IDENTIFICATION, OPTIMIZATION AND USE OF CRYPTIC HLA-B7 EPITOPES FOR IMMUNOTHERAPY

The present invention relates to the field of peptide immunotherapy. In particular, the invention provides novel methods and materials for efficiently treating patients having an HLA- B*0702 phenotype.

Peptide vaccination or immunotherapy is a therapeutic approach which is currently the subject of a great deal of interest 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.

It will be recalled that CTLs do not recognize whole protein antigens, but peptide fragments thereof, generally comprising 8 to 10 amino acids, presented by class I major histocompatibility complex (MHC I) molecules expressed on the surface of cells. The presentation of these peptides is the result of the antigen processing which involves three steps: cytosolic degradation of the antigen by a multienzyme complex called proteasome translocation of the peptides derived from this degradation in the endoplasmic reticulum (ER) by the TAP transporters - association of these peptides with the MHC I molecules and exportation of the peptide/MHC I complexes to the cell surface

The peptide/MHC I complexes interact with the specific T cell receptor (TCR) on CTL inducing the stimulation and amplification of these CTL which become able to attack target cells expressing the antigen from which the peptide is derived. During the antigen processing, a peptide selection takes place, which results in a hierarchy of peptides presentation. Peptides that are preferentially presented by the MHC I molecules are called immunodominant while peptides that are weakly presented are called subdominant/cryptic. Immunodominant peptides exhibit a high affinity for the MHC I and are immunogenic while subdominant/cryptic peptides exhibit a low affinity for MHC I and are non-immunogenic.

Immunodominant peptides have widely been targeted by tumor vaccines in preclinical and clinical studies with disappointing results (Bowne et al., 1999; Colella et al., 2000; Gross et al., 2004; Hawkins et al., 2000; Naftzger et al., 1996; Overwijk et al., 1998; Vierboom et al., 1997; Weber et al., 1998). Tumor antigens are frequently self proteins over-expressed by tumors and expressed at lower levels by normal cells and tissues. 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;

Hernandez et al., 2000; Theobald et al., 1997) thus explaining the incapacity of these peptides to induce a tumor immunity.

Subdominant/cryptic peptides are much less involved in self tolerance process (Anderton et al., 2002; Boisgerault et al., 2000; Cibotti et al., 1992; Friedman et al., 2004; Gross et al., 2004; Moudgil et al., 1999; Overwijk et al., 2003; Sinha et al., 2004) and can therefore induce an efficient tumor immunity providing their immunogenicity is enhanced (Disis et al., 2002; Dyall et al., 1998; Engelhorn et al., 2006; Gross et al., 2004; Grossmann et al., 2001; Lally et al., 2001; Moudgil and Sercarz, 1994a; Moudgil and Sercarz, 1994b; Palomba et al., 2005). The usual strategy for enhancing the immunogenicity of subdominant/cryptic peptides, that because of their low MHC I affinity are non- immunogenic, 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 the peptides having the primary anchor motifs, of a great variability in the binding affinity.

Amino acids substitutions aiming at enhancing affinity for MHC I molecule should preserve the antigenicity of such optimized peptides. CTL generated by optimized peptides should cross-react with the corresponding native peptides. Many teams have succeeded in further enhancing immunogenicity of already immunogenic peptides by increasing their affinity for HLA-A*0201 (Bakker et al., 1997; Parkhurst et al., 1996; Sarobe et al., 1998; 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 et al., 2000). HLA-B*0702 is a frequently expressed molecule (25% of the population). Identification and optimization of HLA-B *0702 restricted tumor cryptic peptides should therefore be necessary in order to develop efficient cancer vaccines for HLA-B*0702 expressing patients.

As described in the experimental part below, the inventors have now found a general strategy to identify, in an antigen, subdominant/cryptic peptides presented by HLA-B *0702 molecule, and to optimize their immunogenicity.

Hence, a first aspect of the present invention is a method for identifying a HLA-B*0702-restricted cryptic epitope in an antigen, comprising a step of selecting, in said antigen, a peptide of 8 to 12 amino acids having a proline (P) in position 2, and an

arginine (R) in position 3, with the proviso that the peptide does not have, simultaneously, an alanine (A) in position 1 and a leucine (L) in C-terminal position.

In what follows, the phrases "HLA-B* 0702 -restricted cryptic epitope", or "native peptide" will be used to designate a peptide selected according to the above method, i.e., a peptide having the sequence X _J PRX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X _S XPXI _O (SEQ ID NO: 119), wherein X ₁ to X ₅ and X ₁₀ are any amino acid, X ₆ to X ₉ are any amino acid or none, and X ₁₀ ≠ L if X ₁ = A. In the present text, the term "peptide" designates not only molecules in which amino acid residues 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. For example, retro-inverso peptidomimetics, in which the peptide bond is reversed, can be advantageously used. These may be made using methods known in the art, for example such as those described in Meziere et al., 1997.

According to a preferred embodiment of the invention, the selected peptide has 9 to 11 amino acids, more preferably 9 or 10 amino acids.

A second aspect of the present invention is a method for increasing the immunogenicity of a HLA-B*0702-restricted cryptic epitope, comprising a step of substituting the N-terminal residue of said epitope with an alanine, and/or substituting the C-terminal residue of said epitope with a leucine. 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-B*0702-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.

The immunogenicity of a HLA~B*0702-restricted cryptic epitope in which the three first residues are APR can be increased only by replacing its last amino- acid by an L (or by adding a leucine at its C-terminus, provided it is not longer than 11 amino acids). When the sequence of the selected HLA-B*0702-restricted cryptic epitope is XIPRX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X _S XPL (SEQ ID NO: 120), wherein X ₁ is any amino acid but A, X ₂ to X ₅ are any amino acid, and X ₆ to X ₉ are any amino acid or none, the substitution of X ₁ by A is sufficient to increase its immunogenicity. In what follows, the expression "optimized peptide" will designate an immunogenic peptide derived from a HLA-B*0702- restricted cryptic epitope by the above methods, and having the general sequence APRXiX ₂ X ₃ X ₄ X ₅ X ₆ XyXsL (SEQ ID No: 121), wherein X ₁ to X ₄ are any amino acid, and X ₅ to X ₈ are any amino acid or none. The inventors have identified a number of HLA-B *0702-restricted cryptic epitopes, which are disclosed in Table 1 below. Accordingly, another aspect of the present invention is a cryptic HLA-B *0702-restricted epitope, selected amongst the peptides of SEQ ID Nos: 1 to 68, disclosed in Table 1.

Table 1 : Selected cryptic HLA-B*0702 restricted peptides

Table 1 (following): Selected cryptic HLA-B*0702 restricted peptides

The present invention also pertains to optimized peptides derived from the cryptic peptides of SEQ ID Nos: 1 to 68, by a method according to the invention. Preferred examples of optimized peptides are APRMHC AVDL (SEQ ID No: 69), APRVSIRLPL (SEQ ID NO: 70), APREYVNAL (SEQ ID No: 71), APRSPLAPL (SEQ ID No: 72), APRALVETL (SEQ ID No: 73), APRRRLGCEL (SEQ ID No: 74), RPRRGAAPEL (SEQ ID NO: 75), APRPWDTPCL (SEQ ID No: 76), APRRLPRLPL (SEQ ID No: 77), and APRRLVQLL (SEQ ID No: 78). Other immunogenic HLA-B*0702 -restricted epitopes obtained according to the invention are those derived from a cryptic HLA-B* 0702 -restricted epitope selected amongst SEQ ID NOs: 8, 9, 29, 34, 62 and 66, by substitution of their C- terminal amino-acid with a leucine, as well as those derived from a cryptic HLA-B*0702- restricted epitope selected amongst SEQ ID NOs: 13, 31 and 32, by substitution of their N-terminal amino-acid with an alanine.

Another immunogenic HLA-B*0702-restricted epitope according to the invention, and derived from the cryptic HLA-B*0702-restricted epitope SEQ ID NO 7, is obtained by adding a leucine at its terminus.

Still other immunogenic HLA-B*0702-restricted epitopes according to the invention are obtained by substituting the N-terminal amino-acid with an alanine and the C-terminal amino-acid with a leucine, starting from a cryptic HLA-B*0702-restricted epitope selected amongst the peptides of SEQ ID NOs: 1 to 6, 10 to 12, 14 to 18, 20 to 28, 30, 33, 37 to 44, 46, 48 to 52, 57, 59, 60, 63 to 65, 67 and 68.

The invention also concerns a chimeric polypeptide, comprising two, three or more HLA-B*0702 -restricted cryptic epitopes or two, three or more immunogenic HLA-B*0702-restricted epitopes as described above. In such a chimeric polypeptide, the epitopes can be different from each other, or the same epitope can be repeated several times. 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.

Another object of the present invention is an isolated nucleic acid molecule designed to cause the expression of a cryptic HLA-B*0702-restricted epitope or an immunogenic epitope or 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 encoding region for 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. Exemplary are adenovirus or vaccinia virus vectors and vectors of the herpes family, especially in a non-replicative form.

Another embodiment of the present invention is a pharmaceutical composition comprising at least, as an active principle, a HLA-B*0702 -restricted cryptic epitope as above-described, or an immunogenic epitope polypeptide derived therefrom as mentioned above, or a chimeric polypeptide according to the invention, or a nucleic acid encoding any of these, and/or a vector carrying said nucleic acid. Formulation of pharmaceutical compositions will accord with contemporary standards and techniques. Medicines intended for human administration will be prepared in adequately sterile conditions, in which the active ingredient(s) are combined with an isotonic solution or other pharmaceutical carrier appropriate for the recommended therapeutic use. Suitable formulations and techniques are generally described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton PA).

In particular, a HLA-B*0702-restricted cryptic epitope, or an immunogenic epitope polypeptide derived therefrom, or a chimeric polypeptide carrying several such immunogenic or cryptic epitopes, or a nucleic acid encoding any of these, included or not in a vector, can be used for the preparation of a composition for preventive or curative immunotherapy, especially, for antiviral or anti-cancer immunotherapy.

In a particular embodiment, a pharmaceutical composition according to the invention is a vaccine. In this latter case, the compositions of this invention can be

combined with an adjuvant to potentiate the immune response. Classic adjuvants include oil emulsions, like Incomplete Freund's Adjuvant, 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.

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 (in the form of promotional material or a package insert) regarding the use of the composition or combination in eliciting an immune response and/or the treatment of cancer.

In a previous patent application (PCT/EP2006/005325), 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 PCT/EP2006/005325 demonstrate that injection of a native peptide corresponding to a sub-dominant/cryptic epitope, following vaccination with its cognate optimized peptide, can maintain the immune response initiated by said optimized peptide.

According to the invention, a HLA-B*0702~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 HLA-B*0702-restricted epitope sequence derived from a HLA- B*0702-restricted cryptic epitope can also be used, for the preparation of a medicinal composition for initiating a CTL immune response against said HLA-B*0702-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 peptide cognate to a native HLA-B*0702-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 two, three or more optimized or cryptic peptides as above-described, instead of single-epitope peptides.

The invention also pertains to a kit of parts comprising, in separate formulations, a first peptide having the sequence of a HLA-B*0702-restricted cryptic epitope, and a second peptide corresponding to its cognate HLA-B*0702-restricted immunogenic epitope. Examples of peptides which can be part of a kit according to the invention are the peptides of SEQ ID NOs: 1 to 68, 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.

Other kits of parts according to the invention comprise at least a chimeric polypeptide. Several variants of such kits are contemplated: in a first embodiment, the kit comprises, in separate formulations, a first chimeric polypeptide comprising two, three or more HLA-B*0702-restricted cryptic epitopes, and a second chimeric polypeptide corresponding to its cognate HLA-B*0702-restricted immunogenic chimeric polypeptide (which means that it comprises optimized HLA-B*0702-restricted immunogenic epitopes cognate to the cryptic epitopes comprised in the first chimeric polypeptide). In a second embodiment, the kit comprises a first chimeric polypeptide comprising two, three or more HLA-B*0702-restricted cryptic epitopes and, in one or several other separate formulations, peptides corresponding to the optimized HLA- B*0702-restricted immunogenic epitopes cognate to the cryptic epitopes comprised in the first chimeric polypeptide. In a third embodiment, the kit comprises two, three or more peptides corresponding to distinct HLA-B *0702-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- B*0702 -restricted immunogenic epitopes cognate to said cryptic peptides.

In the following description of the kits according to the invention, mention will be made only of the peptides (native or optimized), it being understood that chimeric polypeptides (comprising native cryptic epitopes or optimized epitopes) can be enclosed in the kits instead of single-epitope peptides.

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, hi 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 1.5 to 6 months. Therefore, complementary kits, comprising at least 2 doses, and up to 40 or 50 doses of native peptide, are also part of the present invention. Alternatively, the vaccination kit can comprise 2 to 3 doses of optimized peptide, and 3 to 40 or up to 50 doses of native peptide. Of course, said native and optimized peptides present in the kit are as described above.

Each dose comprises between 0.5 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, used as an adjuvant. The skilled artisan can choose any other adjuvant(s) in place of (or in addition to) Montanide. hi a particular embodiment, the doses are in the form of an aqueous

solution. Alternatively, the doses can be in the form of a lyophilized peptide, for extemporaneous preparation of the liquid solution to be injected. Other possible components of said kits are one or several adjuvants, to be added to the peptide compositions before administration, and a notice describing how to use said kits. The invention is further illustrated by the following figures and examples. LEGENDS OF FIGURES

Figure 1: Immunogenicity of HLA-B*0702 restricted peptides. CTL were tested against RMA-B7 targets loaded with peptide as indicated. Figure 2 : Immunogenicity of optimized HLA-B*0702 cryptic peptides. CTL were tested against RMA-B7 targets loaded with peptide as indicated.

Figure 3 : Recognition of endogenous TERT by TERT444 specific murine CTL. A) CTL were tested against RMA-B7 targets loaded with decreasing doses of TERT ₄₄₄ peptide. B) CTL were tested against COS cells transfected with HLA-B*0702 and TERT as indicated.

Figure 4 : Recognition of endogenous TERT by TERT ₄ specific murine CTL. A) CTL were tested against RMA-B7 targets loaded with decreasing doses of TERT ₄ peptide. B) CTL were tested against COS cells transfected with HLA-B* 0702 and TERT as indicated. Figure 5 : Induction of TERT ₄ specific human CTL. A) CTL were tested against T2-B7 targets loaded with TERT ₄ (■) or an irrelevant (•) peptide. B) CTL were tested against the HLA-B* 0702 positive TERT positive SK-MES-I (■), HBL- 100 (•) and the HLA-B*0702 negative TERT positive SW-480 (□), HSS (o) human tumor cell lines. EXAMPLES

The examples have been performed using the following materials and methods:

Transgenic Mice. The HLA-B7 H-2 class-I knockout mice were previously described (Rohrlich et al., 2003). Cells. HLA-B*0702 transfected murine RMA-B7 and human T2-B7 cells were previously described (Rohrlich et al., 2003). COS-7 and WEHI-164 clone 13 cells were provided by F. Jotereau (INSERM 463, Nantes, France). The HLA-B*0201 positive SK-MES-I (lung cancer), HBL- 100 (breast cancer), and the HLA-B*0702 negative SW-480 (colon cancer) and HSS (myeloma) cell lines were used as targets of human CTL. All cell lines were grown in FCS 10% supplemented RPMI 1640 culture medium.

Peptides and Plasmids. Peptides were synthesized by Epytop (Nϊmes, France). HLA-B*0702 plasmid was provided by Dr. Lemonnier (Institut Pasteur, Paris,

France) (Rohrlich et al., 2003) and TERT plasmid was provided by Dr Weinberg (MIT, Boston, MA) (Meyerson et al, 1997).

Measurement of Peptide Relative Affinity to HLA-B*0702. The protocol used has been described previously (Rohrlich et al., 2003). Briefly, T2-B7 cells were incubated at 37°C for 16 hours with peptides concentrations ranging from 100 μM to 0.1 μM, and then stained with ME-I monoclonal antibody (mAb) to quantify the surface expression of HLA-B*0702. For each peptide concentration, the HLA-B*0702 specific staining was calculated as the percentage of staining obtained with 100 μM of the reference peptide CMV _265-274 (RlOV; RPHERNGFTV, SEQ ID NO: 117). The relative affinity (RA) was determined as: RA = (Concentration of each peptide that induces 20 % of HLA-B*0702-expression / Concentration of the reference peptide that induces 20 % of HLA-B*0702 expression).

CTL Induction in vivo in HLA-B*0702 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-A ^b restricted HBVcoreπs T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 118). After 11 days, 5x10 ⁷ spleen cells were stimulated in vitro with peptide (10 μM). On day 6 of culture, the bulk responder populations were tested for specific cytotoxicity.

Peptide Processing Assay on COS-7 Transfected Cells. 2.2x10 ⁴ simian COS-7 cells were plated in flat-bottomed 96 well plates in DMEM+10% FCS, in triplicate for each condition. Eighteen hours later, cells were transfected with 100 ng of each DNA plasmid with DEAE Dextran. After 4 hours, PBS+10% DMSO was added for 2 minutes. Transfected COS cells were incubated in DMEM+10% FCS during 40 hours and then used to stimulate murine CTL in a TNFα secretion assay. TNFa Secretion Assay. Transfected COS-7 cells at day 4 were suspended in 50μl of RPMI+10% FCS and used as stimulating cells. 5x10 ⁴ murine T cells were then added in 50 μl RPMI 10% FCS and incubated for 6 hours. Each condition was tested in triplicate. 50 μl of the supernatant was collected to measure TNF-α. Standard dilutions were prepared in 50μl with final doses of TNF-α ranging from 104 to 0 pg/ml. On both the supernatants and the standard dilutions, 3x10 ⁴ TNF-α sensitive WEHI-164cl 3 cells in 50μl were added. They were incubated for 16h at 37°C. Inhibition of cell proliferation was evaluated by the MTT colorimetric method (Espevik and Nissen-Meyer, 1986).

Generation of CTL from human PBMC. PBMC were collected by leukapheresis from healthy HLA-A*0201 volunteers. Dendritic cells (DC) were produced from adherent cells cultured for seven days (2x10 ⁶ cells/ml) in the presence of 500 IU/ml GM-CSF (Leucomax®, Schering-Plough, Kenilworth, NJ) and 500IU/ml IL-4 (R&D Systems, Minneapolis, MN) in complete medium (RPMI- 1640 supplemented with 10% heat inactivated human AB serum, 2 μM L-Glutamine and antibiotics). On day seven, DC

were pulsed with 10 μM peptides for 2 hrs; maturation agents Poly I:C (Sigma, Oakville, Canada) at 100 ng/ml and anti-CD40 mAb (clone G28-5, ATCC, Manassas, VA) at 2 μg/ml were added in the culture and DCs were incubated at 37°C overnight or up to 48 hours. Mature DC were then irradiated (3500 rads). CD8+ cells were purified by positive selection with CD8 MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. 2x10 ⁵ CD8 ⁺ cells + 6x10 ⁴ CD8 ^" cells were stimulated with 2x10 ⁴ peptide pulsed DC in complete culture medium supplemented with 1000 IU/ml IL-6 and 5 IU/ml IL- 12 (R&D Systems, Minneapolis, MN) in round-bottomed 96 well plates. From day seven, cultures were weekly restimulated with peptide-loaded DC in the presence of 20 IU/ml IL-2 (Proleukin, Chiron Corp., Emeryville, CA) and 10 ng/ml IL-7 (R&D Systems, Minneapolis, MN). After the third in vitro restimulation, bulk cell cultures were tested for cytotoxicity.

Cytotoxic assay. Targets were labeled with 100 μCi of Cr ⁵¹ for 60 min, plated in 96-well V-bottomed plates (3x10 ³ cell/well in 100 μL of RPMI 1640 medium) and, when necessary, pulsed with peptides (1 μM) at 37°C for 2 hours. Effectors were then added in the wells and incubated at 37 ⁰ C for 4 hours. Percentage of specific lysis was determined as: Lysis = (Experimental Release - Spontaneous Release) / (Maximal Release - Spontaneous Release) x 100. Example 1: Affinity of Peptides Eight peptides with the HLA-B*0702 specific anchor motifs (P2 and preferentially L/V at C-terminal position), (Sidney et al., 1996), derived from the Hsp70 (Hsp70 _π5 , Hsp70 ₁₃₇ , Hsp70 ₃₉₇ ), TERT (TERT ₄ and TERT ₄₄₄ ) and MAGE-A (MAGE- A _121-1 , MAGE-A _121-2 and MAGE-A _121-4 .) antigens were tested for binding to the HLA- B*0702 molecule. Only TERT ₄ bound to HLA-B*0702 with a high affinity, the remaining seven peptides were very weak or non binders (Table 2). This demonstrates that the presence of anchor motifs is not sufficient to ensure a high binding affinity to HLA- B*0702. Given their low affinity, peptides Hsp70 ₁₁₅ , Hsp70 ₁₃₇ , Hsp70 ₃₉₇ , TERT ₄₄₄ , MAGE-Aj _2I-1 , MAGE-Ai _21-2 and MAGE-A _121-4 are considered cryptic peptides.

peptide sequence RA SEQ ID No

1 Hsp70 115 YPEEISSMVL >10 79 115A1 APEEISSMVL >10 80 115A1 R3 APREISSMVL 1.6 81

2 Hsp70 137 (10) YPVTNAVITV >10 82

3 Hsp70 397 APLSLGLET >10 83 397R3L9 APRSLGLEL 1 84

4 TERT4 APRCRAVRSL 0.74 85

5 TERT444 DPRRLVQLL >10 61 444A1 APRRLVQLL 1.4 78

6 MAGE-A121.1 EPVTKAEML >10 86 121.1 A1 APVTKAEML >10 87 121.1 A1 R3 APRTKAEML 2.36 88

7 MAGE-A121.2 EPFTKAEML >10 89

8 MAGE-A121.4 EPITKAEIL >10 90

Table 2: HLA-B*0702 affinity of peptides

Example 2: In vivo Immunogenicity of Peptides

HLA-B7 transgenic mice were vaccinated with the selected peptides, and eleven days later, their spleen cells were in vitro stimulated with the peptide. Peptide specific CTL were detected in mice vaccinated with the high affinity TERT ₄ peptide but not in mice vaccinated with the low affinity Hsp70 ₁₃₇ , Hsp70 ₁₁₅ , Hsp70 ₃₉₇ , and TERT ₄₄₄ peptides (Figure 1). This confirms that there is a correlation of binding affinity and immunogenicity for the HLA-B*0702 restricted peptides. Example 3: Enhancement of Immunogenicity of the Nonimmunogenic Low Affinity Peptides

To enhance HLA-B*0702 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 should however preserve the conformation of the peptide segment that interacts with the TCR (position 4 to position 8). The interest was, therefore, focused on secondary anchor positions 1 and 3. Aliphatic aminoacids are favourable motifs at position 1 (Sidney, Southwood et al., 1996). However, peptides Hsp70;u ₅ and Hsp70 ₁₃₇ that have a Y at position 1 are non binders. Moreover, the substitution of the aminoacid at position 1 by an A (present in the strong binder TERT ₄ ) enhances the affinity of the TERT ₄₄₄ but not of the Hsp70n ₅ and the MAGE-A ₁₂₁ j peptides. This indicates that the presence of favourable aa at position 1 and anchor positions 2 and 9/10 cannot ensure by itself a high binding affinity of all peptides. The R has been described to be favourable at position 3 (Sidney et al., 1996) and eight out of 26 identified tumor and HIV derived immunogenic peptides have an R at position 3 (Table 3). This is also the case of the high affinity TERT ₄ and TERT ₄₄₄ Ai peptides.

Table 3 : Tumor and HIV derived HLA-B*0702 restricted epitopes

Hsp70 ₁₁₅ , Hsp70 ₃ 9 ₇ and MAGE-A ₁₂ ] _{. IA1R3} , were therefore modified at position 1 (X-»A) and/or position 3 (X->R). For peptide Hsp70 ₃₉₇ an additional modification at C-terminal position (T- >L) has been introduced. All modified peptides 5 (Hsp70i I ₅ AiR ₃ , Hsp70 ₃ 97R ₃ L9, MAGE-A ₁₂₁ . !AiR3) exhibited a strong affinity for HLA- B*0702 (Table 2). High affinity Hsp70n ₅ AiR3, Hsp70 _397R3L 9 and TERT ₄₄₄ Ai peptide variants have been tested for their in vivo immunogenicity in HLA-B7 mice. All peptides induced an immune response in the majority of vaccinated mice. However, for all peptides but TERT ₄ 44AI, generated CTL recognized the optimized peptide but not the

10 corresponding native peptide (Figure 2). This strongly suggests that substitution of the aa at position 3 by an R may change the conformation of the peptide segment that interacts with the TCR and guarantees TCR cross-recognition. Since a) all tested peptides with A1P2R3L9/10 have a high affinity and are immunogenic and b) substitution of the aa at position 3 by an R may break the cross-recognition of the native peptide, the inventors

15 selected peptides having P2R3 and they substituted aa at position 1 by an A and at C- terminal position with an L when necessary. Table 1 above shows the list of selected peptides derived from different tumor antigens. Eleven of these peptides have been modified by replacing the first aa with an A and the C-terminal aa with a L when necessary (Table 4). All peptides in their native form had a low affinity for HLA-B*0702 0 (cryptic peptides) while all modified peptides were strong binders.

Table 4: Affinity of optimized HLA-B*0702 restricted peptides. - = RA>10, + 1<RA<1O. ++ = RA<1

In conclusion, the inventors describe a method to optimize affinity and immunogenicity of HLA-B*0702 restricted cryptic peptides. It consists of a) selecting peptides with P2R3 and b) substitute the first residue with an A and the C-terminal residue with a L when this later substitution is necessary.

Example 4: CTL Induced by TERlWu Peptide Recognize Endogenous TERT

HLA-B7 transgenic mice were immunized with the TERT _444A i and eleven days later their spleen cells were in vitro stimulated with the native TERT ₄₄₄ peptide. Generated CTL killed RMA-B7 targets loaded with decreasing concentrations of

TERT ₄₄₄ Ai and TERT ₄₄₄ peptides. The half maximal lysis of TERT ₄₄₄ loaded and

TERT ₄₄₄ Ai loaded targets was obtained with 5.5nM and InM respectively (figure 3A).

CTL were then tested for their capacity to recognize COS-7 cells expressing HLA-B*0702 and endogenous TERT. Results presented in figure 3B show that CTL recognized COS-7 cells transfected with both HLA-B *0702 and TERT but not COS-7 cells transfected with either HLA-B*0702 or TERT demonstrating that TERT ₄₄₄ is an HLA-B*0702 restricted cryptic epitope naturally processed from endogenous TERT.

Example 5: CTL Induced by the TERT ₄ Peptide Recognize Endogenous TERT

HLA-B7 transgenic mice were immunized with the TERT ₄ and eleven days later their spleen cells were in vitro stimulated with the peptide. Generated CTL killed RMA-B7 targets loaded with decreasing concentrations OfTERT ₄ peptide. The half maximal lysis of TERT ₄ loaded targets was obtained with 1.5nM (Figure 4A). CTL were then tested for their capacity to recognize COS-7 cells expressing HLA-B*0702 and endogenous TERT. Results presented in figure 4B show that CTL recognized COS-7 cells transfected with both HLA-B*0702 and TERT but not COS-7 cells transfected with either HLA-B*0702 or TERT, demonstrating that TERT ₄ is an HLA-B*0702 restricted epitope naturally processed from endogenous TERT.

Example 6: TERT ₄ Stimulates CTL from Healthy Donors

CD8 cells from healthy donors were in vitro stimulated with autologous dendritic cells loaded with TERT ₄ peptide. After four stimulations, CTL were tested for cytotoxicity against TERT ₄ loaded T2-B7 targets. Three donors were tested and CTL were induced in two of them. Results from one responding donor are presented in Figure 5A. CTL killed T2-B7 targets presenting TERT ₄ but not T2-B7 cells presenting the irrelevant Nef peptide. Interestingly, CTL killed the HLA-B* 0201+TERT+ SK-MES-I and HBL- 100 but not the HLA-B*0702-TERT+ SW-480 and HSS human tumor cell lines confirming the HLA-B*0702 restricted presentation and the endogenous processing of the TERT ₄ epitope (Figure 5B).

REFERENCES

Anderton, S. M., Viner, N. J., Matharu, P., Lowrey, P. A., and Wraith, D. C. (2002). Influence of a dominant cryptic epitope on autoimmune T cell tolerance. Nat Immunol 3, 175-181. 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.

Boisgerault, F., Anosova, N. G., Tarn, R. C, Uligens, B. M., Fedoseyeva, E. V., and Benichou, G. (2000). Induction of T-cell response to cryptic MHC determinants during allograft rejection. Hum Immunol 61, 1352-1362.

Bowne, W. B., Srinivasan, R., Wolchok, J. D., Hawkins, W. G., Blachere, N. E., Dyall, R., Lewis, J. J., and Houghton, A. N. (1999). Coupling and uncoupling of tumor immunity and autoimmunity. J Exp Med 190, 1717-1722. Cibotti, R., Kanellopoulos, J. M., Cabaniols, J. P., Halle-Panenko, O.,

Kosmatopoulos, K., Sercarz, E., and Kourilsky, P. (1992). Tolerance to a self-protein involves its immunodominant but does not involve its subdominant determinants. Proc Natl Acad Sci U S A 89, 416-420.

Colella, T. A., Bullock, T. N., Russell, L. B., Mullins, D. W., Overwijk, W. W., Luckey, C. J., Pierce, R. A., Restifo, N. P., and Engelhard, V. H. (2000). Self- tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy. J Exp Med 191, 1221-1232.

Disis, M. L., Gooley, T. A., Rinn, K., Davis, D., Piepkorn, M., Cheever, M. A., Knutson, K. L., and Schiffman, K. (2002). Generation of T-cell immunity to the HER-2/neu protein after active immunization with HER-2/neu peptide-based vaccines. J Clin Oncol 20, 2624-2632.

Dyall, R., Bowne, W. B., Weber, L. W., LeMaoult, J., Szabo, P., Moroi, Y., Piskun, G., Lewis, J. J., Houghton, A. N., and Nikolic-Zugic, J. (1998). Heteroclitic immunization induces tumor immunity. J Exp Med 188, 1553-1561. 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.

Espevik, T., and Nissen-Meyer, J. (1986). A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J Immunol Methods 95, 99-105.

Friedman, R. S., Spies, A. G., and Kalos, M. (2004). Identification of naturally processed CD8 T cell epitopes from prostein, a prostate tissue-specific vaccine candidate. Eur J Immunol 34, 1091-1101.

Gross, D. A., Graff-Dubois, S., Opolon, P., Cornet, S., Alves, P., Bennaceur-Griscelli, A., Faure, O., Guillaume, P., Firat, H., Chouaib, S., et al. (2004). High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy. J Clin Invest 113, 425-433. Grossmann, M. E., Davila, E., and Celis, E. (2001). Avoiding Tolerance

Against Prostatic Antigens With Subdominant Peptide Epitopes. J Immunother 24, 237- 241.

Hawkins, W. G., Gold, J. S., Dyall, R., Wolchok, J. D., Hoos, A., Bowne, W. B., Srinivasan, R., Houghton, A. N., and Lewis, J. J. (2000). Immunization with DNA coding for gplOO results in CD4 T-cell independent antitumor immunity. Surgery 128, 273-280.

Hernandez, J., Lee, P. P., Davis, M. M., and Sherman, L. A. (2000). The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self tolerance on the TCR repertoire. J Irnmunol 164, 596-602. Lally, K. M., Mocellin, S., Ohnmacht, G. A., Nielsen, M. B., Bettinotti,

M., Panelli, M. C, Monsurro, V., and Marincola, F. M. (2001). Unmasking cryptic epitopes after loss of immunodominant tumor antigen expression through epitope spreading. Int J Cancer 93, 841-847.

Meyerson, M., Counter, C. M., Eaton, E. N., Ellisen, L. W., Steiner, P., Caddie, S. D., Ziaugra, L., Beijersbergen, R. L., Davidoff, M. J., Liu, Q., et al (1997). hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90, 785-795.

Meziere, C, Viguier, M., Dumortier, H., Lo-Man, R., Leclerc, C, Guillet, J. G., Briand, J. P., and Muller, S. (1997). In vivo T helper cell response to retro- inverso peptidomimetics. J Immunol 159, 3230-3237.

Moudgil, K. D., and Sercarz, E. E. (1994a). Can antitumor immune responses discriminate between self and nonself? Immunol Today 15, 353-355.

Moudgil, K. D., and Sercarz, E. E. (1994b). The T cell repertoire against cryptic self determinants and its involvement in autoimmunity and cancer. Clin Immunol Immunopathol 73, 283-289.

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. Naftzger, C, Takechi, Y., Kohda, H., Hara, L, Vijayasaradhi, S., and

Houghton, A. N. (1996). Immune response to a differentiation antigen induced by altered antigen: a study of tumor rejection and autoimmunity. Proc Natl Acad Sci U S A 93, 14809-14814.

Overwijk, W. W., Theoret, M. R., Finkelstein, S. E., Surman, D. R., de Jong, L. A., Vyth-Dreese, F. A., Dellemijn, T. A., Antony, P. A., Spiess, P. J., Palmer, D. C, et al. (2003). Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J Exp Med 198, 569-580. Overwijk, W. W., Tsung, A., Irvine, K. R., Parkhurst, M. R., Goletz, T.

J., Tsung, K., Carroll, M. W., Liu, C, Moss, B., Rosenberg, S. A., and Restifo, N. P. (1998). gplOO/pmel 17 is a murine tumor rejection antigen: induction of "self -reactive, tumoricidal T cells using high-affinity, altered peptide ligand. J Exp Med 188, 277-286.

Palomba, M. L., Roberts, W. K., Dao, T., Manukian, G., Guevara- Patino, J. A., Wolchok, J. D., Scheinberg, D. A., and Houghton, A. N. (2005). CD8+ T- cell-dependent immunity following xenogeneic DNA immunization against CD20 in a tumor challenge model of B-cell lymphoma. Clin Cancer Res 11, 370-379.

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 gplOO modified at HLA-A*0201- binding residues. J Immunol 157, 2539-2548.

Paterson, Y., and Maciag, P. C. (2005). Listeria-based vaccines for cancer treatment. Curr Opin MoI Ther 7, 454-460.

Rohrlich, P. S., Cardinaud, S., Firat, H., Lamari, M., Briand, P., Escriou, N., and Lemonnier, F. A. (2003). HLA-B*0702 transgenic, H-2KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus. Int Immunol 15, 765-772.

Sarobe, P., Pendleton, C. D., Akatsuka, T., Lau, D., Engelhard, V. H.,

Feinstone, S. M., and Berzofsky, J. A. (1998). Enhanced in vitro potency and in vivo immunogenicity of a CTL epitope from hepatitis C virus core protein following amino acid replacement at secondary HLA- A2.1 binding positions. J Clin Invest 102, 1239-1248.

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., et al. (2002). HER-

2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy. J Immunol 168, 5900-5906.

Sidney, J., Southwood, S., del Guercio, M. F., Grey, H. M., Chesnut, R. W., Kubo, R. T., and Sette, A. (1996). Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules. J Immunol 157, 3480-3490.

Sinha, P., Chi, H. H., Kim, H. R., Clausen, B. E., Pederson, B., Sercarz, E. E., Forster, L, and Moudgil, K. D. (2004). Mouse lysozyme-M knockout mice reveal how the self-determinant hierarchy shapes the T cell repertoire against this circulating self antigen in wild-type mice. J Immunol 173, 1763-1771.

Theobald, M., Biggs, J., Hernandez, J., Lustgarten, J., Labadie, C, and Sherman, L. A. (1997). Tolerance to p53 by A2.1 -restricted cytotoxic T lymphocytes. J Exp Med 185, 833-841.

Tourdot, S., Scardino, A., Saloustrou, E., Gross, D. A., Pascolo, S., Cordopatis, P., Lemonnier, F. A., and Kosmatopoulos, K. (2000). A general strategy to enhance immunogenicity of low-affinity HLA-A2. 1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol 30, 3411-3421.

Valmori, D., Fonteneau, J. F., Lizana, C. M., Gervois, N., Lienard, D., Rimoldi, D., Jongeneel, V., Jotereau, F., Cerottini, J. C, and Romero, P. (1998). Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan- A/MART- 1 immunodominant peptide analogues. J Immunol 160, 1750-1758.

Vierboom, M. P., Nijman, H. W., Offringa, R., van der Voort, E. L, van Hall, T., van den Broek, L., Fleuren, G. J., Kenemans, P., Kast, W. M., and Melief, C. J. (1997). Tumor eradication by wild-type p53-specific cytotoxic T lymphocytes. J Exp Med 186, 695-704.

Weber, L. W., Bowne, W. B., Wolchok, J. D., Srinivasan, R., Qin, J., Moroi, Y., Clynes, R., Song, P., Lewis, J. J., and Houghton, A. N. (1998). Tumor immunity and autoimmunity induced by immunization with homologous DNA. J Clin Invest 102, 1258-1264.