# Patent

## Title
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, analogs thereof and polyepitopes containing such epitopes and/or analogs. 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.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national phase entry of PCT/EP2006/007571, filed Jul. 31, 2006, which claimed priority of European Patent Application No. 05291627.7 filed Jul. 29, 2005.
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, vectors and cells comprising such polynucleotides. The present invention also concerns compositions comprising hTERT polypeptides, corresponding polynucleotides, vectors and cells, for use in the treatment and/or prevention of cancer.
In light of deficits in current anticancer therapeutic approaches, antitumor therapy has enjoyed renewed interest. Recent studies have enhanced our understanding of anti melanoma immune responses associated with tumor regression. Collectively these data suggest that activated tumor-specific CD8 CTL are immunological weapon of choice for potent anti-tumor therapy. The research of over-expressed proteins permitting to trigger cytotoxic T lymphocytes to tumours of different origins is also progressing.
Telomerase is a ribonucleoprotein complex, consisting of a protein component, TERT, and an RNA component (TR) containing the template for the synthesis of the repeat unit (T2AG3) added onto the ends of chromosomes, that stabilizes the chromosomes during replication and prevent end-to-end fusion. Maintenance of a constant telomere length prevents cells from aging, and confers immortality (Hahn et al. Nat Med 1999; 5:1164-70). High hTERT activity was found in more than 85% of human cancers, whereas most normal adult human tissues show no or little telomerase activity (Counter et al. Blood 1995; 85:2315-20).
The widespread expression of telomerase in tumors indicates that peptide fragments of hTERT could serve as tumor specific antigen(s) and this has been confirmed in several reports (Vonderheide et al. Immunity 1999; 10:673-9). Recent data from Phase I clinical trials demonstrate the feasibility of vaccine against hTERT in HLA-A2+ patients, opening the way for use of hTERT for therapeutic vaccination (Vonderheide et al. Clin Cancer Res 2004; 10:828-39; Parkhurst et al. Clin Cancer Res 2004; 10:4688-98). Nevertheless, the immunogenic hTERT peptides identified to date are restricted to one MHC allele HLA-A2.1, with only two initial reports on two HLA-supertypes, HLA-A3 and HLA-A24, represented respectively in 44.2% and 40% of the population.
Hence, the following publications reported the identification of hTRET peptides:

    
    
        hTERT peptide ILAKFLHWL (SEQ ID NO: 1), restricted to HLA-A2 (Vonderheide et al. Immunity 1999; 10:673-9),
        hTERT peptides MPRAPRCRA (SEQ ID NO: 2), RPAEEATSL (SEQ ID NO: 6), RPSFLLSSL (SEQ ID NO: 10) and APRCRA VRS (SEQ ID NO: 2) identified by informatics prediction within the HLA-A context WO 00/02581). However, these peptides have never been confirmed by experimental results to be efficient epitopes, neither in a HLA-87 context nor in any HLA context.
        hTERT peptide KLFGVLRLK (SEQ ID NO: 3) (K973), restricted to HLA-A3 (Vonderheide et al. Clin Cancer Res 2001; 7:3343-8), and
        hTERT peptides VYAETKHFL (SEQ ID NO: 4) (TEL324) and VYGFVRACL (SEQ ID NO: 5) (TEL 461), restricted to HLA-A24 (Arai et al. Blood 2001; 9:2903-7)
    
    


Consequently, the hTERT peptides identified so far do not cover all the population, thus excluding a large segment of patients.
In order to overcome this failure, at least in part and to thus better cover the genetic diversity of the human population, the present invention identifies new epitopes derived from hTERT, restricted to a particular HLA which is different from HLA-A3 and HLA-A24. Among the many different alleles, the present application is interested in the HLA-B7 supertype which is expressed in about 25% of the population, and particularly to the second allele the most expressed in the human HLA-B population: the HLA-*B0702 (allele present in 15-20% of individuals in human population).
The gene of the isoform-1 of the telomerase is 4015 base pairs (bp) long (NCBI Accession number AF015950) and encodes a protein of 1132 amino acids (NCBI Accession number AAC51672.1) (FIG. 1).
The invention concerns a polynucleotide encoding a human telomerase reverse transcriptase (hTERT) peptide. In a particular embodiment of the invention, the encoded peptides are 9 amino acids in length (nonamer) or 10 amino acids in length (decamer), and the polynucleotide has hence 27 or 30 nucleotides. In general, the encoded peptide is less than 15 amino acids and the polynucleotide has less than 45 nucleotides.
The invention also concerns a polynucleotide encoding hTERT peptides that are epitopes, restricted to MHC class I molecule, especially epitopes suitable to induce an immune response restricted to HLA-87. The nucleotide sequence of the polynucleotide of the invention is, in a particular embodiment, limited to the sequence encoding the hTERT peptide. Such peptides can be chosen from the group consisting of MPRAPRCRA (SEQ ID NO: 6) (p1; amino acid residues 1 to 9), APRCRAVRSL (SEQ ID NO: 7) (p4; amino acid residues 4 to 13), APSFRQVSCL (SEQ ID NO: 8) (p68; amino acid residues 68 to 77), RPAEEATSL (SEQ ID NO: 9) (p277; amino acid residues 277 to 285), RPSFLLSSL (SEQ ID NO: 10) (p342; amino acid residues 342 to 350), RPSLTGARRL (SEQ ID NO: 11) (p351; amino acid residues 351 to 360), DPRRLVQLL (SEQ ID NO: 12) (p444, amino acid residues 444 to 452), FVRACLRRL (SEQ ID NO: 13) (p464, amino acid residues 464 to 472), AGRNMRRKL (SEQ ID NO: 141 (p966, amino acid residues 966 to 974), LPGTTLTAL (SEQ ID NO: 15) (p1107, amino acid residues 1107 to 1115) and LPSPKFTIL (SEQ ID NO: 16) (p1123, amino acid residues 1123 to 1131). All these polynucleotides may be used to induce a HLA-87-restricted immune response. In a particular embodiment, the invention especially concerns a polynucleotide encoding a HLA-87-restricted hTERT epitope, chosen from the group consisting of RPSL TGARRL (SEQ ID NO: 11) (p351), APSFRQVSCL (SEQ ID NO: 8) (p68), APRCRAVRSL (SEQ ID NO: 7) (p4), DPRRLVQLL (SEQ ID NO: 12) (p444), FVRACLRRL (SEQ ID NO: 13) (p464), AGRNMRRKL (SEQ ID NO: 14) (p966), LPGTTL TAL (SEQ ID NO: 15) (p1107) and h LPSPKFTIL (SEQ ID NO: 16) (p1123).
As defined herein, an “epitope” is an antigenic determinant, i.e. the peptide site recognized by cells of the immune system (immune cells) and especially the site necessary to elicit an immune response. The term epitope encompasses both linear epitope for which the consecutive amino acids (especially, 9 or 10) are recognized by immune cells and, conformational epitope for which immune cells recognize amino acids to the extent they adopt a proper configuration or conformation. Consequently, in some epitopes, the conformation (three dimensional structure) is as important as the amino acid sequence (primary structure).
The expression “MHC class I-restricted” refers to the capacity for a particular peptide or epitope to have an affinity for a MHC (major histocompatibility complex) molecule of class I. Similarly; the expression “HLA-B7-restricted” refers to the capacity for a particular peptide or epitope to have an affinity for this type of HLA molecule.
Briefly, MHC genes encode cell surface polymorphic molecules that do not bind only foreign peptides but also can bind overexpressed or not self peptides or mutated self peptides, to display them on the cell surface of cell enabling their recognition by appropriate immune cells, especially T-cells. Said MHC molecules, referred to as H-2 in mice and HLA (Human Leucocyte Antigen) in humans, are classified as either class I molecules (designated HLA-A, B, or C) or class II molecules (designated DP, DQ or DR).
Accordingly, MHC class I molecules specifically bind CD8 molecules expressed on cytotoxic T lymphocytes (also named TCD8+), whereas MHC class II molecules specifically bind CD4 molecules expressed on helper T lymphocytes (TCD4+).
MHC class I molecules bind peptides derived from proteolytically degraded proteins especially endogenously synthesized proteins, by a cell. Small peptides obtained accordingly are transported into the endoplasmic reticulum where they associate with nascent MHC class I molecules before being routed through the Golgi apparatus and displayed on the cell surface for recognition by cytotoxic T lymphocytes.
In the present invention, the above-identified peptides have been shown on the one hand to bind either with high or medium affinity to MHC class I molecule, and on the other hand to be efficiently transported as a MHC/epitope complex to the cell surface of cells. In a preferred embodiment, the MHC class I molecule is an MHC allele of the HLA-B7 supertype family; the hTERT epitope is said HLA-B7 supertype-restricted. Said family encompasses alleles B0702, B0703, B0704, B0705, B1508, B3501, B3502, B3503, B51, B5301, B5401, B5501, B5502, B5601, B5602, B6701 and B7801, family from which the HLA-B0702 is preferred (HLA-B0702-restricted hTERT epitope).
A MHC stabilization assay may be used to test the affinity of a peptide for a particular HLA class I molecule (relative avidity), such as the one described in Firat et al. (1999. Eur. J. of Immunol. 29: 3112-3121), incorporated herein by reference. Briefly, MHC class I molecule-transfected cells are incubated overnight at 2×105 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 of peptides (negative control),
        in the presence of a reference peptide (positive control), and
        in the presence of the peptides to be tested (hTERT peptides in the present case).

Peptides are incubated at various final concentrations ranging from 0.1 to 100 μM (with intermediate concentrations of 1 and 10 μM). The transfected cells are then labelled with a saturating concentration of an antibody recognizing the particular HLA MHC class I molecule, then washed twice and finally stained with a secondary antibody before flow cytometry. Results are expressed as values of relative avidity, that is the ratio of concentration of tested peptide necessary to reach 20% of the maximal binding by the reference to that of the reference peptide. Therefore, the lower the value, the stronger the binding. Following this method, a peptide is said to have a high relative affinity for a particular HLA class I molecule, when RA<1. In contrast, a medium relative affinity is concluded when RA is comprised between 1 and 5, and preferably between 1 and 3.

    
    


Among the hTERT nonamers and decamers, and particularly the above-identified peptides, the following MHC class I-restricted hTERT epitopes can be classified as having high relative affinity for MHC class I molecule: MPRAPRCRA (SEQ ID NO: 6 (p1), APRCRAVRSL (SEQ ID NO: 7) (p4) and APSFRQVSCL (SEQ ID NO: 81 (p68). Using the same approach, the following MHC class I-restricted hTERT epitopes can be classified as having a medium relative affinity for MHC class I molecule: RPAEEATSL (SEQ ID NO: 9) (p277), RPSFLLSSL (SEQ ID NO: 10) (p342), DPRRL VQLL (SEQ ID NO: 12) (p444) and RPSLTGARRL (SEQ ID NO: 11) (p351).
The present invention also relates to a polynucleotide encoding a MHC class I-restricted epitope analogue, i.e., epitopes having at least one amino acid substitution compared to a class I-restricted hTERT epitope as described above, especially HLA-B7-restricted epitope analogue.
The term “analogue” as defined herein relates to a peptide whose sequence is derived from a hTERT peptide as described above by at least one amino acid substitution, either conservative, semi-conservative or non-conservative. An analogue is opposed to an epitope by the fact that its nucleotide and/or amino acid sequence is not found in the reference hTERT gene or protein disclosed in FIG. 1 which are considered within the present application as the molecules of reference to define the so-called wild type peptides or polynucleotides. Such an analogue may result from the substitution in the corresponding nucleotide sequence of one or several nucleic base(s) in the codon encoding said amino acid. Therefore, a polynucleotide analogue differs from its wild type counterpart (polynucleotide encoding hTERT peptide with the reference sequence from which the peptide analogue is derived from) by at least one substitution, and preferably one, two or three substitutions in the codon(s) encoding the amino acid residue to be substituted. As an example, the APRRLVQLL (SEQ ID NO: 17) peptide (called p444*) is derived from the p444 peptide by the substitution of the first amino acid residue (D→A).
A particular analogue of a HLA-B7-restricted analogue has the same length or is shorter than the epitope from which it derives.
As a particular embodiment, the hTERT epitopes described above or the analogues always conserve in their primary structure a proline (P) in position 2, and/or one of the following amino acids in the last C-terminal position: A, L, I, M, V, F, W or Y. Therefore, the hTERT peptides, including the analogues, have the following consensus sequence: X-P-X6-7-[ALIMVFWY], wherein X refer to any amino acid, X6-7 refers to the number of amino acids and [ALIMVFWY] refers to one of these amino acids.
Therefore, to provide an epitope analogue, the amino acid substitution or the corresponding codon substitution in the polynucleotide is not located in the second position (or second codon). In a preferred embodiment, no substitution is carried out in the C-terminal position, even though the last C-terminal amino acid can be replaced by an equivalent amino acid, i.e. either A, L, I, M, V, F, W or Y. Finally, in a preferred embodiment, the substitution is located in the first amino acid position, wherein any amino acid is replaced by an alanine (A).
In a further embodiment of the invention, the last C-terminal amino acid of a decamer is deleted to give a nonamer, provided that the resulting nonamer maintains or adopts the X-P-X6-7-[ALIMVFWY] consensus sequence. In the same way, an amino acid selecting among A, L, I, M, V, F, W and Y is added at the C-terminal end of a nonamer to give rise to decamer, provided that the resulting decamer maintains or adopts the X-P-X6-7-[ALIMVFWY] consensus sequence.
In case of substitution, deletion or addition, including especially those illustrated above, taken individually or as combinations, the tridimensional conformation of the peptide analogue must be the same or slightly modified with respect to the one of the wild type counterpart, to ensure a correct folding of the analogue and its correct binding to the MHC class I molecule. The MHC stabilization assay described above can be used to check that such constraints are fulfilled.
The resulting analogue has at least the same characteristics as its wild type counterpart, in terms of affinity for a particular MHC class I molecule, especially HLA-B7 molecule, and has essentially the same capacity to be transported as an epitope/MHC complex on the cell surface and/or has essentially the same capacity to elicit an immunogenic response when tested in the same conditions.
In a preferred embodiment, the starting peptide is a hTERT epitope having a medium affinity, and the resulting analogue has a higher affinity than its wild type counterpart. In another embodiment, the analogue has a higher immunogenicity than its wild type counterpart. As an example, the p444* peptide analogue quoted above has an increased affinity for MHC class I molecule compared to the p444 peptide from which it is derived from.
This is an object of the invention to provide hTERT epitope or analogue, able to elicit an immune response, and particularly a CTL response (Cytotoxic T Lymphocyte). However, the T lymphocytes do not recognize said analogue that is used to stimulate said lymphocytes only, via antigen presenting cells. In an embodiment of the invention, the analogue as described above keeps its immunogenic behaviour, and is able to elicit an immune response against cells overexpressing hTERT epitopes, i.e. that CTLs recognize the wild type epitope, even if stimulated with an epitope analogue. In a particular embodiment, the lymphocytes stimulated by an epitope analogue of the invention do not react against cells, which do not overexpressed hTERT epitopes. Therefore, stimulated lymphocytes do not react with cells overexpressing other epitopes (cross reaction) or with cells expressing hTERT epitope as basal level (healthy cells). In a particular embodiment, all the characteristics mentioned above are in a HLA-B7 environment, and preferably in a HLA-B0702 context.
A conventional cytotoxicity assay may be performed by using a standard 4-5 h 51Cr release assay to test the capacity of stimulated lymphocytes to react against target cells, such as in the Firat publication (1999. Eur. J. of Immunol. 29: 3112-3121) incorporated herein by reference. Briefly, cell suspension containing CTLs, are activated with peptide (hTERT peptide or analogue in the present case) plus self MHC Class I molecule in vivo. Target cells (expressing or not the corresponding HTERT epitope) previously incubated with 51Cr, are then incubated with activated lymphocytes. The recognition of target cells by activated CTL leads to the apoptosis of target cells and the release of 51Cr, wherein said release is proportional to the number of target cells killed. An incubation of target cells with a control peptide is used as a negative control to calculate the spontaneous release of 51Cr. Specific percentage of lysis is calculated by subtracting non-specific lysis observed with the control peptide to lysis obtained with the peptides to be tested. The higher the percentage, the more targets have been killed by the CTL. Specific lysis is determined at several ratios of Effector (CTL) to target cells (E:T). The specific lysis is calculated as the ratio between [experimental release−spontaneous release] and [total release−spontaneous release].
The present invention also concerns a polynucleotide encoding a polyepitope. A polyepitope is defined as a polypeptide having at least two epitopes, chosen among the MHC class I-restricted, especially HLA-B7-restricted hTERT epitopes (p1, p4, p68, p277, p342, p351, p444, p464, p966, p1107 and p1123) and/or MHC class I-restricted, especially HLA-B7-restricted, epitope analogues of the invention. The polynucleotide of the invention comprises or consists of at least two polynucleotide units encoding said epitopes or analogues. A polynucleotide unit is defined as the coding sequence for an epitope or analogue of the invention as disclosed herein.
The invention particularly concerns a polynucleotide encoding a polyepitope, comprising at least two epitopes chosen among (a) RPSLTGARRL (SEQ ID NO: 11) (p351), (b) APSFRQVSCL (SEQ ID NO: 8) (p68), (c) APRCRAVRSL (SEQ ID NO: 7) (p4), (d) DPRRLVQLL (SEQ ID NO: 12) (p444), (e) FVRACLRRL (SEQ ID NO: 13) (p464), (f) AGRNMRRKL (SEQ ID NO: 14) (p966), (g) LPGTTLTAL (SEQ ID NO: 15) (p1107) and (h) LPSPKFTIL (SEQ ID NO: 16) (p1123), or their analogues as defined above. Another polynucleotide, encoding a polyepitope, comprises at least one polynucleotidic unit chosen in the group of either a polynucleotide encoding a HLA-B7-restricted hTERT epitope corresponding to (a) RPSLTGARRL (SEQ ID NO: 11) (p351), (b) APSFRQVSCL (SEQ ID NO: 8) (p68), (c) APRCRAVRSL (SEQ ID NO: 7) (p4), (d) DPRRLVQLL (SEQ ID NO: 12) (p444), (e) FVRACLRRL (SEQ ID NO: 13) (p464), (f) AGRNMRRKL (SEQ ID NO: 14) (p966), (g) LPGTTLTAL (SEQ ID NO: 15) (p1107) and (h) LPSPKFTIL (SEQ ID NO: 16) (p1123) and/or their analogues as defined above, and at least one polynucleotidic unit chosen in the group of the polynucleotides encoding the sequence MPRAPRCRA (SEQ ID NO: 6) (p1), RPAEEATSL (SEQ ID NO: 9) (p277) or RPSFLLSSL (SEQ 10 NO: 10) (p342), or their analogues as defined above.
None of the polyepitope-encoding polynucleotides of the invention coincides with the coding sequence of the full length hTERT.
In a particular embodiment of the invention, the number of MHC class I restricted hTERT epitopes and/or analogues in the prepared polyepitope is limited to 30. In another embodiment, the number of HLA-B7 restricted hTERT epitopes and/or analogues is limited to approximately 30, and is preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30. In another embodiment, the number of HLA-B0702 restricted hTERT epitopes and/or analogues is limited to approximately 10, and is preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10.
Accordingly, the polynucleotide encoding a polyepitope has 30 or less polynucleotide units, especially 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 or any number within this range.
In a particular embodiment the polynucleotide units (and accordingly the epitopes) of the polynucleotide are consecutive.
In such a particular embodiment of the invention, the size of the nucleic sequence encoding the consecutive hTERT epitopes or analogues is less than 3000 bp, and preferably less than 2000 bp, 1000 bp, 500 bp, 400 bp, 300 bp, 200 bp or 100 bp.
In a particular embodiment, the polynucleotide encoding the multiple epitopes (polyepitope) consists of a nucleic acid molecule encoding a truncated or mutated form of the hTERT protein. In a preferred embodiment, the truncated or mutated form of the hTERT protein is deprived of its catalytic activity, i.e., is not capable to direct the synthesis of the repeat unit (T2AG3) at the ends of chromosomes, participating in the maintenance of telomere length. Such a hTERT protein deprived of its catalytic activity i.e., the retrotranscriptase activity, is said non-functional. Therefore, in another particular embodiment, the nucleic acid molecule encoding a truncated or mutated form of the hTERT protein lacks the catalytic activity domain of hTERT. In a particular embodiment, the nucleic acid molecule encoding a truncated form of the hTERT protein encodes a protein consisting of at least the 500 last C-terminal amino acids.
In a particular embodiment, the polynucleotide encodes a hTERT protein that is deleted for amino acids 867 to 869 (VDD sequence), corresponding to nucleotides 2654 to 2662 of FIG. 1 (wild-type) and providing nucleotide 2657 to be contiguous to nucleotide 2658 in FIG. 9 representing the deletion site, or alternatively for amino acids 864 to 872, corresponding to nucleotides 2645 to 2671 of FIG. 1 (wild-type) and providing nucleotide 2648 to be contiguous to nucleotide 2649 in FIG. 10 representing the deletion site. In a particular embodiment, the encoded hTERT protein has a deletion that comprises at least the amino acid residues 867 to 869 i.e., that the deletion is larger than the 3 amino acid residues (VDD sequence). As an example are the 864-872 deletion described above as well as a 22 amino acid deletion starting from amino acid residue 857 to 879 (according to FIG. 1) or a deletion comprising the 5 amino acids N-terminal and the 5 amino acids C-terminal to the VDD sequence (from amino acid 862 to amino acid 874 according to FIG. 1, corresponding to nucleotides 2639 to 2679). In a particular embodiment, the invention concerns a polynucleotide comprising or consisting of the nucleotide sequence as set forth in FIG. 9 or FIG. 10.
The polynucleotide units encoding the multiple epitopes of the invention can be arranged consecutively, i.e., the 3′ end of the first polynucleotide unit is directly linked to the 5′ end of the second polynucleotide unit (and so on), resulting in a polynucleotide encoding a peptidic sequence exclusively composed of consecutive epitopes. Such a polynucleotide can encode a polyepitope comprising or consisting of the p1, p4, p68, p277, p342, p351, p444, p464, p966, p1107 and p1123 peptides. In a particular embodiment, the polynucleotide encodes the following peptidic sequence MPRAPRCRAAPRCRAVRSLAPSFRQVSCLRPAEEATSLRPSFLLSSLRPSLTGARRL (SEQ ID NO: 20), comprising thus 6 MHC class I-restricted, particularly HLA-B7 restricted, hTERT epitopes.
The multiple epitopes of the invention can alternatively be separated by a one-amino acid spacer or a peptide spacer, i.e., meaning that the different polynucleotide units are separated by one or several codon(s) encoding respectively one or several amino acid(s). As spacers improving the processing of multiple epitopes, 4 amino acid-peptides composed of an arginine (R) in the C terminal position and hydrophilic residues (A, K, D and/or T) in other positions are preferred. Especially, 4 amino acid-peptides having a positively charged residue or an acidic residue in the C terminal position may be used, dependently or independently of hydrophilic residues (A, K, D and/or T) in other positions. In a particular embodiment, said spacers are internal processing sequences such as endosomal or lysosomal processing sequences, enabling the better processing of the multiple epitopes and avoiding the processing of new peptides resulting from overlapping cutting. Such a separation having recourse to a spacer can be used to separate all or, to the contrary, part of the polynucleotide units and accordingly, all or part of the epitopes.
The order in which the epitopes are arranged can be determined by the man skilled in the art, according to the following criteria: some orders may facilitate either the transcription and/or the translation of the polynucleotide, may facilitate the transport of the resulting expressed polyepitope in the endoplasmic reticulum (ER), especially if the tridimensional conformation impacts the properties, and may facilitate the processing of the polyepitope in several epitopes or analogues and avoid the processing of overlapping epitopes.
The polyepitope of the invention enables the elicitation of a CTL response against at least one, especially against several epitopes or analogues contained in the polyepitope, simultaneously, in a single animal or human.
In a particular embodiment, the polynucleotide encoding the polyepitope of the invention further comprises a polynucleotide encoding a target signal, operably linked to the polynucleotidic unit encoding the most N-terminal epitope of the at least two epitopes. “Operably linked” as used herein means that the target signal (upstream sequence) is linked to the N-terminal epitope (downstream sequence) in a way enabling the targeted signal to be operational, i.e., enabling to target the polyepitope to the correct cellular compartment or domain. Therefore, the link between the two sequences allows each sequence to play its own function in different locations and/or different stages. In a particular embodiment, said target signal is an endoplasmic reticulum signal sequence, and allows the polyepitope to be directed to the ER, for proper processing and association with the MHC class I molecule.
In a further embodiment, a codon encoding a methionine residue is added upstream of the sequence encoding the most N-terminal epitope, to enable the correct translation of the polynucleotide, if the translational process requires an initiation codon. Such a codon is added, only when the most N-terminal epitope does not possess a methionine residue in its first position.
The present invention also relates to a polynucleotide, according to the definitions given above, comprising or consisting of at least two polynucleotide units selected from the group consisting of











SEQ ID NO: 21




a. ATGCCGCGCGCTCCCCGCTGCCGAGCC (n1),



 




SEQ ID NO: 22




b. GCTCCCCGCTGCCGAGCCGTGCGCTCCCTG (n4),



 




SEQ ID NO: 23




c. GCCCCCTCCTTCCGCCAGGTGTCCTGCCTG (n68),



 




SEQ ID NO: 24




d. AGACCCGCCGAAGAAGCCACCTCTTTG (n277),



 




SEQ ID NO: 25




e. CGGCCCTCCTTCCTACTCAGCTCTCTG (n342),



 




SEQ ID NO: 26




f. AGGCCCAGCCTGACTGGCGCTCGGAGGCTC (n351),



 




SEQ ID NO: 27




g. GACCCCCGTCGCCTGGTGCAGCTGCTC (n444),



 




SEQ ID NO: 28




h. TTCGTGCGGGCCTGCCTGCGCCGGCTG (n464),



 




SEQ ID NO: 29




i. GCTGGGAGGAACATGCGTCGCAAACTC (n966),



 




SEQ ID NO: 30




j. CTCCCGGGGACGACGCTGACTGCCCTG (n1107),



 




SEQ ID NO: 31




k. CTGCCCTCAGACTTCAAGACCATCCTG (n1123),




and



 




SEQ ID NO: 32




l. GCCCCCCGTCGCCTGGTGCAGCTGCTC (n444*).






The following polynucleotides make also part of the invention:

    
    
        a polynucleotide, comprising at least one polynucleotidic unit selected from:
    
    


a. AGGCCCAGCCTGACTGGCGCTCGGAGGCTC (SEQ ID NO: 26) (n351)
b. GCCCCCTCCTTCCGCCAGGTGTCCTGCCTG (SEQ ID NO: 23) (n68),
c. GCTCCCCGCTGCCGAGCCGTGCGCTCCCTG (SEQ ID NO: 22) (n4),
d. GACCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 27) (n444),
e. TTCGTGCGGGCCTGCCTGCGCCGGCTG (SEQ ID NO: 28) (n464),
f. GCTGGGAGGAACATGCGTCGCAAACTC (SEQ ID NO: 29) (n966),
g. CTCCCGGGGACGACGCTGACTGCCCTG (SEQ ID NO: 30) (n1107),
h. CTGCCCTCAGACTTCAAGACCATCCTG (SEQ ID NO: 31) (n1123), and
i. GCCCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 32) (n444*), or their analogues as defined above, and at least one polynucleotidic unit selected from:
j. ATGCCGCGCGCTCCCCGCTGCCGAGCC (SEQ ID NO: 21) (n1),
k. AGACCCGCCGAAGAAGCCACCTCTTTG (SEQ ID NO: 24) (n277),
l. CGGCCCTCCTTCCTACTCAGCTCTCTG (SEQ ID NO: 25) (n342), or their analogues as defined above.

    
    
        a polynucleotide, comprising at least one polynucleotidic unit selected from:
    
    


a. AGGCCCAGCCTGACTGGCGCTCGGAGGCTC (SEQ ID NO: 26) (n351)
b. GCCCCCTCCTTCCGCCAGGTGTCCTGCCTG (SEQ ID NO: 23) (n68),
C. GCTCCCCGCTGCCGAGCCGTGCGCTCCCTG (SEQ ID NO: 22) (n4),
d. GACCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 27) (n444),
e. TTCGTGCGGGCCTGCCTGCGCCGGCTG (SEQ ID NO: 28) (n464),
f. GCTGGGAGGAACATGCGTCGCAAACTC (SEQ ID NO: 29) (n966),
g. CTCCCGGGGACGACGCTGACTGCCCTG (SEQ ID NO: 30) (n1107),
h. CTGCCCTCAGACTTCAAGACCATCCTG (SEQ ID NO: 31) (n1123), and
i. GCCCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 32) (n444*), or their analogues as defined above.
Are also included in the present invention, polynucleotides or polynucleotide units encoding an epitope, analogue or polyepitope of the invention, taking into consideration the degeneracy of the genetic code. Therefore, each amino acid can be encoded by the codon from the hTERT nucleotide reference sequence, or by any codon encoding said amino acid.
The invention also relates to a polynucleotide comprising or consisting of any combination of at least two of these polynucleotide units or analogues thereof, selected from the above group, wherein said polynucleotide unit or analogue encodes a MHC class I-restricted, especially a HLA-B7-restricted, hTERT epitope.
All features given above concerning epitopes, analogues, polyepitopes, combination thereof, spacers, target signal sequences . . . are applicable to any polypeptide of the invention as well as to the corresponding polynucleotide sequences.
A recombinant vector, comprising or consisting of a polynucleotide of the invention, as defined above, is also one object of the present invention. The recombinant vector can be a vector for eucaryotic or procaryotic expression, such as a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector. An expression vector as defined herein is chosen to enable the production of an epitope or analogue or polyepitope as defined above, either in vitro or in vivo.
Therefore, besides the polynucleotide, the vector of the invention can further comprise transcription regulation regions (including promoter, enhancer, ribosome binding site (RBS), polyA signal), a termination signal, a prokaryotic or eukaryotic origin of replication and/or a selection gene. The features of the promoter can be easily determined by the man skilled in the art in view of the expression needed, i.e., constitutive, transitory or inducible, strong or weak, tissue-specific and/or developmental stage-specific promoter. Therefore, tissue-specific promoters can be chosen depending on the organ in which a composition containing this vector is administered, for example injected, and depending on the intensity of expression required. In a particular embodiment, the promoter is the CMV promoter (human cytomegalovirus). Said vector can also comprise sequence enabling conditional expression, such as sequences of the Cre/Lox system or analogue systems.
The expression vectors of the invention may be viral vectors, and particularly viral expression vector, such as retroviral-derived, especially lentiviral-derived vectors such as HIV-, FIV- or SIV-derived vectors. More particularly, the lentiviral-derived vector is a human lentiviral-derived vector such as an HIV expression vector, particularly HIV-1 or HIV-2-derived vector. A retroviral-derived vector comprises a retroviral vector genome, usually included in a DNA construct, such as a plasmid, and expressed in viral particles, wherein said retroviral vector genome comprises the elements necessary for the retrotranscription, particularly the LTRs possibly mutated including deleted in part, especially deleted in the U3 region. In no case, the retroviral-derived vector contains all the nucleotide sequences encoding the full-length retroviral proteins. Possibly, it contains part of one or several of said nucleotide sequences providing it does not encode said proteins or functional fragments thereof. Said DNA construct comprising said retroviral vector genome further comprises a DNA of interest recombined with the retroviral nucleotide sequences, said DNA of interest comprising or consisting of a polynucleotide of the invention.
In a preferred embodiment, the retroviral-derived vector genome comprises a DNA flap as described below and at least one polynucleotide of the invention. In a preferred embodiment, the retroviral-derived vector is a HIV expression vector comprising a DNA flap as described below and at least one polynucleotide of the invention. HIV vectors express therefore only the nucleic acid(s) of interest, including the polynucleotides of the invention, contained between the two HIV LTRs and can thus accommodate large sequences up to 5-6 kb. A particular embodiment of the invention is a HIV expression vector, and most particularly a HIV-1 or HIV-2 expression vector, wherein a HIV-1 LTR or respectively the HIV-2 LTR is deleted for the promoter and the enhancer of the U3 domain (ΔU3). This particular deletion has been previously shown to increase the expression of the nucleic acid(s) contained in the vector, and particularly when associated with a promoter. Another particular embodiment, the vector comprises a LTR deleted in the promoter and the enhancer of the U3 domain, a promoter such as a CMV or EF1α promoter and a polynucleotide of the invention.
In another embodiment, the polynucleotide of the invention introduced in the retroviral-derived vector is included in an expression cassette.
A DNA flap (or Triplex as disclosed in WO 99/55892, WO 01/27300 and WO 01/27304) is a nucleotide sequence of retroviral or retroviral-like origin comprising two essential regions, i.e., the cPPT (central polypurine tract) and the CTS (cis-acting termination region) regions, wherein the cPPT and CTS regions induce a three-stranded DNA structure. A DNA flap suitable for the invention may be obtained from a retrovirus or retrovirus-like organism such as retrotransposon, prepared synthetically (chemical synthesis) or by amplification of the DNA flap from any retrovirus nucleic acid such as Polymerase Chain Reaction (PCR). The retrovirus, from which the DNA flap may be obtained, is particularly a retrovirus or a lentivirus, especially a human retrovirus or lentivirus and is in particular a HIV retrovirus, the CAEV (Caprine Arthritis Encephalitis Virus) virus, the EIAV (Equine Infectious Anaemia Virus) virus, the VISNA virus, the SIV (Simian Immunodeficiency Virus) virus or the FIV (Feline Immunodeficiency Virus) virus. In a more preferred embodiment, the DNA flap is obtained from an HIV retrovirus, for example HIV-1 or HIV-2 virus or any different isolate of these two types. It is noteworthy that the DNA flap is used isolated from its natural (viral genome) nucleotide context isolated from the pol gene in which it is naturally contained in a retrovirus. Therefore, the DNA flap is used, in the present invention, deleted from the unnecessary 5′ and 3′ parts of the pol gene and is recombined with sequences of different origin.
The DNA flap acts as a cis-determinant of the vector nuclear import, and is of great interest for the recombination and the integration of nucleic acid(s) into both non-dividing and dividing cells. Expression retroviral-derived vector, especially HIV derived-vectors, including the DNA flap sequence (TRIP vectors) are able to transduce primary B and T cells, macrophages, dendritic cells, etc with a tenfold higher efficiency than other HIV vectors that lack the DNA flap. A transduction of 80-90% of cells can be routinely obtained.
In a preferred embodiment, a vector suitable for an in vivo expression and vaccine strategy is a retroviral and especially a lentiviral vector (see WO 99/55892, WO 01/27300 and WO 01/27304). Such vectors have been shown to be particularly efficient and secure, when their genome is modified (Firat et al. 2002, The Journal of Gene Medicine 4: 38-45). Indeed, these vectors have the ability to efficiently and stably transfer therapeutic or reporter genes, in a large variety of cells and tissues, such as hematopoietic stem cells, brain, liver and retina. Moreover, this high transduction efficiency is irrespective of the proliferative status of the target cells. In particular, these vectors have been shown to efficiently induce CD8+ T cell responses both in vivo in mice and ex vivo in humans, due to their capacity to transduce antigen presenting cells such as dendritic cell (DC) with high efficiency, ex vivo as well as in vivo (Esslinger et al. Hum Gene Ther 2002; 13:1091-100; Breckpot et al. J Gene Med 2003; 5:654-67; Esslinger et al. J Clin Invest 2003; 111:1673-81).
The vector, defined in FIG. 7, has been used for in vitro or in vivo expression of epitopes, analogues or polyepitopes of the invention. As examples of vectors, from which the vectors of the invention can be derived, are the following, all deposited with the CNCM (Collection Nationale de Culture de Microorganismes at Institut Pasteur, Paris, France):
















Described in



Accession
Date of
patent


Vector name
number
deposition
application







pTRIP.EGFP
I-2005
Apr. 15, 1998
WO


PTRIP-MEL.IRES-GFP
I-2185
Apr. 20, 1999
99/55892


PTRIP-TEL/AML-IRES-GFP
I-2326
Oct. 11, 1999
WO


PTRIP-TEL/ILKE-IRES-GFP
I-2327
Oct. 11, 1999
01/27300


PTRIP-DES-IRES-GFP
I-2331
Oct. 11, 1999









The vectors described in the patent application WO 01/27304, and especially TRIP ΔU3 Efα1 GFP and TRIP ΔU3 PL CMV GFP, can also be used to derive the vectors of the present invention.
A particular vector of the invention is the pTRIP-CMV-ΔhTERT vector, deposited at the CNCM (Institut Pasteur, Paris, France) under the number CNCM I-3660 on Jul. 28, 2006. A suitable growth medium for cultivating this vector is a TB medium, optionally supplemented with hygromycin. Another expression vector of the invention is the pTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number CNCM I-3660 on Jul. 28, 2006, in which the deleted hTERT sequence has been substituted by any polynucleotide of the invention.
The present invention also relates to cells comprising the polynucleotides or polynucleotide units of the invention.
In one embodiment, the cell is transfected with a vector of the invention, by methods well known to the man skilled in the art, i.e. by chemical transfection (calcium phospate, lipofectamine), lipid-based techniques (liposome), electroporation, photoporation, use of viral vectors . . . . In another embodiment, a cell is transformed or transduced with a polynucleotide of the invention, in a way enabling integration of the polynucleotide in the cell genome either by a recombination with the homologous cellular sequence or by insertion in the cellular genome. The transfection, infection or transduction can occur ex vivo, i.e. in an artificial environment outside the living organism.
Among cells particularly interesting in the vaccine strategy are cells of the immune system, and especially antigen presenting cells (APC). In a particular embodiment, these cells are APCs involved either in MHC class I recognition, like dendritic cells (DC) or in MHC class II recognition such as macrophages or lymphocytes B. Among DCs, ex vivo fully maturated DCs, i.e., DCs that have been in vitro maturated by epitopes or analogues, are preferred.
As used herein, the terms “transfected”, “transformed” or infected” refer to a cell comprising a vector of the invention (transient expression), whereas the term “genetically transformed” refers to a cell whose genome has been definitively modified by a polynucleotide of the invention (permanent expression).
Said transitory or stably transformed cells can be any prokaryotic (bacteria) or eukaryotic (yeast, animal including mammal especially human) cells. In an embodiment, cells are non-human cells. In a particular embodiment, cells of the invention are isolated human cells, “isolated” meaning outside of its natural environment.
A particular host is the E. coli strain deposited at the CNCM under the number CNCM I-3660 on Jul. 28, 2006.
The invention also relates to epitopes, analogues or polyepitope as defined above when describing the polynucleotides of the invention and particularly to any polypeptide encoded by a polynucleotide or polynucleotide units of the invention. Particular polypeptides are MHC class I-restricted, especially HLA-87-restricted, hTERT epitope, chosen from the group consisting of:











SEQ ID NO: 6




a. MPRAPRCRA (p1),



 




SEQ ID NO: 7




b. APRCRAVRSL (p4),



 




SEQ ID NO: 8




c. APSFRQVSCL (p68),



 




SEQ ID NO: 9




d. RPAEEATSL (p277),



 




SEQ ID NO: 10




e. RPSFLLSSL (p342),



 




SEQ ID NO: 11




f. RPSLTGARRL (p351),



 




SEQ ID NO: 12




g. DPRRLVQLL (p444),



 




SEQ ID NO: 13




h. FVRACLRRL (p464),



 




SEQ ID NO: 14




i. AGRNMRRKL (p966),



 




SEQ ID NO: 15




j. LPGTTLTAL (p1107),




and



 




SEQ ID NO: 16




k. LPSPKFTIL (p1123).






Particular HLA-87-restricted hTERT epitopes, are chosen from the group consisting of:











SEQ ID NO: 11




a. RPSLTGARRL (p351),



 




SEQ ID NO: 8




b. APSFRQVSCL (p68),



 




SEQ ID NO: 7




c. APRCRAVRSL (p4),



 




SEQ ID NO: 12




d. DPRRLVQLL (p444),



 




SEQ ID NO: 13




e. FVRACLRRL (p464),



 




SEQ ID NO: 14




f. AGRNMRRKL (p966),



 




SEQ ID NO: 15




g. LPGTTLTAL (p1107),



 




SEQ ID NO: 16




h. LPSPKFTIL (p1123),




and



 




SEQ ID NO: 17




i. APRRLVQLL (p444*).






A particular group consists of the following MHC class I-restricted, especially HLA-87-restricted, hTERT epitopes:











SEQ ID NO: 6




a. MPRAPRCRA (p1),



 




SEQ ID NO: 7




b. APRCRAVRSL (p4),



 




SEQ ID NO: 8




c. APSFRQVSCL (p68),




and



 




SEQ ID NO: 11




d. RPSLTGARRL (p351).






Another group consists of the following HLA-87-restricted hTERT epitopes: RPAEEATSL (SEQ ID NO: 9) (p277) and RPSFLLSSL (SEQ ID NO: 10) (p342). A preferred HLA-87 allele targeted by these epitopes is HLA-80702.
The invention also concerns analogues of the peptides disclosed above, and having at least one amino acid substitution. Features pertaining to these analogues have especially been described in the above pages. A particular peptide analogue is p444* having the following peptide sequence APRRLVQLL (SEQ ID NO: 17).
Finally, the invention also concerns any polynucleotide encoding a hTERT epitope, analogue or polyepitope as described in the present specification.
The invention also relates to a polyepitope comprising at least two epitopes and/or analogues as described above. The polyepitopes of the invention are not the full-length hTERT protein. The size of said polyepitope can range from 15 to 1000 in particular from 50 or from 100 to 1000 amino acids, especially and particularly about 100, 200, 300, 400, 500 or 1000 amino acids. Such an epitope comprises or consists of 2 to 30 epitopes or analogues, and particularly 2 to 20 or 2 to 10 epitopes and/or analogues. A particular polyepitope comprises or consists of 6 consecutive epitopes and has the following sequence: MPRAPRCRAAPRCRAVRSLAPSFRQVSCLRPAEEATSLRPSFLLSSLRPSLTGARRL (SEQ ID NO: 20). Another particular polyepitope comprises or consists of the p1, p4, p68, p277, p342 and p351 epitopes, the epitopes being either consecutive to each other in the polyepitope obtained or all or part of them being separated by peptide spacers.
Another polyepitope of the invention comprises at least two epitopes, at least one being chosen from the group consisting of:











SEQ ID NO: 11




a. RPSLTGARRL (p351),



 




SEQ ID NO: 8




b. APSFRQVSCL (p68),



 




SEQ ID NO: 7




c. APRCRAVRSL (p4),



 




SEQ ID NO: 12




d. DPRRLVQLL (p444),



 




SEQ ID NO: 13




e. FVRACLRRL (p464),



 




SEQ ID NO: 14




f. AGRNMRRKL (p966),



 




SEQ ID NO: 15




g. LPGTTLTAL (p1107),



 




SEQ ID NO: 16




h. LPSPKFTIL (p1123),




and



 




SEQ ID NO: 17




i. APRRLVQLL (p444*),






or analogues thereof obtained by substitution of at least one amino acid residue, and at least one being chosen from the group consisting of











SEQ ID NO: 6




j. MPRAPRCRA (p1),



 




SEQ ID NO: 9




k. RPAEEATSL (p277),



 




SEQ ID NO: 10




l. RPSFLLSSL (p342),






or analogues thereof obtained by substitution of at least one amino acid residue, wherein said polyepitope is not the full length hTERT.

Polypeptides of the invention can be synthesized chemically, or produced either in vitro (cell free system) or in vivo after expression of the corresponding nucleic acid sequence in a cell system.
The full-length hTERT protein (SEQ ID NO: 37) as represented in FIG. 1 is excluded from the invention, as well as the corresponding full-length coding sequence (SEQ ID NO: 36). Also excluded from the present invention, the RPALL TSRL (SEQ ID NO: 42) peptide.
These peptides are excluded particularly in the context of HLA-87 recognition.
The invention also concerns an epitope, analogue, polyepitope or polynucleotide, an expression vector or host cell as defined above, for use to elicit or participate in providing a HLA-B7-restricted immune response against hTERT.
The invention also concerns a composition comprising a polynucleotide, a vector, a host cell and/or a polypeptide of the invention. In a particular embodiment, said composition is suitable for in vivo administration, i.e., said composition is prepared for injection, or more generally for association with physiologically-acceptable liquid solutions or emulsions for administration. Said composition may be used either for systemic or local administration, especially by injection, and may further comprises a pharmaceutically suitable excipient (including water, saline, dextrose, glycerol, ethanol, and combinations thereof) or a carrier and/or a vehicle.
In a particular embodiment, said composition comprises a polynucleotide of the invention encoding an epitope, analogue or encoding a polyepitope as described above. Said composition can comprise other nucleic acid molecules encoding at least one hTERT epitope or analogue thereof or polyepitope, restricted to a different MHC class I allele from that of HLA-B7. The combination of hTERT epitopes restricted to different HLA supertypes or alleles enables covering a larger population of patients in need of treatment than a sole supertype or allele. To this end, HLA-A1, -A2, -A3 and -A24 are preferred.
In another embodiment, the composition comprises nucleic acid molecules encoding at least one hTERT epitope or analogue thereof or polyepitope, restricted to MHC class II. Said composition can comprise any combination of nucleic acid molecules as described above, with at least one HLA-B7-restricted hTERT epitope, analogue or polyepitope of the present invention. The combination, in a composition of nucleic acid molecules, of polynucleotides encoding Class I and Class II-restricted epitopes enabling the reaction of various immune cells (T lymphocytes or NK cells for class I versus helper lymphocytes for class II), and/or the elicitation of various immune responses (humoral versus cellular response) is also within the present invention.
In another embodiment, the composition comprises nucleic acid molecules comprising at least such molecules encoding one tumour-specific antigen (TSA) and/or at least one tumour-associated antigen (TAA), such as prostate specific antigen (PSA), prostate-specific membrane antigen (PSMA) or prostatic acid phosphatase (PAP) (Tartour et al. 2000 Immunol Lett September 15; 74(1): 1-3; Tartour et al. 1996 Presse Med. November 16; 25(25): 1717-22).
Several types of therapeutic compositions can be used to elicit an immune response against an epitope or analogue of the invention.
A composition comprising a polynucleotide of the invention is administered to a host, for instance injected (known as DNA vaccination) and said nucleic acid expresses in vivo a polypeptide comprising or consisting of multiple epitopes according to the invention. Such DNA vaccines usually consist of plasmid vectors as described above. The delivery of naked DNA has shown to be poorly efficient, and some carriers are needed to improve the delivery and uptake of DNA into cells. Two types of carriers have been yet developed: (1) viral carriers (adenoviruses, lentiviruses, measles virus), or (2) non-viral carriers such as polymers (and especially cationic polymers), encapsulated-DNA (liposomes, comprising cationic lipids interact spontaneously and rapidly with polyanions, such as DNA and RNA, resulting in liposome/nucleic acid complexes) or DNA linked to gold microparticles. Moreover, agents, which assist in the cellular uptake of nucleic acid, such as calcium ions, bacterial proteins (Shigella proteins) viral proteins and other transfection facilitating agents, may advantageously be used. Another type of composition according to the invention is a composition comprising lentiviral pseudoparticles comprising a vector or vector genome as mentioned above.
Another type of composition comprises an epitope, analogue or polyepitope of the invention. Such a composition is immunogenic, i.e., it is capable of eliciting an immune response in a host in which it is administered. However, to increase the immunogenic properties of the polypeptides of the invention, an adjuvant can be administered with the polypeptide, to elicit or improve the immune response. An adjuvant is defined as any substance that enhances the immunogenicity of an antigen mixed with said adjuvant. Some adjuvants convert soluble antigens into small particles, such as aluminium hydroxide gel, oil in water emulsion or immune stimulatory complexes (ISCOMs). Another class of adjuvants comprises sterile constituents of bacteria such as cell wall or polysaccharides, Freund adjuvant. Finally, emulsifying agents or pH buffering agents can also be used to enhance the immunogenic behaviour of the epitope or analogue.
All compositions quoted above can be injected in a host via different routes: subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, oral administration and mucosal administration, especially intranasal administration or inhalation. The quantity to be administered (dosage) depends on the subject to be treated, including the condition of the patient, the state of the individual's immune system, the route of administration and the size of the host. Suitable dosages range from 200 μg to 1 mg, and can be modified by one skilled in the art, depending on circumstances.
The compositions of the invention are useful for the prophylaxis and or treatment of malignant states in patients, resulting from uncontrolled cell proliferation, including tumors, resulting from the over-expression of hTERT, as well for the treatment of detrimental consequences accompanying such malignant state, e.g., cancer. The expression “treatment” encompasses the curative effect achieved with compositions of the invention and also the beneficial effect for the patient undergoing the treatment, said effect being either obtained at cellular level or clinical level, including as a result, an improvement of the condition of the patient and/or a remission state or a recovery of a health state. In a particular embodiment, the composition of the invention further comprises additional active compounds useful for the prophylaxis or the treatment of tumors, either general compounds or compounds proved to be active in a tissue-specific cancer.
The invention also concerns a process to activate T lymphocytes against class I-restricted, particularly HLA-B7-restricted, hTERT epitopes:

    
    
        a. providing T lymphocytes, and,
        b. in vitro cultivating said T lymphocytes with at least one epitope or epitope analogue or polyepitope of the invention, in conditions enabling the activation of said lymphocytes.

In a particular embodiment, activated T lymphocytes are cytotoxic T lymphocytes (CTL). Conventional conditions to activate T lymphocytes use interleukine (IL) 2, IL-7, IL-12 and/or IL-15, and are described in Minev et al. (2000 PNAS; 97(9): 4796-4801) incorporated herein by reference.

    
    


The invention also relates to a process to check the immunogenic behaviour of a hTERT peptide, comprising:

    
    
        a. activating T lymphocytes, by in vitro cultivating said T lymphocytes with at least one epitope or epitope analogue or polyepitope of the invention, in appropriate conditions,
        b. in vitro cultivating said activated lymphocytes with target cells expressing, at their cell surface, a hTERT epitope of the invention bound to a MHC-class I molecule, in suitable conditions, and
        c. determining whether said activated lymphocytes react against said target cells.
    
    


In a particular process to check the immunogenic behaviour of a hTERT peptide, the epitope when used individually i.e., not under the form of a polyepitope, is chosen among (a) RPSLTGARRL (SEQ ID NO: 11) (p351), (b) APSFRQVSCL (SEQ ID NO: 8) (p68), (c) APRCRAVRSL (SEQ ID NO: 7) (p4), (d) DPRRLVQLL (SEQ ID NO: 12) (p444), (e) FVRACLRRL (SEQ ID NO: 13) (p464), (f) AGRNMRRKL (SEQ ID NO: 14) (p966), (g) LPGTTL TAL (SEQ ID NO: 15) (p1107) and (h) LPSPKFTIL (SEQ ID NO: 16) (p1123); an example of analogue is the p444* as defined above.
A particular vector of the invention is the pTRIP-CMV-ΔhTERT vector, deposited at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) (Institut Pasteur, 25 rue du Docteur Roux F-75724 Paris, France) under the number CNCM 1-3660 on Jul. 28, 2006. A suitable growth medium for cultivating this vector is a TB medium, optionally supplemented with hygromycin. Another expression vector of the invention is the pTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number CNCM 1-3660 on Jul. 28, 2006, in which the deleted hTERT sequence has been substituted by any polynucleotide of the invention.
The activation of lymphocytes includes the presentation of said epitopes or analogues by antigen presenting cells to naïve (not activated) T lymphocytes. Once naïve T lymphocytes have recognized the epitope or analogue of the invention, in the context of a particular class-I HLA molecule, they are said “activated” and ready to recognize said epitope on the cell surface of a target cell. The contact between said activated lymphocytes (effector cells) and target cells (expressing the epitope, from which the lymphocyte has been activated), leads to the secretion of molecules and killing of the target cells. If an epitope or analogue used to activate lymphocytes leads to an efficient destruction of a target cell bearing said epitope by said activated lymphocytes, said epitope can be considered as immunogenic enough to allow not only in vitro but also in vivo T cell reaction against cells expressing said epitopes. Suitable conditions for target cells/lymphocytes recognition are a 4 hour-contact at 37° C. in RPMI medium.
The invention also relates to a process to in vitro maturate cells, and especially antigen presenting cells (APC), B cells, T cells and/or dendritic cells, against MHC class I-restricted, particularly HLA-B7-restricted, hTERT epitopes. Such a process to in vitro maturate cells comprises:

    
    
        a. providing cells,
        b. enabling the maturation of said cells with at least one MHC class I-restricted, particularly HLA-B7-restricted, hTERT epitope or epitope analogue or polyepitope of the invention, and
        c. optionally, favouring the expansion of said maturated cells.
    
    


As described above, the activation of lymphocytes requires epitope presentation by maturated antigen presenting cells. In a preferred embodiment of the invention, said cells express at least one HLA-B7 allele. One of them, dendritic cells (DC), are particularly efficient in presentation of endogenous epitopes restricted to MHC class I, to T lymphocytes. One of the objectives in the maturation of said cells is their administration once maturated, to a patient in need of treatment. The administration of said maturated DCs would result in vivo in the activation of patient's lymphocytes, and rapid reaction against cell expressing the epitope (the one, the DCs have been transformed with).
In a particular embodiment, a process to in vitro maturate dendritic cells comprises:

    
    
        a. providing dendritic cells,
        b. enabling the maturation of said dendritic cells with at least one MHC class I-restricted hTERT epitope or epitope analogue or polyepitope of the invention, and
        c. optionally, favouring the expansion of said maturated dendritic cells.
    
    


In a particular embodiment, said dendritic cells are isolated from either circulating blood or bone marrow cells. In another embodiment, dendritic cells are isolated from the patient in need of treatment or from an HLA-matched donor, to avoid rejection after the administration to said patient.
The maturation of DCs can be achieved by genetic transformation of said dendritic cells with a polynucleotide of the invention, by transfection of said dendritic cells with a vector of the invention or by contacting said dendritic cells with at least one epitope, epitope analogue or polyepitope of the invention. The genetic transformation is preferred because of its efficiency and the permanent expression of the epitope, analogue or polyepitope encoded by the polynucleotide inserted in the DC genome.
The invention also concerns a polynucleotide encoding a HLA-87-restricted hTERT epitope for use in the prevention and/or treatment of cancer. In a particular embodiment, said polynucleotide, for use in the prevention and/or treatment of cancer encodes a HLA-87-restricted hTERT epitope or analogue thereof or a polyepitope comprising at least one HLA-87-restricted hTERT epitope or analogue thereof as described in the present application. As far as the polyepitope-encoding polynucleotide is concerned, it does not coincide with the coding sequence of the full-length hTERT. On the other hand, it can coincide with a mutated or deleted version of HTERT, said mutation or deletion suppressing the catalytic activity of the human telomerase. In a particular embodiment, said polynucleotide comprises at least one polynucleotide unit encoding a HLA-87-restricted hTERT epitope, chosen from the group consisting of:











SEQ ID NO: 6




a. MPRAPRCRA  (p1),



 




SEQ ID NO: 7




b. APRCRAVRSL (p4),



 




SEQ ID NO: 8




c. APSFRQVSCL (p68),



 




SEQ ID NO: 9




d. RPAEEATSL  (p277),



 




SEQ ID NO: 10




e. RPSFLLSSL  (p342),



 




SEQ ID NO: 11




f. RPSLTGARRL (p351)



 




SEQ ID NO: 12




g. DPRRLVQLL  (p444),



 




SEQ ID NO: 13




h. FVRACLRRL  (p464),



 




SEQ ID NO: 14




i. AGRNMRRKL  (p966),



 




SEQ ID NO: 15




j. LPGTTLTAL  (p1107),




and



 




SEQ ID NO: 16




k. LPSPKFTIL  (p1123).






or at least one analogue of said HLA-87-restricted hTERT, such as APRRLVQLL (SEQ ID NO: 17) (p444*), or any combination forming a polynucleotide having at least two polynucleotide units encoding said HLA-87 epitopes and/or analogues.

The invention concerns a HLA-B7 hTERT epitope, and especially a HLA-B0702 restricted epitope for use in the prevention and/or treatment of cancer. The invention relates also to a polynucleotide, a vector, a host cell or a polypeptide of the invention for use in the prevention and/or treatment of cancer.
The invention also relates to the use of a HLA-B7 hTERT epitope, (or corresponding polynucleotide) for the manufacture of a drug for the prevention and/or treatment of cancer. Particular HLA-B7 hTERT epitopes (or corresponding polynucleotides) are those described above as well as vectors, cells or compositions comprising or consisting of them. In a particular embodiment, the use of polynucleotide, vector, host cell or polypeptide comprising or consisting of a polyepitope of the invention in the manufacture of a drug for the prevention and/or treatment of cancer is intended for patients having at least one HLA-B7 allele as defined above, and particularly at least one HLA-B0702 allele.
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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: 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.
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 (▪) or control (□) peptides as described in the material and methods. Percentage of lysis at a 60/1 ratio are shown (results from two independent experiments).
FIG. 3: Induction of CTL response against hTERT in PBMC from health blood donors.
T-lymphocyte cells from HLA-*B0702+ 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 (▪) or control (□) peptides, were essayed for lytic activity against 51Cr-labeled T2-B7 cells. Percentage of lysis at a 20/1 effector-target ratio is shown. Results from 8 out of 10 donors are presented (d1 to d8).
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+ 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 II mAb (HLA-DR)) was determined by standard 51Cr-release assay at effector-target ratio of 10/1.
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.
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.
FIG. 6: Priming of specific CD8+ 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 bars) or control (2 last horizontal vertical stripe 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.
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).
FIG. 8:
A) DNA pTRIP-CMV-ΔhTERT immunization primed hTERT-specific CD8+ 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 HLA-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.
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 51Cr-release assay against HHD-transfected EL4 cells loaded with either the relevant peptide or the irrelevant peptide.
FIG. 9: 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.
FIG. 10: 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 3438. 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
Peripheral bloods samples were obtained following written informed consent from adult healthy platelet donors (centre de transfusion sanguine de l'hôpital Mondor, Créteil, 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
HLA-*B0702 transgenic (Tg) mice, expressing an HLA-B0702 α1α2, H2-Kd α3 chimeric construct, in combination with constitutive murine β2-m molecule (HLA-B7mα3) and HHD transgenic mice expressing a chimeric HLA-A2.1/H2-Db molecule, were deleted of their H2-Db and H2-kb genes as previously described (Pascolo et al. J Exp Med 1997; 185:2043-51; Rohrlich et al. Int 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
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).
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
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-87-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).
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
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. Int Immunol 2003; 15:765-72). Briefly, T2-B7 were incubated overnight at 2×105 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.
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
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 re-activated in vitro with relevant peptide in six wells plate. Effector CTL cells were tested in a standard 4-5 h 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
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
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-SaII 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 293 T 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).
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
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.
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.
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
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 (5×105, 2.5×105 and 1.25×105 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/106 cells.
Cytolytic Assay
Cytotoxicity assays were performed by using standard 4-5 h 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
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 FICOLL®-purified human PBMCS were thawed and incubated (4×106/well) in 24-well plates in RPMI 1640, 1 mM sodium pyruvate, 100 IU/m1 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.
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+ 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 51Cr-labeled targets: T2-B7 pulsed with tested hTERT peptides or irrelevant peptide, or tumor cell lines.
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
1. Immunogenicity of HLA-*B-0702 Predicted Peptides Derived from hTERT
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.
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 1








Immunogenicity of selected hTERT HLA-*B0702 binding peptide














SEQ ID


Immunogenicity‡













Peptide*
Sequence
NO:
Score§
Relative avidity†
R/T**
Specific Lysis (%)





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.


§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.


‡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 4-5h 51Cr release assay using peptide loaded RMA-B7 target cells. The Specific lysis was calculated by subtracting non-specific lysis observed with the R10TV control peptide. Specific lysis at a 75:1 effector/target ratio was showed.


**R/T: responder (specific lysis ≧ 10%) versus tested mice.






2. hTERT-Derived Peptides were Processed in HLA-B0702 Transgenic Mice
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 intracellularly 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.
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
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 (d1 to d8), 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+ tumor cells.
4. Specific hTERT CTL were Able to Lyse Tumors of Differents Origins
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+ tumour cells, whereas no cytotoxicity against HLA-B0702− tumors was detected. (See for example CTLp351 in d1, d2 and d3 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).
As shown in FIG. 4, the cytotoxic activity of CTLp351 line toward HLA-B7+ 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+ 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
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.
As shown in FIG. 5A, peptide-specific CD8+ 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+ 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.
As shown in FIG. 8, hTERT peptide-specific CD8+ 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+ 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.
Collectively, these results showed that a single injection of TRIP-hTERT resulted in the induction of a potent multi-specific anti-hTERT CD8+ T-cells response in both HLA transgenic mice groups.







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
d 1
d 6
d 8
d 1
d 6
d 8
d 1
d 6
d 8
d 1
d 6
d 8
d 1
d 6
d 8
d 1
d 6
d 8






























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§
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 51Cr-labeled release assay. Specific lysis: for a 30:1 effector:target ratio were shown


§Autologous B lymphocytes from normal donors were activated for 48 h with a trimeric CD40 L (40 μg/ml).













TABLE 3







Induction of CTL responses following Lv-hTERT immunization


Flap+ Lv-hTRT immunization








HLA-B7 Tg mice
HHD mice












Restimulating


Restimulating




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
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+ PBL from healthy donors induce specific CTL responses recognizing hTERT+ 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 non-functional telomerase gene inserted in a new generation of lentiviral derived flap vector. A strong hTERT specific CD8+ 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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## Claims

1. A method of inducing an HLA-B7-restricted immune response against at least one hTERT epitope, comprising:
administering to an animal a lentiviral vector comprising a polynucleotide sequence that encodes a modified hTERT protein comprising the amino acid sequence of SEQ ID NO: 39;
expressing the modified hTERT protein; and
inducing an HLA-B7-restricted immune response against at least one hTERT epitope selected from:












SEQ ID NO: 6




a. MPRAPRCRA  (p1),



 




SEQ ID NO: 7




b. APRCRAVRSL (p4),



 




SEQ ID NO: 8




c. APSFRQVSCL (p68),



 




SEQ ID NO: 9




d. RPAEEATSL  (p277),



 




SEQ ID NO: 10




e. RPSFLLSSL  (p342),



 




SEQ ID NO: 11




f. RPSLTGARRL (p351).

2. The method of claim 1, wherein the lentiviral vector comprising the polynucleotide sequence that encodes a modified hTERT protein is the pTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number 1-3660 on Jul. 28, 2006.

3. The method of claim 1, 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.

4. The method of claim 3, wherein the APCs are dendritic cells.

5. The method of claim 1, wherein the animal is a human.

6. The method of claim 1, the method comprising inducing an HLA-B7-restricted immune response against at least two hTERT epitopes selected from:











SEQ ID NO: 6




a. MPRAPRCRA  (p1),



 




SEQ ID NO: 7




b. APRCRAVRSL (p4),



 




SEQ ID NO: 8




c. APSFRQVSCL (p68),



 




SEQ ID NO: 9




d. RPAEEATSL  (p277),



 




SEQ ID NO: 10




e. RPSFLLSSL  (p342),




and



 




SEQ ID NO: 11




f. RPSLTGARRL (p351).

7. The method of claim 6, wherein the lentiviral vector comprising the polynucleotide sequence that encodes a modified hTERT protein is the pTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number 1-3660 on Jul. 28, 2006.

8. The method of claim 6, 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.

9. The method of claim 8, wherein the APCs are dendritic cells.

10. The method of claim 6, wherein the animal is a human.

11. The method of claim 1, the method comprising inducing an HLA-B7-restricted immune response against each of hTERT epitopes:











SEQ ID NO: 6




a. MPRAPRCRA  (p1),



 




SEQ ID NO: 7




b. APRCRAVRSL (p4),



 




SEQ ID NO: 8




c. APSFRQVSCL (p68),



 




SEQ ID NO: 9




d. RPAEEATSL  (p277),



 




SEQ ID NO: 10




e. RPSFLLSSL  (p342),




and



 




SEQ ID NO: 11




f. RPSLTGARRL (p351).

12. The method of claim 11, wherein the lentiviral vector comprising the polynucleotide sequence that encodes a modified hTERT protein is the pTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number 1-3660 on Jul. 28, 2006.

13. The method of claim 11, 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.

14. The method of claim 13, wherein the APCs are dendritic cells.

15. The method of claim 11, wherein the animal is a human.