POLYNUCLEOTIDES ENCODING MHC CLASS I-RESTRICTED HTERT EPITOPES, ANALOGUES THEREOF OR POLYEPITOPES

Abstract

This invention relates to the field of anticancer therapy, and to the identification of immunogenic peptides derived from the human telomerase reverse transcriptase (hTERT). The present invention relates to polynucleotides encoding hTERT epitopes restricted to MHC class I molecule, analogues thereof and polyepitopes containing such epitopes and/or analogues. Are also included in the present invention, vector and cell comprising such polynucleotides. The present invention also concerns composition comprising hTERT polypeptides, corresponding polynucleotides, vectors and cells, for use in the treatment and/or prevention of cancer.

Claims

1-74. (canceled)

75. A method of inducing an HLA-A2 and/or HLA-B7-restricted immune response against at least one hTERT epitope, comprising: A) administering to an animal a vector comprising a polynucleotide sequence that encodes a modified hTERT protein comprising the amino acid sequence of SEQ ID NO: 39; B) expressing the modified hTERT protein; and C) inducing: 1) an HLA-A2-restricted immune response against at least one hTERT epitope selected from: a) p540, and b) pY572; and/or 2) inducing an HLA-B7-restricted immune response against at least one hTERT epitope selected from: TABLE-US-00015 a) (SEQ ID NO: 6) MPRAPRCRA (p1), b) (SEQ ID NO: 7) APRCRAVRSL (p4), c) (SEQ ID NO: 8) APSFRQVSCL (p68), d) (SEQ ID NO: 9) RPAEEATSL (p277), e) (SEQ ID NO: 10) RPSFLLSSL (p342), and f) (SEQ ID NO: 11) RPSLTGARRL (p351).

76. The method of claim 75, wherein the vector is a viral vector.

77. The method of claim 75, further comprising providing antigen presenting cells (APCs), transforming the APCs in vitro with the vector comprising the polynucleotide sequence that encodes a modified hTERT protein, and administering the APCs to the animal.

78. The method of claim 77, wherein the APCs are dendritic cells.

79. The method of claim 75, wherein the animal is a human.

80. The method of claim 75, the method comprising inducing an HLA-A2-restricted immune response against the hTERT epitopes a) p540 and b) pY572.

81. The method of claim 80, wherein the vector is a viral vector.

82. The method of claim 80, further comprising providing antigen presenting cells (APCs), transforming the APCs in vitro with the lentiviral vector comprising the polynucleotide sequence that encodes a modified hTERT protein, and administering the APCs to the animal.

83. The method of claim 82, wherein the APCs are dendritic cells.

84. The method of claim 80, wherein the animal is a human.

85. The method of claim 75, the method comprising inducing an HLA-B7-restricted immune response against at least two hTERT epitopes selected from: TABLE-US-00016 a. (SEQ ID NO: 6) MPRAPRCRA (p1), b. (SEQ ID NO: 7) APRCRAVRSL, (p4) c. (SEQ ID NO: 8) APSFRQVSCL (p68), d. (SEQ ID NO: 9) RPAEEATSL (p277), e. (SEQ ID NO: 10) RPSFLLSSL (p342), and f. (SEQ ID NO: 11) RPSLTGARRL (p351).

86. The method of claim 85, wherein the vector is a viral vector.

87. The method of claim 85, further comprising providing antigen presenting cells (APCs), transforming the APCs in vitro with the vector comprising the polynucleotide sequence that encodes a modified hTERT protein, and administering the APCs to the animal.

88. The method of claim 87, wherein the APCs are dendritic cells.

89. The method of claim 85, wherein the animal is a human.

90. The method of claim 75, the method comprising inducing an HLA-B7-restricted immune response against each of hTERT epitopes: TABLE-US-00017 a. (SEQ ID NO: 6) MPRAPRCRA (p1), b. (SEQ ID NO: 7) APRCRAVRSL, (p4) c. (SEQ ID NO: 8) APSFRQVSCL (p68), d. (SEQ ID NO: 9) RPAEEATSL (p277), e. (SEQ ID NO: 10) RPSFLLSSL (p342), and f. (SEQ ID NO: 11) RPSLTGARRL (p351).

91. The method of claim 90, wherein the vector is a viral vector.

92. The method of claim 90, further comprising providing antigen presenting cells (APCs), transforming the APCs in vitro with the vector comprising the polynucleotide sequence that encodes a modified hTERT protein, and administering the APCs to the animal.

93. The method of claim 92, wherein the APCs are dendritic cells.

94. The method of claim 90, wherein the animal is a human.

Description

[0123] Each definition provided in the specification applies to each and any peptide (epitope, analogue or polyepitope) as well as to each and any polynucleotide, taken individually (as such) or encompassed in a group.

[0124] FIGS. 1A to 1F: Gene encoding the hTERT protein (SEQ ID NO: 36) and corresponding amino acid sequence (SEQ ID NO: 37). The coding sequence is located between the nucleotide 56 and 3454. Initiation and termination codons are underlined. First line is the nucleotide sequence; second line is the corresponding amino acid sequence. Third line is the numerotation of the hTERT coding sequence, starting from the initiation codon as the first amino acid.

[0125] FIG. 2: hTERT derived peptides are processed in HLA-B0702 transgenic mice. HLA-B7 Tg mice and one naïve mice (N) were immunized with 100 μg DNA encoding Htert. On day 14, spleen cells from each mouse were separately in vitro stimulated with different hTERT-derived peptides. Effector cells were essayed 6 days later against RMA-B7 targets loaded with relevant [[(.square-solid.)]] (shaded) or control [[(□)]] (unshaded) peptides as described in the material and methods. Percentage of lysis at a 60/1 ratio are shown (results from two independent experiments).

[0126] FIG. 3: Induction of CTL response against hTERT in PBMC from health blood donors. T-lymphocyte cells from HLA-*B0702.sup.+ healthy donors were activated with each of the six hTERT peptide-pulsed autologous PBMC as detailed in materials and Method. After four rounds of weekly stimulation, effector cells, pulsed with relevant [[(.square-solid.)]] (shaded) or control [[(□)]] (unshaded) peptides, were essayed for lytic activity against .sup.51Cr-labeled T2-B7 cells. Percentage of lysis at a 20/1 effector-target ratio is shown. Results from δ out of 10 donors are presented (d1 to dδ).

[0127] FIG. 4: Effect of an anti-HLA class I mAb on cytotoxicity of CTLp351 against tumor cells. The cytotoxicity of the CTLp351 line against HLA-*B0702.sup.+ tumor cell lines Mamo and U293T pre-treated either in absence (none) or presence of HLA mAbs (anti-HLA class I mAb or an anti-class I1 mAb (HLA-DR)) was determined by standard .sup.51Cr-release assay at effector-target ratio of 10/1.

[0128] FIG. 5: Ex-vivo detection of hTERT-specific T-cell response after Lv-hTERT immunization. A) HLA-B7 transgenic mice were immunized with recombinant Trip-hTERT particles or control Trip-GFP (1500 ng). After 12 days, hTERT peptide-specific T cells producing IFNγ of each mouse were detected ex vivo by IFNγ-ELISPOT assay within freshly spleen cells. The number of IFNγ SFCs was calculated after subtracting negative control values. Results from three independent experiments are represented.

[0129] B) HHD mice were immunized with Trip-hTERT as described above. hTERT peptide-specific T cells producing IFNγ were detected ex vivo by ELISPOT as described above. Results from two independent experiments are represented.

[0130] FIG. 6: Priming of specific CD8.sup.+ T cells responses in HLA-B*0702 transgenic mice following Trip-hTERT immunization. HLA-B*0702 transgenic mice were immunized either with Trip-hTERT (4 first [[black]] shaded bars) or control (2 last unshaded bars). 12 days later, IFN-γ producing-single cells within splenocytes of each mouse were detected ex vivo by IFN-γ ELISPOT assay. Ficoll purified lymphocytes from freshly isolated splenocytes of individual immunized mice were directly cultured with or without 5 μg/ml of each HLA-B*0702-restricted hTERT-derived peptides for 24 h. The number of specific-IFN-γ SFCs was calculated after subtracting non-specific values obtained with control without peptide (<15 SFC), and the responses were considered positive for SFC>30.

[0131] FIG. 7: Schematic representation of the pTRIP-hTERT. This lentiviral-derived vector contains the psi sequence, the cPPT and CTS central cis-active sequences (Flap) of the HIV-1 genome and the CMV promoter which allows the expression of the gene of interest. Moreover, the U3 domain is deleted in the 3′LTR (ΔU3).

[0132] FIG. 8: A) DNA pTRIP-CMV-ΔhTERT immunization primed hTERT-specific CD8.sup.+ T cells responses in HHD mice. HHD mice (HLA-A2.1 Tg) were DNA immunized with a DNA encoding a non-functional form of HTERT (pTRIP-CMV-ΔhTERT). Ten days later, IFN-γ producing peptide-specific T cells were detected ex vivo by IFN-γ-ELISPOT assay. Ficoll purified lymphocytes from splenocytes of individual immunized mouse were directly cultured for 24 h, with or without 5 μg/ml of each H LA-A2.1-restricted hTERT-derived peptide. The number of specific—IFN-γ SFCs was calculated as described above. Responses were considered positive for SFC>30.

[0133] B) Induction of short CTL responses in HHD mice after pTRIP-CMV-ΔhTERT immunization. HHD mice were immunized with a DNA encoding a non-functional form of HTERT (pTRIP-CMV-ΔhTERT) for 10 days. Spleen cells from individual mice were restimulated in vitro with HLA-A2.1-restricted hTERT-derived p540 and pY572 peptides for 6 days. Effector cells were tested in a .sup.51Cr-release assay against HHD-transfected EL4 cells loaded with either the relevant peptide or the irrelevant peptide.

[0134] FIGS. 9A to 9F: Sequence of a non-functional hTERT protein (deletion of amino acids 867 to 869). DNA sequence disclosed as SEQ ID NO: 38 and amino acid sequence disclosed as SEQ ID NO: 39. The coding sequence is located between the nucleotide 59 and 3348. Initiation and termination codons are underlined. First line is the nucleotide sequence; second line is the corresponding amino acid sequence. Third line is the numerotation of the hTERT coding sequence, starting from the initiation codon as the first amino acid.

[0135] FIGS. 10A to 10F: Sequence of a non-functional hTERT protein (deletion of amino acids 864 to 872). DNA sequence disclosed as SEQ ID NO: 40 and amino acid sequence disclosed as SEQ ID NO: 41. The coding sequence is located between the nucleotide 59 and 3430. Initiation and termination codons are underlined. First line is the nucleotide sequence; second line is the corresponding amino acid sequence. Third line is the numerotation of the hTERT coding sequence, starting from the initiation codon as the first amino acid.

EXAMPLES

I—Materials and Methods

Blood Donors

[0136] Peripheral bloods samples were obtained following written informed consent from adult healthy platelet donors (centre de transfusion sanguine de I′hópital Mondor, Creteil, France). HLA typing of peripheral blood donors was performed in the HLA laboratory of the H. Mondor. Hospital Creteil (France). The study was approved by the French Blood Bank Institute.

Mice

[0137] HLA-*B0702 transgenic (Tg) mice, expressing an HLA-B0702 α1α2, H2-Kd α3 chimeric construct, in combination with constitutive murine β2-m molecule (HLA-B7.sup.mα3) and HHD transgenic mice expressing a chimeric HLA-A2.1/H2-Db molecule, were deleted of their H2-Db and H2-k.sup.b genes as previously described (Pascolo et al. J Exp Med 1997; 185:2043-51; Rohrlich et al. lnt Immunol 2003; 15:765-72). These mice are on a C57BL/6 background and were bred and maintained under specific pathogen-free conditions in our animal facility.

Tumor Cells Lines

[0138] The T-B hybrid T1, EBV-transformed B cell JY, renal cancer cell line U293T and Burkitt lymphoma cell Raji were from American type Culture Collection (ATCC). Melanoma cell lines (SK23MEL, LB34, and KUL68) were kindly provided by P. Coulie (Bruxell, Belgium) and EBV-transformed B cell BBG.1 and BC3 were kindly provided by H. Collandre (R.A.H.P., Grenoble, France).

[0139] HLA-*B0702 transfected TAP deficient T2 cells (T2-B7) were kindly provided by P. Cresswell (Smith et al. J Immunol 1996; 156:3755-64). Murine lymphoma cell lines RMA, and EL4 were from ATCC; theses cells were also transfected with HLA-*B0702 gene and used as target cells.

Epitope Selection Peptide Synthesis

[0140] We used predictive algorithm “SYFPEITHI” (Lu and Celis E Cancer Res 2000; 60:5223-7) to analyse amino acid sequence of hTERT for the existence of 9-amino acid (nonamer) or 10-amino acid (decamer) peptides, predicted to bind to HLA-*80702. We selected candidate peptides that contain canonical HLA-B7-binding anchors, Pro at position 2 and hydrophobic aliphatic (Ala or Leu) at carboxyl-termini, and according to their highest predictive score. Six peptides were retained and synthesized, three 9-amino acid peptides named p1, (MPRAPRCRA (SEQ ID NO: 6), residues 1-9), p277 (RPAEEATSL (SEQ ID NO: 9), residues 277-285) and p342 (RPSFLLSSL (SEQ ID NO: 10), residues 342-350), and three 10-amino acid peptides, p4 (APRCRAVRSL (SEQ ID NO: 7), residues 4-13) p68, (APSFRQVSCL SEQ ID NO: 8, residues 68-77), and p351 (RPSLTGARRL (SEQ ID NO: 11), residues 351-360) (anchor positions are underlined).

[0141] Peptides derived from human cytomegalovirus pp65, RPHERNGFYV (SEQ ID NO: 33) (R1OTV), and human immunodeficiency virus type 1 IPRRIRQGL (SEQ ID NO: 34) were synthesized and were used as control peptides. Peptide derived from hepatitis B virus core 128-140 (TPPATRPPNAPIL (SEQ ID NO: 35)) was used as helper peptide for peptide immunization in mice. Peptides were purchased from PRIM to a minimum purity 80% and were reconstituted in distilled water or DMSO at a concentration of 2 mg/ml.

HLA-B0702 Binding/Stabilization Assay

[0142] The relative avidity of hTERT derived peptides for HLA-*B0702 was measured by using a MHC stabilization assay on HLAB0702 transfected T2 (T2-B7) cells in comparison with a reference peptide (R10TV) as described (Rohrlich et al. lnt Immunol 2003; 15:765-72). Briefly, T2-B7 were incubated overnight at 2×10.sup.5 cells/well in 96-well plates in serum free medium AIM-V (Invitrogen Corp., Gibco), supplemented with 100 ng/ml of human β2-microglobulin, in the absence (negative control) or in the presence of either reference peptide R10V or hTERT peptides at various final concentrations (100, 10, 1 and 0.1 μM). T2-B7 cells were labelled with a saturating concentration of ME.1 an anti-HLA-B7 mAb, then washed twice and finally stained with FITC-conjugated F(ab′)2 goat anti-mouse Ig before flow cytometry.

[0143] Results are expressed as values of relative avidity, that is the ratio of concentration of test peptide necessary to reach 20% of the maximal binding (obtained with the reference peptide) over the concentration of the reference peptide. Therefore, the lower the value, the stronger the binding.

Peptide Immunization of HLA-*B0702 Transgenic Mice for CTL Induction

[0144] Female HLA-*B0702 transgenic mice at 8-10 weeks of age were injected subcutaneously (s.c.) at the base of the tail with 50 μg of individual HLA-B0702 restricted hTERT peptides supplemented with 140 μg of helper peptide co-emulsified in incomplete Freund's adjuvant (Difco, Detroit, Mich.). Ten days later, spleen cells of individual mouse were reactivated in vitro with relevant peptide in six wells plate. Effector CTL cells were tested in a standard 4-5 h .sup.51Cr-release assay, using relevant or negative control peptide-pulsed, HLA-*B0702 transfected RMA cells (RMA-B7). Mice were considered as responders, when specific lysis≧10% was observed.

DNA Immunization in HLA-*B0702 Transgenic Mice

[0145] The LvCMV-hTERT plasmid vector encoding the hTERT gene under the control of CMV promotor was purified on plasmid Giga kit columns under endotoxin-free conditions (Qiagen). Anesthetized HLA-*B0702 Transgenic mice were injected with said plasmid (50 μg each side) into regenerating tibialis anterior muscles. 14 days after, spleen cells of individual mouse were re-activated in vitro with peptide-pulsed (10 μg/ml), syngenic γ-irradiated (50 Gy) LPS-lymphoblast in complete medium, supplemented with 10% supernatant from Con A-activated rat spleens cells. Cytotoxicity assays were performed 6 days as described.

Lentiviral Vector Construct and Production

[0146] The pTRIP-deltaU3-CMV-hTERT (referred as TRIPLv-hTERT or Lv-hTERT or pTrip-hTERT) (FIG. 7) construct was created by first subcloning an EcoRI-Sall hTERT insert derived from the pBABE-hygro-hTERT plasmid (Counter et al. Proc Natl Acad Sci U.S.A. 1998; 95: 14723-8) into the pSP73 vector (Promega). A BgIII-SaII fragment was then inserted into the pTRIP-CMV plasmid cut with BamH1 and XhoI. Pseudo typed recombinant retroviral particles were produced by transient (48 h) transfection of 293T cells as described (Zennou et al. Cell 2000; 14; 101:173; Firat et al. J Gene Med 2002; 4:38-45). The recombinant retroviral particles were concentrated by ultra-centrifugation and resuspended in PBS. The amount of vector particles was estimated from that of p24 protein in a commercially available ELISA assay (NEN, DUPONT, France Perkin Elmer).

[0147] The pTRIP-CMV-ΔhTERT vector, deposited at the CNCM (Institut Pasteur, Paris, France) under the number CNCM 1-3660 on Jul. 28, 2006, was carried out as described in the paragraph above. However, the hTERT protein was rendered non-functional by deletion of amino acids 867 to 869, corresponding to nucleotides 2654 to 2662 of FIG. 1 (wild-type). The catalytically dead hTERT RT mutant (ΔhTERT) was generated by creating a deletion of amino acid residues 867 to 869 using the QUICKCHANGE XL Site-Directed Mutagenesis Kit (Stratagene) and verified by sequencing.

Immunization in MHC Class I Transgenic Mice and CTL Detection

[0148] Immunization with TRIPLv-hTERT was performed as a single subcutaneously (at the base of the tail) injection of 1500 ng of TripLv-hTERT suspension or control vector.

[0149] Immunization was performed in HLA.A2 transgenic mice as a single intraperitoneal injection of recombinant lentiviral particles, pTRIP-CMV-ΔhTERT or Trip-GFP as a control, equivalent to 1500 ng of p24 antigen in 500 μl of PBS.

[0150] 12 days later, hTERT peptide-specific T among splenocytes were detected by an ELISPOT assay (see below). Cytotoxicity assays were performed on the same immune splenocyte populations after in vitro stimulation with peptide-pulsed as described above.

Evaluation of T-Cell Response by Ex Vivo IFN-γ ELISPOT Assay

[0151] Peptide-specific T cells from immunized mice were detected by IFN-γ ELISPOT assay as previously described (Miyahira et al. J Immunol Methods 1995; 181:45-54). Anti-mouse IFN-γ mAb's (3 μg/ml; Pharmigen, Becton Dickinson biosciences) were coated onto 96-well nitrocellulose microplates (multi screen; Millipore corp, Molsheim, France). After red cell lysis, freshly isolate spleen lymphocytes of individual mouse (5×10.sup.5, 2.5×10.sup.5 and 1.25×10.sup.5 cells/well) were directly cultured with or without 5 μg of native hTERT peptide for 18 h at 37° C. After washings, the plates were incubated 2 hours with biotinylated anti-mouse IFN-γ (2 μg/ml; Pharmigen, Becton Dickinson biosciences). Finally, the plates were washed and incubated at 37° C. for 1 h with alkaline phosphatase-conjugate streptavidin (Roche molecular biochemicals, Mannheim, germany). Positive controls include cells stimulated with phorbol myristate acetate (100 ng/ml, Sigma)) and ionomycin (1 μg/ml). IFNγ spot-forming cells (SFCs) were developed by adding peroxidase substrates (BCIP/NBT, Promega Corp, Madison W; USA) and counted using automated image analysis system a BIOREADER 2000 (Biosys, Karben, germany). The number of specific SFCs was calculated after subtracting negative control values (<10 SFC). Responses were positive if the mean of SFCs in stimulated well was greater than the mean+2 S. D. of the SFCs in the negative control wells and greater than 50 SFC/10.sup.6 cells.

Cytolytic Assay

[0152] Cytotoxicity assays were performed by using standard 4-5 h .sup.51Cr release assay as previously described (Firat et al. J Gene Med 2002; 4:38-45). Specific lysis in % was calculated by subtracting non-specific lysis observed with the control peptide. Mice were considered as responsers when specific lysis≧10% was observed.

Generation of hTERT Peptide-Specific CTL in Human

[0153] Human CTL from donors were obtained after in vitro reactivated PBMC for 4 weeks with hTERT peptide HLA-B0702 restricted as described previously (Hernandez et al. Proc Natl Acad Sci U.S.A. 2002; 99:12275-80). Briefly, Ficoll-purified human PBMCS were thawed and incubated (4×10.sup.6/well) in 24-well plates in RPMI 1640, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 5× 10-5M 2-mercaptoethanol supplemented with heat inactivated 10% human serum (Institut Jacques Boy, Reims, France). They were stimulated with each hTERT peptide (10 μg/ml) and recombinant human IL-7 (20 ng/ml; R&D Systems) was added.

[0154] On day 7, lymphocytes were re-activated with peptide-pulsed γ-irradiated autologous PBMCs (50 Gy). The next day, 20 IU/ml human IL-2 (Roche, Mannheim, Germany) was added to the culture. CTL lines were re-activated weekly during four cycles. For some donors, CD8.sup.+ T cells were purified after three round cycle, using CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's recommendations and activated once before functional test. Cytolytic assays were performed 6 days after the last re-activation against various .sup.51Cr-labeled targets: T2-B7 pulsed with tested hTERT peptides or irrelevant peptide, or tumor cell lines.

[0155] In some experiments, tumors cells were incubated with an anti-HLA class I framework mAb, w6/32 (BD Pharmigen) or anti-HLA-*B0702 mAb, ME.1, or an anti-HLA-DR mAbB, G46.6 (BD Pharmigen) at an optimal concentration (10 μg/mL) for 30 minutes to determine whether cytotoxicity was restricted to HLA class I.

II— Results

[0156] 1. Immunogenicity of HLA-*B-0702 Predicted Peptides Derived from hTERT.

[0157] Using a T-cell epitope prediction program, we analysed the hTERT protein sequence and retained six peptides (3 nonamers and 3 decamers) due to their high predictive score (Table I). We next tested their ability to bind HLA-B0702 molecule using antigen-transporter (TAP)-deficient T2 cells transfected with HLA-B0702 gene (T2-B7). Three peptides p1, p4 and p68 respectively show a high relative affinity (RA≦1) and the three others p277, p342 and p351 exhibited medium RA>1 (Table I). These data indicate that these six peptides are excellent binders to HLA-B0702. Therefore, one might expect such complexes, formed in the endoplasmic reticulum, to reach the surface of tumor cells and be available for CTL recognition.

[0158] To test if these peptides are immunogenic in vivo, we have immunized HLA-*B0702 transgenic mice. The six peptides tested were shown to be able to induce CTL responses, although differences were noticed (Table I). Two peptides p4 and p68, binding with a high affinity to the HLA-B7 molecules, induce strong CTL response in all mice tested. In contrast, peptides (p277 and p342), having lower affinity for the HLA-B7 molecules, enable the generation of moderate specific CTL, in only 50% of mice tested. CTL lines specific for hTERT epitopes, generated from transgenic mice, recognized human T2-B7 pulsed with their respective specific peptides (data not shown), demonstrating the high affinity of their TCR for the complex MHC/peptide. Thus, there is an overall correlation between the results of binding/stabilization of HLAB0702 and the in vivo CTL response in HLA-B7 transgenic mice.

TABLE-US-00012 TABLE 1 Immunogenicity of selected hTERT HLA-*80702 binding peptide* Immunogenicity.sup.‡ Specific SEQ ID Relavive Lysis Peptide* Sequence NO Score.sup.§ Avidity+ R/T** (%) p1 MPRAPRCRA 6 23 0.4 4/6 30, 39, 35, 28 P4 APRCRAVRSL 7 25 0.3 6/6 52, 53, 48, 51, 46, 49 p68 APSFRQVSCL 8 25 0.4 6/6 63, 78, 75, 69, 70, 72 p277 RPAEEATSL 9 23 4.7 3/6 29, 33, 29 p342 RPSFLLSSL 10 23 2.5 3/6 26, 39, 32, p351 RPSLTGARRL 11 23 1.5 4/6 47, 31, 37, 26 *The figure represents the first amino acid of the peptide. Therefore, “p4” indicates that the alanine residue is the fourth amino acid of the coding sequence. .sup.§Algorithm score obtained by using SYFPEITHI predictive program +The relative avidity of hTERT peptides for HLA-B*0702 was measured by using a MHC stabilization assay in comparison with a reference peptide as detailed in material and methods. .sup.‡HLA-B′0702 transgenic mice were immunized with candidate peptides (six mice for each peptide) as detailed above. Ten days later, spleen cells of individual mice were restimulated in vitro with each hTERT peptide. Cytolytic activities were assayed by 4-5 h 51Cr release assay using peptide loaded RMA-87 target cells. The Specific lysis was calculated by subtracting non-specific lysis observed with the R1OTV control peptide. Specific lysis at a 75:1 effector/target ratio was showed. **RIT: responder (specific lysis 10%) versus tested mice.
2. hTERT-Derived Peptides were Processed in HLA-B0702 Transgenic Mice.

[0159] To assess the presentation of endogenously synthesized hTERT peptides in the context of the HLA-B0702 molecule against these six hTERT epitopes identified, HLA-*B0702 transgenic mice were immunised with cDNA encoding hTERT, forty days after peptide-specific CTL responses within spleen cells of individual mice were evaluated. As shown in FIG. 2, hTERT peptide-specific CTLs were induced in most immunized mice (M), from 50 to 80% of mice for p4, p68, p1, p277 and p351. In contrast, p342-specific CTLs can be induced in about 15% tested mice. No significant hTERT-specific CTL responses were also induced from non-immunized naïve mice. Thus, these six hTERT epitopes are effectively intracellular̂ processed. Moreover, natural peptides similar in term of amino acid sequence or structure to the synthetic ones, are presented by the corresponding HLA-*B0702 molecules on the cell surface.

[0160] Further, these data show that multiple CTL specificity can be induced simultaneously against several hTERT epitopes in a single mice and validate our HLA-Class I transgenic mouse model for their potential to test candidate vaccines.

3. Induction of Primary CTL Responses from Healthy Donors by hTERT Peptides.

[0161] We studied whether hTERT peptides would be effective in raising HLA-B7-restricted CTLs, using PBMCs of HLA-B0702 healthy donors in an in vitro immunization protocol. CTL responses were generated in eight out of ten individuals (d.sub.1 to d.sub.8), and peptide-specific CTL responses were obtained in at least 50% of donors, except for p342 (20%) (FIG. 3). hHTERT epitope recognition by in vitro generated CTLs varies among donors, depending upon their genetic background (FIG. 3). Therefore, by random testing of HLA-B0702 healthy donors, it was clearly established that these hTERT peptides are immunogenic in human, implying that specific CTL precursors for hTERT are not deleted in the peripheral adult repertoire. Therefore, we asked whether CTL lines generated from healthy donors would be able to kill HLA-matched hTERT.sup.+ tumor cells.

4. Specific hTERT CTL were Able to Lyse Tumors of Differents Origins

[0162] hTERT-specific CTLs from donors were tested for their capacity to lyse human tumour cell lines of different origins. The results presented in Table 2 show that, CTL lines generated in vitro from healthy donors killed HLA-B0702.sup.+ tumour cells, whereas no cytotoxicity against HLA-B0702′ tumors was detected. (See for example CTLp351 in d.sub.1, d.sub.2 and d.sub.3 in KU L268 or 293-UT target (respectively 52, 25, 20 and 34, 41 and 19%) versus T1 or BBG1 target (respectively 9, 2, 6 and 0, 0, 2%). Differences were observed in tumor recognition according to the CTL specificity; this could be explained by differential presentation of hTERT peptides on the surface of the tumor cells. Importantly, p351-specific CTL lines generated from different donors recognize the majority of tumor cell lines tested (Table 2). In contrast, all p4-specific CTL lines do not lyse all the type of tested tumors. CTL lines, specific for p1 and p68 peptides, only recognize the T1-B7 targets. p342-specific CTL lines recognize only melanoma cells (LB 34 and KU 268 target). Finally, p277-specific CTL lines recognize renal cancer (293 UT) but neither melanoma nor lymphoid tumor cells. On the other hand, normal PBMCs and CD40 activated B cells were not lysed by these hTERT peptide-specific CTL lines, regardless of HLA type (two last lines of Table 2).

[0163] As shown in FIG. 4, the cytotoxic activity of CTLp351 line toward HLA-B7.sup.+ tumor cells is inhibited by an anti-class I mAb anti-HLA-B0702, but not by an anti-HLADR mAb (MHC class II). Similar data were obtained with other peptide-specific CTL lines (data not shown) and suggest that CTL lines exert cytotoxicity against hTERT.sup.+ tumor cells in an HLA-B0702-restricted manner. Collectively, these results show that these six hTERT derived peptides are not equally naturally expressed at the tumor cell surface and that hTERT peptide-specific CTLs can discriminate between tumor cells and normal cells, through the recognition of hTERT peptide in context of HLA-B0702 molecules.

5. Lentiviral Vector Encoding hTERT Vaccination Induces Efficient Peptide-Specific T Cell Responses in Mice.

[0164] We next tested candidate vaccines, comprising either a full-length hTERT gene or a non-functional hTERT gene, inserted in a HIV-derived flap vector (FIG. 7). Previous data have shown that lentiviral vectors of this type target dendritic cells in vitro and in vivo, and induce strong poly-specific anti tumor CTL responses in animals. Therefore, we immunized HLA-*B0702 transgenic mice with either recombinant Lv-hTERT or with pTRIP-CMV-ΔhTERT. Twelve days after, spleen cells of individual mice were evaluated by an ex vivo ELISPOT assay.

[0165] As shown in FIG. 5A, peptide-specific CD8.sup.+ T cell responses were obtained against HLA-B0702 restricted hTERT epitopes, as compared with mice that received Lv-GFP control vector. Functional analysis of the induced peptide-specific CD8.sup.+ cells in chromium release assay after in vitro stimulation confirmed ex vivo ELISPOT data (Table 3) and show that efficient specific CTL response is generated against these six peptides in about 50-70% of mice after a single injection of Lv-hTERT and in 100% of the mice after a boost with TRIPLv-hTERT (FIG. 6). This was also associated with strong CTL responses in all mice (Table 3). Additionally, as show in Table 3, immunization of HHD mice transgenic for HLA-A2.1 with the same vector induced potent CTL responses specific for two HLA-A2.1.1 restricted epitopes previously classified as dominant (p540) and cryptic (p572). Collectively, these results clearly show that administration of Lv-hTERT result in the induction of very efficient multi-specific T cell response in mice, supporting that hTERT could serve as polyepitope and polyallelic TAA for cancer immunotherapy.

[0166] As shown in FIG. 8, hTERT peptide-specific CD8.sup.+ T cell responses were detected ex vivo in HLA-A2 transgenic (Tg) mice after a single injection of recombinant pTRIP-CMV-ΔhTERT. We showed that CD8.sup.+ T cells specific for p540 and PY572 epitopes were induced at least in 50% of immunized mice (FIG. 8). These results clearly showed that the two epitopes were correctly endogenously processed and presented in HLA-A2 Tg mice after immunization with pTRIP-CMV-ΔhTERT.

[0167] Collectively, these results showed that a single injection of TRIP-hTERT resulted in the induction of a potent multi-specific anti-hTERT CD8.sup.+ T-cells response in both HLA transgenic mice groups.

TABLE-US-00013 TABLE 2 anti-hTRT CTL from normal donors lyses tumor cells of various types Percent lysis* CTL p1 CTL p4 CTL p68 CTL p277 CTL p342 CTL p351 Cell target Cell type HLA-B7 d1 d6 d8 d1 d6 d8 d1 d6 d8 d1 d6 d8 d1 d6 d8 d1 d6 d8 T1 T-B hybrid − 9 10 5 8 6 nd 0 7 6 3 5 9 3 6 4 9 2 6 T1-B7 T-B hybrid + 29 26 14 5 7 nd 19 30 4 56 30 12 2 17 5 38 24 31 Sk23mel Melanoma − 0 1 0 0 2 0 10 3 4 1 4 0 4 4 3 5 2 1 LB34 Melanoma + 5 4 0 0 4 2 13 7 11 0 0 2 28 22 14 52 25 20 KUL68 Melanoma + 9 4 7 6 3 0 0 nd 0 7 4 nd 26 14 9 34 41 19 293-UT Renal cell + 2 8 4 1 6 7 3 0 0 36 17 22 9 17 4 28 22 25 BBG1 EBV-B cell − 0 4 3 0 0 2 2 0 1 0 0 5 1 1 0 0 0 2 JY EBV-B cell + 2 0 4 0 6 nd 0 0 9 3 0 1 8 10 6 27 18 15 Raji B lymphoma − 4 1 nd 5 0 nd 0 0 nd 6 nd 1 0 nd 0 4 0 nd Autologous PBMC Normal cell + 0 0 0 4 0 1 0 0 0 0 0 1 0 2 0 0 0 1 Autologous B cell CD40.sup.§ Normal cell + 0 0 0 3 0 2 2 0 0 0 0 6 0 2 3 0 0 4 *hTERT peptide specific-CTL lines (CTLp1, CTLp4, CTLp68, CTLp277, CTLp342, CTLp351) were obtained from healthy donors that were responder after subsequent in vitro immunization as described in material and methods. Cytotoxicity was measured in a standard .sup.51Cr-labeled release assay. Specific lysis: for a 30:1 effector: target ratio were shown .sup.§Autologous B lymphocytes from normal donors were activated for 48 h with a trimeric C040 L (40 μg/ml).

TABLE-US-00014 TABLE 3 Induction of CTL responses following Lv-hTERT immunization Flap+ Lv-hTRT immunization HLA-B7 Tg mice HHD mice Restim- Restim- ulating ulating peptide R/T Specific lysis (%) peptide R/T Specific lysis (%) p1 4/8 27, 30, 29, 32 P540 2/6 21, 18 p4 6/8 18, 25, 54, 33, 16 pY572 5/6 22, 19, 14, 35, 24 p68 4/8 15, 64, 24, 16, p277 5/8 21, 25, 23, 52, 33 p342 4/8 18, 24, 20, 37 p351 5/8 17, 20, 18, 36, 19

III—Conclusion

[0168] New hTERT epitopes, which are in vivo immunogenic and processed in H-2-class I knockout HLA-B0702 transgenic mice have identified. Further, in vitro, hTERT peptide immunization using HLA-B702.sup.+ PBL from healthy donors induce specific CTL responses recognizing hTERT.sup.+ tumors from various origins, implying that there is no deletion in the human T cell repertoire for these epitopes. Moreover, it was shown that depending upon the tumor origins, peptides repertoire expressed on the cell surface could be qualitatively different, underlining, the utility to characterize hTERT as polyepitope tumor associated antigens for circumvent antigenic variability of cancer cells. Finally, a humanized HLA-*B0702 and HLA-A2 1 transgenic mice were used, to test a candidate vaccine consisting of a nonfunctional telomerase gene inserted in a new generation of lentiviral derived flap vector. A strong hTERT specific CD8.sup.+ T cell responses were observed in all the HLA-transgenic mice. These data support the use for therapeutic vaccination in cancer patients and extend the potential applicability of hTERT as a therapeutic target to cover a large population of cancer patients.

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