Branched oncolytic peptides and uses thereof

Abstract

The invention concerns branched oncolytic peptides and uses thereof in the treatment of cancer diseases. Pharmaceutical compositions comprising them are also described. The peptides of the invention were found to be active in the treatment of particular tumors called "cold tumors", which cannot benefit from the most advanced antibody therapies.

Description

[0001] BRANCHED ONCOLYTIC PEPTIDES AND USES THEREOF

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The invention concerns branched oncolytic peptides and uses thereof in the treatment of cancer diseases. Pharmaceutical compositions comprising them are also described. The peptides of the invention were found to be active in the treatment of particular tumors called “cold tumors”, which cannot benefit from the most advanced antibody therapies.

[0005] STATE OF THE ART

[0006] Immunologically inactive tumors, such as pancreatic or colon cancer, are typically more difficult to combat and cannot benefit from new immunotherapeutic drugs, particularly immune checkpoint inhibitors (ICIs). The matrix of “cold” tumors is, in fact, rich in infiltrating immunosuppressive cells, including tumor-associated macrophages (TAMs), regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSCs), and poor in immunosupportive CD8+ T cells. Furthermore, these tumors have a low mutational level, with low levels of neoantigens and a reduced ability of T cells to recognize the tumors.

[0007] Furthermore, “cold” tumors are largely surrounded by connective tissue and poorly perfused; therefore, the stroma acts as a physical barrier that makes it difficult for treatments and immune cells to reach the cancer. Therefore, effective stimulation of the tumor microenvironment is necessary to achieve an effective antitumor immune response. In 2005, the concept of “immunogenic cell death” (ICD) was introduced to indicate cases of regulated cell death inducing antigen-specific immune responses that culminate in immunological memory.

[0008] Cellular stress triggered by chemotherapy, radiotherapy, and targeted anticancer drugs can generate neoantigens, thus favoring the expression of normally silenced genes.

[0009] Furthermore, anticancer drugs can enhance the expression of class I and II major histocompatibility complex molecules on the surface of tumor cells, thereby increasing their antigenicity. However, antigenicity alone cannot trigger adaptive immunity. Indeed, antigenic peptide presentation to T cells, in the absence of co-stimulatory signals, generally leads to T cell anergy linked to peripheral tolerance. In fact, antigen-presenting cells (APCs) have to acquire antigenic material while scanning the extracellular microenvironment, so as to promote T cell activation, providing co-stimulation and pro-inflammatory cytokines.

[0010] A variety of endogenous adjuvant signals, collectively known as danger-associated molecular patterns (DAMPs), are released by cancer cells during immunogenic stress or death, and this induces maturation of antigen-presenting cells. DAMPs are sensed by a set of receptors highly expressed by myeloid cells, collectively known as molecular pattern recognition receptors (PRRs).

[0011] Among the different DAMPs, endoplasmic reticulum proteins, such as heat shock proteins HSP70 and HSP90, calreticulin (CRT), ATP secretion, high mobility group box 1 (HMGB1 ), and type I interferon (type I IFN) are widely described. Collectively, DAMPs act as “eat-me” and “find-me” signals, recruiting antigen-presenting cells to the site of ICD induction and stimulating the uptake, processing, and cross-presentation of tumor- associated antigens (TAAs), resulting in an adaptive immune response.

[0012] ICD-inducing anticancer agents are extremely attractive, especially for “cold tumors”, such as colon or pancreatic cancer, because they exhibit a “dual-action strategy”: while directly killing most tumor cells, dying tumor cells act as a sort of vaccine that can trigger a specific immune response aimed at eradicating the remaining tumor cells.

[0013] Not all chemotherapeutics can induce intracellular stress pathways that activate the release of DAMPs in dying tumor cells. Among conventional chemotherapeutics, those endowed with this property are cyclophosphamide, anthracyclines, some platinum derivatives, but not cisplatin. Photodynamic therapy (PDT) and radiotherapy with y irradiation have also shown efficacy in inducing ICD.

[0014] LTX-315 is a nonameric oncolytic peptide in the clinical development phase for the treatment of solid neoplasms, which has the ability to induce ICD. Preclinical and clinical evidence indicates that the anticancer properties of LTX-315 arise not only from its ability to selectively kill the tumor cells, but also from its ability to promote immune responses directed at the tumor target. Treatment of breast cancer in mice with LTX- 315 is accompanied by a reconfiguration of the immunological tumor microenvironment that favors the activation of anti-cancer immunity and can be enhanced by radiation therapy.

[0015] The literature contains numerous examples of “anti-cancer peptides” that may act similarly to LTX-315. These are all derived from cytolytic sequences, most of which are of natural origin, i.e. host defense peptides (HDPs). HDPs are highly conserved peptides synthesized by nearly all living organisms, including bacteria, plants, fungi, animals, and mammals. They typically consist of 10-50 amino acid residues with a net positive charge resulting from the high prevalence of cationic (e.g. Lys, Arg) and hydrophobic (e.g. Ala, Vai, Gly) amino acids, conferring an overall amphipathic conformation that allows the interaction with biological membranes. These oncolytic peptides can also trigger immunogenic cell death by inducing the release of cell death-related danger-associated molecular patterns (DAMPs). Tumor cells sensitive to the synthetic peptide LTX-315 have been shown to release ATP, HMGB1 exposing calreticulin on the outer surface of the plasma membrane and secrete type I IFNs. These signals, together with the release of tumor antigens by dying tumor cells, drive the recruitment of APC cells into the tumor environment, then the migration of APCs to tumor-draining lymph nodes, and ultimately the establishment of a CTL-dependent anti-cancer immune response.

[0016] One of the limitations in the use of peptides in clinical contexts is their short in vivo halflife due to rapid degradation by peptidases and proteases. The peptide LTX-135, for example, can only be used intratumorally.

[0017] The particular biological aggressiveness that characterizes these tumors, together with the difficulties in combating them and the fact that they cannot benefit from immunotherapy drugs, are the basis of the substantial absence of therapeutic approaches with curative intent.

[0018] The aim of the present invention is therefore to provide peptides effective in the treatment of “cold tumors”, such as colon or pancreatic cancer, which do not have the disadvantages noted above for peptides and drugs known for the same application.

[0019] SUMMARY OF THE INVENTION

[0020] The present invention originates from having identified selected biologically active peptides.

[0021] The invention therefore concerns a peptide or pharmacologically acceptable salts thereof, said peptide having the general formula (I): wherein residue R1 is selected from the group consisting of:

[0022] SEQ ID NO: 1 : LLKKKFKKLQ (BOP7);

[0023] SEQ ID NO: 2 KKKLKFKKLQ (BOP9);

[0024] SEQ ID NO: 3 LLLKKFKKLQ (BOP10);

[0025] SEQ ID NO: 4 SLLSLIRKLIT (BOP12); and

[0026] SEQ ID NO: 5 FLGALFKWASK (BOP2); and wherein residue R2 is Ala (A), beta-Ala (beta-A), PEG, or is absent.

[0027] In a second aspect, the present invention relates to a pharmaceutical composition comprising one or more peptides, the same or different from each other, having the general formula (I), and pharmacologically acceptable excipients.

[0028] In a third aspect, the present invention concerns the use of the composition according to the invention, as a medicament.

[0029] In a fourth aspect, the use of the composition according to the invention, in the treatment of a tumor, is described.

[0030] The dependent claims describe particular embodiments of the invention.

[0031] DESCRIPTION OF THE FIGURES

[0032] The invention will now be described in detail and with reference to the attached Figures.

[0033] Figure 1 shows the image prepared with Biorender, which shows the mechanism of immunogenic cell death in cancer.

[0034] Figure 2. Tetrabranched structure in a schematic form.

[0035] Figure 3. Flow cytometry of peptides at different concentrations. 10pM, 2pM, 0.4pM.

[0036] Figure 4. Immunofluorescence analysis of BOP binding on PANC-1 : A) untreated cells, B) BOP2, C) BOP7, D) BOP9; and on MiaPaca E) untreated cells, F) BOP2, G) BOP7, H) BOP9. The green signal distinguishes the peptides, the red signal marks the plasma membrane, the blue signal highlights the nuclei.

[0037] Figure 5. Peptide cytotoxicity against PANC-1 cells, pancreatic adenocarcinoma, n=6; one way ANOVA, p<0.001 ; Dunnet post test; *** p < 0.001 , ** p < 0.01 , * p < 0.5.

[0038] Figure 6. A) HMGB1 release by PANC-1 after 24 hours of treatment with BOP, daunorubicin and irinotecan; **** p < 0.0001 , *** p < 0.001 , ** p < 0.01 , * p < 0.5 (n = 3); t-tests (unpaired, parametric, two-tailed p-values); B) IFN-|3 secretion by PANC-1 after 24 h of treatment with BOP, daunorubicin and irinotecan, “ p < 0.01 , * p < 0.5 (n = 3); t-tests (unpaired, parametric, two-tailed p-values); C) ATP release over 24 h by PANC- 1 ; D) ATP release at 24 h, one way Anova, Dunnett post-test *** p< 0.0001 .

[0039] Figure 7. Hemolytic activity of BOP peptides incubated with red blood cells at 37°C for 2 h.

[0040] Figure 8. Viability of PANC-1 cells after treatment with linear / monomeric peptides

[0041] All experiments were performed at least three times (n = 3), statistical analysis was performed using two-tailed paired t-test. The asterisk indicates significant differences with respect to control.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] Tetrabranched dendrimeric peptides were identified, which have a much improved resistance profile against peptidases and proteases with respect to their monomeric counterparts. Without being tied to any theory, this peculiarity is due to the steric hindrance of branched peptides that limits the interaction with the peptidase cleavage site. Furthermore, branched peptides allow a higher binding avidity thanks to their ability to form polyvalent bonds.

[0044] The branched structure, with multiple active sequences on the same molecule, also determines a higher local concentration that cannot be achieved with linear homologs. For the above reasons, dendrimeric peptides are considered potential candidates for anticancer therapy and were selected for their cationic and amphipathic chemical characteristics and for their biological activity against cancer cells.

[0045] The invention therefore concerns a peptide or pharmacologically acceptable salts thereof, said peptide having the general formula (I): wherein residue R1 is selected from the group consisting of:

[0046] SEQ ID NO: 1 : LLKKKFKKLQ (BOP7);

[0047] SEQ ID NO: 2: KKKLKFKKLQ (BOP9);

[0048] SEQ ID NO: 3: LLLKKFKKLQ (BOP10);

[0049] SEQ ID NO: 4: SLLSLIRKLIT (BOP12); and

[0050] SEQ ID NO: 5: FLGALFKWASK (BOP2); and wherein residue R2 is Ala (A), beta-Ala (beta-A), PEG, or is absent.

[0051] In one embodiment, in the peptide according to the present invention, residue R1 is always the same.

[0052] In a further embodiment, in the peptide according to the present invention, residue R1 is selected from the group consisting of:

[0053] SEQ ID NO: 1 : LLKKKFKKLQ (BOP7);

[0054] SEQ ID NO: 2: KKKLKFKKLQ (BOP9); and

[0055] SEQ ID NO: 3: LLLKKFKKLQ (BOP10) and has the general formula (II): X1KFKKLQ (II), (SEQ ID NO:14) wherein Xi is selected from the group consisting of:

[0056] SEQ ID NO: 6: LLKK;

[0057] SEQ ID NO: 7: KKKL; and

[0058] SEQ ID NO: 8: LLLK.

[0059] In one preferred embodiment, the peptide is tetrabranched and has a sequence selected from the group consisting of:

[0060] SEQ ID NO: 9:

[0061] LLKKKFKKLQ^

[0062] LLKKKFKKLQ

[0063] K

[0064] LLKKKFKKLQ^

[0065] ' K

[0066] LLKKKFKKLQ

[0067] SEQ ID NO: 10:

[0068] KKKLKFKKLQ.

[0069] ' >K KKKLKFKKLQ ' \

[0070] K

[0071] KKKLKFKKLQ^ / " K

[0072] KKKLKFKKLQ SEQ ID NO: 1 1 :

[0073] LLLKKFKKLQ .

[0074] >K LLLKKFKKLQ

[0075] LLLKKFKKLQ \ K

[0076] LLLKKFKKLQ '

[0077] SEQ ID NO: 12: SLLSLIRKLIT

[0078] SLLSLIRKLIT

[0079] K

[0080] SLLSLIRKLIT

[0081] SLLSLIRKLIT

[0082] SEQ ID NO: 13:

[0083] FLGALFKWASK

[0084] ";K

[0085] FLGALFKWASK \

[0086] K

[0087] FLGALFKWASK ^

[0088] K

[0089] FLGALFKWASK

[0090] In the peptides of SEQ ID NO:9 - SEQ ID NO:13 residue R2 is absent. However, R2 may be Alanine, beta-alanine or PEG (polyethylene glycol or polyethylenglycol).

[0091] Peptides of SEQ ID NO:1 - SEQ ID NO:5 can also be dimers or octamers.

[0092] For example, in the case of the dimer, the peptide has the general formula (III): wherein residue R1 is selected from the group consisting of:

[0093] SEQ ID NO: 1 LLKKKFKKLQ (BOP7);

[0094] SEQ ID NO: 2 KKKLKFKKLQ (BOP9);

[0095] SEQ ID NO: 3 LLLKKFKKLQ (BOP10);

[0096] SEQ ID NO: 4 SLLSLIRKLIT (BOP12); and

[0097] SEQ ID NO: 5 FLGALFKWASK (BOP2); and wherein residue R2 e Ala (A), beta-Ala (beta-A), PEG, or is absent. In one embodiment, in the peptide according to the present invention, residue Ri is always the same and R2 is absent.

[0098] As will be apparent from the experimental part, the peptides of the invention were tested for their selectivity towards cancer and for the ability to induce the release of immunogenic cell death markers, comparing them with two antineoplastic drugs known to be potent inducers of immunogenic cell death (daunorubicin and irinotecan). Furthermore, as can be seen from the data shown in Example 11 , surprisingly, the peptides of the invention (tetrabranched BOP peptides) have an unexpected cytotoxic activity and stability in serum, with respect to the corresponding linear / monomeric peptides.

[0099] In a second aspect, the present invention relates to a pharmaceutical composition comprising one or more peptides, the same or different from each other, having the general formula (I), and pharmacologically acceptable excipients.

[0100] The characteristics of stability in serum and plasma (Example 2) make the peptides of the invention suitable for use in a pharmaceutical composition.

[0101] In a third aspect, the present invention concerns the use of the composition according to the invention, as a medicament.

[0102] In a fourth aspect, the use of the composition according to the invention in the treatment of cancer is described.

[0103] The peptides of the invention were found to be active in binding to various cancer cells (Example 3 and Example 4).

[0104] In one preferred embodiment, the use of the composition according to the invention is in the treatment of an immunologically inactive tumor.

[0105] In a further preferred embodiment, the use of the composition according to the invention is in the treatment of pancreatic cancer or colon cancer.

[0106] Examples of embodiments of the present invention, provided for illustrative purposes, are reported below.

[0107] EXAMPLES

[0108] Example 1 : Peptide selection and synthesis

[0109] Some oncolytic peptides having a branched structure were synthesized by solid phase synthesis (Figure 2 and Table 1 ).

[0110] Table 1 : These peptides were selected for their cationic and amphipathic chemical characteristics and for their biological activity against cancer cells and tested for their selectivity towards cancer and for the ability to induce the release of immunogenic cell death markers, comparing them with two antineoplastic drugs known to be potent inducers of immunogenic cell death (daunorubicin and irinotecan).

[0111] Example 2: Stability of tetrabranched peptides in plasma and serum.

[0112] Tetrabranched peptides BOP2, -6, -7, -8, -9, -10, -12 and -14 were incubated for 4 and 16 hours at 37°C in human serum. By HPLC analysis followed by mass spectrometry, the presence of the peak corresponding to the intact peptide was assessed (Table 2).

[0113] TABLE 2 - Stability of peptides in serum

[0114] Example 3: Peptide binding to pancreatic adenocarcinoma cells and healthy cells.

[0115] Flow cytometry experiments showed that BOP2, BOP7 and BOP9 peptides bind to the pancreatic cancer cell lines PANC-1 and Mia PaCa-2 in a dose-dependent manner. Conversely, when an immortalized murine macrophage cell line of non-tumor origin was analyzed, the binding of BOP7 and BOP9 was drastically reduced, thus demonstrating good tumor selectivity. BOP2, on the other hand, was not able to discriminate between tumor and non-tumor cell lines (Figure 3).

[0116] Example 4: Immunofluorescence analysis of BOP binding to PANC-1 and MIA PaCa-2 To qualitatively evaluate the ability of BOPs to bind the plasma membranes of pancreatic cancer cells, immunofluorescence experiments were performed on PANC-1 and MIA PaCa-2 cell lines. For the immunofluorescence experiments, BOPs were synthesized in a biotinylated form so that their localization could be traced by fluorescent streptavidin. After 30 min of treatment (2 pM), all three branched oncolytic peptides were localized at the cell membrane level in both PANC-1 (Figure 4 A-D) and MIA PaCa-2 (Figure 4 E- H) tumor cell lines, as can be visualized by the green signal reported in the images below. The blue signals represent the nuclei, the red signal represents the plasma membrane, and the green signal represents the biotinylated peptides.

[0117] Example 5:

[0118] Cell Viability Assays for BOPs

[0119] The cytotoxicity of BOPs was tested in the human pancreatic adenocarcinoma cell line PANC-1 . The peptides BOP 2, 7, 9, 10 and 12 showed the best cytotoxicity profile with statistical significance and a IC50 from 1 .8 to 7.1 pM (Figura 5).

[0120] Table 3. Cytotoxicity as inhibition of viability (IC50) on pancreatic adenocarcinoma cells, PANC-1 , by BOP peptides. Example 6: Peptide toxicity in non-cancer cells

[0121] The peptides BOP7 and BOP9 were analyzed in hemolytic activity assays and showed a non-toxic profile even at concentrations 100 times higher than those used against cancer cells (Figure 7).

[0122] Furthermore, the peptides BOP7 and BOP9 were tested on CHO-K1 mammalian cells, in the same conditions used for cancer cells, 24 hours at 37°C, and it was possible to measure the therapeutic index that confirms the relevant specificity of the peptides against cancer cells (Table 4).

[0123] Table 4. Cytotoxicity as inhibition of viability (IC50) on Chinese Ovary Cells, CHO-K1 , by BOP peptides and therapeutic index (IT)

[0124] Example 7: Immunogenic Cell Death Experiments

[0125] To evaluate the ability of branched oncolytic peptides to trigger the release of immunogenic cell death markers, PANC-1 cells were treated with different concentrations of BOPs, comparing them with two chemotherapeutic agents used in clinical practice: daunorubicin, belonging to the anthracycline class, and irinotecan, an antineoplastic agent used in the palliative therapy of pancreatic cancer, both widely demonstrated to be a potent inducer of immunogenic cell death.

[0126] Example 8: HMGB1 Release

[0127] ICD inducers trigger the release of HMGB1 , which, by binding to TLR4, promotes the processing and presentation of tumor antigens by inhibiting their lysosomal degradation. HMGB1 release was assessed by an ELISA test using supernatants of PANC-1 cells treated for 24 h with different compounds at various concentrations (from 300 pM to 2 pM) (Figure 6A). BOP2 at 300 pM caused the highest release of HMGB1 compared to all other compounds. BOP7 and BOP9 induced a higher release of HMGB1 at the concentrations of 300 pM and 50 pM compared to irinotecan, although lower than daunorubicin which was the most effective inducer even at the lowest concentrations.

[0128] Example 9: IFN-3 Secretion

[0129] ICD inducers can stimulate the production of type 1 interferon, which acts on type 1 interferon receptors to stimulate the transcription of a series of target genes culminating in the activation of signal transduction cascades that stimulate T cell recruitment. The secretion of IFN-|3, a member of the type I IFN family, was assessed by an ELISA test using the supernatants of PANC-1 cells treated for 24 hours with the different compounds at various concentrations as samples (Figure 6B).

[0130] Only BOP7 and BOP9 at concentrations of 300 pM and 50 pM were able to stimulate IFN-[3 secretion at statistically significant levels with respect to untreated cells. Therefore, BOP7 and BOP9 were more effective in inducing IFN-p release than daunorubicin and irinotecan.

[0131] Example 10: ATP Release

[0132] ATP release is a major hallmark of immunogenic cell death because, once in the extracellular space, it acts as a “find me” signal to recruit antigen-presenting cells (APCs).

[0133] Measurement of ATP release was examined by bioluminescence assays, using the following biochemical reaction:

[0134] “ATP + d-Luciferin + 02 — > Oxyluciferin + AMP + PPi + CO2 + Light (560nm)”.

[0135] Then, the enzyme was added in conjunction with the treatments of PANC-1 cells with various compounds. When ATP is present, the enzyme catalyzes the reaction, resulting in the emission of light at 560 nm in proportion to the amount of the nucleotide. The intensity of the light, and therefore the levels of ATP released, were measured using a luminometer at different time intervals. The tested compounds were compared based on the concentration at which they were used to treat the cells.

[0136] At 50 pM (Figure 6C-D), daunorubicin causes a very strong release of ATP between 4 and 8 hours after the treatment. At the same concentration, BOP7 and BOP9 release, over time, a smaller amount of ATP, but sustained over time; in fact, at 24 hours, the release of ATP is about 10 times higher than that caused by daunorubicin. Irinotecan is less effective at all concentrations and times. At 20 pM (Figure 6C-D), BOP7 is the most effective, at all-time intervals, and together with BOP9 at 24h it causes a release equal to 5-fold that of daunorubicin.

[0137] Example 1 1 : Comparison of tetrabranched BOP peptides with their corresponding linear / monomeric peptides

[0138] Cell viability assays of linear / monomeric peptides

[0139] For comparison with the BOP cytotoxicity assays described in Example 5, the same assays were performed on linear peptides.

[0140] Specifically, the linear peptides tested were: L2 (SEQ ID NO: 5), L7 (SEQ ID NO: 1 ), L9 (SEQ ID NO: 2), L10 (SEQ ID NO: 3) and L12 (SEQ ID NO: 4).

[0141] As apparent from Figure 8, the linear peptides did not show any cytotoxic activity in the PANC-1 cell line over 24 hours.

[0142] Surprisingly, the data demonstrate that the oncolytic efficacy of tetrabranched BOP peptides was not simply enhanced, but transformed. In fact, linear peptides show no efficacy in the 24-hour cell viability model, while using the tetrabranched BOP peptides, unexpectedly, efficacy at a concentration more than 20 times lower than the maximum ineffective concentration used for linear homologues is obtained (Figure 8).

[0143] Stability of linear / monomeric peptides in serum

[0144] For comparison with the properties of branched BOP peptides, the stability of linear peptides was evaluated by reproducing the experiment of Example 2, incubating the peptides for 4 and 16 hours at 37°C in human serum.

[0145] The presence of the peak corresponding to the intact peptide was evaluated by HPLC analysis followed by mass spectrometry, (Table 5).

[0146] Table 5 - Stability of peptides in serum

[0147] Surprisingly, all branched BOP peptides showed improved stability to serum action with respect to their linear counterparts.

[0148] From the detailed description and the Examples reported above, the advantages achieved by the peptides of the present invention, which have proven to be surprisingly and advantageously effective in the treatment of immunologically inactive tumors, are apparent.

Claims

CLAIMS1 . A peptide or pharmacologically acceptable salts thereof, said peptide having the general formula (I):wherein residue R1 is selected from the group consisting of:SEQ ID NO: 1 : LLKKKFKKLQ (BOP7);SEQ ID NO: 2: KKKLKFKKLQ (BOP9);SEQ ID NO: 3: LLLKKFKKLQ (BOP10);SEQ ID NO: 4: SLLSLIRKLIT (BOP12); andSEQ ID NO: 5: FLGALFKWASK (BOP2); and wherein residue R2 is Ala (A), beta-Ala (beta-A), PEG, or is absent.

2. The peptide according to claim 1 , wherein R1 is always the same and R2 is absent.

3. The peptide according to any one of claims 1 or 2, wherein R1 is selected from the group consisting of:SEQ ID NO: 1 : LLKKKFKKLQ (BOP7);SEQ ID NO: 2: KKKLKFKKLQ (BOP9); andSEQ ID NO: 3: LLLKKFKKLQ (BOP10) and has the general formula (II): X1KFKKLQ (II), wherein Xi is selected from the group consisting of:SEQ ID NO: 6: LLKK;SEQ ID NO: 7: KKKL; andSEQ ID NO: 8: LLLK.

4. The peptide according to any one of claims 1 to 3, wherein said peptide has a sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12 and SEQ ID NO: 13.

5. A pharmaceutical composition comprising one or more peptides, the same or different from each other, according to any one of claims 1 to 4, and pharmacologically acceptable excipients.

6. A pharmaceutical composition according to claim 5, for use as a medicament.

7. A pharmaceutical composition according to claim 5, for use in the treatment of cancer.

8. The pharmaceutical composition for use according to claim 7, wherein said cancer is an immunologically inactive tumor.

9. The pharmaceutical composition for use according to claim 8, wherein said cancer is pancreatic cancer or colon cancer.

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