Identification, Optimization And Use Of Cryptic HLA-B7 Epitopes For Immunotherapy

Abstract

The invention provides methods for identifying a HLA-B*0702-restricted cryptic epitope in an antigen, as well as methods for increasing the immunogenicity of HLA-B*0702-restricted cryptic epitopes. The HLA-B*0702-restricted cryptic epitopes and their cognate immunogenic epitopes are useful for stimulating an immune reaction against the cryptic epitopes in a subject. Accordingly, the invention further provides pharmaceutical compositions comprising a HLA-B*0702-restricted cryptic epitope or a cognate immunogenic epitope thereof, and vaccination kits comprising such epitopes. The novel materials of the invention are particularly useful for efficiently treating patients having an HLA-B*0702 phenotype.

Claims

1-20. (canceled)

21. An immunogenic HLA-B*0702-restricted epitope having the sequence APRRLVQLL (SEQ ID NO: 5).

22. A chimeric polypeptide, comprising the peptide DPRRLVQLL (SEQ ID NO: 1) and one, two or more HLA-B*0702-restricted cryptic epitopes selected from the group consisting of GPKHSDCLA (SEQ ID NO: 4), SPKANKEIL (SEQ ID NO: 3), APRSPLAPS (SEQ ID NO: 2), and DPRRLVQLL (SEQ ID NO: 1).

23. A chimeric polypeptide, comprising SEQ ID No: 5 and one, two or more immunogenic HLA-B*0702-restricted epitopes selected from the group consisting of APKHSDCLA (SEQ ID NO: 8), APKANKEIL (SEQ ID NO: 7), APRSPLAPL (SEQ ID NO: 6), and APRRLVQLL (SEQ ID NO: 5).

24. A pharmaceutical composition comprising at least, as an active principle, an immunogenic HLA-B*0702-restricted epitope polypeptide according to claim 21.

25. A pharmaceutical composition comprising at least, as an active principle, a chimeric polypeptide according to claim 22.

26. A pharmaceutical composition comprising at least, as an active principle, a chimeric polypeptide according to claim 23.

27. The pharmaceutical composition of any one of claims 24 to 26, which is a vaccine.

28. A kit of parts comprising, in separate formulations, a first chimeric polypeptide according to claim 22, and a second chimeric polypeptide according to claim 23.

29. The kit according to claim 28, which is a vaccination kit, wherein said first and second chimeric polypeptides are in separate vaccination doses.

30. The vaccination kit according to claim 29, which comprises 2 or 3 doses of second chimeric polypeptide, and 3, 4, 5, 6 or up to 50 doses of first chimeric polypeptide.

31. The vaccination kit according to claim 29, wherein each dose comprises 1 to 20 mg of chimeric polypeptide.

32. The vaccination kit according to claim 29, wherein the vaccination doses are formulated for subcutaneous injection.

33. A kit of parts comprising, in separate formulations, a chimeric polypeptide according to claim 22 and an immunogenic peptide according to claim 21.

34. The kit according to claim 33, which is a vaccination kit, wherein said chimeric polypeptide and immunogenic peptide are in separate vaccination doses.

35. The vaccination kit according to claim 33, which comprises 2 or 3 doses of immunogenic peptide, and 3, 4, 5, 6 or up to 50 doses of chimeric polypeptide.

36. The vaccination kit according to claim 35, wherein each dose comprises 1 to 20 mg of chimeric polypeptide or of immunogenic peptide.

37. The vaccination kit according to claim 33, wherein the vaccination doses are formulated for subcutaneous injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1: Immunogenicity of HLA-B*0702 restricted peptides. CTL were tested against RMA-B7 targets loaded with peptide as indicated.

[0048] FIG. 2: Immunogenicity of optimized HLA-B*0702 cryptic peptides. CTL were tested against RMA-B7 targets loaded with peptide as indicated.

[0049] FIG. 3: In vivo immunogenicity of optimized HLA-B*0702 Her2/neu.sub.1069L9 (A) and Her2/neu.sub.1069 (B) peptides in HLA-B*0702 transgenic mice. CTL were tested against RMA-B7 targets loaded with peptide as indicated. CTL population induced was diluted 3 (1), 10 (2), 30 (3) and 100 (4) fold.

[0050] FIG. 4: TERT.sub.4 induces TERT specific CTL in HLA-B7 mice and in healthy donors. (A) TERT.sub.4 immunogenicity in HLA-B*0702 transgenic mice. CTL were tested against RMA-B7 targets loaded with decreasing doses of TERT.sub.4 peptide. (B) Recognition of endogenous TERT by TERT.sub.4 specific murine CTL. CTL were tested against COS cells transfected with HLA-B*0702 and TERT as indicated. (C) Induction of TERT.sub.4 specific human CTL. CTL were tested against T2-B7 targets loaded with TERT.sub.4 (.square-solid.) or an irrelevant () peptide using the Effector/Target ratio as indicated (left graph), and against the HLA-B*0702 positive TERT positive SK-MES-1 (.square-solid.), HBL-100 () and the HLA-B*0702 negative TERT positive SW-480 (), HSS () human tumor cell lines (right graph).

[0051] FIG. 5: Recognition of endogenous TERT by TERT.sub.444 specific murine CTL. (A) CTL were tested against RMA-B7 targets loaded with decreasing doses of TERT.sub.444 or TERT.sub.444A1 peptides as indicated. (B) CTL were tested against COS cells transfected with HLA-B*0702 and/or TERT as indicated.

[0052] FIG. 6: Induction of TERT.sub.444A1 specific human CTL. CTL were tested against T2-B7 targets loaded with peptides as indicated. CTL maximal activation is obtained by PMA/ionomycin treatment.

DETAILED DESCRIPTION

[0053] The examples have been performed using the following materials and methods:

[0054] Transgenic Mice. The HLA-B7 H-2 class-I knockout mice were previously described (Rohrlich et al., 2003).

[0055] 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*0702 positive SK-MES-1 (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 RPMI1640 culture medium.

[0056] Peptides and Plasmids. Peptides were synthesized by Epytop (Nimes, 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, Mass.) (Meyerson et al, 1997).

[0057] 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 M to 0.1 M, and then stained with ME-1 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 M of the reference peptide CMV.sub.265-274 (R10V; RPHERNGFTV, SEQ ID NO: 9). 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).

[0058] CTL Induction in vivo in HLA-B*0702 Transgenic Mice. Mice were injected subcutaneously with 100 g of peptide emulsified in Incomplete Freund's Adjuvant (IFA) in the presence of 150 g of the I-A.sup.b restricted HBVcore.sub.128 T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 10). After 11 days, 510.sup.7 spleen cells were stimulated in vitro with peptide (10 M). On day 6 of culture, the bulk responder populations were tested for specific cytotoxicity.

[0059] Peptide Processing Assay on COS-7 Transfected Cells. 2.210.sup.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 TNF secretion assay.

[0060] TNF Secretion Assay. Transfected COS-7 cells at day 4 were suspended in 500 of RPMI+10% FCS and used as stimulating cells. 510.sup.4 murine T cells were then added in 50 l RPMI 10% FCS and incubated for 6 hours. Each condition was tested in triplicate. 50 l of the supernatant was collected to measure TNF. Standard dilutions were prepared in 50 l with final doses of TNF ranging from 104 to 0 pg/ml. On both the supernatants and the standard dilutions, 310.sup.4 TNF sensitive WEHI-164c13 cells in 50 l were added. They were incubated for 16 h at 37 C. Inhibition of cell proliferation was evaluated by the MTT colorimetric method (Espevik and Nissen-Meyer, 1986).

[0061] Generation of CTL from human PBMC. PBMC were collected by leukapheresis from healthy HLA-B*0702 volunteers. Dendritic cells (DC) were produced from adherent cells cultured for seven days (210.sup.6 cells/ml) in the presence of 500 IU/ml GM-CSF and 500 IU/ml IL-4 (R&D Systems, Minneapolis, Minn.) in complete medium (RPMI-1640 supplemented with 10% heat inactivated human AB serum, 2 M L-Glutamine and antibiotics). On day seven, DC were pulsed with 10 M 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 g/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, Calif.) according to the manufacturer's instructions. 210.sup.5 CD8.sup.+ cells+610.sup.4 CD8.sup. cells were stimulated with 210.sup.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, Minn.) 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, Calif.) and 10 ng/ml IL-7 (R&D Systems, Minneapolis, Minn.). After the third in vitro restimulation, bulk cell cultures were tested for cytotoxicity (TERT.sub.4) or for IFN intracellular staining (TERT.sub.444A1).

[0062] Cytotoxic assay. Targets were labelled with 100 Ci of Cr.sup.51 for 60 min, plated in 96-well V-bottomed plates (310.sup.3 cell/well in 100 L of RPMI 1640 medium) and, when necessary, pulsed with peptides (1 M) at 37 C. for 2 hours. Effectors were then added in the wells and incubated at 37 C. for 4 hours. Percentage of specific lysis was determined as: % Lysis=(Experimental ReleaseSpontaneous Release)/(Maximal ReleaseSpontaneous Release)100.

[0063] IFN intracellular staining. T cells (10.sup.5) were incubated with 210.sup.5 T2 cells loaded with stimulating peptide in the presence of 20 g/ml Brefeldin-A (Sigma, Oakville, Canada). Six hours later they were washed, stained with r-phycoerythrin-conjugated anti-CD8 antibody (Caltag Laboratories, Burlingame, Calif., USA) in PBS for 25 min at 4 C., washed again, and fixed with 4% PFA. The cells were then permeabilized with PBS, 0.5% BSA, 0.2% saponin (Sigma, Oakville, Canada), and labeled with an allophycocyanin-conjugated anti-IFN mAb (PharMingen, Mississauga, Canada) for 25 min at 4 C. before analysis with a FACSCalibur flow cytometer.

Examples

Example 1: Affinity of Peptides

[0064] Eight peptides with the HLA-B*0702 specific anchor motifs, i.e. P2 and preferentially L/V at C-terminal position (Sidney et al., 1996) belonging to Hsp70 (Hsp70.sub.115, Hsp70.sub.137, Hsp70.sub.397), TERT (TERT.sub.4 and TERT.sub.444), and MAGE-A (MAGE-A.sub.121.1, MAGE-A.sub.121.2 and MAGE-A.sub.121.4) antigens were tested for binding to the HLA-B*0702 molecule. Only TERT.sub.4 bound to HLA-B*0702 with a high affinity, the remaining seven peptides were very weak or non binders (Table II). 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.sub.115, Hsp70.sub.137, Hsp70.sub.397, TERT.sub.444, MAGE-A.sub.121.1, MAGE-A.sub.121.2, MAGE-A.sub.121.4, are considered cryptic peptides.

TABLE-US-00002 TABLEII HLA-B*0702affinityofpeptides SEQID Peptide Sequence RA NO: 1 Hsp70115 YPEEISSMVL >10 11 Hsp70115A1 APEEISSMVL >10 12 2 Hsp70137(10) YPVTNAVITV >10 13 3 Hsp70397 APLSLGLET >10 14 4 TERT4 APRCRAVRSL 0.74 15 5 TERT444 DPRRLVQLL >10 1 TERT444A1 APRRLVQLL 1.4 5 6 MAGE-A121.1 EPVTKAEML >10 16 MAGE-121.1/A1 APVTKAEML >10 17 7 MAGE-A121.2 EPFTKAEML >10 18 8 MAGE-A121.4 EPITKAEIL >10 19

Example 2: Immunogenicity of Selected Peptides

[0065] The low affinity Hsp.sub.137, Hsp.sub.115, Hsp.sub.397, TERT.sub.444 and the high affinity TERT.sub.4 peptides have been tested for their capacity to induce a specific CTL immune response in HLA-B*0702 transgenic mice. Only the high affinity TERT.sub.4 was immunogenic confirming that immunogenicity of peptides is strongly related to their affinity for HLA (FIG. 1).

Example 3: Enhancement of Affinity of Low Affinity Peptides

[0066] Since all these cryptic peptides had favourable primary anchor motifs, enhancement of their affinity is a prerequisite for them to be immunogenic. It required the identification of unfavourable secondary anchor motifs and their substitution 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 amino acids are favourable motifs at position 1 (Sidney, Southwood et al., 1996). However, peptides Hsp70.sub.115 and Hsp70.sub.137 that have a Y (tyrosine) at position 1 are non binders. Moreover, the substitution of the amino acid at position 1 by an A (alanine) that is also favourable at this position. (Parker et al, 1994) enhances the affinity of the TERT.sub.444 but not of the Hsp70.sub.115 and the MAGE-A.sub.121.1 peptides (Table II). This indicates that the presence of favourable amino acids at position 1 and anchor positions 2 and 9/10 cannot ensure by itself a high binding affinity of all peptides. In the other hand, positively charged peptides (R/H/K) have been described to be favourable at position 3 (Sidney et al., 1996) and ten out of 26 identified tumor and HIV derived immunogenic peptides have an R/K/H at position 3 (Table III).

TABLE-US-00003 TABLEIII TumorandHIVderivedHLA-B*0702restrictedepitopes SEQID Antigen Sequence NO: Reference NY-ESO-1 APRGVRMAV 20 Slageretal,2004 ICE SPRWWPTCL 21 Ronsinetal.,1999 RAGE-1 SPSSNRIRNT 22 Gaugleretal.,1996 RU2AS LPRWPPPQL 23 VanDenEyndeetal.,1999 RBAF500 RPHVPESAF 24 Lennerzetal.,2005 SSX2fusionprotein QPRYGYDQIM 25 Worleyetal,2001 HIVp17 RPGGKKRYKL 26 HIVMolecularImmunology HIVp24 SPRTLNAWV 27 Database(OperatedbyLos HIVp24 HPVHAGPIA 28 AlamosNationalSecurity,LLC, HIVp24 PPIPVGEIY 29 fortheU.S.Departmentof HIVp24 GPGHKARVL 30 Energy'sNationalNuclear HIV-RT SPIETVPVKL 31 SecurityAdministration) HIV-RT GPKVKQWPLT 32 HIV-RT SPAIFQSSM 33 HIV-RT IPLTEEAEL 34 HIV-RT QPDKSESELV 35 HIV-Vif HPRISSEVHI 36 HIV-Vif KPPLPSVKKL 37 HIV-Vif FPRTWLHGL 38 HIVgp160 KPCVKLTPLC 39 HIVgp160 KVVSTQLLL 40 HIVgp160 RPWNNTRKSI 41 HIVgp160 IPRRIRQGL 42 HIVnef FPVTPQVPL 43 HIVnef TPQVPLRPM 44

[0067] According to all these observations, peptides with the sequence APX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11 (SEQ ID NO: 61) should have a high affinity for HLA-B*0702. This is confirmed by results shown in Table IV. All eighteen peptides with the above cited sequence had a high affinity and/or were immunogenic in HLA-B*0702 transgenic mice.

TABLE-US-00004 TABLEIV AffinityandimmunogenicityofAPX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11 HLA-B*0702restrictedpeptides.-= RA> 10, + = 1< RA< 10,++ = RA< 1,Immunogenicitywas testedasdescribedinExemplein2.+:means thataspecificimmunoresponsewasgeneratedat leastoneHLA-B*0702transgenicmice,ND:Not Determined Sequence SEQIDNO: RA Immunogenicity APRRLVQLL 5 + + APRSPLAPL 6 ++ + APKANKEIL 7 ND + APKHSDCLA 8 ND + APRCRAVRSL 15 + + APRMHCAVDL 45 ++ + APRVSIRLPL 46 ++ ND APREYVNAL 47 + + APRGVPQIEL 48 ND + APRALVETL 49 + + APRMPEAAL 50 ND + APRRRLGCEL 51 + + APRPWTPCL 52 + + APRASSPLL 53 ND + APRQLGREL 54 ND + APREISSMVL 55 + + APRSLGLEL 56 ++ + APRTKAEML 57 + +

Example 4: In Vivo Immunogenicity of Peptides with Enhanced Affinity and Recognition of the Native Counterpart

[0068] HLA-B7 transgenic mice were vaccinated with the selected peptides, and eleven days later, their spleen cells were in vitro stimulated with the peptide.

[0069] In this context, Hsp70.sub.115, Hsp70.sub.397 and TERT.sub.444, were therefore modified at position 1 (substitution of the amino acid by an A) and/or position 3 (substitution of the amino acid by an R). For peptide Hsp70.sub.397 an additional modification at C-terminal position (substitution of the T by an L) has been introduced. Modified peptides i.e. Hsp70.sub.115A1R3 (SEQ ID NO: 55), Hsp70.sub.397R3L9 (SEQ ID NO: 56), TERT.sub.444A1 (SEQ ID NO: 5) exhibited a strong affinity for HLA-B*0702 (Table IV) and induced an immune response in the majority of vaccinated mice (FIG. 2). However, for all peptides but TERT.sub.444A1, generated CTL recognized the optimized peptide but not the corresponding native peptide (FIG. 2). This strongly suggests that substitution of the amino acid at position 3 by an R may change the conformation of the peptide segment that interacts with the TCR and guarantees TCR cross-recognition.

[0070] Since a) all tested peptides with APX.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11 have a high affinity and are immunogenic (Table IV and FIG. 1, 2) and b) substitution of the amino acid at position 3 by an R may break the cross-recognition of the native peptide, the inventors selected native peptides having a P and R at positions 2 and 3 respectively, and they substituted the amino acid at position 1 by an A if the last amino acid was favourable (L, A, I, V or M). Given the high importance of position 3 in both affinity and CTL recognition of HLA-B*0702 restricted peptides inventors selected peptides with the sequence X.sub.1PX.sub.3 (wherein X.sub.1 is any amino acid and X.sub.3 is K, R, H or M; these amino acids have been described as being favourable residues at position 3) and a favourable amino acid (A/I/L/V) at C-terminal position. Peptides with this sequence and low affinity for HLA-B*0702 have been modified by substitution of the first residue by an A. This is the case of TERT.sub.444, Her-2/neu.sub.760 and Her-2/neu.sub.246. Inventors also selected peptides with the sequence APX.sub.3 (wherein X.sub.3 is K, R, H or M) and a non favourable residue at C-terminal position (i.e., an amino acid other than L, A, I, V or M). Peptides with this sequence and low affinity for HLA-B*0702 have been modified by substituting residue at C-terminal position with a L. This is the case of Her-2/neu.sub.1069. All these modified peptides had a strong affinity for HLA-B*0702.

Example 5: Immunogenicity of Optimized Peptides and Cross-Recognition of the Native Counterpart

[0071] Native Her2/neu.sub.246, Her2/neu.sub.760, Her2/neu.sub.1069 and TERT.sub.444 peptides were not immunogenic, whereas the optimized peptides were immunogenic in HLA-B*0702 transgenic mice. Moreover, CTL induced by all these optimized peptides were able to cross-react with the corresponding native peptide (FIG. 3 and Table V).

TABLE-US-00005 TABLEV ImmunogenicityofnativeandoptimizedHLA-B*0702restrictedpeptides. + forimmunogenicityornativepeptidecrossrecognitionmeansthat thepeptideinducedaspecificresponseinatleastoneHLA-B*0702 transgenicmouse,abletorecognizedthecorrespondingnative peptide. Correspondingnative SEQID Immuno- peptide Peptide Sequence NO: genicity cross-reconnaissance TERT.sub.444 DPRRLVQLL 1 - TERT.sub.444A1 APRRLVQLL 5 + + Her2/neu.sub.760 SPKANKEIL 3 - Her2/neu.sub.760A1 APKANKEIL 7 + + Her2/neu.sub.246 GPKHSDCLA 4 - Her2/neu.sub.246A1 APKHSDCLA 8 + + Her2/neu.sub.1069 APRSPLAPS 2 - Her2/neu.sub.1069L9 APRSPLAPL 6 + +

[0072] In conclusion, the inventors have described a method to optimize immunogenicity (and also affinity) of HLA-B*0702 restricted cryptic peptides. It consists in a) substituting the residue at position 1 with an A in all peptides comprising the sequence X.sub.1PX.sub.3 (wherein X.sub.1 is any amino acid except A and X.sub.3 is R or K or H or M), a favourable amino acid at C-terminal position (i.e., L or A or I or V or M), and a low affinity for HLA-B*0702, or b) substituting the residue at C-terminal position with a L in peptides comprising the sequence APX.sub.3 (X.sub.3 being defined as above), a non favourable residue at C-terminal position (i.e., an amino acid other that L or A or I or V or M), and a low affinity for HLA-B*0702.

Example 6: TERT.SUB.4 .Immunodominant Peptide Induces TERT Specific CTL

[0073] HLA-B7 transgenic mice were then immunized with the TERT.sub.4 (SEQ ID NO: 15) and eleven days later their spleen cells were in vitro stimulated with the peptide. Generated CTL killed RMA-B7 targets loaded with decreasing concentrations of TERT.sub.4 peptide (FIG. 4A). The half maximal lysis of TERT.sub.4 loaded targets was obtained with 1.5 nM (FIG. 4A). CTL were then tested for their capacity to recognize COS-7 cells expressing HLA-B*0702 and endogenous TERT. Results presented in FIG. 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.sub.4 dominant peptide is an HLA-B*0702 restricted epitope naturally processed from endogenous TERT.

[0074] Moreover, CD8 cells from healthy donors were in vitro stimulated with autologous dendritic cells loaded with TERT.sub.4 peptide. After four stimulations, CTL were tested for cytotoxicity against TERT.sub.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 FIG. 4C. CTL killed T2-B7 targets presenting TERT.sub.4 but not T2-B7 cells presenting the irrelevant Nef peptide (left graph). Interestingly, CTL killed the HLA-B*0702 TERT+SK-MES-1 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.sub.4 epitope (right graph).

Example 7: CTL Induced by TERT.SUB.444A1 .Peptide Recognize Endogenous TERT

[0075] TERT.sub.444A1 (SEQ ID NO: 5) was tested for its ability to induce CTL able to recognize endogenous TERT and to induce CTL in healthy donors (Example 6). HLA-B*0702 transgenic mice were then immunized with the TERT.sub.444A1 and eleven days later their spleen cells were in vitro stimulated with the native TERT.sub.444 peptide (SEQ ID NO: 1). Generated CTL killed RMA-B7 targets loaded with decreasing concentrations of TERT.sub.444A1 and TERT.sub.444 peptides. The half maximal lysis of TERT.sub.444 loaded and TERT.sub.444A1 loaded targets was obtained with 5.5 nM and 1 nM respectively (FIG. 5A). CTL were then tested for their capacity to recognize COS-7 cells expressing HLA-B*0702 and endogenous TERT. Results presented in FIG. 5B 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.sub.444 is an HLA-B*0702 restricted cryptic epitope naturally processed from endogenous TERT.

Example 8: TERT.SUB.444A1 .Stimulates CTL from Healthy Donors

[0076] CD8 cells from healthy donors were in vitro stimulated with autologous dendritic cells loaded with TERT.sub.444A1 peptide. After four stimulations, proliferating cells were divided into 4 pools. Each pool was then tested for intracellular IFNg production upon stimulation with T2-B7 cells loaded with optimized TERT.sub.444A1 or native TERT.sub.444. Results from D5609 responding donor are presented in FIG. 6. IFNg producing CTL were detected in pools 2 and 4 after stimulation with either TERT.sub.444 or TERT.sub.444A1 loaded T2B7 cells (FIG. 6).

REFERENCES

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