SEQUENCE LISTING

The instant application contains a Sequence Listing which is being submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 31, 2020, is named 069818-0364_SL.txt and is 15,272 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and immunology. In particular it relates to novel HPV epitopes that may be used in the prevention, therapy and/or diagnosis of HPV associated diseases.

BACKGROUND OF THE INVENTION

Cervical cancer is the second most common cancer worldwide (Bosch et al. 2003). High risk human papilloma virus (HPV) type 16 and 18 are the cause of cervical cancer in around two third of all patients (Bosch et al. 1995, Munoz et al. 2003). The HPV genome encodes two oncoproteins, E6 and E7, which are constitutively expressed in high grade cervical lesions and cancer because they are required for the onset and maintenance of the malignant cellular phenotype (Zur Hausen, 1996).

The tumor-specific expression of these oncoproteins as well as the presence of low levels of circulating E6- and E7-specific T cells detected in the peripheral blood of almost half of patients with cervical cancer (de Jong et al. 2004, van der Berg et al. 2001, Welters et al. 2003, Welters et al. 2006, Ressing et al. 1996, Bontkes et al. 2000, Luxton et al. 1996) suggested that they could serve as tumor rejection antigens. However, the existence of circulating HPV-specific T cells does not imply that they contribute to the anti-tumor response. In order to control the disease, these T cells should at least be able to home to the tumor sites. Indeed, a proportion of cervical carcinomas are infiltrated by lymphocytes (Bethwaite et al. 1996, Chao et al. 1999, Piersma et al. 2007) but in-depth knowledge on the specificity and type of the T cells infiltrating these cervical tumors is still lacking, probably due to the relative difficulties to establish T cell cultures from tumor tissue. Nonetheless, a few early pioneers were able to isolate HPV-specific tumor infiltrating lymphocytes (TIL) from tumors, resulting in the identification of two single CD8+T cell epitopes of HPV16 (Evans et al. 1997, Oerke et al. 2005) and two CD4 T cell epitopes specific for the less prevalent high risk subtypes HPV59 and HPV33 (Hohn et al. 1999, Hohn et al. 2000). However, larger studies on cervical tissue-infiltrating lymphocytes are urgently needed to comprehend the contribution and role of the HPV-specific adaptive immune response in cervical cancer. In addition, this will allow the rational design of successful immune intervention strategies.

Recent studies showed that two cytokines, IL-7 and IL-15, have a major role in the expansion and survival of CD4+and CD8+effector memory T cells. IL-7 provides survival signals for effector T cells (Li et al. 2003). IL-15 is a critical growth factor in initiating T cell divisions, and in contrast to IL-2—which is generally used to expand TIL cultures—does not limit continued T-cell expansion (Li et al. 2001). Furthermore, IL-15 can also act as an antigen-independent activator of CD8(+) memory T cells (Liu et al. 2002). Together, IL-7 and IL-15 can expand with very high efficiency effector memory T cells, while central memory T cells are less responsive and naive T cells fail to respond to stimulation with these cytokines (Geginat et al. 2001, McKinlay et al. 2007, Bacchetta et al. 2002).

A number of previous studies have reported MHC class II restricted recognition of synthetic peptides consisting of sequences from in HPV16 E6 and/or E7 proteins by T cell from peripheral blood mononuclear cells (PBMC).

WO 02/070006 discloses a DR1 restricted response against a peptide consisting of amino acids 127-142 of HPV16 E6 protein, a DQ2 restricted response against a peptide consisting of amino acids 35-50 of HPV16 E7 protein, a DR3 restricted response against a peptide consisting of amino acids 43-77 of HPV16 E7 protein and a DR15 restricted response against a peptide consisting of amino acids 50-62 of HPV16 E7 protein.

Strang et al. disclose a DR7 restricted response in PBMC from asymptomatic individuals against a synthetic peptide consisting of amino acids 42-57 of HPV16 E6 protein.

Altmann et al. discloses a response in PBMC from asymptomatic individuals that are DR1/DR11-typed against a synthetic peptide consisting of amino acids 5-18 of HPV16 E7 protein, a response in PBMC from asymptomatic individuals that are DR4/DR13-typed against a synthetic peptide consisting of amino acids 17-34 of HPV16 E7 protein and a response in PBMC from asymptomatic individuals that are DR4/DR13-typed against a synthetic peptide consisting of amino acids 69-82 of HPV16 E7 protein.

WO 02/090382 discloses the binding affinities for a series of overlapping peptides from HPV16 E6 and E7 proteins for HLA-DR molecules that are most prevalent in the caucasian population. WO 02/090382 further reports responses against a number of the HPV16 E6 and E7 peptides in CD8-depleted PBMC from patients with bowenoid papulosis.

There is however still a need for knowledge about the presence, type and specificity of tumor infiltrating lymphocytes in HPV-associated malignancies, preferably for the more prevalent high risk subtypes such as HPV16, 18, 31, 33 and 45. It is an object of the present invention to provide for HPV epitopes that are targets for tumor infiltrating lymphocytes and that may be used in the prevention, therapy and/or diagnosis of HPV associated diseases.

DESCRIPTION OF THE INVENTION

The present invention provides novel T cell epitopes that are identified on the basis of our analysis of the presence and HPV16 or HPV18 specificity of cervix infiltrating T cells in a large group of 70 patients with cervical malignancies. We found that these infiltrating lymphocytes comprise HPV-specific T cells. In more detailed analysis we identified 17 novel CD4+and CD8+T cell epitopes and their HLA-restriction elements but also revealed that HPV-specific immune response directed towards all parts of the E6 and E7 oncoproteins. Unexpectedly, the vast majority of the CD4+T cell epitopes were presented in the context of the less abundantly expressed HLA-DQ and HLA-DP molecules. Since the identified T cell epitopes constitute physiological targets in the immune response to HPV16 and HPV18 positive tumors they are valuable targets for optimization of prevention against HPV-related diseases and immunotherapy in patients with HPV related diseases.

In one aspect, the present invention thus relates to amino acid sequences of newly identified CD4+Th and CD8+CTL cell epitopes of HPV, as well as HPV derived synthetic peptides and immunogenic compositions comprising these are also part of the present invention. Such peptides result in a much improved, enhanced and prolonged CD8+CTL effector and memory response upon administration in a wide range of patients with HPV associated disease, including HPV related malignancies. Such peptides can also induce a much improved pro-inflammatory microenvironment that is more likely to be infiltrated by effector cells, as the result of this CD4+Th response.

Since the peptides of the invention are preferably used as a vaccine alone or in combination or as part of an immunogenic composition, the peptides are preferably named vaccine peptides and the composition vaccine compositions.

The use of relatively short peptides is highly preferred for medical purposes as these can be synthesized in vitro efficiently, which is not possible or uneconomical for native proteins larger than about 100 amino acids. Chemical synthesis of peptides is routine practice and various suitable methods are known to the skilled person. Chemical synthesis of peptides also overcomes the problems associated with recombinant production of intact proteins, which is difficult to standardize and requires extensive purification and quality control measures. Peptides with a length that exceeds the length of HLA class I and class II epitopes (e.g. having a length as indicated below herein) are particularly advantageous for use as vaccine component because they are large enough to be taken up by professional antigen presenting cells, in particular DC, as explained in WO02/070006 and processed in the DC before cell surface presentation of the contained HLA class I and class II epitopes takes place. Therefore, the disadvantageous induction of T cell tolerance by the systemic presentation of minimal HLA class I epitopes on non-antigen presenting cells (as shown in Toes et al., 1996, Proc. Natl. Acad. Sci. U.S.A 93:7855 and Toes et al., 1996, J. Immunol. 156:3911), is prevented by the application of peptides of the invention having a length as indicated herein (as shown in Zwaveling et al., 2002, J. Immunol. 169:350). Peptides comprising epitopes which are to be presented to T cell receptors of CTL and/or Th cells preferably have sufficient length to contain both HLA class I and HLA class II epitopes

In a first aspect of the invention there is provided a peptide comprising a contiguous amino acid sequence selected from the full length amino acid sequences of at least one of the HPV E6 and E7 proteins. Preferably, the contiguous amino acid sequence selected from the full length amino acid sequences of the HPV E6 and E7 proteins from a high risk HPV serotype, such as serotypes 16, 18, 31, 33 or 45, more preferably from the amino acid sequences of the HPV E6 and E7 serotypes 16, 18, 31 or 33, most preferably from serotypes 16 or 18, of which 16 is most preferred. The amino acid sequence of the HPV serotype 16 E6 and E7 proteins are depicted in SEQ ID No. 1 and 2, respectively. The amino acid sequence of the HPV serotype 18 E6 and E7 proteins are depicted in SEQ ID No. 3 and 4, respectively.

Preferably, the peptide comprises at least one HLA class II Th cell epitope and/or at least one HLA class I cytotoxic T cell epitope, preferably an epitope as herein defined below in more detail. Preferably the peptide has a length of no more than 100 amino acids and comprises at least 19 contiguous amino acids selected from the amino acid sequence of one of the above-defined HPV proteins, wherein the peptide preferably comprises at least one of an HLA class II epitope and an HLA class I epitope, more preferably both at least one HLA class II epitope and at least one HLA class I epitope and most preferably (but not necessarily) both from the amino acid sequence of one of the above-defined HPV proteins. More preferably, in the peptide at least one HLA class II epitope and at least one HLA class I epitope are present within a contiguous amino sequence from the amino acid sequence of one of the above-defined HPV proteins. For the sake of clarity, the peptides of the invention preferably comprise HLA class I presented epitopes and/or HLA class II presented epitopes. Each of these epitopes are presentable and will bind to the corresponding specific HLA molecule present on the cells after having been processed as described herein. In the context of the invention, an HLA-haplotype specific epitope may therefore also be referred to as an epitope binding to, presented by and/or being restricted by that HLA-haplotype.

Within the context of the invention, “a peptide has a length of no more than 100 amino acids” preferably means that the number of consecutive amino acids originating from a HPV protein and present in a peptide as defined herein, is 100, 98, 96, 94, 92, 90 or less. Therefore, by definition, a peptide as defined herein is distinct from a full length HPV protein. Such a peptide may comprise additional amino acids than the ones originating from a HPV protein or may entirely be made of or consist of an amino acid sequence originating from a HPV protein. The length of the contiguous amino acid sequence from one of the above-defined HPV proteins comprised within the peptide, preferably is at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids and/or preferably no more than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 60, 50, 45, 40, 35, 33 or 30 amino acids, more preferably the length of the contiguous amino acid sequence from one of the above-defined HPV proteins comprised within the peptide is 19-45, even more preferably 22-40 amino acids, even more preferably 30-35 and most preferably 33-35 amino acids. In another preferred embodiment, the peptide of the invention consists of any of the contiguous amino acid sequence from the HPV proteins as defined herein, whereby it is understood that no amino acids are appended to either end of the contiguous amino acid sequence from the HPV protein that are not contiguous with this amino acid sequence in the sequence of the native HPV protein. The peptides of the invention may be easily synthesized and are large enough to be taken up by professional antigen presenting cells, processed by the proteasome and have sufficient physical capacity and length to contain at least one HLA class I and/or at least one HLA class II epitope. Optionally a peptide may comprise N- or C-terminal extensions, which may be amino acids, modified amino acids or other functional groups that may for instance enhance bio-availability, cellular uptake, processing and/or solubility.

A preferred peptide of the invention has a length of no more than 100, 98, 96, 94, 92 amino acids and comprises at least 19 contiguous amino acids from the amino acid sequence of at least one of an HPV E6 and E7 protein, wherein the contiguous amino acid sequence comprises an epitope that is recognized by a T cell that infiltrates a cervical neoplastic lesion or by a T cell that is present in or isolated from a lymph node from the pelvic region, that is draining from the cervical neoplastic lesion, preferably a T cell that is present in or isolated from a draining lymph node comprising metastatic tumor cells. A peptide according to the invention is preferably used to induce a T-cell response.

In a further preferred peptide of the invention the contiguous amino acid sequence comprises an epitope that is selected from the group consisting of amino acids 11-32 of an HPV E6 protein, amino acids 37-68 of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein, amino acids 51-72 of an HPV6 protein, amino acids 55-86 of an HPV E6 protein, amino acids 61-82 of an HPV E6 protein, amino acids 71-92 of an HPV E6 protein, amino acids 73-105 of an HPV E6 protein, amino acids 91-112 of an HPV E6 protein, amino acids 101-122 of an HPV E6 protein, amino acids 121-142 of an HPV E6 protein, amino acids 129-138 of an HPV E6 protein, amino acids 1-32 of an HPV E7 protein, amino acids 21-42 of an HPV E7 protein, amino acids 51-72 of an HPV E7 protein, amino acids 76-86 of an HPV E7 protein; amino acids 13-22 of an HPV E6 protein, amino acids 29-38 of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein, amino acids 129-138 of an HPV E6 protein, amino acids 137-146 of an HPV E6 protein, amino acids 149-158 of an HPV E6 protein, and amino acids 11-19 of an HPV E7 protein. In yet a further preferred peptide of the invention the contiguous amino acid sequence comprises an epitope that is selected from the group consisting of SEQ ID No.'s 5-26.

A preferred peptide of the invention comprises at least an HPV-specific class II CD4+Th cell epitope. Preferably, a class II CD4+Th cell epitope comprised in a peptide according to the invention is capable of inducing or activating a CD4+Th cell in human patient with an HPV associated disease and/or a healthy control. The activation is preferably assessed ex vivo or in vivo, more preferably in a human patient with an HPV associated disease, such as an HPV associated malignancy, whose infected and/or tumor cells express an HPV protein as defined above. Most preferably, the HLA class II epitope is capable of activating a CD4+Th memory and/or CD4+Th-effector response, i.e. activation of a CD45RO-positive CD4+Th cell. This will lead, by virtue of the ‘license to kill’ signal through CD40-triggering of DC (Lanzavecchia, 1998) to a more robust CD8+effector and memory CTL response. In another setting the activated CD4+Th-cells may activate non-HLA restricted killer cells of the immune system.

A preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is selected from the group consisting of amino acids 11-32 of an HPV E6 protein, amino acids 37-68 of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein, amino acids 51-72 of an HPV E6 protein, amino acids 55-86 of an HPV E6 protein, amino acids 61-82 of an HPV E6 protein, amino acids 71-92 of an HPV E6 protein, amino acids 73-105 of an HPV E6 protein, amino acids 91-112 of an HPV E6 protein, amino acids 101-122 of an HPV E6 protein, amino acids 121-142 of an HPV E6 protein, amino acids 129-138 of an HPV E6 protein, amino acids 1-32 of an HPV E7 protein, amino acids 21-42 of an HPV E7 protein, amino acids 51-72 of an HPV E7 protein, and amino acids 76-86 of an HPV E7 protein. A more preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is selected from the group consisting SEQ ID No.'s 5-21.

Another preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is an epitope that is restricted by a haplotype selected from the group consisting of DR4, DR7, DR12, DR15, DP1, DP0201, DP4, DP14, DP1401, DP17, DQ5, DQ6, DP1901, DQ*0301, DQ*0302, DQ*0308, DQ*0501. A further preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is an epitope that is restricted by a DP or DQ haplotype, of which DP1, DP0201, DP4, DP14, DP1401, DP17, DQ5, DQ6, DP1901, DQ*0301, DQ*0302, DQ*0308, and DQ*0501 are more preferred. One previously disclosed HLA-DQ restricted epitope (WO02/070006) consists of amino acid 35-50 of the HPV16 E7 protein. This epitope is however recognized epitope by peripheral T cells and not by a T cell that infiltrates a cervical neoplastic lesion or by a T cell that is present in or isolated from a lymph node from the pelvic region, that is draining from the cervical neoplastic lesion. The contiguous sequence in a peptide of the invention therefore preferably does not comprise an epitope consisting of amino acid 35-50 of the HPV16 E7 protein. Thus, a preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is an epitope that is restricted by a DP or DQ haplotype and not by a DR haplotype. Expression of HLA-DR molecules is known to be upregulated on tumor cells. Presentation in that context may, as presentation of antigens on non-professional Antigen Presenting Cells (APC), lead to induction of tolerance. Expression of HLA-DP or -DQ molecules is much lower but HLA-DQ and HLA-DP epitopes when presented on professional APC, such as e.g. DC, may nonetheless lead to effective immune responses.

Yet another preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is an epitope that is restricted by a DP or DQ haplotype and that is an epitope of an HPV E6 or E7 protein, more preferably an E6 or E7 protein of HPV serotypes 16, 18, 31, 33 or 45, and most preferably of HPV serotypes 16 or 18, of which 16 is most preferred.

Yet a further preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is an epitope selected from the group consisting of amino acids 11-32 of an HPV E6 protein, amino acids 37-68 of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein, amino acids 51-72 of an HPV E6 protein, amino acids 61-82 of an HPV E6 protein, amino acids 71-92 of an HPV E6 protein, amino acids 73-105 of an HPV E6 protein, amino acids 91-112 of an HPV E6 protein, amino acids 101-122 of an HPV E6 protein, amino acids 121-142 of an HPV E6 protein, amino acids 1-32 of an HPV E7 protein, and amino acids 51-72 of an HPV E7 protein. A more preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is selected from the group consisting SEQ ID No.'s 5, 6, 7, 9, 10, 11, 12, 13, 16, 18, 19, 20 and 21.

In another preferred embodiment, a peptide of the invention comprises at least an HPV-specific class I CD8+CTL epitope. In addition, said HLA class I epitope is preferably capable of activating a CD8+CTL response. Most preferably, the CTL activating capability has been demonstrated ex vivo and/or in vivo, in human healthy control individuals or even more preferably in a human patient with an HPV associated disease, such as an HPV associated malignancy, whose infected and/or tumor cells express an HPV protein as defined above. The presence of both an HLA class I and class II epitope within one peptide has been observed to be particularly advantageous due to synergy in mounting and maintaining an effective CTL cell response (as shown in Zwaveling et al., 2002).

Peptides comprising epitopes which are to be presented to T cell receptors of CTL and/or Th cells preferably fulfill a number of requirements. The peptides preferably have sufficient length to contain both HLA class I and HLA class II epitopes. Furthermore, the peptides preferably comprise anchor residues within their HLA class I binding parts to enable binding to the class I molecules, respectively. The stability of the interaction between peptide and presenting MHC molecule preferably is sufficient in order to generate a significant and effective immune response. In the context of the present invention, the stability of the interaction between peptide and presenting MEW molecule therefore preferably is such that the peptide has an intermediate to high affinity binding, whereby an IC50≤about 5 μM is considered high affinity binding, about 5 μM<IC50≤about 15 μM is considered intermediate affinity binding, about 15 μM<IC50≤100 μM is judged low affinity binding and IC50>about 100 μM was regarded as no binding, whereby the binding affinity of a peptide for an MHC molecule is determined as described in van der Burg et al., 1995 and Kessler et al., 2003.

A specific proteasomal cleavage site generating the C-terminus of the epitope, preferably is present exactly after the epitope amino acid sequence in order to be liberated from the larger peptide and presented on the HLA class I molecule. Length requirements are much less strict for HLA class II presented epitopes, therefore a need for precise enzymatic generation of the class II binding peptide is less absolute. These requirements have been used in the present invention to localize and design peptides in the full length sequences of HPV proteins, particularly in the HPV E6 and E7 proteins, which comprise preferred CTL and Th cell epitopes and/or combinations thereof and are thus highly suitable peptides for vaccination purposes.

Moreover, in vitro and ex vivo T cell experiments are preferably used to confirm the capability of peptides according to the invention to induce substantial CD4+Th and CD8+CTL responses. The peptides of the present invention thereby provide a marked improvement in the selection of relatively short peptides that may be chemically synthesized, comprising the most potent and most widely applicable HLA class I and/or class II presented T cell epitopes derived from the HPV E6 and E7 tumor antigens. The peptides are particularly optimized with respect to their proteasomal cleavage and preferably contain at least one of HLA class I and class II epitopes and more preferably both HLA class I and class II epitopes. The liberation of the C-termini of CTL epitopes contained within the peptides of the invention by the 20S proteasome provides HLA class I binding fragments with CD8+CTL stimulatory capacity.

The HLA class I epitopes in the HPV peptides of the invention are preferably capable of being presented on HLA alleles that are predominant in the population of human subjects to be treated. Preferred HLA class I epitopes in HPV derived peptides of the invention are epitopes capable of binding to HLA-A2, HLA-B7, HLA-B14, HLA-B27, HLA-B57, and HLA*0201. The most preferred HLA class I CTL epitopes are the HLA-B binding HPV epitopes, of which HLA-B7, HLA-B14, HLA-B27, HLA-B57 are most preferred. The HLA class I epitope preferably has a high peptide binding capacity (IC50<about 5 μM peptide) or at least intermediate affinity (5 μM<IC50<about 15 μM peptide). A preferred class I CTL epitope comprised in (a contiguous sequence in) a peptide according to the invention is an epitope that is restricted by class I haplotype as indicated above and that is an epitope of an HPV E6 or E7 protein, more preferably an E6 or E7 protein of HPV serotypes 16, 18, 31, 33 or 45, and most preferably of HPV serotypes 16 or 18, of which 16 is most preferred.

A preferred class I CTL epitope comprised in (a contiguous sequence in) a peptide according to the invention is selected from the group consisting of amino acids 13-22 of an HPV E6 protein, amino acids 29-38 of an HPV E6 protein, amino acids 52-61 of an HPV E6 protein, amino acids 129-138 of an HPV E6 protein, amino acids 137-146 of an HPV E6 protein, amino acids 149-158 of an HPV E6 protein and amino acids 11-19 of an HPV E7 protein. A more preferred class II CD4+Th cell epitope comprised in (a contiguous sequence in) a peptide according to the invention is selected from the group consisting SEQ ID No.'s 7, 14, 22-26.

A preferred epitope comprised in a peptide according to the invention is an epitope that is presented by an HLA-B molecule. Preferably, the HLA-B molecule is an HLA-B7, HLA-B14, HLA-B27 or HLA-B57 molecule. Such epitope is selected from the group consisting of SEQ ID No.'s 7, 22, 24, 25 and 26.

Another preferred epitope comprised in a peptide according to the invention is an epitope that is presented by an HLA-A molecule. Preferably the HLA-A molecule is an HLA-A2, or HLA*0201 molecule. Such epitope is selected from the group consisting of SEQ ID No.'s 23 and 26.

According to a more preferred embodiment, peptides of the invention have a length of no more than 100, 98, 96, 94,94, 92 amino acids and comprise a contiguous amino acid sequence from an HPV protein selected from the group consisting of amino acids 1-32 of an HPV E6 protein (SEQ ID NO: 27), amino acids 19-50 of an HPV E6 protein (SEQ ID NO: 28), amino acids 41-65 of an HPV E6 protein (SEQ ID NO: 29), amino acids 55-80 of an HPV E6 protein (SEQ ID NO: 30), amino acids 71-95 of an HPV E6 protein (SEQ ID NO: 31), amino acids 85-109 of an HPV E6 protein (SEQ ID NO: 32), amino acids 91-122 of an HPV E6 protein (SEQ ID NO: 33), amino acids 109-140 of an HPV E6 protein E6 (SEQ ID NO: 34), amino acids 127-158 of an HPV E6 protein (SEQ ID NO: 35), amino acids 1-35 of an HPV E7 protein (SEQ ID NO: 36), amino acids 22-56 of an HPV E7 protein (SEQ ID NO: 37), amino acids 43-77 of an HPV E7 protein (SEQ ID NO: 38), and amino acids 64-98 of an HPV E7 protein (SEQ ID NO: 39). More preferably the peptides of the invention consist of a contiguous amino acid sequence from an HPV protein selected from the group consisting of amino acids 1-32 of an HPV E6 protein (SEQ ID NO: 27), amino acids 19-50 of an HPV E6 protein (SEQ ID NO: 28), amino acids 41-65 of an HPV E6 protein (SEQ ID NO: 29), amino acids 55-80 of an HPV E6 protein (SEQ ID NO: 30), amino acids 71-95 of an HPV E6 protein (SEQ ID NO: 31), amino acids 85-109 of an HPV E6 protein (SEQ ID NO: 32), amino acids 91-122 of an HPV E6 protein (SEQ ID NO: 33), amino acids 109-140 of an HPV E6 protein E6 (SEQ ID NO: 34), amino acids 127-158 of an HPV E6 protein (SEQ ID NO: 35), amino acids 1-35 of an HPV E7 protein (SEQ ID NO: 36), amino acids 22-56 of an HPV E7 protein (SEQ ID NO: 37), amino acids 43-77 of an HPV E7 protein (SEQ ID NO: 38), and amino acids 64-98 of an HPV E7 protein (SEQ ID NO: 39). The contiguous amino acid sequence from the HPV E6 or E7 proteins are preferably of HPV serotypes 16, 18, 31, 33 or 45, and most preferably of HPV serotypes 16 or 18, of which 16 is most preferred.

It is clear to a skilled person that a peptide as defined herein will have a desired and advantageous property linked to the presence of an epitope in said peptide (for example an epitope which is identified in the invention as being presented by at least one of an HLA-DQ and HLA-DP molecule and/or as being recognized by a T cell that infiltrates a cervical neoplastic lesion or by a T cell from a draining lymph node) as soon as this epitope is present in said peptide. A peptide according to the invention is preferably used to induce a T-cell response. The skilled person will understand that even if this application does not identify each peptide that can be designed as comprising or consisting of a desired epitope as identified herein, nevertheless the invention encompasses any peptide as defined herein comprising or consisting of an epitope as identified herein. In a preferred embodiment, a peptide is distinct from a HPV protein. In another preferred embodiment, a peptide does not comprise or consist of amino acid 35-50 of the HPV16 E7.

For example, one preferred epitope is SEQ ID NO:5 (aa 11-32 of HPV16 E6). This paragraph is illustrative and may be applied for each epitope as identified herein. Any peptide comprising SEQ ID NO:5 is encompassed by the present invention and may be used according to the present invention. In this preferred embodiment, a peptide is distinct from a HPV protein. Preferred amino acid length for a peptide of the invention has already been defined herein. When designing a peptide of the invention, a peptide may start at the N-terminal site of a given epitope as identified herein or end at the C-terminal site of a given epitope as identified herein. Alternatively, a given epitope (for example SEQ ID NO:5) may be comprised within a peptide of the invention. Using SEQ ID NO:5 as example, if we design a peptide having a length of 45 amino acids, such peptide may consist or comprise 11-56, 1-45, 2-46, 3-47, 4-48, 5-49, 5-50 from HPV16 E6. A peptide of the invention may further comprise any other HPV epitope as defined herein or as already known to the skilled person.

In this preferred embodiment (SEQ ID NO:5 as epitope), a peptide does not comprise or consist of amino acid 9-33 of the HPV16 E6 as disclosed in US2005/0142541. In this preferred embodiment, a peptide does not comprise or consist of amino acid 1-37 of the HPV16 E6 as disclosed in EP 451 550. In this preferred embodiment, a peptide does not comprise or consist of amino acid 8-37 of the HPV16 E6 as disclosed in U.S. Pat. No. 5,629,161. In a preferred embodiment, a peptide comprising SEQ ID NO:5 consists of or comprises 10-32, 1-32, 1-45, 11-56, 2-46, 3-47, 4-48, 5-49, 5-50 the numbers indicating the starting and ending amino acid from HPV16 E6
In another preferred embodiment (SEQ ID NO:8 as epitope, aa 55-86 from HPV16 E6), a peptide does not comprise or consist of a fragment of HPV16 E6 as disclosed on uniprot having the following accession number Q919B2 (1-99, numbers indicating the starting and ending amino acid from HPV16 E6) or Q80882 (1-84). For this embodiment also, a peptide comprising SEQ ID NO:8 may start at the N-terminal site of this epitope, or end at the C-terminal site of this epitope, or this epitope may be present within the peptide. For example if we design a peptide having a length of 45 amino acids, such peptides may consist or comprise 55-100, 41-86, 45-90. In a preferred embodiment, a peptide comprising SEQ ID NO:8 consists of or comprises 55-100, 41-86, 45-90, the numbers indicating the starting and ending amino acid in the HPV16 E6 protein amino acid sequence.

The HPV-derived peptides of the invention may be modified by deletion or substitution of one or more amino acids, by extension at the N- and/or C-terminus with additional amino acids or functional groups, which may improve bio-availability, targeting to T-cells, or comprise or release immune modulating substances that provide adjuvant or (co)stimulatory functions. The optional additional amino acids at the N- and/or C-terminus are preferably not present in the corresponding positions in the native amino acid sequence of the HPV protein, more preferably they are not from any of the HPV E6 or E7 amino acid sequences (e.g. SEQ ID No.'s 1-4). The skilled person will appreciate that HPV amino acid sequences of the various HPV serotypes are expressly included in the invention.

The HPV-derived peptides of the invention are obtainable by chemical synthesis and subsequent purification (e.g. see Example 1). The HPV-derived peptides of the invention are preferably soluble in physiologically acceptable watery solutions (e.g. PBS) comprising no more than 35, 20, 10, 5 or 0% DMSO. In such a solution the peptides are preferably soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml. More preferably, a mixture of more than one different HPV-derived peptides of the invention is soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml in such solutions.

A preferred use of the peptides according to the invention is their use as a medicament, whereby more preferably the peptides are used as a vaccine or an active component thereof. Each peptide may be either used alone or preferably in combinations of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 15 and up to 20 different peptides of the invention, in the treatment and/or prevention of cancer, for the manufacture of medicaments, preferably vaccine for the treatment or prevention of an HPV associated disease. Such a medicament and/or anti-tumor vaccine according to the invention may be used to treat patients suffering from or at risk of developing the following, non extensive list of cervical intraepithelial neoplasia of the cervix (CIN), vulva (VIN), vagina (VaIN), anus (AIN), and penis (PIN), as well as cancer of the cervix, vulva, vagina, anus, penis, and head & neck.

In a further aspect, the current invention further relates to compositions which may be useful for treatment and/or vaccination of human subjects, comprising at least at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 15 and up to 20 different peptides of the invention as defined above and optionally one or more pharmaceutically acceptable excipients, in particular adjuvants and immune modulators. Preferably, the composition is a pharmaceutical composition and/or intended for use as a medicament. The pharmaceutical composition is preferably intended for vaccination. The pharmaceutical composition are preferably used for the treatment and/or prevention of cancer, for the manufacture of medicaments, preferably vaccine for the treatment or prevention of an HPV associated disease. A non-exhaustive list of an HPV associated diseases has already been given herein.

Thus, in one aspect the invention relates to the use of a peptide for the manufacture of a medicament for the prevention and/or treatment of an HPV associated disease, wherein the peptide has a length of no more than 100, 98, 96, 94, 92 amino acids and comprises at least 19 contiguous amino acids from the amino acid sequence of at least one of an HPV E6 and E7 protein, wherein the contiguous amino acid sequence comprises an epitope that is presented by at least one of an HLA-DQ and HLA-DP molecule. Preferably, the epitope is not the epitope presented in the context of HLA-DQ2 and consisting of amino acid 35-50 of the HPV16 E7 protein. Alternatively or in combination with previous preferred embodiment in another preferred embodiment, the contiguous amino acid sequence comprises an epitope that is recognized by a T cell that infiltrates a cervical neoplastic lesion or by a T cell from a draining lymph node. The peptides, contiguous amino acid sequences and epitopes are preferably as defined herein above.

In another aspect the invention relates to the use of a peptide for the manufacture of a medicament for the prevention and/or treatment of an HPV related disease, wherein the peptide has a length of no more than 100, 98, 96, 94, 92, amino acids and comprises at least 19 contiguous amino acids from the amino acid sequence of at least one of an HPV E6 and E7 protein, wherein the contiguous amino acid sequence comprises an epitope that is recognized by a T cell that infiltrates a cervical neoplastic lesion or by a T cell from a draining lymph node. The peptides, contiguous amino acid sequences and epitopes are preferably as defined herein above.

Formulation of medicaments, ways of administration and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21stEdition 2005, University of Sciences in Philadelphia. Pharmaceutical compositions and medicaments of the invention are preferably formulated to be suitable for intravenous or subcutaneous, or intramuscular administration, although other administration routes can be envisaged, such as mucosal administration or intradermal and/or intracutaneous administration, e.g. by injection. Intradermal administration is preferred herein. Advantages and/or preferred embodiments that are specifically associated with intradermal administration are later on defined in a separate section entitled “intradermal administration”.

It is furthermore encompassed by the present invention that the administration of at least one peptide and/or at least one composition of the invention may be carried out as a single administration. Alternatively, the administration of at least one peptide and/or at least one composition may be repeated if needed and/or distinct peptides and/or compositions of the invention may be sequentially administered.

The pharmaceutically compositions (also referred to as medicaments) according to the invention may preferably comprise at least one immune response stimulating compound or adjuvant. Advantageously the pharmaceutical composition according to the invention may additionally comprise one or more synthetic adjuvants. These adjuvants may be admixed to the pharmaceutical composition according to the invention or may be administered separately to the mammal or human to be treated. Particularly preferred are those adjuvants that are known to act via the Toll-like receptors and/or via a RIG-1 (Retinoic acid-Inducible Gene-1) protein and/or via an endothelin receptor. Immune modifying compounds that are capable of activation of the innate immune system can be activated particularly well via Toll like receptors (TLR's), including TLR's 1-10. Compounds capable of activating TLR receptors and modifications and derivatives thereof are well documented in the art. TLR1 may be activated by bacterial lipoproteins and acetylated forms thereof, TLR2 may in addition be activated by Gram positive bacterial glycolipids, LPS, LPA, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or from the host, and Mycobacterial lipoarabinomannans. TLR3 may be activated by dsRNA, in particular of viral origin, or by the chemical compound poly(I:C). TLR4 may be activated by Gram negative LPS, LTA, Heat shock proteins from the host or from bacterial origin, viral coat or envelope proteins, taxol or derivatives thereof, hyaluronan containing oligosaccharides and fibronectins. TLR5 may be activated with bacterial flagellae or flagellin. TLR6 may be activated by mycobacterial lipoproteins and group BStreptococcusheat labile soluble factor (GBS-F) orStaphylococcusmodulins. TLR7 may be activated by imidazoquinolines. TLR9 may be activated by unmethylated CpG DNA or chromatin—IgG complexes. In particular TLR3, TLR7 and TLR9 play an important role in mediating an innate immune response against viral infections, and compounds capable of activating these receptors are particularly preferred for use in the methods of treatment and in the compositions or medicaments according to the invention. Particularly preferred adjuvants comprise, but are not limited to, synthetically produced compounds comprising dsRNA, poly(I:C), unmethylated CpG DNA which trigger TLR3 and TLR9 receptors, IC31, a TLR 9 agonist, IMSAVAC, a TLR 4 agonist, Montanide ISA-51, Montanide ISA 720 (an adjuvant produced by Seppic 7, France). RIG-1 protein is known to be activated by ds-RNA just like TLR3 (Immunity, (2005), 1:19-28). In another preferred embodiment, the synthetic adjuvant compounds are physically linked to the peptides of the invention. Physical linkage of adjuvants and costimulatory compounds or functional groups, to the HLA class I and HLA class II epitope comprising peptides provides an enhanced immune response by simultaneous stimulation of antigen presenting cells, in particular dendritic cells, that internalize, metabolize and display antigen. Another preferred immune modifying compound is an inhibitor of an endothelin receptor such as BQ-788 (Buckanovich R J et al. Nature Medicine (2008), 14:28-36, Ishikawa K, PNAS (1994) 91:4892). BQ-788 is N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine. However any derivative of BQ-788 or modified BQ-788 compound is also encompassed within the scope of this invention.

Furthermore, the use of antigen presenting cell (co)stimulatory molecules, as set out in WO99/61065 and in WO03/084999, in combination with the peptides and compositions of the invention is preferred. In particular the use of 4-1-BB and/or CD40 ligands, agonistic antibodies, OX40 ligands or functional fragments and derivates thereof, as well as synthetic compounds with similar agonistic activity are preferably administered separately or combined with the peptides of the invention to subjects to be treated in order to further stimulate the mounting of an optimal immune response in the subject.

In addition a preferred embodiment comprises delivery of the peptides, with or without additional immune stimulants such as TLR ligands and/or anti CD40/anti-4-1 BB antibodies in a slow release vehicle such as mineral oil (e.g. Montanide ISA 51) or PLGA. Alternatively, the peptides of the invention may be delivered by intradermally, e.g. by injection, with or without immune stimulants (adjuvants). Preferably for intradermal delivery the peptides of the invention are administered in a composition consisting of the peptides and one or more immunologically inert pharmaceutically acceptable carriers, e.g. buffered aqueous solutions at physiological ionic strength and/or osmolarity (such as e.g. PBS).

Intradermal Administration

In a preferred embodiment, a peptide or a composition comprising a peptide or a medicament used in the invention all as defined herein are formulated to be suitable for intradermal administration or application. Intradermal is known to the skilled person. In the context of the invention, intradermal is synonymous with intracutaneous and is distinct from subcutaneous. A most superficial application of a substance is epicutaenous (on the skin), then would come an intradermal application (in or into the skin), then a subcutaneous application (in the tissues just under the skin), then an intramuscular application (into the body of the muscle). An intradermal application is usually given by injection. An intradermal injection of a substance is usually done to test a possible reaction, allergy and/or cellular immunity to it. A subcutaneous application is usually also given by injection: a needle is injected in the tissues under the skin.

In another further preferred embodiment, the medicament used in the invention does not comprise any adjuvant such as Montanide ISA-51, it means the formulation of the medicament is more simple: an oil-water based emulsion is preferably not present in the medicament used. Accordingly, the medicament used in the invention does not comprise an adjuvant such as Montanide ISA-51 and/or does not comprise an oil-in-water based emulsion. Therefore, in a preferred embodiment, the medicament used in the invention is a buffered aqueous solutions at physiological ionic strength and/or osmolarity, such as e.g. PBS (Phosphate Buffer Saline) comprising or consisting of one or more peptide as defined earlier herein. The skilled person knows how to prepare such a solution.

The medicament as used in the invention has another advantage, which is that by intradermally administering low amounts of a peptide as earlier herein defined, an immunogenic effect may still be achieved. The amount of each peptide used is preferably ranged between 1 and 1000 μg, more preferably between 5 and 500 μg, even more preferably between 10 and 100 μg.

In another preferred embodiment, the medicament comprises a peptide as earlier defined herein and at least one adjuvant, said adjuvant being not formulated in an oil-in water based emulsion and/or not being of an oil-in-water emulsion type as earlier defined herein. This type of medicament may be administered as a single administration. Alternatively, the administration of a peptide as earlier herein defined and/or an adjuvant may be repeated if needed and/or distinct peptides and/or distinct adjuvants may be sequentially administered. It is further encompassed by the present invention that a peptide of the invention is administered intradermally whereas an adjuvant as defined herein is sequentially administered. The adjuvant may be intradermally administered. However any other way of administration may be used for the adjuvant.

The intradermal administration of a peptide is very attractive since the injection of the vaccine is realized at or as close by as possible to the site of the disease resulting in the local activation of the disease draining lymph node, resulting in a stronger local activation of the immune system. In particular for VIN, VAIN, AIN, PIN, Penile cancer, Vulva cancer, Anal cancer, Head and Neck cancers.

In a preferred embodiment, the intradermal administration is carried out directly at the site of the lesion or disease. At the site of the lesion is herein understood to be within less than 5, 2, 1, 0.5, 0.2 or 0.1 cm from the site of the lesion.

Upon intradermally administering a medicament as defined herein, not only Th2 but also Th1 responses are triggered. This is surprising since it was already found that cutaneous antigen priming via gene gun lead to a selective Th2 immune response (Alvarez D. et al, 2005 Furthermore, the immune response observed is not only restricted to the skin as could be expected based on (Alvarez D. et al, 2005). We demonstrate that specific T cells secreting IFNγ circulate through the secondary lymph system as they are detected in the post challenged peripheral blood.

Another crucial advantage of the medicament of the invention is that relatively low amounts of peptides may be used, in one single shot, in a simple formulation and without any adjuvant known to give undesired side-effects as Montanide ISA-51. Without wishing to be bound by any theory, we believe the HPV intradermal peptide(s) used in the invention specifically and directly targets the epidermal Langerhans cells (LC) present in the epithelium. Langerhans cells are a specific subtype of DC which exhibit outstanding capacity to initiate primary immune responses (Romani N. et al 1992). These LC may be seen as natural adjuvants recruited by the medicament used in the invention.

In another preferred embodiment, the invention relates to the use of a peptide derived from HPV-E2, -E6 and/or -E7 protein for the manufacture of a medicament for the treatment or prevention of an HPV related disease, wherein the medicament is for intradermal administration as earlier defined and wherein in addition a peptide derived from HPV-E2, -E6 and/or -E7 protein is further used for the manufacture of a medicament for the treatment or prevention of an HPV related disease, wherein the medicament is for subcutaneous administration.

The medicament for intradermal administration has already been defined herein. The peptide used for subcutaneous administration is the same as the one used for intradermal administration and has already been defined herein. The skilled person knows how to formulate a medicament suited for subcutaneous administration. Preferably, the medicament suited for subcutaneous administration comprises a peptide as already herein defined in combination with an adjuvant. Preferred adjuvants have already been mentioned herein. Other preferred adjuvants are of the type of an oil-in water emulsions such as incomplete Freund's adjuvant or IFA, Montanide ISA-51 or Montanide ISA 720 (Seppic France). In a further preferred embodiment, the medicament suited for subcutaneous administration comprises one or more peptides, an adjuvant both as earlier defined herein and an inert pharmaceutically acceptable carrier and/or excipients all as earlier defined herein. Formulation of medicaments, and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21ndEdition 2005, University of Sciences in Philadelphia. The second medicament used in the invention is formulated to be suitable for subcutaenous administration.

In this preferred embodiment, the medicament suited for intradermal administration may be simultaneously administered with the medicament suited for subcutaneous administration. Alternatively, both medicament may be sequentially intradermally and subsequently subcutaneously administered or vice versa (first subcutaneous administration followed by intradermal administration). In this preferred embodiment as in earlier preferred embodiment dedicated to the intradermal administration, the intradermal and/or subcutaneous administration of a peptide as earlier herein defined and/or of an adjuvant may be repeated if needed and/or of distinct peptides and/or of distinct adjuvants may be sequentially intradermally and/or subcutaneously administered. It is further encompassed by the present invention that a peptide of the invention is administered intradermally and/or subcutaneously whereas an adjuvant as defined herein is sequentially administered. The adjuvant may be intradermally and/or subcutaneously administered. However any other way of administration may be used for the adjuvant.

We expect the combination of an intradermal and a subcutaneous administration of a medicament according to the invention is advantageous. DC in the epidermis are clearly different from DC in the dermis and in the subcutis. The intracutaneous (intradermal) immunization will cause antigen processing and activation of epidermal DC (Langerin-positive langerhans cells) that through their dendritic network are in close contact with the keratinocytes. This will also optimally activate inflammatory pathways in the interactions between Langerhans cell and keratinocytes, followed by trafficking of antigen loaded and activated Langerhans cell to the skin-draining lymph nodes.

The subcutaneous administration will activate other DC subsets, that will also become loaded with antigen and travel independently to the skin-draining lymph nodes. Conceivably, the use of a medicament which may be administered both intradermally and subcutaneously may lead to a synergistic stimulation of T-cells in these draining nodes by the different DC subsets.

In another aspect, the invention relates to nucleic acids encoding the peptides and/or epitopes as defined herein above. Preferably the nucleic acids do not encode the wild type full length HPV E6 or E7 proteins but rather encode the peptides and/or epitopes of the invention as such, or flanked by amino acid sequence that are not contiguous with the wild type HPV E6 or E7 proteins. Such flanking amino acids may be from proteins other than the wild type HPV E6 or E7 proteins and/or they may be from other locations within the wild type HPV E6 or E7 proteins that are not contiguous with the peptide/epitope they flank. In a preferred embodiment the nucleic acids encode two or more peptides and/or epitopes of the invention arranged as beads-on-string, whereby the peptides and/or epitopes of the invention (the beads) are linked directly together and/or are linked through linker sequences that are from proteins other than the wild type HPV E6 or E7 proteins and/or from other locations within the wild type HPV E6 or E7 proteins that are not contiguous with the peptide/epitope they flank. The amino acid sequences flanking or linking the peptides/epitopes may comprise proteolytic cleavage sites. Such nucleic acids may be applied to deliver the peptides/epitopes of the invention in various ways. They may e.g. be used in the production of recombinant protein in a suitable host cell (e.g.E. coli) from which the may be purified. Alternatively the nucleic acid may be operably linked to expression regulatory sequences (promoters and the like) and incorporated in expression constructs for human cells. Such (autologous) cells may be transfected or transduced ex vivo to be (re)-administered to a subject in need thereof. Alternatively the expression construct may be incorporated into suitable gene therapy vector. Viral vector (based on a defective virus) are more efficient agents for gene transfer as compared to the non-viral agents. Suitable viral expression constructs include e.g. vectors that are based on adenovirus, adeno-associated virus (AAV), retroviruses or modified vaccinia Ankara (MVA).

In another embodiment, the present invention provides a tool to isolate HPV-specific T cell receptor (TCR) molecules from T cells capable of interacting with an HPV epitope of the invention as herein described. A TCR according to this invention will preferably be capable of interacting with the HPV epitope comprising peptides when they are in the context of and/or displayed by an HLA molecule, preferably on a living cell in vitro or in vivo. T cell receptors and in particular nucleic acids encoding TCR's according to the invention may for instance be applied to transfer such a TCR into T cells from patients, whom are otherwise not capable to raise T cell immunity against an HPV epitopes of the invention as herein described. By this TCR cloning method, T cell clones may be provided that essentially are isogenic with the recipient to be treated with the T cell clones, i.e. the TCR expression T cell clones are autologous to the patient suffering from an HPV associated disease. The method thus provides T cell clones capable of recognizing an HPV epitope according to the invention that may be generated for and can be specifically targeted to tumor and/or HPV-infected cells expressing an HPV epitope in a subject in need thereof. In a preferred embodiment T-cells from the subject are isolated and transduced with the TCR recognizing the HPV epitopes of the invention as herein described. Following selection and expansion, known to the skilled artisan, these autologous T cells that are now expressing a TCR which can recognize HPV-induced tumor cells or HPV infected cells, can be re-infused into the patient where they specifically target to the tumor and HPV infected cells. Hence, the invention provides T lymphocytes encoding and expressing a T cell receptor capable of interacting with an HPV epitope as defined herein, preferably in the context of an HLA molecule. Said T lymphocyte may be a recombinant or a naturally selected T lymphocyte. T lymphocytes of the invention may also be used for or in the methods and pharmaceutical compositions of the invention. This specification thus provides at least two methods for producing a cytotoxic T lymphocyte of the invention, comprising the step of bringing undifferentiated lymphocytes into contact with an HPV epitope of the invention (or a peptide comprising the epitope) under conditions conducive of triggering an immune response, which may be done in vitro or in vivo for instance in a patient receiving a graft, using peptides according to the invention. Alternatively, it may be carried out in vitro by cloning a gene encoding the TCR specific for interacting with an HPV epitope of the invention, which may be obtained from a cell obtained from the previous method or which may be obtained from a subject exhibiting an immune response against the epitope, into a host cell and/or a host lymphocyte, preferably a autologous lymphocyte, and optionally differentiate to cytotoxic T lymphocyte (CTL). Details of the methods in this embodiment are described in e.g. De Witte et al. 2006 and Schumacher et al. 2002.

In a further embodiment the invention pertains to the use of the nucleic acids encoding the peptides and/or epitopes of the invention, T cell receptors recognizing the epitopes of the invention, nucleic acids encoding such T cell receptors, T cell (clones) expressing such nucleic acids as a medicament. Preferably the medicament is used in the treatment and/or prevention of an HPV associated disease. Such a medicament according to the invention may be used to treat patients suffering from or at risk of developing the following, non extensive list of cervical intraepithelial neoplasia of the cervix (CIN), vulva (VIN), vagina (VaIN), anus (AIN), and penis (PIN), as well as cancer of the cervix, vulva, vagina, anus, penis, and head & neck.

In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a peptide or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way

