Methods and compositions for treating cancer

Abstract

To provide a novel therapeutic strategy for treating cancers expressing embryonic antigens.SOLUTION: The inventors have developed a metastatic 4T1 breast tumor model in BALB / c mice. The inventors have showed that vaccination with xenogeneic embryonic stem cells in combination with valproic acid (VPA) generates a higher antitumor response against a breast cancer and inhibits metastasis development. The inventors have demonstrated that these responses are only achieved by the addition of valproic acid in a therapeutic regimen compared to the use of ESC or iPSC alone. Thus, the inventors provide a novel therapeutic strategy for treating cancers expressing embryonic antigens. The present invention therefore relates to a method for treating a subject suffering from cancer, comprising the steps of administering to the subject simultaneously, separately or sequentially a therapeutic amount of i) a population of pluripotent cells and ii) a compound selected from the group that activates MHC expression as a combined preparation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to the field of oncology, and more specifically, to combination therapy with anti-cancer vaccines.

[0002] In particular, the present invention relates to a method for producing a composition comprising pluripotent cells that present multiple neoantigens, and such compositions are useful in preparing cancer cell vaccines.

[0003] Background of the invention:

[0004] The majority of cancers result from random mutations that occur during DNA replication in normal stem cells required for development and tissue maintenance. Cancer stem cells (CSCs) are heterogeneous and epigenetically plastic in a dynamic state. Tumor cells arising from CSCs are driven by the simultaneous accumulation of mutations in oncogenes, tumor suppressor genes, and signaling pathways, leading to a clonal wave of tumor evolution. In the "clonal evolution model," the type of mutation changes as cancer develops, so individual cancer cells gradually become transformed and invasive. Mutations acquired in early tumorigenesis are maintained in advanced disease.

[0005] Over time, these mutations, in conjunction with gradual immunoediting and attrition, lead to oligoclonal tumor expansion and cancer cell resistance. Mutational signatures in cancer are highly associated with genomic instability. Mutations generate novel non-self neoantigens with immunogenic epitopes. However, because the tumor microenvironment is generally immunosuppressive, the host immune system is generally unable to adequately destroy these cells and fight these cancers. Clinical benefits reported with T-cell responses using immune checkpoint inhibitors correlate well with tumor mutation rates and mutational landscapes.

[0006] Cancer stem cells (CSCs) represent a small population of self-renewing cancer cells that contribute to tumor persistence and recurrence because they are often resistant to conventional treatments. First discovered in hematopoietic malignancies, CSCs have been described in solid tumors of various origins, including breast cancer, glioblastoma, prostate cancer, colon cancer, head and neck squamous cell carcinoma, ovarian cancer, bladder cancer, lung cancer, and pancreatic cancer. CSCs are characterized by their ability to form xenograft tumors and initiate tumor formation in mice. They are also radioresistant and chemoresistant, contributing to a lack of therapeutic response in patients. The persistence of CSCs leads to tumor recurrence and / or metastasis after completion of treatment. Several studies have shown a molecular link between tumor pathogenesis and embryonic stem cell (ESC) status. CSCs express numerous embryonic antigens, which are also expressed in human embryonic stem cells (hESCs) and human inducible pluripotent stem cells (hiPSCs).

[0007] The OCT4, NANOG, and SOX2 transcription factors are the primary regulators, working together as part of a highly integrated network (related to the c-myc and Polycomb networks) to drive the transition from somatic cells to either CSCs or iPSCs using epigenetic mechanisms, and to remodel chromatin through histone modification and DNA methylation. These factors are not present in normal adult stem cells. Embryonic stem cell-like gene expression and Polycomb regulatory gene underexpression, which define the identity of human pluripotent stem cells (ESCs / IPSCs), are associated with poorly differentiated human tumors with poor clinical outcomes and distant recurrence after chemoradiotherapy, regardless of cancer origin (breast, pancreas, bladder, lung, prostate, medulloblastoma, etc.). Poorly differentiated tumors with a “stem cell-like” profile are associated with mesenchymal phenotype in cancer cells with epithelial-mesenchymal “EMT” markers, low levels of MHC-I expression, and an immunosuppressive tumor microenvironment with preneoplastic inflammatory leukocytes, stromal cells, and macrophages. Tumor cells undergoing EMT acquire stem cell-like characteristics, becoming CSCs (Cellular Stem Cells) with the ability to migrate very early throughout the organism and persist in prolonged dormancy. CSCs act as reservoirs for seeding and replenishing tumor compartments. They also proliferate through autoregeneration, propagate to different tissues, and generate metastases. These CSCs share a pluripotent embryonic genetic signature and are resistant to anticancer drugs and radiation therapy. They also evade immunosuppressive antitumor defenses for the reasons mentioned above (immunosuppressive microenvironment).

[0008] It has been reported that mice can be immunized using fetal tissue, and that this can induce rejection of transplanted tumors, including cancers of the skin, liver, and gastrointestinal tract. This response has been explained by the fact that these tumor cells express a large number of oncoemulsional antigens.

[0009] To date, several human cancer vaccine trials have been designed to target embryonic antigens, such as oncoembryonic antigens (CEAs), alpha-fetoproteins, or cancer / testicular antigens. Unfortunately, targeting a single antigen alone has been shown to be insufficiently efficient in generating a strong antitumor immune response that mediates tumor rejection, due to the rapid emergence of evasive variants, which leads to the common inefficiency of monovalent cancer vaccines.

[0010] Recent interest in the potential of stem cells in regenerative medicine has led to the widespread availability of well-defined ESC lines and iPSCs that are phenotypic and functionally similar to ESCs.

[0011] Cancer-related epigenetic abnormalities are characteristic traits of cancer stem cells, encompassing all components of the epigenetic mechanism (DNA methylation, histone modification, non-coding RNA, specifically microRNA expression).

[0012] Several epigenetic modifiers with tumor inhibitory activity are currently used clinically in oncology, including hypomethylating agents (e.g., azacitidine or decitabine) and histone deacetylase inhibitors (e.g., vorinostat or romidepsin).

[0013] It has been possible to reprogram cancer cells using such drugs. Furthermore, epigenetic reprogramming of the tumor microenvironment with epigenetic drugs is an attractive operational approach to cancer treatment, given the clear evidence of cancer-stromal interactions in cancer development.

[0014] Thus, there continues to be a need for new approaches for preventing and / or treating cancers having a stem cell signature. These cancers express a set of embryonic genes common to ESCs / IPSCs (i.e., also called neoantigens), including in particular pancreatic cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, medulloblastoma, cholangiocarcinoma, liver cancer, chronic and acute leukemia, and myeloma. Cancers of this classification are most associated with a mesenchymal-like signature for which there is a need to develop treatments that specifically target CSCs to improve the survival of cancer patients and enhance the quality of life. In particular, such strategies should lead to the restoration of a permissive anti-tumor microenvironment combined with an immune anti-CSC response (the tumor microenvironment is generally immunosuppressive and should thus be re-engineered to be immunoresponsive). This and other needs are addressed in whole or in part by the presently disclosed subject matter.

[0015] Summary of the Invention:

[0016] The present invention is based on the determination by the inventors that HDACi (histone deacetylase inhibitor) can be used to stimulate an immune response in a patient against an antigen of interest when an immunogenic composition comprising or targeting the antigen of interest is administered to the patient in combination with HDACi (optionally followed by further treatment with HDACi). The immunogenic composition is intended to enable the initiation of an immune response against the antigen of interest. The use of HDACi as an adjuvant is of particular interest for the treatment of cancers, particularly cancers having a stem cell signature. The present invention is defined particularly by the claims.

[0017] Detailed Description of the Invention

[0018] The inventors have shown that the use of HDACi together with a population of pluripotent cells leads to a synergistic effect and an efficient response of the immune system against tumor cells.

[0019] In fact, the inventors further hypothesized that pluripotent stem cells (e.g., hESCs or hiPSCs) could be used as vaccines to generate immune responses against various embryonic antigens shared by tumor cells. The inventors found that vaccination of mice with hESCs or hiPSCs in combination with compounds that can induce MHC I (e.g., valproic acid) could induce efficient immune and antitumor responses against breast cancer without side effects or signs of autoimmune disease. The inventors also found that this combination regimen was associated with significant inhibition of lung metastases. Surprisingly, the inventors demonstrated that these responses were greatly improved by the addition of HDACi, particularly valproic acid, to the treatment regimen compared to the use of ESCs or iPSCs alone.

[0020] HDACi to improve immune response

[0021] The present invention relates to a method for increasing the efficacy of a vaccine composition in a patient, comprising the step of administering HDACi to the patient together with the vaccine composition. In particular, HDACi can be added to the vaccine composition.

[0022] The increase in efficacy can be understood as an increase in the immunogenicity of the vaccine composition, an increase in the immune response to the vaccine composition, or an increase in the immune response generated by the vaccine composition. This can be compared to the immune response generated in the absence of HDACi.

[0023] The vaccine composition contains immunogenic elements intended to induce an immune response in a patient to one or more target antigens. The target antigen is any antigen for which an immune response is desired and may include any peptide, protein, or exogenous antigen from the body (e.g., antigens from cancer cells), or other types of antigens, such as proteins from bacteria, viruses, or parasites, or nucleic acids, sugars, lipopolysaccharides, etc.

[0024] The present invention thus relates to the use of HDACi as an adjuvant, particularly for increasing the immune response to a vaccine composition, and to HDACi for use as an adjuvant or for increasing the immune response to a vaccine composition. The present invention also relates to the use of HDACi for the manufacture of a vaccine composition comprising one or more target antigens, intended to induce an immune response in a patient against the target antigen.

[0025] The methods and uses disclosed herein are of particular interest when the vaccine composition is a cancer vaccine composition, i.e., when it contains an antigen of interest expressed by cancer cells. In particular, these methods and uses are well suited for solid tumor cancers. Indeed, in these types of cancers, the immune microenvironment is particularly immunosuppressive (i.e., there is the expression of cytokines and molecular signals, as well as the recruitment of such CD4 cells, which reduces the efficacy of immune cells against cancer antigens). Without being bound by this theory, it is hypothesized that the presence of HDACi modifies the microenvironment, perhaps by altering the expression of immunosuppressive genes in cells present in, near, or around the tumor, thereby enhancing immune cells and enabling them to fight cancer cells.

[0026] The methods described herein may also include a step of administering HDACi over several days following administration of the vaccine composition. This series of HDACi administrations may be useful in maintaining microenvironmental modifications for a sufficiently long period of time for immune cells to “take over” the tumor. Generally, this further series of HDACi administrations consists of daily administrations of an appropriate dose of HDACi for at least three days and up to one month after vaccine administration. It is preferable, however, if the further HDACi administration is carried out for at least one week, more preferably at least about two weeks.

[0027] The vaccine composition contains immunogenic elements (compounds) to induce an immune response in a patient to one or more target antigens.

[0028] This immunogenic element may be an antigen (or multiple antigens). This antigen may be in any form, depending on the target cell (which is intended to include host cells, as well as bacterial cells, parasitic pathogens, or viral particles), as seen above. It may also be formulated with any adjuvant (immunostimulant) known in the art, such as alum or Freund's complete or incomplete adjuvant.

[0029] In another embodiment, the immunogenic compound is an extract from a cell composition, the cells of which express the antigen of interest. The cell extract is lysed cells centrifuged to remove insoluble substances (e.g., membrane fragments, vesicles, and nuclei), and thus consists mostly of cytosol. In another embodiment, the extract may be prepared using specific techniques to deplete or concentrate specific components (e.g., using sonication to break down large membrane fragments into smaller particles remaining in the extract, or high-speed centrifugation to remove minimal insoluble components). The cell extract is obtained by any chemical or mechanical action (e.g., pressure, distillation, evaporation, etc.).

[0030] In another embodiment, the immunogenic element is a cellular composition, and the cells of the composition express the antigen of interest. In this embodiment, it is preferable to preserve the cell membrane (so that antigen presentation occurs via the MHC-1 pathway). It is also preferable to inactivate the cells, as described below.

[0031] In these embodiments, the cells may be pluripotent cells (described below), cancer cells, virus-infected cells, or bacterial cells.

[0032] In another embodiment, the immunogenic element is a cell composition comprising antigen-presenting cells (APCs) pre-stimulated in vitro with the antigen of interest. This composition is an antigen-presenting cell vaccine comprising an antigen and antigen-presenting cells (APCs). Antigen-presenting cells are cells that present an antigen complexed with major histocompatibility complex (MHC) on their surface. Dendritic cells (DCs) may be cited, and are preferred in the context of the present invention, because they can present antigens to both helper T cells and cytotoxic T cells, macrophages, or B cells. These APCs may be native or engineered cells. In particular, Eggermont et al. (Trends in Biotechnology, 2014, 32, 9, 456-465) may be cited, which outlines advances in the development of artificial antigen-presenting cells. Methods for developing anti-cancer vaccines using APCs have been widely proposed in the art and are known to those skilled in the art.

[0033] In another embodiment, the immunogenic element does not actually contain an antigen, but consists of a composition of T lymphocytes that are pre-stimulated in vitro to a target antigen, for example, by exposure to antigen-presenting cells that present the target antigen. Consequently, this composition can initiate an immune response in vivo to the target antigen. This strategy can be called “T cell adoptive transfer,” and it is known that such adoptively transferred T cells can persist in vivo for extended periods and readily migrate between lymphoid and vascular compartments (Bear et al, J Biomed Biotechnol. 2011; 2011:417403; Melief et al, J Clin Invest. 2015; 125(9):3401-3412).

[0034] In all of these embodiments, HDACi is administered in combination with a vaccine composition containing an immunogenic element. Such administration may be simultaneous, separate, or sequential, as disclosed below for embodiments in which the immunogenic element is a composition of pluripotent cells. Note that all subsequent descriptions disclosing a composition of pluripotent cells are equally applicable to any of the immunogenic element-containing vaccines disclosed above.

[0035] This specification emphasizes HDAC inhibitors (particularly valproic acid) in conjunction with pluripotent cell compositions, because such pluripotent cells express neoantigens, which, as recalled above, are also found in highly invasive cancers. Consequently, whatever the immunogenic element may be, it is preferred when the antigen of interest is a neoantigen expressed by cancer cells, as described above and also below.

[0036] In particular, the immunogenic elements are cellular compositions, which are obtained by the proliferation and inactivation of pluripotent cells, as will be disclosed in more detail below.

[0037] A method for treating cancer patients using a compound preparation.

[0038] The present invention relates to a method for treating a subject suffering from cancer, comprising the step of simultaneously, separately, or sequentially administering to the subject a therapeutic dose of a compound selected from i) a population of pluripotent cells and ii) a compound that activates MHC expression and / or an immune response, as a complex preparation.

[0039] Under favorable conditions, cells were cultured to present neoantigens via the MHC I pathway, and in particular, some cells in the population presented mutations. The compounds used in combination with the cells may also preserve the pluripotency of the cells. It is highly preferable to enhance the immune response by administering a compound that activates MHC expression and / or the immune response (preferably the same as, but potentially different from, the one initially administered in the combination) following the administration of the cells.

[0040] As used herein, the terms “treatment” or “treatment” refer to both prophylactic and preventive treatments, as well as curative or disease-modifying treatments, for subjects at high risk of having or suspected of having cancer (e.g., hereditary familial cancer syndrome), and for subjects diagnosed with the disease or suffering from cancer or a medical condition, including the suppression of clinical recurrence. Treatment is administered to subjects who have cancer or are likely to acquire cancer in order to prevent, cure, delay the onset, reduce the severity of, or improve one or more symptoms of cancer or recurrent cancer, or to extend the subject’s survival beyond what would be expected in the absence of such treatment. “Treatment regimen” means a pattern of treatment for the disease, e.g., a pattern of medication used between treatments. A treatment regimen may include an induction regimen and a maintenance regimen. The phrase “induction regimen” or “induction period” refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of the disease. The general goal of an induction regimen is to provide the subject with a high level of medication during the initial period of the treatment regimen. An induction regimen may (in part or in whole) be a “loading regimen,” which may include administering a greater amount of medication than the physician may administer during the maintenance regimen, administering medication more frequently than the physician may administer during the maintenance regimen, or both. The terms “maintenance regimen” or “maintenance period” refer to a treatment regimen (or part of a treatment regimen) used to maintain the subject between treatments for the disease, for example, to keep the subject in remission for an extended period (months or years). A maintenance regimen may use continuous treatment (e.g., administering medication at regular intervals (e.g., weekly, monthly, yearly)) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment on relapse, or treatment upon achievement of specific predetermined criteria (e.g., pain, disease signs)).

[0041] As used herein, the term "simultaneous administration" refers to the administration of two active ingredients simultaneously or substantially simultaneously via the same route. The term "separate administration" refers to the administration of two active ingredients simultaneously or substantially simultaneously via different routes. The term "sequential administration" refers to the administration of two active ingredients at different times, with the same or different routes of administration.

[0042] As used herein, the term “subject” refers to any mammal, such as rodents, cats, dogs, and non-human and human primates. In particular, in the present invention, the subject is a human being who has or is susceptible to cancer expressing pluripotent embryoid stem cell antigen.

[0043] As used herein, the term “population” refers to a population of cells in which the majority of the total number of cells (e.g., at least about 20%, preferably at least about 50%, more preferably at least about 70%, and even more preferably at least about 80%, and even more preferably at least about 90%) have the specific characteristics of the cells of interest (e.g., pluripotency markers for iPSCs, ESCs as defined by the International Stem Cell Initiative, including at least 96 markers (Adewumi et al, Nat Biotech 2007), and gene expression-based assays (PluriTest) (FJ Muller, Nature Methods 2011)).

[0044] In particular, the term "population of pluripotent cells" refers to a population of cells whose characteristic feature is the expression of pluripotency markers for iPSCs or ESCs. These cells are preferably selected from the group consisting of human embryonic stem cells (hESCs), induced human pluripotent stem cells (hiPSCs), allogeneic, xenogeneic, or syngeneic / autologous stem cells.

[0045] As used herein, the term “pluripotency” refers to a cell that, under appropriate conditions, can differentiate into all cell types derived from the three germ layers (endoderm, mesoderm, and ectoderm) and possess the ability to produce offspring, with characteristics of a particular cell lineage. The term “pluripotency” includes all sources of adult somatic cells (ASCs) and normal embryonic stem cells (ESCs) reprogrammed from cellular origin, or very small embryo-like stem cells (VSELs) or engineered inducible pluripotent stem cells (iPSCs).

[0046] Pluripotent stem cells contribute to the tissues of organisms before, after, or in adulthood. Using standard, accepted tests in the art, we establish the pluripotency of cell populations, e.g., their ability to form teratomas in 8-12 week old SCID mice, and the various characteristics of pluripotent stem cells. More specifically, human pluripotent stem cells express at least some (at least three, more commonly at least four or five), and sometimes all, of the following non-limiting list of markers: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, alkaline phosphatase (ALP), Sox2, E-cadherin, UTF-I, Oct4, Lin28, Rex1, Nanog, TERC, TERT.

[0047] Pluripotent stem cells traditionally arise from the blastocyst stage of embryonic development and have the ability to develop into all types of fetal cells and adult cells, perhaps with the exception of the placenta. Embryonic pluripotent stem cells (ESCs) can generally be isolated from 50–150 cells (post-fertilized blastocysts at 4–5 days of age). While ESCs can be proliferated indefinitely ex vivo, they exist only transiently in vivo during embryonic development. Various animal (including human) ESC lines (e.g., the NIH-approved cell line WAO9 human ESC) are commercially available from the WiCell Research Institute (Madison, Wisconsin). Human ESC lines (e.g., Cecol-14) are commercially available from, for example, Cecolfes (Bogotá, Colombia). Of course, other embryonic stem cell lines may be used if desired.

[0048] As used herein, the term “embryonic stem cells” refers to human pluripotent cells (i.e., hESCs). hESCs are isolated from pre-blastocyst stage embryos. In another embodiment, hES cells are prepared by dedifferentiation of at least partially differentiated cells (e.g., pluripotent cells) and are actually totipotent. Methods for preparing hESCs are well known and are taught, for example, in U.S. Patents 5,843,780, 6,200,806, 7,029,913, 5,453,357, 5,690,926, 6,642,048, 6,800,480, 5,166,065, 6,090,622, 6,562,619, 6,921,632, and 5,914,268, U.S. Publication No. 2005 / 0176707, and International Application No. WO2001085917. In the context of the present invention, human embryonic stem cells (hESCs) are generated without embryonic disruption according to the technique described in Chung et al 2008.

[0049] As used herein, the term “inducible pluripotent stem cell” refers to pluripotent stem cells artificially derived from non-pluripotent cells by a reprogramming procedure using a method known in the art and first disclosed by Yamanaka (in particular WO2012 / 060473, PCT / JP2006 / 324881, PCT / JP02 / 05350, US 9,499,797, US 9,637,732, US 8,158,766, US 8,129,187, US 8,058,065, US 8,278,104). In summary, somatic cells are reprogrammed into inducible pluripotent stem cells (iPSCs) by ectopic expression of defined factors (e.g., Oct4, Sox2, Klf4, and c-My, or Oct4, Sox2, Lin28, and Nanog). In certain embodiments, the inducible pluripotent stem cells are derived from mammals, particularly (but not limited to) rodents, pigs, cats, dogs, and non-human primates, as well as humans.

[0050] iPSCs have been successfully generated from somatic cells of various origins (fibroblasts, hematopoiesis, keratinocytes, etc.) using variable techniques with or without small chemical compounds (e.g., embedded lentiviruses / retroviruses and non-embedded vectors, e.g., Sendai virus, episomal vectors, synthetic mRNA, adenovirus, rAAV, recombinant proteins, etc.).

[0051] By using small molecules and acting as epigenetic modifiers (i.e., modifying the expression of certain genes), the induction and quality of mouse and human iPSCs can be enhanced. For example, BIX01294 (BIX, a G9a histone methyltransferase inhibitor), sodium butyrate (NaB, a histone deacetylase HDAC inhibitor), or S-adeno-sylhomocysteine ​​(SAH, a DNA demethylating agent), 5-azacitidine (5-AZA, a DNA methyltransferase inhibitor), and valproic acid (VPA, another histone deacetylase inhibitor) also improve the reprogramming and quality of normal iPSCs.

[0052] Fully reprogrammed true iPSCs express pluripotency genes similar to embryonic stem cells with self-renewal capabilities, representing an unlimited source of stem cells (or stem cell-like cells).

[0053] ESCs and IPSCs can be repeatedly amplified over multiple and unlimited passages, enabling an expandable stem cell supply. Pluripotency is actively maintained under tolerable culture conditions by preserving high levels of pluripotency gene expression. These methods are known in the art.

[0054] While it is possible to replicate stable genomes under specific culture conditions and methods, some exome mutations and epigenetic modifications have nevertheless been described (Gore A and al. Nature 2011).

[0055] As used herein, the term “somatic cell” refers to any cell of the body except germline cells (sperm and egg).

[0056] As used herein, the term “allogeneic cells” refers to cells from the same species, but which are genetically distinct.

[0057] As used herein, the terms “syngenetic or autologous cells” refer to cells from the same species and the same genetic background.

[0058] As used herein, the term “heterogeneous cells” refers to genetically distinct cells from different species.

[0059] In certain embodiments, stem cells may be derived from mammals (but not limited to rodents, pigs, cats, dogs, and primates (including humans)).

[0060] Method for producing a population of pluripotent cells:

[0061] In a first embodiment, the present invention is a method for producing a cell composition, comprising the following steps: i) Proliferating pluripotent cells in the presence of a drug that induces MHC-I presentation of antigens in the population during the proliferation process, in the presence of conditions that maintain the pluripotency of the cells; ii) Expose the proliferated cells to an inactivating agent that deactivates the cells; iii) Collect the proliferated inactivated cells and condition them. In certain embodiments, the integrity of the cell envelope is maintained in step ii). In another embodiment, cells are inactivated to obtain cell-derived products, such as cell extracts. Cell compositions prepared according to the above method can be used for cancer treatment according to the methods disclosed herein.

[0062] Drugs for MHC I antigen presentation

[0063] Pluripotent cells are grown in the presence of a drug that enhances antigen presentation via the MHC I pathway. Such improved expression can be checked by comparing the number of MHC I molecules on the cell surface in the presence or absence of the drug. Such drugs are known in the art, and in particular histone deacetylase inhibitors (HDACi) can be cited. Many products having this activity are known in the art, and among these HDACi, valproate (VPA or valproic acid, CAS number 99-66-1) can be cited in particular. Other HDACi that can be used (with the same mechanism of action as VPA) include, in particular, vorinostat, romidepsin-kidamide, panobinostat, belinostat, panobinostat, mosetinostat, avexinostat, entinostat, SB939, resminostat, zivinostat, or quidinostat. These drugs are present in the cell culture (growth) medium during the proliferation of pluripotent cells.

[0064] Maintains the cellular pluripotency.

[0065] Cell proliferation is carried out under conditions (culture medium, temperature) that maintain the pluripotency of the cells. These culture conditions are known in the art. Maintaining the pluripotency of the cells ensures that such cells express (and therefore have) all embryonic antigens, thereby increasing the cells' ability to present such antigens on their surface via the MHC I pathway. The more embryonic antigens are presented on the surface of pluripotent cells, the greater the likelihood that at least one of these antigens will also be present on the surface of cancer cells, which will then be recognized and targeted by an immune system that may be pre-stimulated by the vaccine composition of the present invention. Therefore, the maintenance of cellular pluripotency of the compositions according to the present invention obtained by the methods disclosed herein leads to the presentation of a wide variety of embryonic antigens, thus resulting in the ubiquitous efficacy of the vaccine compositions of the present invention in the treatment methods disclosed herein. Cell proliferation under conditions that maintain pluripotency is well known in the art. This is particularly described in all iPSC proliferation protocols described to date (Shi Y and al, Nat Rev Drug Discovery 2017; Chen KG and al, Cell Stem Cell. 2014). The following conditions are preferable: - Use of E8 medium or all clinical-grade ES / iPSC medium supplemented with VPA and / or mutagenic agents (e.g., ENU, see below) as needed. - A temperature of 37°C, with or without hypoxia. - Daily medium changes using the same medium with the addition of VPA (0.1 mM to 5 mM) and / or ENU (0.1 μg / ml to 100 μg / ml) and / or a p53 inhibitor and / or a compound that enhances cell viability (e.g., Y-27632 Rock inhibitor). Cells are typically cultured for 8 weeks, maintaining an optimal density of 90% through regular weekly subculturing using enzymatic exfoliation (collagenase, trypsin).

[0066] It inactivates cells.

[0067] In a preferred embodiment, pluripotent cells used in the treatment methods disclosed herein are inactivated. The term “inactivated” and its grammatical variations are used herein to refer to cells (e.g., pluripotent cells) that are viable but rendered unable to proliferate (i.e., mitotically inactivated). Those skilled in the art may use techniques known in the art, including, but not limited to, exposure to chemical agents, irradiation and / or lyophilization. Pluripotent cells can be inactivated so that they are unable to divide upon administration to a subject, and thus unable to form teratomas in the subject. In a multi-cell situation, it is understood that not all cells need to be unable to proliferate. Thus, as used herein, the phrase “inactivated to a degree sufficient to prevent teratoma formation in a subject” refers to the degree of inactivation in the population as a whole, so that teratomas do not form after administration to the subject because the exposed pluripotent stem cells no longer divide, as confirmed by in vitro culture. Even if one or more cells in a group of cells are actually capable of proliferating in the subject, it is assumed that these cells are destroyed by the host's immune system before teratoma formation occurs. Such inability to proliferate and form teratomas may be confirmed by testing in mice with functional and non-functional immune systems.

[0068] In some embodiments, "inactivated" cells are killed cells. That is Inactivated pluripotent stem cells can still stimulate an immune response when mice are vaccinated using hESCs or hiPSCs in combination with valproic acid or another HDACi. This vaccination can induce an efficient immune and antitumor response against 4T1 breast cancer without evidence of side effects or autoimmune disease.

[0069] Typically, to inactivate stem cells, they can be exposed to a lethal dose of radiation (e.g., a single fraction of 5–100 Gy). The exact dose and duration of the radiation delivered to the cells are not critical as long as the cells become unviable.

[0070] Cell harvesting and conditioning.

[0071] The recovery process of this method involves one or more steps of washing the cell culture and resuspending the cells in any suitable medium (e.g., X-Vivo / Stemflex medium or any other clinical-grade cell medium).

[0072] Cell conditioning may include freezing or lyophilizing cells so that the cell composition can be stored before use.

[0073] We mutate pluripotent cells to induce the expression of neoantigens.

[0074] It is thought that pluripotent cells are genetically very stable cells. In fact, they are present very early in embryonic development, and because they must proliferate for embryonic development, it is important that these cells are homogeneous in the embryo and therefore less prone to mutation.

[0075] Consequently, cells within a population of pluripotent cells are generally very homogeneous, considering their gene content (i.e., more than 95% of the cells in the population present the same genetic background).

[0076] When preparing iPSCs, selective advantages of some cells arise between multiple passages, leading to a population of iPSC clones that exhibit specific mutations in later passages, while the cell genome sequences remain nearly 100% similar.

[0077] However, after several passages, iPSCs are as stable as hESCs (Hussein SM et al, Nature 2011). Culture-induced (adaptive) mutations will likely be acquired with very few genetic changes during long-term culture (Hussein SM et al, Bioessays, 2012).

[0078] However, it is preferable to induce mutations in cells to increase the variability of fetal / embryonic neoantigens on treated cell material, as is found in invasive cancers. Thus, this would increase the likelihood that the immune system will generate T cells against these mutated cells, which can fight against cancer cells as well as cancer cells that may undergo subsequent changes during tumor growth.

[0079] This may help in combating cancer caused by the accumulation of genetic changes resulting from DNA replication errors and / or environmental damage during the proliferation of cancer stem cells. These changes include cancer-driven mutations that initiate oncogenic and genomically destabilizing mutations. This increased genomic instability leads to clonal evolution, resulting in the selection of more invasive clones with increased drug resistance.

[0080] Consequently, in certain embodiments, cells are grown under conditions that induce mutations in the cells' genes.

[0081] Cells can thus be exposed to mutagenic agents, that is, physical or chemical substances that alter the genetic material of an organism, usually DNA, and thus increase the frequency of mutations above the natural background level.

[0082] Mutagens can be selected from the group consisting of physical mutagens and chemical mutagens.

[0083] Among physical mutagens, the following can be cited: - Ionizing radiation that can cause DNA damage and other damage (e.g., X-rays, gamma rays, and alpha particles). In particular, radiation from cobalt-60 and cesium-137 can be cited. The level of irradiation must be much lower than the level used for cell inactivation and can be designed by those skilled in the art. - Ultraviolet light with wavelengths exceeding 260 nm (if left uncorrected, this may cause errors in replication). -or radioactive decay (e.g., 14C in DNA).

[0084] Among chemical mutagens, the following can be cited: - Reactive oxygen species (ROS), such as superoxide, hydroxyl radicals, hydrogen peroxide, etc. - Deamination agents that can induce transition mutations by converting cytosine to uracil (e.g., nitrite); -Polycyclic aromatic hydrocarbons (PAHs) (can bind to DNA when activated into diol-epoxides); - Alkylating agents, such as ethyl nitrosourea (ENU, CAS number 759-73-9), mustard gas, or vinyl chloride; - Aromatic amines and amides, such as 2-acetylaminofluorene; - Alkaloids from plants, such as alkaloids from vinca seeds; - Bromine and certain compounds containing bromine; - Sodium azide; -Bleomycin; - Psoralens combined with ultraviolet light; -benzene; - A base analogue that can substitute DNA bases during replication, causing transition mutations; - Inserts, such as ethidium bromide, proflavin, daunorubicin, etc. - Metals, such as arsenic, cadmium, chromium, nickel, and their mutagenic compounds.

[0085] In this embodiment, the cells will obtain a population of pluripotent stem cells that have random mutations (generally different from cell to cell, thereby leading to a heterogeneous population) particularly in cancer-associated neoantigens.

[0086] The inventors have shown that it is possible to design culture conditions that enable the induction of DNA replication errors in pluripotent stem cells without inducing DNA damage-dependent apoptosis.

[0087] This is particularly surprising because, as shown above, pluripotent cells are naturally very stable, as they should contain as few mutations as possible that were introduced during the early stages of embryonic development. From this, it follows that DNA repair mechanisms are highly efficient in these cells, thereby correcting most defects and / or inducing apoptosis when it is not possible to correct these defects on their own.

[0088] In one embodiment, pluripotent cells of an initial population (e.g., ESCs or IPSCs) are grown and maintained in a pluripotency-tolerant culture medium (as known in the art) to preserve the pluripotency stage during repeated passages. In these states, a small number of exome mutations (5-10 mutations per exome) may generally be observed.

[0089] Pluripotent cells were then cultured in vitro using mutagenic compound methods to induce and increase genomic instability within pluripotent stem cells, as listed above. DNA damage was clearly confirmed by phosphorylation of γH2AX as a marker for double-strand breaks (DSBs). Both the percentage of γH2AX-positive cells and the frequency of γH2AX lesions increased in ESCs or IPSCs and in a greater number of micronuclei as a marker of genomic instability.

[0090] Preferred agents include bleomycin, ENU, alkylating agents, actinomycin D, ROS regulators, UV, H2O2, and ionizing radiation (gamma rays, X-rays), which enable the induction and enhancement of mutation rates in pluripotent stem cells accumulated during culture.

[0091] In preferred embodiments, N-ethyl-N-nitrosourea (ENU) has been shown to induce novel mutations and enhance neoantigen levels in treated pluripotent stem cells at doses of <50 μg / ml during long-term culture for at least 7–60 days. These mutations are similar to those reported in cancer.

[0092] Thus, particularly when cells are cultured in culture medium using HDACi, it is possible to maintain cellular pluripotency while accumulating diverse mutations in response to DNA damage during pluripotent cell proliferation, which is accompanied by a high mutation rate due to the selective advantage during long-term culture. The presence of HDACi in culture leads to an increase in active histones (H3K4me3 and H3K9ac) and pluripotency epigenetic marks, as well as the preservation of replication and proliferation rates between passages, in response to the induction of DNA damage.

[0093] In another embodiment of the compositions and methods described herein, mutations are induced in pluripotent cells through genetic modification of cells using genes that promote high levels of genomic instability. In particular, appropriate inhibitors (e.g., NER / BER / DSBR / MMR inhibitors) can be used to delete or reduce the activity of genes or signaling pathways involved in DNA repair and replication. These methods for inducing increased DNA damage-related genomic instability may be carried out by using “vectors” or “genetic modification” that inactivate or knock down DNA repair-related genes or signaling pathways, such as DNA polymerase delta complex, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR), DSBR, or NEJH. Other examples of DNA repair genes include DNApkC, Ku70, Rad51, Brca1, or Brca2.

[0094] In other embodiments, pluripotent cells are modified to suppress apoptosis-related genes (e.g., p53) by genetic modification or chemical p53 (e.g., Pifithrin-mu, Nutlin-3, etc.), or by using a cell viability-enhancing compound, such as Y-27632 (a selective inhibitor of p160-Rho-related coiled kinase (ROCK)).

[0095] In certain embodiments, the population of pluripotent cells consisted of inducible pluripotent stem cells (iPSCs) generated from somatic cells (e.g., cells isolated from a patient), which already contained the relevant genomic alterations: i) DNA repair disorders, including, for example, inflammatory telangiectasia, Bloom's syndrome, Cockayne syndrome, Fanconi anemia, Werner syndrome, xeroderma pigmentosum, and Nijmemen's rupture syndrome; ii) Hereditary familial cancer syndromes with genomic instability, such as Lynch syndrome (hereditary nonpolyposis colorectal cancer with mutations in the MMR genes, including MLH1, MSH2, MSH6, PMS1, and PMS2), Li-Fraumeni syndrome with mutations in the TP53 gene or CHEK2, hereditary breast and ovarian cancer (HBOC) syndrome with deletions or mutations in the BRCA1 / 2 genes, and familial adenomatous polyposis (FAP) with mutations in the APC gene; iii) Somatic cell carcinogenesis-inducible genomic instability, such as CML, accompanied by translocation (T9;22).

[0096] In a preferred embodiment, the population of mutant pluripotent cells is generated from inducible pluripotent stem cells, which are derived from somatic cells containing disease-associated genomic alterations. Typically, these genomic alterations may be translocations (T9:22), deletions (BRCA1 / 2), or mutations (BRCA, RET).

[0097] In certain embodiments, the population of pluripotent stem cells consists of iPSCs generated from cancer cell lines or patient-specific cancer cells.

[0098] In another embodiment, a population of ESCs or IPSCs is genetically modified to overexpress multiple non-random cancer stem-associated neoantigens by using a “vector.” In a specific embodiment, a population of ESCs or IPSCs is genetically modified to express multiple mutations and cancer stem cell-specific neoantigens (at least five) in pluripotent stem cells using “genome editing” technology. The present invention provides compositions and methods for providing ESCs or IPSCs by introducing these multiple neoantigens by RNA-guided multiplex genome editing, modification, inhibition of expression, and other RNA-based technologies.

[0099] As used herein, the term “genome editing” refers, in particular, to RNA-mediated gene manipulation involving a guide RNA for Cas9-mediated genome editing. This guide RNA (gRNA) is transfected together with the endonuclease Cas9. The guide RNA provides a scaffold and a spacer sequence complementary to the target. In another embodiment, the gene-manipulated sequence may be an siRNA or microRNA sequence designed for gene silencing according to standard methods in the art using the Crispr-Cas 9 system. Compositions and methods for constructing and using the Crispr-Cas system are known in the art, in particular, as described in U.S. Patent No. 8,697,359.

[0100] In certain embodiments, a population of pluripotent cells is treated with an alkylating agent. As used herein, the term “alkylating agent” refers to a substance that adds one or more alkyl groups from one molecule to another. This treatment introduces novel mutations in neoantigens that provide a superior immune response by increasing the oligoclonal proliferation of TILs and Th1 / Th2 cell-mediated immunity.

[0101] In the present invention, the alkylating agent is selected from the group consisting of nitrogen mustard, nitrosourea, alkylsulfonic acid, triazine, ethyleneimine, and combinations thereof. Non-limiting examples of nitrogen mustard include mechloretamine (Lundbeck), chlorambucil (GlaxoSmithKline), cyclophosphamide (Mead Johnson Co.), bendamustine (Astellas), ifosfamide (Baxter International), melphalan (Ligand), melphalanfluphenamide (Oncopeptides), and pharmaceutically acceptable salts thereof. Non-limiting examples of nitrosourea include streptozosin (Teva), carmustine (Eisai), lomustine (Sanofi), and pharmaceutically acceptable salts thereof. Non-limiting examples of alkylsulfonic acid include busulfan (Jazz Pharmaceuticals) and pharmaceutically acceptable salts thereof. Non-limiting examples of triazines include dacarbazine (Bayer), temozolomide (Cancer Research Technology), and pharmaceutically acceptable salts thereof. Non-limiting examples of ethyleneimines include thiotepa (Bedford Laboratories), altoretamine (MGI Pharma), and pharmaceutically acceptable salts thereof.Other alkylating agents include ProLindac (Access), Ac-225BC-8 (Actinium Pharmaceuticals), ALF-2111 (Alfact Innovation), thromphosphamide (Baxter International), MDX-1203 (Bristol-Myers Squibb), thioureidobutyronitrile (CellCeutix), mitobronitol (Chinoin), mitractol (Chinoin), nimustine (Daiichi Sankyo), gluphosphamide (Eleison Pharmaceuticals), combination of HuMax-TAC ​​and PBD ADC (Genmab), BP-C1 (Meabco), treosulfan (Medac), nifurtimox (Metronomx), improsulfan tosylate (Mitsubishi Tanabe Pharma), ranimustine (Mitsubishi Tanabe Pharma), ND-01 (NanoCarrier), and HH-1 (Nordic). This includes Nanovector, 22P1G cells and ifosfamide combination (Nuvilex), estramustine phosphate (Pfizer), prednimastine (Pfizer), lurubinectedin (PharmaMar), trabectedin (PharmaMar), althreamin (Sanofi), SGN-CD33A (Seattle Genetics), fotemustine (Servier), nedaplatin (Shionogi), heptaplatin (Sk Holdings), apadiquon (Spectrum Pharmaceuticals), SG-2000 (Spirogen), TLK-58747 (Telik), laromustine (Vion Pharmaceuticals), procarbazine (Alkem Laboratories Ltd.), and pharmaceutically acceptable salts thereof. In another embodiment, the alkylating agent is selected from the group consisting of mechloretamine (Lundbeck), chlorambucil (GlaxoSmithKline), cyclophosphamide (Mead Johnson Co.), streptozocin (Teva), dacarbazine (Bayer), thiotepa (Bedford Laboratories), altretamine (MGI Pharma), pharmaceutically acceptable salts thereof, and combinations thereof.In another embodiment, the alkylating agent may be ProLindac (Access), Ac-225BC-8 (Actinium Pharmaceuticals), ALF-2111 (Alfact Innovation), bendamustine (Astellas), ifosfamide (Baxter International), trophosfamide (Baxter International), MDX-1203 (Bristol-Myers Squibb), temozolomide (Cancer Research Technology), thioureidobutyronitrile (CellCeutix), mitobronitol (Chinoin), mitractol (Chinoin), nimustine (Daiichi Sankyo), carmustine (Eisai), gluphosphamide (Eleison Pharmaceuticals), a combination of HuMax-TAC ​​and PBD ADC (Genmab), or busulfan (Jazz Pharmaceuticals), melphalan (Ligand), BP-C1 (Meabco), treosulfan (Medac), nifurtimox (Metronomx), improsulfan tosylate (Tanabe Mitsubishi Pharma), ranimustine (Tanabe Mitsubishi Pharma), ND-01 (NanoCarrier), HH-1 (Nordic Nanovector), combination of 22P1 G cells and ifosfamide (Nuvilex), melphalanflufenamide (Oncopeptides), estramustine phosphate (Pfizer), prednimustine (Pfizer), lurubinectedin (PharmaMar), trabectedin (PharmaMar), altreatamin (Sanofi), lomustine (Sanofi), SGN-CD33A (Seattle Genetics), fotemustine (Servier), nedaplatin (Shionogi), heptaplatin (Sk Select from the group consisting of (Holdings), Apadiquon (Spectrum Pharmaceuticals), SG-2000 (Spirogen), TLK-58747 (Telik), Laromustine (Vion Pharmaceuticals), Procarbazine (Alkem Laboratories Ltd.), pharmaceutically acceptable salts thereof, and combinations thereof.

[0102] In certain embodiments, a population of pluripotent cells is treated with N-ethyl-N-nitrosourea (ENU, CAS number 759-73-9). ENU has the chemical formula C3H7N3O2 and is a highly potent mutagen that transfers an ethyl group to a nucleic acid base in nucleic acids.

[0103] As shown above, the purpose of mutagenic drugs is to introduce random mutations into the genes of pluripotent cells during proliferation (the introduction of mutations occurs during cell replication and division). The population of pluripotent stem cells acquires mutations that are selected to promote culture adaptation, which can provide growth advantages. Passaging of ESCs or iPSCs is subject to a high level of selective pressure, and multiple clonal mutation populations may be preferred during proliferation.

[0104] It should be noted that because pluripotent cells are very stable, mutagen application may need to be carried out over a long period of time. For example, if an ENU is used, it may be applied for at least 7 days, more preferably at least 15 days, more preferably at least 20 days, more preferably at least 30 days, more preferably at least 40 days, preferably at least 50 days, or even further at least 60 days. After mutagen application, the cells can be washed (if the mutagen is a chemical agent) and further grown in the presence of an agent favorable to MHC-1 expression, particularly HDACi. This agent is preferably present even during the application of the mutagenic agent.

[0105] Thus, it can be observed that mutagens induce mutations (i.e., non-synonyms, nonsense, frameshift, arrest, acquisition, splice variants, CNVs, SNVs) in some embryogenetic genes expressed by pluripotent cells, and therefore increase the diversity of these antigens (novel neoantigens within the whole genome). This increases the possibility of vaccine compositions with enhanced immunogenicity that can stimulate a broad immune response against invasive cancers with rapid and frequent mutations.

[0106] An efficient immune response may be difficult to obtain for some cancers, where clonal proliferation of cancer cells is accompanied by mutations in antigens expressed by tumor cells. The immune response will thus depend on the mutational burden of the cancer. The generation of random mutations in pluripotent cell populations through the use of mutagens leads to the expression of mutated embryonic antigens, increasing the diversity of antigens presented to the immune system at the time of vaccination.

[0107] Consequently, there may already be pre-stimulated T cells in cancer cells that appear during the division of such cells and can accelerate and improve the immune response against these cells by targeting mutated antigens.

[0108] Modification of pluripotent cells.

[0109] In certain embodiments, a population of pluripotent stem cells is genetically modified to overexpress compounds that stimulate an immune response by using gene integration within the pluripotent cell genome. Typically, in a first step, the stem cell population is isolated and grown. In a second step, the gene of interest is packaged in an embedded viral vector (e.g., a retrovirus or lentivirus). In a third step, the embedded viral vector containing the gene of interest is transferred into the stem cell population.

[0110] In certain embodiments, a population of pluripotent cells is modified using genes for proteins that stimulate MHC expression and / or immune responses. These compounds are selected from the group consisting of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), their functional fragments, and combinations thereof.

[0111] The interferons (IFNs) studied in this invention include common types of IFN, IFN-alpha (IFN-α), IFN-beta (IFN-β), and IFN-gamma (IFN-γ). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior, and / or increasing their antigen production, thus facilitating the immune system to recognize and destroy cancer cells. IFNs can also act indirectly on cancer cells, for example, by slowing angiogenesis, strengthening the immune system, and / or stimulating natural killer (NK) cells, T cells, and macrophages. Recombinant IFN-alpha is commercially available as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).

[0112] The interleukins studied in this invention include IL-2, IL-4, IL-11, and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Washington) is currently testing a recombinant form of IL-21, which is also being considered for use in the combination of the present invention.

[0113] The colony-stimulating factors (CSFs) studied in this invention include granulocyte colony-stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony-stimulating factor (GM-CSF or salgramostim), and erythropoietin (epoetin alfa, darbepoetin). Treatment with one or more growth factors can help stimulate the generation of new blood cells in subjects undergoing conventional chemotherapy. Therefore, treatment with CSFs can help reduce the side effects associated with chemotherapy and allow for the use of higher doses of chemotherapy agents. Various recombinant colony-stimulating factors are commercially available, such as Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Ortho Biotech (erythropoietin; Amgen), and Arnesp (erytropoietin).

[0114] In its broadest sense, a “vector” is any medium capable of facilitating the transfer of oligonucleotides into cells. Preferably, the vector transports nucleic acids to cells with reduced degradation compared to the degree of degradation that would result in the absence of the vector. Generally, vectors useful in the present invention include naked plasmids, non-viral delivery systems (electroporation, sonoporation, cation transfection agents, liposomes, etc.), phagemids, viruses, and other media derived from viral or bacterial sources manipulated by insertion or incorporation of nucleic acid sequences. Viral vectors are preferred types of vectors, but are not limited to, those containing nucleic acid sequences from the following viruses: RNA viruses, e.g., retroviruses (e.g., Moloney mouse leukemia virus and lentivirus-derived vectors), Harvey mouse sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40 virus; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia viruses; and polioviruses. Although not named, other vectors known in the art can be easily used.

[0115] Typically, in the context of the present invention, viral vectors include adenoviruses and adeno-associated (AAV) viruses, which are DNA viruses already approved for human use in gene therapy. In practice, 12 different AAV serotypes (AAV1-12) are known, each with a different histotropy (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAVs are derived from AAV-dependent parvoviruses (Choi, VW J Virol 2005; 79:6801-07). Adeno-associated virus types 1-12 can be manipulated to be replication-deficient and can infect a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316-27). It also has advantages such as thermal and lipid solvent stability; high transduction frequency in diverse cell lineages, including hematopoietic cells; and the absence of re-infection inhibition, which allows for multiple transduction sequences. Furthermore, wild-type adeno-associated virus infection can persist for more than 100 passages in tissue culture in the absence of selective pressure, indicating that adeno-associated virus genome integration is a relatively stable phenomenon. Adeno-associated viruses can also function in extrachromosomal ways.

[0116] Other vectors include plasmid vectors. Plasmid vectors are extensively described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., 1989. For the past few years, plasmid vectors have been used as DNA vaccines for delivering antigen-coding genes to cells in vivo. They are particularly advantageous for this purpose because they do not have the same safety concerns as many viral vectors. However, these plasmids, which have promoters compatible with host cells, can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. In addition, plasmids may be custom-designed using restriction enzyme and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by various parenteral, mucosal, and local routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or by other routes. It may also be administered by intranasal spray or nasal drops, rectal suppositories, and orally. Preferably, the DNA plasmid is injected through an intraocular method (such as intravitreous, subretinal, or choroidal). It may also be administered into the epidermal or mucosal surface using a gene gun. The plasmid may be given in aqueous solution, dried on gold particles, or associated with another DNA delivery system (including, but not limited to, liposomes, dendrimers, spirals, and microencapsulation).

[0117] In a specific embodiment, a population of stem cells is modified by homologous recombination through the introduction of a transgene (e.g., siRNA) into the AAVS1 locus on chromosome 19.

[0118] As used herein, the term "homologous recombination" refers to gene targeting methods for artificially modifying specific genes on a chromosome or genome. It refers to recombination that occurs based on nucleotide sequence homology between a genomic fragment and its corresponding chromosomal locus when a genomic fragment containing a portion homologous to a target sequence on a chromosome is introduced into a cell.

[0119] Furthermore, the term "genetic modification" refers to the insertion of exogenous DNA at a desired locus on a chromosome, the replacement of part or all of a gene using exogenous DNA, or the deletion of a gene. More specifically, genetic modification refers to the insertion of an exogenous DNA fragment (i.e., "knock-in"), while the endogenous DNA sequence is preserved in the form of a replacement, deletion, or disruption (i.e., "knock-out") of part or all of the gene sequence, either by the fragment being expressed or constitutively expressed in conjunction with the expression of the gene at a specific locus, or by an alteration of the endogenous DNA sequence.

[0120] Examples of methods for introducing artificial chromosomes into cells include calcium phosphate precipitation (Graham et al., (1978) Virology 52: 456-457, Wigler et al., (1979) Proc. Natl. Acad. Sci. USA 76 1373-1376 and Current Protocols in Molecular Biology Vol.1, Wiley Inter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)), fusion methods using polyethylene glycol (US Patent No. 4,684,611), and methods using lipid carriers (e.g., lipofectin) (Teifel et al., (1995) Biotechniques 19: 79-80, Albrecht et al., (1996) Ann. Hematol. 72: 73-79; Holmen et al., (1995) In Vitro Cell Dev. Biol. Anim. 31: 347-351, Remy et al., (1994) Bioconjug. Chem. 5: 647-654, Le Bolc'het al., (1995) Tetrahedron Lett. 36: 6681-6684, Loeffler et al., (1993) Meth. Enzymol, 217: 599-618 and Strauss (1996) Meth. Mol. Biol. 54: 307-327), methods for electroporation and fusion with micronuclei (U.S. Patents No. 5,240,840, No. 4,806,476, No. 5,298,429, and No. 5,396,767, Fournier (1981) Proc. Natl. Acad. Sci. USA 78: This includes 6349-6353 and Lambert et al., (1991) Proc. Natl. Acad. Sci. USA 88: 5907-59).

[0121] A group of cells

[0122] Thus, using the method described above, the inventors have obtained a population of pluripotent cells that express novel embryonic epitopes in some or all of the embryonic genes, thereby inducing more efficient antitumor immunity. In particular, the inventors have shown that a population of pluripotent cells treated with N-ethyl-N-nitrosourea (ENU) exhibits random mutations compared to a population of pluripotent cells not treated with ENU. Therefore, this population is also subject to the present invention.

[0123] The present invention relates to a cell composition comprising pluripotent cells, wherein the cells in the population exhibit a mutation rate of at least 0.1%, preferably at least 1%, more preferably at least 2%, more preferably at least 5%, more preferably at least 10%, more preferably at least 15%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, or even more preferably at least 50% in at least three genes selected from the following group consisting of TP53, P2RY8, CRLF2, CRTC3, BLM, ASXL1, IDH2, NTRK3, MALAT1, EXT1, NCOA2, IKF1, PIK3R1, EP300, AKT2, PPP2R1A, CDK12, BRCA1, ERB2, CDH1, TBX3, SMARCD1, HSP90AA1, EZH2, SUZ12, STAT5B, and POUF5F1, more preferably at least four genes, more preferably at least five genes, more preferably at least six genes, more preferably at least seven genes.

[0124] The mutation rate of this gene should be tested in the cell population after exposure to mutagenic agents, or before or after further proliferation, if such further proliferation is to be carried out.

[0125] Due to the fact that pluripotent cells are genetically very stable, the presence of large mutations in at least three of the genes listed above demonstrates the existence of a new population of pluripotent cells that did not exist before and would not have been observed in the absence of mutagenic conditions.

[0126] Exposure of cells to mutagenic drugs induces the emergence of random mutations in the genomes of such cells. Populations resulting from such exposure are thus heterogeneous compared to populations of pluripotent cells, due to their low spontaneous mutation rates during long-term growth and culture. The populations obtained in this specification are, in particular, the following: - The cells are pluripotent (i.e., they possess markers of pluripotency). As shown above, there are multiple differences in the genomes of cells within a population, meaning that it is possible to detect the rate of the mutated genes listed above (as shown above) within the cells of the population.

[0127] As an example, a 5% mutation rate in the TP53 gene means that 95% of the TP53 sequences in a cell population are identical (referred to as the TP53 reference sequence), and the last 5% of the TP53 sequence is different from the TP53 reference sequence.

[0128] In a further embodiment, the present invention thus relates to a cell composition comprising pluripotent cells, wherein the cells in the population present a mutational landscape in a population of ESCs or IPSCs having one or more of the following characteristics: i) At least >3 (as seen above, or more) cancer-associated neoantigen mutations genetically introduced into ESCs or IPSCs by genome modification. ii) A combination of mutants enriched by selective advantages in cultured embryonic pluripotent stem cells, limited to cancer genomes and induced by mutagenic agents.

[0129] Mutagenic processes result in increased levels of novel genomic mutations and genetic mosaicism in the resulting late-passage human iPS cell lines.

[0130] Analysis of gene mutations is preferably performed by large-scale genomic analysis of inducible cancer-associated "mutanome" signatures in each ESS and IPSC population using NGS (exome, RNA-seq, or whole-genome sequencing), CGH arrays, and SNP arrays. Whole-exome sequencing in combination with transcriptome profiling allows for the description of mutantomes encoding expressed proteins.

[0131] Genomic abnormalities are identified by using at least two algorithms for bioinformatics analysis known in the art. The prevalence of all mutations in the whole genome after application of a mutagenic agent confirms higher mutation and / or CNV loading in the output ESC or IPSC.

[0132] Qualitative and quantitative criteria make it possible to define each cell population within a genetic mosaic in ESCs or IPSCs as described below: The qualitative criteria include: - Identification of acquired novel molecular somatic changes (mutations, CNVs, or SNVs) related to their presence in ESCs or iPSC genomes after mutagenesis and their absence in ESCs or iPSCs without mutagenesis during similar culture passages. - Classification of novel mutations (i.e., non-synonyms, nonsense, splice variants, CNVs, SNVs) by detecting their duplications in cancer genomes (from databases, i.e., TCGA, ICGC, COSMIC) and in pluripotency genes and embryonic pathways (following pluripotency genes, i.e., the Plurinet gene).

[0133] Such quantitative criteria include: - The prevalence of these novel somatic mutations (confidence level FDR ≤ 0.05) and novel CNVs / SNVs (FDR < 10%) in the whole genome is defined for each ESC or iPS cell. - The presence of verified mutations in at least >3 different genes. - Mutation rates of each novel stable somatic mutation with allele frequencies from at least >0.1% or other percentages seen above, with respect to clonal selection and proliferation or passage rate (from 50 × depth to 100 × depth, and 80–98% of target exome coverage). - Expression of pluripotency markers with an expression rate of at least >90% compared to input ESCs or IPSCs before mutagenesis or genetic modification, and gene expression-based assay (PluriTest). - Increased expression of MHC I molecules on the cell surface (as determined, e.g., by FACS) by at least 50%, and generally up to 90%, compared to cell populations grown in the absence of HDACi, particularly VPA.

[0134] The present invention relates to a vaccine composition comprising a population of pluripotent cells as disclosed above, and an agent that stimulates an immune response and / or MHC I expression.

[0135] In particular, such pluripotent cells are preferably inactivated and optionally mutated ESCs or IPSCs, as disclosed above.

[0136] The agent that stimulates the immune response may be an adjuvant (immunostimulant) known in the art. Preferably, it is an HDACi (used in a dose range of 0.2 mM to 4 mM). When using such an HDACi, another adjuvant may be used.

[0137] The present invention also relates to a device (e.g., a syringe) containing such a vaccine composition, which can be used for the simultaneous administration of an HDACi compound (coumpound) and a cell composition.

[0138] Such vaccine compositions can be used as therapeutic vaccines against stem cell cancer (cancer in which the cells express neoantigens) for the cure of patients, or as prophylactic vaccines, particularly to prevent the development of such cancers in patients who are susceptible to them.

[0139] The predisposing genes are, for example, as follows (see also Lindor et al, 2008 Journal of the National Cancer Institute Monographs, No. 38, Concise Handbook of Familial Cancer Susceptibility Syndromes, Second Edition). Breast / Ovary: BRCA1, BRCA2, PALB2, RAD51 Lynch syndrome: MLH1, MSH2, MSH6, PMS2, EPCAM Hereditary papillary renal cell carcinoma: FH, MET Cowden disease: PTEN, PIK3CA Fanconi disease: FANC Von Hippel-Lindau disease: VHL Malignant melanoma: CDKN2A, MITF, BAP1, CDK4 Endocrine tumors: MEN1, RET, CDKN1B Neurofibromatosis: NF1, NF2, LZTR1, SMARCB1, SPRED1 Hereditary paraganglioma pheochromocytoma: SDH, TMEM127, MAX, EPAS1 Familial adenomatous polyposis: APC, MUTYH Retinoblastoma: RB1 Birt-Hogg-Dubé syndrome: FLCN Bloom syndrome: BLM Carney syndrome: PRKAR1A Gorlin syndrome: PTCH1 Li-Fraumeni syndrome: TP53, CHEK2 Nijmegen syndrome: NBN Peutz-Jeghers syndrome: STK11 Familial juvenile polyposis: BMPR1A, SMAD4 Xeroderma pigmentosum: XP This list is not exhaustive.

[0140] In certain embodiments, the cancer stem cell vaccine product comprises a mixture of lyophilized cell lysates, a mixture of concentrated pluripotent stem neoantigens, purified cancer stem neoantigens, exosomes, DNA, RNA, proteins, or multiple peptides from engineered ESCs or IPSCs derived from ESCs or IPSCs. These are the immunogenic agents disclosed above, which are formulated in the presence of HDACi.

[0141] In another embodiment, the cancer stem cell vaccine product is mixed with supernatant GMP medium from an engineered irradiated ESC or IPSC, which is used as an adjuvant effector.

[0142] In a preferred embodiment, the cells in this composition are inactivated (i.e., they can no longer divide).

[0143] The cell composition of the present invention is easily obtained by any of the methods disclosed above.

[0144] Note that the cells in this composition are heterogeneous in properties if they are not cultured using a mutagen and are therefore different in properties from homogeneous pluripotent cell compositions cultured according to methods known in the art.

[0145] When cultured in the absence of mutagens, the resulting cell population differs from that obtained by methods known in the art. This is because the presence of agents in the culture medium that maintain the expression of pluripotent genes and increase MHC I presentation leads to cells having more of these MHC I molecules on their surface.

[0146] As used herein, the term “compounds selected from the group that activate MHC expression and / or immune response” refers to compounds capable of stimulating immunogenicity. Such compounds are called activators of MHC expression and / or immune response. The term “MHC” refers to the major histocompatibility complex, which is present on the cell surface to recognize foreign molecules (called antigens). MHCs bind to antigens and present them to immune molecules (e.g., lymphocytes T and B). The term “immune response” refers to the immunological response of the immune system to antigens. By activating the immune response, the populations of FoxP3 subpopulations and myeloid-derived suppressor cells (MDSCs) are reduced, while the NK population is increased. In the context of the present invention, the immune response to a tumor includes a cytotoxic T cell response to antigens present in or on tumor cells. In some embodiments, the cytotoxic T cell response is mediated by CD8+ T cells. Typically, in the context of the present invention, antigens that activate MHC expression and / or immune response correspond to molecules present on the populations of pluripotent cells described above. Compounds that activate MCH expression and / or the immune system are neoantigens. The term “neoantigen” or “neoantigenicity” refers to a class of antigens that arise from at least one mutation that alters the amino acid sequence of a genome-encoded protein.

[0147] In the context of the present invention, the compound is selected from the group consisting of cytokines, histone deacetylase inhibitors, DNA methyltransferase inhibitors, and histone-lysine N-methyltransferase enzyme inhibitors.

[0148] In certain embodiments, the activator of MHC expression and / or immune response is a histone deacetylase inhibitor.

[0149] As used herein, the term histone “histone deacetylase inhibitor” is also known as HDACi and refers to a class of compounds that interfere with the function of histone deacetylase. Histone deacetylase (HDAC) plays a crucial role in the transcriptional regulation and pathogenesis of cancer. Typically, HDACi inhibitors regulate transcription and induce cell growth arrest, differentiation, and apoptosis. HDACi also enhance the cytotoxic effects of therapeutic agents used in cancer treatment, including radiotherapeutic and chemotherapeutic drugs.

[0150] In certain embodiments, the histone deacetylase inhibitor is valproic acid (VPA).

[0151] The term "valproic acid" is 2-propylpentanoic acid (C8H 16 O2) refers to the following CAS number and formula 99-66-1 in the art: [ka]

[0152] Valproic acid has multiple biological activities (Chateauvieux et al, J. Biomed. Biotechnol, 2010, pii: 479364. doi: 10.1155 / 2010 / 479364). Valproic acid affects the enhancement and inhibitory activity of the neurotransmitter GABA (gamma-aminobutyric acid). Several mechanisms of action have been suggested. Valproic acid is particularly involved in GABA metabolism: it inhibits the breakdown of GABA and GABA-transaminobutyric acid (LAMP), the adaptation of GABA synthesis, and modifies its metabolic turnover. Valproic acid also blocks specific ion channels, reducing excitation mediated by N-methyl-D-aspartate and blocking the activity of ion channels containing Na+ and Ca2+ (voltage-gated L-type CACNA1 type C, D, N, and F).

[0153] In the context of the present invention, valproic acid is used as an immunostimulant to enhance the immune response against cancers expressing pluripotent antigens shared with human embryonic stem cells (ESCs) or inducible pluripotent stem cells (IPSCs).

[0154] In particular, VPA is used to stimulate and enhance MHC-1 expression on cancer stem cell compartments, increasing the neoantigen content in CSC compartments. Higher expression of MHC-1 in ESCs and IPSCs, as well as in CSCs, enhances the presentation of MHC-1-related neoantigens to APCs / dendritic cells, enabling the induction of a TH1 immune response. Higher levels of chemokines (CXCL9, CXCL10, etc.) can enhance the recruitment of T cells into tumors.

[0155] The present invention relates to a method for increasing neoantigen content in the CSC compartment expression of embryonic antigens in CSCs and tumor cells through chromatin remodeling and chemokine expression (CXCL9, CXCL10, etc.) by proliferating pluripotent cells in the presence of HADCi (e.g., VPA and / or 5-azacitidine).

[0156] In particular, when used to treat patients in vivo, the compositions and vaccines of the present invention enable the modification of the tumor microenvironment and the promotion of T cell recruitment into the tumor, thereby achieving a long-term, sustained reduction in tumor volume.

[0157] This is due to the synergistic effect of co-administration of cancer pre-stimulated pluripotent cell vaccine and VPA when HDACi is administered to the patient further after vaccine injection (for example, for at least 15 days).

[0158] This example demonstrates that combined treatment with both cancer stem cell vaccine and VPA provides a superior antitumor response by increasing TILs with Th1 / Th2 cell-mediated immunity, decreasing FoxP3 TReg subpopulations, activating NK cells, and reducing the suppressive effect on MDSCs, thereby reversing tumor immunosuppression, reducing TRegs (in tumors and spleens), and recruiting T CD4+ and CD8+ lymphocytes into tumors with lower proportions of T CD4 and CD8-expressing PD-1.

[0159] VPA may downregulate c-Myc expression levels and potentially induce apoptosis and autophagy in cancer cells and CSCs. VPA may enhance adaptive immune responses through autophagosome cross-presentation.

[0160] The known effects of VPA include a reduction in inflammatory cytokines in lymph nodes, such as IL-6, IL-8, TNFα interleukin (IL)-1β, and IL-17.

[0161] In certain embodiments, the histone deacetylase inhibitor is suberoylanilide hydroxamic acid, also known as vorinostat (N-hydroxy-N'-phenyloctanediamide), which was the first histone deacetylase inhibitor approved by the U.S. Food and Drug Administration (FDA) in 2006 (Marchion DC et al 2004; Valente et al 2014).

[0162] In certain embodiments, the histone deacetylase inhibitor is panobinostat (LBH-589), which was approved by the FDA in 2015 and has the structure described in Valente et al 2014.

[0163] In certain embodiments, the histone deacetylase inhibitor is gibinostat (ITF2357), which is approved as an orphan drug in the European Union (Leoni et al 2005; Valente et al 2014).

[0164] In certain embodiments, the histone deacetylase inhibitor is vorinostat, also known as belinostat (PXD-101), which received FDA approval in 2014 (Ja et al 2003; Valente et al 2014).

[0165] In certain embodiments, the histone deacetylase inhibitor is entinostat (as SNDX-275 or MS-275). This molecule has the following chemical formula (C 21 H 20 N4O3) and has the structure described in Valente et al 2014.

[0166] In certain embodiments, the histone deacetylase inhibitor is mocetinostat (MGCD01030) having the following chemical formula (C 23 H 20 N6O) (Valente et al 2014).

[0167] In certain embodiments, the histone deacetylase inhibitor is pracinostat (SB939) having the following chemical formula (C 20 H 30 N4O2) and the structure described in Diermayr et al 2012.

[0168] In certain embodiments, the histone deacetylase inhibitor is chidamide (CS055 / HBI-8000) having the following chemical formula (C 22 H 19 FN4O2).

[0169] In certain embodiments, the histone deacetylase inhibitor is quisinostat (JNJ-26481585) having the following chemical formula (C 21 H 26 N6O2).

[0170] In certain embodiments, the histone deacetylase inhibitor is of the following chemical formula (C 21 H 23Abexinostat (PCI24781) having N3O5) (Valente et al 2014).

[0171] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula (C 20 H 19 FN6O2) and is CHR-3996 (Moffat D et al 2010; Banerji et al 2012).

[0172] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula (C 18 H 20 N2O3) and is AR-42 (Lin et al 2012).

[0173] In certain embodiments, the activator of MHC expression is a DNA methyltransferase inhibitor.

[0174] As used herein, the term "DNA methyltransferase inhibitor" refers to a compound that can interact with DNA methyltransferases (DNMTs) and inhibit their activity. DNMTs are enzymes that catalyze the transfer of methyl groups to DNA. DNA methylation serves a variety of biological functions. All known DNA methyltransferases use S-adenosylmethionine (SAM) as a methyl donor.

[0175] In certain embodiments, the DNA methyltransferase inhibitor is also known as azacitidine, which has the following chemical formula (C8H 12 N4O5) and structure (Kaminskas et al 2004; Estey et al 2013).

[0176] In certain embodiments, the DNA methyltransferase inhibitor has the following formula (C8H 12This is decitabine, also known as 5-aza-2'-deoxycytidine (N4O4) (Kantarjian et al 2006).

[0177] In certain embodiments, the activators of MHC expression and / or immune response are histone-lysine N-methyltransferase enzyme inhibitors or DNA methyltransferase inhibitors. As used herein, the term “histone-lysine N-methyltransferase enzyme inhibitor” refers to a compound capable of interacting with the histone-lysine N-methyltransferase enzyme encoded by enhancers of the zeste homolog 1 (EZH1) and 2 (EZH2) genes involved in DNA methylation. EZH2 catalyzes the addition of a methyl group to histone H3 at lysine 27 by using the cofactor S-adenosyl-L-methionine.

[0178] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is 3-deazanepranosin A (DZNep, C-c3Ado). DZNep, C-c3Ado is known in the art as having the following chemical formula C 12 H 14 It contains N4O3 and has CAS number 102052-95-9.

[0179] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is UNC1999 and an inactive analog compound. UNC1999 is known in the art as having the following chemical formula C 33 H 43 It contains N7O2 and has CAS number 1431612-23-5.

[0180] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is UNC2400 and an inactive analogue. UNC2400 is known in the art as having the following chemical formula C 35 H 47 It contains N7O2 and CAS number 1433200-49-7.

[0181] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is tazemetostat (EPZ6438, E7438). Tazemetostat is known in the art as having the following chemical formula C 34 H 44 It has N4O4 and CAS number 1403254-99-8.

[0182] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is trifluoroacetate (EPZ011989). Trifluoroacetate has the following chemical formula CF3COONa and CAS number 2923-18-4 in the art.

[0183] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is EPZ005687. EPZ005687 is known in the art as having the following chemical formula C 32 H 37 It contains N5O3 and has CAS number 1396772-26-1.

[0184] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is GSK343. GSK343 ​​is known in the art as having the following chemical formula C 31 H 39 It has N7O2 and CAS number 1346704-33-3.

[0185] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is GSK126. GSK126 is known in the art as having the following chemical formula C 31 H 38 It contains N6O2 and has CAS number 1346574-57-9.

[0186] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is GSK2816126. GSK2816126 is known in the art as having the following chemical formula C 31 H 38 It contains N6O2 and has CAS number 1346574-57-9.

[0187] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is ZLD1039. ZLD1039 is known in the art as having the following chemical formula C 36 H 48 It contains N6O3 and has CAS number 1826865-46-6.

[0188] The use of both HDACi and DNA methyltransferase inhibitors is also considered.

[0189] In fact, it has been shown that the combined use of VPA and 5-azacitidine (an analog of nucleoside cytidine that can be incorporated into DNA and RNA) leads to a synergistic effect on the re-expression of neo-anti-embryonic antigens.

[0190] HDACi should be administered in a therapeutically effective dose. For VPA, this can be 10–15 mg / kg / day, up to 60 mg / kg / day. Plasma levels of VPA should preferably be within the normally acceptable therapeutic range (50–100 μg / ml).

[0191] In a further embodiment, the methods according to the present invention are suitable for treating cancers expressing numerous embryonic antigens shared with human embryonic stem cells (hESCs) or human inducible pluripotent stem cells (hiPSCs) (e.g., embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, TRA-1-81, Oct4, Sox2, Klf4, Nanog, Lin28, etc.).

[0192] As used herein, the term “human stem cell-expressing cancer” refers to cancers that are more preferably targeted by the methods, vaccines, and compositions disclosed herein, and which express a number of embryonic antigens shared with human embryonic stem cells or inducible pluripotent stem cells (iPSCs), and which are cancer stem cells (hESCs). Typically, cancers are selected from the group consisting of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, gastric sarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphocytic and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, kidney cancer, head and neck tumors, and solid tumors.

[0193] As used herein, the term “administer” or “dosage” means the act of injecting a substance into a subject when the substance is extracorporeally present (e.g., a compound preparation), or otherwise physically delivering it, including by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, the administration of the substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the administration of the substance typically occurs before the onset of the disease or its symptoms.

[0194] In a preferred embodiment, the vaccine composition (pluripotent cells + a drug that stimulates MHC presentation) is administered by subcutaneous injection. The injections may be administered simultaneously, sequentially, or separately, at the same time of injection or at different time of injection, in the same syringe or in different syringes...

[0195] In a preferred embodiment, follow-up treatment (administration of MHC I and / or immune system-stimulating compounds, such as HDACi, particularly those that stimulate VPA) is administered orally.

[0196] The term "therapeutably effective dose" refers to the minimum amount of active agent required to provide a therapeutic benefit to the subject. For example, a "therapeutably effective dose" to a subject is the amount that causes improvement or tolerance in pathological symptoms, disease progression, or physiological conditions associated with suffering from the disorder. It will be understood that the total daily dose of the compound of the present invention is determined by the attending physician within the bounds of sound medical judgment. A specific therapeutically effective dose level for any particular subject depends on a variety of factors, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used, the subject's age, weight, overall health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field. For example, it is well within the scope of the art to start with a dose of the compound at a level lower than required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product may vary over a wide range of 0.01 to 1000 mg per adult per day. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient for symptomatic dose adjustment to the subject being treated. The pharmacopoeia typically contains about 0.01 mg to about 500 mg of the active ingredient, preferably about 1 mg to about 100 mg of the active ingredient. The effective amount of the drug is usually supplied at dose levels of 0.0002 mg / kg to about 20 mg / kg body weight / day, particularly about 0.001 mg / kg to 7 mg / kg body weight / day.

[0197] In certain embodiments, the methods according to the present invention further include one or more radiotherapy, targeted therapy, immunotherapy, or chemotherapy. Typically, a physician may choose to administer to a subject a compound selected from the group that activates MHC expression and / or immune response, as a complex preparation with i) a population of pluripotent cells and ii) radiotherapy, targeted therapy, immunotherapy, or chemotherapy.

[0198] In some embodiments, subjects are administered with i) a population of pluripotent cells and ii) a compound selected from the group that activates MHC expression and / or the immune response, as a complex preparation and chemotherapeutic agent.

[0199] The term "chemotherapeutic agent" refers to a chemical compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclophosphamide); alkyl sulfonic acids (e.g., busulfan, improsulfan, and biposulfan); aziridines (e.g., benzodopa, carboquan, metsuredopa, and uredopa); ethyleneamines and methylamelamines (including altoretamine, triethylenemelamine, trimethylenephosphoramide, triethylenethiophosphalamamide, and trimethylolmelamine); acetogenins (especially bratacin and bratacinone); carnoptothecin (including its synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adzeresin, carzeresin, and bizeresin synthetic analogs); and cryptophycin (especially cryptophycin 1 and Cryptophycin 8); Dorastatin; Duocalmycin (including synthetic analogues, KW-2189, and CBI-TMI); Eleuterobin; Pancratistatin; Sarcodithiine; Spongestatin; Nitrogen mustard (e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembicin, fenesterine, prednimustine, thromphosphamide, uracil mustard, etc.); Nitrosourea (e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, ranimustine); Antibiotics, e.g., engine antibiotics (e.g., calicheamicin, especially calicheamicin (11 and calicheamicin 211, e.g., Agnew See Chem Intl. Ed. Engl. 33:183-186 (1994); Dyne sewing machine (including Dyne sewing machine A); Espera sewing machine;Furthermore, neocarcinostatin chromophores and related pigment proteins (endiin antibiotic chromophores), acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinonomycin, carabicin, canninomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin ( Morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalarnycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptomgrin, streptozocin, tubercidine, ubenimex, dinostatin, zolbicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folate analogs (e.g., denopterin, methotrexate, ptepteterin, trimethrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine) , thioguanine etc); pyrimidine analogs (e.g., azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU etc; androgens (e.g., cartestron, drostanolone propionate, epithiostanol, mepitostatane, testolactone etc); antiadrenal drugs (e.g., aminoglutethimide, mitotane, trilostane etc); folic acid supplements (e.g., humic acid etc); acegraton; aldofarnide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisanthren; edatrexate; defofamine; demecolsin; diazicon; elfornithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamine; mytansinoids, e.g., meitansin and Anthamitocin; Mitoguazone; Mitoxantrone; Mopidamole; Nitracin; Pentostine; Fenamet; Pirarubicin;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; schizophyllan; spirogennanium; tenuazonic acid; triadiquan; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, beracrine A, loridine A, and angidin); urethanes; vindesine; dacarbazine; mannomustine; mitobromitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C") cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, New Jersey) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, France, Antony); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs (e.g., cisplatin and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormone agents that act to regulate or inhibit the hormonal effects on tumors, such as anti-estrogens (e.g., tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston)); and anti-androgen agents, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0200] In some embodiments, subjects are administered i) a population of pluripotent cells and ii) a compound selected from the group that activates MHC expression and / or immune responses as a target cancer treatment.

[0201] Targeted cancer therapy is a drug or other substance that blocks cancer growth and propagation by interfering with specific molecules ("molecular targets") involved in the growth, progression, and transmission of cancer. Targeted cancer therapy may also be referred to as "molecular targeted drugs," "molecular targeted therapy," "high-precision medicine," or similar names. In some embodiments, targeted therapy consists of administering a tyrosine kinase inhibitor to a subject. The term "tyrosine kinase inhibitor" refers to any of the various therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and / or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and are described in U.S. Patent Application Publication 2007 / 0254295, which is incorporated herein by reference in its entirety. It will be understood by those skilled in the art that compounds related to tyrosine kinase inhibitors can replicate the effects of tyrosine kinase inhibitors. For example, related compounds may act on different components of the tyrosine kinase signaling pathway and have the same effect as a tyrosine kinase inhibitor of that tyrosine kinase. Examples of suitable tyrosine kinase inhibitors and related compounds for use in the methods of embodiments of the present invention include, but are not limited to, dasatinib (BMS-354825), PP2, BEZ235, salakatinib, gefitinib (Iressa), sunitinib (Sutent;SU11248), erlotinib (Tarceva;OSI-1774), lapatinib (GW572016;GW2016), canertinib (CI 1033), semaxinib (SU5416), batalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec;STI571), and leflunomide. This includes (SU101), vandetanib (Zactima; ZD6474), bevacizumab (avastin), MK-2206 (8-[4-aminocyclobutyl]phenyl)-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthirizine-3(2H)-one hydrochloride) derivatives, analogs thereof, and combinations thereof.Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are, for example, U.S. Patent Application Publication No. 2007 / 0254295, U.S. Patents No. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, and 6,329,380. These are described in Patent Nos. 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated herein by reference in their entirety. In certain embodiments, the tyrosine kinase inhibitor is an orally administered small molecule kinase inhibitor that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical trial, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological sign. Examples of such inhibitors include, but are not limited to, gefitinib, erlotinib, lapatinib, canertinib, BMS-599626 (AC-480), neratinib, KRN-633, CEP-11981, imatinib, nilotinib, dasatinib, AZM-475271, CP-724714, TAK-165, sunitinib, batalanib, CP-547632, vandetanib, bosutinib, restaurtinib, tandetonib, midostaurin, enzastaurin, and AEE. Includes -788, pazopanib, actinivib, motacenib, OSI-930, sediranib, KRN-951, dovitinib, sericiclib, SNS-032, PD-0332991, MKC-I (RO-317453, R-440), sorafenib, ABT-869, brivanib (BMS-582664), SU-14813, teratinib, SU-6668, (TSU-68), L-21649, MLN-8054, AEW-541, and PD-0325901.

[0202] In some embodiments, subjects are administered with i) a population of pluripotent cells and ii) a compound selected from the group that activates MHC expression and / or the immune response, as an immune checkpoint inhibitor.

[0203] As used herein, the term “immune checkpoint inhibitor” refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins modulate the activation or function of T cells. Numerous checkpoint proteins are known, e.g., CTLA-4 and its ligands CD80 and CD86; and PD1 with its ligands PDL1 and PDL2 (Pardoll, Nature Reviews Cancer 12: 252-264, 2012). These proteins are involved in co-stimulating or inhibitory interactions of the T cell response. Immune checkpoint proteins modulate and maintain self-tolerance and the duration and amplitude of the physiological immune response. Immune checkpoint inhibitors contain or are derived from antibodies. In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies (e.g., nivolumab, pembrolizumab), anti-PDL1 antibodies, anti-TIM3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies. Examples of anti-CTLA-4 antibodies are described in U.S. Patents 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; ​​6,682,736; 6,984,720; and 7,605,238. One anti-CTLA-4 antibody is tremelimumab (tisilimumbab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010), a fully human monoclonal IgG antibody that binds to CTLA-4. Another immune checkpoint protein is programmed cell death 1 (PD-1). Examples of PD-1 and PD-L1 blockers are described in U.S. Patents 7,488,802; 7,943,743; 8,008,449; 8,168,757; and PCT published patent applications WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.In some embodiments, the PD-1 blocker includes an anti-PD-L1 antibody. In certain other embodiments, PD-1 blockers include anti-PD-L1 antibodies and similar binding proteins, e.g., nivolumab (MDX 1106, BMS 936558, ONO 4538) (a fully human IgG4 antibody that binds to a PD-1 blocker and blocks PD-1 activation by its ligands PD-L1 and PD-L2); ramlolituzumab (MK-3475 or SCH 900475) (a humanized monoclonal IgG4 antibody against PD-1; CT-011 (a humanized antibody that binds to PD-1), etc.); AMP-224 is a B7-DC fusion protein; antibody Fc portion; BMS-936559 (MDX-1105-01) for PD-L1 (B7-H1) blockade. Other immune checkpoint inhibitors include lymphocyte activator gene 3 (LAG-3) inhibitors, e.g., IMP321 (a soluble Ig fusion protein) etc. (Brignone et al., (2007, J. Immunol. 179:4202-4211). Other immune checkpoint inhibitors include B7 inhibitors (e.g., B7-H3 and B7-H4 inhibitors). In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM3 (T cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). In some embodiments, the immunotherapeutic treatment consists of adoptive immunotherapy, as described by Nicholas P. Restifo, Mark E. Dudley and Steven A. Rosenberg (Adoptive immunotherapy for cancer: harnessing the T cell This is as described in response, Nature Reviews Immunology, Volume 12, April 2012.In adoptive immunotherapy, the patient's circulating lymphocytes or tumor-infiltrating lymphocytes are isolated in vitro, activated with a lymphokine (e.g., IL-2), and re-administered (Rosenberg et al., 1988; 1989). The activated lymphocytes are most preferably the patient's own cells, which have been isolated earlier from a blood sample and activated (or "proliferated") in vitro.

[0204] In some embodiments, subjects are administered with i) a population of pluripotent cells and ii) a compound selected from the group that activates MHC expression and / or the immune response, as a complex preparation and radiotherapy agent.

[0205] As used herein, the term “radiotherapy agent” means any radiotherapy agent known to those skilled in the art to be effective in treating or relieving cancer, but not limited to such agents. For example, a radiotherapy agent may be an agent administered in close-range radiotherapy or radionuclide therapy. Such a method may, depending on the circumstances, further include the administration of one or more additional cancer therapies (e.g., chemotherapy and / or other radiotherapy).

[0206] Pharmaceuticals and vaccine compositions

[0207] The compounds that activate MHC expression and / or immune responses as described above, as well as populations of pluripotent cells, may be combined with pharmaceutically acceptable excipients and, optionally, with sustained-release matrices such as biodegradable polymers to form pharmaceutical compositions.

[0208] "Medically" or "medically acceptable" refers to molecular entities and compositions that, when administered appropriately to mammals, particularly humans, do not produce harmful, allergic, or other adverse reactions. A medically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration can be administered to animals and humans in unit dose forms, as mixtures with conventional pharmaceutical carriers, with the active ingredient alone or in combination with other active ingredients. Appropriate unit dose forms include oral route forms, e.g., tablets, gel capsules, powders, granules, and oral suspensions or solutions; sublingual and buccal administration forms, aerosols, implants; subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, subarachnoid, and intranasal administration forms; and rectal administration forms. Typically, the pharmaceutical compositions include medically acceptable media for injectable formulations. These may be, in particular, isotonic sterile salines (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts) or dry, especially lyophilized compositions, which, depending on the circumstances, allow for the composition of the injectable solution upon addition of sterile water or saline. Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that simple injection is possible. It must be stable under manufacturing and storage conditions and must be preserved against microbial contamination (e.g., bacteria and fungi). Solutions containing the compounds of the present invention as free bases or pharmacokinetically acceptable salts can be prepared in water appropriately mixed with a surfactant (e.g., hydroxypropyl cellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.Polypeptides (or nucleic acids encoding them) can be formulated into neutral or salt-form compositions. pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and are formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine. The carrier may also be a solvent or dispersion containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings (e.g., lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents (e.g., sugars or sodium chloride). Sustained absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions are prepared by incorporating the required amount of active polypeptide in a suitable solvent with some of the other components listed above, as required, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterilized medium containing a basic dispersion medium and other required components from those listed above. In the case of sterilized powders for the preparation of sterilized injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques that produce a powder of the active ingredient + any additional desired components from the previously sterilized filtered solution. At prescription, the solution is administered in a therapeutically effective amount in a manner compatible with the drug formulation. The preparation can be easily administered in various dosage forms (such as the injectable solution forms described above), but drug-releasing capsules can also be used.For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered as needed, and the liquid diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the presented injection site. Some variation in the dose will inevitably occur depending on the condition of the subject being treated. The person administering the drug will determine the appropriate dose for each individual subject in any given event.

[0209] In particular, compounds that activate populations of pluripotent cells and MHC expression and / or immune responses are formulated in vaccine compositions. Accordingly, the present invention relates to vaccine compositions comprising compounds selected from the group i) populations of pluripotent cells and ii) compounds that activate MHC expression and / or immune responses.

[0210] In certain embodiments, a vaccine composition according to the present invention comprises i) human embryonic stem cells and ii) valproic acid.

[0211] A vaccine composition according to the present invention, comprising, in a particular embodiment, i) inducible pluripotent stem cells (iPSCs) expressing a neoantigen, particularly enhanced by a mutagenic agent or genetic modification, and ii) valproic acid.

[0212] The composition may also contain 5-azacytidine.

[0213] Furthermore, the vaccine composition of the present invention can be used in subjects suffering from cancer, as described above.

[0214] Vaccine compositions according to the present invention can be formulated using the above-mentioned physiological excipients in a manner similar to that of immunogenic compositions. For example, pharmaceutically acceptable media include, but are not limited to, phosphate-buffered saline, distilled water, emulsions (e.g., oil / water emulsions), various types of wetting agents, and sterile solutions. Adjuvants such as muramil peptide (e.g., MDP), IL-12, aluminum phosphate, aluminum hydroxide, alum, and / or montanide® can be used in the vaccine.

[0215] The vaccine composition according to the present invention can be administered by subcutaneous (sc), intradermal (id), intramuscular (im), or intravenous (iv) injection, oral administration, or intranasal administration or inhalation. The vaccine is usually administered as a single dose. Alternatively, the administration of the vaccine of the present invention is first produced (first vaccination), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 This is followed by recalls (subsequent administrations) of 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, using the same population of stem cells, immune system stimulating compounds or combinations thereof, and / or combinations with one or more further therapies of radiotherapy, targeted therapy, immunotherapy, or chemotherapy.

[0216] The vaccine composition is also provided in the kit. The kit includes the vaccine composition and an information pamphlet providing instructions for immunization. The kit also includes all materials for administering the product.

[0217] The present invention is further illustrated by the following drawings and embodiments. However, these embodiments and drawings should not be construed as limiting the scope of the present invention. [Brief explanation of the drawing]

[0218] [Figure 1] Vaccination studies using mESCs, hESCs, miPSCs, and 4T1 cells in a 4T1 mammary tumor model. Study design: Mice (5 per group) received two booster immunizations of 7 and 14 days of vaccination using 105 irradiated cells; mouse embryonic stem cells (mESCs), mouse inducible pluripotent stem cells (miPSCs), human embryonic stem cells (hESCs), or 4T1 cells. After 14 days, 5 × 10⁴ 4T1 cells were injected into the mammary fat pads of the mice. Five mice were injected with 4T1-CSCs (4T1 cell growth using additional cytokines, e.g., TGF-beta and TNF-alpha, to generate CSC growth in mammoth sphere morphology). [Figure 2A] Immunoprotection after vaccination. A; Quantification of CD4-positive tumor-infiltrating lymphocytes (TILs) by flow cytometry. B; Quantification of CD25-positive regulatory T cells within CD4+ TILs by flow cytometry. C; Quantification of PD1 regulatory T cells within CD8+ T cells in a mouse population by flow cytometry. [Figure 2B] Immunoprotection after vaccination. A; Quantification of CD4-positive tumor-infiltrating lymphocytes (TILs) by flow cytometry. B; Quantification of CD25-positive regulatory T cells within CD4+ TILs by flow cytometry. C; Quantification of PD1 regulatory T cells within CD8+ T cells in a mouse population by flow cytometry. [Figure 2C] Immunoprotection after vaccination. A; Quantification of CD4-positive tumor-infiltrating lymphocytes (TILs) by flow cytometry. B; Quantification of CD25-positive regulatory T cells within CD4+ TILs by flow cytometry. C; Quantification of PD1 regulatory T cells within CD8+ T cells in a mouse population by flow cytometry. [Figure 3A]Vaccination study using hESCs combined with VPA in a 4T1 mouse model. Study design: Mice (n=5 / group) received two booster immunizations for 7 and 14 days with 105 irradiated cell hESCs, with or without VPA. After 14 days, 5 × 10⁴ 4T1 cells were injected into the mammary fat pads of the mice. A: Tumor volume for each group: 1 / control (PBS), 2 / vaccinated with hESCs, 3 / vaccinated with hESCs combined with VPA, 4 / mice that received VPA only. B: Tumor weight at day 44. [Figure 3B] Vaccination study using hESCs combined with VPA in a 4T1 mouse model. Study design: Mice (n=5 / group) received two booster immunizations for 7 and 14 days with 105 irradiated cell hESCs, with or without VPA. After 14 days, 5 × 10⁴ 4T1 cells were injected into the mammary fat pads of the mice. A: Tumor volume for each group: 1 / control (PBS), 2 / vaccinated with hESCs, 3 / vaccinated with hESCs combined with VPA, 4 / mice that received VPA only. B: Tumor weight at day 44. [Figure 4A] Immunoprotection after hESC vaccination and VPA: A; Decrease in PD1 cells in the spleen within the CD4 and CD8 populations. B; Increase in CD4+ and CD8+ T cells in tumors. C; Increase in CD4+ and CD8+ T cells in the spleen. [Figure 4B] Immunoprotection after hESC vaccination and VPA: A; Decrease in PD1 cells in the spleen within the CD4 and CD8 populations. B; Increase in CD4+ and CD8+ T cells in tumors. C; Increase in CD4+ and CD8+ T cells in the spleen. [Figure 4C] Immunoprotection after hESC vaccination and VPA: A; Decrease in PD1 cells in the spleen within the CD4 and CD8 populations. B; Increase in CD4+ and CD8+ T cells in tumors. C; Increase in CD4+ and CD8+ T cells in the spleen. [Figure 5] Quantification of luciferase reporter genes in the lungs of each of the four animal groups (control, hESC, hESC+ VPA, VPA) after dissection using the IVIS spectral system. [Figure 6A] Real-time PCR of pluripotent gene expression in 4T1 cells and 4T1 CSCs (mammoth spheres) treated with 0.5 mM VPA for 5 days. [Figure 6B] Real-time PCR of pluripotent gene expression in 4T1 cells and 4T1 CSCs (mammoth spheres) treated with 0.5 mM VPA for 5 days. [Figure 7A] Oct4 expression in 4T1 cells treated with VPA and 5Aza by RT-PCR and real-time PCR. [Figure 7B] Oct4 expression in 4T1 cells treated with VPA and 5Aza by RT-PCR and real-time PCR. [Figure 8] Detection of gene variants altering the protein sequence of neoantigens in ENU-treated and untreated iPSCs by exome sequencing. Quantification of variants in ENU-treated and untreated iPSCs (NS = non-synonym, FS = frameshift, SG = obtained stop). [Figure 9] A schematic diagram of the process according to the present invention.

[0219] Examples:

[0220] Example 1

[0221] Fetal tissue has been reported to be usable to immunize mice capable of rejecting transplantable tumors, including cancers of the skin, liver, and gastrointestinal tract. This response is explained by the fact that these tumor cells express a large number of oncoembryonic antigens. To date, several human cancer vaccine trials have been set up to target embryonic antigens, such as oncoembryonic antigen (CEA) and alpha-fetoprotein or cancer / testicular antigens. Unfortunately, targeting a single antigen alone has been shown to be inefficient enough to generate a strong anti-tumor immune response that mediates tumor rejection, due to the rapid emergence of evasive mutants and the general inefficiency of monovalent cancer vaccines. Recent interest in the potential of stem cells in regenerative medicine has made well-defined undifferentiated ESC lines, as well as undifferentiated iPSCs that are phenotypic and functionally similar to ESCs, widely available. In our trials, we hypothesized that undifferentiated stem cells could be used as a multivalent vaccine to generate an immune response against various embryonic antigens shared by tumor cells and CSCs. The inventors have for the first time discovered that ESCs or iPSCs can induce immune and clinical responses against breast cancer. Surprisingly, the inventors have found that the addition of valproic acid to the treatment regimen can induce a higher immune and antitumor response compared to the use of ESCs or iPSCs alone.

[0222] Materials and methods

[0223] The inventors developed a metastatic 4T1 breast cancer model in BALB / c mice. To confirm embryonic ES-like markers in 4T1 mouse TNBC breast cancer cell lines, meta-analysis was performed using embryonic cell samples (D3 stem cells - GSE51782, annotated in Affymetrix plate form GPL16570) and incorporating different datasets: TNBC cell line 4T1 cultured in vitro (GSE73296, annotated in Affymetrix plate form GPL6246), TNBC cell line 4T1 xenografted in the mouse model (GSE69006, annotated in Affymetrix plate form GPL6246), and mammary gland samples (GSE14202, annotated in Affymetrix plate form GLP339) (Padovani et al. 2009). Microarray analysis of balb / c mice transplanted with 4T1 revealed 1304 different genes (TRAP1A, TET1, TSLP, FAM169A, ETV5, MOXD1, PHLDA2, CRIP1, ADAMDEC1, NID1, EPCAM, H2-EA-PS, GPA33, IBSP, KANK3, MEST, MMP9, SPRY4, CLDN4, PRSS22, DDAH2, SPRY2, USP1) It was shown that several genes (including CTNNAL1, ZFP532, GRB10, CACNG7, ST14, CTH, RCN1, PECAM1, TMEFF1, PPP1R1A, GPR97, KIF2C, BRCA2, SLAIN1, CSRP2, DOCK6, HUNK, RAD51, ESYT3, SKP2, CCL24, SFRP1, HMGB2, ITM2A, ASPN, MSH2, SUGT1, ARHGAP8, etc.) are shared with mouse ESCs. All of these genes were found to be generally upregulated in 4T1 and mESCs compared to normal mouse mammary gland cells. It was also shown that xenografted 4T1 cells expressed CSC markers (e.g., CD44) more highly than cells collected in vitro. high CD24 low 39% vs. 0.27% (not shown).

[0224] Using the same format, whole-genome expression profile analysis was performed on triple-negative breast cancer (TNBC) patients. To identify embryonic ES-like markers in patients with TNBC, meta-analyses were performed using embryonic cell samples and human breast samples by mixing sample data from different datasets: dataset GSE18864 (Silver et al. 2010) containing 84 breast cancer samples and annotated with Affymetrix plate form GPL570; dataset GSE20437 (Graham et al. 2010) containing 42 samples of normal human breast and annotated with Affymetrix platform GPL96; dataset GSE23402 (Guenther et al. 2010) containing 42 samples of human embryonic stem cells and induced pluripotent stem cells and annotated with Affymetrix plate form GPL570; dataset of breast cancer cell lines (Maire et al. 2013); and dataset GSE36953 (Yotsumoto et al. 2010) containing cell culture samples of TNBC cell lines and annotated with Affymetrix plate form GPL570. 2013).Supervised one-way ANOVA was used to analyze the three grades of breast cancer across groups and identified 4288 key genes that enable the classification of the majority of triple-negative breast cancer (TNBC) grade III using iPS and ES samples (CDC20, KRT81, NCAPG, MELK, DLGAP5, AURKA, ADAM8, CCNB1, RRM2, QPRT, SLAMF8, EZH2, CENPF, HN1, CENPA, SLC19A1, SLC39A4, CDK1, SEPHS1, GMDS, TUBB, SCRIB, DDX39A, YBX1, MKI67, TKT, WDR1, SKP2, ISG2). (Including 0, NRTN, SEC14L1, GAPDH, ILF2, PSMB2, DHTKD1, TPX2, CCNB2, IL27RA, NADK, H2AFX, MRPS18A, AURKA, MCM7, MCAM, NOP2, KIF23, JMJD4, YIPF3, CDH3, TALDO1, BID, C16orf59, HMMR, BIRC5, ZNF232, RANBP1, CDK1, SHMT2, KIF20A, EPHB4, SPAG5, PPARD, ORC6, TUBB4B, LYZ, TK1, PDXK, NAA10, BAG6, SF3B3, MARCKSL1, MCM3, PSRC1, NUSAP1, etc.) All of these genes were thus found to be generally upregulated in TNBC tumors and hESCs compared to normal human mammary gland tissue.

[0225] result

[0226] Result 1. Vaccination using xenogenic embryonic stem cells generates immunological and antitumor responses against breast cancer.

[0227] The inventors first investigated whether vaccination with irradiated mouse ESCs (mESCs), mouse inducible pluripotent stem cells (mouse iPSCs), human embryonic stem cells (hESCs), or 4T1 cells is effective against breast cancer in a syngeneic 4T1 mouse model. Following this vaccination, mice were attacked using two different types of 4T1 cells: 4T1 cells normally cultured in 10% DMEM of SVEM to generate cancer stem cells (CSCs) that proliferate in the form of mammoth spheres, or 4T1 cells grown with additional cytokines (e.g., TGF beta and TNF alpha). The inventors found that, in contrast to unvaccinated mice, mice vaccinated with hESCs, mESCs, mouse iPSCs, and 4T1 cells generated a consistent cellular immune response against 4T1 cancer, which correlated with a significant reduction in breast tumor volume (p<0.05) (Figure 1). The inventors found that while tumors progressively proliferated in the PBS control group, immunization with mESCs, miPS, or hESCs significantly delayed tumor growth, resulting in statistically significant differences in average tumor size between each group compared to the PBS group (Figure 1). The inventors observed dramatic inhibition of tumor growth when mice were attacked with CSC-derived 4T1 compared to mice attacked with 4T1, which is normal growth under normal conditions indicating that vaccination with syngeneic mESCs preferentially targets CSCs. To further investigate the cellular immune mechanisms mediating the antitumor effect, the inventors analyzed the phenotypes of tumor-infiltrating lymphocytes from different groups and quantified CD4, CD8, CD25, and PD1 subpopulations. The antitumor effect correlated with 1 / an increase in CD4+ TILs that significantly correlated with tumor size (p=0.0039) (Figure 2A), 2 / a decrease in the percentage of CD25-positive cells that was inversely correlated with tumor size (Figure 2B), and 3 / a decrease in PD1-positive cells, which was more pronounced in mice with a better response to the vaccine regimen (vaccination using hECS, 4T1, or mESC) (Figure 2C).

[0228] Result 2. Vaccination using xenoembryonic stem cells combined with valproic acid (VPA) generates a higher antitumor response against breast cancer and inhibits metastasis.

[0229] To evaluate metastatic sites in the 4T1 mammary model, 4T1 cells were genetically modified to express both GFP and luciferase reporter protein (4T1Luc-GFP), enabling in vivo tracking of these cells in deeper organs (spleen, lungs, bones, liver) using bioluminescence imaging (Ivis spectra). This experiment was conducted as previously described, but using only irradiated hESCs as the vaccine; 5 mice per group were given 10 doses with or without VPA at a dose of 0.40 mM in drinking water. 5 The cells received two additional immunizations using irradiated hESC cells, at 7 and 14 days. After 14 days, 5 × 10⁶ cells were immunized. 4 4T1Luc-GFP cells were injected into the mammary fat pads of mice. The inventors found that mice vaccinated with hESC combined with VPA generated a higher cellular immune response against 4T1 cancer in contrast to unvaccinated mice, which correlated with a significant reduction in mammary tumor volume (p<0.05) (Figure 3A) and a reduction in tumor weight (Figure 3B). The antitumor response correlated with a dramatic decrease in PD-1 expression in both CD4+ T cells and CD8+ T cells in mice treated with hESC and VPA (Figure 4A). Furthermore, the antitumor response correlated with a significant increase in the percentage of CD4+ T and CD8+ T cells in tumors (Figure 4B) and spleens (Figure 4C) in mice treated exclusively with the combination therapy (hESC and VPA) compared to the control group (PBS). The inventors also found that all mice treated with the hESC vaccine and VPA showed a significant reduction in lung metastatic tumors (Figure 5). In summary, these results indicate that heterozygous stem-based vaccination (hESC) using VPA is the most potent compared to the use of hESC or VPA alone. These results suggest that heterozygous stem-based vaccination may be an efficient treatment for reducing tumor recurrence in breast cancer.

[0230] Example 2 - Valproic acid regulates the expression of MHC class 1 and embryonic genes.

[0231] Major histocompatibility complexes (MHCs) are a set of cell surface proteins essential for the acquired immune system to recognize foreign molecules that play an essential role in the acquired immune system. The main function of MHC molecules is to bind to novel and foreign antigens and present them on the cell surface for recognition by appropriate T cells: By interacting with CD4 molecules on the surface of helper T cells, MHC class II mediates the establishment of specific immunity called acquired immunity or adaptive immunity. By interacting with CD8 molecules on the surface of cytotoxic T cells, MHC class I mediates infection or destruction of malignant host cells.

[0232] Immune tolerance is a crucial mechanism by which growing tumors with mutated proteins and altered antigen expression are prevented from being eliminated by the host immune system. Tumor immune tolerance can be partially explained by the absence of β2-m on the cell surface and / or the absence of MHC class I on tumor cells. VPA has been shown to increase MHC class I expression on 4T1 cells at doses between 0.2 mM and 2 mM. MHC class I expression on 4T1 and 4T1 mammothspheres (CSCs induced by treatment with TNFα and TGFb) increases 2-3 times after 24 to 72 hours of exposure with 2 mM VPA.

[0233] In particular, VPA was shown to increase the expression of HLA ABC MHC class I (63% vs. 92%) and pluripotency markers (e.g., SSEA4 and Tra1-60) (55% vs. 72%) in iPSCs at a dose of 0.5 mM.

[0234] These markers decreased after ENU exposure (60 days after treatment) and recovered when cells were treated with 0.5 mM VPA (28%–92% of HLA ABC-positive iPSCs-ENU and iPSCs, respectively) and (48%–69% of SSEA4 / Tra-1-60-positive iPSCs-ENU and iPSCs, respectively).

[0235] HDAC inhibitors, acting as VPAs, can selectively alter gene transcription, partly through chromatin remodeling and structural changes in transcription factors. We investigated whether VPAs could regulate the expression of pluripotency genes in breast cancer cells. For this purpose, we treated 4T1 and 4T1 mammospheres (CSCs induced by treatment with TNFα and TGFb) with 1 mM–2 mM VPA. In all cases, VPA increased the expression of three distinct key transcription factors highly expressed in ESCs or iPSCs (e.g., Oct4, Sox2, and Nanog) by 2–3-fold (Figure 6). Importantly, a significant synergistic effect of these transcription factor expressions was observed when tumor cells were treated with a combination of VPA and 5-azacitidine (5aza) (used at doses that inhibit DNA methyltransferase and cause DNA hypomethylation). In particular, treatment of 4T1 cells with VPA and 5aza resulted in a seven-fold increase in oct-4 transcripts (Figures 7A and B).

[0236] Example 3 - Induction of DNA damage and DNA repair errors due to genomic instability in iPSCs exposed to mutagenic drugs (e.g., N-ethyl-N-nitrosourea (ENU)).

[0237] Ethyl-N-nitrosourea (ENU) is a mutagenic alkylating agent that induces base transfers, single point mutations, and double-strand breaks (DSBs).

[0238] ENU made it possible to confirm that DNA damage occurs in iPSCs.

[0239] The amount of phosphorylated gamma-H2AX attracted to DSB sites within cells was evaluated. In this experiment, iPSCs were detached from the stromal culture using collagenase and treated in vitro with ENU at the indicated concentration (50 μg / ml) and time, followed by Western blot analysis using an anti-phospho-gamma-H2AX antibody. An increase in gamma-H2AX levels was observed at the early stages (2-10 minutes), followed by a recovery to basal levels.

[0240] The protocol was designed to induce genomic instability in iPSCs by sequential treatment with ENU to accumulate DNA repair errors over a wide range of growth. Cells were treated for 60 days with daily medium changes, accompanied by daily addition of ENU at a concentration of 10 μg / ml. VPA was added during culture.

[0241] On day 61, the iPSCs are evaluated for the genomic outcomes of the mutagenesis procedure by karyotyping, RNA sequencing, CGH array, exon sequencing, and WGS. Genomic changes accumulated in cultured iPSCs are compared to iPSCs that have not been treated with ENU.

[0242] After ENU exposure, mutated iPSCs were maintained and grown in culture with VPA. CGH array and exome sequencing were performed sequentially via different pathways to confirm the genocopy prevalence of somatic mutations in ENH-iPSCs during their growth. The phenotype of the iPSCs was assessed by evaluating the expression of pluripotency (Pluritest) and Oct4, Sox2, Nanog, Tra-1 60, and SSEA4. The inventors found that the replication rate and population doubling were similar to those of iPSCs not exposed to ENU.

[0243] iPSCs were treated with 10 μg / ml ENU, and exome sequencing was performed to detect mutated neoantigens. Forty-eight changes were found in ENU-treated iPSCs compared to eight changes in untreated iPSCs (Figure 8). These loci were combined with the cBioPortal program (http: / / www.cbioportal.org) to access cancer genomics datasets from human tumor samples from different cancer studies. Using the cBioPortal program, it was found that 10–75% of these changes were also deregulated in cancers (pancreas, lung, prostate, etc.). For all exome sequencing, the inventors estimated a depth of 30–400 readouts for all mutations detectable in ENU-treated iPSCs.

[0244] Example 4 - Haploinsufficiency of BRCA1 leads to DNA repair changes and genomic instability in iPSCs, accompanied by the accumulation of CNVs during culture.

[0245] Alterations in BRCA1 / 2 are included in hereditary breast and ovarian cancer (HBOC) syndromes. BReast CAncer1 (BRCA1) is a tumor suppressor gene that plays a central role in maintaining genomic stability by regulating DNA repair in homologous recombination, double-strand break repair, S phase and G2 / M, spindle checkpoint, and centrosome regulation.

[0246] Fibroblasts with a deletion in exon 17 of BRCA1 were reprogrammed using Sendai virus containing Oct3 / 4, Sox2, Klf4, and cMyc (CytoTune®-iPS Sendai Reprogramming Kit, Life technologies). Cells were cultured in human pluripotent stem cell medium (hPSC medium) based on DMEM / F12 supplemented with 20% Knock Out Serum Replacer, 1 mM L-glutamine, 1% penicillin / streptomycin, 100 μM 2-mercaptoethanol (Life technologies), and 12.5 ng / ml basic FGF (Miltenyi Biotech). On day 26, fully reprogrammed colonies were manually picked based on FACS analysis, Q RT-PCR of Nanog, Oct4, Sox2, teratoma formation in NOD-SCID mice, and their morphology and pluripotency markers by Pluritest. The karyotype was normal.

[0247] The levels and activities of DNA damage response (DDR) in normal (WT) and BRCA1+ / - iPSCs were compared. γH2AX foci were determined by immunofluorescence in proliferating iPSCs after irradiation or ENU exposure. IPSC BRCA1+ / - showed significantly higher levels of phosphorylated ATM / ATR substrates as well as γH2AX recruitment to DNA compared to normal WT-iPSCs, indicating that proliferating IPSC BRCA1+ / - undergoes increased DNA damage compared to WT-IPSCs.

[0248] Since iPSC BRCA1+ / - presented increased levels of DDR at early passages, it was verified whether this could be associated with the accumulation of genomic changes during repeated passages and proliferation.

[0249] CGH arrays in proliferative pluripotent stem cells were analyzed after long-term passage of iPSCs in HDACi (VPA) supplemented medium. For this purpose, Agilent CGHarray experiments were performed using the Roche-Nimbelgen aCGH platform with DNA from iPSC samples. Signal extraction and genomic spacing were identified using Agilent cytogenetics and Nexus Roche-Nimbelgen software on the human genome's HG18. Loci were converted to HG19 coordinates using the Roche-Nimbelgen annotation file (Genes_July_2010_hg19, Roche-Nimbelgen website). European copy number variation (CNV) polymorphisms were excluded from the experiments using the scandb database (Gamazon et al. 2010). Array CGH CNV ratios were plotted as a heatmap using MEV version 4.9.0 standalone software (red: acquisition, green: loss, and dark zero) (Saeed et al. 2003). The affected loci in the origin cell were subtracted from the affected loci in each IPSC sample. The resulting filtered CNVs, specific to each iPSC, were mixed with the COSMIC census database (Futreal et al. 2004). Genomic Circosplots on HG19 were performed using oncogenes found to be affected in each IPSC after filtration. This genome plotting was performed using the OmicCircos R-package in R environment version 3.0.2 (Hu et al. 2014).

[0250] The culture of iPSC BRCA1+ / - (>100 days) was shown to lead to the accumulation of genomic abnormalities concomitant with an increase in genomic instability without ENU exposure at late passages. The karyotype at late passages was normal. Agilent aCGH experiments were performed on DNA extracts from iPSC cells and their respective origin cells. Genomic mapping of intervals affected during these reprogramming events showed that BRCA1+ / - iPSCs were affected by a significant number of loci compared to WT ones. After filtering for CNV polymorphisms in WT iPSCs, only 58 loci were still affected (polymorphisms represent 1.69% of all affected loci), similarly, 5273 loci were still found in BRCA1- / + iPSCs after polymorphism filtering. Most of the loci affected in BRCA1+ / - iPSCs were involved in DNA gain.

[0251] Among these loci affected by genomic instability, some of them are known as driver cancer genes in the consensus COSMIC database. WT iPSCs showed only one cancer locus affected in aCGH (CDK4). BRCA1- / + iPSCs were affected by changes regarding 131 cancer loci, among which 11 genes are known to be affected in breast cancer: MSH2, SMARCD1, TBX3, CDH1, TP53, ERBB2, CDK12, BRCA1, PPP2R1A, AKT2, EP300. These changes were found to be particularly overexpressed on small chromosomes 19 and 17 (chromosome 17 where the BRCA1 locus is located).

[0252] In summary, the majority of iPSC BRCA1+ / - cells showed higher levels of indels (deletions or amplifications) compared to WT-IPSCs. 8% of CNVs in 5273 genes were identified and confirmed. Bioinformatics analysis revealed the expression of 131 genes identified in the cosmic database as being involved in carcinogenesis, essentially in leukemia, epithelial tumors, and mesenchymal tumor cells. Some altered genes were similarly observed in breast and ovarian cancers.

[0253] Replication rate, pluripotency (Pluritest, cell surface marker), and MHC I are maintained and stable throughout the entire culture time in the presence of VPA.

[0254] In conclusion, deletion or inactivation of DNA repair-related genes (e.g., BRCA-1) can induce genomic instability, making it possible to generate mutations associated with multiple CNVs, indels, and MHC I.

[0255] Example 5: N-ethyl-N-nitrosourea (ENU) increases the load of mutant neoantigens in CML-iPSCs.

[0256] Characterization of iPSCs generated from leukemic blood cells of Philadelphia-positive chronic myeloid leukemia (CML) patients. iPSCs were generated using Sendai virus-mediated transfer of pluripotency genes Oct4, c-Myc, Klf4, and Sox2. Cells with pluripotent iPSC morphology were amplified and characterized by cell surface pluripotency markers (Tra-1-60 and SSEA4) and the ability to generate teratomas after intramuscular injection into NSG mice. These iPSCs possessed the Philadelphia chromosome characteristic of CML. CML iPSc cells were exposed to ENU for 60 days. Cell derivative colonies from ENU-treated CML-IPS were compared to IPSCs not treated with ENU.

[0257] DNA from CML iPSCs was analyzed using CGH arrays. Several genomic abnormalities were observed in blast colonies derived from ENU-treated iPSCs, accompanied by the detection of loss of heterogeneity among the genomic abnormalities selected by ENU pressure in CML iPSCs (CB32, which included copy number variation CNVs with 332 loci). After filtering with the European Caucasian genome polymorphism database, 225 loci remained present in these genomic abnormalities. The majority of the genomic abnormalities involved loss of genomic DNA (71%) and loss of heterozygosity (23%). Matching these genomic abnormalities with transcription factor databases, oncogene databases, and pluripotency gene databases revealed that these key deregulated operators were primarily affected on chromosomes 7, 8, 15, Y, and X. Circoplots allowed for the correlation of most of these abnormalities with transcription factors (e.g., MESP and IKZF1, which are involved in mesodermal cell migration). While some pluripotent genes and some oncogenes already described as being involved in Ph1-positive leukemia, such as IDH2, NCOA2, IKZF1, and BLM, were affected, this suggests a correlation with abnormalities generated by ENU-induced mutagenesis.

[0258] This analysis reveals that several genetic abnormalities (e.g., acquisition and loss) and some of the identified abnormalities are oncogenes identified in the Cosmic Database.

[0259] By comparing the abnormalities identified in ENU-iPSCs, it becomes possible to reproduce the invasive acute leukemia phase abnormalities already identified in CML patients in the acute leukemia phase, suggesting that ENU-treated CML iPSCs are a unique tool for reproducing these genomic abnormalities in this particular cancer in vitro.

[0260] Throughout this application, various references describe cutting-edge technologies relating to the present invention. The disclosures of these references are incorporated into this disclosure by reference.

[0261] References: Table 1 TIFF0007880390000003.tif237165 TIFF0007880390000004.tif249165 TIFF0007880390000005.tif60165

Claims

1. A pharmaceutical product for the preventive or therapeutic treatment of cancer, a. A vaccine composition containing a population of pluripotent cells inactivated by radiation; and b. Valproic acid, a histone deacetylase inhibitor. Pharmaceuticals, including medicines.

2. A pharmaceutical product for the preventive or therapeutic treatment of cancer, (a) A vaccine composition comprising a population of pluripotent cells inactivated by radiation, (b) valproic acid, which is administered simultaneously with or in succession to histone deacetylase, Pharmaceuticals.

3. A pharmaceutical product for the preventive or therapeutic treatment of cancer, (b) Contains histone deacetylase which is valproic acid, (a) A vaccine composition containing a population of pluripotent cells inactivated by radiation. Administered simultaneously or consecutively, Pharmaceuticals.

4. The pharmaceutical product according to any one of claims 1 to 3, wherein the pluripotent cells include mutagenic pluripotent cells.

5. The pharmaceutical product according to any one of claims 1 to 4, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, gastric sarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphocytic and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, kidney cancer, head and neck tumors, and solid tumors.

6. A kit for the prophylactic or therapeutic treatment of cancer, a. A vaccine composition containing a population of pluripotent cells inactivated by radiation; and b. Valproic acid, a histone deacetylase inhibitor. A kit that includes this.

7. A kit for the prophylactic or therapeutic treatment of cancer, (a) A vaccine composition comprising a population of pluripotent cells inactivated by radiation, The vaccine composition (a) is administered simultaneously with or consecutively with histone deacetylase which is (b) valproic acid. kit.

8. A kit for the prophylactic or therapeutic treatment of cancer, (b) Contains histone deacetylase which is valproic acid, The histone deacetylase (b) is administered simultaneously with or consecutively with (a) a vaccine composition containing a population of pluripotent cells inactivated by radiation. kit.

9. The kit according to any one of claims 6 to 8, wherein the pluripotent cells include mutagenic pluripotent cells.

10. The kit according to any one of claims 6 to 9, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, gastric sarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphocytic and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, kidney cancer, head and neck tumors, and solid tumors.

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