive amino acids of the human cyclin B1 sequence of SEQ ID NO: 1 comprising at least residues 115-123, 146-154, 166-174, 172-180,187-195, 191-199, 197-205, 239-247, 266-274, 271-279, 277-285, 293-301, 329-337, 361-369 or 387-395 of said sequence SEQ ID NO: 1, and b) the sequences of 11 to 30 amino acids having at least 70% identity with a sequence in a).

8. The method as claimed in claim 7, characterized in that the said second peptide is selected from the group consisting of the sequences SEQ ID NO: 25, 29, 32, 34, 36, 37, 38, 47, 53, 55, 56, 59, 66, 72 and 78.

9. The method as claimed in claim 4, characterized in that the said peptides are linked to one another in the form of a multi-epitope polypeptide.

10. The method as claimed in claim 1, characterized in that the said peptide is a modified peptide derived from the peptide as claimed in claim 1 through the introduction of a chemical modification, said modified peptide comprising at least one human cyclin B1 CD4.sup.+ T epitope which is immunodominant in vitro and being capable of stimulating a cyclin 1-specific human CD4.sup.+ T lymphocyte response.

11. The method as claimed in claim 9, characterized in that the said multi-epitope polypeptide is a modified polypeptide derived from the multi-epitope polypeptide as claimed in claim 9 through the introduction of a chemical modification, said modified polypeptide comprising at least one human cyclin B1 CD4.sup.+ T epitope which is immunodominant in vitro and being capable of stimulating a cyclin B1-specific human CD4.sup.+ T lymphocyte, response.

12. The method as claimed in claim 1, characterized in that the said peptide is comprised within an immunogenic or vaccine composition.

13. The method as claimed in claim 1, characterized in that the said subject has a cancer.

14. The method as claimed in claim 13, characterized in that the said cancer is selected from the group consisting of breast, colon, prostate, esophagus, stomach, lung, head and neck cancers.

15. A method of immunotherapy in a subject having a cancer associated with the overexpression of cyclin B1, comprising the induction of a cyclin B1-specific human CD4.sup.+ T lymphocyte response in the subject according to the method of claim 1.

16. The method as claimed in claim 15, characterized in that the said cancer is selected from the group consisting of breast, colon, prostate, esophagus, stomach, lung, head and neck cancers.

Description

(1) In addition to the above arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the subject of the present invention, with reference to the appended drawings in which:

(2) FIG. 1 illustrates the specificity and the magnitude of the response of CD4.sup.+ T lymphocytes with respect to CCNB1. The CD4.sup.+ T lymphocyte lines were obtained by in vitro stimulation with autologous dendritic cells pre-loaded with the CCNB1 protein. A. Examples of CD4.sup.+ T lymphocyte lines specific for CCNB1 peptides. B. Frequencies of CD4.sup.+ T lymphocytes specific for KLH and for CCNB1 in donor blood;

(3) FIG. 2 illustrates the recognition of the CCNB1 protein by the CD4.sup.+ T lymphocyte lines directed against the peptides. The CD4.sup.+ T lymphocyte lines were obtained by in vitro stimulation with autologous dendritic cells pre-loaded with CCNB1 peptides. The CD4.sup.+ T lymphocyte lines were brought into contact with autologous dendritic cells pre-loaded with CCNB1 and the activation thereof was tested by IFN-gamma Elispot after 24 h of incubation. A. Examples of CCNB1-peptide-specific CD4.sup.+ T lymphocyte lines recognizing CCNB1. The name of the CD4.sup.+ T lymphocyte line and its peptide specificity are indicated in the quadrant. B. Number of peptide-specific CD4.sup.+ T lymphocyte lines recognizing CCNB1.

EXAMPLE 1: MATERIALS AND METHODS

(4) 1) Peptide Synthesis

(5) The peptides were synthesized according to the Fmoc strategy in solid-phase parallel synthesis by means of a resin of Rink amide type, purified by HPLC and controlled by mass spectrometry (ES-MS). The N-terminal of all the peptides is free and the C-terminal of all the peptides is amidated.

(6) 2) Production and Purification of the Recombinant Human CCNB1 Protein

(7) E. coli bacteria of the BL 21 strain were transformed by electroporation (2500 V, 4.8 ms) with the pET-28a-(+) plasmid (Novagen) into which the human CCNB1 gene had been cloned. After preculture in LB medium for 20 min at 37 C., the transformed bacteria were cultured for 48 h at 37 C. in LB/kanamycin selective medium, and then expression of cyclin B1 was induced by incubation for 21 h at 15 C. in the presence of IPTG. The protein produced, which has a His tag, was extracted from the bacteria by cell lysis (treatment with lysozymes followed by mechanical grinding and then a treatment with benzonases), and then purified by several successive chromatography operations: a step of affinity chromatography on a HisTrap column (nickel column, GE Healthcare), a step of exclusion chromatography on a Superdex 75 column (GE Healthcare), a step of ion exchange chromatography on a HiTrap SP HP column (GE Healthcare), and a step of removal of the endotoxins by affinity chromatography on an EndoTrap Blue column (Lonza). The concentration of the protein in the final solution was determined by measuring the absorbance at 280 nm, and its purity was verified by mass spectrometry.

(8) 3) Cell Preparation

(9) The blood samples (buffy coat) come from the Etablissement Francais du Sang [French blood bank] (Rungis center). The peripheral blood mononuclear cells (PBMCs) were isolated by means of a ficoll gradient. The immature dendritic cells (DCs) were obtained from the PBMCs by differentiation of the adherent cells after 5 days of culture in AIM-V medium (Life Technologies) containing 1000 U/ml of IL-4 and 1000 U/ml of GM-CSF (R&D Systems). The mature DCs were obtained from the immature DCs after having been cultured for two days in the presence of lipopolysaccharide (LPS). The CD4.sup.+ T lymphocytes were purified from the PBMCs using magnetic microbeads coupled to anti-CD4 antibodies (Miltenyi Biotec). The HLA-DRB1 genotyping of the donors was carried out by sequence-specific PCR using the All Set.sup.+ Gold SSP HLA-DRB1 kit (Invitrogen).

(10) 4) Production of CD4.sup.+ T Lymphocyte Lines Specific for CCNB1 or for the Peptides

(11) The CD4.sup.+ T lymphocytes (1 to 310.sup.5) were cultured in round-bottomed 96-well plates with autologous immature DCs preloaded with 1.2 M of CCNB1 and matured in the presence of LPS, or with autologous mature DCs preloaded with pools of peptides (10 g/ml). The culturing is carried out in IMDM medium (Life Technologies) supplemented with human serum of group AB (complete IMDM medium) containing 1000 U/ml of IL-6 (R&D Systems) and 10 ng/ml of IL-12 (R&D Systems). The culture is stimulated at the end of one week by adding 10 000 to 30 000 DCs loaded with the CCNB1 protein or the pools of peptides, 20 U/ml of IL-2 (R&D Systems) and 10 ng/ml of IL-7 (R&D Systems). Another stimulation is carried out after 14 and optionally 21 days of culture. Between 5 and 7 days after the final stimulation, a specificity test is carried out by EliSpot. Each culture well constitutes a CD4.sup.+ T lymphocyte line.

(12) 5) Evaluation of the Specificity of CD4.sup.+ T Lymphocytes Cultured with CCNB1 or Peptides by EliSpot

(13) The anti-human IFN- antibody 1-D1K (Mabtech) is adsorbed at 2.5 g/ml in 1PBS onto 96-well Multiscreen-HA plates (Millipore) by overnight incubation at 4 C. The plates are then saturated by incubation for 2 hours at 37 C. with complete IMDM medium. The CD4.sup.+ T lymphocytes are incubated for 16 hours at 37 C. in these plates after having been washed in AIM-V/IL-7 medium (0.5 ng/ml of IL-7). The autologous immature DCs (5000 cells/well) preloaded for 4 hours with the CCNB1 protein or KLH (0.5 to 3 M) or PBMCs (50 000 cells/well) incubated in the presence of 10 g/ml of peptides are used as presenting cells. After incubation, the plates are washed with distilled water, PBS/0.05% Tween and PBS. 100 l/well of biotinylated anti-human IFN- Ab 7-B6-1 (Mabtech) at 0.25 g/ml in 1PBS/1% BSA is added to the plates, which are incubated for 1 h 30 min at 37 C. After several washes in PBS or PBS/0.05% tween, the IFN- secretion is demonstrated by adding extravidin-alkaline phosphatase (Sigma) and NBT/BCIP. After 5 to 10 minutes of incubation, the reaction is stopped by washing with running water. After drying, the spots are counted on an Elispot reader (AID). A CD4.sup.+ T lymphocyte line is considered to be antigen-specific if the number of spots in the wells containing the antigen is at least two times higher than the well not containing the antigen, the difference between the 2 types of well being at least greater than 25. The frequency of the CD4.sup.+ T lymphocytes present in the donor blood is estimated using the Poisson's law distribution according to the formula: frequency=Ln ((number of nonspecific lines/total number of lines tested))/(number of CD4.sup.+ lymphocytes incubated in each well).

(14) 6) Purification of Class II HLA Molecules

(15) Lymphoblastoid cells transformed with the EBV virus, homozygous for class II HLA molecules, are used as a source of class II HLA molecules (Texier et al., J. Immunol., 2000, 164, 3177-3184; Texier et al., Eur. J. Immunol., 2001, 31, 1837-1846; Castelli et al., J. Immunol., 2002, 169, 6928-6934). The HLA-DR and HLA-DP molecules are purified by affinity chromatography by means of the L243 anti-HLA-DR (ATCC, Rockville, USA) and B7/21 anti-HLA-DP monoclonal antibodies coupled to a protein A sepharose CL 4B gel (Pharmacia).

(16) 7) Measurement of the Relative Affinity of the Peptides for the Class II HLA Molecules

(17) The affinity of the peptides is evaluated by competition between a biotinylated peptide and the test peptide with respect to the class II HLA molecules. Complexes are revealed by ELISA. These tests are described in American patent U.S. Pat. No. 6,649,166 and PCT international application WO 03/040299, respectively for the HLA-DR and HLA-DP4 molecules. The use of these tests to measure the activity with respect to binding of peptides derived from various antigens is illustrated in American patent U.S. Pat. No. 6,649,166 and PCT international applications WO 02/090382, WO 03/040299 and WO 2004/014936.

(18) More specifically, the class II HLA molecules are diluted in a 10 mM phosphate buffer containing 150 mM NaCl, 1 mM dodecylmaltoside (DM) and 10 mM citrate with a fixed concentration of the biotinylated peptide and dilutions of the test peptide. The plates are incubated for 24 h to 72 h. After neutralization with 50 l of 450 mM Tris-HCl buffer, pH=7.5, containing 0.003% thimerosal, 0.3% BSA and 1 mM DM, the samples are transferred onto 96-well ELISA plates (Maxisorp, Nunc), onto which the L243 (anti-HLA-DR) or B7/21 (anti-HLA-DP) antibodies have been adsorbed at a concentration of 10 g/ml. These plates were also saturated with a 100 mM Tris-HCl buffer, pH=7.5, containing 0.3% BSA and 0.003% thimerosal. After 2 hours of incubation, the ELISA plates are washed. The presence of the biotinylated peptide in the wells is revealed by successive incubation of a streptavidin-alkaline phosphatase conjugate (GE Healthcare) and of 4-methylumbelliferyl phosphate as substrate (Sigma). The fluorescence emitted is measured at 450 nm after excitation at 365 nm by means of a Gemini spectrofluorimeter (Molecular Devices). The maximum binding is given by the wells not containing competitor, and the background noise is measured in the wells not containing HLA molecule. For each peptide, the concentration inhibiting 50% of the maximum binding (IC50) is evaluated. Each experiment is controlled using a reference peptide which is the non-biotinylated form of the tracer peptide. Their sequence and their IC50 value are the following: HA 306-318 (PKYVKQNTLKLAT; SEQ ID No. 2) for DRB1*01:01 (1 nM), DRB1*04:01 (6 nM), DRB1*11:01 (5 nM) and DRB5*01:01 (3 nM), YKL (AAYAAAKAAALAA; SEQ ID No. 3) for DRB1*07:01 (10 nM), A3 152-166 (EAEQLRAYLDGTGVE; SEQ ID No. 4) for DRB1*15:01 (30 nM), MT 2-16 (AKTIAYDEEARRGLE; SEQ ID No. 5) for DRB1*03:01 (121 nM), B1 21-36 (TERVRLVTRHIYNREE; SEQ ID No. 6) for DRB1*13:01 (51 nM), LOL 191-210 (ESWGAVWRIDTPDKLTGPFT; SEQ ID No. 7) for DRB3*01:01 (28 nM), E2/E168 (AGDLLAIETDKATI; SEQ ID No. 8) for DRB4*01:01 (10 nM) and Oxy 271-287 (EKKYFAATQFEPLAARL; SEQ ID No. 9) for DPB1*04:01 (7 nM) and DPB1*04:02 (6 nM). The data are expressed in the form of relative affinity: IC50 of the peptide/IC50 of the reference peptide. A relative affinity value of less than 100 corresponds to a peptide which is a ligand of the class II HLA molecule. Each value is the average of at least two independent experiments.

EXAMPLE 2: CHOICE OF THE SEQUENCE OF THE CNBB1 PEPTIDES

(19) The class II HLA molecules and in particular the HLA-DR and HLA-DP molecules bind the antigenic peptides via several anchoring residues present on the sequence of the peptides. These peptides have a hydrophobic or aromatic residue in their N-terminal portion which constitutes the main anchoring residue, housed in the pocket P1 of the class II HLA molecule. In order to optimize the binding of the peptides to the class II HLA molecules, the overlaps of the peptides which cover the entire CCNB1 sequence were chosen in such a way that they all have a hydrophobic or aromatic residue in their N-terminal portion. These peptides, the sequence of which is presented in Table III, were synthesized.

(20) TABLE-US-00003 TABLEIII OverlappingpeptidesderivedfromtheCCNB1protein (SEQIDNos.10to84) Numbering* Sequence 1-15 MALRVTRNSKINAEN 9-23 SKINAENKAKINMAG 17-31 AKINMAGAKRVPTAP 25-39 KRVPTAPAATSKPGL 37-51 PGLRPRTALGDIGNK 43-57 TALGDIGNKVSEQLQ 50-64 NKVSEQLQAKMPMKK 54-68 EQLQAKMPMKKEAKP 58-72 AKMPMKKEAKPSATG 71-85 TGKVIDKKLPKPLEK 77-91 KKLPKPLEKVPMLVP 81-95 KPLEKVPMLVPVPVS 87-101 PMLVPVPVSEPVPEP 96-110 EPVPEPEPEPEPEPV 108-122 EPVKEEKLSPEPILV 113-127 EKLSPEPILVDTASP 118-132 EPILVDTASPSPMET 128-142 SPMETSGCAPAEEDL 140-154 EDLCQAFSDVILAVN 144-158 QAFSDVILAVNDVDA 147-161 SDVILAVNDVDAEDG 151-165 LAVNDVDAEDGADPN 164-178 PNLCSEYVKDIYAYL 168-182 SEYVKDIYAYLRQLE 172-186 KDIYAYLRQLEEEQA 179-193 RQLEEEQAVRPKYLL 185-199 QAVRPKYLLGREVTG 189-203 PKYLLGREVTGNMRA 195-209 REVTGNMRAILIDWL 199-213 GNMRAILIDWLVQVQ 202-216 RAILIDWLVQVQMKF 207-221 DWLVQVQMKFRLLQE 212-226 VQMKFRLLQETMYMT 216-230 FRLLQETMYMTVSII 221-235 ETMYMTVSIIDRFMQ 225-239 MTVSIIDRFMQNNCV 231-245 DRFMQNNCVPKKMLQ 237-251 NCVPKKMLQLVGVTA 241-255 KKMLQLVGVTAMFIA 244-258 LQLVGVTAMFIASKY 250-264 TAMFIASKYEEMYPP 256-270 SKYEEMYPPEIGDFA 260-274 EMYPPEIGDFAFVTD 264-278 PEIGDFAFVTDNTYT 267-281 GDFAFVTDNTYTKHQ 269-283 FAFVTDNTYTKHQIR 275-289 NTYTKHQIRQMEMKI 280-294 HQIRQMEMKILRALN 285-299 MEMKILRALNFGLGR 291-305 RALNFGLGRPLPLHF 299-313 RPLPLHFLRRASKIG 303-317 LHFLRRASKIGEVDV 310-324 SKIGEVDVEQHTLAK 315-329 VDVEQHTLAKYLMEL 320-334 HTLAKYLMELTMLDY 323-337 AKYLMELTMLDYDMV 327-341 MELTMLDYDMVHFPP 332-346 LDYDMVHFPPSQIAA 338-352 HFPPSQIAAGAFCLA 342-356 SQIAAGAFCLALKIL 347-361 GAFCLALKILDNGEW 351-365 LALKILDNGEWTPTL 359-373 GEWTPTLQHYLSYTE 363-377 PTLQHYLSYTEESLL 366-380 QHYLSYTEESLLPVM 369-383 LSYTEESLLPVMQHL 374-388 ESLLPVMQHLAKNVV 377-391 LPVMQHLAKNVVMVN 385-399 KNVVNVNQGITKHMT 392-406 QGLTKHMTVKNKYAT 396-410 KHMTVKNKYATSKHA 402-416 NKYATSKHAKISTLP 410-424 AKISTLPQLNSALVQ 416-430 PQLNSALVQDLAKAV *the numbering is done with reference to the human CNBB1 sequence (SEQ ID No. 1) **the hydrophobic or aromatic residue is in bold

EXAMPLE 3: PRODUCTION OF CCNB1-PROTEIN-SPECIFIC CD4+ T LYMPHOCYTE LINES

(21) 8 healthy donors comprising HLA molecules which are varied and most of which are very frequent in the Caucasian population were selected. CD4.sup.+ T lymphocyte lines were obtained by coculture of the CD4.sup.+ T lymphocytes with autologous dendritic cells preloaded with the CCNB1 protein, produced recombinantly in E. coli. At the end of the culture, each line was tested by Elispot for its capacity to recognize pools of peptides which cover the sequence of the CCNB1 protein, and then, in a second test, the individual peptides contained in the pool recognized by the lines. In order to be sure that each donor had the capacity to respond, KLH-specific CD4.sup.+ T lymphocyte lines were also produced.

(22) Table IV indicates the peptides recognized by the cyclin-B1-specific CD4.sup.+ T lymphocytes and the number of specific lines which are associated therewith.

(23) TABLE-US-00004 TABLE IV Peptide specificity of the CCNB1-specific T lymphocyte lines peptides Typing 1- 17- 25- 118- 280- 285- 303- 315- 320- 323- 374- 377- 410- 416- 419- Donors HLA-DRB1 15 31 39 132 294 299 317 329 334 337 388 391 424 430 433 TOTAL 693 01:02/ 3 1 4 13:01 694 04:01/ 1 1 1 1 4 15:01 703 07:01/ 1 2 3 13:02 721 03:01/ 2 2 11:01 593 01:01/ 1 1 1 3 08:01 784 07:01/ 2 1 1 5 9 11:01 795 11:01/ 1 1 15:01 803 04:04/ 2 1 1 4 04:01 Total 4 1 1 1 1 7 1 1 1 1 1 1 3 1 5

(24) 15 different peptides corresponding to CCNB1 CD4.sup.+ T epitopes which are immunodominant in vitro were identified (Table IV). 3 peptides are common to at least two individuals (Table IV). These 15 T epitopes which are immunodominant in vitro are different than the known T epitopes (epitopes 215-229, 219-233 and 223-234; Vella et al., Proc. Natl. Acad. Sci. USA, 2009, 106, 14010-14015; application US 2011/0280897) and are located outside the corresponding region of CNBB1 (region 215-233; Vella et al., Proc. Natl. Acad. Sci. USA, 2009, 106, 14010-14015; application US 2011/0280897). In addition, the identified peptides participate in the response against CCNB1, given that the CD4.sup.+ T lymphocytes were generated by stimulation with the CCNB1 protein. Examples of a line of CD4.sup.+ T lymphocytes specific for CCNB1 and for peptides of this protein are presented (FIG. 1A).

(25) On the basis of the number of lines obtained, the frequency of the CD4.sup.+ T lymphocytes pre-existing in the blood of the donors was evaluated. For these 8 donors, this average frequency is 0.82 for CCNB1 and at least 9.2 for KLH, all the donors being responders to the 2 proteins (FIG. 1B). This precursor frequency value corresponds to immunogenic proteins (Delluc et al., Blood, 2010, 116, 4542-4545; Delluc et al., Faseb J., 2011, 25, 2040-2048).

EXAMPLE 4: AFFINITY OF THE CCNB1 PEPTIDES FOR THE CLASS II HLA MOLECULES

(26) All of the overlapping peptides covering the cyclin B1 sequence were tested for their capacity to bind to class II HLA molecules by means of a competitive binding assay as described in Example 1. The data are presented in 3 different tables according to the peptides: the peptides having been found to be of the CCNB1 T epitopes which are immunodominant in vitro during the previous experiments (Table V), the additional peptides having a broad binding specificity with respect to class II HLA molecules (Table VI), and the rest of the peptides (Table VII). For each peptide, the concentration inhibiting 50% of the maximum binding (IC50) is evaluated. The data are expressed in the form of relative affinity: IC50 of the peptide/IC50 of the reference peptide. A relative affinity value of less than 100 corresponds to a peptide which is a ligand of the class II HLA molecule. A relative affinity value of less than 20 corresponds to a peptide which has a strong affinity (value in bold). Each value is the average of at least two independent experiments. The peptides in bold were selected for the subsequent experiments.

(27) TABLE-US-00005 TABLE V Class II HLA molecule-binding activity of the T epitopes which are immunodominant in vitro Peptides DR1 DR3 DR4 DR7 DR11 DR13 DR15 1-15 100 >825 46 2 22 58 5 17-31 5 833 >1771 3 2 21 120 280-294 1225 0.2 0.4 462 2000 >1949 0.3 285-299 1 51 7 0.1 4 1 0.03 303-317 495 3 1394 849 1 1 17 320-334 16 1 1 18 2 10 3 323-337 4 2 0.2 8 0.4 >1 949 2 374-388 837 764 91 3 0.3 6 19 410-424 89 >825 408 >1036 1 94 12 416-430 693 9 54 47 123 >1949 71 419-433 155 1 333 183 3 37 7 25-39 >11 262 >825 >1771 >1036 >1949 >1949 >329 118-132 6359 0.4 0.3 >1036 >1949 >1949 400 315-329 2449 >825 149 6 632 211 7 377-391 894 177 373 13 3 2 >329 Number Peptides DRB3 DRB4 DRB5 DP401 DP402 HLA II 1-15 >362 >961 374 >1357 >1733 6 17-31 >362 >961 11 >1357 >1733 5 280-294 53 >961 4 >1357 >1733 5 285-299 >362 18 2 30 4 11 303-317 >362 >961 1333 >1357 >1733 4 320-334 24 18 17 8 3 12 323-337 41 7 2 1 6 11 374-388 >362 424 91 >1357 >1733 6 410-424 >362 966 >3467 >1357 1118 4 416-430 3 1800 548 59 74 7 419-433 7 >961 112 >1357 148 5 25-39 >362 >961 >3467 >1357 >1733 0 118-132 12 >961 >3467 >1357 >1733 3 315-329 >362 >961 224 >1357 >1733 2 377-391 >362 >961 4082 >1357 >1733 3

(28) Table V shows that, out of the 15 peptides identified as being immunodominant in vitro, 11 peptides bind to at least 4 different class II HLA molecules, 3 peptides binding to more than 11 molecules out of the 12 tested.

(29) Table VI presents the results of other peptides which also have a broad class II HLA molecule-binding specificity. These peptides bind to at least 6 different molecules.

(30) TABLE-US-00006 TABLE VI Class II HLA molecule-binding activity of peptides other than the peptides which are immunodominant in vitro, said other peptides having a broad binding specificity Num- pep- ber tides DR1 DR3 DR4 DR7 DR11 DR13 DR15 DRB3 DRB4 DRB5 DP401 DP402 HLA II 168-182 22 97 163 346 3 12 12 30 >961 3 159 127 7 199-213 27 1 56 10 77 >1 949 1 9 9 58 36 24 11 202-216 1 225 1 59 9 400 >1 949 4 2 20 55 47 14 9 207-221 38 0.5 >1771 7 0.4 1 7 160 49 5 >1357 171 8 212-226 1 1 2 40 5 1 46 34 683 2 110 9 10 216-230 7 1 2 0.5 14 >1949 3 11 22 50 2 1 11 221-235 89 0.5 37 1 1 59 0.3 22 7 1 59 68 12 225-239 24 1 100 5 31 >1949 2 9 6 79 72 35 11 241-255 3 15 1 0.1 90 >1949 1 >362 141 1 424 35 8 244-258 2 9 141 1 23 >1949 1 267 98 1 77 32 9 250-264 22 4 70 4 6 3 9 34 >961 82 0.1 1 11 267-281 1500 0.2 1 22 1333 62 86 38 >961 24 >1357 >1733 7 299-313 63 168 624 35 0.1 0.2 200 >362 13 26 >1357 >1733 6 342-356 46 >825 163 20 3 >1949 21 239 57 163 79 25 7 363-377 28 1 76 1 45 >1949 1 11 5 24 22 50 11 366-380 32 119 65 1 55 >1949 47 32 >961 471 0.2 0.3 8

(31) Table VII presents the results of the remaining peptides.

(32) TABLE-US-00007 TABLE VII Class II HLA molecule-binding activity of the rest of the peptides covering cyclin B1 Num- pep- ber tides DR1 DR3 DR4 DR7 DR11 DR13 DR15 DRB3 DRB4 DRB5 DP401 DP402 HLA II 9-23 >11 262 22 >1 771 >1 036 490 146 >329 >362 >961 1000 >1 357 >1 733 1 37-51 >11 262 >825 >1 771 >1 036 >1 949 422 >329 >362 >961 >3 467 >1 357 >1 733 0 43-57 >11 262 >825 >1 771 >1 036 >1 949 1 789 300 >362 >961 >3 467 >1 357 >1 733 0 50-64 >11 262 >825 >1 771 >1 036 1549 258 >329 >362 >961 51 >1 357 >1 733 1 54-68 3742 >825 >1 771 808 1414 105 >329 >362 >961 35 >1 357 >1 733 1 58-72 >11 262 >825 >1 771 >1 036 40 683 >329 >362 >961 >3 467 >1 357 >1 733 1 71-85 >11 262 228 >1 771 >1 036 >1 949 >1 949 >329 >362 >961 250 >1 357 >1 733 0 77-91 >11 262 >825 >1 771 >1 036 1633 >1 949 >329 >362 75 >3 467 212 474 1 81-95 524 >825 500 >1 036 >1 949 >1 949 >329 >362 141 >3 467 >1 357 >1 733 0 87-101 1500 >825 96 231 >1 949 >1 949 134 >362 748 >3 467 >1 357 >1 733 1 96-110 >11 262 >825 >1 771 >1 036 >1 949 >1 949 >329 >362 >961 >3 467 >1 357 >1 733 0 108-122 894 >825 >1 771 >1 036 >1 949 >1 949 >329 >362 >961 >3 467 >1 357 >1 733 0 113-127 6000 6 55 >1 036 >1 949 >1 949 >329 151 231 >3 467 72 71 4 128-142 3586 >825 >1 771 >1 036 >1 949 >1 949 >329 >362 >961 >3 467 >1 357 >1 733 0 140-154 >11 262 161 1107 >1 036 >1 949 >1 949 207 >362 >961 >3 467 >1 357 >1 733 0 144-158 1 732 8 12 >1 036 >1 949 >1 949 46 18 >961 >3 467 51 129 5 147-161 >11 262 >825 54 >1 036 >1 949 >1 949 7 >362 >961 >3 467 >1 357 >1 733 2 151-165 >11 262 >825 667 >1 036 >1 949 >1 949 >329 >362 >961 >3 467 >1 357 >1 733 0 164-178 2828 >825 527 73 >1 949 >1 949 28 53 40 500 274 625 4 172-186 42 33 187 1 600 1 5 11 >362 >961 775 >1 357 >1 733 5 179-193 9000 >825 >1 771 271 >1 949 943 >329 >362 >961 2 160 >1 357 >1 733 0 185-199 59 >825 260 >1 036 22 4 >329 >362 >961 1 667 >1 357 >1 733 3 189-203 17 764 34 >1 036 3 >1 949 >329 >362 >961 129 >1 357 >1 733 3 195-209 55 1 260 173 1183 45 30 20 23 1 414 725 791 5 231-245 11 >825 52 >1 036 2 000 >1 949 >329 >362 >961 1 581 >1 357 >1 733 2 237-251 4 >825 179 115 87 >1 949 65 >362 1 400 112 >1 357 >1 733 3 256-270 3 >825 456 61 1366 >1 949 153 >362 >961 603 894 >1 733 2 264-278 7000 85 2 86 1667 >1 949 300 19 >961 61 >1 357 >1 733 5 269-283 1342 0.1 0.4 577 2191 >1 949 17 34 >961 42 >1 357 >1 733 5 275-289 173 >825 456 43 4 158 85 >362 80 50 >1 357 >1 733 5 291-305 1 >825 1 414 3 1000 >1 949 65 >362 >961 4 >1 357 >1 733 4 310-324 >11 262 306 >1 771 >1 036 190 1800 >329 >362 >961 1500 >1 357 >1 733 0 327-341 418 32 22 126 216 >1 949 10 >362 139 163 2 47 5 332-346 224 >825 289 20 511 >1 949 36 >362 >961 1 581 >1 357 >1 733 2 338-352 598 >825 >1 771 1400 >1 949 >1 949 257 >362 >961 >3 467 >1 357 188 0 347-361 >11 262 >825 >1 771 >1 036 9 >1 949 1 >362 >961 >3 467 >1 357 >1 733 2 351-365 648 >825 1 000 624 2333 >1 949 52 >362 >961 3055 >1 357 510 2 359-373 548 >825 500 0.4 22 >1 949 0.2 >362 >961 224 >1 357 >1 733 3 369-383 212 >825 58 91 45 >1 949 350 72 >961 1379 4 1 6 385-399 4500 228 986 >1 036 766 37 0.02 >362 >961 52 116 2 4 392-406 >11 262 408 577 533 693 7 129 >362 >961 47 >1 357 >1 733 2 396-410 7483 >825 408 533 7 9 229 >362 >961 187 >1 357 >1 733 2 402-416 367 >825 707 9 4 >1 949 >329 >362 >961 1 732 >1 357 >1 733 2

(33) Some of the peptides of Table VII are capable of binding to 4 or 5 class II HLA molecules.

(34) The peptides of Table VI and the 11 peptides which are immunodominant in vitro and bind to at least 4 different class II HLA molecules (Table V) were retained for additional studies. On the other hand, none of the peptides of Table VII was retained, since the number of peptides that could be evaluated was limited.

EXAMPLE 5: INDUCTION OF CD4+ T LYMPHOCYTE LINES SPECIFIC FOR CYCLIN B1 PEPTIDES

(35) The peptides retained for the CD4.sup.+ T lymphocyte line induction tests are described in Table VIII. For 7 peptides, the overlapping sequences were combined with one another, forming longer peptides ranging up to 20 amino acids.

(36) TABLE-US-00008 TABLEVIII Sequencesofthepeptidestestedfortheircapacitytoinduce specificCD4.sup.+Tlymphocytes Sequences Combination Sequence SEQIDNO: Size 1-15 MALRVTRNSKINAEN 10 15 17-31 AKINMAGAKRVPTAP 12 15 168-182 SEYVKDIYAYLRQLE 33 15 199-216 199-213,202-216 GNMRAILIDWLVQVQMKF 85 18 207-221 DWLVQVQMKFRLLQE 41 15 212-226 VQMKFRLLQETMYMT 42 15 216-230 FRLLQETMYMTVSII 43 15 221-235 ETMYMTVSIIDRFMQ 44 15 225-239 241-255,244-258 MTVSIIDRFMQNNCV 45 15 241-258 KKMLQLVGVTAMFIASKY 86 18 250-264 TAMFIASKYEEMYPP 50 15 267-281 280-294,285-299 GDFAFVTDNTYTKHQ 54 15 280-299 299-313,303-317 HQIRQMEMKILRALNFGLGR 87 20 299-317 320-334,323-337 RPLPLHFLRRASKIGEVDV 88 19 320-337 HTLAKYLMELTMLDYDMV 89 18 342-356 363-377,366-380 SQIAAGAFCLALKIL 69 15 363-380 PTLQHYLSYTEESLLPVM 90 18 374-388 ESLLPVMQHLAKNVV 76 15 410-424 416-430,419-433 AKISTLPQLNSALVQ 85 15 416-433 PQLNSALVQDLAKAVAKV 91 18

(37) 13 healthy donors comprising HLA molecules which are varied and most of which are very frequent in the Caucasian population were selected (Table IX).

(38) CD4.sup.+ T lymphocyte lines were obtained by coculture of the CD4.sup.+ T lymphocytes with autologous dendritic cells preloaded with pools of peptides. After amplification of the T lymphocytes, each line was tested by Elispot (Table IX). The results are reported in the form of the number of times when a response to a peptide is observed. All the peptides induce a T lymphocyte response, but the strength of the response and the number of responders are very variable. 9 peptides activate T lymphocytes in more than half the donors.

(39) However, notably, the most effective peptides, capable of inducing the most effective specific CD4.sup.+ T response (response strength greater than 2.5% and which can reach 6.1% and responder frequency greater than 65% and which can reach 85%) in a population of individuals carrying varied HLA II molecules, comprising all the HLA-DRB1 molecules that are the most frequent in the Caucasian population, covering by themselves 80% of the individuals of the Caucasian population, correspond to peptides which are immunodominant in vitro.

(40) TABLE-US-00009 TABLE IX Analysis of the average strength and the frequency of the CD4.sup.+ T response induced by the peptides Number of specific responses HLA-DRB1 1- 17- 168- 199- 207- 212- 216- 221- 225- 241- 250- Donor Typing 15 31 182 216 221 226 230 235 239 258 264 877 01:01/13:01 1 1 4 1 829 07:01/15:01 1 3 876 11:01/11:04 3 1 2 996 08:04/14:08 2 5 2 1 1 1 14 07:01/13:01 1 1 1 1 2 1 1 22 01:01/09:01 1 2 2 1 3 1 3 23 04:01/13:01 1 1 1 1 1 1 1 26 11:04/15:01 4 3 1 2 30 07:01/11:01 1 1 1 2 1 1 32 03:01 1 62 01:01/04:05 1 3 4 1 2 31 13:01/16:01 1 4 1 8 1 56 07:01/11:01 3 2 1 3 1 1 1 2 total 18 14 13 8 6 5 15 10 8 15 3 Average 2.8 2.2 2.0 1.2 0.9 0.8 2.4 1.5 1.2 2.3 0.5 strength (%) responders 10 6 8 5 5 3 5 7 7 7 2 frequency 77% 46% 62% 38% 38% 23% 38% 54% 54% 54% 15% Number of specific responses HLA-DRB1 267- 280- 299- 320- 342- 363- 374- 410- 416- Donor Typing 281 299 317 337 356 380 388 424 433 Total 877 01:01/13:01 2 2 1 1 13 829 07:01/15:01 2 2 1 9 876 11:01/11:04 1 4 2 2 3 18 996 08:04/14:08 4 1 1 2 20 14 07:01/13:01 2 1 1 1 2 7 13 35 22 01:01/09:01 3 8 2 3 2 31 23 04:01/13:01 2 1 1 1 2 1 5 20 26 11:04/15:01 3 1 1 15 30 07:01/11:01 1 3 1 1 1 3 1 2 20 32 03:01 3 1 2 1 1 1 10 62 01:01/04:05 1 1 13 31 13:01/16:01 1 3 6 2 1 5 33 56 07:01/11:01 1 6 1 2 7 1 5 37 total 10 23 33 6 5 7 28 8 39 274 Average 1.5 3.5 5.0 1.0 0.8 1.1 4.3 1.2 6.1 strength (%) responders 6 11 10 5 4 5 10 6 10 frequency 46% 85% 77% 38% 31% 38% 77% 46% 77% The peptides which are immunodominant in vitro are indicated in bold. The most immunogenic peptides (highest frequency of responders and highest number of lines) are shaded.

EXAMPLE 6: RECOGNITION OF CCNB1 BY THE CD4+ T LYMPHOCYTES

(41) In order to evaluate the capacity of the CD4.sup.+ T lymphocytes to recognize the CCNB1 protein, a part of the CD4.sup.+ T lymphocyte lines was cultured in the presence of autologous dendritic cells preloaded with CCNB1 and then the activation thereof was tested by IFN-gamma Elispot. Examples of responses to the CCNB1 protein are presented in FIG. 2A. All of the peptides which generated CD4.sup.+ T lymphocytes capable of recognizing the CCNB1 protein are presented in FIG. 2B. Not all the peptides could be tested.

(42) TABLE-US-00010 TABLEX Aminoacidsequences SEQ ID Name Sequence No. CCNB1 MALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTAL 1 GDIGNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLV PVPVSEPVPEPEPEPEPEPVKEEKLSPEPILVDTASPSPMETSGC APAEEDLCQAFSDVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQ LEEEQAVRPKYLLGREVTGNMRAILIDWLVQVQMKFRLLQETMYM TVSIIDRFMQNNCVPKKMLQLVGVTAMFIASKYEEMYPPEIGDFA FVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKIGEV DVEQHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGE WTPTLQHYLSYTEESLLPVMQHLAKNVVMVNQGLTKHMTVKNKYA TSKHAKISTLPQLNSALVQDLAKAVAKV HA306-318 PKYVKQNTLKLAT 2 YKL AAYAAAKAAALAA 3 A3152-166 EAEQLRAYLDGTGVE 4 MT2-16 AKTIAYDEEARRGLE 5 B121-36 TERVRLVTRHIYNREE 6 LOL191-210 ESWGAVWRIDTPDKLTGPFT 7 E2/E168 AGDLLAIETDKATI 8 Oxy271-287 EKKYFAATQFEPLAARL 9 CCNB11-15 MALRVTRNSKINAEN 10 CCNB19-23 SKINAENKAKINMAG 11 CCNB117-31 AKINMAGAKRVPTAP 12 CCNB125-39 KRVPTAPAATSKPGL 13 CCNB137-51 PGLRPRTALGDIGNK 14 CCNB143-57 TALGDIGNKVSEQLQ 15 CCNB150-64 NKVSEQLQAKMPMKK 16 CCNB154-68 EQLQAKMPMKKEAKP 17 CCNB158-72 AKMPMKKEAKPSATG 18 CCNB171-85 TGKVIDKKLPKPLEK 19 CCNB177-91 KKLPKPLEKVPMLVP 20 CCNB181-95 KPLEKVPMLVPVPVS 21 CCNB187-101 PMLVPVPVSEPVPEP 22 CCNB196-110 EPVPEPEPEPEPEPV 23 CCNB1108-122 EPVKEEKLSPEPILV 24 CCNB1113-127 EKLSPEPILVDTASP 25 CCNB1118-132 EPILVDTASPSPMET 26 CCNB1128-142 SPMETSGCAPAEEDL 27 CCNB1140-154 EDLCQAFSDVILAVN 28 CCNB1144-158 QAFSDVILAVNDVDA 29 CCNB1147-161 SDVILAVNDVDAEDG 30 CCNB1151-165 LAVNDVDAEDGADPN 31 CCNB1164-178 PNLCSEYVKDIYAYL 32 CCNB1168-182 SEYVKDIYAYLRQLE 33 CCNB1172-186 KDIYAYLRQLEEEQA 34 CCNB1179-193 RQLEEEQAVRPKYLL 35 CCNB1185-199 QAVRPKYLLGREVTG 36 CCNB1189-203 PKYLLGREVTGNMRA 37 CCNB1195-209 REVTGNMRAILIDWL 38 CCNB1199-213 GNMRAILIDWLVQVQ 39 CCNB1202-216 RAILIDWLVQVQMKF 40 CCNB1207-221 DWLVQVQMKFRLLQE 41 CCNB1212-226 VQMKFRLLQETMYMT 42 CCNB1216-230 FRLLQETMYMTVSII 43 CCNB1221-235 ETMYMTVSIIDRFMQ 44 CCNB1225-239 MTVSIIDRFMQNNCV 45 CCNB1231-245 DRFMQNNCVPKKMLQ 46 CCNB1237-251 NCVPKKMLQLVGVTA 47 CCNB1241-255 KKMLQLVGVTAMFIA 48 CCNB1244-258 LQLVGVTAMFIASKY 49 CCNB1250-264 TAMFIASKYEEMYPP 50 CCNB1256-270 SKYEEMYPPEIGDFA 51 CCNB1260-274 EMYPPEIGDFAFVTD 52 CCNB1264-278 PEIGDFAFVTDNTYT 53 CCNB1267-281 GDFAFVTDNTYTKHQ 54 CCNB1269-283 FAFVTDNTYTKHQIR 55 CCNB1275-289 NTYTKHQIRQMEMKI 56 CCNB1280-294 HQIRQMEMKILRALN 57 CCNB1285-299 MEMKILRALNFGLGR 58 CCNB1291-305 RALNFGLGRPLPLHF 59 CCNB1299-313 RPLPLHFLRRASKIG 60 CCNB1303-317 LHFLRRASKIGEVDV 61 CCNB1310-324 SKIGEVDVEQHTLAK 62 CCNB1315-329 VDVEQHTLAKYLMEL 63 CCNB1320-334 HTLAKYLMELTMLDY 64 CCNB1323-337 AKYLMELTMLDYDMV 65 CCNB1327-341 MELTMLDYDMVHFPP 66 CCNB1332-346 LDYDMVHFPPSQIAA 67 CCNB1338-352 HFPPSQIAAGAFCLA 68 CCNB1342-356 SQIAAGAFCLALKIL 69 CCNB1347-361 GAFCLALKILDNGEW 70 CCNB1351-365 LALKILDNGEWTPTL 71 CCNB1359-373 GEWTPTLQHYLSYTE 72 CCNB1363-377 PTLQHYLSYTEESLL 73 CCNB1366-380 QHYLSYTEESLLPVM 74 CCNB1369-383 LSYTEESLLPVMQHL 75 CCNB1374-388 ESLLPVMQHLAKNVV 76 CCNB1377-391 LPVMQHLAKNVVMVN 77 CCNB1385-399 KNVVMVNQGLTKHMT 78 CCNB1392-406 QGLTKHMTVKNKYAT 79 CCNB1396-410 KHMTVKNKYATSKHA 80 CCNB1402-416 NKYATSKHAKISTLP 81 CCNB1410-424 AKISTLPQLNSALVQ 82 CCNB1416-430 PQLNSALVQDLAKAV 83 CCNB1419-433 NSALVQDLAKAVAKV 84 CCNB1199-216 GNMRAILIDWLVQVQMKF 85 CCNB1241-258 KKMLQLVGVTAMFIASKY 86 CCNB1280-299 HQIRQMEMKILRALNFGLGR 87 CCNB1299-317 RPLPLHFLRRASKIGEVDV 88 CCNB1320-337 HTLAKYLMELTMLDYDMV 89 CCNB1363-380 PTLQHYLSYTEESLLPVM 90 CCNB1416-433 PQLNSALVQDLAKAVAKV 91 survivin LTLGEFLKL 92 96-104 survivin ELTLGEFLKL 93 95-104 survivin-2B AYACNTSTL 94 80-88 PADRE KXVAAWTLKAA 95 P1 AGYLMELCV 96 P2 AGYLMELCM 97 P3 AGYLMELCF 98 P4 AGYLMELCC 99 P5 AGYLMELCMA 100 P6 AGYLMELCFA 101 CB9 AKYLMELTM 102 CB10 AKYLMELTML 103 CB9L2 ALYLMELTM 104 CB9M2 AMYLMELTM 105 CB204 ILIDWLVQV 106 CB215-229 KFRLLQETMYMTVSI 107 CB219-233 LQETMYMTVSIIDRF 108 CB223-234 MYMTVSIIDRFM 109