Patent Application
20240082392
The present invention is related to cancer immunotherapy and antiviral therapy field. In particular, it is based on the use of the peptide identified as SEQ ID NO: 1 for inducing antitumor and antiviral immunity by increasing immunogenic cell death. Also, it relates to the combination of the peptide with cancer immunotherapy.
Cancer is one of the leading causes of death worldwide. The global incidence of cancer continues to grow, with new cases estimated to exceed 21.7 million by 2030 and there will be 13 million deaths from cancer per year (Tartari F and cols. Cancer Treat Rev 2016; 48:20-24). One of the strategies in the development of new cancer treatment in the last 20 years has been the development of drugs whose mechanism is target-specific to interfere with the biochemical events involved with the viability of the tumor cell, thus producing apoptosis in the same. Generally, this class of drug is employed in combination with standard chemotherapy in first or second line of treatment of cancer patients, both in advanced and early stages.
The synthetic peptide identified as SEQ ID NO: 1 is a therapeutic candidate for cancer treatment, its primary action mechanism is to inhibit protein kinase CK2-mediated phosphorylation, thus leading to tumor cell death by apoptosis. It has shown antitumor effect in experimental oncology animal models (Perea S E and cols. Cancer Res 2004; 64:7129-9). Recently, direct antiviral effect of the peptide identified as SEQ ID NO: 1 has also been observed in different in vitro viral models. From a mechanistic point of view, it has been found that the peptide identified as SEQ ID NO: 1 interacts with B23/Nucleophosmin protein in the tumor cell nucleolus, inhibits its phosphorylation and induces nucleolar disassembly as apoptosis prelude (Perera Y and cols. Mol Cancer Ther 2009; 8(5)). Consistent with such molecular events, the peptide identified as SEQ ID NO: 1 modulates a series of proteins linked to different cellular processes and inhibits lung metastases colonization and tumor angiogenesis in preclinical cancer models (Farina H G and cols. Exp Cell Res 2011; 317:1677-1688) (Benavent Acero F and cols. Lung Cancer 2017; 107:14-21). Furthermore, the peptide identified as SEQ ID NO: 1 inhibits phosphorylation of other CK2 substrates such as AKT and PTEN in chronic lymphocytic leukemia cells (Martins L R and cols. Oncotarget 2014; 5:258-263). In clinical field, exploration of safety and tolerability of the peptide identified as SEQ ID NO: 1 in cancer patients has begun, where a trend of increased survival has been observed in patients with advanced disease, and above life expectancy (Batista-Albuerne N and cols. J Med Oncol 2018; 1:4). These studies have made it possible to know pharmacokinetics and biodistribution of the peptide identified as SEQ ID NO: 1, after its local or intravenous administration, as well as safe dose ranges and toxicity linked to research drug (Solares A M and cols. BMC Cancer 2019; 9(1):146) (Sarduy M R and cols. Br J Cancer 2015; 112:1636-43).
The highest aspiration of clinical immunologists and oncologist for many years has been trying to involve the immune system of the body in the neoplastic disease control. In line with this thinking, immunotherapy is presented as a promising option that is revolutionizing cancer treatment through discovery and development of new approaches that activate and strengthen antitumor immunity in cancer patients (Zugazagoitia J and cols. Clin Ther 2016; 38(7):1551-1566) (Yang Y. J Clin Invest 2015; 125(9):3335-3337).
At present, main immunotherapy options for cancer treatment are: monoclonal antibodies, immune checkpoint inhibitors, cancer vaccines and cell-based immunotherapy (Kenderian S S and cols. Biol Blood Transplant 2017; 23:235-246) (Ruella M and Kenderian S S. BioDrug 2017; 31(6):473-481). In addition, there are other non-specific immunotherapies, which activate the immune system in a general way, and this could thus attack tumor cells (Klener P Jr and cols. Curr Pharm Biotechnol 2015; 16(9):771-781) (Lee V C. P&T 2017; 42(6):375-383). Although chemotherapeutic drugs and radiotherapy do not have a direct stimulatory effect on cellular immunity, today it is known that some of these approaches can increase it indirectly, by inducing immunogenic cell death in the tumor cell. In the last years, the number of drugs that induce immunogenic cell death has been increased. Those include: anthracyclines (doxorubicin (DOX), epirubicin, idarubicin), oxaliplatin, cyclophosphamide, bortezomib, mitoxantrone and bleomycin (Garg A D and cols. Oncoimmunology 2017; 6(12): e1386829). Additionally, physical therapeutic methods such as radiotherapy, hypericin-based photodynamic therapy and high hydrostatic pressures, as well as some oncolytic viruses and microtubular inhibitors have been also shown to induce this type of cell death (Li X. Tumori Journal 2018; 104(1):1-8).
In the past, necrosis was the only type of cell death considered immunogenic, producing unwanted inflammatory reactions, due to rapid release of various intracellular factors, cytokines and other inflammatory mediators (Rock K L and Kono H. Annu Rev Pathol 2008; 3:99-126). Conversely, apoptosis was considered a physiological process of cell death that is mostly tolerogenic or silent from immunological point of view (Matzinger P. Science 2002; 296:301-305). During late-phase apoptosis, cells are rapidly and specifically recognized by macrophages and other phagocytic cells, due to 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 concomitant suppression of “don't eat me” signals, such as CD47 expression (Liu X and cols. Journal of Hematology & Oncology 2017; 10:12). The latter is a molecule that is expressed on tumor cells and has been considered as an immune checkpoint for tumor evasion, as it constitutes an antiphagocytic signal for macrophages and dendritic cells (Tong B and Wang M. Future Oncol. 2018; 14(21):2179-2188) (Weiskopf K and cols. Science 2013; 341(6141):88-91).
Compared to classic tolerogenic apoptosis, immunogenic cell death activates the host's immune system and increases the immune response to immunotherapy, such as the response to dendritic cells-based cancer vaccines (Vandenberk L and cols. Front Immunol 2016; 6:663). In fact, tumor cells in process of immunogenic cell death, induced by drugs in vitro, are capable to induce an anticancer vaccine effect when they are 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 in the initiation of an effective antitumor immune response (Ma Y and cols. Immunity 2013; 38:729-41). One of fundamental characteristics of damaged and dying cells is the exposition on membrane or secretion of molecules normally hidden in living and healthy cells, which acquire immunostimulatory properties. These molecules belong to well-known damage associated molecular pattern and could 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 inability of anticancer drugs to produce this type of death at concentration that are lower than maximum tolerated dose in vivo (Montico B and cols. Int J Mol Sci. 2018; 19:594-609). This obstacle has been overcome with ex vivo treatment of tumor cell from patient, with high doses of drug that induce of immunogenic apoptosis, for generating autologous dendritic cells for vaccination purposes (Chen H M and cols. Cancer Immunol Immunother. 2012; 61:1989-2002) (Montico B and cols. Oncoimmunology. 2017; 6:e1356964). However, surgical resection of patient tumor is not always possible to achieve ex vivo procedure that allows obtaining autologous dendritic cells, especially in advanced stages of disease, or due to tumor anatomical location.
Therefore, at present, a major limitation of using immunogenic apoptosis inducers as adjuvant to dendritic cells-mediated vaccines consists of impossibility of using such drugs in vivo without the previous ex vivo activation procedure.
The present invention solves the aforementioned problem by providing the use of the peptide identified as SEQ ID NO: 1 to manufacture a medicine for inducing antitumor and antiviral immunity by increasing immunogenic cell death. The peptide identified as SEQ ID NO: 1 is a proapoptotic peptide, comprising the synthetic peptide initially called P15 fused to the cell-penetrating peptide Tat (amino acids 48-60), to facilitate internalization into cells (Perea S E and cols. Cáncer res. 2004; 64: 7127-7129). Unexpectedly, it is shown that this peptide is able to induce antitumor immunity activation, by activating “eat me” signals and reducing “don't eat me” signals. In particular, the invention demonstrates that there is activation of antitumor adaptive immune response, where there is maturation and activation of dendritic cells and cellular immune response mediated by CD8+ cytotoxic T lymphocytes. The effect described for the peptide identified as SEQ ID NO: 1 in the present invention is not observed for other inhibitors of CK2-mediated phosphorylation, such as the chemical compound CX4945, which directly block catalytic subunit of CK2 protein kinase. This effect is neither observed when cell-penetrating peptide Tat is employed, which has been evaluated as a negative control.
In one embodiment of the invention, the peptide identified as SEQ ID NO: 1 is used for the manufacture of a drug 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 in culture of tumor cells with the peptide identified as SEQ ID NO: 1. Ex vivo treatment refers to in vitro co-incubation of tumor cells from patients treated with the peptide identified as SEQ ID NO: 1 in the laboratory, together with dendritic cells from de same patients. In relation to in vivo treatment, an increase in “eat me” signals and a decrease in “don't eat me” signals are observed in the tumor, with subsequent activation of cellular immune response, both at systemic and intratumoral level.
In the invention, it is shown for first time that the peptide identified as SEQ ID NO: 1 is capable of inducing immunogenic cell death of tumor cells, which administered in a vaccination schedule in mice induces in vivo immune response that protects against further challenge with live tumor cells, impairing tumor debut and growth.
In an embodiment of the invention, the induction of antitumor immunity by the use of the peptide identified as SEQ ID NO: 1 comprises the enhancement of the NK or cytotoxic T cell activity at tumor site. The use of the peptide identified as SEQ ID NO: 1 allows increasing the activity of cytotoxic T cells through adequate maturation and activation of dendritic cells. The peptide identified as SEQ ID NO: 1 is capable of increasing the secretion of HMGB-1 and adenosine triphosphate (ATP) by tumor cells after treatment, which act as chemoattractant for antigen presenting cells, favoring their recruitment, maturation and activation at the tumor site. This make the peptide identified as SEQ ID NO: 1 a suitable compound to enhance the effect of vaccines based on dendritic cells or to contribute to in situ vaccination with these cells.
Contrary to other antitumor agents that induce immunogenic cell death, which only achieve activation of dendritic cells when they are incubated ex vivo with tumor cells, the use of the peptide identified as SEQ ID NO: 1 solves said limitation by inducing in vivo activation of dendritic cells, without need to isolate cells from the patients to do the procedure. This effect is observed when the peptide identified as SEQ ID NO: 1 is administered both intratumorally and systemically, which makes it an effective treatment for both leukemias and solid tumors, including those hard to access.
In a materialization of the invention, the induction of antitumor immunity by the used of the peptide identified as SEQ ID NO: 1 comprises the reversion of immunosuppressive to immunostimulatory tumor microenvironment. This synthetic peptide enhances the recruitment of immune cells capable of orchestrating a specific antitumor response and decreasing the present of regulatory T cells.
In a realization of the invention, the peptide achieves the induction of antitumor and antiviral immunity by increasing pro-inflammatory cytokines such as TNF-α, IL-6 and IL-12, capable of increasing the expression of MHC-I on antigen-presenting cells, and promote T cells differentiation and NK cells activation. The increase in pro-inflammatory cytokines is accompanied by the decrease in anti-inflammatory cytokines, such as IL-10 which limits the immune response generated and contributes to tumor progression or viral infection.
In a particular embodiment, the induction of antiviral immunity comprises the enhancement of specific humoral immune response against viral antigens, as demonstrated for the first time in the present invention. The ability of the peptide identified as SEQ ID NO: 1 to enhance the immune response makes it an ideal candidate to treat infectious diseases, particularly during viral infections.
The invention also provides a method for treating cancer or viral infection by using therapeutically effective amounts of a drug that induces antitumor and antiviral immunity by immunogenic cell death where the drug comprises the synthetic peptide of the invention SEQ ID NO: 1.
The method of the present invention permits to decrease “don't eat me” signals like CD47 expression, which, together with the increase in “eat me” signals, favor phagocytosis of tumor cells by dendritic cells and M1 macrophages. This M1 macrophage function is relevant to their antiangiogenic activity that finally inhibits tumor progression. Therefore, the effect of the peptide of the invention eliminates the need to use additional CD47 inhibitors for enhancing the antitumor effect of certain drugs and reduces the number of compounds required to achieve an effective antitumor response. Thus, in one embodiment of the method of the invention, the induction of antitumor and antiviral immunity comprises the activation and differentiation of dendritic cells in vivo and in vitro.
In another aspect, the use of the peptide identified as SEQ ID NO: 1 increase “eat me” signals such as CRT. This 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 favors the diversification of peptide-induced response and increases the antitumor efficiency. Therefore, in a materialization of the method of the invention, the induction of antitumor immunity comprises the enhancement of NK and cytotoxic T cells activity at tumor site, as well as the reversion of immunosuppressive to immunostimulatory tumor microenvironment, and increased secretion of pro-inflammatory cytokines. In another embodiment of the method of the invention, the induction of antiviral immunity comprises the enhancement of a specific humoral immune response against viral antigens.
In one embodiment of the 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 in the course of the same 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 (TILs).
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 that, the peptide can be administered prior to vaccination by intratumoral or systemic route, which allows it to be used for both leukemias and solid tumor. It is necessary to highlight that the adjuvant effect was only observed for the peptide identified as SEQ ID NO: 1 and not for another inhibitor of phosphorylation mediated by CK2, like CX4945.
Another object of the present invention is a pharmaceutical combination that comprises the peptide identified as SEQ ID NO: 1 and a vaccine for cancer immunotherapy. In a materialization of the invention, the vaccine used in cancer immunotherapy is a vaccine based on dendritic cells, cytotoxic T cells or TILs.
The ability to increase tumor infiltration of cytotoxic T lymphocytes, and decrease the presence of regulatory cells, makes the peptide identified as SEQ ID NO: 1 a suitable compound enhancing the effect of vaccines based on cytotoxic T lymphocytes (CTL) and TILs, thus leading to a more effective immunotherapeutic approach to treat cancer.
In one embodiment of the invention, the drug comprising the peptide identified as SEQ ID NO: 1 enhances the effect of a PDL1 immune checkpoint inhibitor used in cancer immunotherapy. In a materialization of the invention, the peptide identified as SEQ ID NO: 1 and the PDL1 immune checkpoint inhibitor are administered sequentially or simultaneously in the course of the same treatment. In a particular embodiment, the PDL1 immune checkpoint inhibitor is an anti-PDL1 monoclonal antibody.
In another aspect, the invention discloses a pharmaceutical combination comprising the peptide identified as SEQ ID NO: 1 and a PDL1 immune checkpoint inhibitor used in cancer immunotherapy. In an embodiment of the invention, in the pharmaceutical combination, the PDL1 immune checkpoint inhibitor is an anti-PDL1 monoclonal antibody.
For instance, in patients with non-small cell lung cancer lacking actionable mutations, the most effective therapy is PDL1 immune checkpoint inhibitors. However, its efficacy depends on the level of expression of this molecule in the tumor. The use of the peptide identified as SEQ ID NO: 1 in combination with a PDL1 inhibitor solves the problem by increasing PDL1 expression and therefore increasing the clinical efficacy of the PDL1 inhibitor. This combination offers a novel therapeutic strategy for cancer treatment.
In order to corroborate whether the peptide identified as SEQ ID NO: 1 was capable of inducing cell death in tumor cell lines, mouse lymphocytic leukemia cells L1210 were treated with 10 μM peptide identified as SEQ ID NO: 1 for 20 minutes at 4° C. and 37° C. After that time, cells were labeled with Annexin V-FITC, which bind to phosphatidylserine residues exposed on the outer membrane of apoptotic cells, and 7-AAD, which only penetrates into dead cells. Finally, samples were analyzed by flow cytometry.
Treatment with the peptide identified as SEQ ID NO: 1 at 37° C. elicited an increase of up to 15 folds the percentage of apoptotic cells (Annexin V+/7-AAD−). This increase was not detected when the cells were incubated at 4° C., which could indicate the induction of apoptosis, since this is an energy-dependent process (
In ex vivo evaluations carried out with samples from patients with acute myeloid leukemia, induction of cell death was also observed when cells were treated with 40 μM of peptide identified as SEQ ID NO: 1 during 24 hours. This phenomenon was observed in 6 out of 10 patients evaluated (
CRT exposure at the tumor cell membrane constitutes a phagocytic signal (“eat me” signal) for dendritic cells, which also occurs accompanied by Erp57 expression. Taking this into account, we decide to evaluate whether the peptide identified as SEQ ID NO: 1 was capable of causing the translocation of these molecules in L1210 cells. For this purpose, 2.5×105 cells were seeded in 12-wells plate and treated with 40 μM peptide identified as SEQ ID NO: 1 for 3 and 5 hours. Subsequently, the cells were collected, washed twice with cold PBS and fixed with 0.2% paraformaldehyde for 5 minutes at 4° C. After Fc receptor blocking (with PBS containing 2% fetal bovine serum (FBS) for 30 minutes at 4° C.), the cells were labeled with the primary antibodies for 30 minutes at 4° C., followed by two washes and incubation with a secondary monoclonal antibody conjugated to Alexa 488 in blocking solution for 30 minutes at 4° C. After repeating washes, the cells were analyzed by Flow Cytometry. In addition, the effect of the CK2 inhibitor called CX4945 was also evaluated after treating with 5 μM for 24 hours. Cells without treatment constituted the negative control of the test.
Treatment with the peptide identified as SEQ ID NO: 1 at the two times evaluated did induce CRT and Erp57 translocation at the cell surface in L1210 cells (
Recently, a strong association has been reported between anti-phagocytic signals, also known as “don't eat me” signals, with tumor growth and progression. In this sense, we evaluated whether the peptide identified as SEQ ID NO: 1 had any effect on these molecules, specifically on CD47. To do that, two leukemia cell lines were employed: 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 8 experimental groups in both cell lines, treated with 40 μM of peptide identified as SEQ ID NO: 1 for 30 minutes and 3 hours, with untreated cell controls at each times. Three experimental replicates were used per group in 12-well plates, for a total of 24 independent samples as shown in Table 1.
After 30 minutes and 3 hours of incubation, culture medium was removed, cells were washed once with PBS buffer and each well was collected in Lysis Buffer (RNeasy Plus Minikit, Qiagen, EU) with 1% β-mercaptoethanol and homogenized. Purification proceeded according to manufacture 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 validated by qPCR. Oligonucleotides used are shown in Table 2. GAPDH, DDX5 and ABL1 were employed as reference genes. Reactions were carried out on LightCycler®480II equipment (Roche, Germany) in 96-well plates in SYBR Green Probe II mode.
Fold change of each gene was determined with respect to untreated condition, normalized with the three reference genes GAPDH, DDX5 and ABL1. As shown in
During the immunogenic cell death, expression and release of certain danger signals involved in the activation of an effective antitumor immune response is induced. Taken this into account, we 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 murine tumor cell lines and in serum from patients treated with the peptide.
For ATP, HSP70 and HMGB-1 in vitro detection, 6×104 cells were seeded in 24-well plates and treated with: 40 μM peptide identified as SEQ ID NO: 1, 40 μM Tat both for 5 and 24 hours, and 5 μM CX4945 for 24 hours. Cells treated with 5 μM DOX for 24 hours as positive control. Untreated cells and serum from patients taken at zero time constituted the negative controls. ATP secretion was measured by luciferin-based ENLITEN ATP Assay (Promega), HSP70 were stained with an 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 manufacturer's instructions.
Treatment with the peptide identified as SEQ ID NO: 1 for 5 and 24 hours elicited HMGB-1 release to culture medium. Levels detected were statistically higher than those found in untreated cells or treated with either Tat peptide or the CK2 inhibitor, CX4945 (
ATP levels also increased significantly in supernatants of tumor cell cultures after treating with the peptide identified as SEQ ID NO: 1 for 5 and 24 hours, compared to untreated cells and those treated with CX4945 (
To evaluate whether tumor cells treated with the peptide identified as SEQ ID NO: 1 are capable of stimulating phagocytosis and maturation of dendritic cells, L1210 and 3LL cells were treated with 40 μM and 130 μM, respectively, of the peptide identified as SEQ ID NO: 1 in T25 flasks, for 3 hours. For the phagocytosis experiment, tumor cells were previously stained with 1 μM of Carboxyfluorescein succinimidyl ester (CFSE). After treatment, tumor cells were washed and seeded in U-bottom 96-well plates, 1:2 ration with respect to dendritic cells, for 48 hours. Dendritic cells were differentiated using FLT3, from bone marrow precursors of DBA/2 mice, for co-culture with L1210 cells, and C57/BL6 mice for the case of the experiment with 3LL cells. The co-culture was labeled with anti-CD11c antibody, for the case of phagocytosis experiments, or with anti-CD11c, anti-CD86, anti-MHCII and anti-CD40 antibodies for dendritic cells maturation experiments. Untreated cells constituted the negative control of experiment. DOX-treated cells were used as positive control. The effect of Tat peptide and the CK2 inhibitor called CX4945 on the stimulation of phagocytosis was also evaluated.
Additionally, the ability of activating CD8+ T cells in vitro by these dendritic cells matured with peptide-treated 3LL cells was evaluated. For this purpose, dendritic cells previously co-incubated with tumor cells, under the conditions described above, were washed and loaded with the SIINFELK peptide from OVA, MHC class I restricted, at different concentrations (0, 10, 30 and 100 pg/mL) during three hours. After that time, they were washed again and co-incubated with RagOT-I cells in a 1:1 ratio for 48 hours. IFN-γ production was finally quantified by ELISA. RagOT-I cells co-incubated with dendritic cells previously incubated with untreated 3LL cells constituted the negative control of experiment.
As shown in
Dendritic cells matured with peptide- and DOX-treated 3LL cells, were capable to activate CD8+ OT-I cells as determined by an increase in IFN-γ production, which was significantly higher than negative control and cells treated with CX4945 and Tat peptide (
To study whether the peptide identified as SEQ ID NO: 1 was capable of modulating the tumor microenvironment, female DBA/2 mice, 6-8 weeks old, and between 17-19 grams were inoculated with 0.5×106 L1210 cells, subcutaneously, into right flank (inguinal inoculation). Seven days after tumor challenge, 42 mice with palpable and non-measurable tumor were selected, which were random assigned to six groups of seven animals each. The mice received the treatment as shown in Table 3. Treatment with the 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, with 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, a daily inoculation for five days. The compound CX4945 (75 mg/kg) was administered in 25 mM NaH2PO4 buffer, twice a day, orally, for five days. Seven days after the end of the treatment, the mice were euthanized to obtain the tumors (day 18 of the procedure). Before sacrificing the mice, blood samples were obtained for the cytokines (IL-6, IL-10, IL-12p70, TNF-α) detection by ELISA. Tumors were cut into small pieces and transferred to gentleMACS tubes with RPMI medium, supplemented with an enzyme cocktail from Tumor Dissociation kit (Miltenyi Biotech, Germany). Tumor dissociation was performed using the gentleMACS™ Dissociator kit and a 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.
Treatment with the peptide identified as SEQ ID NO: 1 administered both intratumoral and systemically, induced a significant increase of IL12p70, TNF-α and IL-6 levels in peripheral blood, compared to the rest of the groups. Otherwise, peptide treatment significantly reduced IL-10 levels. This modulation in cytokine levels was not observed when mice were treated with Tat, DOX or CX4945 (
Similarly, treatment with the peptide identified as SEQ ID NO: 1, by the two administration routes caused a significant increase in the intratumoral levels of CD8+ and CD4+ T cells, as well as in situ maturation of dendritic cells, as determined by increase of CD86 and MHC-II maturation markers. Additionally, a significant decrease in intratumoral Foxp3+ T cell levels was observed after treatment with the peptide identified as SEQ ID NO: 1 and DOX, obtaining a greater reduction after application of the peptide. This effect was not observed when mice were treated with Tat or CX4945 (
In order to explore whether the cell death induced by the peptide identified as SEQ ID NO: 1 had any immunomodulatory effect, a vaccination schedule was performed in mice with tumor cells previously treated with the peptide identified as SEQ ID NO: 1. For this purpose, 1×106 L1210 cells treated in vitro with 40 μM of the peptide identified as SEQ ID NO: 1, or time-matched untreated cells during 3 hours were subcutaneously injected in 0.1 mL in the inguinal area into right flank of 6-8 weeks female DBA/2 mice. After seven days, 1×106 live L1210 cells were inoculated on opposite flank, and tumor growth and volume were evaluated until day 45. Tumors were measured with Vernier calipers, three times a week, tumor volume was estimated using the formula: Volume=width2 (mm)×length/2 (mm). As a positive control, cells treated with 15 μM DOX for 24 hours were used for vaccination. A group vaccinated with 5 μM CX4945-treated cells for 24 hours was also included. In total 4 groups were formed of 10 mice each group.
As showed in
The induction of cellular immune response by tumor cells treated with the peptide identified as SEQ ID NO: 1 was evaluated. For this purpose, 1×106 3LL-OVA cells treated or untreated in vitro with one of following variants: a) 40 μM of the peptide identified as SEQ ID NO: 1 for three hours, b) 15 μM DOX for 24 hours, c) 5 μM CX4945 and d) 40 μM Tat for three hours, were inoculated subcutaneously, in inguinal area of the right leg of 6-8 weeks female C57/BL6 mice in a volume of 0.1 mL. After 5 days, five mice from each group were euthanized to get lymphoid nodules which were subsequently macerated to acquire the cells.
IFN-γ secretion was evaluated by enzyme linked immunospot assay (ELISPOT), following the procedure described below. 96-well microplates with nitrocellulose membranes (Millipore, Bedfors, MA, USA) were coated with 100 μL/well of murine anti-IFN-γ monoclonal antibody (BD Biosciences, Canada) at a concentration of 5 μg/mL in PBS, for 16 hours at 4° C. After three washes with PBS the plates were blocked with RPMI medium supplemented with 10% FBS, for 1 hour at 37° C. in a humid atmosphere with 5% CO2. A total of 2×105 cells/well were stimulated, in duplicate, with OVA protein and SIINBELF peptide from OVA, at a final concentration of 6 μg/mL, in a final volume of 0.2 mL. Concanavalin A at 5 μg/mL, in RPMI medium with 10% FBS, was used as positive control. The plates were incubated for 48 hours at 37° C., in humid atmosphere of 5% CO2. After incubation, a standard ELISPOT assay was performed (Vázquez-Blomquist D et. al. (2002) Viral Immunol. 5(2):337-356). Counting of spots was carried out in automatic counter (Immunospot Analyzer, Cellular Technology Ltd., Shaker Heights, OH, USA). Cells incubated with RPMI medium with 10% FBS were taken as negative controls.
Results were considered positive when the number of spots was at least three times higher than the average number of spot in the negative control group and there were at least three specific spots (value obtained after subtracting the number of spots from an individual animal in a stimulation condition minus the number of spots of the same in the non-stimulation condition) per 106 cells.
As shown in
The release of danger signals such as HSP70 and CRT has been reported to be involved in increasing the susceptibility of tumor cells toward cytotoxic activity of NK cells. Therefore, the effect of the peptide identified as SEQ ID NO: 1 on the susceptibility of tumor cells toward NK cytotoxic activity was evaluated. To do that, the classic target cells to NK activity, K562 cells, were employed. Target cells were labeled with Cr51 (approximate specific activity: 100 μCi/μg) by incubation for 70 minutes at 37° C. Subsequently, target cells were washed three times with RPMI medium supplemented with 2% FBS and seeded in 96-well plates, at a target:effector ratio of 1:25. On the other hand, NK cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors, by negative selection, using magnetic beads (Miltenyi Biotec). Thus, CD56+CD3− NK cells were obtained with more than 96% purity. 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, supernatant was collected for the measurement of Cr51 release using a scintillation counter. The percentage of specific lysis was calculated as follows:
Target cells without coincubation with effector cells were employed as spontaneous lysis control. Target cells with the peptide identified as SEQ ID NO: 1 constitute the toxicity control of the peptide.
As shown in
Treatment with the peptide identified as SEQ ID NO: 1, as described in examples 2 and 3, is capable of increasing “eat me” signals and decreasing “don't eat me” signals. Therefore, it fostered to evaluate whether the treatment with the peptide identified as SEQ ID NO: 1 facilitate phagocytosis of tumor cells by macrophages, and whether this is performed by M1 or M2 macrophages. For that purpose, HL-60 and OCI-AML3 leukemia tumor cell lines were previously stained with 0.5 μM CFSE and treated with 10 or 40 μM of the peptide identified as SEQ ID NO: 1, for five hours, 5 μM CX4945 for 24 hours or 40 μM Tat during 5 hours. Subsequently, cells were washed and co-cultured with each type of M1 and M2 macrophages, at a 1:1 ratio for three hours. Different types of macrophages were obtained from PBMC of healthy donors by Ficoll™ gradient. Monocytes were purified by positive selection, using CD14 beads with an MS column (Miltenyi Biotech, Germany), obtaining 96% purity. Monocytes were seeded at 0.25×106 cells/mL/well, in 12-wells plates and were induced with 100 ng/mL of GM-CSF, for seven days, and with 50 ng/mL of IFN-γ for last 24 hours, to obtain M1 macrophages. M2 macrophages were induced with 50 ng/mL of M-CSF for seven days and 100 ng/mL of IL-10 for last 24 hours. Subsequently, supernatants were collected and cells were stained with anti-CD33 (to determine corresponding macrophages population), anti-CD86 (to determine M1 population) and anti-CD163 (to determine M2 population).
Pretreatment of OCI-AML3 and HL-60 tumor cells with the peptide identified as SEQ ID NO: 1, both at IC50 dose (40 μM) and suboptimal dose (10 μM), increased their susceptibility to be phagocytosed by M1 macrophages and not M2 macrophages (
Taken into account the capacity of the peptide identified as SEQ ID NO: 1 for inducing immunogenic cell death, the antitumor response generated by the peptide identified as SEQ ID NO: 1 combined with the CTL vaccine candidate CIGB550-E7+VSSPs (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 a Human Papillomavirus (HPV)-E7 mutein to the cell-penetrating peptide CIGB550, formulated with synthetic NAcGM/VSSP adjuvant.
For that purpose, 6-8 weeks old C57/BL6 female mice were subcutaneously injected with 5×104 TC-1 cells into right flank (inguinal inoculation). Seven days after tumor challenge, 48 mice with palpable and non-measurable tumor were randomly divided into six groups with 10 mice per group. Mice received different treatment as shown in Table 4. Treatment with the peptide identified as SEQ ID NO: 1 (at 40 mg/kg) was administered intratumoral and intravenously, in a final volume of 50 μL and 100 μL, respectively. CX4945 (at 75 mg/kg) was administrated in 25 mM NaH2PO4, twice daily orally. The vaccine candidate CIGB550-E7+VSSPs was injected subcutaneously into right flank in a final volume of 0.2 mL. Tumor growth was followed up and tumor volume was determined and calculated as described in Example 7.
Results obtained showed that the peptide identified as SEQ ID NO: 1, injected both intratumoral and systemically prior to administration of vaccine, is capable of enhancing antitumor effect of CIGB550-E7+VSSPs vaccine candidate, as determined by tumor growth control and mice survival (
Taken into account the ability of the peptide identified as SEQ ID NO: 1 to induce immunogenic cell death, it was decided to evaluate its capacity to enhance a B cells immune response. For this, 30 female BALB/c mice, eight weeks old and weighing 18-20 grams were divided into three groups of 10 mice per group. The first group was immunized with 10 μg OVA protein formulated in aluminum phosphate. The second group received 10 μg OVA protein and 100 μg of the peptide identified as SEQ ID NO: 1. The third group constituted the control group of the study that was inoculated only with Aluminum Phosphate. The OVA administration was made on days 0, 14 and 28, subcutaneously, in a final volume of 100 μL. On days 21 and 42 blood samples were obtained to evaluate the induction of IgG response against OVA protein.
As shown in
Taking into account the results observed in OVA protein formulated with the peptide identified as SEQ ID NO: 1, the ability of this peptide to enhance specific antibody response against viral antigens during an in vivo infection, was evaluated. For that purpose, 14 female BALB/c mice, 8 weeks old and weighing 18-20 grams, were challenged with a recombinant vaccinia virus that compresses the structural Hepatitis C virus (HCV) protein (Alvarez-Lajonchere et al. Biotecnología Aplicada 2007; 24(3)). The virus was inoculated intraperitoneally at a dose of 106 plaque-forming units (pfu) in 200 μL of PBS. The following day mice were divided into two groups of seven mice per group. The mice of the first group were treated with 3 mg/kg of the 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. On sixth day after the viral challenge a total blood extraction was carried out to evaluate humoral immune response against HCV structural proteins.
The 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. Antibody titers were significantly higher than those of the control group (
Induction of Antibodies in Patients Infected by Anti-SARS-CoV-2 Treated with the Peptide Identified as SEQ ID NO: 1
A randomized phase I/II clinical trial in 20 SARS-CoV-2 infected patients treated with the peptide identified as SEQ ID NO: 1, was performed. Ten patients were treated intravenously with 2.5 mg of the peptide per kg of body weight, once a day, for five consecutive days along with the standard therapy consisting of Kaletra™, Chloroquine and IFN-α2b. On the other hand, control group enrolled 10 patients that only received the standard therapy. Specific anti-NP and anti-RBD IgG levels were determined by using an in home ELISA.
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Currently, one of the potentialities in cancer immunotherapy is constituted by dendritic cell-based vaccines. Taking this into account, it was explored whether the peptide identified as SEQ ID NO: 1 was capable of enhancing the effect of a dendritic cell vaccine. For that purpose, C57/BL6 mice were euthanized to obtain spleens, which were macerated and digested with collagenase and DNAse. Low density mononuclear cells were separated using a gradient medium density. Dendritic cells were purified by murine Pan-DC magnetics beads mediated cell drawing (Miltenyi Biotech, Bergisch-Gladbach, Germany), according to manufacturer's recommendations. Dendritic cells were obtained with more than 90% purity as determined by cytometric analyzed 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 hours at 37° C. Non-adherent dendritic cells, loaded with OVA protein (DC+OVA) were collected for inoculation into the animals. Dendritic cells alone were used as negative control.
To evaluate whether the peptide identified as SEQ ID NO: 1 is capable to enhance the effect of DC+OVA in a murine model, 5×105 3LL-OVA cells were inoculated into 6-8 week C57/BL6 female mice with 17-19 grams of body weight. These 3LL-OVA cells were subcutaneously injected, into right flank, in 50 μL final volume. Seven days after tumor challenge, 42 mice with palpable and non-measurable tumors were selected, which were randomly divided into six groups with seven mice per group. Animals received different treatments as shown in Table 5. Treatment with the peptide identified as SEQ ID NO: 1 (40 mg/kg) was intratumorally applied in 50 μL of final volume. CX4945 (75 mg/kg) was orally administered in 25 mM NaH2PO4 buffer twice daily. The administration of 5×105 dendritic cells loaded or unloaded with OVA protein was carried out intratumorally, in 50 μL of final volume. Tumor growth was followed up during time and tumor volume determined and calculated as described in Example 7. The survival of animals was recorded until the tumor volume reached 4000 mm3, time at which animals were euthanized by cervical dislocation. Throughout the experimentation, mice were kept in a pathogen-free environment and the procedures were conducted according to institutional guidelines.
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The used of TILs for cancer treatment is one of the novel immunotherapeutic strategies currently in application. According to previously obtained results (Example 6), the 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 capable of enhancing the antitumor effect of TILs in a cancer murine model. For that purpose, 6-8 weeks old DBA/2 female mice with 17-19 grams of body weight were subcutaneously inoculated with 5×104 L1210 cells in right flank (inguinal administration). Seven days after tumor challenge, 42 animals with palpable and non-measurable tumors were randomized into six groups with seven mice per group. The different treatments were administrated as shown in Table 3, Example 6. Seven days after the end of the treatment, mice were euthanized to obtain the tumors. They were cut into small pieces with an approximate size of 1-3 mm3 and seeded in 24-well plates in RPMI medium supplemented with 10% FBS and 2000 IU/mL recombinant murine IL-2. The cultures were expanded in 12-well plates. TILs from fragments of the same tumor were joined and counted. 5×106 TILs were intravenously inoculated into other DBA/2 mice that were subsequently challenged with 5×104 L1210 cells at 24 hours. Tumor growth was followed up during the time and tumor volume was measured and calculated as described in Example 7.
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PDL1 Increase in H460 and A549 Cells After the In Vitro Treatment with the Peptide Identified as SEQ ID NO: 1
Immune checkpoints are currently an important target in cancer immunotherapy, due to their immunosuppressive role during the course of this disease. Taking this into account, effect of the peptide identified as SEQ ID NO: 1 on modulation of PDL1 levels in two non-small cell lung cancer cell lines: A549 and H549, was evaluated. For that, 5×104 cells were seeded in 24-well plates. The day after, cells were treated with 30 μM peptide identified as SEQ ID NO: 1; 5 μM CX4945 or 30 μM Tat peptide, for 24 hours. After treatment, cells were collected, staining with an anti-PDL1 antibody and analyzed by flow cytometry.
Table 6 shows percentage of PDL1 positive cells and mean fluorescence intensity (MFI). Treatment with the peptide identified as SEQ ID NO: 1 elicited a significant increase of PDL1 expression in both cell lines. Differences were not observed when cells were treated with CX4945 or Tat peptide.
These results suggest that is possible to use the combination of the peptide identified as SEQ ID NO: 1 with PDL1 immune checkpoint inhibitors.
Taking into account the results obtained in the in vitro test, it was decided to evaluate whether the use of the peptide identified as SEQ ID NO: 1 improved the antitumor efficacy of anti-PDL1 therapy in a murine model of lung cancer. For that, 2×105 Lewis Lung Carcinoma cells were subcutaneously implanted (day 0) in 6-8 weeks old C57/BL6 female mice with 17-19 grams of body weight. Nine days after the tumor challenge, 42 animals with 40 mm3 tumors were randomized into six groups with seven mice each. The different treatments were administrated as shown in Table 7. CX4945 (75 mg/kg) was orally administered in 25 mM NaH2PO4 buffer twice daily. 40 mg/kg of the peptide identified as SEQ ID NO: 1 and 100 μg anti-PDL1 antibody (clone 10F.9G2, isotype rat IgG2b kappa) were intraperitoneally applied. Tumor volume and body weight were determined 3 times a week for 40 days. Tumor volume was measured and calculated as described in Example 7.
As shown in
These results indicate that the peptide identified as SEQ ID NO: 1, by inducing PDL1 expression in tumor cells, increases the probability of blocking an anti-PDL1 therapy and thus enhances the induction of a cytotoxic immune response against the tumor.
A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via Patent Center encoded as XML in UTF-8 text. The electronic document, created on Jun. 16, 2023, is entitled “976-119_PCTUS”, and is 1.81 KB bytes in size.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-0103 | Dec 2020 | CU | national |
This application is the U.S. National Phase of International Patent Application Number PCT/CU2021/050013, filed 23 Nov. 2021, which claims priority from CU 2020-0103, filed 18 Dec. 2020, each of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CU2021/050013 | 11/23/2021 | WO |
