Use of synthetic peptides for induction of antitumor and antiviral immunity

Abstract

Use of the peptide identified as SEQ ID NO: 1 for the manufacture of a medicament that induces antitumor and antiviral immunity through immunogenic cell death. The present invention also provides a method for treating cancer or viral infections by using a therapeutically effective amount of a medicament comprising a synthetic peptide of the present invention, which induces antitumor and antiviral immunity through immunogenic cell death. The present invention also includes pharmaceutical combinations of the peptide identified as SEQ ID NO: 1 with cancer immunotherapy approaches to achieve more powerful immunotherapeutic management to combat the disease.

Description

[Technical field]

[0001] The present invention relates to the field of cancer immunotherapy and antiviral therapy. In particular, the present invention is based on the use of a peptide identified as SEQ ID NO:1 to induce antitumor and antiviral immunity by increasing immunogenic cell death. The present invention also relates to the combination of said peptide with cancer immunotherapy. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide. The global incidence of cancer continues to increase, with new cases estimated to exceed 21.7 million by 2030, resulting in 13 million cancer deaths per year (Tartari F and cols.Cancer Treat Rev 2016;48:20-24).

[0003] One of the strategies in the development of new cancer treatments in the past two decades has been the development of drugs whose mechanisms are target specific to interfere with the biochemical events involved in tumor cell survival, thus inducing apoptosis in tumor cells. Generally, this class of drugs is used in combination with standard chemotherapy in the first or second line of treatment for cancer patients, both in advanced and early stages.

[0004] The synthetic peptide identified as SEQ ID NO:1 is a therapeutic candidate for cancer treatment, whose main mechanism of action is the inhibition of phosphorylation mediated by protein kinase CK2, resulting in tumor cell death by apoptosis. The synthetic peptide identified as SEQ ID NO:1 has shown antitumor effects in experimental oncology animal models (Perea SE and cols. Cancer Res 2004;64:7129-9). Recently, a direct antiviral effect of the peptide identified as SEQ ID NO:1 has also been observed in different in vitro virus models. From a mechanistic point of view, the peptide identified as SEQ ID NO:1 has been found to interact with the B23 / nucleophosmin protein in the nucleoli of tumor cells, inhibiting their phosphorylation and inducing dissociation of the nucleoli as a precursor to apoptosis (Perera Y and cols. Mol Cancer Ther 2009;8(5)). Consistent with these molecular events, peptide identified as SEQ ID NO:1 regulates a series of proteins associated with different cellular processes and inhibits lung metastasis colonization and tumor angiogenesis in preclinical cancer models (Farina HG and cols. Exp Cell Res 2011;317:1677-1688) (Benavent Acero F and cols. Lung Cancer 2017;107:14-21). Furthermore, peptide identified as SEQ ID NO:1 inhibits phosphorylation of other CK2 substrates such as AKT and PTEN in chronic lymphocytic leukemia cells (Martins LR and cols. Oncotarget 2014;5:258-263). In the clinical arena, investigations into the safety and tolerability of the peptide identified as SEQ ID NO:1 in cancer patients have begun, with a trend toward increased survival observed in patients with advanced disease and above average life expectancy (Batista-Albuerne N and cols. J Med Oncol 2018;1:4).These studies made it possible to determine the pharmacokinetics and biodistribution of the peptide identified as SEQ ID NO:1 following local or intravenous administration of the peptide identified as SEQ ID NO:1, as well as the safe dose range and toxicity associated with the studied drug (Solares AM and cols. BMC Cancer 2019;9(1):146) (Sarduy MR and cols. Br J Cancer 2015;112:1636-43).

[0005] The greatest desire of clinical immunologists and oncologists for many years has been to involve the body's immune system in neoplastic disease control. In line with this idea, immunotherapy has been presented as a promising option to revolutionize cancer treatment through the discovery and development of new approaches to activate and enhance antitumor immunity in cancer patients (Zugazagoitia J and cols. Clin Ther 2016;38(7):1551-1566) (Yang YJ Clin Invest 2015;125(9):3335-3337).

[0006] Currently, the main immunotherapeutic options for cancer treatment are monoclonal antibodies, immune checkpoint inhibitors, cancer vaccines and cell-based immunotherapies (Kenderian SS and cols.Biol Blood Transplant 2017;23:235-246) (Ruella M and Kenderian SS.BioDrug 2017;31(6):473-481). In addition, there are other non-specific immunotherapies that activate the immune system in a general manner, which can therefore attack tumor cells (Klener P Jr and cols.Curr Pharm Biotechnol 2015;16(9):771-781) (Lee VC.P&T 2017;42(6):375-383). Although chemotherapy drugs and radiotherapy do not have a direct stimulatory effect on cellular immunity, it is known today that some of these approaches can indirectly increase the stimulatory effect on cellular immunity by inducing immunogenic cell death in tumor cells. In recent years, the number of drugs inducing immunogenic cell death has increased. They include anthracyclines (doxorubicin (DOX), epirubicin, idarubicin), oxaliplatin, cyclophosphamide, bortezomib, mitoxantrone and bleomycin (Garg AD and cols. Oncoimmunology 2017;6(12):e1386829). In addition, physical therapeutic methods such as radiation therapy, hypericin-based photodynamic therapy and high hydrostatic pressure, as well as some oncolytic viruses and microtubule inhibitors, have also been shown to induce this type of cell death (Li X. Tumori Jornal 2018;104(1):1-8).

[0007] In the past, necrosis was the only type of cell death that was considered immunogenic and caused undesirable inflammatory responses due to the rapid release of various intracellular factors, cytokines and other inflammatory mediators (Rock KL and Kono H. Annu Rev Pathol 2008;3:99-126). Conversely, apoptosis was considered a physiological process of cell death that was mostly tolerogenic or silent from an immunological point of view (Matzinger P. Science 2002;296:301-305). During late apoptosis, cells are rapidly and specifically recognized by macrophages and other phagocytes due to the expression of various "eat me" signals such as calreticulin (CRT), Erp57 and HSP90 on the membrane (Hou W and cols. Cell Death and Disease 2013;4,e966) and the concomitant suppression of "don't eat me" signals such as CD47 expression (Liu X and cols. Journal of Hematology & Oncology 2017;10:12). CD47 is a molecule expressed on tumor cells and constitutes an anti-phagocytic signal for macrophages and dendritic cells, and has therefore been considered an immune checkpoint for tumor evasion (Tong B and Wang M. Future Oncol. 2018;14(21):2179-2188) (Weiskopf K and cols. Science 2013;341(6141):88-91).

[0008] Compared to classical tolerogenic apoptosis, immunogenic cell death activates the host immune system and increases immune responses to immunotherapy, such as the response to dendritic cell-based cancer vaccines (Vandenberk L and cols. Front Immunol 2016;6:663). Indeed, tumor cells undergoing drug-induced immunogenic cell death in vitro can induce anticancer vaccine effects when implanted subcutaneously in immunocompetent mice (Keep O and cols. Oncoimmunology. 2014,3:9). In this case, dendritic cells play a central role in the recognition of apoptotic cells and initiation of effective antitumor immune responses (Ma Y and cols. Immunity 2013;38:729-41). One of the fundamental characteristics of damaged and dying cells is the exposition or secretion of molecules that are normally hidden in living and healthy cells, which acquire immune-stimulating properties. These molecules belong to the well-known damage-associated molecular patterns and can exert several effects on antigen-presenting cells, including maturation, activation and antigen processing / presentation (Zitvogel L and cols. Cell. 2010; 140: 798-804). However, antitumor immunity associated with immunogenic cell death is rarely achieved in cancer patients, due to the inability of anticancer drugs to cause this type of death at concentrations lower than the maximum tolerated dose in vivo (Montico B and cols. Int J Mol Sci. 2018; 19: 594-609). This obstacle has been overcome by ex vivo treatment of tumor cells from patients with high doses of drugs that induce immunogenic apoptosis to generate autologous dendritic cells for vaccination purposes (Chen HM and cols. Cancer Immunol Immunother. 2012; 61: 1989-2002) (Montico B and cols. Oncoimmunology. 2017; 6: e1356964). However, surgical resection of the patient's tumor is not always possible, especially at advanced stages of the disease, or due to the anatomical location of the tumor, to achieve an ex vivo procedure that makes it possible to obtain autologous dendritic cells.

[0009] Thus, currently, the main limitation of using immunogenic apoptosis inducers as adjuvants for dendritic cell-mediated vaccines consists of the inability to use such drugs in vivo without a prior ex vivo activation procedure. Summary of the Invention

[0010] The present invention solves the above problems by providing the use of a peptide identified as SEQ ID NO:1 for the manufacture of a medicament for inducing antitumor and antiviral immunity by increasing immunogenic cell death. The peptide identified as SEQ ID NO:1 is a proapoptotic peptide comprising a synthetic peptide, originally called P15, fused to the cell-penetrating peptide Tat (amino acids 48-60) to facilitate internalization into cells (Perea SE and cols. Cancer res. 2004;64:7127-7129). Unexpectedly, it has been shown that this peptide can induce antitumor immune activation by activating "eat me" signals and reducing "don't eat me" signals. In particular, the present invention demonstrates that there is maturation and activation of dendritic cells and activation of an antitumor adaptive immune response in which there is a cellular immune response mediated by CD8+ cytotoxic T lymphocytes. The effect described for the peptide identified in this invention as SEQ ID NO:1 is not observed with other inhibitors of CK2-mediated phosphorylation, such as the chemical compound CX4945, which directly blocks the catalytic subunit of the CK2 protein kinase, nor when using the cell-permeable peptide Tat, which has been evaluated as a negative control.

[0011] In one aspect of the invention, the peptide identified as SEQ ID NO:1 is used for the manufacture of a medicament for the induction of antitumor and antiviral immunity by in vivo and in vitro activation and differentiation of dendritic cells. In vitro treatment is understood to mean the incubation of tumor cells in culture with the peptide identified as SEQ ID NO:1. Ex vivo treatment refers to the in vitro co-incubation of tumor cells from a patient treated with the peptide identified as SEQ ID NO:1 with dendritic cells from the same patient in the laboratory. In connection with the in vivo treatment, an increase in "eat me" signals and a decrease in "don't eat me" signals are observed in the tumor, followed by activation of the cellular immune response at both the systemic and intratumoral levels.

[0012] In the present invention, it is shown for the first time that the peptide identified as SEQ ID NO:1 is capable of inducing immunogenic cell death of tumor cells which, when administered in a vaccination schedule in mice, induces an in vivo immune response that protects against further challenge by live tumor cells and inhibits tumor initiation and growth.

[0013] In one aspect of the present invention, the induction of antitumor immunity by the use of the peptide identified as SEQ ID NO:1 includes the enhancement of NK or cytotoxic T cell activity at the tumor site. The use of the peptide identified as SEQ ID NO:1 allows to increase the activity of cytotoxic T cells through sufficient maturation and activation of dendritic cells. The peptide identified as SEQ ID NO:1 can increase the secretion of HMGB-1 and adenosine triphosphate (ATP) by tumor cells after treatment, which act as chemoattractants for antigen-presenting cells and favor their recruitment, maturation and activation at the tumor site. This makes the peptide identified as SEQ ID NO:1 a suitable compound for enhancing the effect of dendritic cell-based vaccines or for contributing to in situ vaccination by these cells.

[0014] In contrast to other anti-tumor agents that induce immunogenic cell death, which only achieve activation of dendritic cells when the cells are incubated with tumor cells ex vivo, the use of the peptide identified as SEQ ID NO:1 overcomes this limitation by inducing in vivo activation of dendritic cells without the need to isolate the cells from the patient to perform the manipulation. This effect is observed when the peptide identified as SEQ ID NO:1 is administered both intratumorally and systemically, making the peptide identified as SEQ ID NO:1 an effective treatment for both leukemias and solid tumors, including those that are difficult to access.

[0015] In an embodiment of the present invention, the induction of anti-tumor immunity by use of the peptide identified as SEQ ID NO:1 involves the reversion of an immunosuppressive tumor microenvironment to an immunostimulatory tumor microenvironment. This synthetic peptide enhances the recruitment of immune cells that can direct a specific anti-tumor response and reduce the presence of regulatory T cells.

[0016] In realizing the present invention, the peptide can increase the expression of MHC-I on antigen-presenting cells, and achieves the induction of anti-tumor and anti-viral immunity by increasing pro-inflammatory cytokines such as TNF-α, IL-6 and IL-12, which promote T cell differentiation and NK cell activation. The increase in pro-inflammatory cytokines is accompanied by a decrease in anti-inflammatory cytokines such as IL-10, which limits the immune response generated and contributes to tumor progression or viral infection.

[0017] In certain embodiments, the induction of anti-viral immunity includes enhancing a specific humoral immune response to a viral antigen, as demonstrated for the first time in the present invention. The ability of the peptide identified as SEQ ID NO:1 to enhance an immune response makes the peptide identified as SEQ ID NO:1 an ideal candidate for treating infectious diseases, particularly during viral infections.

[0018] The present invention also provides a method for treating cancer or viral infection by using a therapeutically effective amount of a drug that induces anti-tumor and anti-viral immunity through immunogenic cell death, the drug comprising a synthetic peptide of the present invention of SEQ ID NO:1.

[0019] The method of the present invention allows for the reduction of "don't eat me" signals, such as CD47 expression, which promotes the phagocytosis of tumor cells by dendritic cells and M1 macrophages, along with an increase in "eat me" signals. This M1 macrophage function is related to their anti-angiogenic activity, which ultimately inhibits tumor progression. Thus, the effect of the peptides of the present invention eliminates the need to use additional CD47 inhibitors to enhance the anti-tumor effect of certain drugs, reducing the number of compounds required to achieve an effective anti-tumor response. Thus, in one embodiment of the method of the present invention, the induction of anti-tumor and anti-viral immunity includes the activation and differentiation of dendritic cells in vivo and in vitro.

[0020] In another aspect, the use of the peptide identified as SEQ ID NO:1 increases "eat me" signals such as CRT, which increases the ability of antigen-presenting cells to capture apoptotic tumor cells. The increase in "eat me" signals induced by the peptide identified as SEQ ID NO:1 also increases the susceptibility of tumor cells to NK cytotoxicity, which promotes diversification of the peptide-induced response and increases antitumor efficiency. Thus, in an embodiment of the method of the present invention, the induction of antitumor immunity includes enhanced NK and cytotoxic T cell activity at the tumor site, as well as the reversion of an immunosuppressive tumor microenvironment to an immunostimulatory tumor microenvironment and increased secretion of proinflammatory cytokines. In another embodiment of the method of the present invention, the induction of antiviral immunity includes enhanced specific humoral immune response to viral antigens.

[0021] In one embodiment of the present invention, the drug comprising the peptide identified as SEQ ID NO:1 enhances the effect of a vaccine used in cancer immunotherapy. The drug comprising the peptide identified as SEQ ID NO:1 and the vaccine used in cancer immunotherapy can be administered sequentially or simultaneously during the same course of treatment. In a particular embodiment, the vaccine used in cancer immunotherapy is a vaccine based on dendritic cells, cytotoxic T cells or tumor infiltrating lymphocytes (TIL).

[0022] In the present invention, the use of the peptide identified as SEQ ID NO:1 as an adjuvant to enhance the cellular immune response of antitumor vaccines is described for the first time. To do so, the peptide can be administered by intratumoral or systemic route before vaccination, which allows the peptide to be used against both leukemia and solid tumors. It should be emphasized that the adjuvant effect was only observed for the peptide identified as SEQ ID NO:1, but not for other inhibitors of phosphorylation mediated by CK2, such as CX4945.

[0023] Another object of the present invention is a pharmaceutical combination comprising a peptide identified as SEQ ID NO:1 and a vaccine for cancer immunotherapy. In an embodiment of the present invention, the vaccine used in cancer immunotherapy is a dendritic cell, cytotoxic T cell or TIL based vaccine.

[0024] The ability to increase tumor infiltration of cytotoxic T lymphocytes and reduce the presence of regulatory cells makes the peptide identified as SEQ ID NO:1 a suitable compound to enhance the efficacy of cytotoxic T lymphocyte (CTL) and TIL-based vaccines, thus resulting in a more effective immunotherapeutic approach to treat cancer.

[0025] In one embodiment of the present invention, a drug comprising the peptide identified as SEQ ID NO:1 enhances the effect of a PDL1 immune checkpoint inhibitor used in cancer immunotherapy. In an embodiment of the present invention, the peptide identified as SEQ ID NO:1 and the PDL1 immune checkpoint inhibitor are administered sequentially or simultaneously during the same course of treatment. In a particular embodiment, the PDL1 immune checkpoint inhibitor is an anti-PDL1 monoclonal antibody.

[0026] In another aspect, the present invention discloses a pharmaceutical combination comprising a peptide identified as SEQ ID NO:1 and a PDL1 immune checkpoint inhibitor for use in cancer immunotherapy. In one embodiment of the present invention, in the pharmaceutical combination, the PDL1 immune checkpoint inhibitor is an anti-PDL1 monoclonal antibody.

[0027] For example, in patients with non-small cell lung cancer that lacks actionable mutations, the most effective treatment is PDL1 immune checkpoint inhibitors. However, its effectiveness depends on the level of expression of this molecule in tumors. The use of peptide identified as SEQ ID NO:1 in combination with PDL1 inhibitors solves this problem by increasing PDL1 expression, thus increasing the clinical effectiveness of PDL1 inhibitors. This combination provides a novel therapeutic strategy for cancer treatment. [Brief description of the drawings]

[0028] [Figure 1] Assessment of induction of cell death in L1210 (A) and 3LL (B) cell lines by peptide identified as SEQ ID NO: 1. Bar graphs show the frequency of dead cells (Annexin V+ / 7AAD+) and dying cells (Annexin V / 7AAD-) following treatment with peptide identified as SEQ ID NO: 1. Positive error bars indicate standard deviation from the mean of replicates. [Diagram 2]Evaluation of ex vivo apoptosis induction by the peptide identified as SEQ ID NO: 1 in cells from patients with hematological malignancies. This figure shows the percentage of Annexin V / PI+ cells from 10 patients with hematological malignancies after 48 hours of ex vivo treatment with the peptide identified as SEQ ID NO: 1. [Diagram 3] Flow cytometry analysis of CRT translocation on the cell membrane in L1210 cells treated with peptide identified as SEQ ID NO:1. The figure shows the percentage of CRT+ cells upon treatment with peptide identified as SEQ ID NO:1 and CX4945. Graphs are presented as the mean ± standard deviation of six replicates of three independent experiments. Asterisks indicate statistically significant differences in the percentage of CRT+ cells for untreated and CX4945-treated cells (Fisher's exact test, p<0.05). [Figure 4] Flow cytometry analysis of Erp57 translocation on the membrane of L1210 cells upon treatment with peptide identified as SEQ ID NO:1. The figure shows the percentage of Erp57+ cells upon treatment with peptide identified as SEQ ID NO:1 and CX4945. Graphs are presented as the mean ± standard deviation of six replicates of three independent experiments. Asterisks indicate statistically significant differences in the percentage of Erp57+ cells for untreated cells and cells treated with CX4945 (Fisher's exact test, p<0.05). [Diagram 5] CD47 expression on HL60 and OCI-AML3 cells upon treatment with peptide identified as SEQ ID NO:1. Microarray analysis (A) and quantitative PCR (qPCR) (B). Bars represent fold change in CD47 gene expression compared to untreated cells, and results were normalized to three reference genes GAPDH, DDX5 y ABL1. Data were processed using the REST 2009 v2.0.13 program. Asterisks indicate statistical significance. [Figure 6]Evaluation of HMGB-1 secretion in culture supernatants of several tumor cell lines treated with peptide identified as SEQ ID NO:1. The graph represents HMGB-1 levels determined by ELISA from culture supernatants of different tumor cell lines. Bar graphs represent the mean ± standard deviation of three independent replicates. Statistical significance was determined using the Kruskal-Wallis test and Dunn's multiple comparisons. Different letters indicate statistically significant differences: p<0.05. [Figure 7] Detection of HMGB-1 in serum from patients with acute myeloid leukemia treated with peptide identified as SEQ ID NO: 1. The graph represents HMGB-1 levels determined by ELISA from serum of five patients treated with peptide. Samples were taken at 0, 3 and 6 weeks after treatment. [Figure 8] Detection of ATP in culture supernatants of several tumor cell lines treated with peptide identified as SEQ ID NO:1. The graph represents ATP levels as relative light units (RLU) detected in each condition. Bar graphs represent the mean ± standard deviation of three independent replicates. Statistical significance was determined using the Kruskal-Wallis test and Dunn's multiple comparisons. Different letters indicate statistically significant differences: p<0.05. [Figure 9] Surface expression of HSP70 on several tumor cell lines following treatment with the peptide identified as SEQ ID NO:1. Graph represents the percentage of HSP70+ cells. Bars represent the mean ± standard deviation of three independent replicates. Statistical differences were determined by one-way ANOVA, *p<0.05; **p<0.01. [Figure 10] Phagocytosis of L1210 and 3LL cell lines by dendritic cells after treatment with peptide identified as SEQ ID NO:1. The graph shows the percentage of CD11c+ / CFSE+ cells. Error bars indicate standard deviation from the mean of six replicates from three independent experiments. Different letters indicate statistically significant differences (Fisher's exact test, p<0.05). [Figure 11]Expression of maturation markers of dendritic cells after co-culture with L1210 and 3LL cells treated with peptide identified as SEQ ID NO: 1. Bar graphs show expression of maturation markers of dendritic cells after co-culture with L1210 (A) and 3LL (B) tumor cell lines. [Figure 12] In vitro activation of OT-I CD8+ cells by dendritic cells. The graph shows IFN-γ concentrations detected by ELISA in the supernatants of cultures of dendritic cells co-cultured with tumor cells pretreated for 48 h with peptide identified as SEQ ID NO:1, CX4945, Tat peptide or DOX. Error bars indicate standard deviation from the mean of six replicates of three independent experiments. Different letters indicate statistically significant differences in IFN-γ concentrations (Kruskal-Wallis test and Dunn's multiple comparisons, p<0.05). SEQ1: peptide identified as SEQ ID NO:1. The x-axis represents OVA class I peptide concentration of sequence SIINFELK. [Figure 13] Quantification of IL-12p70 (A), TNF-α (B), IL-6 (C) and eIL-10 (D) levels in peripheral blood serum of DBA / 2 mice challenged with L1210 cells after 7 days of treatment with peptide identified as SEQ ID NO:1. Graphs show cytokine levels of individual mice (mean ± standard deviation). Different letters indicate statistically significant differences (Kruskal-Wallis test and Dunn's multiple comparisons, p<0.01). [Figure 14] Study of different intratumoral cell populations 7 days after treatment of DBA / 2 mice with peptide identified as SEQ ID NO:1. The figure shows the percentage of intratumoral CD8+ T cells (A), CD4+ T cells (B), CD11c / CD86 / MHC-II+ dendritic cells (C) and Foxp3+ T cells (D) in DBA / 2 mice treated with peptide identified as SEQ ID NO:1, CX4945, DOX or Tat peptide. Bars represent the mean ± standard deviation of each group (n=7). Statistical differences were determined by one-way ANOVA, p<0.05. [Figure 15]Evaluation of in vivo immunogenicity of L1210 cells treated with peptide identified as SEQ ID NO: 1 in DBA / 2 mice. The graphs correspond to tumor growth curves (mean ± standard deviation) (A) and survival (Kaplan-Meier curves) (B). Different letters indicate statistically significant differences (p<0.05) in comparisons made between groups using one-way ANOVA test and Tukey's multiple comparison test for tumor volume and log-rank test for survival analysis. [Figure 16] Assessment of cellular immune responses by IFN-γ secretion against OVA (A) and SIINFELK peptide from MHC class I-restricted OVA (B) in C57 / BL6 mice inoculated with 3LL-OVA cells. Results are shown as the mean number of puncta per million cells in each group ± standard deviation. Different letters indicate statistically significant differences in the number of puncta per million cells (Kruskal-Wallis test and Dunn's multiple comparisons, p<0.05). [Figure 17] Cytotoxic activity of NK cells against K562 after treatment with peptide identified as SEQ ID NO:1. Graph shows mean ± standard deviation of three independent replicates. Asterisks (*) indicate statistically significant differences in percent specific lysis versus baseline control (One-way ANOVA, Tukey's multiple comparison test, p<0.05). SEQ1: Peptide identified as SEQ ID NO:1. [Figure 18] In vitro evaluation of the effect of peptide identified as SEQ ID NO:1 on the phagocytic capacity of macrophage subtypes M1 and M2. OCI-AML3 (A) and HL-60 (B) cells were treated with peptide identified as SEQ ID NO:1, CX4945 or Tat. Graphs show the mean ± standard deviation of three independent experiments. Asterisks (*) indicate statistically significant differences in the percentage of CFSE+ cells in each macrophage subtype evaluated versus cells without treatment (One-way ANOVA, Tukey's multiple comparison test, p<0.05). [Figure 19]Evaluation of the antitumor effect of the combination of the peptide identified as SEQ ID NO:1 with the vaccine candidate CIGB550-E7+VSSP. The figure shows graphs corresponding to tumor growth curves (mean ± standard deviation) (A) and survival (Kaplan-Meier curves) (B). Different letters indicate statistically significant differences (p<0.05) in comparisons made between dose groups using one-way ANOVA test and Tukey's multiple comparison test for tumor volume and log-rank test for survival analysis. [Figure 20] Evaluation of humoral responses to OVA protein. Results are shown as the mean reciprocal of antibody titers against OVA protein determined by ELISA on days 21 (A) and 42 (B) of the immunization schedule. Error bars indicate the standard deviation of the mean titers for each group. Different letters indicate statistically significant differences in antibody titers (ANOVA, Tukey's multiple comparison test, p<0.01). [Figure 21] Evaluation of humoral responses against Core.120, E1.340 and E2.680 proteins. Results are shown as the reciprocal of the mean antibody titers against Core.120, E1.340 and E2.680 proteins determined by ELISA 6 days after challenge with recombinant vaccinia virus expressing structural proteins of Hepatitis C virus (HCV). Error bars indicate the standard deviation of the mean antibody titers against each protein. "a": indicates a statistically significant difference from the control group in terms of antibody titers (ANOVA, Tukey's multiple comparison test, p<0.01). [Figure 22] Evaluation of humoral responses against the nucleocapsid (NP) (A) and S protein receptor binding domain (RBD) (B) of the SARS-CoV-2 virus. The graph shows IgG antibody levels in samples from patients treated with peptide identified as SEQ ID NO:1 in combination with standard of care and from a control group treated with standard of care alone. Significance level p<0.05 (Wilcoxon test). DO / CO: optical density of samples between the cutoff values ​​of the assay. [Figure 23]Evaluation of the ability of the peptide identified as SEQ ID NO:1 to enhance the effect of dendritic cell-based treatment. The figure shows the graphs corresponding to tumor growth curves (mean ± standard deviation) (A) and survival (B). Different letters indicate statistically significant differences (p<0.05) in comparisons made between groups using one-way ANOVA test and Tukey's multiple comparison test for tumor volume and log-rank test for survival analysis. [Figure 24] Evaluation of the ability of the peptide identified as SEQ ID NO:1 to enhance the effect of TIL-based antitumor treatment. The figure shows the corresponding graphs of tumor growth curves (mean ± standard deviation) (A) and survival (B). Different letters indicate statistically significant differences (p<0.05) between groups using one-way ANOVA test and Tukey's multiple comparison test for tumor volume and log-rank test for survival analysis. [Diagram 25] Evaluation of the ability of the peptide identified as SEQ ID NO:1 to enhance the antitumor effect of anti-PDL1 treatment. The figure shows a graph corresponding to the tumor growth curve (mean ± standard deviation). Different letters indicate statistically significant differences (p<0.05) between groups using one-way ANOVA test and Tukey's multiple comparison test for tumor volume.

[0029] example Example 1. Cell death induced by the peptide identified as SEQ ID NO:1. To determine whether the peptide identified as SEQ ID NO:1 can induce cell death in tumor cell lines, mouse lymphocytic leukemia cells L1210 were treated with 10 μM of the peptide identified as SEQ ID NO:1 at 4° C. and 37° C. for 20 minutes. The cells were then labeled with Annexin V-FITC, which binds to phosphatidylserine residues exposed on the outer membrane of apoptotic cells, and 7-AAD, which penetrates only into dead cells. Finally, the samples were analyzed by flow cytometry.

[0030] Treatment with peptide identified as SEQ ID NO:1 at 37°C induced up to a 15-fold increase in the percentage of apoptotic cells (Annexin V+ / 7-AAD-). This increase was not detected when cells were incubated at 4°C, which may indicate induction of apoptosis since apoptosis is an energy-dependent process (Figure 1A). Similar results were observed when 3LL mouse lung carcinoma cells were treated with 130 μM of peptide identified as SEQ ID NO:1 for 3 and 5 hours (Figure 1B).

[0031] In an ex vivo evaluation performed with samples from patients with acute myeloid leukemia, induction of cell death was also observed when cells were treated with 40 μM of the peptide identified as SEQ ID NO:1 for 24 hours. This phenomenon was observed in 6 of the 10 patients evaluated (FIG. 2). These results demonstrate that the peptide identified as SEQ ID NO:1 is capable of inducing tumor cell death both in vitro and ex vivo.

[0032] Example 2. Translocation of CRT and Erp57 induced by the peptide identified as SEQ ID NO:1. CRT exposure at the tumor cell membrane constitutes a phagocytic signal ("eat me") for dendritic cells that also occurs with Erp57 expression. Taking this into account, we decided to evaluate whether the peptide identified as SEQ ID NO:1 is capable of inducing the translocation of these molecules in L1210 cells. For this purpose, 2.5×10 5Cells were seeded in 12-well plates and treated with 40 μM of the peptide identified as SEQ ID NO:1 for 3 and 5 hours. Afterwards, cells were collected, washed twice with cold PBS and fixed with 0.2% paraformaldehyde for 5 minutes at 4° C. After Fc receptor blocking (in PBS containing 2% fetal bovine serum (FBS) for 30 minutes at 4° C.), cells were labeled with primary antibodies for 30 minutes at 4° C., followed by washing twice and incubation with a secondary monoclonal antibody conjugated to Alexa 488 in blocking solution for 30 minutes at 4° C. After repeated washing, cells were analyzed by flow cytometry. In addition, the effect of a CK2 inhibitor called CX4945 was also evaluated after treatment at 5 μM for 24 hours. Cells without treatment constituted the negative control of the study.

[0033] Treatment with peptide identified as SEQ ID NO:1 at the two time points evaluated induced CRT and Erp57 translocation to the cell surface in L1210 cells (Figures 3 and 4). The percentage of CRT+ and Erp57+ cells was significantly increased compared to untreated cells and cells treated with the inhibitor CX4945 (Figures 3 and 4). These results demonstrate that peptide identified as SEQ ID NO:1 causes the translocation to the cell surface of molecules that constitute the "eat me" signal, which constitutes an essential stimulus for phagocytosis and capture of tumor antigens.

[0034] Example 3. Treatment with the peptide identified as SEQ ID NO:1 reduces CD47 "don't eat me" signaling in tumor cells. Recently, a strong association between anti-phagocytic signals, also known as "don't eat me" signals, and tumor growth and progression has been reported. In this sense, we evaluated whether the peptide identified as SEQ ID NO:1 has any effect on these molecules, especially on CD47. To do so, two leukemia cell lines were used: HL60 (ATCC® CCL240™) and OCI-AML3 (DSMZ ACC 582). Cells were maintained in RPMI-1640 medium (Sigma, EEUU) supplemented with 10% FBS (Capricorn Scientific GmbH, Alemania), 2 mM L-glutamine and 50 μg / mL gentamicin (Sigma, EEUU). The experimental design consisted of eight experimental groups for both cell lines, where at each time point, non-treated cells were treated with 40 μM of the peptide identified as SEQ ID NO:1 as a control for 30 minutes and 3 hours. As shown in Table 1, three experimental replicates were used per group in 12-well plates for a total of 24 independent samples.

[0035] [Table 1]

[0036] After 30 min and 3 h of incubation, the culture medium was removed, the cells were washed once with PBS buffer, collected in each well in Lysis Buffer (RNeasy Plus Minikit, Qiagen, EU) containing 1% β-mercaptoethanol, and homogenized. Purification proceeded according to the manufacturer's instructions QiaCube (Qiagen, EEUU). Total ribonucleic acid (RNA) samples were resuspended in nuclease-free water and stored at -70°C until microarray differential gene expression was analyzed. The results were further verified by qPCR. The oligonucleotides used are shown in Table 2. GAPDH, DDX5, and ABL1 were used as reference genes. Reactions were performed in 96-well plates on a LightCycler® 480II instrument (Roche, Germany) in SYBR Green Probe II mode.

[0037] [Table 2]

[0038] The fold change of each gene was determined relative to the untreated condition and normalized to three reference genes GAPDH, DDX5 and ABL1. As shown in Figure 5, a 3 hour treatment with peptide identified as SEQ ID NO:1 induced a significant decrease in CD47 messenger RNA (mRNA) levels in both cell lines evaluated. This was observed by both microarray (Figure 5A) and qPCR (Figure 5B). These results indicate that peptide identified as SEQ ID NO:1 modulates the levels of "don't eat me" signals such as CD47.

[0039] Example 4. Induction of danger signals by the peptide identified as SEQ ID NO:1. During immunogenic cell death, the expression and release of certain danger signals are induced that are involved in the activation of an effective antitumor immune response. With this in mind, the inventors evaluated the ability of the peptide identified as SEQ ID NO:1 to induce the release of ATP, HSP70 and HMGB-1 in different human and mouse tumor cell lines and in serum from patients treated with the peptide.

[0040] 6 × 10 for in vitro detection of ATP, HSP70, and HMGB-1 4Cells were seeded in 24-well plates and treated with 40 μM of peptide identified as SEQ ID NO:1, 40 μM of Tat, both for 5 and 24 h, and 5 μM of CX4945 for 24 h. As a positive control, cells were treated with 5 μM DOX for 24 h. Untreated cells and serum from patients taken at time 0 constituted negative controls. ATP secretion was measured by the luciferin-based ENLITEN ATP Assay (Promega), HSP70 was stained with anti-HSP70 antibody (clone C92F3A-5, Enzo Life Sciences), and HMGB-1 release was assessed by enzyme-linked immunosorbent assay (ELISA, IBL International) according to the manufacturer's instructions.

[0041] Treatment with the peptide identified as SEQ ID NO:1 for 5 and 24 hours induced HMGB-1 release into the culture medium. The levels detected were statistically higher than those seen in untreated cells or in cells treated with either the Tat peptide or the CK2 inhibitor CX4945 (Figure 6). At 24 hours, the HMGB-1 levels secreted into the culture medium by cells treated with the peptide identified as SEQ ID NO:1 were significantly higher than the HMGB-1 levels of cells treated with DOX. An increase in HMGB-1 levels was observed in the serum of patients treated with the peptide identified as SEQ ID NO:1 (Figure 7). This was observed in 4 of the 5 patients evaluated, mainly at 6 weeks of treatment.

[0042] ATP levels were also significantly increased in the supernatants of tumor cell cultures after treatment with peptide identified as SEQ ID NO:1 for 5 and 24 hours compared to untreated cells and cells treated with CX4945 (Figure 8). The levels were even higher than those detected after treatment with DOX, a classical immunogenic cell death inducer. Similar results were observed when evaluating HSP70. Treatment with peptide identified as SEQ ID NO:1 for 5 and 24 hours induced a significant increase in the percentage of HSP70 positive cells compared to the remaining evaluated conditions (Figure 9). These results demonstrate that peptide identified as SEQ ID NO:1, unlike CX4945 and Tat peptides, causes the induction of danger signals involved in the recruitment and activation of immune cells.

[0043] Example 5. In vitro phagocytosis, maturation and activation of dendritic cells by tumor cells treated with the peptide identified as SEQ ID NO:1. To assess whether tumor cells treated with the peptide identified as SEQ ID NO:1 can stimulate phagocytosis and maturation of dendritic cells, L1210 and 3LL cells were treated with the peptide identified as SEQ ID NO:1 at 40 μM and 130 μM, respectively, in T25 flasks for 3 hours. For phagocytosis experiments, tumor cells were pre-stained with 1 μM carboxyfluorescein succinimidyl ester (CFSE). After treatment, tumor cells were washed and seeded in U-bottom 96-well plates at a ratio of 1:2 to dendritic cells for 48 hours. Dendritic cells were differentiated from bone marrow precursors of DBA / 2 mice using FLT3 for co-culture with L1210 cells, and C57 / BL6 mice for experiments with 3LL cells. Co-cultures were labeled with anti-CD11c antibody for phagocytosis experiments, or with anti-CD11c, anti-CD86, anti-MHCII, and anti-CD40 antibodies for dendritic cell maturation experiments. Untreated cells constituted the negative control of the experiment. Cells treated with DOX were used as a positive control. The effects of Tat peptide and a CK2 inhibitor called CX4945 on stimulating phagocytosis were also evaluated.

[0044] Furthermore, the ability of these dendritic cells matured with peptide-treated 3LL cells to activate CD8+ T cells in vitro was evaluated. For this purpose, dendritic cells previously co-incubated with tumor cells under the above conditions were washed and loaded with the MHC class I-restricted, OVA-derived SIINFELK peptide at different concentrations (0, 10, 30 and 100 pg / mL) for 3 h. They were then washed again and co-incubated with RagOT-I cells in a 1:1 ratio for 48 h. Finally, IFN-γ production was quantified by ELISA. RagOT-I cells co-incubated with dendritic cells previously incubated with non-treated 3LL cells constituted the negative control of the experiment.

[0045] As shown in Figure 10, treatment with peptide identified as SEQ ID NO:1 stimulated phagocytosis of tumor cells by bone marrow-derived dendritic cells. The percentage of CD11c+ / CFSE+ cells in the case of co-culture of dendritic cells with tumor cells treated with peptide identified as SEQ ID NO:1 was significantly higher than the rest of the evaluated conditions (p<0.05). Under these same conditions, induction of dendritic cell maturation was observed by an increase in CD40, CD86 and MHC-II markers (Figure 11).

[0046] Dendritic cells treated with peptide and matured by DOX-treated 3LL cells were able to activate CD8+OT-I cells as determined by increased IFN-γ production, which was significantly higher than negative control and cells treated with CX4945 and Tat peptides ( FIG. 12 ). These results demonstrate that treatment with the peptide identified as SEQ ID NO:1 induces the expression of molecules that stimulate phagocytosis and maturation of dendritic cells and increases their ability to activate CD8+ T cells. This effect may indicate the initiation of anti-tumor response induction.

[0047] Example 6. Evaluation of the effect of the peptide identified as SEQ ID NO:1 on the tumor microenvironment in DBA / 2 mice. To study whether the peptide identified as SEQ ID NO:1 can modulate the tumor microenvironment, 0.5×10 mAbs were injected subcutaneously into the right flank of female DBA / 2 mice, 6-8 weeks of age and 17-19 grams. 6 L1210 cells were inoculated (groin inoculation). Seven days after tumor challenge, 42 mice with palpable and non-measurable tumors were selected and randomly assigned to six groups of seven animals each. Mice received the treatments shown in Table 3. Treatment with peptide identified as SEQ ID NO:1 (20 mg / kg) was administered intratumorally and intravenously in a volume of 50 μL and 100 μL, respectively, in a daily inoculation for five days. DOX (10 mM) and Tat peptide (20 mg / kg) were administered intratumorally in a final volume of 50 μL, in a daily inoculation for five days. Compound CX4945 (75 mg / kg) was administered orally twice a day for five days in 25 mM NaH2PO4 buffer. Seven days after the end of treatment, mice were euthanized to obtain tumors (18th day of operation). Before sacrificing the mice, blood samples were obtained for cytokine (IL-6, IL-10, IL-12p70, TNF-α) detection by ELISA. Tumors were cut into small pieces and transferred into gentleMACS tubes containing RPMI medium supplemented with enzyme cocktail from the Tumor Dissociation Kit (Miltenyi Biotech, Germany). Tumor dissociation was performed using the gentleMACS™ Dissociator Kit and the Tumor Dissociation Kit (Miltenyi Biotech, Germany). The digested suspension was filtered, centrifuged and labeled with specific antibodies against CD8, CD4, Foxp3, CD11c, CD86 and CD40.

[0048] [Table 3]

[0049] Treatment with peptide identified as SEQ ID NO:1, administered both intratumorally and systemically, induced a significant increase in IL12p70, TNF-α and IL-6 levels in peripheral blood compared to the remaining groups. Additionally, peptide treatment significantly reduced IL-10 levels. This modulation in cytokine levels was not observed when mice were treated with Tat, DOX or CX4945 (Figure 13).

[0050] Similarly, treatment with peptide identified as SEQ ID NO:1 by two routes of administration caused a significant increase in intratumoral levels of CD8+ and CD4+ T cells, as well as in situ maturation of dendritic cells, as determined by an increase in CD86 and MHC-II maturation markers. Furthermore, a significant decrease in intratumoral Foxp3+ T cell levels was observed after treatment with peptide identified as SEQ ID NO:1 and DOX, with a greater decrease obtained after application of the peptide. This effect was not observed when mice were treated with Tat or CX4945 (Figure 14). These results indicate that treatment with peptide identified as SEQ ID NO:1 can revert an immunosuppressive tumor environment to an immunostimulatory one, accompanied by a significant increase in dendritic cell maturation and effector cell recruitment.

[0051] Example 7. Evaluation of the vaccine efficacy of inoculation of tumor cells treated with the peptide identified as SEQ ID NO:1 in DBA / 2 mice. To determine whether cell death induced by the peptide identified as SEQ ID NO:1 has any immunomodulatory effect, a vaccination schedule was performed in mice bearing tumor cells pretreated with the peptide identified as SEQ ID NO:1. To this end, 1×10 mice treated in vitro with 40 μM of the peptide identified as SEQ ID NO:1 were vaccinated. 6 L1210 cells or time-matched untreated cells were injected subcutaneously at 0.1 mL into the groin and right flank of 6-8 week-old female DBA / 2 mice for 3 h. Seven days later, 1 × 10 6Viable L1210 cells were inoculated into the contralateral flank and tumor growth and volume were assessed up to day 45. Tumors were measured three times a week with Vernier calipers and calculated using the formula: volume = width 2 Tumor volume was estimated using (mm) × length / 2 (mm). As a positive control, cells treated with 15 μM DOX for 24 h were used for vaccination. A group vaccinated with cells treated with 5 μM CX4945 for 24 h was also included. In total, four groups were formed with 10 mice in each group.

[0052] As shown in Figure 15, mice vaccinated with tumor cells treated with peptide identified as SEQ ID NO:1 showed a clear tumor growth delay that may be due to the induction of anti-tumor immune responses. The tumor volume in this group was smaller than the remaining groups, a difference that reached statistical significance (Figure 15A, p<0.05). Importantly, in the group inoculated with cells treated with peptide identified as SEQ ID NO:1, 7 out of 10 mice remained tumor-free until the end of the study, which was significantly better than the remaining evaluated conditions (Figure 15B, p<0.01). These results confirm that cell death induced by peptide identified as SEQ ID NO:1 is immunogenic, as it generates an immune response that prevents the growth of subsequent tumor challenge. Cells treated with CX4945 did not induce a vaccine effect.

[0053] Example 8. Cellular immune responses generated by tumor cells treated with the peptide identified as SEQ ID NO:1 in C57 / BL6 mice. The induction of a cellular immune response by tumor cells treated with the peptide identified as SEQ ID NO:1 was evaluated. For this purpose, 1×10 tumor cells were treated or not in vitro with one of the following different conditions: a) 40 μM of the peptide identified as SEQ ID NO:1 for 3 hours, b) 15 μM of DOX for 24 hours, c) 5 μM of CX4945 and d) 40 μM of Tat for 3 hours. 63LL-OVA cells were inoculated subcutaneously in the inguinal region of the right leg of 6-8 week-old female C57 / BL6 mice in a volume of 0.1 mL. Five days later, five mice from each group were euthanized to obtain lymph nodes, which were then macerated in liquid to obtain cells.

[0054] IFN-γ secretion was evaluated by enzyme-linked immunospot assay (ELISPOT) according to the procedure described below. 96-well microplates with nitrocellulose membranes (Millipore, Bedfors, MA, USA) were coated with 100 μL / well of mouse anti-IFN-γ monoclonal antibody (BD Biosciences, Canada) at a concentration of 5 μg / mL in PBS for 16 h at 4°C. After three washes with PBS, the plates were blocked with RPMI medium supplemented with 10% FBS for 1 h at 37°C in a humidified atmosphere containing 5% CO2. A total of 2 × 10 IFN-γ secretion was performed with OVA protein and OVA-derived SIINFELF peptide at a final concentration of 6 μg / mL in a final volume of 0.2 mL. 5 Cells / well were stimulated in duplicate. Concanavalin A at 5 μg / mL in RPMI medium containing 10% FBS was used as a positive control. Plates were incubated for 48 h at 37°C in a humidified atmosphere of 5% CO2. After incubation, a standard ELISPOT assay was performed (Vazquez-Blomquist D et.al.(2002)Viral Immunol.5(2):337-356). Spot counting was performed in an automatic counter (Immunospot Analyzer, Cellular Technology Ltd., Shaker Heights, OH, USA). Cells incubated with RPMI medium containing 10% FBS were taken as a negative control.

[0055] The number of spots was at least 3 times higher than the average number of spots in the negative control group and 10 6 Results were considered positive if at least three specific spots per cell (a value obtained after subtracting the number of spots obtained from each animal in unstimulated conditions from the number of spots obtained from each animal in stimulated conditions) were present.

[0056] As shown in Figure 16, inoculation of 3LL-OVA cells treated with peptide identified as SEQ ID NO:1 induced a significantly higher IFN-γ secreting effector cell response than the remaining groups. Tumor cells treated with CX4945 and Tat were unable to generate this type of response. These results demonstrate that peptide identified as SEQ ID NO:1, but not CX4945 or another CK2 inhibitor such as Tat peptide, induces a type of cell death that can generate an effective CD8+ T lymphocyte response.

[0057] Example 9. Evaluation of the effect of the peptide identified as SEQ ID NO:1 on tumor cell susceptibility to NK activity. It has been reported that the release of danger signals such as HSP70 and CRT is involved in increasing the susceptibility of tumor cells to the cytotoxic activity of NK cells. Therefore, the effect of the peptide identified as SEQ ID NO:1 on the susceptibility of tumor cells to NK cytotoxic activity was evaluated. To do so, K562 cells, which are classical target cells for NK activity, were used. The target cells were sensitized to Cr by incubation at 37°C for 70 minutes. 51 (approximate specific activity: 100 μCi / μg). Target cells were then washed three times with RPMI medium supplemented with 2% FBS and seeded in 96-well plates at a target:effector ratio of 1:25. Meanwhile, NK cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors by negative selection using magnetic beads (Miltenyi Biotec). As a result, CD56+CD3- NK cells were obtained with a purity of more than 96%. The peptide identified as SEQ ID NO:1 was added to the co-culture at 12.5, 25 and 50 μM. After 4 hours of incubation, Cr was counted using a scintillation counter. 51 The supernatant was collected for measurement of release. The percentage of specific lysis was calculated as follows:

number

[0058] Target cells without co-incubation with effector cells were used as a spontaneous lysis control. Target cells bearing the peptide identified as SEQ ID NO:1 constitute a toxicity control for this peptide.

[0059] As shown in Figure 17, the presence of peptide identified as SEQ ID NO:1 in co-cultures of K562 cells with NK cells enhanced the cytotoxicity of NK cells. The 12.5 and 25 μM doses significantly increased the percentage of specific lysis compared to baseline cytotoxicity. Since the 50 μM dose of peptide was toxic to the target cells, it cannot be concluded that the observed lysis was due to an effect on NK cells in that case. The data from these experiments indicate that peptide identified as SEQ ID NO:1 can sensitize tumor cells to the direct action of NK cells, which helps generate a diverse and effective immune response.

[0060] Example 10. Evaluation of the effect of the peptide identified as SEQ ID NO:1 on tumor cell susceptibility to macrophage cytotoxicity. As described in Examples 2 and 3, treatment with the peptide identified as SEQ ID NO:1 can increase the "eat me" signal and decrease the "don't eat me" signal. This therefore prompted us to evaluate whether treatment with the peptide identified as SEQ ID NO:1 promotes phagocytosis of tumor cells by macrophages, and whether this is done by M1 or M2 macrophages. To that end, HL-60 and OCI-AML3 leukemia tumor cell lines were pre-stained with 0.5 μM CFSE and treated with 10 or 40 μM of the peptide identified as SEQ ID NO:1 for 5 hours, with 5 μM of CX4945 for 24 hours, or with 40 μM of Tat for 5 hours. Afterwards, the cells were washed and co-cultured with each type of M1 and M2 macrophage in a 1:1 ratio for 3 hours. The different types of macrophages were obtained from PBMCs of healthy donors by Ficoll™ gradient. Monocytes were purified by positive selection using CD14 beads with MS columns (Miltenyi Biotech, Germany) to obtain a purity of 96%. 6 Monocytes were seeded at cells / mL / well and induced with 100 ng / mL GM-CSF for 7 days and 50 ng / mL IFN-γ for the final 24 hours to obtain M1 macrophages. M2 macrophages were induced with 50 ng / mL M-CSF for 7 days and 100 ng / mL IL-10 for the final 24 hours. Afterwards, supernatants were collected and cells were stained with anti-CD33 (to determine the corresponding macrophage population), anti-CD86 (to determine the M1 population) and anti-CD163 (to determine the M2 population).

[0061] Pretreatment of OCI-AML3 and HL-60 tumor cells with peptide identified as SEQ ID NO:1 at both IC50 (40 μM) and suboptimal (10 μM) doses increased their susceptibility to be phagocytosed by M1 macrophages, but not M2 macrophages (FIG. 18). The increase in phagocytosis was statistically significant versus co-culturing macrophages with untreated tumor cells. This effect was not observed when tumor cells were treated with CX4945 or Tat. These results demonstrate that the reduction in the "don't eat me" signal provided by peptide identified as SEQ ID NO:1, but not by CX4945 or Tat, enhances the susceptibility of tumor cells to M1 macrophages, but not to M2 macrophages.

[0062] Example 11. Antitumor effect of the peptide identified as SEQ ID NO:1 in combination with the vaccine candidate CIGB550-E7+VSSP in C57BL6 mice (TC-1 therapeutic scenario). Given the ability of the peptide identified as SEQ ID NO:1 to induce immunogenic cell death, the antitumor response generated by the peptide identified as SEQ ID NO:1 in combination with the CTL vaccine candidate CIGB550-E7+VSSP (Granadillo M. et al., (2019) Vaccine 37(30):3957-3960) was evaluated. The vaccine candidate contains the recombinant fusion protein CIGB550-E7, based on the fusion of the human papillomavirus (HPV)-E7 mutein to the cell-penetrating peptide CIGB550, formulated with a synthetic NAcGM / VSSP adjuvant.

[0063] For that purpose, 5 × 10 cells were injected into the right flank of 6–8 week-old C57 / BL6 female mice. 4TC-1 cells were injected subcutaneously (inguinal inoculation). Seven days after tumor challenge, 48 mice with palpable and non-measurable tumors were randomized into 6 groups with 10 mice per group. Mice received different treatments as shown in Table 4. Treatment with peptide identified as SEQ ID NO:1 (40 mg / kg) was administered intratumorally and intravenously in a final volume of 50 μL and 100 μL, respectively. CX4945 (75 mg / kg) was administered orally twice daily in 25 mM NaH2PO4. Vaccine candidate CIGB550-E7+VSSP was injected subcutaneously in the right flank in a final volume of 0.2 mL. Tumor growth was followed and tumor volumes were determined and calculated as described in Example 7.

[0064] The results showed that both peptides identified as SEQ ID NO:1, injected intratumorally and systemically prior to administration of the vaccine, were able to enhance the antitumor efficacy of the CIGB550-E7+VSSP vaccine candidate as determined by tumor growth control and mouse survival ( FIG. 19 ). This data supports the use of peptides identified as SEQ ID NO:1 to enhance the efficacy of antitumor vaccines.

[0065] [Table 4]

[0066] Example 12. Evaluation of the ability of the peptide identified as SEQ ID NO:1 to enhance B cell immune responses. Induction of antibodies against OVA protein Given the ability of the peptide identified as SEQ ID NO:1 to induce immunogenic cell death, it was decided to evaluate its ability to enhance B cell immune responses. For this purpose, 30 female BALB / c mice, 8 weeks of age and weighing 18-20 grams, were divided into three groups of 10 mice per group. The first group was immunized with 10 μg of OVA protein formulated in aluminum phosphate. The second group received 10 μg of OVA protein and 100 μg of the peptide identified as SEQ ID NO:1. The third group constituted the control group of the study, inoculated with aluminum phosphate only. OVA administration was performed subcutaneously on days 0, 14 and 28, in a final volume of 100 μL. On days 21 and 42, blood samples were obtained to evaluate the induction of an IgG response against the OVA protein.

[0067] As shown in Figure 20, all mice immunized with OVA protein elicited specific antibodies against the protein. The highest antibody titers were induced in the group immunized with OVA protein formulated with the peptide identified as SEQ ID NO:1, which were significantly higher than those obtained in the remaining study groups. These results suggest that the peptide identified as SEQ ID NO:1 can enhance B cell immune responses.

[0068] Induction of antibodies against HCV antigens in a surrogate virus model Given the results observed with OVA protein formulated with the peptide identified as SEQ ID NO:1, the ability of this peptide to enhance the specific antibody response against viral antigens during in vivo infection was evaluated. For that purpose, 14 female BALB / c mice, 8 weeks of age and weighing 18-20 grams, were challenged with a recombinant vaccinia virus compressing structural Hepatitis C virus (HCV) proteins (Alvarez-Lajonchere et al., Biotecnologia Aplicada, 2007;24(3)). 100 μL of OVA in 200 μL of PBS was used to challenge the mice. 6Virus was inoculated intraperitoneally at a dose of plaque forming units (pfu). The next day, mice were divided into two groups of 7 mice per group. The first group of mice was treated with 3 mg / kg of peptide identified as SEQ ID NO:1 and the second group was inoculated with PBS, both intraperitoneally on days 1, 2, 3 and 4 of the study. Whole blood extraction was performed 6 days after virus challenge to evaluate humoral immune responses against HCV structural proteins.

[0069] Treatment with the peptide identified as SEQ ID NO:1 enhanced the induction of specific antibodies against Core.120, E1.340 and E2.680 protein antigens. The antibody titers were significantly higher than those of the control group (FIG. 21).

[0070] Induction of Antibodies in Patients Infected with Anti-SARS-CoV-2 Treated with a Peptide Identified as SEQ ID NO:1 A randomized Phase I / II clinical trial was conducted in 20 SARS-CoV-2 infected patients treated with the peptide identified as SEQ ID NO:1. Ten patients were treated intravenously with 2.5 mg of the peptide per kg of body weight once daily for five consecutive days along with standard of care consisting of Kaletra™, chloroquine, and IFN-α2b. Meanwhile, the control group enrolled 10 patients who received only standard of care. Specific anti-NP and anti-RBD IgG levels were determined by using in home ELISA.

[0071] As shown in Figure 22, a significant increase in IgG antibody levels against the NP and RBD of the SARS-CoV-2 virus was detected after treatment with the peptide identified as SEQ ID NO: 1. In the control group, no significant difference was observed between the levels of antibodies detected at both evaluation times. These results suggest that the peptide identified as SEQ ID NO: 1 can enhance specific humoral immune responses against viral antigens during viral infection.

[0072] Example 13. Enhancement of the efficacy of dendritic cell vaccines with the peptide identified as SEQ ID NO:1. Currently, one of the possibilities in cancer immunotherapy is constituted by dendritic cell-based vaccines. With this in mind, it was investigated whether the peptide identified as SEQ ID NO:1 could enhance the effect of dendritic cell vaccines. For that purpose, C57 / BL6 mice were euthanized and spleens were obtained, macerated and digested with collagenase and DNAse. Gradient medium density was used to separate low density mononuclear cells. Dendritic cells were purified by mouse Pan-DC magnetic bead-mediated cell sorting (Miltenyi Biotech, Bergisch-Gladbach, Germany) according to the manufacturer's recommendations. Dendritic cells were obtained with a purity of more than 90%, as judged by cytometric analysis of CD11c expression. The purified cells were loaded with OVA protein (Sigma-Aldrich) (100 μg / mL) in IMDM medium supplemented with 10% FBS for 16 h at 37°C. Non-adherent dendritic cells loaded with OVA protein (DC+OVA) were collected for inoculation into animals. Dendritic cells alone were used as a negative control.

[0073] To evaluate whether the peptide identified as SEQ ID NO:1 can enhance the effect of DC+OVA in a mouse model, 5×10 5 3LL-OVA cells were inoculated into the right flank in a final volume of 50 μL. Seven days after tumor challenge, 42 mice with palpable and non-measurable tumors were selected and randomly divided into six groups with 7 mice per group. The animals received different treatments as shown in Table 5. Treatment with peptide identified as SEQ ID NO:1 (40 mg / kg) was applied intratumorally in a final volume of 50 μL. CX4945 (75 mg / kg) was administered orally twice daily in 25 mM NaH2PO4 buffer. 5×105 The administration of dendritic cells was performed intratumorally in a final volume of 50 μL. Tumor growth was followed during that time and tumor volume was determined and calculated as described in Example 7. When the tumor volume was 4000 mm 3 Animal survival was recorded until the animal reached a survival time of 0.05 mg / kg, at which point the animal was euthanized by cervical dislocation. Throughout the experiments, mice were housed in a pathogen-free environment and procedures were performed in accordance with institutional guidelines.

[0074] [Table 5]

[0075] As shown in Figure 23, the peptide-based treatment identified as SEQ ID NO: 1 combined with dendritic cell vaccine induced tumor growth delay in C57 / BL6. The tumor volume in this group was significantly smaller than the remaining groups (Figure 23A). A significant difference was also observed in terms of survival of animals treated with the combination versus the remaining groups (Figure 23B). These results demonstrate that the peptide identified as SEQ ID NO: 1 can enhance the efficacy of dendritic cell-based vaccine.

[0076] Example 14. Evaluation of the ability of the peptide identified as SEQ ID NO:1 to enhance the efficacy of tumor infiltrating lymphocyte-based antitumor treatments. The use of TILs for cancer treatment is one of the novel immunotherapeutic strategies currently being applied. According to the results obtained previously (Example 6), treatment with the peptide identified as SEQ ID NO:1 increases the tumor infiltration of CD8+ T cells. Taking this into account, it was decided to evaluate whether the peptide identified as SEQ ID NO:1 is able to enhance the antitumor effect of TILs in a mouse model of cancer. For that purpose, 5 × 10 4L1210 cells were inoculated subcutaneously (inguinal injection). Seven days after tumor challenge, 42 animals with palpable and non-measurable tumors were randomly assigned to six groups with seven mice per group. Different treatments were administered as shown in Table 3, Example 6. Seven days after the end of treatment, mice were euthanized to obtain tumors. Tumors were grown to approximately 1-3 mm 3 The tumors were cut into small pieces of 10 cm in size and seeded in 24-well plates in RPMI medium supplemented with 10% FBS and 2000 IU / mL recombinant mouse IL-2. Cultures were grown in 12-well plates. TILs from the same tumor fragments were combined and counted. At 24 hours, 5×10 4 Other DBA / 2 mice were subsequently challenged with 5 × 10 L1210 cells. 6 TILs were inoculated intravenously. Tumor growth was followed during that time, and tumor volumes were measured and calculated as described in Example 7.

[0077] As shown in Figure 24, TILs from mice treated with peptide identified as SEQ ID NO:1 induced antitumor activity after challenge, both intratumorally and systemically. The mean tumor volume was significantly smaller than the remaining groups (Figure 24A). Significant differences were also observed in the survival of animals treated with TILs from mice treated with peptide identified as SEQ ID NO:1 compared to TILs from mice treated with DOX, CX4945 or Tat (Figure 24B). These results demonstrate that peptide identified as SEQ ID NO:1 can enhance the antitumor effect of TIL-based vaccines.

[0078] Example 15. Evaluation of the ability of the peptide identified as SEQ ID NO:1 to modulate levels of the immune checkpoint PDL1. Increased PDL1 in H460 and A549 cells following in vitro treatment with peptide identified as SEQ ID NO:1 Immune checkpoints are currently important targets in cancer immunotherapy due to their immunosuppressive role in the cancer process. With this in mind, the effect of the peptide identified as SEQ ID NO: 1 on the regulation of PDL1 levels in two non-small cell lung cancer cell lines: A549 and H549 was evaluated. To this end, 5×10 4 The cells were seeded in a 24-well plate. The next day, the cells were treated with 30 μM of the peptide identified as SEQ ID NO:1, 5 μM CX4945 or 30 μM Tat peptide for 24 hours. After treatment, the cells were harvested, stained with anti-PDL1 antibody and analyzed by flow cytometry.

[0079] Table 6 shows the percentage of PDL1 positive cells and the mean fluorescence intensity (MFI). Treatment with peptide identified as SEQ ID NO:1 induced a significant increase in PDL1 expression in both cell lines. No differences were observed when cells were treated with CX4945 or Tat peptide.

[0080] [Table 6]

[0081] These results suggest that the peptide identified as SEQ ID NO:1 may be used in combination with a PDL1 immune checkpoint inhibitor.

[0082] Evaluation of the ability of the peptide identified as SEQ ID NO:1 to enhance the effect of anti-PDL1 therapy in a mouse model of lung cancer. Taking into account the results obtained in the in vitro tests, it was decided to evaluate whether the use of the peptide identified as SEQ ID NO:1 improves the antitumor effect of anti-PDL1 therapy in a mouse model of lung cancer. To this end, 2 × 10 5 Lewis lung carcinoma cells were implanted subcutaneously (day 0). Nine days after tumor challenge, 40 mm 3Forty-two animals bearing tumors of 10 ...

[0083] [Table 7]

[0084] As shown in Figure 25, treatment with the peptide identified as SEQ ID NO: 1 significantly reduced tumor progression. Furthermore, when administration of the peptide was combined with anti-PDL1 antibody, a more significant reduction in tumor volume was observed. This effect was not observed when mice were treated with CX4945 alone or in combination with anti-PDL1 therapy.

[0085] These results indicate that the peptide identified as SEQ ID NO:1 increases the probability of blocking anti-PDL1 therapy by inducing PDL1 expression in tumor cells, thereby enhancing the induction of a cytotoxic immune response against tumors.

Claims

1. 2. Use of a peptide identified as SEQ ID NO: 1 for the manufacture of a medicament for inducing anti-tumor and anti-viral immunity mediated by immunogenic cell death.

2. The use according to claim 1, wherein the induction of anti-tumor and anti-viral immunity comprises in vivo and in vitro activation and differentiation of dendritic cells.

3. The use according to claim 1, wherein the induction of anti-tumor immunity comprises enhancing the cytotoxic activity of NK cells or the activity of cytotoxic T cells at the tumor site.

4. The use according to claim 1, wherein the induction of anti-tumor immunity comprises the reversion of an immunosuppressive tumor microenvironment to an immunostimulatory tumor microenvironment.

5. The use according to claim 1, wherein the induction of anti-tumor and anti-viral immunity comprises increasing the secretion of pro-inflammatory cytokines.

6. The use according to claim 1, wherein the induction of anti-viral immunity comprises enhancing a specific humoral immune response against a viral antigen.

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