Methods and compositions for treating cancers

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] The present invention is in the field of oncology, more specifically, the present invention relates to anti-cancer vaccine combination therapy.

[0002] In particular, the present invention relates to methods for producing compositions comprising pluripotent cells that present multiple neoantigens and which 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, which are required during 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 present in oncogenes, tumor suppressor genes, and signaling pathways, leading to clonal waves of tumor evolution. In the "clonal evolution model," the types of mutations fluctuate as cancer develops, so that individual cancer cells become progressively transformed and invasive. Mutations acquired in early tumor development are maintained in advanced disease.

[0005] Evolving over time, these mutations, in conjunction with progressive immune editing and exhaustion, lead to oligoclonal tumor expansion and cancer cell resistance. Cancer mutation signatures are highly associated with genomic instability. Mutations generate novel non-self neo-antigens with immunogenic epitopes. However, because the tumor microenvironment is generally immunosuppressive, the host immune system is generally unable to adequately destroy these cells and combat these cancers. The reported clinical benefits of T cell responses with immune checkpoint inhibitors correlate well with the mutation rate and mutation landscape of the tumor.

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

[0007] Primarily OCT4, NANOG, and SOX2 transcription factors are master regulators that work together as part of a highly integrated network (associated with c-myc and Polycomb networks) to drive the transition from somatic cells to either CSCs or iPSCs using epigenetic mechanisms, remodeling chromatin through histone modifications and DNA methylation. These factors are not present in normal adult stem cells. Embryonic stem cell-like gene expression and under-expression of Polycomb-regulated genes that define the identity of human pluripotent stem cells (ESCs / IPSCs) are associated in 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-likeness” profile are associated with mesenchymal traits in cancer cells with epithelial-mesenchymal “EMT” markers, low levels of MHC-I expression, and an immunosuppressive tumor microenvironment with pro-tumor inflammatory leukocytes, stromal cells, and macrophages. Tumor cells undergoing EMT acquire stem cell-like properties and become CSCs with the ability to migrate very early throughout the organism and persist in dormancy for long periods of time. CSCs act as reservoirs to seed and replenish the tumor compartment. They also proliferate by self-renewal, disseminate to different tissues, and generate metastases. These CSCs share a pluripotent embryonic genetic signature and are resistant to anticancer drugs and radiotherapy. They also evade immune antitumor defenses for the reasons shown above (immunosuppressive microenvironment).

[0008] It has been reported that fetal tissue can be used to immunize mice and induce rejection of transplanted tumors, including those of the skin, liver, and gastrointestinal tract, and this response has been explained by the fact that the tumor cells express multiple carcinoembryonic antigens.

[0009] To date, several human cancer vaccine trials have been set up to target embryonic antigens, such as carcinoembryonic antigen (CEA), alpha-fetoprotein, or cancer / testis antigens. Unfortunately, targeting one antigen alone has been shown to be inefficient enough to generate strong antitumor immune responses mediating tumor rejection, due to the rapid emergence of escape mutants, which leads to the general 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 as well as iPSCs that are phenotypically and functionally similar to ESCs.

[0011] Cancer-associated epigenetic abnormalities are a hallmark trait of cancer stem cells, including all components of the epigenetic machinery (DNA methylation, histone modifications, and non-coding RNAs, specifically microRNA expression).

[0012] Several epigenetic modifiers with tumor-inhibitory activity are currently in clinical use in oncology, including hypomethylating agents (such as azacytidine or decitabine) and histone deacetylase inhibitors (such as vorinostat or romidepsin).

[0013] Using such drugs, it was possible to reprogram cancer cells, and epigenetic reprogramming of the tumor microenvironment by epigenetic drugs is an attractive engineering approach for cancer therapy, since there is clear evidence of cancer-stroma interactions in cancer development.

[0014] Thus, there remains a need for new approaches to prevent and / or treat cancers with stem cell signatures. These cancers express a set of embryonic genes (i.e., also called neoantigens) in common with ESC / IPSC and include, among others, pancreatic, breast, ovarian, colon, lung, kidney, prostate, medulloblastoma, cholangiocarcinoma, liver cancer, chronic and acute leukemia, and myeloma. This class of cancers is most associated with a mesenchymal-like signature that requires the development of therapies that specifically target CSCs to improve survival and enhance the quality of life of cancer patients. In particular, such strategies should lead to the restoration of a permissive anti-tumor microenvironment (the tumor microenvironment is generally immunosuppressive and should thus be remodeled to be immunoresponsive) combined with an immune anti-CSC response. 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 immune response in a patient against an antigen of interest, when an immunogenic composition that comprises or targets said 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 allow the initiation of an immune response against the antigen of interest. The use of HDACi as an adjuvant is particularly interesting for the treatment of cancer, particularly cancer with stem cell signature. The present invention is particularly defined 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 and efficient response of the immune system against tumor cells.

[0019] Indeed, the inventors further hypothesized that pluripotent stem cells (such as 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 capable of inducing MHC I (such as valproic acid) could induce efficient immune and antitumor responses against breast cancer without side effects and signs of autoimmune disease. The inventors also found that this combination regimen was associated with significant inhibition of lung metastasis. Surprisingly, the inventors demonstrated that these responses were greatly improved by the addition of HDACi, especially valproic acid, in the treatment regimen, compared to the use of ESCs or iPSCs alone.

[0020] HDACi for improving immune response

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

[0022] Increased efficacy can be understood as increased immunogenicity of the vaccine composition, increased immune response to the vaccine composition, or increased immune response generated by the vaccine composition, which can be compared to the immune response generated in the absence of the HDACi.

[0023] Vaccine compositions contain immunogenic elements intended to induce a patient to generate an immune response against one or more antigens of interest. The antigen of interest can be any antigen against which an immune response is desired, including any peptide, protein, either from the self (e.g., an antigen from a cancer cell), or exogenous, e.g., bacterial, viral, or parasitic proteins, or other types of antigens, e.g., nucleic acids, sugars, lipopolysaccharides, etc.

[0024] The present invention thus relates to the use of HDACi as adjuvant, in particular for increasing the immune response to a vaccine composition, as well as to HDACi for its 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 antigens of interest, intended to generate an immune response in a patient against said antigens.

[0025] The method and use disclosed herein is particularly interesting when the vaccine composition is a cancer vaccine composition, i.e., comprises the antigen of interest expressed by cancer cells.In particular, this method and use is highly suitable for solid tumor cancer.Indeed, in these types of cancer, the immune microenvironment is particularly immunosuppressive (i.e., there is the expression of cytokines and molecular signals, and 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, possibly by modifying the expression of genes that have immunosuppressive effects in cells present in, near, or around the tumor, allowing immune cells to be enhanced and fight against cancer cells.

[0026] The method described herein can also include administering HDACi for several days after administering vaccine composition.This continuous administration of HDACi can be useful to maintain microenvironment modification for a long enough time for immune cells to "take over" tumor.Generally, this further continuous administration of HDACi consists of daily administration of HDACi at a suitable dose for at least 3 days and up to 1 month after vaccine administration.However, it is preferred if further HDACi administration is carried out for at least 1 week, more preferably at least about 2 weeks.

[0027] Vaccine compositions contain immunogenic elements (compounds) for generating an immune response in a patient against one or more antigens of interest.

[0028] This immunogenic element can be an antigen (or antigens). This antigen can be in any form, depending on the target cells (which is intended to include host cells, as well as bacterial cells, parasitic pathogens, or viral particles), as seen above. It can 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. Cell extracts are lysed cells that have been centrifuged to remove insoluble material (such as membrane fragments, vesicles, and nuclei), and thus consist mostly of cytosol. In another embodiment, the extract may be made using certain techniques to deplete or enrich for certain components (e.g., using sonication to break up large membrane fragments into small particles that remain in the extract, or high-speed centrifugation to remove the smallest insoluble components). Cell extracts can be obtained by any chemical or mechanical action (e.g., pressure, distillation, evaporation, etc.).

[0030] In another embodiment, the immunogenic element is a cellular composition, the cells of which express an antigen of interest. This embodiment is preferred if the membranes of the cells are preserved (so that antigen presentation is via the MHC-1 pathway), or if the cells are inactivated, as described below.

[0031] In these embodiments, the cells may be pluripotent cells (described below), cancer cells, virally 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 an antigen of interest. This composition is an antigen-presenting cell vaccine consisting of an antigen and an antigen-presenting cell (APC). Antigen-presenting cells are cells that present antigens complexed with major histocompatibility complexes (MHC) on their surface. Dendritic cells (DCs) can be cited, which are preferred in the context of the present invention, because they are able to present antigens to both helper and cytotoxic T cells, macrophages, or B cells. These APCs can be natural or engineered cells. In particular, Eggermont et al. (Trends in Biotechnology, 2014, 32, 9, 456-465) can be cited, which provides an overview of the progress 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 by 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 cells lymphocytes that have been prestimulated in vitro against an antigen of interest, for example, by exposure to antigen-presenting cells that present the antigen of interest. As a result, this composition can initiate an immune response in vivo against the antigen of interest. This strategy can be called "adoptive transfer of T cells," and it is known that such adoptively transferred T cells persist in vivo for long periods and readily migrate between lymphatic 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 these embodiments, the HDACi is administered in combination with a vaccine composition comprising an immunogenic element. Said administration can be simultaneous, separate or sequential, as disclosed below for the embodiment in which the immunogenic element is a composition of pluripotent cells. Note that all the following statements disclosing compositions of pluripotent cells are equally applicable to vaccines comprising any of the immunogenic elements disclosed above.

[0035] Herein, HDAC inhibitors (especially valproic acid) are emphasized in conjunction with the composition of pluripotent cells, because such pluripotent cells express neoantigens that, as recalled above, are also found in highly aggressive cancers. Consequently, whatever the immunogenic element, 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 element is a cellular composition, which has been obtained by proliferation and inactivation of pluripotent cells, as disclosed in more detail below.

[0037] Method of treating a subject suffering from cancer using the combined preparation

[0038] The present invention relates to a method for treating a subject suffering from cancer, comprising the step of administering to said 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 and / or immune response, as a combined preparation.

[0039] In favorable circumstances, the cells are cultured to present neoantigens through the MHC I pathway, with a particular subset of cells in the population presenting mutations. Compounds used in combination with the cells may also preserve the pluripotency of the cells. Following administration of the cells, it is highly preferred to enhance the immune response by administration of a compound (preferably the same as that first administered in the combination, but potentially another) that activates MHC expression and / or the immune response.

[0040] As used herein, the term "treat" or "treatment" refers to both prophylactic or preventative treatments of subjects at high risk of suffering from or suspected of suffering from cancer (such as hereditary familial cancer syndromes), as well as curative or disease-modifying treatments of subjects who are ill or diagnosed as suffering from cancer or a medical condition, including suppression of clinical recurrence. Treatment is administered to subjects who have cancer or who may eventually acquire cancer to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of cancer or recurrent cancer, or to prolong the survival of the subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of treatment of a disease, e.g., a pattern of dosing used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the subject during the initial period of the treatment regimen. An induction regimen may use (in part or in whole) a "loading regimen", which may involve administering a larger amount of drug than the physician would use during a maintenance regimen, administering a drug more frequently than the physician would use during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a subject during disease treatment, for example, to keep the subject in remission for an extended period of time (months or years). A maintenance regimen may use continuous treatment (e.g., administering a drug at regular intervals (e.g., weekly, monthly, yearly, etc.)) or intermittent treatment (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., pain, disease symptoms, etc.)).

[0041] As used herein, the term "concurrent administration" refers to administration of two active ingredients by the same route at the same time or substantially the same time. The term "separate administration" refers to administration of two active ingredients by different routes at the same time or substantially the same time. The term "sequential administration" refers to administration of two active ingredients at different times, where the routes of administration are the same or different.

[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 suffering from or susceptible to a cancer having expression of a pluripotent embryonic-like stem cell antigen.

[0043] As used herein, the term "population" refers to a population of cells, where a majority (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 preferentially at least about 90%) of the total number of cells have a particular characteristic of a cell of interest (e.g., pluripotency markers for iPSCs, ESCs as defined by the International Stem Cell Initiative which includes 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 cellular characteristics are 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 "pluripotent" refers to cells with the ability to give rise to progeny that can, under appropriate conditions, differentiate into all cell types derived from the three germ layers (endoderm, mesoderm, and ectoderm) with characteristics of a particular cell lineage. The term "pluripotent" includes all sources of adult somatic cells (ASCs) and normal embryonic stem cells (ESCs) reprogrammed from cellular origins, or very small embryonic-like stem cells (VSELs) or engineered induced pluripotent stem cells (iPSCs).

[0046] Pluripotent stem cells contribute to tissues of prenatal, postnatal, or adult organisms. Standard art-accepted tests are used to establish the pluripotency of a cell population, such as the ability to form teratomas in 8-12 week old SCID mice, and various pluripotent stem cell characteristics. More specifically, human pluripotent stem cells express at least some (at least three, more commonly at least four or five), and possibly all, of the markers from the following non-limiting list: 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 capacity to develop into all types of fetal and adult cells, possibly except for the placenta. Embryonic pluripotent stem cells (ESCs) can typically be isolated from 50-150 cells (4-5 day old post-fertilization blastocysts). Although ESCs are capable of indefinite ex vivo expansion, they only exist transiently in vivo during embryonic development. A variety of animal (including human) ESC lines (such as the NIH approved cell line WAO9 human ESC) can be obtained commercially from WiCell Research Institute (Madison, WI). Human ESC lines (such as Cecol-14) can be obtained commercially from, for example, Cecolfes (Bogota, 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 totipotent in nature. Methods for preparing hESCs are well known and are taught, for example, in U.S. Patent Nos. 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. Published Application No. 2005 / 0176707, International Application No. WO2001085917. In the context of the present invention, human embryonic stem cells (hESCs) are generated without embryo destruction according to the technique described in Chung et al 2008.

[0049] As used herein, the term "induced pluripotent stem cells" refers to pluripotent stem cells artificially derived from non-pluripotent cells by reprogramming procedures using methods known in the art and first disclosed by Yamanaka (especially 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 induced pluripotent stem cells (iPSCs) by ectopic expression of defined factors (such as Oct4, Sox2, Klf4 and c-My, or Oct4, Sox2, Lin28 and Nanog). In certain embodiments, the induced 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 different origins (fibroblasts, blood cells, keratinocytes, etc.) using variable technologies (e.g. integrative lentivirus / retrovirus and non-integrative vectors such as Sendai virus, episomal vectors, synthetic mRNA, adenovirus, rAAV, recombinant proteins, etc.) with or without small chemical compounds.

[0051] Small molecules can be used to enhance the derivation and quality of mouse and human iPSCs by acting as epigenetic modifiers (i.e., modifying the expression of some genes). By way of illustration, BIX01294 (BIX, a G9a histone methyltransferase inhibitor), sodium butyrate (NaB, a histone deacetylase HDAC inhibitor), or S-adenosylhomocysteine ​​(SAH, a DNA demethylating agent), 5-azacytidine (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, bona fide iPSCs express pluripotency genes similar to embryonic stem cells with self-renewal capacity and represent an unlimited source of stem cells (or stem cell-like cells).

[0053] ESCs and IPSCs can be repeatedly expanded for multiple and unlimited passages allowing for an expandable stem cell source. Pluripotent capacity is actively maintained in permissive culture conditions by preserving high level expression of pluripotency genes. These methods are known in the art.

[0054] Although certain culture conditions and methods allow for stable genome duplication, some exome mutations and epigenomic 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 germ line cells (sperm and eggs).

[0056] As used herein, the term "allogeneic cells" refers to cells from the same species, but that are genetically different.

[0057] As used herein, the term "syngeneic or autologous cells" refers to cells from the same species and same genetic background.

[0058] As used herein, the term "heterologous cells" refers to genetically distinct cells from different species.

[0059] In certain embodiments, the stem cells may be derived from mammals, including but not limited to rodent, porcine, feline, canine, and primates (including humans).

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

[0061] In a first aspect, the present invention provides a method for producing a cell composition, comprising the steps of: i) expanding pluripotent cells in the presence of an agent that induces MHC-I presentation of antigen in said population during the expansion step, in the presence of conditions that maintain the pluripotent capacity of the cells; ii) exposing the expanded cells to an inactivating agent that inactivates the cells; iii) The expanded inactivated cells are harvested and conditioned. In certain embodiments, the integrity of the cell envelope is maintained in step ii). In another embodiment, the cells are inactivated and a cell-derived product, such as a cell extract, is obtained. Cell compositions produced according to the above methods can be used for the treatment of cancer according to the methods disclosed herein.

[0062] Agents for MHC I antigen presentation

[0063] Pluripotent cells are grown in the presence of an agent that improves the presentation of antigens through the MHC I pathway. Such improved expression can be checked by comparing the number of MHC I molecules at the surface of the cells in the presence and absence of the agent. Such agents are known in the art and may in particular be mentioned as histone deacetylase inhibitors (HDACi). Many products with this activity are known in the art and among these HDACi, may in particular be mentioned as valproate (VPA or valproic acid, CAS number 99-66-1). Other HDACi that may be used (with the same mechanism of action as VPA) are in particular vorinostat, romidepsin-quidamide, panobinostat, belinostat, panobinostat, mocetinostat, abexinostat, entinostat, SB939, resminostat, divinostat or quisinostat. These agents are present in the cell culture (growth) medium during the expansion of the pluripotent cells.

[0064] Maintains the pluripotency of cells.

[0065] The cells are grown under conditions (medium, temperature) that maintain the pluripotent potential of the cells. These culture conditions are known in the art. Maintaining the pluripotent potential of the cells ensures that such cells express (and therefore are present) all embryonic antigens, thereby increasing the ability of the cells to present such antigens on their surface through 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 the immune system, which may be primed by the vaccine composition of the present invention. Therefore, maintenance of pluripotency of the cells of the composition according to the invention obtained by the method disclosed herein leads to the presentation of a wide variety of embryonic antigens and thus to the ubiquitous efficacy of the vaccine composition of the invention in the treatment methods disclosed herein. The proliferation of cells under conditions that maintain pluripotency is known in the art. It is described in particular 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). It is preferred to use the following conditions: Use of E8 medium or any clinical grade ES / iPSC medium, optionally supplemented with VPA and / or mutagenic agents (e.g. ENU, see below). - Temperature of 37°C with or without hypoxia. - Daily medium change 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 p53 inhibitors and / or compounds that enhance cell survival (such as Y-27632 Rock inhibitor). Cells are typically cultured for 8 weeks and 90% optimal density is maintained by regular passaging once a week using enzymatic detachment (collagenase, trypsin).

[0066] Inactivate the cells.

[0067] In a preferred embodiment, the pluripotent cells used in the methods of treatment disclosed herein are inactivated. The term "inactivated" and grammatical variations thereof are used herein to refer to cells (e.g., pluripotent cells) that are viable but rendered incapable of proliferation (i.e., mitotically inactivated). One skilled in the art may use techniques known in the art, including, but not limited to, exposure to chemical agents, irradiation, and / or freeze-drying. Pluripotent cells can be inactivated such that upon administration to a subject, the pluripotent cells are incapable of division, and thus are incapable of forming teratomas in the subject. In the context of multiple cells, it is understood that not all cells need to be incapable of proliferation. Thus, as used herein, the phrase "inactivated to a degree sufficient to prevent teratomas from forming in a subject" refers to the degree of inactivation in the population as a whole, such that after administration to a subject, teratomas do not form, because the exposed pluripotent stem cells no longer divide, as confirmed by in vitro culture. It is hypothesized that even if one or more of the cells in the plurality are in fact capable of proliferation in a subject, the host's immune system will destroy those cells before they can form a teratoma. Such inability to proliferate and form a teratoma may be confirmed by testing in mice with functional and non-functional immune systems.

[0068] In some embodiments, the "inactivated" cells are killed cells, and in some embodiments, the inactivated cells are whole cell lysates, exosomes derived from pluripotent stem cells, concentrated cancer stem neo-antigens, whole purified cancer stem neo-antigens, DNA, RNA and protein extracts, lyophilized whole cell suspensions, fractions of cell lysates (such as membrane fractions, cytoplasmic fractions, or combinations thereof). Inactivated pluripotent stem cells are still capable of stimulating immune responses when vaccination of mice is performed with hESCs or hiPSCs in combination with valproic acid or another HDACi. This vaccination can induce efficient immune and anti-tumor responses against 4T1 breast cancer without side effects and evidence of 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 amount of radiation delivered to the cells and the length of the dose are not important, so long as it renders the cells nonviable.

[0070] Cell harvesting and conditioning.

[0071] The harvesting step of this method involves one (or more) steps of washing the cell culture and resuspending the cells in any suitable medium (such as X-Vivo / Stemflex medium or any other clinical grade cell culture medium).

[0072] Conditioning the cells may include freezing or lyophilizing the cells so that the cell composition can be stored prior to use.

[0073] Multipotent cells are mutated to express neoantigens.

[0074] It is recalled that pluripotent cells are genetically very stable cells, in fact they are present very early in the embryonic development process, and since they must proliferate for the embryonic development, it is important that these cells are not prone to mutations in order to have homogeneity in the embryo.

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

[0076] When preparing iPSCs, a selective advantage of some cells arises over multiple passages, leading to a population of iPSC clones that display specific mutations at later passages, but whose cellular genomes are nearly 100% similar in sequence.

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

[0078] However, it would be preferable to be able to induce mutations in cells to increase the variability of fetal / embryonic neoantigens on the treated cellular material found in aggressive cancers, thus increasing the likelihood that the immune system will be able to generate T cells against these mutated cells and fight the cancer cells as well as cancer cells that may undergo subsequent changes during tumor growth.

[0079] This can help to combat cancers caused by the accumulation of genetic alterations due to DNA replication errors and / or environmental insults during the proliferation of cancer stem cells. These alterations include cancer-driving mutations that initiate carcinogenesis and genome destabilizing mutations. This increased genome instability leads to clonal evolution that leads to the selection of more aggressive clones with increased drug resistance.

[0080] Consequently, in certain embodiments, cells are grown in conditions that induce mutations in genes of said cells.

[0081] Cells can thus be exposed to mutagenic agents, i.e., physical or chemical agents that alter the genetic material of an organism, usually DNA, thus increasing the frequency of mutations above natural background levels.

[0082] The mutagen may be selected from the group consisting of physical mutagens and chemical mutagens.

[0083] Among the physical mutagens the following can be cited: - Ionizing radiation (such as X-rays, gamma rays, and alpha particles) that can cause DNA breakage and other damage. In particular, radiation from Cobalt-60 and Cesium-137 can be cited. The level of radiation must be much lower than that used for cell inactivation and can be designed by the skilled artisan. - Ultraviolet light with wavelengths above 260 nm (which can cause errors in reproduction if left uncorrected). -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 radical, hydrogen peroxide, etc.; -Deaminating agents capable of producing transition mutations by converting cytosine to uracil (e.g. nitrous acid); -Polycyclic aromatic hydrocarbons (PAHs) (which can bind to DNA when activated to diol-epoxides); - alkylating agents, such as ethylnitrosourea (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 species; -Bromine and some compounds containing bromine; -Sodium azide; - Bleomycin; -Psoralen in combination with ultraviolet light; -benzene; - Base analogues that can substitute for DNA bases during replication and cause transition mutations; -Intercalating agents, such as ethidium bromide, proflavine, daunorubicin, etc.; - Metals, such as arsenic, cadmium, chromium, nickel, and their compounds which are mutagenic.

[0085] In this embodiment, one would obtain a population of pluripotent stem cells whose cells carry random mutations (which generally vary from cell to cell, thereby leading to a heterogeneous population) specifically in cancer-associated neo-antigens.

[0086] The inventors have shown that it is possible to design culture conditions that allow 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, since there should be as few mutations as possible introduced during the early stages of embryonic development, which leads to DNA repair mechanisms being very efficient in these cells, thereby correcting most defects and / or inducing apoptosis if it is not possible to correct these defects.

[0088] In one embodiment, a starting population of pluripotent cells (such as ESCs or IPSCs) are grown and maintained in a pluripotency-permissive culture medium (as known in the art) to preserve the pluripotent stage during repeated passaging. In these conditions, typically a small amount of exome mutations (5-10 mutations per exome) can be observed.

[0089] The pluripotent cells are then cultured in vitro using mutagenic compound methods to induce and increase genomic instability in pluripotent stem cells as listed above. DNA damage is fully 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 foci are increased in ESCs or IPSCs as well as a higher number of micronuclei as a mark of genomic instability.

[0090] Preferred drugs are bleomycin, ENU, alkylating agents, actinomycin D, ROS modulators, UV, H2O2, ionizing radiation (gamma rays, X-rays), which allow to induce and enhance the mutation rate in pluripotent stem cells accumulating in culture.

[0091] In a preferred embodiment, N-ethyl-N-nitrosourea (ENU) has been shown to create novel mutations and enhance the levels of neo-antigens 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, it is possible to accumulate multiple mutations in response to DNA damage during pluripotent cell proliferation with high mutation rates from selective advantage in long-term culture, while maintaining cellular pluripotency, especially when cells are cultured with HDACi in the medium. The presence of HDACi in culture preserves the increase in active histones (H3K4me3 and H3K9ac) and epigenetic marks of pluripotency in response to induction of DNA damage, as well as replication and proliferation rates between passages.

[0093] In another embodiment of the compositions and methods described herein, mutations are induced in pluripotent cells through genetic modification of cells with genes that promote high levels of genomic instability. In particular, suitable inhibitors (such as 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 of inducing genomic instability associated with increased DNA damage can be performed by using "vectors" or by "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 are DNApkC, Ku70, Rad51, Brca1, or Brca2.

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

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

[0096] In a preferred embodiment, a population of mutant pluripotent cells is generated from somatic cells that contain a genomic alteration associated with a disease, typically a translocation (T9:22), deletion (BRCA1 / 2), or mutation (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 neo-antigens by using a "vector". In a particular embodiment, a population of ESCs or IPSCs is genetically modified to express multiple mutations and cancer stem cell specific neo-antigens (at least 5) in pluripotent stem cells by "genome editing" techniques. The present invention provides compositions and methods to provide ESCs or IPSCs by introducing those multiple neo-antigens by RNA-guided multiplex genome editing, modification, inhibition of expression, and other RNA-based techniques.

[0099] The term "genome editing" as used herein refers to RNA-mediated genetic engineering, in particular including guide RNA for cas9-mediated genome editing. The guide RNA (gRNA) is transfected with endonuclease cas9. The guide RNA provides a scaffold and a spacer sequence that is complementary to the target. In another embodiment, the genetic engineering sequence can be an siRNA or microRNA sequence designed for gene silencing according to standard methods in the art by using the Crispr-Cas 9 system. Compositions and methods for making and using the Crispr-Cas system are known in the art and are described, in particular, in U.S. Patent No. 8,697,359.

[0100] In certain embodiments, the population of pluripotent cells is treated with an alkylating agent. As used herein, the term "alkylating agent" refers to an agent that adds one or more alkyl groups from one molecule to another. This treatment creates new mutations in neo-antigens that provide a superior immune response by increasing oligoclonal expansion of TILs and Th1 / Th2 cellular immunity.

[0101] In the present invention, the alkylating agent is selected from the group consisting of nitrogen mustards, nitrosoureas, alkylsulfonic acids, triazines, ethylenimines, and combinations thereof. Non-limiting examples of nitrogen mustards include mechlorethamine (Lundbeck), chlorambucil (GlaxoSmithKline), cyclophosphamide (Mead Johnson Co.), bendamustine (Astellas), ifosfamide (Baxter International), melphalan (Ligand), melphalan flufenamide (Oncopeptides), and pharmaceutically acceptable salts thereof. Non-limiting examples of nitrosoureas include streptozocin (Teva), carmustine (Eisai), lomustine (Sanofi), and pharmaceutically acceptable salts thereof. Non-limiting examples of alkylsulfonic acids include busulfan (Jazz Pharmaceuticals) and pharmaceutically acceptable salts thereof. Non-limiting examples of triazines include dacarbazine (Bayer), temozolomide (Cancer Research Technology), and pharma- ceutically acceptable salts thereof. Non-limiting examples of ethylenimines include thiotepa (Bedford Laboratories), altretamine (MGI Pharma), and pharma-ceutically acceptable salts thereof.Other alkylating agents include ProLindac (Access), Ac-225BC-8 (Actinium Pharmaceuticals), ALF-2111 (Alfact Innovation), thoronphosphamide (Baxter International), MDX-1203 (Bristol-Myers Squibb), thioureidobutyronitrile (CellCeutix), mitobronitol (Chinoin), mitolactol (Chinoin), nimustine (Daiichi Sankyo), glufosfamide (Eleison Pharmaceuticals), HuMax-TAC ​​and PBD ADC combination (Genmab), BP-C1 (Meabco), threosulfan (Medac), nifurtimox (Metronomx), improsulfan tosylate (Mitsubishi Tanabe Seiyaku), ranimustine (Mitsubishi Tanabe Seiyaku), ND-01 (NanoCarrier), HH-1 (Nordic Nanovector), a combination of 22P1G cells and ifosfamide (Nuvilex), estramustine phosphate (Pfizer), prednimustine (Pfizer), lurbinectedin (PharmaMar), trabectedin (PharmaMar), altreatam (Sanofi), SGN-CD33A (Seattle Genetics), fotemustine (Servier), nedaplatin (Shionogi), heptaplatin (Sk Holdings), apaziquone (Spectrum Pharmaceuticals), SG-2000 (Spirogen), TLK-58747 (Telik), laromustine (Vion Pharmaceuticals), procarbazine (Alkem Laboratories Ltd.), and pharma- ceutical acceptable salts thereof. In another embodiment, the alkylating agent is selected from the group consisting of mechlorethamine (Lundbeck), chlorambucil (GlaxoSmithKline), cyclophosphamide (Mead Johnson Co.), streptozocin (Teva), dacarbazine (Bayer), thiotepa (Bedford Laboratories), altretamine (MGI Pharma), pharma- ceutical acceptable salts thereof, and combinations thereof.In another embodiment, the alkylating agent is selected from the group consisting of ProLindac (Access), Ac-225BC-8 (Actinium Pharmaceuticals), ALF-2111 (Alfact Innovation), bendamustine (Astellas), ifosfamide (Baxter International), trofosfamide (Baxter International), MDX-1203 (Bristol-Myers Squibb), temozolomide (Cancer Research Technology), thioureidobutyronitrile (CellCeutix), mitobronitol (Chinoin), mitolactol (Chinoin), nimustine (Daiichi Sankyo), carmustine (Eisai), glufosfamide (Eleison Pharmaceuticals), HuMax-TAC ​​and PBD ADC combination (Genmab), busulfan (Jazz Pharmaceuticals, melphalan (Ligand), BP-C1 (Meabco), treosulfan (Medac), nifurtimox (Metronomx), improsulfan tosylate (Mitsubishi Tanabe Pharma), ranimustine (Mitsubishi Tanabe Pharma), ND-01 (NanoCarrier), HH-1 (Nordic Nanovector), combination of 22P1 G cells and ifosfamide (Nuvilex), melphalan flufenamide (Oncopeptides), estramustine phosphate (Pfizer), prednimustine (Pfizer), lurbinectedin (PharmaMar), trabectedin (PharmaMar), altreatam (Sanofi), lomustine (Sanofi), SGN-CD33A (Seattle Genetics), fotemustine (Servier), nedaplatin (Shionogi), heptaplatin (Sk Holdings), apaziquone (Spectrum Pharmaceuticals), SG-2000 (Spirogen), TLK-58747 (Telik), laromustine (Vion Pharmaceuticals), procarbazine (Alkem Laboratories Ltd.), pharma- ceutical acceptable salts thereof, and combinations thereof.

[0102] In certain embodiments, the population of pluripotent cells is treated with N-ethyl-N-nitrosourea (ENU, CAS number 759-73-9). ENU has the following chemical formula C 3 H 7 N 3 O 2 and is a highly potent mutagen by transferring an ethyl group to nucleobases in nucleic acids.

[0103] As shown above, the purpose of the mutagenic agent is to introduce random mutations into the genes of pluripotent cells during expansion (introduction of mutations occurs during cell replication and division). A population of pluripotent stem cells acquires mutations that may provide a growth advantage and are selected for to promote culture adaptation. Passaging of ESCs or iPSCs is subject to high levels of selection pressure, and multiple clonal mutant populations may be selected for during expansion.

[0104] Note that pluripotent cells are very stable, so application of the mutagen may have to be carried out for a long period of time. As a matter of example, if 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 at least 60 days. After application of the mutagen, the cells can be washed (if the mutagen is a chemical agent) and further grown in the presence of an agent favoring MHC-1 expression, in particular HDACi. This agent is preferably also present during application of the mutagenic agent.

[0105] Thus, it can be observed that mutagens induce mutations (i.e. nonsynonymous, nonsense, frameshift, stop, gain, splice variants, CNV, SNV) in some of the embryogenesis genes expressed by pluripotent cells, thus increasing the diversity of these antigens (new neo-antigens within the whole genome). This increases the possibility of vaccine compositions with enhanced immunogenicity that can stimulate a broad immune response against aggressive cancers where rapid and frequent mutations exist.

[0106] Effective immune responses may be difficult to obtain for some cancers, where the clonal proliferation of cancer cells is accompanied by mutations in antigens expressed by tumor cells. Immune responses will thus depend on the mutation load of cancer. The generation of random mutations in pluripotent cell populations by using mutagens leads to the expression of mutated embryonic antigens, which increases the diversity of antigens presented to the immune system upon vaccination.

[0107] As a result, there will already be primed T cells against the mutated antigens that appear in cancer cells during the division of such cells and that can accelerate and improve the immune response against these cells.

[0108] Engineering pluripotent cells.

[0109] In certain embodiments, a population of pluripotent stem cells is genetically modified to overexpress a compound that stimulates immune response by using gene integration in the genome of pluripotent cells.Typically, in the first step, a population of stem cells is isolated and expanded.In the second step, a gene of interest is packaged in an integrative viral vector (such as retrovirus or lentivirus).In the third step, the integrative viral vector containing the gene of interest is transferred into a population of stem cells.

[0110] In certain embodiments, the population of pluripotent cells is engineered with a gene for a protein that stimulates MHC expression and / or an immune response, the compound being 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), functional fragments thereof, and combinations thereof.

[0111] Interferons (IFNs) contemplated by the present invention include the general types of IFNs, 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 production of antigens, thus making it easier for the immune system to recognize and destroy the cancer cells. IFNs can also act indirectly on cancer cells, for example, by slowing down 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] Interleukins contemplated by the present 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] Colony stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte macrophage colony stimulating factor (GM-CSF or sargramostim), and erythropoietin (epoetin alfa, darbepoetin). Treatment with one or more growth factors can help stimulate the production of new blood cells in subjects undergoing traditional chemotherapy. Thus, treatment with CSFs can help reduce the side effects associated with chemotherapy and allow for the use of higher doses of chemotherapy agents. A variety of recombinant colony-stimulating factors are commercially available, e.g., Neupogen® (G-CSF; Amgen), Neulasta (pegfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Ortho Biotech (erythropoietin; Amgen), Arnesp (erytropoietin).

[0114] In the broadest sense, a "vector" is any vehicle capable of facilitating the transfer of an oligonucleotide into a cell. Preferably, the vector transports the nucleic acid to the cell with reduced degradation compared to the extent of degradation that would result in the absence of the vector. In general, vectors useful in the present invention include naked plasmids, non-viral delivery systems (electroporation, sonoporation, cationic transfection agents, liposomes, etc.), phagemids, viruses, other vehicles derived from viruses or bacterial sources that have been engineered with the insertion or incorporation of nucleic acid sequences. Viral vectors are a preferred type of vector, including, but not limited to, nucleic acid sequences from the following viruses: RNA viruses, such as retroviruses (e.g., Moloney murine leukemia virus and lentivirus-derived vectors), Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma virus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; polio virus. Other vectors not named, but known in the art, can readily be 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 fact, 12 different AAV serotypes (AAV1-12) are known, each with a different tissue tropism (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAV is derived from the dependent parvovirus AAV (Choi, VW J Virol 2005; 79:6801-07). Adeno-associated virus types 1-12 can be engineered to be replication-deficient and are capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316-27). It further has advantages, such as heat and lipid solvent stability; high transduction frequency in cells of diverse lineages, including hematopoietic cells; lack of superinfection inhibition, allowing multiple rounds of transduction. Also, wild-type adeno-associated virus infections have persisted for more than 100 passages in tissue culture in the absence of selective pressure, implying that adeno-associated virus genome integration is a relatively stable event. Adeno-associated virus can also function in an extrachromosomal fashion.

[0116] Other vectors include plasmid vectors. Plasmid vectors have been described extensively in the art and are well known to those of skill in the art. See, for example, Sambrook et al., 1989. Over the past few years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as many of the viral vectors. However, these plasmids, which have a promoter compatible with the host cell, 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 of skill in the art. In addition, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or by other routes. It may also be administered by intranasal spray or drops, rectal suppository and orally. Preferably, the DNA plasmid is injected via intraocular methods (intravitreal, subretinal, suprachoroidal, etc.). It may also be administered into the epidermis or mucosal surfaces using a gene gun. The plasmid may be provided in an aqueous solution, dried onto gold particles, or associated with another DNA delivery system (including but not limited to liposomes, dendrimers, cochleates, and microencapsulation).

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

[0118] The term "homologous recombination" as used herein refers to a gene targeting method for artificially modifying a specific gene on a chromosome or genome. When a genomic fragment having a portion homologous to a portion of a target sequence on a chromosome is introduced into a cell, the recombination occurs based on the nucleotide sequence homology between the introduced genomic fragment and the corresponding gene locus on the chromosome.

[0119] The term "genetic modification" also refers to the insertion of exogenous DNA at a desired locus on a chromosome, the replacement of a part or the whole of a gene with 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"), where the endogenous DNA sequence is maintained in such a manner that the fragment is expressed together with the expression of the gene at a particular locus or is constitutively expressed, or the replacement, deletion, or disruption of a part or the whole of the gene sequence (i.e., "knock-out") that modifies 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 (U.S. Pat. No. 4,684,611), and methods using lipid carriers (e.g., lipofectin, etc.) (Teifel et al., (1995) Biotechniques 19: 79-80, Albrecht et al., (1996) Ann. Hematol. 72: 73-79; Holmen et al., (1995) In Vitro Cell 19: 74-75). 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), electroporation and methods for fusion with microcells (U.S. Pat. Nos. 5,240,840, 4,806,476, 5,298,429, and 5,396,767; Fournier (1981) Proc. Natl. Acad. Sci. USA 78: 6349-6353 and Lambert et al., (1991) Proc. Natl. Acad. Sci. USA 88: 5907-59).

[0121] Population of cells

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

[0123] The invention thus relates to a composition of cells comprising pluripotent cells, wherein the cells in said 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 at least 50% in at least three genes selected from the 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 in at least four genes, more preferably in at least five genes, more preferably in at least six genes, more preferably in at least seven genes.

[0124] This mutation rate of the gene is tested in the cell population after exposure to the mutagenic agent, before or after further expansion, if such further expansion is performed.

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

[0126] Exposure of cells to mutagenic agents induces the appearance of random mutations in the genome of such cells, and the population resulting from such exposure is thus heterogeneous compared to a population of pluripotent cells that is essentially homogenous due to the low spontaneous mutation rate during long-term growth and culture. The populations obtained herein are thus in particular: - The cells are pluripotent (i.e., have markers of pluripotency). - As shown above, there are multiple differences in the genome of the cells in the population, i.e. it is possible to detect the rates of the mutant genes listed above (as shown above) in the cells of the population.

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

[0128] In a further embodiment, the invention thus relates to a composition of cells comprising pluripotent cells, wherein the cells in said population display a mutation landscape in a population of ESCs or IPSCs comprising one or more of the following characteristics: i) At least >3 (as seen above, or more) cancer-associated neo-antigenic mutations genetically introduced into ESCs or IPSCs by genomic modification. ii) A combination of mutations restricted to the cancer genome and induced by mutagenic drugs, enriched by selective advantage in cultured embryonic pluripotent stem cells.

[0129] The mutagenic process gives rise to novel genomic mutations and increased levels of genetic mosaicism in the resulting late passage human iPS cell lines.

[0130] The analysis of mutations in genes is preferably performed by large-scale genomic analysis of inducible cancer-associated "mutanome" signatures in each ESS and IPSC population by NGS (exome, RNAseq or whole genome sequencing), CGH arrays, SNP arrays. Whole exome sequencing in combination with transcriptome profiling allows the description of the mutagenesis that codes for 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 entire genome after application of the mutagenic agent confirms a higher mutation and / or CNV load in the output ESCs or IPSCs.

[0132] Qualitative and quantitative criteria make it possible to define each cell population within a genetic mosaic in ESCs or IPSCs as described: Qualitative criteria include: -Identification of acquired novel molecular somatic changes (mutations, CNVs, or SNVs) with respect to their presence in the ESC or IPSC genome after mutagenesis and their absence in ESC or IPSC without mutagenesis in similar culture passages. - Classification of each novel mutation (i.e. nonsynonymous, nonsense, splice variants, CNV, SNV) by their overlap detection present in the cancer genome (from databases i.e. TCGA, ICGC, COSMIC) and in pluripotency genes and embryonic pathways (following pluripotency genes i.e. Plurinet genes).

[0133] Such quantitative criteria include: - The prevalence of these novel somatic mutations (false discovery rate confidence value FDR ≤ 0.05) and novel CNVs / SNVs (FDR < 10%) in the whole genome will be defined for each ESC or IPS. - Presence of verified mutations in at least >3 different genes. - Mutation rates of each novel stable somatic mutation of at least >0.1% after clonal selection and expansion or for passage number, or other percentages as seen above, with allele frequencies from up to 50% (50x depth to 100x depth and 80-98% of targeted exome coverage). - Expression of pluripotency markers with an expression rate of at least >90% compared to the input ESCs or IPSCs before mutagenesis or genetic modification and gene expression-based assays (PluriTest). - an increased expression of MHC I molecules at the cell surface (for example as determined by FACS) of at least 50%, typically up to 90%, compared to a cell population grown in the absence of HDACi, in particular 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 immune response can be an adjuvant (immunostimulant) known in the art. It is preferably HDACi (used in a dose range of 0.2mM to 4mM). When using such HDACi, another adjuvant may be used.

[0137] The invention also relates to devices (eg syringes, etc.) containing such vaccine compositions, which can be used for the co-administration of HDACi compounds and cell compositions.

[0138] Such vaccine compositions can be used as therapeutic vaccines against stem cell cancers (cancers whose cells express neo-antigens) to cure patients, or as prophylactic vaccines to prevent the onset of such cancers, particularly in patients who are susceptible to these cancers.

[0139] Predisposing genes are, for example, the following (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 / Ovarian: 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 pheochromocytoma paragallium: 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 cell lysates after lyophilization, a mixture of concentrated pluripotency stem neo-antigens, purified cancer stem neo-antigens, exosomes derived from ESCs or IPSCs, DNA, RNA, proteins, or multiple peptides from engineered ESCs or IPSCs, which are 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 engineered irradiated ESCs or IPSCs used as an adjuvant effector.

[0142] In a preferred embodiment, the cells in the composition are inactivated (ie, no longer able to divide).

[0143] The composition of cells of the present invention is susceptible to being obtained by any of the methods disclosed above.

[0144] Note that the cells in this composition are heterogeneous in nature when a mutagen has been used and therefore differ from pluripotent cell compositions that have been cultured according to methods known in the art and are homogenous.

[0145] If it is cultured in the absence of a mutagen, the population of cells will differ from the population of cells cultured according to methods known in the art, because the presence of an agent that maintains expression of pluripotency genes and increases MHC I presentation in the culture medium will lead to cells that have 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 complexes present on the cell surface to recognize foreign molecules (called antigens). MHC binds antigens and presents them to immune molecules (such as lymphocytes T and B). The term "immune response" refers to the immunological response of the immune system to an antigen. Activating the immune response reduces the FoxP3 subpopulation and the population of myeloid-derived suppressor cells (MDSC) and conversely increases the NK population. In the context of the present invention, the immune response against the tumor comprises a cytotoxic T cell response against antigens present in or on the cells of the tumor. In some embodiments, the cytotoxic T cell response is mediated by CD8+ T cells. Typically, in the context of the present invention, the antigens that activate MHC expression and / or immune response correspond to molecules present on the population of pluripotent cells described above. Compounds that activate MCH expression and / or the immune system are neoantigens. The term "neoantigen" or "neoantigenic" refers to a class of antigens that result from at least one mutation that changes the amino acid sequence of a genomically 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 inhibitors", also known as HDACi, refers to a class of compounds that interfere with the function of histone deacetylase. Histone deacetylase (HDAC) plays an important role in the transcription regulation and pathogenesis of cancer. Typically, inhibitors of HDAC regulate transcription and induce cell growth arrest, differentiation, and apoptosis. HDACi also enhance the cytotoxic effect of therapeutic agents used in cancer treatment (including radiotherapeutic agents and chemotherapy drugs).

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

[0151] The term "valproic acid" refers to 2-propyl pentanoic acid (C 8 H 16 O 2 ), which has in the art the following CAS number and formula 99-66-1: [ka]

[0152] The biological activities of valproic acid are multiple (Chateauvieux et al, J. Biomed. Biotechnol, 2010, pii: 479364. doi: 10.1155 / 2010 / 479364). Valproic acid affects the neurotransmitter GABA (gamma aminobutyric acid) enhancing inhibitory activity. Several mechanisms of action have been suggested. Valproic acid specifically affects GABA metabolism: it inhibits the degradation of GABA, GABA transaminobutyric acid (LAMP), the adaptation of GABA synthesis, and alters its turnover. Valproic acid also blocks certain ion channels, reduces excitation mediated by N-methyl-D-aspartate, and blocks the activity of ion channels containing Na+ and Ca2+ (voltage-gated L-type CACNA1 types 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 pluripotency antigens shared with human embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs).

[0154] In particular, VPA is used to stimulate and enhance the expression of MHC-I on the cancer stem cell compartment, increasing the neoantigen content in the CSC compartment. Higher expression of MHC I in ESCs and IPSCs as well as in CSCs allows for enhanced presentation of neoantigens associated with MHC-I to APCs / dendritic cells and induce TH1 immune responses. Higher levels of chemokines (CXCL9, CXCL10...) allow for enhanced recruitment of T cells into the tumor.

[0155] The present invention relates to methods for increasing neoantigen content in CSC compartment expression of embryonic antigens in CSCs and tumor cells through chromatin remodeling, as well as chemokine expression (CXCL9, CXCL10...), by expanding pluripotent cells in the presence of HADCi (such as VPA and / or 5-azacytidine).

[0156] In particular, when used to treat patients in vivo, the compositions and vaccines of the invention make it possible to modify the tumor microenvironment and promote the recruitment of T cells into the tumor so as to obtain a long-lasting reduction in tumor volume.

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

[0158] This example shows that combined treatment with both cancer stem cell vaccine and VPA provides superior anti-tumor responses by increasing TILs with Th1 / Th2 cellular immunity, decreasing FoxP3 TReg subpopulation, activating NK cells, and decreasing the suppressive effect of MDSCs, reversing tumor immune suppression, decreasing TRegs (in tumor and spleen), and recruiting T CD4+ and CD8+ lymphocytes into the tumor with a lower percentage of T CD4 and CD8 expressing PD-1.

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

[0160] A known effect of VPA is the reduction of inflammatory cytokines in lymph nodes, such as IL6, IL8, TNFα, interleukin (IL)-1β, IL-17, etc.

[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 US 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 FDA approved in 2015 and has the structure described in Valente et al. 2014.

[0163] In certain embodiments, the histone deacetylase inhibitor is gibinostat (ITF2357), which has been 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 belinostat, also known as beleodaq (PXD-101), which was FDA approved in 2014 (Ja et al., 2003; Valente et al., 2014).

[0165] In a particular embodiment, the histone deacetylase inhibitor is entinostat (as SNDX-275 or MS-275). This molecule has the following chemical formula: 21 H 20 N 4 O 3 ) and has the structure described in Valente et al. 2014.

[0166] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 23 H 20 N 6 and mocetinostat (MGCD01030) with 5-HT2A (O) (Valente et al 2014).

[0167] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 20 H 30 N 4 O 2 ) and Practinostat (SB939), which has the structure described in Diermayr et al. 2012.

[0168] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 22 H 19 FN 4 O 2 ) and Chidamide (CS055 / HBI-8000).

[0169] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 21 H 26 N 6 O 2) and Quisinostat (JNJ-26481585).

[0170] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 21 H 23 N 3 O 5 ) and abexinostat (PCI24781) (Valente et al., 2014).

[0171] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 20 H 19 FN 6 O 2 ) (Moffat D et al., 2010; Banerji et al., 2012).

[0172] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 18 H 20 N 2 O 3 ) and 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 capable of interacting with DNA methyltransferases (DNMTs) and inhibiting their activity. DNMTs are enzymes that catalyze the transfer of methyl groups to DNA. DNA methylation serves a wide variety of biological functions. All known DNA methyltransferases use S-adenosylmethionine (SAM) as the methyl donor.

[0175] In certain embodiments, the DNA methyltransferase inhibitor is a compound known in the art to have the following chemical formula: 8 H 12 N4 O 5 ) and azacitidine, also known as 5-aza-2-deoxycytidine, which has the structure (Kaminskas et al., 2004; Estey et al., 2013).

[0176] In certain embodiments, the DNA methyltransferase inhibitor has the following formula: 8 H 12 N 4 O 4 ) and decitabine, also known as 5-aza-2'-deoxycytidine (Kantarjian et al 2006).

[0177] In certain embodiments, the activator of MHC expression and / or immune response is a histone-lysine N-methyltransferase enzyme inhibitor or a DNA methyltransferase inhibitor. 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 enhancer of 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 a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is 3-deazaneplanocin A (DZNep, C-c3Ado). DZNep, C-c3Ado is known in the art as having the following chemical formula C 12 H 14 N 4 O 3 and has CAS number 102052-95-9.

[0179] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitors are UNC1999 and inactive analog compounds. UNC1999 is known in the art as having the following chemical formula C: 33 H43 N 7 O 2 and CAS number 1431612-23-5.

[0180] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitor is UNC2400 and inactive analogues. UNC2400 is known in the art as having the following chemical formula C: 35 H 47 N 7 O 2 and CAS number 1433200-49-7.

[0181] In a particular embodiment, 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 N 4 O 4 and CAS number 1403254-99-8.

[0182] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is trifluoroacetate (EPZ011989). Trifluoroacetate is known in the art as having the following chemical formula: CF 3 It has COONa and CAS number 2923-18-4.

[0183] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is EPZ005687, which is known in the art as having the following chemical formula: 32 H 37 N 5 O 3 and has CAS number 1396772-26-1.

[0184] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is GSK343. GSK343 ​​is known in the art as having the following chemical formula: 31 H 39 N7 O 2 and has 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: 31 H 38 N 6 O 2 and CAS number 1346574-57-9.

[0186] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is GSK2816126, which is known in the art as having the following chemical formula: 31 H 38 N 6 O 2 and CAS number 1346574-57-9.

[0187] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is ZLD1039, which is known in the art as having the following chemical formula: 36 H 48 N 6 O 3 and has CAS number 1826865-46-6.

[0188] The use of both an HDACi and a DNA methyltransferase inhibitor is also envisioned.

[0189] Indeed, it has been shown that the combined use of VPA and 5-azacytidine, an analogue of the 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] The HDACi is administered in a therapeutically effective amount. For VPA, it can be 10-15 mg / kg / day, up to 60 mg / kg / day. Plasma levels of VPA should preferably be in the normally tolerated therapeutic range (50-100 μg / ml).

[0191] In a further aspect, the methods according to the invention are suitable for treating cancers that express a number of embryonic antigens that share expression with human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) (e.g., embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, TRA-1-81, Oct4, Sox2, Klf4, Nanog, Lin28...).

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

[0193] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance to a subject when it is present outside the body (e.g., in a complex preparation), such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When treating a disease or a symptom thereof, administration of the substance typically occurs after the onset of the disease or a symptom thereof. When preventing a disease or a symptom thereof, administration of the substance typically occurs before the onset of the disease or a symptom thereof.

[0194] In a preferred embodiment, the vaccine composition (pluripotent cells plus agents stimulating MHC presentation) is injected subcutaneously. The injections may be simultaneous, sequential, separate, at the same injection time point or at different injection time points, in the same syringe, in different syringes...

[0195] In a preferred embodiment, the follow-up treatment (administration of a compound that stimulates MHC I and / or the immune system, such as an HDACi, in particular VPA) is administered by the oral route.

[0196] "Therapeutically effective amount" refers to the minimum amount of active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" for a subject is an amount that causes an improvement in or resistance to pathological symptoms, disease progression, or physiological conditions associated with suffering from a disorder. It will be understood that the total daily use of the compounds of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or concomitantly with the specific compound used; and similar factors well known in the medical arts. For example, it is well within the scope of one of ordinary skill in the art to start the dose of a compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. However, the daily dosage 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 active ingredient, for symptomatic adjustment of the dosage to the treated subject. The medicament typically contains about 0.01 mg to about 500 mg of active ingredient, preferably about 1 mg to about 100 mg of active ingredient. An effective amount of the drug is usually supplied at a dosage level of 0.0002 mg / kg to about 20 mg / kg body weight / day, in particular about 0.001 mg / kg to 7 mg / kg body weight / day.

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

[0198] In some embodiments, the subject is administered with i) a population of pluripotent cells and ii) a compound selected from the group of combination preparations and chemotherapeutic agents that activate MHC expression and / or an immune response.

[0199] The term "chemotherapeutic agent" refers to a chemical compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents (such as thiotepa and cyclophosphamide); alkylsulfonic acids (such as busulfan, improsulfan, and piposulfan); aziridines (such as benzodopa, carboquone, meturedopa, and uredopa); ethyleneamines and methylamelamines (including altretamine, triethylenemelamine, trimethylenephosphoramide, triethylenethiophosphamide, and trimethylolmelamine); acetogenins (particularly bullatacin and bullatacinone); carnoptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8; dolastatins; duocarmycins (including synthetic analogs, KW-2189, and CBI-TMI); eleutherobin; pancratistatin; sarcodithiins; spongistatins; nitrogen mustards (e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicine, phenesterine, prednimustine, thoronphosphamide, uracil mustard, and the like); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicins (11 and calicheamicin 211, e.g., Agnew See Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicins (including dynemicin A); esperamicins;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinonomycin, carabicin, canninomycin, carzinophilin, 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, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (such as denopterin, methotrexate, peptepterin, trimetrexate); purine analogs (such as fludarabine, 6-mercaptopurine, thiamiprine, etc.) , thioguanine, etc.); pyrimidine analogues (e.g. azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, etc.); androgens (e.g. cartestrone, drostanolone propionate, epithiostanol, mepitostanol, testolactone, etc.); antiadrenal drugs (e.g. aminoglutethimide, mitotane, trilostane, etc.); folic acid supplements (e.g. humic acid, etc.); aceglatone; aldophospharnide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diazicon; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, e.g. maytansine and Ansamitocin;Mitoguazone;Mitoxantrone;Mopidamol;Nitracin;Pentostine;Phenamet;Pirarubicin;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; schizophyllan; spirogenanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C") cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues (such as 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 pharmaceutical acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens (including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyfene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharma- ceutical acceptable salts, acids, or derivatives of any of the above;

[0200] In some embodiments, the subject is administered a compound selected from the group consisting of i) a population of pluripotent cells and ii) a combination preparation and a compound that activates MHC expression and / or an immune response as a targeted cancer therapy.

[0201] Targeted cancer therapy is a drug or other substance that blocks the growth and spread of cancer by interfering with specific molecules ("molecular targets") involved in the growth, progression, and spread of cancer. Targeted cancer therapy may also be referred to as "molecularly targeted drugs," "molecularly targeted therapy," "precision medicine," or similar names. In some embodiments, targeted therapy consists of administering to a subject with a tyrosine kinase inhibitor. The term "tyrosine kinase inhibitor" refers to any of a variety of 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 No. 2007 / 0254295, which is incorporated herein by reference in its entirety. Those skilled in the art will appreciate that compounds related to tyrosine kinase inhibitors will replicate the effects of tyrosine kinase inhibitors. For example, related compounds will act on different components of the tyrosine kinase signaling pathway and have the same effects as a tyrosine kinase inhibitor of that tyrosine kinase. Examples of suitable tyrosine kinase inhibitors and related compounds for use in the methods of the present embodiments include, but are not limited to, dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide. (SU101), vandetanib (Zactima; ZD6474), bevacizumab (avastin), MK-2206 (8-[4-aminocyclobutyl]phenyl)-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-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 described, for example, in U.S. Patent Application Publication No. 2007 / 0254295; U.S. Patent Nos. 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; 6,329,380; and 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 by reference herein in their entireties. In certain embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that is orally administered, 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 has been approved by the FDA for at least one hematological or oncological indication. 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, vatalanib, CP-547632, vandetanib, bosutinib, lestaurtinib, tandetonib, midostaurin, enzastaurin, AEE -788, pazopanib, actinib, motasenib, OSI-930, cediranib, KRN-951, dovitinib, seliciclib, SNS-032, PD-0332991, MKC-I (RO-317453, R-440), sorafenib, ABT-869, brivanib (BMS-582664), SU-14813, telatinib, SU-6668, (TSU-68), L-21649, MLN-8054, AEW-541, and PD-0325901.

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

[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 regulate the activation or function of T cells. Many checkpoint proteins are known, such as 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 costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance as well as the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies. In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD1 antibody (e.g., nivolumab, pembrolizumab), an anti-PDL1 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-BTLA antibody, and an anti-B7H6 antibody. Examples of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 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 (ticilimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (10D1, also known as 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. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; PCT published patent applications WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.In some embodiments, the PD-1 blocking agent comprises an anti-PD-L1 antibody. In certain other embodiments, PD-1 blockade includes anti-PD-L1 antibodies and similar binding proteins, such as nivolumab (MDX 1106, BMS 936558, ONO 4538) (a fully human IgG4 antibody that binds to PD-1 blockade and blocks activation of PD-1 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); AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX-1105-01) for PD-L1 (B7-H1) blockade. Other immune checkpoint inhibitors include lymphocyte activation gene 3 (LAG-3) inhibitors, such as IMP321 (a soluble Ig fusion protein) (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune checkpoint inhibitors include B7 inhibitors, such as 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 The present invention is as described by Roberts et al., J. Med. Pharmacology, 2012, 111:1311-1315, 2012.In adoptive immunotherapy, the patient's circulating or tumor-infiltrating lymphocytes are isolated in vitro, activated with lymphokines (e.g., IL-2), and readministered (Rosenberg et al., 1988; 1989). The activated lymphocytes are most preferably the patient's own cells, isolated earlier from a blood sample and activated (or "expanded") in vitro.

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

[0205] The term "radiotherapy agent" as used herein refers to any radiotherapy agent known to those skilled in the art to be effective in treating or ameliorating cancer, including but not limited to. For example, the radiotherapy agent may be an agent administered in brachytherapy or radionuclide therapy. Such methods may optionally further include administration of one or more additional cancer therapies, including but not limited to chemotherapy and / or another radiation therapy.

[0206] Pharmaceutical and vaccine compositions

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

[0208] "Pharmaceutically" or "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered appropriately to a mammal, particularly a human. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary. 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 a unit dosage form, in admixture with a conventional pharmaceutical support, with the active ingredient alone or in combination with other active ingredients. Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal, and intranasal dosage forms, and rectal dosage forms. Typically, the pharmaceutical composition includes a pharmaceutically acceptable vehicle for a formulation that can be injected. These may be, in particular, isotonic sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts) or dry, especially lyophilized compositions, which allow the constitution of an injectable solution upon addition of sterile water or saline, depending on the case. 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 extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions containing the compounds of the invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.The polypeptide (or the nucleic acid encoding it) can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the 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, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating (such as lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents that delay absorption, such as aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the required amount of the active polypeptide in the appropriate solvent with some of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution thereof. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be used.For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous medium 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 infusion or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0209] In particular, the population of pluripotent cells and a compound that activates MHC expression and / or an immune response are formulated in a vaccine composition.Thus, the present invention relates to a vaccine composition comprising i) a population of pluripotent cells and ii) a compound selected from the group that activates MHC expression and / or an immune response.

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

[0211] In a particular embodiment, a vaccine composition according to the invention comprising i) induced pluripotent stem cells (iPSCs) expressing a neo-antigen, in particular enhanced by a mutagenic agent or genetic modification, and ii) valproic acid.

[0212] The composition may also include 5 azacytidine.

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

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

[0215] The vaccine composition according to the invention can be administered by subcutaneous (sc), intradermal (id), intramuscular (im) or intravenous (iv) injection, orally and intranasally or by inhalation. The administration of the vaccine is usually a single dose. Alternatively, the first administration of the vaccine of the present invention is made (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 , 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 recalls (subsequent administrations) follow using the same population of stem cells, a compound that stimulates the immune system or a combination thereof, and / or using a combination with one or more further of radiation therapy, targeted therapy, immunotherapy, or chemotherapy.

[0216] The vaccine composition may also be provided in a kit. The kit includes the vaccine composition and an information pamphlet providing instructions for immunization. The kit also includes all materials for administration of the product.

[0217] The present invention is further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the invention in any way. [Brief description of the drawings]

[0218] [Figure 1] Vaccination study with mESC, hESC, miPSC, and 4T1 cells in breast tumor 4T1 model. Study design: Mice (5 per group) received two boosts of vaccine at 7 and 14 days with 105 irradiated cells; mouse embryonic stem cells (mESC), mouse induced pluripotent stem cells (miPSC), human embryonic stem cells (hESC), or 4T1 cells. After 14 days, 5x104 4T1 cells were injected into the mammary fat pad of the mice. Five mice were injected with 4T1-CSC (4T1 cells grown with additional cytokines such as TGF beta and TNF alpha to generate CSC growth in the form of mammospheres). [Figure 2A] Immune protection after vaccination. A, Quantification of CD4 positive tumor infiltrating lymphocytes (TILs) by flow cytometry. B, Quantification of CD25 positive regulatory T cells by flow cytometry within CD4+ TILs. C, Quantification of PD1 regulatory T cells by flow cytometry within CD8+ T cells for groups of mice. [Figure 2B] Immune protection after vaccination. A, Quantification of CD4 positive tumor infiltrating lymphocytes (TILs) by flow cytometry. B, Quantification of CD25 positive regulatory T cells by flow cytometry within CD4+ TILs. C, Quantification of PD1 regulatory T cells by flow cytometry within CD8+ T cells for groups of mice. [Figure 2C] Immune protection after vaccination. A, Quantification of CD4 positive tumor infiltrating lymphocytes (TILs) by flow cytometry. B, Quantification of CD25 positive regulatory T cells by flow cytometry within CD4+ TILs. C, Quantification of PD1 regulatory T cells by flow cytometry within CD8+ T cells for groups of mice. [Figure 3A]Vaccination study with hESC combined with VPA in 4T1 mouse model. Study design: Mice (n=5 / group) received two boosts of vaccine with 105 irradiated hESC with or without VPA on days 7 and 14. After 14 days, 5x104 4T1 cells were injected into the mammary fat pad of the mice. A; Tumor volume for each group: 1 / control (PBS), 2 / vaccinated with hESC, 3 / vaccinated with hESC combined with VPA, 4 / mice that received VPA only. B: Tumor weight at day 44. [Figure 3B] Vaccination study with hESC combined with VPA in 4T1 mouse model. Study design: Mice (n=5 / group) received two boosts of vaccine with 105 irradiated hESC with or without VPA on days 7 and 14. After 14 days, 5x104 4T1 cells were injected into the mammary fat pad of the mice. A; Tumor volume for each group: 1 / control (PBS), 2 / vaccinated with hESC, 3 / vaccinated with hESC combined with VPA, 4 / mice that received VPA only. B: Tumor weight at day 44. [Figure 4A] Immune protection after hESC vaccination and VPA: A, Reduction of PD1 cells in the spleen within the CD4 and CD8 populations. B, Increase of CD4+ and CD8+ T cells in the tumor. C: Increase of CD4+ and CD8+ T cells in the spleen. [Figure 4B] Immune protection after hESC vaccination and VPA: A, Reduction of PD1 cells in the spleen within the CD4 and CD8 populations. B, Increase of CD4+ and CD8+ T cells in the tumor. C: Increase of CD4+ and CD8+ T cells in the spleen. [Figure 4C] Immune protection after hESC vaccination and VPA: A, Reduction of PD1 cells in the spleen within the CD4 and CD8 populations. B, Increase of CD4+ and CD8+ T cells in the tumor. C: Increase of CD4+ and CD8+ T cells in the spleen. [Diagram 5] Quantification of luciferase reporter gene by IVIS Spectrum system in the lungs after dissection for animals in each of the four groups (control, hESC, hESC+VPA, VPA). [Figure 6A] Expression of puripotent genes by real-time PCR in 4T1 cells and 4T1 CSCs (mammospheres) treated with 0.5 mM VPA for 5 days. [Figure 6B] Expression of puripotent genes by real-time PCR in 4T1 cells and 4T1 CSCs (mammospheres) treated with 0.5 mM VPA for 5 days. [Figure 7A] Expression of Oct4 in 4T1 treated with VPA and 5Aza by RT PCR and real-time PCR. [Figure 7B] Expression of Oct4 in 4T1 treated with VPA and 5Aza by RT PCR and real-time PCR. [Figure 8] Detection by exome sequencing of genetic variants that alter the protein sequence of neoantigens in iPSCs treated or not with ENU. Quantification of variants (NS = nonsynonymous, FS = frameshift, SG = stop obtained) in ENU-treated and non-treated iPSCs. [Figure 9] 1 is a schematic representation of a process according to the present invention.

[0219] Working Example:

[0220] Example 1

[0221] It has been reported that fetal tissues can be used to immunize mice capable of rejecting transplantable tumors, including those of the skin, liver, and gastrointestinal tract. This response is explained by the fact that those tumor cells express multiple carcinoembryonic antigens. To date, several human cancer vaccine trials have been set up to target embryonic antigens, such as carcinoembryonic antigen (CEA) and alpha-fetoprotein or cancer / testis antigens. Unfortunately, targeting one antigen alone has been shown to be inefficient enough to generate a strong antitumor immune response that mediates tumor rejection, due to the rapid emergence of escape mutants and the general inefficiency of monovalent cancer vaccines. Recent interest in the potential of stem cells in regenerative medicine has led to the widespread availability of well-defined undifferentiated ESC lines as well as undifferentiated iPSCs that are phenotypically and functionally similar to ESCs. In our study, we hypothesized that undifferentiated stem cells can be used as a multivalent vaccine to generate immune responses against various embryonic antigens shared by tumor cells and CSCs. The present inventors have found for the first time that immune and clinical responses against breast cancer can be induced by ESCs or iPSCs. Surprisingly, the present inventors have found that the addition of valproic acid in a treatment regimen can induce higher immune and antitumor responses compared to the use of ESCs or iPSCs alone.

[0222] Materials and Methods

[0223] We developed a metastatic 4T1 breast cancer model in BALB / c mice. To validate embryonic ES-like markers in 4T1 mouse TNBC breast cancer cell lines, a meta-analysis was performed using embryonic cell samples (D3 stem cells - GSE51782, annotation on Affymetrix plate form GPL16570) and incorporating different datasets: TNBC cell line 4T1 cultured in vitro (GSE73296, annotation on Affymetrix plate form GPL6246), TNBC cell line 4T1 xenografted in mouse model (GSE69006, annotation on Affymetrix plate form GPL6246), and mammary gland samples (GSE14202, annotation on Affymetrix plate form GLP339) (Padovani et al. 2009). For this 4T1 model, microarray analysis of 4T1 transplanted BALB / c mice revealed that 1304 distinct 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 (e.g., 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 these genes were thus found to be commonly upregulated in 4T1 and mESCs compared to normal mouse mammary glands. It was also shown that xenografted 4T1 highly expressed CSC markers (e.g., CD44) compared to cells collected in vitro (CD44 high CD24 low 39% vs. 0.27%) (not shown).

[0224] In the same manner, whole genome expression profile analysis was performed on triple-negative breast cancer (TNBC) from patients. To validate embryonic ES-like markers in patients with TNBC, a meta-analysis was performed using embryonic cell samples and human breast samples by mixing sample data from different datasets: dataset GSE18864, which contains 84 breast cancer samples and is annotated with the Affymetrix plate form GPL570 (Silver et al. 2010), dataset GSE20437, which contains 42 samples of human normal breast and is annotated with the Affymetrix platform GPL96 (Graham et al. 2010), dataset GSE23402, which contains 42 samples of human embryonic stem cells and induced pluripotent stem cells and is annotated with the Affymetrix plate form GPL570 (Guenther et al. 2010), a dataset of breast cancer cell lines (Maire et al. 2013), and dataset GSE36953, which contains cell culture samples of TNBC cell lines and is annotated with the Affymetrix plate form GPL570 (Yotsumoto et al. 2013). 2013).By supervised one-way ANOVA, 4288 significant genes were identified that allowed to analyze between the three groups of breast cancer grades and to classify 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). 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 these genes were thus found to be commonly upregulated in TNBC tumors and hESCs compared to normal human mammary gland.

[0225] result

[0226] Results 1. Vaccination with xenogeneic embryonic stem cells generates immunological and antitumor responses against breast cancer.

[0227] We first investigated whether vaccination with irradiated mouse ESCs (mESCs), mouse induced pluripotent stem cells (mouse iPSCs), human embryonic stem cells (hESCs), or 4T1 cells was effective against breast cancer in a syngeneic 4T1 mouse model. Following this vaccination, mice were challenged with two different types of 4T1 cells: 4T1 normally cultured in DMEM 10% of SVMC, or 4T1 cells grown with additional cytokines (such as TGF beta and TNF alpha) to generate cancer stem cells (CSCs) that grow in the form of mammospheres. We found that mice vaccinated with hESCs, mESCs, mouse iPSCs, and 4T1 generated consistent cellular immune responses against 4T1 cancer, which correlated with a significant reduction in breast tumor volume (p<0.05) (Figure 1), in contrast to non-vaccinated mice. We found that, whereas tumors grew progressively in the PBS control group, notably, immunization with mESCs, miPS, or hESCs resulted in a delay in tumor growth, with statistically significant differences in the mean tumor size of each group compared to the PBS group (Figure 1). We observed a dramatic inhibition of tumor growth when mice were challenged with CSC-derived 4T1 compared to mice challenged with 4T1, which grows under normal conditions, indicating that vaccination with syngeneic mESCs preferentially targets CSCs. To further test the cellular immune mechanism mediating the antitumor effect, we analyzed the phenotype of tumor-infiltrating lymphocytes from the different groups and quantified CD4, CD8, CD25, and PD1 subpopulations. The antitumor effect correlated with 1) an increase in CD4+ TILs that correlated significantly (p=0.0039) with tumor size (Figure 2A), 2) a decrease in the percentage of CD25 positive cells that inversely correlated with tumor size (Figure 2B), and 3) a decrease in PD1 positive cells that was more pronounced in mice with a better response to the vaccine regimen (vaccination with hECS, 4T1, or mESC) (Figure 2C).

[0228] Result 2. Vaccination with xenogeneic embryonic stem cells in combination with valproic acid (VPA) generates a higher antitumor response against breast cancer and inhibits metastasis development.

[0229] To assess metastatic sites in the 4T1 breast model, 4T1 cells were genetically modified to express both GFP and luciferase reporter proteins (4T1Luc-GFP), allowing their tracking in vivo using bioluminescence imaging (Ivis spectrum) in deeper organs (spleen, lung, bone, liver). The experiment was performed as previously described, but using only irradiated hESCs as vaccine; five mice per group received 10 5 The mice were boosted twice at days 7 and 14 with irradiated hESC cells. After 14 days, 5 × 10 4 4T1Luc-GFP cells were injected into the mammary fat pad of mice. We found that mice vaccinated with hESCs combined with VPA, in contrast to non-vaccinated mice, generated a higher cellular immune response against 4T1 cancer, which correlated with a significant reduction in mammary tumor volume (p<0.05) (Figure 3A) and tumor weight (Figure 3B). The anti-tumor response correlated with a dramatic reduction in PD-1 expression in both CD4+ and CD8+ T cells in mice that received hESCs and VPA (Figure 4A). The anti-tumor response also correlated with a significant increase in the percentage of CD4+ and CD8+ T cells in the tumor (Figure 4B) and spleen (Figure 4C) for mice that received exclusively combination treatment (hESCs and VPA) compared to the control group (PBS). We also found that all mice had significantly reduced lung metastatic tumors treated with hESC vaccine and VPA (Figure 5). Taken together, these results show that xenogeneic embryonic stem-based vaccination (hESC) with VPA has the strongest efficacy compared to the use of hESC and VPA alone. These results indicate that xenogeneic embryonic stem-based vaccination may be an effective treatment to reduce tumor recurrence in breast cancer.

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

[0231] The major histocompatibility complex (MHC) is a set of cell surface proteins essential for the acquired immune system to recognize foreign molecules. The main function of MHC molecules is to bind 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 a specific immunity called acquired or adaptive immunity; by interacting with CD8 molecules on the surface of cytotoxic T cells, MHC class I mediates the destruction of infected or malignant host cells.

[0232] Immune tolerance is an important means 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 explained in part by the absence of β2-m on the cell surface and / or the absence of MHC class I on tumor cells. It has been shown that VPA can increase the expression of MHC class I on 4T1 cells at doses between 0.2 mM and 2 mM. Expression of MHC class I on 4T1 and 4T1 mammospheres (CSCs induced by treatment with TNFα and TGFb) increases 2- to 3-fold after 24- to 72-h exposure with 2 mM VPA.

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

[0234] These markers were decreased after ENU exposure (60 days after treatment) and were restored 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 such as VPA can selectively alter gene transcription, in part, by chromatin remodeling and by altering the structure of proteins in transcription factors. We tested whether VPA can modulate the expression of pluripotency genes in breast cancer cells. To this end, we treated 4T1 and 4T1 mammospheres (CSCs induced by treatment with TNFα and TGFb) with 1 mM to 2 mM VPA. In all cases, VPA increased the expression of three different key transcription factors (e.g. Oct4, Sox2, and Nanog) that are highly expressed in ESCs or iPSCs by 2-3 fold (Figure 6). Importantly, a significant synergistic effect of these transcription factor expression was shown when tumor cells were treated with a combination of VPA and 5-azacytidine (5aza), used at doses that inhibit DNA methyltransferase and cause DNA hypomethylation. Notably, treatment of 4T1 cells with VPA and 5aza demonstrated a seven-fold increase in oct-4 transcripts (Fig. 7A and B).

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

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

[0238] It was possible to confirm that ENU caused DNA damage in iPSCs.

[0239] The amount of phosphorylated gamma-H2AX attracted to sites of DSBs present in cells was assessed. In this experiment, iPSCs were detached from stromal cultures by the use of collagenase and treated in vitro with ENU at the indicated concentrations (50 μg / ml) and times, followed by Western blot analysis using an anti-phospho-gamma-H2AX antibody. An increase in gamma-H2AX levels was seen at early time points between 2 and 10 min, followed by a recovery to basal levels.

[0240] A protocol was designed to induce genomic instability in iPSCs by continuous treatment of iPSCs with ENU to accumulate DNA repair errors during extensive proliferation. 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 the culture.

[0241] At day +61, iPSCs will be karyotyped and the genomic consequences of the mutagenesis procedure assessed by RNA sequencing, CGH arrays, exon sequencing, and WGS. Genomic changes accumulated in cultured iPSCs will be compared to iPSCs not treated with ENU.

[0242] After ENU exposure, the mutated iPSCs were maintained and expanded in culture with VPA. CGH arrays and exome sequencing were performed sequentially by different routes to confirm the genocopy of somatic mutation prevalence in ENH-iPSCs during their expansion. The phenotype of iPSCs was performed by evaluating pluripotency Pluritest and the expression of Oct4, Sox2, Nanog, Tra-1 60, SSEA4. We found that the replication rate and population doubling were similar to iPSCs without ENU exposure.

[0243] iPSCs were treated with 10 μg / ml ENU and exome sequencing for detection of mutated neo-antigens was performed. 48 alterations in ENU-treated iPSCs were found compared to 8 alterations in iPSCs without ENU (Figure 8). These loci were mixed with the cBioPortal program (http: / / www.cbioportal.org), which allows access to cancer genomics datasets from human tumor samples from different cancer studies. Using the cBioPortal program, more than 10-75% of these alterations were found to be deregulated in cancers (pancreatic, lung, prostate, etc.). For all exome sequencing, we estimate a depth of coverage of 30-400 reads for all mutations that can be detected in ENU-treated iPSCs.

[0244] Example 4 - Haploinsufficiency for BRCA1 leads to DNA repair alterations and genomic instability with accumulation of CNVs in iPSCs during culture.

[0245] BRCA1 / 2 alterations 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 controlling homologous recombination, double-strand break repair, DNA repair in S phase and G2 / M, spindle checkpoint, and centrosome regulation.

[0246] Fibroblasts carrying a deletion of exon 17 in 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 their morphology and pluripotency markers by FACS analysis, Q RT-PCR of Nanog, Oct4, Sox2, teratoma formation in NOD-SCID mice, and Pluritest. Karyotype was normal.

[0247] The levels and activity of DNA damage response (DDR) in normal (WT) and BRCA1+ / - iPSCs were compared. Gamma H2AX 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 gamma H2AX recruitment to DNA compared to normal WT-iPSCs, indicating that proliferating IPSC BRCA1+ / - undergo increased DNA damage compared to WT-IPSCs.

[0248] As iPSC BRCA1+ / - displayed increased levels of DDR at early passages, we examined whether this could be associated with the accumulation of genomic alterations during repeated passaging and expansion.

[0249] CGH arrays in proliferating pluripotent stem cells were analyzed after long-term passaging of iPSCs in medium supplemented with HDACi (VPA). For this purpose, Agilent CGHarray experiments were performed on DNA from IPSC samples using the Roche-Nimbelgen aCGH platform. Signal extraction and genomic intervals were identified using Agilent Cytogenetics and Nexus Roche-Nimbelgen software on HG18 of the human genome. Genetic loci were transformed in 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 experiment in the scandb database (Gamazon et al. 2010). Array CGH CNV ratios were depicted as heatmaps using MEV version 4.9.0 standalone software (red: gain, green: loss, and dark zero) (Saeed et al. 2003). The loci affected in the cell of origin were subtracted from the loci affected in each IPSC sample. The resulting filtered CNVs specific to each iPSC were mixed with the COSMIC census database (Futreal et al. 2004). A Genomic Circosplot on HG19 was performed with the cancer genes found to be affected in each IPSC after filtering. This genomic depiction was performed using the OmicCircos R-package in the R environment version 3.0.2 (Hu et al. 2014).

[0250] It was shown that culturing iPSCs BRCA1+ / - (>100 days) leads to the accumulation of genomic aberrations at later passages, concomitant with increased genomic instability without ENU exposure. Karyotypes at later passages are normal. Agilent aCGH experiments were performed on DNA extracts from iPSC cells and their respective cells of origin. Genomic mapping of the intervals affected during their pluripotency induction showed that BRCA1+ / - iPSCs were affected by a significant number of affected loci compared to those of WT. After filtering out CNV European polymorphisms in WT iPSCs, only 58 loci were still affected (polymorphisms represent 1.69% of the total affected loci), similarly in BRCA1- / + iPSCs 5273 loci were still found after filtering out polymorphisms. The majority of affected loci in BRCA1+ / - iPSCs were involved in DNA acquisition.

[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 alterations involving 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 alterations were found to be particularly overexpressed in small chromosomes 19 and 17 (chromosome 17, the chromosome of the BRCA1 locus).

[0252] In summary, the majority of iPSC BRCA1+ / - showed higher amounts of indels (deletions or amplifications) compared to WT-IPSC. An 8% rate of CNVs in 5273 genes was identified and confirmed. Bioinformatics analysis revealed the expression of 131 genes identified in the Cosmic database that are involved in cancer development essentially in leukemia, epithelial tumors, and in mesenchymal tumor cells. Some altered genes are similarly observed in breast and ovarian cancer.

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

[0254] In conclusion, deletion or inactivation of DNA repair-related genes (such as BRCA-1) induces genomic instability and allows the generation of multiple CNVs, indels, and MHC I-associated mutations.

[0255] Example 5 - N-ethyl-N-nitrosourea (ENU) increases mutant neoantigen load 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 the pluripotency genes Oct4, c-Myc, Klf4, and Sox2. Cells with pluripotent iPSC morphology were expanded 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 iPSCs were exposed to ENU for 60 days. Cell outgrowth colonies from CML-IPS treated with ENU were compared to IPSCs not treated with ENU.

[0257] DNA of CML iPSCs was analyzed by CGH array. Several genomic abnormalities were observed in blast colonies derived from ENU-treated iPSCs, with the detection of loss of heterogeneity among genomic abnormalities selected by ENU pressure in CML iPSCs (CB32, which included copy number variation CNVs involving 332 loci). After filtering with the European Caucasian genome polymorphism database, 225 loci were still present in these genomic abnormalities. The majority of genomic abnormalities involved loss of genomic DNA (71%) and were accompanied by loss of heterozygosity (23%). By matching these genomic abnormalities with transcription factor databases, cancer gene databases, and pluripotency gene databases, it was observed that these important deregulated actors were mainly affected on chromosomes 7, 8, 15, Y, and X. Circoplots also allowed us to relate most of these abnormalities to transcription factors, such as MESP and IKZF1, involved in mesodermal cell migration. Some pluripotency genes as well as some oncogenes already described to be involved in Ph1-positive leukemia, such as IDH2, NCOA2, IKZF1, and BLM, were affected, suggesting the relevance of the abnormalities generated by ENU-induced mutagenesis.

[0258] This analysis shows that several genetic abnormalities (eg, gains and losses) and some of the identified abnormalities were found to be cancer genes identified in the COSMIC database.

[0259] Comparison of the abnormalities identified in ENU-iPSCs allowed us to recapitulate aggressive acute leukemic stage abnormalities already identified in acute leukemic stage CML patients, suggesting that ENU-treated CML iPSCs are a unique tool to recapitulate in vitro these genomic abnormalities in this particular cancer.

[0260] Throughout this application various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure.

[0261] References:

Table 1

Claims

1. A method of treating a subject comprising the steps of simultaneously, separately or sequentially administering to the subject therapeutic amounts of a vaccine composition comprising i) a histone deacetylase inhibitor (HDACi) and ii) an immunogenic element.

2. The method of claim 1 , further comprising administering an HDACi for a period of time following administration of the vaccine composition.

3. 3. The method of claim 1 or 2, wherein the immunogenic element in the vaccine composition is selected from the group consisting of: a. Antigen of interest b. An extract from a cellular composition, wherein the cells of the composition express the antigen of interest. c. A cell composition, wherein the cells of the composition express an antigen of interest. d. Cellular compositions comprising antigen-presenting cells prestimulated in vitro with an antigen of interest. e. T cell lymphocytes that have been primed in vitro against an antigen of interest by exposure to antigen-presenting cells that present the antigen of interest.

4. The method according to any one of claims 1 to 3, wherein the antigen of interest is an antigen expressed by a cancer cell, in particular a neo-antigen expressed by a cancer cell.

5. The method according to any one of claims 1 to 4, wherein the treatment is a therapeutic treatment.

6. The method according to any one of claims 1 to 4, wherein the treatment is a prophylactic treatment.

7. A method for producing a cell composition comprising the steps of: i) expanding the pluripotent cells in the presence of conditions that maintain the pluripotent capacity of the cells in the presence of an agent that induces MHC-I presentation of the antigen in the population during the expansion step. ii) exposing the proliferating cells to an inactivating agent that inactivates the cells while maintaining the integrity of the cell envelope; iii) harvesting and conditioning the expanded inactivated cells;

8. 8. The method of claim 7, wherein the population of pluripotent cells is selected from the group consisting of human embryonic stem cells, induced pluripotent stem cells (iPS), allogeneic, xenogeneic, autologous, or syngeneic stem cells.

9. 9. The method of claim 7 or 8, wherein the pluripotent cells are exposed to a mutagenic agent during propagation to induce gene mutagenesis in the cells of the population.

10. 10. The method of claim 9, wherein the mutagenic agent is selected from the group consisting of chemical mutagenic agents and radiation mutagenic agents (X-rays, ultraviolet light).

11. The method of any one of claims 7 to 10, wherein the mutagenic agent is selected from the group consisting of ENU, reactive oxygen species, deaminating agents, polycyclic aromatic hydrocarbons, aromatic amines, and sodium azide.

12. 10. The method of claim 9, wherein the mutagenic agent is an alkylating agent, in particular ENU, and exposure of the pluripotent cells to the mutagenic agent is carried out for a period of at least 15 days, more preferably at least 30 days, even more preferably at least 45 days, more preferably at least 60 days.

13. The method according to any one of claims 7 to 12, wherein the agent that induces MHC-I presentation of the antigen is a histone deacetylase inhibitor (HDACi).

14. 14. The method of claim 13, wherein the histone deacetylase inhibitor is selected from the group consisting of valproic acid (VPA), vorinostat, panobinostat, divinostat, belinostat, entinostat, mocetinostat, pracinostat, chidamide, xinostat, and abexinostat.

15. The method according to any one of claims 7 to 14, wherein the composition comprises a DNA methyltransferase inhibitor, in particular 5-azacytidine.

16. The method according to any one of claims 7 to 15, wherein the cells are inactivated by exposure to a lethal dose of radiation.

17. The method according to any one of claims 7 to 16, wherein the harvesting comprises washing and resuspending the cells in a suitable buffer.

18. The method according to any one of claims 7 to 17, wherein conditioning comprises freezing or lyophilizing the cells.

19. 1. A composition of cells comprising pluripotent cells, wherein the cells in the population, after expansion, display a mutation rate of at least 0.1% in at least three genes selected from the 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.

20. A vaccine composition comprising: a. A pluripotent cell population; and b. Histone deacetylase inhibitors.

21. 21. The vaccine composition of claim 20, wherein the pluripotent cells comprise mutagenized pluripotent cells.

22. 22. A vaccine composition according to claim 20 or 21 for its use for the treatment of a subject suffering from cancer.

23. 23. A vaccine composition for use thereof according to claim 22, wherein the cancer has a stem cell signature (expresses embryonic antigens).

24. A kit comprising a vaccine composition according to any one of claims 20 to 23 and an information pamphlet providing instructions for immunization.

25. 1. A method of treating a subject suffering from cancer comprising administering to the subject simultaneously, separately or sequentially therapeutic amounts of i) a population of pluripotent cells and ii) a compound that activates MHC expression and / or an immune response as a combined preparation.

26. 26. The method of claim 25, wherein the population of pluripotent cells is selected from the group consisting of human embryonic stem cells, induced pluripotent stem cells (iPS), allogeneic, xenogeneic, autologous, or syngeneic stem cells.

27. 27. The method of claim 25 or 26, wherein the cancer is selected from the group consisting of cancers that express embryonic antigens.

28. 28. The method of any one of claims 25 to 27, wherein the cancer is selected from the group consisting of 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, renal cancer, head and neck tumors, and solid tumors.

29. The method of any one of claims 25 to 28, wherein the pluripotent cells are genetically modified to overexpress a compound that stimulates MHC expression and / or an immune response.

30. 30. The method of claim 29, wherein the compound that stimulates MHC expression and / or immune response is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), interferon gamma (IFN-gamma), interleukin 2 (IL-2), and combinations thereof.

31. The method of any one of claims 25 to 30, wherein the pluripotent cells have been treated with a mutagenic agent.

32. The method of any one of claims 24 to 31, further comprising administering to the patient an immune checkpoint inhibitor.

33. The method of any one of claims 25 to 31, further comprising administering chemotherapy to the patient.

34. The method of any one of claims 25 to 31, further comprising administering radiation therapy to the patient.

35. The method of any one of claims 25 to 31, further comprising administering to the patient both chemotherapy and radiation therapy.

36. The method according to any one of claims 25 to 35, wherein the administration of the pluripotent cells is carried out by subcutaneous injection.

37. 37. The method of any one of claims 25 to 36, further comprising administering a histone deacetylase inhibitor for a period of time following administration of the population of pluripotent cells.

38. 38. The method of claim 37, wherein the period of time comprises between 3 days and 3 weeks.

39. The method of any one of claims 25 to 38, wherein the compound that activates MHC expression and / or immune response is a histone deacetylase inhibitor.

40. The method of any one of claims 25 to 39, wherein the activator of MHC expression is valproic acid.

41. The method according to any one of claims 25 to 38, wherein the activator of MHC expression is a DNA methyltransferase inhibitor.

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