Methods and compositions for treating cancer

Abstract

To provide a new therapeutic strategy for treating a cancer expressing an embryonic antigen.SOLUTION: There is provided a combination of (i) a histone deacetylase inhibitor (HDACi) and (ii) a vaccine composition comprising a population of inactivated fetal cells, for use in the treatment of cancer in a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of oncology, more particularly the present invention relates to anti-cancer vaccine combination therapy.

[0002] In particular, the present invention relates to methods for producing compositions comprising fetal stem cells that present multiple neoantigens and which are useful in the preparation of cancer cell vaccines. [Background technology]

[0003] Cancer stem cells (CSCs) are a minority population of self-renewing cancer cells responsible for tumor persistence and recurrence and may be resistant to conventional treatments. These CSCs have recently been demonstrated in solid tumors of various origins, including breast, colon, and head and neck carcinomas, and represent a novel therapeutic target. These CSCs have been shown to express numerous embryonic antigens that share expression with human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). Expression of some of these embryonic antigens has also been found in differentiated cancer cells associated with tumorigenesis and / or tumor progression. Furthermore, cancers also express fetal antigens that are not expressed in pluripotent cells.

[0004] Over the past decade, cancer treatment approaches have advanced from targeted therapy to immune intervention strategies, resulting in unprecedented gains in survival and cancer-related morbidity and mortality. However, despite the proven efficacy and clinical benefits of immune checkpoint inhibitors, there are numerous partial responders and primary resistant tumors ("innate resistance") caused by immune modulators that influence tumor-specific immune responses and cancer cell-autonomous cues. After an initial response to PD-1 / PD-L1 blockade, acquired resistance occurs in many cancers during progression and recurrence. The underlying mechanism of acquired resistance to PD1 / PD-L1 blockade is caused by the evolution of the neoantigen landscape (evolving tumor immune microenvironment (TIME)) accompanied by acquired somatic mutations (mutanome) and epigenetic stability of exhausted T cells.

[0005] Cancer germline antigens are proteins expressed during embryonic and fetal development, and these epigenetic control antigens can be re-expressed in various parts of many cancers. To date, several human cancer vaccine trials have been designed to target embryonic antigens, such as carcinoembryonic antigen (CEA), alpha-fetoprotein, or cancer / testis antigen (NY-ESO-1). Adoptive cell transfer of autologous lymphocytes genetically engineered to express the T cell antigen receptor (TCR) for the HLA*0201 epitope of the cancer germline antigen NY-ESO-1 resulted in durable tumor regression in several patients with metastatic melanoma. Unfortunately, the rapid emergence of escape mutants and novel somatic neoantigens, as well as the general inefficiency of monovalent cancer vaccines, have demonstrated that targeting only one antigen is insufficient to generate a potent antitumor immune response and mediate tumor rejection.

[0006] 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, which are phenotypically and functionally similar to ESCs.

[0007] Cancers with stem cell signatures exhibit genomic plasticity with profound changes in the chromatin landscape secondary to intrinsic pathways and inducers from the potent immunosuppressive tumor microenvironment. Their ability to dedifferentiate into undifferentiated progenitor cells confers tumor clones with re-expression of fetal-derived genes accompanied by downregulation of CMH class I and upregulation of co-inhibitory molecule expression.

[0008] Therefore, there is a continuing need for new approaches to prevent and / or treat cancers with stem cell signatures. Vaccination against stem cell mutant neoepitopes can be used to enhance the immune response of adoptively transferred T cells or cells activated by immunological checkpoint blockade.

[0009] This and other needs are addressed in whole or in part by the subject matter of the present disclosure. Summary of the Invention

[0010] The present invention relates to a method for treating a subject suffering from cancer, comprising the simultaneous, separate or sequential administration to said subject of therapeutic amounts of (i) a histone deacetylase inhibitor (HDACi) and (ii) a vaccine composition containing an immunogenic element. Preferably, the immunogenic element is a population of inactivated fetal cells, which are advantageously of the same cell lineage as the cancer to be treated. The present invention is defined, inter alia, by the claims.

[0011] In one embodiment, the present invention relates to the combination of (i) histone deacetylase inhibitor (HDACi) and (ii) vaccine composition comprising a group of inactivated fetal cells, for use in treating cancer in subject.In another embodiment, the vaccine is comprised of a group of inactivated fetal cells.In particular, the cells of the group express one or more target antigens that are also expressed by cancer cells of subject.In certain embodiments, the group of inactivated fetal cells is organoid or is derived from organoid (i.e., is obtained by destroying the 3D structure of organoid).

[0012] Fetal stem cells are a. differentiating a population of pluripotent cells towards a pathway associated with a particular cancer in a patient; b. Expanding the differentiated cells; c. optionally, exposing the cells of the population to a mutagenic agent during expansion to induce genetic mutagenesis in the cells of the population; d. verifying that at least 70% of the cells in the population express fetal markers; e. optionally verifying that cells of the population express at least one tumor-associated antigen (TAA) or neoantigen present in cancer cells of the subject; f. inactivating the cells so that they lose their ability to divide. It is preferred if the polymer is obtained by a process comprising:

[0013] When mutagenesis is performed, it is preferred that the mutagen is selected from the group consisting of chemical mutagens and radiation mutagens (X-rays, UV rays), in particular, the mutagen is selected from the group consisting of ENU, reactive oxygen species, deaminating agents, polycyclic aromatic hydrocarbons, aromatic amines and sodium azide.

[0014] In a preferred embodiment, the histone deacetylase inhibitor is selected from the group consisting of valproic acid (VPA), vorinostat, panobinostat, gibinostat, belinostat, entinostat, mocetinostat, practinostat, chidamide, xinostat, and abexinostat.

[0015] The present invention also provides a composition of inactivated cells comprising inactivated fetal stem cells obtained from iPS-derived fetal hematopoietic lineages, wherein the cells in the population, after expansion, [Table 1] The present invention relates to a composition of matter that exhibits a mutation rate of at least 0.1% in at least one gene selected from the group consisting of: These genes are generally expressed in acute leukemia, particularly acute myeloid leukemia.

[0016] The present invention also provides a composition of inactivated fetal cells comprising inactivated fetal stem cells in iPS-derived kidney organoids, wherein the cells in the population are selected from the following groups: [Table 2] The present invention relates to a composition of matter expressing at least one fetal antigen selected from the following: These genes are commonly expressed in primary adult renal carcinomas, whether or not associated with c-Met mutations.

[0017] The present invention also relates to the composition of inactivated fetal cells, comprising inactivated fetal stem cells in iPS-derived lung organoid, wherein the cell in the group expresses at least one fetal antigen selected from the following group: AIM2, AQP4, AURKA, BMP5, CDCA7, CEP55, CYP4B1, DACH1, EMP2, EPB41L4A, GJB2, MAOA, MELK, MKI67, NEBL, NFIA, PHF19, RNF144B and UHRF1.These genes are generally expressed in adult lung carcinoma.

[0018] The present invention also provides a vaccine composition comprising: A population of inactivated fetal stem cells and b. Agents that stimulate immune responses and / or MHC I expression The present invention relates to a vaccine composition comprising:

[0019] In particular, the inactivated fetal stem cells include mutagenized fetal stem cells, which can be used to treat cancer in a subject, especially if the cancer has a fetal stem cell signature.

[0020] Also part of the invention is a kit comprising a vaccine composition disclosed herein and an information leaflet providing instructions for immunization.

[0021] The present invention also relates to a combined preparation of i) a population of inactivated fetal stem cells and ii) a compound that activates MHC expression and / or immune response for use in treating cancer in a subject by simultaneous, separate or sequential administration, where the cancer is selected from the group consisting of bladder carcinoma, breast carcinoma, cervical carcinoma, bile duct carcinoma, colorectal carcinoma, gastric sarcoma, glioma, lung carcinoma, lymphoma, acute and chronic lymphocytic and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, gastric carcinoma, renal carcinoma, head and neck tumors, and all subtypes of solid tumors and hematopoietic malignancies.

[0022] A method of treatment in which a therapeutic amount of the composition (inactivated fetal cell population and adjuvant) is administered to a patient in need thereof is also disclosed and is part of the present invention.

[0023] In this application, all genes are referred to by their names known to those skilled in the art. From these names, gene and protein sequences can be found by using any search engine (including general search engines) or in specific databases for maintaining libraries of cancer genes, such as the COSMIC database (Catalogue Of Somatic Mutations In Cancer, developed by the Sanger Institute in the UK) or the Cancer Genome Atlas (TCGA, maintained by NCBI in the USA). These databases compile various sequences encoding antigens expressed in cancer cells. [Brief explanation of the drawings]

[0024] [Figure 1] Transcriptome identified during kidney organoid specification of c-MET mutant IPSCs. Expression heatmap (Euclidean distance) of differentially expressed genes between PB56 (c-MET mutant IPSCs) and EB56 (embryonic body-derived fetal kidney organoids derived from parental c-Met IPSCs). [Figure 2] Venn diagram of meta-analysis between the transcriptome of cMET-IPSCs and that of primary papillary renal carcinoma (PRCC) samples. The p-value of embryonic fetal kidney organoid transcriptome signature in PRCC expression profile was calculated by Fisher's hypergeometric test. [Figure 3] Expression of CMH class I and II in cultured human hematopoietic embryonic stem cells (EBs) obtained after differentiation of iPSCs in the presence and absence of HADCi (VPA). [Figure 4]Genomic variants in human-derived hematopoietic fetal stem cells induced by mutagens in BCR-ABL-positive IPSCs. Venn diagram of genes found to be affected by genomic variants by exome sequencing compared to parental BCR-ABL-positive IPSCs. Three different experimental conditions were tested: derived hematopoietic EBs without genomic instability (blue), early-passage derived fetal hematopoietic EBs with ENU-induced genomic instability (green), and late-passage derived fetal hematopoietic EBs with ENU-induced genomic instability (red). [Figure 5] Unsupervised principal component analysis performed on 123 genes in the ENU-treated IPSCP BCR-ABL transcriptome experiment compared to the AML transcriptome (GSE10358). The 123 affected genes in the "blast crisis in a dish" model integrated with the AML patient blast transcriptome analysis predict prognostic differentiation (log-rank p-value = 1E-4). Small gray spots: G2 favorable prognosis AML. Large black spots: G1 poor prognosis AML. Abscissa: first dimension expressed in arbitrary units of factor analysis (principal component analysis); ordinate: second dimension expressed in arbitrary units of factor analysis (principal component analysis). [Figure 6] Overall survival of AML patients with good and poor prognosis signatures. Upper curve: good prognosis AML (G2). Lower curve: poor prognosis AML (G1). Abscissa: probability of overall survival; ordinate: time in months. [Figure 7] Signature between iPSC-derived lung organoids and lung cancer showing 19 common genes. [Figure 8] Concentrations of HDACi used to assess MHC1 HLA-ABC expression. [Figure 9]Expression of MHC1 HLA ABC on CML-derived iPSCs (PB32) in the presence and absence of HDACi. Left panel: RFI mean normalized to DMSO control for iPSCs (PB32) treated with four different HDACi using an MHC I HLA-ABC monoclonal antibody coupled to APC. Ordinate: RF / MHC1 fluorescence magnification. Right panel: % of MHC1 expression normalized to DMSO control for CML-derived iPSCs (PB32) exposed to four different HDACi. Ordinate: % of positive APC fluorescence normalized to DMSO control. [Figure 10] Expression of MHC1 HLA ABC on IPSCs (PB33) without genomic alterations in the presence and absence of HDACi. Left panel: RFI mean normalized to DMSO control for IPSCs (PB33) treated with four different HDACi using an MHC I HLA-ABC monoclonal antibody coupled to APC. Ordinate: RF / MHC1 fluorescence magnification. Right panel: % of MHC1 expression normalized to DMSO control for IPSCs (PB33) exposed to four different HDACi. Ordinate: % of positive APC fluorescence normalized to DMSO control. [Figure 11] Unsupervised principal component analysis (PCA) performed on 392 genes in transcriptome experiments of mouse iPSCs, mouse ESCs, transplanted Pan02, and mouse endodermal progenitor cells (EndoPCs). Abscissa: first dimension expressed in arbitrary units of factorial analysis (PCA); ordinate: second dimension expressed in arbitrary units of factorial analysis (PCA). [Figure 12] Gene expression heatmap and unsupervised classification performed using 359 genes between mouse iPSCs, mouse ESCs, transplanted Pan02 and mouse endodermal progenitor cells (EndoPCs). [Figure 13]Expression of pluripotency genes by RT-PCR in mouse fibroblasts, iPSCs, and EndoPCs. Expression of iPSC-enriched genes by quantitative RT-PCR in EndoPCs compared to mouse iPSCs and primary mouse C57BL / 6 fibroblasts. Seven different factors, including OCT4, SOX2, NANOG, LIN28, cMYC, KLF4, and alkaline phosphatase (ALP), were quantified and subsequently normalized to the mRNA levels found in miPSCs (a value of 100). Ordinate: relative mRNA expression expressed in arbitrary units. Abscissa, left to right, groups of bars: (i) mifibroblasts; (ii) EndoPCs; (iii) miPSCs. For each group of bars, left to right: (i) OCT4, (ii) SOX2, (iii) NANOG, (iv) LIN28, (v) cMYC, (vi) KLF4, (vii) ALP. [Figure 14] Expression of the mESC marker SSEA-1 in EndoPCs compared to ESCs by flow cytometry analysis. Left panel: mESCs (CK35); right panel: EndoPCs. Ordinate: fluorescence signal intensity (log) expressed in arbitrary units. Abscissa: signal magnitude. [Figure 15] Western blot analysis of STAT3, pSTAT3 (Y705), and β-actin on Pan02 cells expanded with or without 100 ng / ml IL6, performed after 0.5 and 4 hours. Pan02 cells were incubated with (columns, left to right): (i) no IL-6, no Jak inhibitor, (ii) no IL-6, no Jak inhibitor, (iii) IL-6, no Jak inhibitor, (iv) IL-6, no Jak inhibitor, (v) IL-6, Jak inhibitor, (vi) IL-6, Jak inhibitor. Rows, top to bottom: (i) p-Stat3 Y705, (ii) Stat3, (iii) β-actin. [Figure 16] Overall survival of mice vaccinated with two booster immunizations of EndoPC compared to untreated mice (n=8). Ordinate: survival rate; abscissa: time in days. [Figure 17]Quantification of bioluminescence regions of interest (ROI) measuring surface intensity in the pancreas of treated mice compared to control mice. Ordinate: region of interest (ROI) surface intensity expressed in arbitrary units. [Figure 18] This diagram represents the different steps to obtain fetal stem cells derived from pluripotent stem cells. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed description of the invention:

[0026] The present inventors have demonstrated that the combination of HDACi and a population of fetal stem cells resulted in a synergistic and efficient immune system response against tumor cells. The present inventors have also demonstrated that various HDAC inhibitors (HDACi), including belinostat, entinestat, leviteracetam, and valproic acid, can act synergistically with a population of fetal stem cells to generate an efficient immune response against tumor cells. The present inventors have also demonstrated that vaccination of individuals suffering from cancer, such as pancreatic cancer, with a combination of (i) fetal stem cells, such as irradiated endodermal progenitor cells (EndoPCs), and (ii) HDACi, such as valproic acid, resulted in dramatic tumor inhibition and significantly improved survival rates.

[0027] Indeed, the inventors hypothesized that induced iPSC-derived fetal stem cells, as a source of fetal neoantigens, could be used as a vaccine to generate an immune response against various fetal antigens shared by tumor cells, which response could be more specific than that obtained with pluripotent cells. The inventors further hypothesized that vaccination of mice with fetal stem cells in combination with HDACi (e.g., valproic acid) would enhance the immune system, and were able to demonstrate that this induced efficient immune and antitumor responses without side effects or evidence of autoimmune disease.

[0028] Description of prior art documents

[0029] WO 2012 / 122629 discloses the combination of an HDACi with a viral oncolytic vaccine expressing an antigen selected from the group consisting of tumor antigens (AFP), carcinoembryonic antigen (CEA), CA125, Her2, dopachrome tautomerase (DCT), GP100, MARTI, MAGE proteins, NY-ESO1, HPV E6, and HPV E7. HDACi are immune modulators that increase the secondary immune response after the primary immune response induced by a viral antigen (derived from or expressed by the virus).

[0030] Bartlett et al. (Molecular Cancer 2013, 12:103) disclose oncolytic viruses (OVs) as therapeutic cancer vaccines. The immunostimulatory genes provided by OVs induce potent antitumor immunity in animal models and human patients, particularly when co-administered with HDAC inhibitors, which temporarily inhibit innate immunity and promote OV infection and spread. Therefore, the role of HDAC inhibitors (HDACi) is to increase the immune response by allowing the vector to replicate more efficiently.

[0031] Bridle et al. (Molecular Therapy vol. 21 no. 4, 887-894 apr. 2013) disclosed that HDAC inhibition suppresses primary immune responses, enhances secondary immune responses, and neutralizes autoimmunity during tumor immunotherapy. However, this result was obtained in the context of oncolytic viruses (OVs) as therapeutic agents applicable to various malignancies, and only demonstrated that HDACi could impair the primary immune response induced against oncolytic vaccine vectors and enhance the resulting secondary response.

[0032] Wu et al (J Ovarian Res. 2015 Oct 24;8:68) used an established ovarian cancer cell line enriched in stem cells as a vaccine against a given cancer without the use of any adjuvant.

[0033] WO 2016 / 065330 discloses the use of modified stem cells containing a cytotoxic payload; (ii) wild-type or genetically modified viruses; (iii) wild-type or genetically modified bacteria; or (iv) a combination of two or more thereof to treat solid tumors or hematological malignancies in a subject. The description provides a list of stem cells that may potentially be used, but does not provide any details regarding the actual nature or characteristics of these cells. The document does not mention or suggest the addition of HDACi to generate an immune response against cancer.

[0034] WO 2017 / 027757 discloses the use of a smallpox vaccine to treat cancer in a subject, and stem cells can be added to the vaccine. This description provides a list of stem cells that can potentially be used, but does not provide any details regarding the actual properties or characteristics of these cells. This document does not describe or suggest the addition of HDACi to generate an immune response against cancer.

[0035] European Patent Application Publication No. 2599860 discloses induced cancer stem cells, which are induced progenitor cancer stem cells or induced malignant stem cells, expressing six genes: POU5F1, NANOG, SOX2, ZFP42, LIN28, and TERT; and having an abnormality that is either (a) a mutation in an endogenous tumor suppressor gene or (b) increased expression of an endogenous cancer-related gene. Considering the expressed genes, these cells are not fetal cells. In particular, POU5F1 and NANOG are markers of non-differentiation and pluripotency.

[0036] Zheng et al. (Oncol Rep. 2017 Mar;37(3):1716-1724) compared vaccination with hepatic stem cells (HSCs) or embryonic stem cells (ESCs). HSCs were isolated from the livers of adult mice and are therefore not fetal cells. Furthermore, no other adjuvants, particularly HDACi, were used in this study.

[0037] WO 2017 / 202949 discloses the use of pluripotent cells in combination with HDACi to treat cancer. Pluripotent cells are distinct from fetal cells and do not express some antigens expressed in fetal cells and cancer.

[0038] In summary, none of the above documents discloses the specific combination of a population of (potentially mutated) inactivated fetal cells with an adjuvant (especially one that stimulates HDACi or MHC I expression) as a therapeutic or prophylactic vaccine against cancer.

[0039] Fetal Cell Compositions and Methods of Use

[0040] The present invention also relates to the use of a population of fetal cells as an immunogen.

[0041] In the present context, a population of fetal cells corresponds to a population of cells maintained as a cell culture, but also encompasses organoids, in which cells have begun to form organs and in which the 3D spatial organization of the cells can be observed.

[0042] Note that differentiation is the process by which more specialized cells are formed from less specialized cells. It is a continuous process. Starting from pluripotent cells (embryonic stem cells or iPS cells), cells lose pluripotency, commit to a differentiation mode, and mature into fully differentiated specialized cells. In some organs, multiple cells will create an organoid during the differentiation process.

[0043] The induction and direction of differentiation of pluripotent cells are known to those skilled in the art. Reference may be made to Wu et al. (Cell. 2016 Jun 16;165(7):1572-1585), Fatehullah et al. (Nat Cell Biol. 2016 Mar;18(3):246-54) or Sasaki and Clevers (Curr Opin Genet Dev. 2018 Sep 24;52:117-122), which describe the development or organoids from pluripotent cells. There are several other papers that describe and teach the methods and conditions for differentiating pluripotent cells into various tissues of interest.

[0044] Definition of fetal cell populations

[0045] Fetal cells are cells that have lost their pluripotency as they have begun to commit to differentiation pathways (endoderm, mesoderm, ectoderm).

[0046] It is possible to determine whether a population of cells is a population of fetal cells, as the cells express fetal markers (see below) and do not express pluripotency markers.

[0047] The populations of the present invention comprise a large number of cells (at least 0.5×10 6 cells, more preferably at least 1 x 10 6 cells, more preferably at least 2 x 10 6 cells or 5 x 10 6 cells or 5 x 10 6 Contains more than 1000 pieces.

[0048] To determine whether a population of cells is a population of fetal cells, (a) it must be determined that the cells of the population do not essentially express pluripotency genes (or markers); (b) The presence of fetal genes (or markers) expressed by cells of the population must be determined.

[0049] In certain embodiments, the cells of the fetal cell population are: (a) no or less than 10% of cells express genes typically expressed in undifferentiated pluripotent self-renewing cells (embryonic stem cells or induced pluripotent stem cells), preferably as determined by flow cytometry, more particularly by FACS (fluorescence-activated cell sorting); and (b) Whether the population is in the form of committed differentiated progenitors derived from the three germ cell layers or 3D organoid tissue, at least 70%, more preferably greater than 75%, more preferably greater than 80% of the cells in the population express progenitor / fetal markers.

[0050] It is also preferred if less than 10% of the cells express an adult tissue marker. An adult tissue marker is a marker (protein or gene) that is expressed in adult cells.

[0051] The percentages refer to the percentage of cells in the population that express a given marker. As an example, low expression (<10%) of a master gene typically expressed in undifferentiated pluripotent self-renewing cells indicates that fewer than 10% of the cells in the population express the gene under investigation, as further explained below.

[0052] Note that the markers expressed vary during the differentiation process. Consequently, some markers associated with the fetal nature of the cells are expressed early in the differentiation process (i.e., shortly after the loss of pluripotency), while some markers are expressed later in the process (i.e., before maturation of adult cells). The lack of expression of these fetal markers indicates that the cells have lost their fetal characteristics and likely acquired a phenotype indicative of their maturation into differentiated adult cells.

[0053] (a) Gene expression and / or immunocytochemistry assessment can be used to determine that pluripotency genes are not expressed by the cells of a population. The goal is to demonstrate the absence or low expression of master genes typically expressed in undifferentiated pluripotent self-renewing cells (embryonic stem cells and induced pluripotent stem cells).

[0054] especially, a) A population of iPS cells can be used as a positive control for markers of pluripotency; b) The expression levels of a set of pluripotency genes in the target population and the iPS cell population can be compared.

[0055] If the expression level of the pluripotency genes is less than 10%, more preferably less than 5%, of the expression level of these genes in the iPS cell population, or if less than 10%, more preferably less than 5% of the cells express the genes, the cells of the target population are considered not to express the pluripotency genes. Any quantification method can be used, such as RT PCR or flow cytometry or immunohistochemistry. It is preferable to use FACS (fluorescence-activated cell sorting) of the cells. In this method, less than 10% of the cells of the population express these pluripotency genes.

[0056] There are several markers expressed by pluripotent cells. Indeed, the expression of these pluripotency markers is correlated, so that when a cell loses its pluripotent characteristics, it will also lose the expression of these markers. Consequently, although several genes expressed by pluripotent cells (pluripotency genes) are known in the art, it is not necessary to study a large number of such.

[0057] More particularly, it is preferred to study the expression of at least one pluripotency gene selected from the group consisting of NANOG, POU5F1(Oct4), SSEA4, Tra-1-81 and Tra-1-60.

[0058] In one embodiment, a combination of one intracellular (e.g., OCT4 or Nanog) and one extracellular (e.g., SSEA-4 or Tra-1-60 or Tra-1-81) may be used to improve the accuracy of the measurement.

[0059] However, three, four or even five of these genes may be investigated.

[0060] Determining the percentage of cells in a population that express these markers is easily performed by FACS using antibodies available in the art. It is even possible to perform this analysis in multiplex experiments.

[0061] When multiple genes are studied, the proportion of cells in a population that are considered pluripotent is determined by taking the average of the proportion of cells with each marker.

[0062] By way of example, if the percentage of cells in a given population that express gene (1) is 6% and the percentage of cells in a given population that express gene (2) is 5%, the population would be considered to contain 5.5% pluripotent cells (the average of 5% and 6%), and the given population would be considered to have satisfied condition (a) above.

[0063] To determine that cells of a population express fetal genes and satisfy condition (b), it is necessary to detect genes (markers, proteins, or antigens) that are expressed by the cells when they enter one of the differentiation pathways.

[0064] Neural fetal cells: Early neuroectodermal precursors: TP63, MASH1, Notch1, Sox1, Sox2, Musashi 2, Musashi 1, Nestin, Pax6, MUC18, BMI1, Mash1, FABP7, Nucleostemin

[0065] Hematopoietic fetal cells Hematopoietic mesoderm precursor: Brachyury (T), MIXL1, cryptic, GATA1, LMO2, ACE, SCL (Tal1), HoxA9, Fli1

[0066] Fetal kidney cells: Renal mesoderm precursors: WT1, HOXD11, SIX2, SALL1, WT1, PAX2, OSR1, PAX8, LHX1, GATA3, HOXB7

[0067] Fetal liver cells: Hepatic endoderm precursors: SOX17, HNF3B, HNF6, Fox-A2, HNF1B, GATA4, AFP, LGR5

[0068] Fetal pancreatic cells: Pancreatic endoderm precursors SOX17, Fox-A2, CXCR4, GATA4, HNF1B, HNF4A, PDX1, HNF6, PROX1, Ngn3, NeuroD1, PAX6, SYP, SOX9, NKX2-2, NKX6-1, P48, LGR5, HB9

[0069] intestinal fetal cells Gut endoderm precursors: CDX2, TCF-2, SOX9, NMYC, ID2, SOX2, PAX8, Nkx2.1, LGR5

[0070] fetal lung cells Pulmonary endoderm precursors: CXCR4, SOX17, FOXA2, NKX2.1, PAX9, TBX1, SOX2, SOX9, ID2, Foxj1, Scgb1a1, Foxj1

[0071] fetal thyroid cells Thyroid endoderm precursors: CXCR4, SOX17, FOXA2, Pax8, HHEX, Nkx2-1

[0072] Other fetal cells Myoblast precursors: Pax7, Pax3, Myf5 Chondrocyte precursors: osteonectin, Sox9 Osteoblast precursors: Runx2, ALP, Osx, osteopontin, osteocalcin

[0073] All of the above genes are known in the art and are specific to each differentiation pathway and each tissue organoid.These fetal genes in early or late precursors are not expressed in fully differentiated adult cells.As shown, their sequences can be found in the public database that is widely available.

[0074] Consequently, these markers are markers of early ontogeny and reflect the fact that cells bearing these markers are not fully mature adult cells: they are still progenitor cells at a fetal stage of development, which means that they can still give rise to various types of mature cells.

[0075] In the context of the present invention, to obtain a fetal cell population, the skilled person will induce the differentiation of pluripotent cells (e.g. embryonic stem cells or iPS cells) in one of the differentiation pathways according to known methods.

[0076] Loss of pluripotency is verified by checking the loss of expression of the above markers in at least 90% of the cells.

[0077] Depending on the differentiation pathway chosen by the person skilled in the art, it is possible to check the presence of the above-mentioned specific fetal markers in the cell population.

[0078] To do this, those skilled in the art will use FACS analysis to measure the percentage of cells that express fetal markers of a given pathway, and calculate the percentage by verifying that at least 70% of cells express at least one of these markers.The use of multiplex FACS analysis also allows the number of cells that express more than one marker to be determined.In other words, this means that the percentage of cells that do not express any of these markers does not exceed 30%.This can also be easily determined by FACS analysis.

[0079] It is also possible to determine whether a cell population is fetal in origin without prior knowledge of the differentiation pathway of the cells.

[0080] To check whether a cell population is a fetal cell population of the present invention, one should first examine whether the cells express one or more of the above pluripotency markers (and the proportion of cells in the population that express said markers). If fewer than 10% of the cells express the above markers, one skilled in the art can examine the expression of fetal markers by the cells of the population.

[0081] Cell morphology / histology can provide information about cell lineage commitment to those skilled in the art, allowing for the selection of several markers for initial checks. However, it is also possible to verify the fetal nature of cells without prior knowledge of cell lineage commitment.

[0082] To do this, RNA from the cells of the population can be extracted, reverse transcribed, optionally amplified, and applied to any DNA chip or array containing the above-mentioned fetal marker probes.In particular, low-density arrays (LDA) can be used.This not only allows determining the presence of fetal markers, but also allows qualifying these markers (i.e., determining the differentiation pathway of the cells of the population) (depending on the probes that are "turned on" by the RNA from the cell population).

[0083] If the differentiation pathway is known, FACS analysis can be performed using specific markers of this particular cell lineage differentiation pathway to quantify the proportion of cells in the population that express these markers.

[0084] Use of fetal cell populations

[0085] It has long been suggested that fetal antigens may be expressed in tumor cells (Ting et al., Proc Natl Acad Sci USA. 1972 Jul; 69(7): 1664-1668).

[0086] The inventors have now demonstrated that the populations of fetal cells disclosed herein can be used for the prophylactic or therapeutic treatment of cancer in a subject. The inventors hypothesize that cancer onset and progression may be driven by or promoted by mutations in the test cells that induce dedifferentiation, causing them to reverse their differentiation pathway to new "fetal-like" characteristics, and leading to their proliferation. As a result, such cells express fetal markers that are not expressed in mature, fully differentiated adult cells. Furthermore, as these cells divide rapidly, this induces mutations that create mutant antigens, also referred to as neoantigens. It should indeed be noted that fetal antigens or neoantigens of tumor cells are generally shared between cancers, at least between cancers of organs derived from the same differentiation pathway (ectoderm, endoderm, or mesoderm).

[0087] From the ectodermal pathway, organs include epidermal skin cells, neurons, glial cells, neural crest; and pigment cells.

[0088] From the mesodermal pathway, organs include cardiac muscle, skeletal muscle cells, kidneys (renal tubules), red blood cells, and smooth muscle (in the intestine).

[0089] From the endodermal pathway can be cited lung cells (particularly alveoli), thyroid cells, pancreatic cells, and liver cells.

[0090] Finally, the microenvironment of cancer cells is generally favorable to the immune system, as it inhibits the action of T lymphocytes.

[0091] By administering these inactivated fetal cell populations, preferably together with HDACi or with compounds that increase the expression of MHC I molecules, it will be possible to induce an immune response in a subject (preferably a human, but which could also be another mammal, such as a dog, cat, cow, or horse) against fetal antigens present on the cells of the population, and therefore against tumor cells, thereby resulting in cancer regression, which is effective for both solid and hematological tumors.

[0092] Indeed, cancer cells may express antigens (markers) such as those expressed by cells of the fetal population disclosed and characterized herein.

[0093] As a result, a population of fetal cells (fetal population) can be used to prime a patient's immune system so that it can adequately and efficiently fight cancer.

[0094] The inventors have noted a synergistic effect when using both HDACi (or agents that increase MHC-I expression) and a population of inactivated fetal cells, which may be due to one or more of the following: i) Increased MHC class I expression on fetal and tumor cells (better presentation of fetal and neoantigens), activating / strengthening the immune response ii) Increased fetal antigens / neoantigens in cancer stem cells (CSCs) and tumor cells by demethylation, which induces specific immune responses against these antigens / neoantigens. iii) Increased chemokine expression, which recruits CD4+ and / or CD8+ and / or CD8+PD1- T lymphocytes to the tumor and makes the tumor immunoreactive. iv) Reduction of regulatory T lymphocytes and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, making the tumor immunoreactive.

[0095] Considering different pathways, fetal cell populations may be used to treat lung, pancreatic, renal, breast, blood, gastrointestinal, thyroid, prostate, brain (particularly glioblastoma), stomach, liver, bone, and ovarian cancers. A fetal cell population should be selected that is committed to the same cell lineage as the cancer to be treated.

[0096] The use of such fetal cell populations allows for the delivery of at least 10, more typically at least 50 or at least 100, 500 or even 1000 fetal or neoantigens that are expressed in a given cancer or that are common to different cancers.

[0097] Fetal cells may contain a mutation that predisposes to familial cancers and that is shared among cancers of a lineage, expressing a fetal gene (BRCA, cMET, RET, APC, etc.) that is deregulated by this mutation; for example, iPS cells obtained from blood cells containing a c-Met mutation can be derived as kidney organoids that contain a c-Met mutation present in kidney cancer.

[0098] The use of mutagenic agents when preparing fetal cell compositions (see below) introduces mutations (e.g., missense or frameshift mutations) into genes of the cells of the population, resulting in the expression of neoantigens.

[0099] In particular, the inventors have shown that iPS cells derived from chronic myeloid leukemia (CML) cells, mutated with ENU, and derived from hematopoietic fetal cells, contain antigens present in acute myeloid leukemia (AML).

[0100] To treat the patient, i) obtaining an antigen-specific signature of such cancer from a biopsy of the cancer in the subject; ii) selecting a population of inactivated fetal cells containing cells expressing at least one of the antigens determined in step i); iii) This population may be administered to the patient together with an HDACi or an agent that increases MHC I expression.

[0101] Step i) is carried out by methods known in the art using tools available in the art.

[0102] The signature, in particular, - determining the genes expressed in cancer cells (exome sequencing), - comparing the gene with databases of cancer-specific genes (in particular, the COSMIC database (Catalogue Of Somatic Mutations In Cancer) developed by the Sanger Institute in the UK) or the Cancer Genome Atlas (TCGA maintained by NCBI in the USA) may be cited) (these databases regroup the various sequences encoding antigens expressed in cancer cells), - Obtained by selecting genes present in both the exome and the database as antigen-specific signatures of cancer.

[0103] Step ii) is performed by performing an exome sequencing of the fetal cell population and verifying that at least one gene of the antigen-specific signature of the cancer is present in the exome obtained from the fetal cell population.

[0104] In another embodiment, i) obtaining an antigen-specific signature of such cancer from a biopsy of the cancer in the subject; ii) selecting a population of inactivated fetal cells containing cells that generally express at least one of the antigens determined in step i); iii) Extracts of this population may be administered to patients together with HDACi or agents that increase MHC I expression.

[0105] In this embodiment, the extract is selected from total RNA, mRNA, DNA, protein extract, lysate, lyophilized extract, lyophilized or dried cells, exosomes, extracellular microvesicles and apoptotic bodies.

[0106] In another embodiment, i) obtaining an antigen-specific signature of such cancer from a biopsy of the cancer in the subject; ii) selecting a population of inactivated fetal cells containing cells that generally express at least one of the antigens determined in step i); iii) A population of T cells or antigen-presenting cells primed in vitro with the population of ii) or an extract of such a population in the presence of an HDACi or an agent that increases MHC I expression may be administered to a patient.

[0107] In certain embodiments, the population comprises: a. Differentiating a population of pluripotent cells toward a pathway associated with a particular cancer in a patient, wherein the pluripotent cells have optionally been expanded in the presence of a mutagenic agent; b. Expansion of the differentiated cells; c. optionally, exposing the cells of the population to a mutagen during expansion to induce genetic mutagenesis in the cells of the population; d. verifying that at least 70% of the cells in the population express fetal markers; e. optionally verifying that cells of the population express at least one cancer or neo-antigen present in cancer cells of the subject; f. inactivating the cells so that they lose their ability to divide. This was obtained by

[0108] The use of fetal cell populations according to the invention is particularly interesting, as these cells contain a number of fetal antigens that are likely to be expressed by cancer cells.

[0109] The present invention also relates to methods for developing and producing populations of cells intended to be used in the treatment of cancer in patients.

[0110] The method includes the following steps: a) optionally performing a biopsy of the cancer; b) analyzing cells recovered from a cancer biopsy from the patient to identify fetal and cancer markers expressed by the cancer cells; c) differentiating the population of pluripotent cells through a pathway associated with the patient's particular cancer (e.g., inducing renal pathway differentiation if the patient has renal cancer); d) Optionally, introducing mutations into the population of differentiated cells: this step is optional but preferably performed. It is intended to increase the diversity of antigens expressed by the cells of the population, improve the ability of the immune system to control cancer cells upon exposure to the cells, even in the presence of mutations in the cells. The mutation rate can be controlled by checking the sequence of one or more genes in the cell population. The presence of mutant sequences of a given gene in the population can be identified and quantified, e.g., compared with the sequences of the gene in the population. For example, a 0.1% mutation rate for a given gene indicates that 0.1% of the sequences identified for this gene in the population have mutations. e) optionally verifying that the cells of the population express at least one cancer or neo-antigen present in cancer cells of the subject; f) Inactivating the cells so that they lose their ability to divide, in order to avoid proliferation of the cells in vivo after administration of all or part of the cell population to a patient.

[0111] Once the cell population is obtained, all or a portion thereof may be administered to an animal (preferably a mammal, more preferably a human), preferably in the presence of an HDACi or a compound that stimulates expression of MHC I. As noted above, in all methods, inactivated fetal cell populations or extracts thereof, or T lymphocytes or antigen-presenting cells primed with said populations or extracts thereof, may be administered.

[0112] In a particular embodiment, the pluripotent cells of step c) are iPS cells (induced pluripotent stem cells) generated from the patient's cells. This may reduce the risk of cross-immunization when administering fetal cells to the patient. Indeed, fetal antigens (present on the cells of the population and on cancer cells) are recognized by the immune system, whereas non-fetal antigens are not.

[0113] Alternatively, the present invention provides a method for treating a patient, comprising: a) optionally performing a biopsy of the cancer; b) analyzing cells recovered from a cancer biopsy from the patient to identify fetal and cancer markers expressed by the cancer cells; c) selecting a population of inactivated fetal cells that are engaged in a differentiation pathway associated with the patient's particular cancer and that are optionally mutagenized; d) administering to the patient an HDACi, or a compound, cell that stimulates or increases MHC I expression. The present invention relates to a method, including:

[0114] In certain embodiments, the fetal cells are committed to the lung differentiation pathway. Thus, they will express the markers set forth above for lung. These cells are particularly suited for the treatment of lung cancer.

[0115] In certain embodiments, the fetal cells are committed to the thyroid differentiation pathway. Thus, they will express the markers set forth above for the thyroid gland. These cells are particularly suited for the treatment of thyroid cancer.

[0116] In certain embodiments, the fetal cells are committed to the renal differentiation pathway. Thus, they will express the markers set forth above for the kidney. These cells are particularly suited for the treatment of kidney cancer.

[0117] In certain embodiments, the fetal cells are committed to the hematopoietic differentiation pathway. Thus, they will express the markers set forth above for hematopoietic cells. These cells are particularly suited for the treatment of blood cancers (leukemia).

[0118] In certain embodiments, the fetal cells are committed to the liver differentiation pathway. Thus, they will express the markers set forth above for the liver. These cells are particularly suited for the treatment of liver cancer.

[0119] In certain embodiments, the fetal cells are committed to the intestinal differentiation pathway. Thus, they will express the markers set forth above for the intestine. These cells are particularly suited for the treatment of gastrointestinal cancers.

[0120] In certain embodiments, the fetal cells are committed to the pancreatic differentiation pathway. Thus, they will express the markers set forth above for the pancreas. These cells are particularly suited for the treatment of pancreatic cancer.

[0121] In certain embodiments, the fetal cells are committed to the neural differentiation pathway. Thus, they will express the markers set forth above for neurons or brain. These cells are particularly suited for the treatment of brain cancers, particularly glioblastoma.

[0122] In certain embodiments, the fetal cells are committed to the bone differentiation pathway. Thus, they will express the markers set forth above for osteoblasts. These cells are particularly suited for the treatment of bone cancer.

[0123] HDACi for improving immune response

[0124] In a first aspect, the present invention relates to a method for increasing the efficacy of a vaccine composition in a subject, comprising administering to said subject an HDACi together with said vaccine composition, in particular wherein said HDACi is added to the vaccine composition.

[0125] The present invention also relates to a combination of (i) a histone deacetylase inhibitor (HDACi) and (ii) a vaccine composition comprising an immunogenic element for use in treating cancer in a subject. In some embodiments, the present invention relates to a combination of (i) a histone deacetylase inhibitor (HDACi) and (ii) a vaccine composition comprising an immunogenic element for use in treating cancer in a subject by simultaneous, separate or sequential administration.

[0126] The present invention also relates to the use of a combination of (i) a histone deacetylase inhibitor (HDACi) and (ii) a vaccine composition containing an immunogenic element to prepare a pharmaceutical composition for treating cancer in a subject. According to some embodiments, the present invention relates to the use of a combination of (i) a histone deacetylase inhibitor (HDACi) and (ii) a vaccine composition containing an immunogenic element, by simultaneous, separate or sequential administration, to prepare a pharmaceutical composition for treating cancer in a subject.

[0127] As used herein, the term "increased efficacy" refers to an increase in the immunogenicity of a vaccine composition, an increase in the immune response to a vaccine composition, or an increase in the immune response generated by a vaccine composition, which can be compared to the immune response generated in the absence of the HDACi.

[0128] Vaccine compositions contain immunogenic elements intended to elicit an immune response in a subject 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, or other type of antigen (e.g., nucleic acid, sugar, lipopolysaccharide), whether of self (e.g., antigens derived from cancer cells) or exogenous (e.g., bacterial, viral, or parasitic proteins).

[0129] The present invention therefore relates to the use of HDACi as adjuvants, in particular for increasing the immune response to vaccine compositions, as well as HDACi for use as adjuvants or for increasing the immune response to vaccine compositions. The present invention also relates to the use of HDACi for the manufacture of vaccine compositions containing one or more antigens of interest, with the intention of generating an immune response in a subject against the antigens of interest.

[0130] The methods and uses disclosed herein are particularly interesting when the vaccine composition is a cancer vaccine composition, i.e., contains a target antigen expressed by cancer cells.In particular, the methods and uses are particularly suitable for tumors with an immunosuppressive tumor microenvironment (i.e., there is the expression of cytokines and molecular signals and the recruitment of immune tolerant cells, reducing the effectiveness of immune cells against cancer antigens).Without being bound by this theory, it is hypothesized that the presence of HDACi modifies the tumor microenvironment, possibly by modifying the expression of genes with immunosuppressive effects in cells present in, near, or around the tumor, thereby enabling immune cells to be enhanced to fight cancer cells.

[0131] The method described herein can also include administering HDACi for several days after administering the vaccine composition. This continuous administration of HDACi can be useful for maintaining microenvironment modification for a long enough time so that immune cells can "take over" tumors. Generally, this further continuous administration of HDACi will consist of daily administration of an appropriate dose of HDACi for at least 3 days and up to 1 month after vaccine administration. However, it is preferred if the further administration of HDACi is carried out for at least 1 week, more preferably at least about 2 weeks.

[0132] Vaccine compositions contain immunogenic elements (also referred to herein as immunogenic compounds) intended to induce an immune response in a subject against one or more antigens of interest.

[0133] This immunogenic element can be an antigen (or multiple antigens). This antigen can be in any form, as seen above, depending on the target cell (which is intended to include host cells as well as bacterial cells, parasitic pathogens, or viral particles). It can also be formulated with any adjuvant (immunostimulant) known in the art, such as alum or Freund's complete or incomplete adjuvant.

[0134] In another embodiment, the immunogenic compound is an extract from a cell composition, the cells of which express the antigen of interest. The cell extract may be lysed cells that have been centrifuged to remove insoluble material, such as membrane fragments, vesicles, and nuclei, and thus consist primarily of cytosol. In another embodiment, the extract is one that has been prepared using a specific technique to deplete or concentrate specific components (e.g., sonication can be used to break down large membrane fragments into small particles that remain in the extract, or high-speed centrifugation can be used to remove the smallest insoluble components). Cell extracts can be obtained by any chemical or mechanical process, such as pressure, distillation, or evaporation.

[0135] In another embodiment, the immunogenic element is a cellular composition, and the cells of the composition express the antigen of interest. In a specific embodiment, the cell membrane is preserved (so that antigen presentation occurs via the MHC-I pathway). In a specific embodiment, the cells are inactivated as described below. In a specific embodiment, the cells are fetal stem cells, cancer stem cells, virally infected cells, or bacterial cells. In another embodiment, the immunogenic element is a cellular composition comprising antigen-presenting cells (APCs) primed in vitro with the antigen of interest. This composition is an antigen-presenting cell vaccine made from the antigen and the antigen-presenting cells (APCs). Antigen-presenting cells are cells that present antigens complexed with major histocompatibility complexes (MHC) on their surface. Dendritic cells (DCs) can present antigens to both helper T cells and cytotoxic T cells, macrophages, or B cells, and are therefore preferred in the context of the present invention. These APCs can be natural or engineered cells. In particular, Eggermont et al. (Trends in Biotechnology, 2014, 32, 9, 456-465) may be cited, which outlines 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 to those skilled in the art.

[0136] In another embodiment, the immunogenic element does not actually contain an antigen, but instead consists of a composition of T cells / lymphocytes that have been primed in vitro against an antigen of interest, e.g., by exposure to antigen-presenting cells that present the antigen of interest. As a result, this composition is capable of initiating an immune response against the antigen of interest in vivo. This strategy can be referred to as "adoptive transfer of T cells," and such adoptively transferred T cells are known to persist long-term in vivo and readily migrate between the lymphoid and vascular compartments (Bear et al., J. Biomed. Biotechnol. 2011;2011:417403; Melief et al., J. Clin. Invest. 2015;125(9):3401-3412).

[0137] In some embodiments, the HDACi is administered in combination with a vaccine composition containing an immunogenic element, which may be simultaneous, separate, or sequential, as disclosed below for embodiments in which the immunogenic element is a fetal stem cell composition. It should be noted that all of the following disclosures regarding fetal stem cell compositions are equally applicable to vaccines containing any of the immunogenic elements disclosed above.

[0138] Because fetal stem cells express neoantigens that are also found in highly aggressive cancers, as discussed above, the present specification emphasizes HDAC inhibitors (particularly valproic acid) in conjunction with such fetal stem cell compositions. Consequently, whatever the immunogenic element, it is preferred when the antigen of interest is a neoantigen expressed by cancer cells, as discussed above and further below.

[0139] In a particular embodiment, the immunogenic element is a cellular composition, and as disclosed in more detail below, the fetal cell composition is derived from the inactivation of pluripotent stem cells and fetal cells.

[0140] As used herein, the term "immunogenic element" refers to a compound that stimulates the immune system. In the context of the present invention, an immunogenic element is: a. the antigen of interest, b. fetal stem cell compositions; b. an extract from a cellular composition, wherein the cells of said composition express an antigen of interest; c. a cellular composition, wherein the cells of said composition express an antigen of interest; d. A cell composition comprising antigen-presenting cells primed in vitro with an antigen of interest, or e. T cell lymphocytes primed in vitro against an antigen of interest by exposure to antigen-presenting cells that present the antigen of interest. is selected from the group consisting of:

[0141] In a particular embodiment, the immunogenic element is a cellular composition, which is obtained by in vitro differentiation of pluripotent stem cells (ESCs and iPSCs). More specifically, the immunogenic element is a population of fetal stem cells obtained by differentiation from ESCs and iPSCs.

[0142] The method of the invention wherein the treatment is a therapeutic treatment.

[0143] The method of the present invention wherein the treatment is a prophylactic treatment.

[0144] Methods of treating subjects suffering from cancer with combined preparations

[0145] In a second aspect, the present invention relates to a method of treating a subject suffering from cancer, comprising administering to said subject simultaneously, separately or sequentially therapeutic amounts of i) a population of fetal stem cells and ii) a compound selected from the group that activates MHC expression and / or immune responses as a combined preparation.

[0146] In certain embodiments, the cells are cultured to present neoantigens through the MHC I pathway, and in particular, some cells present in the population are mutated. The compounds used in combination with the fetal cells also do not preserve the pluripotency of the pluripotent stem cells. In certain embodiments, after administration of the fetal cells, a compound that activates MHC expression and / or an immune response (preferably the same as that initially administered in combination, but possibly a different one) is administered to enhance the immune response.

[0147] As used herein, the term "treat" or "treatment" refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of subjects at high predisposition risk for or suspected of having cancer, such as inherited familial cancer syndromes, as well as subjects who are ill or diagnosed with cancer or a medical condition, including the suppression of clinical recurrence. Treatment may be administered to a subject who has cancer or who may ultimately 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 subject's survival beyond that expected in the absence of such treatment. A "therapeutic regimen" refers to a pattern of disease treatment, e.g., a pattern of medication 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 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. The induction regimen may (partially or entirely) use a "loading regimen" (which may involve administering a higher dose of drug than the physician would use during a maintenance regimen, administering the drug more frequently than the physician would administer the drug during a maintenance regimen, or both). The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part 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., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon the achievement of certain predetermined criteria (e.g., pain, disease symptoms, etc.)).

[0148] As used herein, the term "co-administration" refers to the administration of two active ingredients at the same time or substantially the same time by the same route. The term "separate administration" refers to the administration of two active ingredients at the same time or substantially the same time by different routes. The term "sequential administration" refers to the administration of two active ingredients at different times, where the routes of administration are the same or different.

[0149] 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 an embryonic stem cell antigen.

[0150] As used herein, the term "population" refers to a population of cells in which a majority of the total number of cells (e.g., at least about 20%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 80%, and even more preferentially at least about 90%) have a particular characteristic of the cell of interest (e.g., a fetal stem cell marker).

[0151] As used herein, the term "fetal stem cell population" refers to a population of fetal cells that are transient precursors that appear at an early stage of development. This type of population can be replicated in vitro by differentiation of allogeneic, heterogeneous, or syngeneic pluripotent stem cells (ESCs and iPSCs). Fetal population cells are characterized by the loss of genes related to pluripotency, including the following genes: [Table 3] Notably, fetal stem cells are also characterized by a lack of expression of lineage-specific genes in adult differentiated cells.

[0152] In certain embodiments, fetal stem cell populations can be obtained by dedifferentiation procedures, or by transdifferentiation techniques using small molecules, and / or by direct conversion of adult somatic cells by overexpression of specific transcription factors. These so-called "derived fetal population cells" are characterized by the acquisition of fetal genes and the loss of lineage-specific genes of adult cells. All fetal population cells are derived from three germ layers: ectoderm, endoderm, and mesoderm precursors, respectively. These fetal genes are 1) in endodermal progenitors, by at least SOX17, CXCR4, FOXA1, FOXA2, FOXA3, HHEX, GATA4, GATA6, HNF1B, HNF4A, TF, ALB, TBX3, AFP, TTR, CER1, MIXL1, LHX1, GSC, PAX9, NEPN, SHH, PYY, MNX1, KITL, CLDN4, CLDN8, GFPT2, KRT19, SORCS2, EPPK1, NEDD9, PLAT, VTN, PDX1, TMPRSS4, CLIC6, RIPK4, CLDN8, and ST1A; 2) in ectodermal progenitors, by at least PCGF4, PAX6, PAX7, CXCR4, SOX1, SOX2, SOX10, ITGB1, FABP7, NES, FUT4, PROM1, MELK, MSI1, MAP2, DCX, NCAM1, TUBB3, SLC1A3, CD44, S100B, VIM, GFAP, CNP, OLIG2, CA2, CSPG4, TAZ, MSX1, SPARC, ID2, NES, NKX2.2, NKX6-1, FOXP2, FOXD3, and ZIC1; and 3) In mesodermal progenitors, at least Blackury (T), MIXL1, SNAI1, SNAI2, HLX, EOMES, MESP1, MESP2, TBX6, MEST, NKX2-5, and KDR are involved. is represented.

[0153] Typically, fetal mass cells express fetal development genes that are not expressed in adult stage.These fetal genes are associated with committed fetal cell lineages or differentiated tissues, such as 3D organoid structures or embryoid bodies or spheroids or cell aggregates.These fetal cells can be neural stem cells, neurons, hepatocyte-like cells, hepatoblasts, nephron kidney progenitor cells, pancreatic endoderm precursors, bile duct cells, hematopoietic precursors, hemangioblasts, mesenchymal stem cells, endothelial cells, cardiomyocytes, neural crest precursors, mammary epithelial cells, intestinal or colon organoids, lung organoids, kidney organoids, and brain organoids.

[0154] As used herein, the term "pluripotent" refers to cells that have the ability, under appropriate conditions, to give rise to progeny that can undergo differentiation into all cell types derived from the three germ layers (endoderm, mesoderm, and ectoderm) and possess specific cell lineage characteristics. The term "pluripotent" includes engineered induced pluripotent stem cells (iPSCs) reprogrammed from all sources and cellular origins, including normal embryonic stem cells (ESCs) or very small embryonic-like stem cells (VSELs), or adult somatic cells (ASCs).

[0155] Pluripotent stem cells contribute to fetal development of tissues in prenatal, postnatal, or adult organisms. Standard, art-recognized tests are used to establish the pluripotency of a cell population, e.g., its ability to form teratomas in 8-12 week-old SCID mice, as well as 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 sometimes all of the following non-limiting markers: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, alkaline phosphatase (ALP), Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rex1, Nanog, TERC, and TERT.

[0156] Pluripotent stem cells traditionally arise from the blastocyst stage of embryonic development and have the potential to develop into all types of fetal and adult cells, possibly with the exception of the placenta. Embryonic pluripotent stem cells (ESCs) can generally be isolated from 4- to 5-day-old postfertilization blastocysts containing 50-150 cells. While ESCs are capable of indefinite ex vivo expansion, they only exist transiently in vivo during embryonic development. Various animal (including human) ESC lines, such as the NIH-approved cell line WAO9 human ESC, can be commercially obtained from WiCell Research Institute, Madison, Wis. Human ESC lines, such as Cecol-14, can be commercially obtained from, for example, Cecolfes, Bogota, Colombia. Of course, other embryonic stem cell lines can be used if desired.

[0157] 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. Patent Application Publication No. 2005 / 0176707, and WO 2001085917. In the context of the present invention, human embryonic stem cells (hESCs) are established without embryo destruction according to the technique described in Chung et al. (2008).

[0158] In certain embodiments, the fetal population cells of the present invention are obtained by in vitro methods, for example, by differentiation of pluripotent stem cells, such as ESCs and iPSCs. 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, first disclosed by Yamanaka (see, inter alia, WO 2012 / 060473, International Patent Application No. PCT / JP2006 / 324881, International Patent Application No. PCT / JP02 / 05350, U.S. Pat. Nos. 9,499,797, 9,637,732, 8,158,766, 8,129,187, 8,058,065, and 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.

[0159] iPSCs have been successfully generated from normal somatic cells of various origins (fibroblasts, blood cells, keratinocytes, etc.) and from various diseases, such as somatic or hereditary cancers (leukemia, glioblastoma, melanoma, breast cancer, etc.) and genetic disorders. Cell reprogramming can be performed with or without small compounds by variable techniques (e.g., integrating lentivirus / retrovirus and non-integrating vectors such as Sendai virus, episomal vectors, synthetic mRNA, adenovirus, rAAV, recombinant proteins, etc.). Small molecules can be used to enhance the derivation and quality of mouse and human iPSCs by acting as epigenetic modifiers (i.e., altering the expression of some genes).

[0160] As examples, 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) can also improve the reprogramming and quality of normal iPSCs. Fully reprogrammed, authentic iPSCs express pluripotency genes similar to self-renewing embryonic stem cells and represent an unlimited stem cell (or stem cell-like) source. ESCs and iPSCs can be repeatedly amplified over multiple and unlimited passages, enabling a scalable stem cell source. Pluripotency is actively maintained in permissive culture conditions by preserving high-level expression of pluripotency genes. These methods are known in the art. Certain culture conditions and methods allow for stable genome replication, but nevertheless, some exome mutations and epigenome modifications have been described (Gore A and al. Nature 2011).

[0161] As used herein, the term "somatic cells" refers to any somatic cell except germline cells (sperm and eggs). As used herein, the term "allogeneic cells" refers to cells derived from the same species but that are genetically distinct. As used herein, the term "syngeneic or autologous cells" refers to cells derived from the same species and the same genetic background. As used herein, the term "xenogeneic cells" refers to genetically distinct cells derived from a different species. In certain embodiments, stem cells may be derived from mammals, including but not limited to rodents, pigs, cats, dogs, and primates (including humans).

[0162] Methods for producing fetal stem cell compositions:

[0163] In a third aspect, the present invention provides a method for producing a fetal cell composition, comprising: i) fetal stem cells in the presence of an agent that induces MHC I presentation of antigens in a population following differentiation of pluripotent stem cells; ii) exposing the fetal stem cells to an inactivating agent that inactivates the cells; iii) Recovering and Conditioning the Differentiated, Inactivated Fetal Stem Cells The present invention relates to a method, including:

[0164] In certain embodiments, fetal stem cell envelope integrity is maintained in step ii). In another embodiment, the fetal stem cells are inactivated to obtain a cell-derived product, such as a cell extract. Cell compositions produced according to the above methods can be used for cancer treatment according to the methods disclosed herein.

[0165] Preparation of fetal stem cells

[0166] Fetal stem cells or fetal organoids are obtained from pluripotent stem cells by classical methods (for example, in 2D or 3D culture systems, under defined culture conditions using small molecules, morphogenetic and growth factors) after differentiation is induced.Fetal stem cells or organoids lose pluripotency markers.Usually, fetal stem cells or organoids are depleted of at least 20% of the following genes, which are characteristic of pluripotency: [Table 4]

[0167] Agents for MHC I antigen presentation

[0168] After differentiation, fetal stem cells or organoids derived from induced iPSCs or ESCs are maintained in the presence of a drug that improves antigen presentation through the MHC I pathway. Such improved expression can be checked by comparing the number of MHC I molecules on the surface of the cells in the presence or absence of the drug.

[0169] Such agents are known in the art, and in particular, histone deacetylase inhibitors (HDACi) may be mentioned. Many products with this activity are known in the art, and among these HDACi, valproate (VPA or valproic acid, CAS number 99-66-1) may be mentioned. Other HDACi that may be used (having the same mode of action as VPA) are, in particular, vorinostat, romidepsin-quidamide, panobinostat, belinostat, panobinostat, mocetinostat, abexinostat, entinostat, SB939, resminostat, divinostat, or quisinostat.

[0170] These agents are present in the cell culture medium permissive for fetal stem cells after pluripotent stem cell differentiation.

[0171] Fetal cell inactivation

[0172] The fetal stem cells used in the present invention are inactivated. As used herein, the term "inactivated" and its grammatical variations refer to cells (e.g., fetal cells) that are viable but rendered incapable of proliferation (i.e., mitotically inactivated). Those skilled in the art may use techniques known in the art, including, but not limited to, exposure to chemical agents, irradiation, and / or freeze-drying. Fetal stem cells can be inactivated so that, upon administration to a subject, the fetal cells are incapable of dividing and therefore cannot form fetal tissue in the subject. In the context of a plurality of cells, it is understood that not every cell needs to be incapable of proliferation. Thus, as used herein, the phrase "inactivated to a degree sufficient to prevent tissue formation in a subject" refers to a degree of inactivation in the population as a whole such that, after administration to a subject, fetal tissue does not form because the irradiated fetal stem cells no longer divide, as confirmed by in vitro culture. It should be noted that even if one or more cells in the plurality of cells are actually capable of proliferation in the subject, it is assumed that the host's immune system will destroy those cells before fetal tissue can form. This inability to grow and form tissues can be confirmed by studies in mice with functional and non-functional immune systems.

[0173] In some embodiments, the "inactivated" cells are dead cells. In other embodiments, the inactivated cells are whole cell lysates, fetal stem cell or organoid-derived exosomes, concentrated cancer stem neoantigens, purified whole cancer stem neoantigens, DNA, mRNA and protein extracts, lyophilized whole cell suspensions, cell lysate fractions (e.g., membrane fractions, cytoplasmic fractions), or combinations thereof. When mice are vaccinated with fetal stem cells in combination with valproic acid or another HDACi, the inactivated fetal stem cells can still stimulate an immune response. This vaccination can induce efficient immune and antitumor responses against carcinoma without side effects and evidence of autoimmune disease.

[0174] Typically, to inactivate fetal stem cells, they can be exposed to a lethal dose of radiation (e.g., a single fraction of 5-100 Gy). The exact dose of radiation delivered to the fetal cells and the length of the dose are not important, as long as the cells are rendered nonviable.

[0175] Cell harvesting and conditioning.

[0176] The harvesting step of the method includes one or more steps of washing the cell culture and resuspending the cells in any suitable medium, such as any clinical-grade cell culture medium. Conditioning the cells can include freezing or lyophilizing the cells so that the cell composition can be stored prior to use.

[0177] Mutations and neoantigen expression in fetal stem cells

[0178] It should be noted that pluripotent cells are genetically very stable cells. In fact, they exist very early in the embryonic development process, and since they must increase for embryonic development, it is important that these cells are not prone to mutations in order to have uniformity in the embryo.

[0179] Consequently, the cells present in a population of pluripotent cells are generally highly homogeneous, given their genetic content (ie, greater than 95% of the cells in the population exhibit the same genetic background).

[0180] When preparing iPSCs, a selective advantage of some cells arises during multiple passages, leading to a population of iPSC clones that exhibit specific mutations at later passages, but whose cellular genome sequences are nearly 100% similar.

[0181] However, after multiple passages, iPSCs are stable like 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, 2013).

[0182] However, it would be preferable to be able to induce mutations in pluripotent stem cells to increase the variability of embryonic neoantigens on the treated cell material. Derived fetal stem cells from mutant pluripotent stem cells could be used to reveal tissue-specific fetal neoantigens found primarily in invasive cancers. In this way, it would increase the likelihood that the immune system would generate T cells against fetal neoantigens presented by these mutant fetal cells that can combat cancer cells as well as those that undergo subsequent mutations during tumor development.

[0183] This may be useful for combating cancers caused by the accumulation of somatic genetic alterations resulting from DNA replication errors and / or environmental insults during cancer stem cell proliferation and progression. These alterations include cancer-driving mutations that initiate carcinogenesis and genome-destabilizing mutations. This increased genome instability leads to clonal evolution, which leads to the selection of more aggressive clones with increased drug resistance.

[0184] Thus, cells can be exposed to mutagens, ie, physical or chemical agents that alter the genetic material, usually DNA, of an organism, increasing the frequency of mutations above natural background levels.

[0185] The mutagen may be selected from the group consisting of physical mutagens and chemical mutagens.

[0186] Among the physical mutagens we may cite: - Ionizing radiation, such as X-rays, gamma rays and alpha particles, which can cause DNA breakage and other damage. In particular, mention may be made of radiation from cobalt-60 and cesium-137. The level of irradiation must be significantly lower than that used for cell inactivation and can be designed by one skilled in the art. - Ultraviolet light with wavelengths above 260 nm (which can cause replication errors if left uncorrected). - or radioactive decay, e.g. 14C in DNA.

[0187] Among chemical mutagens, the following may be cited: - reactive oxygen species (ROS), e.g. superoxide, hydroxyl radical, hydrogen peroxide; - deaminating agents, e.g., nitrite (which can cause transition mutations by converting cytosine to uracil); - 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, for example those from Vinca species; - bromine and some compounds containing bromine; -Sodium azide; -bleomycin; -Psoralen in combination with ultraviolet light; -benzene; - base analogs that can substitute for DNA bases during replication and cause transition mutations; -intercalating agents, such as ethidium bromide, proflavine, daunorubicin; Metals such as arsenic, cadmium, chromium, nickel and their compounds which may be mutagenic.

[0188] In certain embodiments, one will obtain a population of pluripotent cells in which the cells have random mutations (which generally vary from cell to cell, thereby leading to a heterogeneous population), particularly in cancer-associated neoantigens.

[0189] We have shown that it is possible to design culture conditions that allow the induction of DNA replication errors in pluripotent cells without triggering DNA damage-dependent apoptosis.

[0190] This is particularly surprising because, as mentioned above, pluripotent cells are naturally very stable, since as few mutations as possible should be introduced during the early stages of embryonic development. As a result, in these cells, DNA repair mechanisms are very efficient, thereby correcting most defects and / or inducing apoptosis when it is not possible to correct these defects.

[0191] In certain embodiments, a starting population of pluripotent stem cells is expanded and differentiated into fetal lineages in 2D or 3D organoid culture systems (as known in the art) using permissive media to induce fetal-specific tissue development. Under these conditions, a low number of exome mutations will generally be observed (5-10 mutations per exome).

[0192] The pluripotent stem cells are then cultured in vitro using mutagenic compounds to induce and increase genomic instability in the pluripotent stem cells, such as those listed above. DNA damage is confirmed by phosphorylation of γH2AX, a marker of double-strand breaks (DSBs). In ESCs or iPSCs, both the percentage of γH2AX-positive cells and the frequency of γH2AX foci are increased, as well as a higher number of micronuclei as indicators of genomic instability. The mutant pluripotent stem cells are then expanded and differentiated into fetal lineages in 2D or 3D organoid culture systems to induce fetal-specific tissue development. During differentiation, a series of somatic mutations are selectively expressed in fetal cells. These tissue- or lineage-specific somatic mutations promote growth and survival advantages and are lineage-specific.

[0193] In one embodiment, fetal cells obtained after differentiation may also be cultured in vitro with a mutagenic compound to induce somatic mutations.

[0194] Preferred agents are bleomycin, ENU, alkylating agents, actinomycin D, ROS modulators, UV, H2O2, ionizing radiation (gamma rays, X-rays), all of which will allow for the induction and enhancement of mutation rates in pluripotent stem cells accumulating in culture.

[0195] In certain embodiments, N-ethyl-N-nitrosourea (ENU) has been shown to generate novel mutations and enhance neoantigen levels in treated pluripotent stem cells during long-term culture for at least 7-60 days at doses of <50 μg / ml. These somatic mutations selectively expressed in fetal stem cells are similar to those reported in cancer. Therefore, it is possible to accumulate diverse mutations in response to DNA damage in pluripotent stem cells with high mutation rates, providing a selective advantage during long-term culture while maintaining cellular pluripotency, particularly when cells are cultured with HDACi in the medium. The presence of HDACi in the culture medium preserves the increase in active histones (H3K4me3 and H3K9ac). After differentiation, fetal cells derived from mutant pluripotent stem cells are maintained in permissive culture medium and HDACi. Cells expressing higher levels of neoantigens are compared to fetal cells derived from non-mutated pluripotent stem cells.

[0196] In another embodiment of the compositions and methods described herein, mutations are induced in pluripotent stem cells through genetic modification of cells with genes that promote high levels of genomic instability. In particular, appropriate inhibitors, such as NER / BER / DSBR / MMR inhibitors, can be used to eliminate 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 carried out by using "vectors" or "genetic modifications" that inactivate or knock down DNA repair-related genes or signaling pathways, such as DNA polymerase delta complex, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR), DSBR, or NEJH. Other examples of DNA repair genes include DNApkC, Ku70, Rad51, Brca1, or Brca2.

[0197] In other embodiments, pluripotent stem cells are engineered to suppress apoptosis-related genes, such as p53, by genetic or chemical modification, e.g., Pifithrin-mu, Nutlin-3, or by using compounds that enhance cell survival, e.g., Y-27632 (a selective inhibitor of p160-Rho-associated coiled kinase (ROCK)).

[0198] In certain embodiments, the population of pluripotent stem cells comprises: i) DNA repair disorders including, for example, inflammatory telangiectasia, Bloom's syndrome, Cockayne's syndrome, Fanconi anemia, Werner's syndrome, xeroderma pigmentosum, Nijmegen breakage syndrome; ii) Hereditary familial cancer syndromes associated with genomic instability, such as Lynch syndrome (hereditary nonpolyposis colorectal cancer associated with mutations in MMR genes including MLH1, MSH2, MSH6, PMS1, and PMS2), Li-Fraumeni syndrome associated with mutations in the TP53 gene or CHEK2, hereditary breast and ovarian cancer (HBOC) syndrome associated with deletions or mutations in the BRCA1 / 2 gene, familial adenomatous polyposis (FAP) associated with mutations in the APC gene; renal cell carcinoma associated with c-Met mutations; medullary thyroid carcinoma associated with RET mutations; iii) Somatic cell carcinogenesis-induced genomic instability, such as CML with translocation (T9;22) and Jak mutations The gene was generated from somatic cells, such as cells isolated from a patient, that already contained genomic alterations associated with the gene.

[0199] In certain embodiments, pluripotent stem cells are derived from somatic cells containing disease-associated genomic alterations. Typically, the genomic alterations can be translocations (t9:22), deletions (BRCA1 / 2), or mutations (BRCA, RET, c-Met). Fetal stem cells derived from these pluripotent stem cells recapitulate the genomic alterations at the fetal level.

[0200] In certain embodiments, a population of pluripotent stem cells (iPSCs) is generated from a cancer cell line or patient-specific cancer cells. The derived fetal stem cells or organoids recapitulate cancer phenotypes and genotypes at the fetal level similar to those reported in primary cancers. In another embodiment, pluripotent stem cells are genetically modified to overexpress multiple non-random cancer stem-associated neoantigens using a "vector." In certain embodiments, a population of pluripotent stem cells, fetal stem cells, or organoids are genetically modified to express multiple mutations and at least one cancer stem cell-specific neoantigen using "genome editing" techniques. The genetically modified fetal stem cells or organoids recapitulate cancer genotypes similar to those reported in primary cancers. The present invention provides compositions and methods for providing pluripotent stem cells and fetal cells or organoids by introducing multiple neoantigens using RNA-guided multiplex genome editing, modification, inhibition of expression, and other RNA-based techniques.

[0201] The term "genome editing" as used herein refers to RNA-mediated genetic manipulation, particularly including a guide RNA for cas9-mediated genome editing. This guide RNA (gRNA) is transfected together with the endonuclease cas9. The guide RNA provides a scaffold and a spacer sequence complementary to the target. In another embodiment, the genetic manipulation sequence can be an siRNA or microRNA sequence designed for gene silencing using the Crispr-Cas9 system according to standard methods in the art. Compositions and methods for making and using the Crispr-Cas system are known in the art and are described, inter alia, in U.S. Patent No. 8,697,359.

[0202] In certain embodiments, the population of pluripotent stem cells or derived fetal cells is treated with an alkylating agent. As used herein, the term "alkylating agent" refers to a substance that adds one or more alkyl groups from one molecule to another. This treatment creates new mutations in neoantigens, which provide a superior immune response by increasing the oligoclonal expansion of TILs and Th1 / Th2 cellular immunity. In the present invention, the alkylating agent is selected from the group consisting of nitrogen mustards, nitrosoureas, alkylsulfonates, triazines, ethyleneimines, 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 their pharmaceutically acceptable salts. Non-limiting examples of alkylsulfonates include busulfan (Jazz Pharmaceuticals) and their pharmaceutically acceptable salts. Non-limiting examples of triazines include dacarbazine (Bayer), temozolomide (Cancer Research Technology), and their pharmaceutically acceptable salts. Non-limiting examples of ethylenimines include thiotepa (Bedford Laboratories), altretamine (MGI Pharma), and their pharmaceutically acceptable salts. Other alkylating agents include ProLindac (Access), Ac-225BC-8 (Actinium Pharmaceuticals), ALF-2111 (Alfact Innovation), thromphosphamide (Baxter International), MDX-1203 (Bristol-Myers Squibb), and others.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 Pharma), ranimustine (Mitsubishi Tanabe Pharma), ND-01 (NanoCarrier), HH-1 (Nordic Nanovector), a combination of 22P1G cells and ifosfamide (Nuvilex), estramustine phosphate (Pfizer), prednimastine (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 pharmaceutically 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), pharmaceutically 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 (AlfactInnovation), bendamustine (Astellas), ifosfamide (Baxter International), thromphosphamide (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), threosulfan (Medac), nifurtimox (Metronomx), improsulfan tosylate (Mitsubishi Tanabe Pharma), ranimustine (Mitsubishi Tanabe Pharma), ND-01 (NanoCarrier), HH-1 (Nordic Nanovector), combination of 22P1G cells and ifosfamide (Nuvilex), melphalan flufenamide (Oncopeptides), estramustine phosphate (Pfizer), prednimastine (Pfizer), lurbinectedin (PharmaMar), trabectedin (PharmaMar), altreatamin (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.), pharmaceutically acceptable salts thereof, and combinations thereof.

[0203] In certain embodiments, the population of pluripotent stem cells is treated with N-ethyl-N-nitrosourea (ENU, CAS number 759-73-9), which has the chemical formula CHNO and is a highly potent mutagen that acts by transferring an ethyl group to nucleobases in nucleic acids.

[0204] As described above, the purpose of the mutagen is to introduce random mutations into the genes of pluripotent stem cells during expansion (the introduction of mutations occurs during cell replication and division). The pluripotent stem cell population acquires mutations that may provide a growth advantage and are selected for to promote culture adaptation. The pluripotent stem cell-derived fetal cell population acquires mutations that promote the growth and survival of fetal cells in permissive culture media.

[0205] In certain embodiments, when 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.

[0206] After application of the mutagen, the cells may be washed (if the mutagen is a chemical agent) and further incubated in the presence of an agent favorable to MHC-I expression, particularly an HDACi, which is also preferably present during application of the mutagen.

[0207] Therefore, it can be observed and checked that mutagens induce mutations (i.e., nonsynonymous, nonsense, frameshift, stop, gain, splice variants, CNV, SNV) in some fetal genes expressed in fetal cells, thus increasing the diversity of fetal antigens (new neo-antigens within the whole genome). This will therefore increase the possibility of vaccine compositions with enhanced immunogenicity that can stimulate a broad immune response against aggressive cancers where rapid and frequent mutations exist.

[0208] Indeed, for some cancers, where clonal evolution occurs with de novo somatic mutations in antigens expressed by tumor cells during progression, obtaining an efficient immune response can be difficult. Therefore, the immune response will depend on the mutation load of the cancer and immunogenic neoantigens. Therefore, generating specific mutations in fetal cell populations by using mutagens will lead to an increase in the diversity of antigens presented to the immune system upon vaccination.

[0209] As a result, there will already be primed T cells against mutant fetal antigens that emerge in cancer cells during the division of such cells and accelerate and improve the immune response against these cells.

[0210] In certain embodiments, pluripotent stem cells can first be differentiated by classical methods (e.g., in defined culture conditions using small molecules, morphogenetic and growth factors in 2D or 3D culture systems) and then treated with a mutagen (e.g., ENU) to express fetal neoantigens.

[0211] Modification of fetal stem cells

[0212] 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 pluripotent stem cells is isolated and expanded.In the second step, a gene of interest is packaged in an integrating virus vector, such as a retrovirus or a lentivirus.In the third step, the integrating virus vector containing the gene of interest is introduced into a population of pluripotent stem cells, and they are differentiated into fetal stem cells.

[0213] In certain embodiments, fetal stem cell population or organoid is modified with the gene of protein that stimulates MHC expression and / or immune response.These compounds are selected from the group consisting of interferon alpha (IFN-α), interferon gamma (IFN-γ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor (GM-CSF), their functional fragments and their combinations.

[0214] Interferons (IFNs) contemplated by the present invention include the common 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 more normally behaving cells, and / or increasing their antigen production, making it easier for the immune system to recognize and destroy them. IFNs can also act indirectly on cancer cells, for example, by slowing angiogenesis, strengthening the immune system, and / or stimulating natural killer (NK) cells, T cells, and macrophages. Recombinant IFN-alpha is commercially available as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).

[0215] 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, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention.

[0216] 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, darbepoietin). 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 chemotherapy-related side effects and allow for the use of higher doses of chemotherapy agents. Various recombinant colony-stimulating factors are commercially available, such as Neupogen® (G-CSF; Amgen), Neulasta (pegfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), and Arnesp (erythropoietin).

[0217] In its 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 into the cell with reduced degradation relative to the extent of degradation that would occur in the absence of the vector. In general, vectors useful in the present invention include, but are not limited to, naked plasmids, non-viral delivery systems (such as electroporation, sonoporation, cationic transfection agents, liposomes, etc.), phagemids, viruses, and 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 derived 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; and poliovirus. Other vectors not named but known in the art can readily be used.

[0218] Typically, in the context of the present invention, viral vectors include adenoviruses and adeno-associated (AAV) viruses, which are DNA viruses already approved for use in human gene therapy. Indeed, 12 different AAV serotypes (AAV1-12), each with a different tissue tropism, are known (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAVs are derived from the parasite-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 can infect a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316-27). They also have advantages such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and the lack of superinfection inhibition, allowing for multiple rounds of transduction. In addition, in the absence of selective pressure, wild-type adeno-associated virus infections have been tracked for over 100 passages in tissue culture, suggesting that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function extrachromosomally.

[0219] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, Sambrook et al., 1989. For the past several years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes to cells in vivo. They are particularly advantageous for this purpose because they do not have the same safety concerns as many viral vectors. However, these plasmids with promoters 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 skilled in the art. In addition, plasmids can be custom designed to remove and add specific fragments of DNA using restriction enzymes and ligation reactions. Plasmids can 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. They can also be administered by nasal spray or drops, rectal suppository, and orally. Preferably, the DNA plasmid is injected via intraocular methods (intravitreal, subretinal, suprachoroidal...). It can also be administered into the epidermis or mucosal surfaces using a gene gun. The plasmid can be provided in an aqueous solution, dried on gold particles, or bound to another DNA delivery system, including but not limited to liposomes, dendrimers, cochleates, and microencapsulation.

[0220] In a particular embodiment, a population of fetal stem cells is modified by introduction of a transgene, eg, an siRNA, into the AAVS1 locus on chromosome 19 by homologous recombination.

[0221] As used herein, the term "homologous recombination" 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 that of a target sequence on a chromosome is introduced into a cell, this term refers to recombination that occurs based on nucleotide sequence homology between the introduced genomic fragment and its corresponding gene locus on the chromosome.

[0222] The term "genetic modification" also refers to the insertion of exogenous DNA at the locus of a desired gene on a chromosome, the replacement of part or all 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") (in which case the endogenous DNA sequence is maintained in such a manner that the fragment is expressed in conjunction with the expression of the gene at a particular locus or is constitutively expressed), or the replacement, deletion, or disruption of part or all of a gene sequence (i.e., "knock-out") that modifies the endogenous DNA sequence.

[0223] Examples of methods for introducing artificial chromosomes into cells include calcium phosphate precipitation (Graham et al., (1973) Virology 52: 456-467; 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 such as lipofectin (Teifel et al., (1995) Biotechniques 19: 79-80; Albrecht et al., (1996) Ann. Hematol. 72: 73-79; Holmen et al., (1995) In Vitro Cell Dev. Biol. Anim. 31: 347-351, Remy et al., (1994) Bioconjug. Chem. 5: 647-654, Le Bolc'h et 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).

[0224] A population of fetal stem cells

[0225] Thus, using the above method, the inventors obtained a population of fetal stem cells expressing new fetal epitopes in some or all fetal genes that will trigger more efficient anti-tumor immunity. Thus, in a fourth aspect, the present invention relates to a population of stem cells obtained according to the above method. A population of fetal stem cells derived from pluripotent stem cells pretreated with N-ethyl-N-nitrosourea (ENU) exhibits an increased number of novel mutations compared to a population of fetal cells derived from non-mutated pluripotent stem cells (i.e., without pretreatment with ENU). These fetal neoantigens are associated with primary cancer. Therefore, this population is also the subject of the present invention.

[0226] The population thus obtained is characterized in that the fetal stem cells have lost at least 20% of the genes related to pluripotency, in particular for the following genes: [Table 5]

[0227] Lack of expression of lineage-specific genes in adult differentiated cells.

[0228] Therefore, the present invention relates to a composition of cells comprising fetal stem cells, wherein the cells in the population present fetal neoantigens. The somatic mutation rate of fetal neoantigens is determined in a fetal stem cell population derived from a master bank of pluripotent stem cells after exposure to a mutagen. If further expansion is performed, the stability of these somatic fetal neoantigens is determined before or after such further expansion. The mutation rate in fetal stem cells or organoids is 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% of at least one gene selected from fetal neoantigens expressed in derived fetal cells or organoids. The mutation rate of a given gene is calculated explicitly by sequencing the DNA of the gene and calculating the proportion of copies containing mutations relative to the native sequence (since the predominant sequence is the native "wild-type" sequence, this is essentially the predominant sequence).

[0229] Human-derived fetal hematopoietic stem cells (i.e., derived from human hematopoietic embryonic bodies after ex vivo treatment with mutagenic agents) expressed fetal neoantigens, which are characterized by at least the following group: [Table 6]

[0230] In certain embodiments, the human fetal kidney organoids derived from pluripotent stem cells express fetal antigens in renal precursors that are generally related to the cancer genes reported in primary adult renal carcinoma.These characterized fetal genes correspond to at least the following groups: [Table 7]

[0231] Exposure of pluripotent cells to mutagens will trigger the appearance of random mutations in the genomes of such cells, and the population resulting from such exposure is therefore heterogeneous compared to the population of fetal stem cells, which is inherently more homogeneous and is limited to cancer neoantigens reported in primary cancer genomes.

[0232] In a further embodiment, the present invention provides a composition of cells comprising fetal stem cells, wherein the cells in said population exhibit the following characteristics: 1) at least one (or more, as seen above) cancer-associated neoantigen mutation genetically introduced into fetal stem cells by genomic modification; 2) A combination of mutation types restricted to the cancer genome, induced by a mutagen, and enriched by a selective advantage in cultured fetal stem cells. The present invention relates to a composition that exhibits a mutational landscape in a population of fetal stem cells comprising one or more of:

[0233] The mutagenic process induces novel genomic mutations and increased levels of genetic mosaicism in fetal stem cell lines. Analysis of genetic mutations is preferably performed by large-scale genomic analysis of induced cancer-associated "mutanome" signatures in fetal stem cell populations using NGS, exome, RNA-seq or whole genome sequencing, CGH arrays, and SNP arrays. Whole exome sequencing combined with transcriptome profiling allows for the description of the mutame encoding expressed proteins.

[0234] Genomic somatic abnormalities and neoantigens are identified by using at least two art-known algorithms for bioinformatics analysis. The prevalence of total mutations in the entire genome after application of a mutagen will confirm a higher mutation and / or CNV load in the output fetal stem cells.

[0235] Qualitative and quantitative criteria will allow defining each cell population within the genetic mosaic in fetal stem cells, as described:

[0236] Qualitative criteria include: -Identification of acquired novel molecular somatic alterations (mutations, CNVs or SNVs), defined in terms of their presence in the fetal stem cell genome after mutagenesis and their absence in the parental pluripotent stem cells with and without mutagenesis; - Classification of each novel mutation (i.e., nonsynonymous, nonsense, splice variant, CNV, SNV) and validation by detecting their overlap between the primary patient-specific cancer or cancer genome (from databases i.e., TCGA, ICGC, COSMIC) and fetal stem cell genes (which are not present in normal adult cells or tissues).

[0237] 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 is defined for each fetal stem cell population or organoid; - Presence of validated mutations in at least three different fetal genes; - mutation rate of each novel stable somatic mutation at an allele frequency of at least >0.1% or other percentages (up to 50%) found above after clonal selection and expansion, or for passage number (50x to 100x depth and 80-98% of target exome coverage); - Expression of stable fetal stem cell markers and gene expression-based assays that are at least 90% greater than the expression rate of the input fetal stem cells prior to mutagenesis or genetic modification; - Expression of MHC I molecules on the cell surface (as determined, for example, by FACS) that is increased by at least 50%, and generally by up to 90%, compared to fetal cell populations maintained in the absence of HDACi, particularly VPA.

[0238] Vaccine Composition

[0239] The population of fetal stem cells may be used in a vaccine composition. Thus, in a fifth aspect, the present invention relates to a vaccine composition comprising a population of fetal stem cells as disclosed above and an agent that stimulates an immune response and / or MHC I expression.

[0240] In particular, such fetal stem cells are inactivated and optionally mutated to inhibit their proliferative capacity and optionally to obtain a cell extract.

[0241] The agent that stimulates immune response can be an adjuvant (immunostimulant) known in the art.It is preferably an HDACi (used in a dose range of 0.2 mM to 4 mM).When such an HDACi is used, another adjuvant can also be used.

[0242] The present invention also relates to devices (eg, syringes) containing such vaccine compositions, which can be used for the co-administration of HDACi compounds and cell compositions.

[0243] Such vaccine compositions can be used as therapeutic vaccines against cancer cells (cancer cells expressing immunogenic neoantigens, driver or passenger mutations; tumor-initiating cells expressing precursor, fetal and embryonic genes, such as epigenetically dedifferentiated cells) for the purpose of curing the subject, or as prophylactic vaccines to prevent the onset of such cancers in subjects particularly susceptible to these cancers.

[0244] Predisposing genes are, for example, the following (see 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 syndrome: PTEN, PIK3CA Fanconi disease: FANC von Hippel-Lindau disease: VHL Melanoma: CDKN2A, MITF, BAP1, CDK4 Endocrine neoplasm: MEN1, RET, CDKN1B Neurofibromatosis: NF1, NF2, LZTR1, SMARCB1, SPRED1 Hereditary pheochromocytoma-paraganglioma: SDH, TMEM127, MAX, EPAS1 Familial adenomatous polyposis: APC, MUTYH Retinoblastoma:RB1 Birt-Hogg-Dube 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

[0245] This list is not exhaustive.

[0246] In certain embodiments, the cancer stem cell vaccine product comprises a lyophilized cell lysate mixture, a concentrated mixture of multiple cancer stem neoantigens, purified cancer stem neoantigens, fetal stem cell-derived exosomes, induced fetal stem cells, and organoid-derived DNA, RNA, protein, or multiple peptides, which are immunogenic agents as disclosed above, formulated in the presence of an HDACi.

[0247] In another embodiment, the cancer stem cell vaccine product is mixed with supernatant GMP medium derived from engineered irradiated fetal stem cells used as an adjuvant effector.

[0248] In certain embodiments, the derived fetal cells in the composition are inactivated (ie, no longer capable of proliferation).

[0249] The derived fetal stem cell and organoid compositions of the present invention are susceptible to being obtained by any of the methods disclosed above.

[0250] It should be noted that the derived fetal cells in this composition are genetically heterogeneous and have specific somatic mutations when mutagens are used, and therefore differ from derived fetal cell compositions produced according to methods known in the art, which are more genetically homogeneous.

[0251] The population of derived fetal cells differs from populations of derived fetal cells produced using methods known in the art when cultured in the absence of a mutagen because the presence in the culture medium of an agent that maintains fetal gene expression and increases MHC I presentation results in cells with more of these MHC I molecules on their surface.

[0252] As used herein, the term "a compound selected from the group that activates MHC I expression and / or immune response" refers to a compound capable of stimulating immunogenicity. Such compounds are referred to as activators of MHC expression and / or immune response. The term "MHC" refers to the major histocompatibility complex present on cell surfaces to recognize foreign molecules (called antigens). MHC binds to antigens and presents them to immune molecules (e.g., T and B lymphocytes). 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 myeloid-derived suppressor cell (MDSC) population, while conversely increasing the NK population. In the context of the present invention, the immune response against a tumor comprises a cytotoxic T cell response against an antigen present in or on tumor cells. In some embodiments, the cytotoxic T cell response is mediated by CD8+ T cells. Typically, in the context of the present invention, the antigen that activates MHC expression and / or immune response corresponds to a molecule present on the fetal stem cell population. Compounds that activate MCH expression and / or the immune system are fetal genes or immunogenic neoantigens. The term "neoantigen" or "neoantigenic" refers to a class of antigens that result from at least one mutation that alters the amino acid sequence of a genomically encoded protein.

[0253] 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.

[0254] In a particular embodiment, the activator of MHC expression and / or immune response is a histone deacetylase inhibitor.

[0255] As used herein, the term "histone deacetylase inhibitors," also referred to as HDACi, refers to a class of compounds that interfere with the function of histone deacetylase. Histone deacetylases (HDACs) play an important role in the transcriptional regulation and pathogenesis of cancer. Typically, inhibitors of HDACs modulate transcription and induce cell growth arrest, differentiation, and apoptosis. HDACi also enhance the cytotoxic effects of therapeutic agents used in cancer treatment (including radiotherapeutic agents and chemotherapy drugs).

[0256] In a particular embodiment, the histone deacetylase inhibitor is valproic acid (VPA).

[0257] The term "valproic acid" refers to 2-propylpentanoic acid (CH 16 O2), which has in the art the following CAS number and formula 99-66-1: [ka]

[0258] Valproic acid has multiple biological activities (Chateauvieux et al., J. Biomed. Biotechnol., 2010, pii: 479364. doi: 10.1155 / 2010 / 479364). Valproic acid exerts an inhibitory effect on the neurotransmitter GABA (gamma-aminobutyric acid). Several mechanisms of action have been suggested. Valproic acid specifically affects GABA metabolism: it inhibits the degradation of GABA, GABA-transaminobutyric acid (LAMP), enhances GABA synthesis, and alters its turnover. In addition, valproic acid blocks certain ion channels, reduces N-methyl-D-aspartate-mediated excitation, and blocks the activity of Na+- and Ca2+-containing ion channels (voltage-gated L-type CACNA1 types C, D, N, and F).

[0259] In the context of the present invention, valproic acid is used as an immunostimulant to enhance the immune response against cancers that express cancer fetal stem cell neoantigens that are shared with fetal stem cells.

[0260] More specifically, VPA is used to stimulate and enhance the expression of MHC I on the cancer stem cell compartment, increasing the neoantigen content in some tumor cells. Higher expression of MHC I on fetal stem cells enhances the presentation of MHC I-associated neoantigens to APCs / dendritic cells, allowing for the induction of TH1 immune responses. Higher levels of chemokines (CXCL9, CXCL10) allow for enhanced recruitment of T cells to tumors.

[0261] The present invention relates to methods for increasing neoantigen content in derived fetal stem cells in the presence of HADCi such as VPA and / or 5-azacytidine, and in tumor cells through expression of fetal antigens by chromatin remodeling and chemokine expression (CXCL9, CXCL1).

[0262] In particular, when used to treat a subject in vivo, the compositions and vaccines of the present 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.

[0263] This is due to the synergistic effect of fetal stem cell vaccine and VPA co-administration, which is further improved if an HDACi is also administered to the patient after vaccine injection (eg, at least 15 days).

[0264] The examples show that combined treatment with both fetal stem cell vaccine and VPA provides superior anti-tumor responses by reversing tumor immune suppression, reducing TRegs (in tumor and spleen), and recruiting T CD4+ and CD8+ lymphocytes into the tumor with a lower proportion of T CD4 and CD8 expressing PD-1 in the spleen, while increasing TILs associated with Th1 / Th2 cellular immunity and reducing the FoxP3 TReg subpopulation.

[0265] VPA can downregulate c-Myc expression levels and potentially induce apoptosis and autophagy in cancer cells and tumor-initiating cells. VPA can enhance adaptive immune responses through autophagosome cross-presentation.

[0266] Another well-known effect of VPA is the reduction of inflammatory cytokines in lymph nodes, such as IL6, IL8, TNFα, interleukin (IL)-1β, and IL-17.

[0267] 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).

[0268] 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.

[0269] In a particular embodiment, 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).

[0270] In a particular embodiment, 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).

[0271] 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 N4O3) and has the structure described in Valente et al. 2014.

[0272] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 23 H 20 The first compound in the study was mocetinostat (MGCD01030) with N6O (Valente et al., 2014).

[0273] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 20 H 30 N4O2) and pracinostat (SB939), which has the structure described in Diermayr et al. 2012.

[0274] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 22 H 19 The compound is chidamide (CS055 / HBI-8000) with FN4O2.

[0275] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 21 H 26 The compound with the formula N6O2 is xinostat (JNJ-26481585).

[0276] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 21 H 23 The compound with the highest potency (N3O5) is abexinostat (PCI24781) (Valente et al. 2014).

[0277] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: 20 H 19 FN6O2) (Moffat D et al 2010; Banerji et al 2012).

[0278] In certain embodiments, the histone deacetylase inhibitor has the following chemical formula: (C 18 H 20 The AR-42 has N2O3 (Lin et al. 2010).

[0279] In a particular embodiment, the activator of MHC expression is a DNA methyltransferase inhibitor.

[0280] As used herein, the term "DNA methyltransferase inhibitor" refers to a compound that can interact with DNA methyltransferases (DNMTs) and inhibit their activity. DNMTs are enzymes that catalyze the transfer of methyl groups to DNA. DNA methylation serves a wide variety of biological functions. All known DNA methyltransferases use S-adenosylmethionine (SAM) as the methyl donor.

[0281] In certain embodiments, the DNA methyltransferase inhibitor is a compound known in the art to have the following chemical formula: 12 N4O5) and azacitidine, also known as 5-aza-2-deoxycytidine, which has the structure (Kaminskas et al., 2004; Estey et al., 2013).

[0282] In certain embodiments, the DNA methyltransferase inhibitor has the following formula: 12 The compound decitabine, also known as 5-aza-2'-deoxycytidine, has the formula N4O4 (Kantarjian et al 2006).

[0283] 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 the enhancer of zeste homolog 1 (EZH1) and 2 (EZH2) genes, which participate 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.

[0284] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is 3-deazaneplanocin A (DZNep, C-c3Ado), which is known in the art as having the following chemical formula C 12 H 14 N4O3 and has CAS number 102052-95-9.

[0285] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitors are UNC1999 and inactive analog compounds. UNC1999 is known in the art to have the following chemical formula: 33 H 43 N7O2 and has CAS number 1431612-23-5.

[0286] In certain embodiments, the histone-lysine N-methyltransferase enzyme inhibitors are UNC2400 and inactive analog compounds. UNC2400 is known in the art to have the following chemical formula C 35 H 47 N7O2 and has CAS number 1433200-49-7.

[0287] 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: 34 H 44N4O4 and has CAS number 1403254-99-8.

[0288] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is trifluoroacetate (EPZ011989), which has the following chemical formula in the art: CF3COONa and CAS number 2923-18-4.

[0289] 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 N5O3 and has CAS number 1396772-26-1.

[0290] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is GSK343, which is known in the art as having the following chemical formula C 31 H 39 N7O2 and has CAS number 1346704-33-3.

[0291] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is GSK126, which is known in the art as having the following chemical formula C 31 H 38 N6O2 and has CAS number 1346574-57-9.

[0292] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is GSK2816126, which is known in the art to have the following chemical formula: 31 H 38 N6O2 and has CAS number 1346574-57-9.

[0293] In a particular embodiment, the histone-lysine N-methyltransferase enzyme inhibitor is ZLD1039, which is known in the art to have the following chemical formula: 36 H 48N6O3 and has CAS number 1826865-46-6.

[0294] In certain embodiments, both HDACi and DNA methyltransferase inhibitors are used. Indeed, the combined use of VPA and 5-azacytidine (an analog of the nucleoside cytidine that can be incorporated into DNA and RNA) has been shown to lead to a synergistic effect on the re-expression of neoanti-embryonic antigens.

[0295] HDACi are administered at therapeutically effective doses. For VPA, this can be 10-15 mg / kg / day, up to 60 mg / kg / day. Plasma levels of VPA should preferably be within the normally tolerated therapeutic range (50-100 μg / ml).

[0296] In a further aspect, the methods of the present invention are suitable for treating cancers that express multiple fetal antigens that share expression with human fetal stem cells (e.g., NACC1, BLM, WDR33, DAZAP1, CDK1, CDC45, ZNF165, XRCC5, SMARCAD1, AIMP2, CKS1B...).

[0297] As used herein, the term "fetal stem cell-expressing cancer" refers to cancer stem cells that express multiple fetal antigens that share expression with human fetal stem cells, and are preferably targeted by the methods, vaccines, and compositions disclosed herein. Typically, the cancer is selected from the group consisting of bladder carcinoma, breast carcinoma, cervical carcinoma, bile duct carcinoma, colorectal carcinoma, gastric sarcoma, glioma, lung carcinoma, lymphoma, acute and chronic lymphocytic and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, gastric carcinoma, kidney carcinoma, head and neck tumors, and all solid tumors and hematopoietic malignancies. It should be noted that the cells in this composition are heterogeneous in nature. More specifically, the use of mutagens therefore differs from pluripotent cell compositions (which are homogeneous) cultured according to methods known in the art.

[0298] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance to a subject when the substance is present outside the body (e.g., in a combined 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.

[0299] In a specific embodiment, the vaccine composition (fetal stem cells plus an agent that stimulates MHC presentation) is injected subcutaneously. The injections can be simultaneous, sequential, or separate, at the same injection time point or at different injection time points, in the same syringe, in different syringes...

[0300] In a particular 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.

[0301] The term "therapeutically effective amount" refers to the minimum amount of an active agent required to provide a therapeutic benefit to a subject. For example, a "therapeutically effective amount" for a subject is an amount that induces, improves, or otherwise 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 dosage 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 with or simultaneously with the specific compound used; and similar factors well known in the medical arts. For example, it is well within the skill of one in the art to start doses of a compound at levels lower than those 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 can vary over a wide range, from 0.01 to 1000 mg per adult per day. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient, allowing for symptomatic adjustment of the dosage to the treated subject. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from about 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg body weight / day, particularly from about 0.001 mg / kg to 7 mg / kg body weight / day.

[0302] In certain embodiments, the methods of the present invention further comprise one or more of radiation therapy, targeted therapy, immunotherapy, or chemotherapy. Typically, a physician may choose to administer to a subject i) a population of fetal stem cells and ii) a compound selected from the group that activates MHC expression and / or immune responses as a combined preparation in conjunction with radiation therapy, targeted therapy, immunotherapy, or chemotherapy.

[0303] In some embodiments, the subject is administered a combined preparation of i) a population of fetal stem cells and ii) a compound selected from the group that activates MHC expression and / or immune response, and a chemotherapeutic agent.

[0304] The term "chemotherapeutic agent" refers to a compound effective for inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclophosphamide); alkylsulfonates (e.g., busulfan, improsulfan, and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa); ethyleneamines and methylolmelamines (including altretamine, triethylenemelamine, trimethylenephosphoramide, triethylenethiophosphamide, and trimethylolmelamine); acetogenins (e.g., bullatacin and bullatacinone); carnoptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (e.g., cryptophycin 1 and cryptophycin 2). Syn 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodithin; spongistatins; nitrogen mustards (e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicine, phenesterine, prednimustine, thromphosphamide, uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin (11) and calicheamicin 211, e.g., Agnew See Chem Intl. Ed. Engl. 33:183-186 (1994)); dynemicins (including dynemicin A); esperamicins;and neocarzinostatin chromophores 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, Sorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, ptepteterin, trimetrexate); purine analogs (e.g., fludarabine, 6-methyl- thiamiprine, thioguanine); pyrimidine analogs (e.g., azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, etc.); androgens (e.g., cartestrone, drostanolone propionate, epithiostanol, mepitotestan, testolactone); antiadrenal drugs (e.g., aminoglutethimide, mitotane, trilostane); folic acid supplements (e.g., humic acid); aceglatone; aldophosphatidyl glycosides; aminolevulinic acid; Amsacrine; Bestravsil; Bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Elfornithine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracin; Pentostine; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark); Razoxane; Rhizoxin; Sizofiran;Spirogenanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C") cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs (e.g., cisplatin and carboplatin); vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within 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, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston)); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0305] In some embodiments, a subject is administered a combined preparation of i) a population of fetal stem cells and ii) a compound selected from the group that activates MHC expression and / or an immune response, and a targeted cancer therapeutic.

[0306] Targeted cancer therapeutics are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") involved in cancer growth, progression, and spread. Targeted cancer therapeutics may also be referred to as "molecularly targeted drugs," "molecularly targeted therapies," "precision medicine," or similar terms. In some embodiments, targeted therapy comprises administering a tyrosine kinase inhibitor to a subject. 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 may act on different members of a tyrosine kinase signaling pathway to produce the same effect as a tyrosine kinase inhibitor of that tyrosine kinase. Examples of suitable tyrosine kinase inhibitors and related compounds for use in the methods of the present invention 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 in, for example, U.S. Patent Application Publication No. 2007 / 0254295, U.S. Pat. No. 5,618,829, U.S. Pat. No. 5,639,757, U.S. Pat. No. 5,728,868, U.S. Pat. No. 5,804,396, U.S. Pat. No. 6,100,254, U.S. Pat. No. 6,127,374, U.S. Pat. No. 6,245,759, U.S. Pat. No. 6,306,874, U.S. Pat. No. 6,313,138, U.S. Pat. No. 6,316,444, U.S. Pat. No. 6,329,380, U.S. Pat. No. Nos. 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated herein by reference in their entireties. In certain embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that is orally administered and 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, AE Examples include E-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.

[0307] In some embodiments, the subject is administered a combined preparation of i) a population of fetal stem cells and ii) a compound selected from the group that activates MHC expression and / or an immune response, and an immune checkpoint inhibitor.

[0308] 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 T cell activation or function. 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 and the duration and magnitude 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-CTLA-4 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; 8,217,149 and WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, and WO 2011161699. In some embodiments, PD-1 blockers include anti-PD-L1 antibodies. In certain other embodiments, PD-1 blockers include 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 and blocks its activation by its ligands PD-L1 and PD-L2; ramlolituzumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1; CT-011, a humanized antibody that binds to PD-1; AMP-224, a fusion protein of B7-DC; an antibody Fc portion; and 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 (e.g., B7-H3 and B7-H4 inhibitors), particularly the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors are also included (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 response," Nature Reviews Immunology, Volume 12, April 2012). In adoptive immunotherapy, a 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.

[0309] In some embodiments, the subject is administered a combined preparation of i) a population of fetal stem cells and ii) a compound selected from the group that activates MHC expression and / or immune response, and a radiotherapeutic agent.

[0310] As used herein, the term "radiotherapeutic agent" is intended to refer to, but is not limited to, any radiotherapeutic agent known to those skilled in the art to be effective for treating or ameliorating cancer. For example, a radiotherapeutic agent may be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods may optionally further include the administration of one or more additional cancer therapies (e.g., but not limited to, chemotherapy and / or another radiation therapy).

[0311] Pharmaceutical and vaccine compositions

[0312] The compound that activates MHC expression and / or an immune response and the population of fetal stem cells may be combined with a pharmaceutically acceptable excipient, and optionally a sustained release matrix, such as a biodegradable polymer, to form a pharmaceutical composition.

[0313] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered appropriately to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. 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, containing the active ingredient alone or in combination with other active ingredients, in admixture with a conventional pharmaceutical support. 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, pharmaceutical compositions contain a pharmaceutically acceptable vehicle for injectable formulations. These can be, in particular, isotonic sterile saline solutions (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or dry, especially lyophilized, compositions (which, as the case may be, allow the constitution of an injectable solution upon addition of sterile water or saline). Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the 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 preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). Solutions containing the compounds of the present invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant (e.g., 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.Polypeptides (or nucleic acids encoding them) can be formulated into compositions 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 (e.g., hydrochloric or phosphoric acid) 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 (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine). The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating (e.g., 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, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it will be preferable to include an isotonic agent (e.g., sugar or sodium chloride). Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptide in the required amount 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, 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 a 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 can be easily administered in a variety of dosage forms, such as the injectable solution types described above, but drug release capsules and the like can also be used.For parenteral administration in an aqueous solution, for example, the solution should be suitably 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, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the indicated 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.

[0314] More specifically, the population of fetal stem cells and the compound that activates MHC expression and / or an immune response are formulated in a vaccine composition. Accordingly, the present invention relates to a vaccine composition comprising i) a population of fetal stem cells and ii) a compound selected from the group that activates MHC expression and / or an immune response.

[0315] In a particular embodiment, the vaccine composition of the present invention comprises i) fetal stem cells and ii) valproic acid.

[0316] In a particular embodiment, the vaccine composition of the present invention comprises i) fetal stem cells expressing neoantigens, particularly enhanced by mutagenic agents or genetic modification, and ii) valproic acid.

[0317] The composition may also include 5 azacytidine.

[0318] The vaccine compositions of the present invention may also be used in subjects suffering from cancer, as described above.

[0319] The vaccine compositions of the present invention can be formulated using the above-mentioned physiological excipients in the same manner as immunogenic compositions. For example, pharmaceutically acceptable vehicles include, but are not limited to, phosphate-buffered saline, distilled water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Adjuvants such as muramyl peptides (e.g., MDP), IL-12, aluminum phosphate, aluminum hydroxide, alum, and / or Montanide® can be used in the vaccine.

[0320] The vaccine composition of the present invention can be administered by subcutaneous (sc), intradermal (id), intramuscular (im) or intravenous (iv) injection, oral administration, intranasal administration or inhalation administration. The administration of the vaccine is usually a single dose. Alternatively, the vaccine of the present invention can be administered first (primary vaccination), followed by the same population of stem cells, a compound that stimulates the immune system or a combination thereof, and / or with one or more additional treatments such as radiation therapy, targeted therapy, immunotherapy or chemotherapy. 5, 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) are administered.

[0321] The vaccine composition may also be provided in a kit, which includes the vaccine composition and an information leaflet providing instructions for immunization. The kit may also include all materials for administration of the product.

[0322] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Example]

[0323] Example 1: Characterization of fetal antigens from derived renal oragnoids harboring the c-MET mutation.

[0324] We established an iPSC line carrying a hereditary c-MET mutation by reprogramming blood cells from a donor with type 1 papillary renal carcinoma (PRCC) using Sendai virus-mediated pluripotency gene transfer. We designed a 3D culture system to induce differentiation of c-MET iPSCs into kidney organoids composed of fetal cells. We demonstrated that iPSC-derived kidney organoids expressed markers of renal progenitors with glomerular and tubular structures. Transmission electron microscopy analysis confirmed the presence of tight junctions in the tubular structures. Gene array analysis was performed on kidney organoids (EB56) and parental pluripotent stem cells (PB56). A supervised analysis by ranking products algorithm between iPSCs (PB56) with the c-met mutation and derived fetal kidney organoids containing committed nephron precursors allowed the identification of 196 differentially expressed gene probes: 148 were found to be downregulated in EB56 compared to iPSCs PB56, and a minority of those, 48, were found to be upregulated in EB56 compared to iPSCs PB56 ( Figure 1 This confirms that fetal kidney organoids have lost pluripotency genes from iPSCs and acquired fetal genes related to committed kidney tissue.

[0325] Supervised machine learning by c-MET status performed on PRCC RNAseq samples allowed us to characterize 1333 predictive genes with minimal misclassification error. Meta-analysis between the c-MET mutant IPSC signature and the PRCC signature revealed a significant enrichment of the IPSC profile to predict c-MET mutant PRCC tumor status (enrichment fold: 5.68; p-value < 2.2E-16) ( Figure 2 Characterized fetal genes from renal precursors that are commonly associated with reported oncogenes present in primary adult renal carcinomas are in at least the following groups: [Table 8]

[0326] These results confirm that fetal kidney organoids derived from c-MET mutant iPSCs are suitable fetal cells to model papillary renal cell carcinoma, expressing at least 77 common cancer-associated fetal neoantigens, allowing for the preparation of cancer cell vaccine products or cell extracts for renal carcinoma associated with c-met mutation.

[0327] Example 2 Common mutagenesis in derived fetal hematopoietic stem cells with acute myeloid leukemia (AML).

[0328] We developed an induced pluripotent cell (IPSC) model of chronic myeloid leukemia (CML) to model disease progression. It is well known that BCR-ABL fusion protein expression increases during CML progression, and this phenomenon induces genomic instability and promotes the emergence of secondary genomic events compatible with blast transformation leading to acute myeloid leukemia.

[0329] Human pluripotent stem cells carrying the Bcr-Abl oncogene were cultured and expanded with a mutagen (ENU) to induce genomic instability and enhance somatic mutations during serial divisions. Committed hematopoietic precursor fetal cells were produced by growing embryonic bodies (EBs) in the presence of growth and morphogens. Embryonic bodies (EBs) at day 2–3, corresponding to the hemangioblast stage, blast colonies, and EBs at day 4–20 were analyzed. Hematopoietic EBs were characterized for hematopoietic markers CD34, CD43, and CD45 by FACS analysis. Hematopoietic capacity was confirmed by colony formation assays by counting the number of CFCs and analyzing the type of CFCs. We demonstrate that ENU induced genomic instability and enabled the recapitulation of blast crisis in culture and in dishes using regenerative hematopoietic myeloid precursors. As demonstrated by FACS analysis, the presence of VPA in cultured hematopoietic EBs enhanced the amount of CMH class I with a lower CMH class II efficiency ( Figure 3 ).

[0330] Gene, exome, and CGH arrays were performed on day 16 derived fetal hematopoietic EB precursors obtained from early-passage (>20 passages) and late-passage (>100 passages) IPSCs after exposure to ENU. Whole-exome analysis was performed on DNA from parental IPSCs compared with differentiated embryoid bodies with and without genomic instability, and EBs derived from IPSCs cultured at early and late passages. Next-generation sequencing was performed by Illumina Technologies paired-end sequencing using the CASAVA pipeline aligned to the HG19 genome version. Genomic variants with frequencies less than 0.01 for the general human population in the EXAC database were selected.

[0331] We compared genomic variations in hematopoietic EBs with parental IPSCs with allele frequency differences of >0.10. Figure 4 and Table 1As shown in , we identified several genomic mutations in hematopoietic EBs generated from BCR-ABL-positive IPSCs: 14 genes were found to be affected by 9 missense single nucleotide mutations and 5 frameshifts.

[0332] On the other hand, after exposure to ENU, IPSCs (<20 and >100 passages) were differentiated into hematopoietic fetal stem cells (EBs), and the mutagenesis signature was compared with that of derived EBs derived from parental IPSCs without ENU. ENU was shown to induce genomic instability, numerous genomic and somatic mutations in hematopoietic EBs. Importantly, we found similar mutations in derived hematopoietic EBs derived from early- and late-passage IPSCs, indicating that the mutagenesis during scalable expansion of iPSCs is stable ( ). Figure 4 and Table 1 ).

[0333] A total of 123 genomic mutations, including coding missense, stop-gain, and frameshift mutations, were found in EBs derived from ENU-treated IPSCs. These genomic mutations are commonly expressed and reported in primary acute leukemias, at least in the following: [Table 9]

[0334] These 123 affected genes in the "blast crisis in a dish" model integrated with AML patient blast transcriptome analysis were prognostic for overall survival (p-value = 0.00000187, Figure 5 ) (log-rank p-value = 1E-4, Figure 6 ).

[0335] These results confirm that neoantigens affected by genomic mutations in IPSC-derived hematopoietic EBs recapitulate similar fetal neoantigens expressed in AML. Therefore, vaccine products, such as irradiated cells or cell extracts (AND, ARN, proteins) or neoepitope and peptide preparations, can be produced from these modified artificial fetal hematopoietic cells. Therefore, these relevant fetal hematopoietic cells can be used to stimulate immune responses against acute leukemia through vaccination strategies. [Table 10]

[0336] Example 3 Common gene expression in derived fetal lung organoids with lung cancer

[0337] We investigated transcriptome analysis of IPSC-derived lung organoids (thus composed of fetal cells) to predict lung cancer signatures: the LIMMA algorithm, after multi-test correction by false discovery rate (FDR), identified 8,372 variable genes between sorted cells and cell cultures at passages 0-5. In combination, the SAM algorithm found 5,619 genes differentially expressed between lung tumors and normal lung tissue (FDR<0.05, n=246 samples). The Stanford-identified leave-one-out algorithm was used for machine learning to adjust nested analyses for crossover between alveolar organoids and lung cancer signatures. A consensus signature of 19 predictive genes was found with a minimum misclassification error of less than 9% ( Figure 7 ).

[0338] Example 4 HDAC inhibitors increase vaccine immunogenicity

[0339] Higher expression of MHC I in cells used as vaccines would enhance the presentation of MHC I-associated neoantigens to APCs / dendritic cells, allowing for the induction of TH1 immune responses. To this end, we tested four different HDACi to check their ability to increase MHC I expression on two independent iPSCs: one iPSC with no genetic alteration (PB33) and one iPSC with a BCR-ABL fusion product (PB32) produced from a patient with CML disease.

[0340] Four HDACi were tested at doses of 1–1.5 μM, including belinostat, entinestat, leviteracetam, and valproic acid ( Figure 8 ) After 24 hours of culture, MHC1 HLA ABC expression was quantified by flow cytometry analysis, showing a 23-52% increase in MHC1 for both types of iPSCs ( Figures 9 and 10, right panels For each cell line, normalization of the mean relative fluorescence intensity (RFI) to the DMSO control shows a 0.84- to 2.45-fold increase in MHC1 ( Figures 9 and 10, left panel ).

[0341] Example 5 Vaccination with autologous endodermal progenitor cells in combination with valproic acid (VPA) generates an antitumor response against pancreatic ductal adenocarcinoma (PDAC).

[0342] We established endodermal progenitor cells (EndoPCs) from mouse tail fibroblast-derived iPSCs and differentiated mouse hepatocytes by using viral vectors expressing Oct4 / Sox2 / cMyc / Kfl4 transcription factors. These precursors are fetal cells. To highlight the pancreatic tumor expression profile of EndoPCs, we compared their transcriptome with that of iIPSCs derived from mouse tail fibroblasts, which are related to mouse embryonic stem cells (D3), and related to Pan02 cells transplanted into syngeneic C57BL / 6 mice. A composite cross-batch normalized transcriptome matrix revealed that 10 -4A supervised ANOVA was performed between the four sample groups, with 500 permutations between groups, with a p-value threshold below 0.01. A list of 3,230 gene identifiers was found to vary significantly between the four experimental conditions (data not shown). In a second run, the SAM supervised algorithm was used on these variable expression profiles, resulting in the identification of genes that were significantly differentially expressed between the following groups: (EndoPC + Pan02 in vivo) vs. (D3-ES + mouse iPSC) (FDR less than 1%). Using these analyses, the pancreatic tumor gene expression profile of EndoPC was analyzed using unsupervised principal component analysis ( Figure 11 ) as well as unsupervised clustering (Pearson distance, complete method, Figure 12 ) was also found to contain 359 gene identifiers that allowed for significantly individualized experimental groups (P value = 1.138249e-10). These results suggest that EndoPC may highlight pancreatic tumor expression profiles.

[0343] We also demonstrated that EndoPCs, processing a unique molecular signature, are distinct from mouse iPSCs and were found to be negative for genes involved in maintaining pluripotency, such as OCT4, SOX2, NANOG, LIN28, CMYC, KLF4, and alkaline phosphatase (ALP), by quantitative RT-PCR ( Figure 13 These latter results were confirmed by flow cytometry analysis showing a lack of stage-specific embryonic antigen (SSEA)-1 expression ( Figure 14 In addition, EndoPCs share several genes with Pan02, including PDX1, HNF4A, HNF1B, HNF1A, FOXA2, and FOXA3 (data not shown), and they depend on the IL-6 / JAK / STAT3 signaling pathway for their proliferation and self-renewal abilities. To evaluate the IL-6 / JAK / STAT3 axis, both Pan02 and EndoPCs were treated with 100 ng / ml IL-6 and IL-6 in the presence of a JAK inhibitor.

[0344] Activation of this pathway in both cell lines correlated with phosphorylation of STAT3 at tyrosine 705 in response to IL-6 ( Figure 15 shows the results for Pan02 cells only. After 30 min and 4 h of exposure to JAK inhibitors, detection of the Tyr-705-phospho-STAT3 form by Western blot analysis was strongly inhibited. In addition, activation of the IL-6 / JAK / STAT3 axis was associated with upregulation of β-catenin and TCF4 mRNA (data not shown).

[0345] We then investigated whether vaccination with irradiated EndoPC in combination with VPA is effective against pancreatic cancer in a syngeneic PDAC mouse model. Vaccination was performed with 2 × 10 EndoPC irradiated at a dose of 80 Gray. 6 The study consisted of injecting two suspensions of EndoPC by the subcutaneous route.

[0346] 2 x 10 expressing the luciferase gene 6 Pan02Luc cells were injected 7 and 14 days before orthotropic injection into the tail of the pancreas. Mice (n=8) that received two vaccine boosts received a 0.40 mM dose of VPA in their drinking water on the day of challenge. Concurrently, unvaccinated mice received the same number of cancer Pan02Luc cells without VPA. We found a significant improvement in survival rate after pre-injection of irradiated EndoPC in mice, in contrast to unvaccinated mice ( Figure 16 ). We also found that while Pon02 tumors grew progressively in the PBS control group, notably, immunization with EndoPC resulted in a delay in tumor growth, with the mean tumor size in the treatment group being statistically significantly different from the control group. Systematic quantification of regions of interest (ROIs) measuring surface intensity by bioluminescence showed a dramatic inhibition of tumor growth in the vaccine-treated group starting from day 4 after tumor challenge. Figure 17 ).

[0347] Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure.

[0348] References:

[0349] Patent documents European Patent Application Publication No. 2599860 International Patent Application No. PCT / JP2006 / 324881, International Patent Application No. PCT / JP02 / 05350 US Patent Application Publication No. 2005 / 0176707 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; 5,914,268; 9,499,797; 9,637,732; 8,158,766; 8,129,187; 8,058,065; 8,278,104; 8,69 Nos. 7,359; 4,684,611; 5,240,840; 4,806,476; 5,298,429; 5,396,767; 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; ​​6,682,736; 6,984,720; 7,605,238; 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149 WO 2012 / 122629; WO 2016 / 065330; WO 2017 / 027757; WO 2017 / 202949; WO 2001 / 085917; WO 2012 / 060473; WO 03 / 042402; WO 2008 / 156712; WO 2010 / 089411; WO 2010 / 036959; WO 2011 / 066342; WO 2011 / 159877; WO 2011 / 082400 and WO 2011 / 161699

[0350] Non-patent literature

Table 11

Claims

1. A combination of (i) a histone deacetylase inhibitor (HDACi) and (ii) a vaccine composition comprising a population of inactivated fetal cells for use in treating cancer in a subject.

2. 2. The combination for its use according to claim 1, wherein the cells of said population express one or more antigens of interest that are also expressed by said cancer cells of said subject.

3. The combination for use according to claim 1 or 2, wherein the population of inactivated fetal cells is an organoid.

4. Fetal stem cells, a. differentiating a population of pluripotent cells towards a pathway associated with a particular cancer in a patient; b. Expanding the differentiated cells; c. Optionally, exposing the cells of the population to a mutagenic agent during expansion to induce genetic mutagenesis in the cells of the population; d. verifying that at least 70% of the cells in the population express fetal markers; e. optionally verifying that cells of the population express at least one tumor-associated antigen (TAA) or neoantigen present in cancer cells of the subject; f. Inactivating the cells so that they lose their ability to divide.

3. The combination for use thereof according to any one of claims 1 to 2, which is obtained by a process comprising:

5. The combination for its use according to claim 4, wherein said mutagen is selected from the group consisting of chemical mutagens and radiation mutagens (X-rays, ultraviolet light).

6. 6. The combination for its use according to any one of claims 4 or 5, 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.

7. 7. The combination for its use according to any one of claims 1 to 6, wherein the histone deacetylase inhibitor is selected from the group consisting of valproic acid (VPA), vorinostat, panobinostat, gibinostat, belinostat, entinostat, mocetinostat, practinostat, chidamide, xinostat and abexinostat.

8. 1. A composition of inactivated cells comprising inactivated fetal stem cells obtained from iPS-derived fetal hematopoietic lineages, wherein the cells in the population, after expansion, are: Table 1 10. A composition exhibiting a mutation rate of at least 0.1% in at least one gene selected from the group consisting of:

9. 1. A composition of inactivated fetal cells comprising inactivated fetal stem cells in iPS-derived kidney organoids, wherein the cells in the population are selected from the following groups: Table 2 A composition expressing at least one fetal antigen selected from:

10. 1. A composition of inactivated fetal cells comprising inactivated fetal stem cells in iPS-derived lung organoids, wherein the cells in the population express at least one fetal antigen selected from the following group: AIM2, AQP4, AURKA, BMP5, CDCA7, CEP55, CYP4B1, DACH1, EMP2, EPB41L4A, GJB2, MAOA, MELK, MKI67, NEBL, NFIA, PHF19, RNF144B, and UHRF1.

11. 1. A vaccine composition comprising: a. A population of inactivated fetal stem cells; b. Agents that stimulate immune responses and / or MHC I expression 10. A vaccine composition comprising:

12. 12. The vaccine composition of claim 11, wherein the inactivated fetal stem cells comprise mutagenized fetal stem cells.

13. 13. A vaccine composition according to claim 11 or 12 for use in treating cancer in a subject.

14. 14. The vaccine composition for use thereof according to claim 13, wherein said cancer has a fetal stem cell signature.

15. A kit comprising a vaccine composition according to any one of claims 11 to 12 and an information leaflet providing instructions for immunisation.

16. A combined preparation of i) a population of inactivated fetal stem cells and ii) a compound that activates MHC expression and / or immune response for use in treating cancer in a subject by simultaneous, separate or sequential administration.

17. 17. The combined preparation of claim 16, wherein the cancer is selected from the group consisting of bladder carcinoma, breast carcinoma, cervical carcinoma, bile duct carcinoma, colorectal carcinoma, gastric sarcoma, glioma, lung carcinoma, lymphoma, acute and chronic lymphocytic and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, gastric carcinoma, renal carcinoma, head and neck tumors, and all subtypes of solid tumors and hematopoietic malignancies.

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