阅读说明：本技术 用于治疗癌症的方法和组合物 (Methods and compositions for treating cancer ) 是由 F·格里瑟利 A·蒂汉 A·本纳瑟尔·格里瑟利 于 2019-08-06 设计创作，主要内容包括：本发明涉及用于治疗癌症的方法。许多癌症具有干性标记以去分化为未成熟的祖细胞,从而赋予肿瘤克隆从胎儿发育中重新表达基因的能力。发明人获得了每组五只小鼠,其分别接受7天和14天的2x106个辐射的hESC细胞的两次加强疫苗接种,这些细胞与3种不同的佐剂混合：500μgTLR3、50μg TLR9激动剂或50μg/mlQuil-疫苗佐剂。14天后,将5x1044T1个细胞注入小鼠的乳腺脂肪垫中,并以4mg/ml的剂量将丙戊酸添加到饮用水中。他们已经表明,与未接种疫苗的小鼠相反,与使用TLR9激动剂或Quil-疫苗佐剂相比,用hESC和TLR3激动剂联合接种的小鼠产生了最大的乳腺肿瘤体积减少(p<0.001)。因此,本发明涉及用i)诱导抗原的MHC-I呈递的试剂,ii)含有免疫原性成分的疫苗组合物和iii)佐剂治疗患有癌症的受试者的方法。(The present invention relates to methods for treating cancer. Many cancers have sternness markers to dedifferentiate into immature progenitor cells, thereby conferring tumor clones the ability to re-express genes from fetal development. The inventors obtained five mice per group that received two booster vaccinations of 2x106 irradiated hESC cells for 7 and 14 days, respectively, mixed with 3 different adjuvants: 500 μ g TLR3, 50 μ g TLR9 agonist or 50 μ g/ml Quil- A vaccine adjuvant. After 14 days, 5x1044T1 cells were injected into the mammary fat pad of mice and valproic acid was added to the drinking water at a dose of 4 mg/ml. They have shown that in contrast to unvaccinated mice, either the TLR9 agonist or Quil-)

1. A method for treating a subject having cancer, comprising the steps of: administering to the subject simultaneously, separately or sequentially a therapeutic amount of (I) an agent that induces MHC-I presentation of an antigen, wherein the agent is a histone deacetylase inhibitor (HDACi), (ii) a vaccine composition comprising an immunogenic component, and iii) an adjuvant, wherein the adjuvant is an agonist of toll-like receptor (TLR) 3.

2. The method of claim 1, wherein the immunogenic component of the vaccine composition is selected from the group consisting of:

a. an antigen of interest is selected from the group consisting of,

b. human embryonic stem cell (hESCs) compositions,

c. human induced pluripotent stem cell (hiPSC) compositions,

d. fetal stem cell compositions

e. An extract from a cellular composition, wherein cells of the composition express an antigen of interest,

f. a cellular composition, wherein cells of the composition express an antigen of interest,

g. a cell composition comprising antigen-presenting cells that have been primed in vitro by an antigen of interest, or

h. T cell lymphocytes that have been primed in vitro against a target antigen by exposure to antigen presenting cells that present the target antigen.

3. The method of claim 1 or 2, wherein the antigen is a fetal antigen or antigens.

4. The method of any one of claims 1-3, wherein the vaccine composition comprises cells expressing a population of one or more antigens of interest that are also expressed by the cancer cells of the subject.

5. The method of claim 4, wherein the vaccine composition comprises an inactivated fetal cell population that expresses a neofetal antigen.

6. The method of claim 4, wherein the vaccine composition comprises a population of inactivated pluripotent cells, such as embryonic stem cells (hESCs) or induced pluripotent stem cells (hipSCs), expressing a neofetal antigen.

7. The method of claim 5, wherein the fetal stem cells are obtained by a method comprising:

a. differentiating the population of pluripotent cells into pathways associated with the patient's particular cancer,

b. the cells thus differentiated are expanded and,

c. optionally exposing to a mutagen during amplification to induce gene mutations in cells of the population,

d. verifying that at least 70% of the cells of the population express a fetal marker,

e. optionally verifying that cells of said population express at least one Tumor Associated Antigen (TAA) or neoantigen present in cancer cells of said subject,

f. inactivating said cells such that said cells lose their ability to divide.

8. The method of claim 6, wherein the stem cells are obtained by a method comprising the steps of:

a. expanding pluripotent cells in the presence of conditions to maintain the pluripotency of the cells, optionally in the presence of an agent that induces MHC-I presentation of antigens in the population during the expanding step,

b. and exposing the expanded cells to an inactivating agent that will inactivate the cells while maintaining the integrity of the cell envelope.

9. The method of any one of claims 1-8, wherein the histone deacetylase inhibitor is selected from the group consisting of: valproic acid (VPA), vorinostat, panobinostat, gevistat, belinostat, levetiracetam, entinostat, mosettatide, Practinostat, cidentamine, quininostat and abetas.

10. The method of any one of claims 1-9, wherein the TLR3 agonist is Poly (aju) or Poly (I: C).

11. The method of any one of claims 1-10, wherein the vaccine composition comprising an immunogenic component and a TLR3 agonist is administered for a first time and multiple administrations of the histone deacetylase inhibitor are performed from the first time.

12. The method of any one of claims 1-11, wherein the cancer is selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, gastric sarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphoid and myeloid leukemias, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, renal cancer, head and neck tumors, and all subtypes of solid and hematopoietic malignancies, and RET-mutated endocrine tumors, including medullary thyroid carcinoma.

13. The method of any one of claims 1-11, wherein the cancer is a hormone-dependent cancer.

14. The method of claim 13, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, uterine cancer, and ovarian cancer.

A combined preparation of (i) a histone deacetylase inhibitor (HDACi), (ii) a vaccine composition containing an immunogenic component, and iii) an agonist of toll-like receptor (TLR)3 for use in the treatment of cancer in a subject by simultaneous, separate or sequential administration.

16. Combined preparation for use according to claim 15, wherein the immunogenic component in the vaccine composition is selected from the group consisting of:

a. an antigen of interest is selected from the group consisting of,

b. human embryonic stem cell (hESCs) compositions,

c. human induced pluripotent stem cell (hiPSC) compositions,

d. fetal stem cell compositions

e. An extract from a cellular composition, wherein cells of the composition express an antigen of interest,

f. a cellular composition, wherein cells of the composition express an antigen of interest,

g. a cell composition comprising antigen-presenting cells that have been primed in vitro by an antigen of interest, or

h. T cell lymphocytes that have been primed in vitro against a target antigen by exposure to antigen presenting cells that present the target antigen.

17. Combined preparation for use according to claim 15 or 16, wherein the antigen is a foetal antigen or a plurality of foetal antigens.

18. The combined preparation for use according to any one of claims 15-17, wherein the vaccine composition comprises cells expressing a population of one or more antigens of interest that are also expressed by cancer cells of the subject.

19. Combined preparation for use according to claim 18, wherein the vaccine composition comprises an inactivated population of foetal cells expressing a neofoetal antigen.

20. Combined preparation for use according to claim 18, wherein the vaccine composition comprises a population of inactivated pluripotent cells, such as embryonic stem cells (hESC) or induced pluripotent stem cells (hiPSC), expressing a neo-fetal antigen.

21. Combined preparation for use according to claim 19, wherein the fetal stem cells are obtained by a method comprising the steps of:

a. differentiating the population of pluripotent cells into pathways associated with the patient's particular cancer,

b. the cells thus differentiated are expanded and,

c. optionally exposing to a mutagen during amplification to induce gene mutations in cells of the population,

d. verifying that at least 70% of the cells of the population express a fetal marker,

e. optionally verifying that cells of said population express at least one Tumor Associated Antigen (TAA) or neoantigen present in cancer cells of said subject,

f. inactivating said cells such that said cells lose their ability to divide.

22. Combined preparation for use according to claim 20, wherein the stem cells are obtained by a method comprising the steps of:

a. expanding pluripotent cells in the presence of conditions to maintain the pluripotency of the cells, optionally in the presence of an agent that induces MHC-I presentation of antigens in the population during the expanding step,

b. and exposing the expanded cells to an inactivating agent that will inactivate the cells while maintaining the integrity of the cell envelope.

23. The combined preparation for use according to any one of claims 15-22, wherein the histone deacetylase inhibitor is selected from the group consisting of: valproic acid (VPA), vorinostat, levetiracetam, panobinostat, gibvista, belinostat, entinostat, mosettatide, Practinostat, cidentamine, quininostat, and abetas.

24. Combination preparation for use according to any one of claims 15-23, wherein the TLR3 agonist is Poly (a: U) or Poly (I: C).

25. Combined preparation for use according to any one of claims 15-24, wherein the vaccine composition comprising an immunogenic ingredient and a TLR3 agonist is subjected to a primary administration and multiple administrations of the histone deacetylase inhibitor are performed from the primary administration.

26. The combination preparation for use according to any one of claims 15-25, wherein the cancer is selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, gastric sarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphoid and myeloid leukemias, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, renal cancer, head and neck tumors, and all subtypes of solid and hematopoietic malignancies, and RET-mutated endocrine tumors, including medullary thyroid carcinoma.

27. The combination preparation for use according to any one of claims 15-25, wherein the cancer is a hormone-dependent cancer.

28. The combination preparation for use according to claim 27, wherein the cancer is selected from breast cancer, prostate cancer, uterine cancer and ovarian cancer.

Technical Field

The present invention is in the field of oncology, and more particularly, the present invention relates to anti-cancer vaccine combination therapy. More particularly, the invention relates to methods of treating cancer with agents that induce MHC-I presentation of antigens, (ii) vaccine compositions containing immunogenic components, and iii) adjuvants.

Background

Cancer Stem Cells (CSCs) represent a small population of self-renewing cancer cells that respond to persistence and recurrence of tumors because they may be resistant to conventional therapies. These CSCs have recently been demonstrated in solid tumors of various origins, including breast, colon and neck cancers, and represent a new therapeutic target. Those CSCs have been shown to express large amounts of embryonic antigens that are expressed in common with human embryonic stem cells (hescs) or human induced pluripotent stem cells (hipscs). Expression of some of those embryonic antigens has also been found in differentiated cancer cells associated with tumorigenesis and/or tumor progression.

Over the past decade, cancer treatment approaches have evolved from targeted therapies to immune intervention strategies, with unprecedented increases in survival and cancer-related morbidity and mortality. However, despite the demonstrated efficacy and clinical benefit of immune checkpoint inhibitors, there are still a large number of local responses and primary drug-resistant tumors ("innate resistance") caused by immunomodulatory factors that affect tumor-specific immune responses and cancer cell autonomous signaling. Following the initial response to the blocking of PD-1/PD-L1, resistance was acquired in both the development and recurrence of many cancers. The underlying mechanism for gaining resistance to PD1/PDL-1 blockade was caused by the evolution of a neoantigenic landscape with acquired somatic mutations (mutants), an evolved Tumor Immune Microenvironment (TIME) with epigenetic stability of depleted T cells.

Cancer germline antigens represent proteins expressed during embryonic and fetal development, and these epigenetic controlled antigens can be re-expressed at different rates for a variety of cancers. Several human cancer vaccine trials have been established to date to target embryonic antigens such as carcinoembryonic antigen (CEA), alpha fetoprotein or cancer/testis antigen (NY-ESO-1). Adoptive cells transferred from autologous lymphocytes using T cell antigen receptors (TCR) genetically engineered to express HLA-A-0201 epitopes directed against the cancer germline antigen NY-ESO-1 cause persistent tumor regression in some metastatic melanoma patients. Unfortunately, targeting only one antigen has proven insufficient to generate a strong anti-tumor immune response to mediate tumor rejection due to the rapid emergence of escape mutants and new somatic neoantigens and the general ineffectiveness of monovalent cancer vaccines.

Recent interest in the potential of stem cells in regenerative medicine has made widely available well-defined undifferentiated ESC lines and undifferentiated ipscs that are phenotypically and functionally analogous to ESCs.

Cancers with sternness exhibit profound changes in genomic plasticity and chromatin landscape secondary to intrinsic pathways and inducers from a powerful immunosuppressive tumor microenvironment. Their ability to dedifferentiate into immature progenitor cells confers on tumor clones the ability to re-express genes from fetal development, wherein the expression of MHC class I is down-regulated and the expression of co-suppressor molecules is up-regulated.

Therefore, there is a continuing need for new approaches to prevent and/or treat cancers with stem cell markers. Vaccination against mutated neo-epitopes of stem cells can be used to enhance the immune response of adoptively transferred T cells or activated cells by blocking immune checkpoints.

This need and other needs are addressed in whole or in part by the subject matter disclosed herein.

Brief description of the invention

The present invention relates to a method for treating a subject suffering from cancer, comprising the steps of: administering to the subject simultaneously, separately or sequentially a therapeutic amount of (I) an agent that induces MHC-I presentation of an antigen, (ii) a vaccine composition comprising an immunogenic component and iii) an adjuvant. The invention is particularly defined by the claims. In particular, the agent inducing MHC-I presentation of an antigen is a histone deacetylase inhibitor (HDACi) and the adjuvant is a toll-like receptor (TLR)3 agonist. Preferably the subject does not have pre-existing immunity to the antigen.

In a specific embodiment, the immunogenic component of the vaccine composition is selected from the group consisting of:

a. an antigen of interest is selected from the group consisting of,

b. human embryonic stem cell (hESCs) compositions,

c. human induced pluripotent stem cell (hiPSC) compositions,

d. fetal stem cell compositions

e. An extract from a cellular composition, wherein cells of the composition express an antigen of interest,

f. a cellular composition, wherein cells of the composition express an antigen of interest,

g. a cellular composition comprising antigen presenting cells that have been primed in vitro by an antigen of interest, and

h. t cell lymphocytes that have been primed in vitro against a target antigen by exposure to antigen presenting cells that present the target antigen.

In particular, the antigen is a fetal antigen or antigens.

In one embodiment, the vaccine composition comprises cells expressing a population of one or more antigens of interest that are also expressed by the cancer cells of the subject.

In one embodiment, the vaccine composition comprises an inactivated fetal cell population that expresses a neofetal antigen.

In particular, the fetal stem cells are obtained by a method comprising the steps of:

a. differentiating the population of pluripotent cells to a pathway associated with a particular cancer in the patient,

b. the cells thus differentiated are expanded and the cells thus differentiated,

c. optionally exposing to a mutagen during amplification to induce mutagenesis of the gene in the cells of the population,

d. verifying that at least 70% of the cells of the population express a fetal marker,

e. optionally verifying that said cells of said population express at least one Tumor Associated Antigen (TAA) or neoantigen present in cancer cells of said subject,

f. inactivating said cells such that said cells lose their ability to divide.

In one embodiment, the vaccine composition comprises a population of inactivated pluripotent cells, such as embryonic stem cells (hescs) or induced pluripotent stem cells (hipscs), that express a neofetal antigen. This population is described in WO 2017202949.

In particular, the stem cells are obtained by a method comprising the steps of:

a. expanding pluripotent cells in the presence of conditions to maintain the pluripotency of the cells, optionally in the presence of an agent that induces MHC-I presentation of antigens in the population during the expanding step,

b. and exposing the expanded cells to an inactivating agent that will inactivate the cells while maintaining the integrity of the cell envelope.

Preferably, the histone deacetylase inhibitor is selected from the group consisting of: valproic acid (VPA), vorinostat, panobinostat, gevistat, belinostat, entinostat, mosetinostat, Practinostat, cidam, quinostat, abetas, and levetiracetam.

Preferably the TLR3 agonist is poly (A: U) or poly (I: C).

In one embodiment, the vaccine composition comprising an immunogenic component and a TLR3 agonist is administered for a first time, and multiple administrations of the histone deacetylase inhibitor are performed from the first administration.

In one embodiment, the cancer is selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, carcinoma of the large intestine, gastrarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphoid and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, renal cancer, head and neck tumors, as well as all subtypes of solid tumors, hematopoietic malignancies, and RET mutated endocrine tumors, including medullary thyroid carcinoma.

In one embodiment, the cancer is a hormone-dependent cancer. In this embodiment, the cancer is selected from the group consisting of breast cancer, prostate cancer, uterine cancer, and ovarian cancer.

The present invention relates to combined preparations of (i) a histone deacetylase inhibitor (HDACi), (ii) a vaccine composition containing an immunogenic component, and iii) an agonist of toll-like receptor (TLR)3 for use in the treatment of cancer in a subject by simultaneous, separate or sequential administration.

In one embodiment, the immunogenic component of the vaccine composition is selected from the same group as described above, in particular a foetal antigen or a plurality of foetal antigens, cells expressing one or more populations of antigens of interest also expressed by the cancer cells of the subject, a population of inactivated foetal cells expressing neofoetal antigens or a population of inactivated pluripotent cells expressing neofoetal antigens, such as embryonic stem cells (hESC) or induced pluripotent stem cells (hiPSC).

Detailed Description

The inventors obtained five mice per group that received two booster vaccinations of 2x106 irradiated hESC cells for 7 and 14 days, respectively, mixed with 3 different adjuvants: 500 μ g TLR3, 50 μ g TLR9 agonist or 50 μ g/mlA Saponin vaccine adjuvant. After 14 days, 5x1044T1 cells were injected into the mammary fat pad of mice and valproic acid was added to the drinking water at a dose of 4 mg/ml. They have shown that in contrast to unvaccinated mice, the use of TLR9 agonists orMice vaccinated with a combination of hESC and TLR3 agonist produced the greatest reduction in breast tumor volume (p) compared to the Saponin vaccine adjuvant<0.001)。

Methods for treating a subject having cancer

The present invention relates to a method for treating a subject suffering from cancer, comprising the steps of: administering to the subject simultaneously, separately or sequentially a therapeutic amount of (I) an agent that induces MHC-I presentation of an antigen, ii) a vaccine composition comprising an immunogenic component and iii) an adjuvant as a combined preparation.

As used herein, the term "treatment" refers to prophylactic or preventative treatment as well as curative or disease modifying treatment, including treatment of subjects at high risk of developing cancer (e.g., hereditary family cancer syndrome), or subjects suspected of having cancer as well as subjects who are ill or have been diagnosed with cancer or a medical condition, and includes inhibition of clinical relapse. A treatment may be administered to a subject having cancer or who may ultimately have cancer, to prevent, cure, delay the onset of, reduce the severity of, or improve one or more symptoms of cancer or recurrent cancer, or to extend the survival of the subject beyond that expected in the absence of such treatment. "treatment regimen" refers to the mode of treatment of a disease, e.g., the dosage mode used during treatment. The treatment regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to a subject during the initial phase of a treatment regimen. The induction regimen may employ a "loading regimen," which may include administering a larger dose of the drug than the physician uses during the maintenance regimen, administering the drug more frequently than the physician uses during the maintenance treatment, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) for maintaining a subject during treatment of a disease, e.g., to maintain the subject in remission for an extended period of time (months or years). Maintenance regimens may employ continuous therapy (e.g., administration of a drug at regular intervals (e.g., weekly, monthly, yearly, etc.)) or intermittent therapy (e.g., treatment interrupted, intermittent, recurrent or when certain predetermined criteria are met (e.g., pain, disease manifestation, etc.)).

As used herein, the term "cancer" refers to abnormal cell growth with the potential to invade or spread to other parts of the body. The cancer is selected from, but not limited to, the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, carcinoma of the large intestine, gastrarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphoid and myeloid leukemia, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, renal cancer, head and neck tumors, as well as all subtypes of solid tumors, hematopoietic malignancies, and RET mutated endocrine tumors, including medullary thyroid carcinoma.

As used herein, the term "subject" refers to any mammal, such as rodents, cats, dogs, and non-human and human primates. In particular, in the present invention, the subject is a human suffering from or susceptible to a cancer having expression of a pluripotent embryonic-like stem cell antigen or a fetal-like stem cell antigen. Typically, the subject is a human suffering from or susceptible to cancer as described above.

As used herein, the term "agent that induces MHC-I presentation of an antigen" refers to a compound that is capable of stimulating immunogenicity. The compounds are referred to as activators of MHC expression and/or immune response in a subject. Induction of MHC-I can be assessed by flow cytometry (FACS) analysis to determine at least 2-fold increase in MHC-I molecules on the surface of cancer cells, such as 4T1 cells or lewis lung carcinoma, LLC cells. The term "MHC" refers to the major histocompatibility complex that exists on the surface of cells to recognize foreign molecules called antigens. The MHC binds antigens and presents them to immune molecules (such as lymphocytes T and B). The term "immune response" refers to the immune response of the immune system to an antigen. By activating an immune response, the population of FoxP3 subpopulations and myeloid suppressor cells (MDSCs) decreased, and conversely, the NK population increased. In the context of the present invention, an immune response against a tumor includes a cytotoxic T cell response against an antigen present in or on the 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 an immune response corresponds to a molecule present in a population of fetal stem cells as described above. In this case, the compound that activates MCH expression and/or the immune system is a fetal gene or an immunogenic neo-antigen. The term "neoantigen" or "neoantigenic" refers to a class of antigens derived from at least one mutation that alters the amino acid sequence of a histone encoded by the genome.

In the context of the present invention, the compound is selected from the group consisting of: cytokines, Histone Deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, and histone lysine N-methyltransferase inhibitors.

In a specific embodiment, the activator of MHC expression and/or immune response is a histone deacetylase inhibitor. HDAC inhibitors (HDACi) are natural or synthetic compounds with a broad range of functions in cells. A variety of HDACi are known and designed to target the catalytic site of HDACs. HDACi can be divided into several classes, based on its structure and specificity, including hydroxamates, cyclic peptides, aliphatic acids, and benzamides. Compounds can be tested for HDACI activity by the methods disclosed by Blackwell et al (Life Sciences 82(2008) 1050-.

As used herein, the term histone "histone deacetylase inhibitor", also known as HDACi, refers to a class of compounds that interfere with the function of histone deacetylases. Histone Deacetylases (HDACs) play an important role in transcriptional regulation and pathogenesis of cancer. Generally, inhibitors of HDACs regulate transcription and induce cell growth arrest, differentiation and apoptosis. HDACs also potentiate the cytotoxic effects of therapeutic agents (including radiation and chemotherapeutic drugs) used in cancer therapy.

In a specific embodiment, the histone deacetylase inhibitor is valproic acid (VPA).

The term "valproic acid" refers to the acid sodium 2-valproate (C)₈H₁₆O₂) It has the following CAS number and formula 99-66-1 in the art:

valproic acid has a variety of biological activities (Chateauvieuux et al, J.biomed.Biotechnol, 2010, pii:479364.doi: 10.1155/2010/479364). Valproic acid affects GABA (gamma aminobutyrate), a neurotransmitter that enhances inhibitory activity. Several mechanisms of action are proposed. Valproic acid is metabolized especially by GABA: inhibit degradation of GABA, GABA transaminoxanthine (LAMP), attenuation of GABA synthesis, and alter its flow. In addition, valproic acid blocks certain ion channels, reduces arousal mediated by N-methyl-D-aspartic acid, and blocks the activity of ion channels including Na + and Ca2+ (voltage-dependent L-CACNA 1C, D, N and F).

In the context of the present invention, valproic acid is used as an immunostimulant to boost the immune response against cancer expressing human embryonic stem cells, pluripotent cells are exposed to mutagens shared with fetal stem cells or fetal stem cell neoantigens.

More specifically, VPA is used to stimulate and enhance MHC-1 expression on the cancer stem cell compartment, increasing neoantigen content in some tumor cells. Higher expression on fetal stem cells allows for enhanced presentation of MHC-I associated neoantigens to APC/dendritic cells to induce a TH1 immune response. Higher levels of chemokines (CXCL9, CXCL10) allow for enhanced T cell recruitment into the tumor.

The present invention relates to methods of increasing the neoantigen content of derived human embryonic stem cells, pluripotent cells exposed to mutagens in the presence of HADCi (such as VPA and/or 5 azacytidine), and tumor cells with expression of fetal antigens by chromatin remodeling, as well as in chemokine expression (CXCL9, CXCL10, CXL 13).

In particular, when used for in vivo treatment of a subject, the compositions and vaccines of the invention make it possible to modify the tumor microenvironment and promote recruitment of T cells into the tumor, thereby obtaining a long lasting reduction in tumor volume.

This is due to the synergistic effect of co-administration of human embryonic stem cells, a mutagenic-exposed pluripotent cell or fetal stem cell vaccine and VPA, which is further improved when HDACi is further administered to the patient for a period of time (such as at least 15 days) after vaccine injection.

In a specific embodiment, the histone deacetylase inhibitor is suberoylanilide hydroxamic acid, also known as vorinostat (N-hydroxy-N' -phenylsuberamide), which is the first histone deacetylase inhibitor approved by the U.S. Food and Drug Administration (FDA) in 2006 (Marchion DC et al 2004; Valentie et al 2014).

In a specific embodiment, the histone deacetylase inhibitor is panobinostat (LBH-589) approved by the FDA in 2015, the structure of which is described in valence et al 2014.

In a particular embodiment, the histone deacetylase inhibitor is Girvester (ITF2357), which has been approved by the European Union as an orphan drug (Leoni et al 2005; Valente et al 2014).

In another embodiment, the histone deacetylase inhibitor is levetiracetam: although this compound has no direct effect on HDAC, its major carboxylic acid metabolite, 2-pyrrolidone-n-butyric acid (PBA), in humans inhibits HDAC with a Ki value of 2.25+/-0.78mM (Eyal S, Epilepsia.2004Jul; 45(7): 737-44). Thus, in this context, it is considered to be HDACin.

In a specific embodiment, the histone deacetylase inhibitor is belinostat, also known as Beleodaq (PXD-101), which was approved by the FDA in 2014 (Ja et al 2003; Valente et al 2014).

In a specific embodiment, the histone deacetylase inhibitor is entinostat (which is SNDX-275 or MS-275). The molecule has the following chemical formula (C)₂₁H₂₀N₄O₃) And has a structure as described by Valent et al 2014.

In a specific embodiment, the histone deacetylase inhibitor is of the formula (C)₂₃H₂₀N₆O) (Valentite et al 2014) of mositensted (MGCD 01030).

In a specific embodiment, the histone deacetylase inhibitor is of the formula (C) as described in Diermayr et al 2012₂₀H₃₀N₄O₂) And Practinostat of structure (SB 939).

In a specific embodiment, the histone deacetylase inhibitor is of the formula (C)₂₂H₁₉FN₄O₂) Sidapamide (CS 055/HBI-8000).

In a specific embodiment, the histone deacetylase inhibitor is of the formula (C)₂₁H₂₆N₆O₂) Quinunostat (JNJ-26481585).

In a specific embodiment, the histone deacetylase inhibitor is of the formula (C)₂₁H₂₃N₃O₅) Abelistat (PCI24781) (Valentie et al 2014).

In a specific embodiment, the histone deacetylase inhibitor is of the formula (C)₂₀H₁₉FN₆O₂) CHR-3996(Moffat D et al 2010; banerji et al 2012).

In a specific embodiment, the histone deacetylase inhibitor is of the formula (C)₁₈H₂₀N₂O₃) AR-42(Lin et al 2012).

In a specific embodiment, the activator of MHC expression is a DNA methyltransferase inhibitor.

As used herein, the term "DNA methyltransferase inhibitor" refers to a compound that is capable of interacting with DNA methyltransferase (DNMT) and inhibiting its activity. DNMT is an enzyme that catalyzes the transfer of a methyl group to DNA. DNA methylation has a variety of biological functions. All known DNA methyltransferases use S-adenosylmethionine (SAM) as methyl donor.

In a specific embodiment, the DNA methyltransferase inhibitor is azacytidine, also known as 5-aza-2-deoxycytidine, having the following formula (C)₈H₁₂N₄O₅) And structures of the art (Kaminskas et al 2004; estey et al 2013).

In a specific embodiment, the DNA methyltransferase inhibitor is decitabine, also known as 5-aza-2' -deoxycytidine, having the formula (C)₈H₁₂N₄O₄) (Kantariian et al, 2006).

In a particular embodiment, the activator of MHC expression and/or immune response is a histone-lysine N-methyltransferase inhibitor or a DNA methyltransferase inhibitor. As used herein, the term "histone-lysine N-methyltransferase inhibitor" refers to a compound capable of interacting with histone-lysine N-methyltransferase encoded by an enhancer of zeste homolog 1(EZH1) and 2(EZH2) genes involved in DNA methylation. EZH2 catalyzes the addition of a methyl group to histone H3 at lysine 27 by using the cofactor S-adenosyl-L-methionine.

In a specific embodiment, histone-lysine N-The methyltransferase inhibitor is 3-deazalinone A (DZNep, C-C3 Ado). DZNep, C-C3Ado has the following chemical formula C in the art₁₂H₁₄N₄O₃And CAS number 102052-95-9.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is UNC1999 and an inactive analogous compound. UNC1999 has the following chemical formula C in the art₃₃H₄₃N₇O₂And CAS number 1431612-23-5.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is UNC2400 and an inactive similar compound. UNC2400 has the following chemical formula C in the art₃₅H₄₇N₇O₂And CAS number 1433200-49-7.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is tazemetostat (EPZ6438, E7438). Tazemetostat has the following chemical formula C in the art₃₄H₄₄N₄O₄And CAS number 1403254-99-8.

In a particular embodiment, the histone-lysine N-methyltransferase inhibitor is trifluoroacetate (EPZ 011989). Trifluoroacetate has the following chemical formula CF in the art₃COONa and CAS number 2923-18-4.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is EPZ 005687. EPZ005687 has the following chemical formula C in the art₃₂H₃₇N₅O₃And CAS number 1396772-26-1.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is GSK 343. GSK343 has the following chemical formula C in the art₃₁H₃₉N₇O₂And CAS number 1346704-33-3.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is GSK 126. GSK126 has the following chemical formula C in the art₃₁H₃₈N₆O₂And CAS number 1346574-57-9.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is GSK 2816126. GSK2816126 has the following chemical formula C in the art₃₁H₃₈N₆O₂And CAS number 1346574-57-9.

In a specific embodiment, the histone-lysine N-methyltransferase inhibitor is ZLD 1039. ZLD1039 has the following chemical formula C in the art₃₆H₄₈N₆O₃And CAS number 1826865-46-6.

As used herein, the term "vaccine composition" refers to a vaccine composition containing an immunogenic component intended to generate an immune response in a subject against one or more antigens of interest. The antigen of interest is any antigen against which an immune response is desired, including any peptide, protein from itself (such as an antigen from a cancer cell) or any protein of foreign origin (such as a bacterial, viral or parasitic protein), other types of antigens (such as nucleic acids, sugars, lipopolysaccharides, etc.).

In a specific embodiment, the vaccine composition contains an immunogenic component (compound) intended to raise an immune response in a subject against one or more antigens of interest.

In another embodiment, the immunogenic compound is an extract from a cellular composition, wherein the cells of the composition express an antigen of interest. The cell extract may be lysed cells that have been centrifuged to remove insolubles such as membrane fragments, vesicles, and nuclei, and thus consist primarily of cytosol. In another embodiment, the extract may be prepared using a particular technique to deplete or enrich for a particular component (e.g., sonication may be used to break large membrane fragments into small particles that remain in the extract, or high speed centrifugation to remove the smallest insoluble components). The cell extract is obtained by any chemical or mechanical action, e.g. by pressure, distillation, evaporation, etc.

As used herein, the term "immunogenic component" refers to a compound that stimulates the immune system. In the context of the present invention, the immunogenic component is selected from, but not limited to, the group of:

a) an antigen of interest is selected from the group consisting of,

b) human embryonic stem cell (hESCs) compositions,

c) human induced pluripotent stem cell (hiPSC) compositions,

d) a composition of fetal stem cells comprising a combination of fetal stem cells,

e) an extract from a cellular composition, wherein cells of the composition express an antigen of interest,

f) a cellular composition, wherein cells of the composition express an antigen of interest,

g) a cell composition comprising antigen-presenting cells that have been primed in vitro by an antigen of interest, or

h) T cell lymphocytes that have been primed in vitro against a target antigen by exposure to antigen presenting cells that present the target antigen.

Methods of obtaining a cell composition:

in another embodiment, the immunogenic component is a cellular composition, wherein the cells of the composition express an antigen of interest.

In a specific embodiment, the cell composition is a human embryonic stem cell (hESCs) composition, a human induced pluripotent stem cell (hiPSCs) composition, or a fetal stem cell composition.

Fetal cell populations and uses

Herein, a fetal cell population corresponds to a cell population maintained as a cell culture, but also organoids are contemplated, wherein the cells start to form an organ and 3D spatial organization of the cells can be observed.

It is reminded that differentiation is a process by which more specific cells are formed from less specific cells. This is a continuous process. Starting from pluripotent cells (embryonic stem cells or iPS cells), these cells will lose pluripotency and participate in a differentiation mode in which it matures in fully differentiated specialized cells. For some organs, multiple cells produce organoids during differentiation.

Inducing and directing differentiation of pluripotent cells is known to those skilled in the art. Wu et al (cell.2016Jun 16; 165(7):1572 1585), Fatehulah et al (Nat Cell biol.2016 Mar; 18(3):246-54) or Sasaki and Clevers (Curr Opin Genet Dev.2018 Sep 24; 52: 117) which describe the development or organoid of pluripotent cells can be cited. There are many other articles that describe the population and teach methods and conditions for differentiating pluripotent cells in various target tissues.

In one embodiment, the immunogenic component is a population of foetal cells that have been inactivated, advantageously being in the same cell differentiation lineage as the cancer to be treated.

In this embodiment, the invention relates to (i) a histone deacetylase inhibitor (HDACi), and (ii) a vaccine composition comprising an inactivated fetal cell population, and iii) an adjuvant, wherein the adjuvant is a combination of an agonist of toll-like receptor (TLR)3, for use in the treatment of cancer in a subject.

In another embodiment, the vaccine consists of an inactivated fetal cell population. In particular, the cells of the population express one or more antigens of interest that are also expressed by the cancer cells of the subject. In a particular embodiment, the inactivated fetal cell population is organoid or derived from an organoid (i.e., has been obtained by disrupting the 3D structure of the organoid).

In general, the term "fetal stem cell" refers to a population of fetal cells, which are transient progenitor cells that appear early in development. This population can be propagated in vitro by differentiation of allogeneic, xenogeneic or syngeneic pluripotent stem cells (ESCs and iPSCs). The cells of the fetal population are characterized by a loss of genes associated with pluripotency, wherein at least 20% of the following genes are lost: NACC1, BLM, WDR33, DAZAP 33, CDK 33, ZNF165, XRCC 33, SMARCAD 33, AIMP 33, CKS1 33, NANOG, ZFP 33, U2AF 33, CCNB 33, DCTPP 33, TGIF 33, SUPT3 33, AURKB, GEMIN 33, SRSF 33, PNP, SIGLEC 33, POU5F 33, PSMA 33, RMND5 33, GDF 33, STXBP 33, BAG 33, GMPS, NME 33, POP 33, RCCP 33, SMARCC 33, HNRNPK, PTMA 33, NPM 33, SNRPPA 33, MYBBP1 33, MYBBP 33, HSPCDT 33, HSP 33, PSNDP 33, PSNCR 33, PSNDP 33, PSN 33, ARMC, COPS, MCM, PPAP2, LSM, NME-NME, EWSR, POLG, BCL, NFKBIB, SALL, PXN, EXOSC, HSPA, HMGB, RUVBL, GOT, PPM1, ATIC, DHCR, APEX, RFC, WDYHV, NTHL, EXOSC, SNRPD, DPPA, MRPS, FBL, POLD, MCM, EXOSC, NOP, TPX, PAK, HNRNPAB, ANXA, BUB1, SEPHS, HSPR, LUC7L, VASP, MCM, PAK, PMAIP, PBX, NOLC, PCYT1, NCL, ORC, GPRIN, EXORC, RADA, ANXA, NUP, SNRPC, HAUS, MYMATK, BIRC, PSIP, DSMC, STP, SMBP, SMIF, TOBE, LSM, LSE, NME-NME, NMAD, PSAF, PSRB, PFRB, PFG, PSRB, PFRB, PSRB, PFRB, RFC, PSRB, POP5, RFC5, CHEK 5, BCCIP, SOCS 5, PHB, PMF 5, MPP 5, NOC 25, HDAC 5, CENPE, RECQL 5, CASP 5, GNL 5, SRSF 5, BRIX 5, MYB, RNMTL 5, DHFR, FEN 5, SNRPF, MUTYH, PRNP, MT 15, PSMD 5, GAR 5, DDX 5, FUBP 5, CDK 5, WRAP 5, CASP 5, RASL11 5, CHAF 15, CCNB 5, CKS 5, CCNA 5, PPAN, WEE 5, HMMR, TDP 5, CHATP 5, or CHA 3654. In particular, the fetal stem cells are also characterized by the absence of expression of lineage specific genes of adult differentiated cells.

More specifically, it is preferable when the fetal stem cells are obtained by a method comprising the steps of:

a. differentiating the population of pluripotent cells into pathways associated with the patient's particular cancer,

b. the cells thus differentiated are expanded and,

c. optionally exposing to a mutagen during amplification to induce mutagenesis of the gene in cells of the population,

d. verifying that at least 70% of the cells of the population express a fetal marker,

e. optionally verifying that cells of said population express at least one Tumor Associated Antigen (TAA) or neoantigen present in cancer cells of said subject,

f. inactivating said cells such that said cells lose their ability to divide.

When mutagenesis is performed, preferably the mutagen is selected from the group consisting of: chemical mutagens and radiation mutagens (X-rays, UV radiation). In particular, the mutagen is selected from the group consisting of: ENU, active oxygen species, deaminating agent, polycyclic aromatic hydrocarbon, aromatic amine, and sodium azide.

In a preferred embodiment, the histone deacetylase inhibitor is selected from the group consisting of: valproic acid (VPA), vorinostat, levetiracetam, panobinostat, gibvista, belinostat, entinostat, mosettatide, Practinostat, cidentamine, quininostat, and abetas. Valproic acid (VPA), vorinostat and levetiracetam are of particular interest.

When the inactivated cell composition comprising inactivated fetal stem cells is obtained from an iPS derived fetal hematopoietic lineage, and when the cells in the population after expansion exhibit a mutation rate of at least 0.1% in at least one gene selected from the group consisting of: ARHGEF10, TRIM, NKAIN, ITGAGGT, PDZD, MUC, NECB, MNT, GLTSCR, COPZ, ZFP, MIB, ABCC, IGFN, LRRK, RIN, GGT, ANK, HDAC, MUC, SDCCAG, DNAI, BTNL, ABTB, MC2, DOCK, FSD1, CRP, PPP1R3, SLC22A, PITPNM, A2, CTDSP, IFNA, KIF5, THNSL, GTF3C, NRXN, MED, FNBP, TMCO, ING, ZNF292, RBL, CD109, FOXRED, SN PLIN, ZNF, SENPE, CENPE, BTBD, STOM, LRF 317, TET, ZNBA, MED, CDC, CDR, HPHPRT, BCMSH. These genes are commonly expressed in acute leukemias, particularly acute myeloid leukemia.

In one embodiment, the inactivated fetal cell composition comprises inactivated fetal stem cells in an iPS-derived kidney organ, wherein cells in the population express at least one fetal antigen selected from the group consisting of: TRAPPC, MX, ITSN, DNAJC, TAF, TMEM, CRYM, PRTG, TYRO C12ORF, FJX, ADM, FAM45, ASS, CA, ZFXX, CLVS, NRG, EZH, SLC22A, MSH, FBN, GTF2H, LIX, HESX, FZD, LRP, RHOQ, NUAK, ILF, ACP, RPL, NMNAT, ID, U2AF, KLHL, CDH, GREB1, ARRDC, THBS, BMP, LRIG, SOX, SF, LGR, MGEA, BCORL, STOM, GLIS, ANXA, KDM4, SDC, TMEM130, MAGI, GLI, HEY, TPBG, ID, MYLIP, ENC, EGR, CDH, NPY1, CDSEL 1L, NOTAT, CLDN, CEP, BHJC, BHE, TAF, LHL, LHIG, HGBG, ARAP, ARF 3, and ARIFAP. These genes are typically expressed in primary adult kidney cancer, either related or unrelated to the c-Met mutation.

In one embodiment, the inactivated fetal cell composition comprises inactivated fetal stem cells in an iPS-derived lung organoid, wherein cells in the population express at least one fetal antigen selected from the group consisting of: AIM2, AQP4, AURKA, BMP5, CDCA7, CEP55, CYP4B1, DACH1, EMP2, EPB41L4A, GJB2, MAOA, MELK, MKI67, NEBL, NFIA, PHF19, RNF144B, and UHRF 1. These genes are commonly expressed in adult lung cancer.

The present invention also relates to a vaccine composition comprising:

a. an inactivated population of fetal stem cells,

b. agents that stimulate immune response and/or MHC I expression, and

c. an adjuvant which is a toll-like receptor 3 agonist, in particular poly I: C or poly A: U.

In particular, the inactivated fetal stem cells comprise mutagenized fetal stem cells. It may be used to treat cancer in a subject, particularly where the cancer is characterized by fetal stem cells.

Another part of the invention is a kit comprising a vaccine composition as described above and an information sheet providing immunological guidance.

The invention also relates to a combined preparation of (i) an inactivated fetal stem cell population, (ii) a compound that activates MHC expression and/or an immune response, and iii) a toll-like receptor 3 agonist, for use in the treatment of cancer in a subject by simultaneous, separate or sequential administration. The combination may be used when the cancer is selected from all subtypes of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, gastric sarcoma, glioma, lung cancer, lymphoma, acute and chronic lymphoid and myeloid leukemias, melanoma, multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, renal cancer, head and neck tumors, and solid and hematopoietic malignancies.

Also disclosed are methods of treatment wherein a therapeutic amount of the composition (inactivated fetal cell population and adjuvant) is administered to a patient in need of treatment and are part of the invention.

In the present application, all genes are referred to by names known to those skilled in the art. According to these names, gene and protein sequences can be found using any search engine, including the talent search engine, or In databases dedicated to maintaining Cancer gene banks (e.g., the COSMIC database, (Catalogue Of viral Mutations In Cancer, developed by Sanger research, UK) or Cancer genomic profiles (TCGA, maintained by NCBI, USA).

Thus, a fetal cell refers to a cell that has lost its pluripotency because it begins to participate in the differentiation pathway (endoderm, mesoderm, ectoderm).

It can be determined whether the cell population is a fetal cell population, since these cells should express a fetal marker (see below) but not a pluripotency marker.

According to the invention, the population comprises a plurality of cells (at least 0.5x 10)⁶A cell, more preferably at least 1x10⁶Individual cells, more preferablyAt least 2x10⁶Single cell or 5x10⁶Single cell or greater than 5x10⁶And (4) cells.

To determine whether a cell population is a fetal cell population, it is necessary to:

(a) determining that the cells of the population do not substantially express the pluripotency gene (or marker),

(b) determining the presence of a fetal gene (or marker) expressed by cells of the population.

In particular embodiments, the cells of the fetal cell population are such that

(a) None or less than 10% of the cells express genes that are typically expressed in undifferentiated pluripotent self-renewing cells (embryonic stem cells or induced pluripotent stem cells). This is preferably determined by flow cytometry, more particularly by FACS (fluorescence activated cell sorting)

And

(b) at least 70%, more preferably greater than 75%, more preferably greater than 80% of the cells in the population express progenitor/fetal markers, regardless of whether the population is in the form of committed differentiated progenitor cells derived from three germline layers or 3D organoid tissue.

This is also preferred when less than 10% of the cells express adult tissue markers. An adult tissue marker is a marker (protein or gene) that is expressed in adult cells.

The above percentages relate to the percentage of cells in the population that express a given marker. By way of illustration, low expression (< 10%) of a major gene typically expressed in undifferentiated pluripotent self-renewing cells indicates that less than 10% of the cells in the population express the gene of interest, as explained further below.

It is reminded that the expressed markers are changed during the differentiation process. Thus, some markers associated with the fetal nature of the cell are expressed early in the differentiation process (i.e., rapidly after loss of pluripotency), while some are expressed late in the differentiation process (i.e., before adult cells mature). The lack of expression of these fetal markers indicates that the cells have lost their fetal properties and may acquire a phenotype indicating that they have matured into differentiated adult cells.

To determine that (a) the pluripotency gene is not expressed by cells of the population, gene expression and/or immunocytochemistry evaluation may be used. The aim is to demonstrate the lack or low expression of major genes typically expressed in undifferentiated pluripotent self-renewing cells (embryonic stem cells and induced pluripotent stem cells).

In particular, it is possible to:

a) using a population of iPS cells as a positive control for pluripotency markers, and

b) the expression levels of a panel of pluripotency genes in the target population and the iPS cell population are compared.

When the expression level of the pluripotency gene is less than 10%, more preferably less than 5%, of the expression level of these genes in the iPS cell population, or when less than 10%, more preferably less than 5%, of the cells express the gene, the cells of the target population are considered not to express the pluripotency gene. Any quantitative method may be used, such as RT PCR or flow cytometry, or immunological tissue labeling. FACS (fluorescence activated cell sorting) of the cells is preferably used. In this way, less than 10% of the cells of the population should express these pluripotency genes.

Pluripotent cells express multiple markers. Indeed, when a cell loses its pluripotency profile, it will also lose expression of these markers, since their expression is correlated. Thus, although multiple genes expressed by pluripotent cells (pluripotency genes) are known in the art, it is not necessary to study a large number of such genes.

In more detail, 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.

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

However, it is also possible to observe three, four or even five of these genes.

The percentage of cells in the population that express these markers can be readily determined by FACS methods using antibodies available in the art. It is even possible to perform this analysis in multiple experiments.

When multiple genes are studied, the percentage of cells in the population considered to be pluripotent is determined by taking the average of the percentage of cells with each marker.

As an 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%, then the population is considered to comprise 5.5% of pluripotent cells (average of 5% and 6%) and the given population will be considered to have passed condition (a) above.

In order to determine that the cells in the population express fetal genes and satisfy condition (b), it is necessary to detect the genes (markers, proteins or antigens) expressed by the cells when they enter one of the differentiation pathways.

Neural fetal cell:

early neuroectodermal progenitor cells: TP63, MASH1, Notch1, Sox1, Sox2, Musashi 2, Musashi 1, Nestin, Pax6, MUC18, BMI1, Mash1, FABP7, Nucleostemin

Hematopoietic fetal cells:

hematopoietic mesodermal progenitor cells: brachyury (T), MIXL1, cryptic, GATA1, LMO2, ACE, SCL (Tal1), HoxA9, Fli1

Renal fetal cell:

renal mesoderm progenitor cells: WT1, HOXD11, SIX2, SALL1, WT1, PAX2, OSR1, PAX8, LHX1, GATA3, HOXB7

Hepatic fetal cell:

hepatic endoderm progenitor cells: SOX17, HNF3B, HNF6, Fox-A2, HNF1B, GATA4, AFP, LGR5

Pancreatic fetal cells:

pancreatic endoderm progenitor cells: SOX17, Fox-A2, CXCR4, GATA4, HNF1B, HNF4A, PDX1, HNF6, PROX1, Ngn3, neuroD1, PAX6, SYP, SOX9, NKX2-2, NKX6-1, P48, LGR5, HB9

Intestinal fetal cells:

enterodermic progenitor cells: CDX2, TCF-2, SOX9, NMYC, ID2, SOX2, PAX8, Nkx2.1, LGR5

Lung fetal cells:

lung endoderm progenitor cells: CXCR4, SOX17, FOXA2, NKX2.1, PAX9, TBX1, SOX2 SOX9, ID2, Foxj1, Scgb1a1, Foxj1

Thyroid fetal cells:

thyroid endoderm progenitor cells: CXCR4, SOX17, FOXA2, Pax8, HHEX, Nkx2-1

Other foetal cells:

myoblast progenitor cell: pax7, Pax3, Myf5

Chondrocyte progenitor cell: osteonectin, Sox9

Osteoblast progenitor cells: runx2, ALP, Osx, osteopontin, osteocalcin.

The above genes are all known in the art and are specific for each differentiation pathway and each tissue organoid. The fetal genes in these early or late progenitor cells are not expressed in fully differentiated adult cells. Their sequences can be found in widely used public databases, as shown.

Thus, these markers are markers of early ontogeny and reflect the fact that cells bearing these markers are not fully adult mature cells. They are still progenitor cells at the fetal developmental stage, which means that they can still give rise to various types of mature cells.

In the context of the present invention, in order to obtain a population of foetal cells, the person skilled in the art should induce differentiation of pluripotent cells (e.g. embryonic foetal stem cells or iPS cells) in one of the differentiation pathways according to known methods.

As described above, loss of pluripotency will be verified by examining at least 90% of the cells for loss of expression of the markers as described above.

Depending on the differentiation pathway chosen by the person skilled in the art, the cell population may be examined for the presence of the specific foetal markers described above.

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

Even without prior knowledge of the cell differentiation pathway, it can be determined whether a cell population is a fetal cell population.

To examine whether a population of cells is a population of fetal cells according to the invention, it should first be observed whether the cells express one or more of the above-described pluripotency markers (and the percentage of cells in the population that express the markers). If less than 10% of the cells express the above markers, one skilled in the art can observe the expression of the fetal markers by the cell population.

The morphology/histology of the cells can provide information to those skilled in the art regarding the submission of cell lineages so that some markers can be selected for first examination. However, it is also possible to verify the fetal natural stage of cells without prior knowledge of the cell lineage commitment.

To this end, RNA from the population of cells can be extracted, reverse transcribed, optionally amplified, and applied to any DNA chip or array comprising probes for fetal markers as described above. In particular, Low Density Arrays (LDA) may be used. This allows not only the presence of fetal markers to be determined, but also qualifies these markers, i.e. the differentiation pathway of the cells of the population can be determined (depending on the probe being "turned on" by RNA from the cell population).

Once the differentiation pathway is known, FACS analysis can be performed using specific markers of the differentiation pathway of that particular cell lineage to quantify the percentage of cells in the population that express those markers.

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

The population of fetal cells as disclosed herein is for use in the prophylactic or therapeutic treatment of cancer in a subject. Indeed the development and progression of cancer may be caused or promoted by mutations in the cells of the subject which induce de-differentiation and cause them to resolve in the differentiation pathway to reach a new "fetal-like" characteristic and cause them to proliferate. Thus, such cells express fetal markers but not mature and fully differentiated adult cells. Furthermore, since these cells divide at a high rate, this induces mutations that produce mutated antigens, also referred to as neoantigens. It is indeed noted that the fetal or neoantigens of tumor cells are usually shared between cancers, at least between cancers of organs derived from the same differentiation pathway (ectoderm, endoderm or mesoderm).

From the ectodermal pathway, the organs are epidermal skin cells, neurons, glial cells, neural crest, pigmented cells.

From the mesodermal pathway, organs are cardiac muscle, skeletal muscle cells, kidney (renal tubules), red blood cells, smooth muscle (intestine).

From the endoderm pathway, reference can be made to lung cells (in particular alveoli), thyroid cells, pancreatic cells, liver cells.

Finally, the microenvironment of cancer cells is often beneficial to the immune system as it will inhibit the effects of T lymphocytes.

Administration of inactivated cells of these foetal cell populations, preferably together with HDACi or a compound that increases expression of an MHC-1 molecule, will induce an immune response against a foetal antigen present on the cells of the population in a subject (preferably a human, but which may be another mammal, such as a dog, cat, cow or horse), thus combating tumour cells, thereby causing regression of the cancer. It is effective against both solid and hematological tumors.

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

Thus, the fetal cell population (fetal population) can be used to prime the immune system of a patient to enable it to adequately and effectively fight cancer.

Given the different approaches, the fetal cell population can be used to treat lung, pancreatic, kidney, breast, hematological, gastrointestinal, thyroid, prostate, brain (especially glioblastoma), stomach, liver, bone, ovarian cancer. A population of fetal cells should be selected that are involved in the same cell differentiation lineage as the cancer to be treated.

The use of such a population of foetal cells makes it possible to deliver at least 10, more typically at least 50, or at least 100, 500 or even 1000 foetal or neoantigens expressed in a given cancer or common among different cancers.

The fetal cells may contain familial cancer-prone mutations that express the fetal genes deregulated by the mutation (BRCA, cMET, RET, APC, etc.) and are shared among lineage cancers, e.g., iPS cells obtained from blood cells containing the c-Met mutation may be derived to renal organoids containing the c-Met mutation present in kidney cancers.

The use of mutagens (see below) in the preparation of fetal cell compositions should introduce mutations (e.g., missense or frameshift mutations) in the genes of the cells of the population, thereby expressing the neoantigens.

In particular, the inventors have shown that iPS cells obtained from Chronic Myelogenous Leukemia (CML), mutated by ENU and derived from hematopoietic fetal cells comprise antigens present in Acute Myelogenous Leukemia (AML).

To treat the patient, one may:

i) obtaining antigen-specific characteristics of the cancer of the subject from a biopsy of such cancer,

ii) selecting a population of inactivated foetal cells comprising cells expressing at least one antigen determined in step i),

iii) administering the population to the patient with HDACI or an agent that increases MHC-I expression and a TLR3 agonist.

Step i) is performed by methods known in the art using tools available in the art.

Said features are obtained in particular by:

determining the genes expressed in cancer cells (exome sequencing),

comparing the genes with a database of cancer-specific genes (reference may be made, inter alia, to the COSMIC database (catalogue of cancer somatic mutations developed by Sanger research institute, UK) or to the cancer genome map (TCGA, maintained by NCBI, USA.) these databases recombine various sequences encoding antigens expressed in cancer cells,

-selecting genes present in both the exome and the database as antigen-specific characteristics of the cancer.

Step ii) is performed by performing exome of the fetal cell population and verifying the presence or absence of at least one gene of an antigen-specific characteristic of the cancer in the exome obtained from the fetal cell population.

In another embodiment, the method can

i) Obtaining antigen-specific characteristics of the cancer of the subject from a biopsy of such cancer,

ii) selecting a population of inactivated foetal cells comprising cells which normally express at least one antigen determined in step i),

iii) administering to the patient an extract of the population together with HDACI or an agent that increases MHC-I expression and a TLR3 agonist.

In this embodiment, the extract is selected from the group consisting of total RNA, mRNA, DNA, protein extracts, lysates, lyophilized extracts, lyophilized or desiccated cells, exosomes, extracellular microvesicles, and apoptotic bodies.

In another embodiment, the method can

i) Obtaining antigen-specific characteristics of the cancer of the subject from a biopsy of such cancer,

ii) selecting a population of inactivated foetal cells comprising cells which normally express at least one antigen determined in step i),

iii) administering to the patient a population of T cells or antigen presenting cells that have been primed in vitro with the population of ii) or an extract of that population and a TLR3 agonist in the presence of HDACI or an agent that increases MHC-I expression.

In a specific embodiment, the population is obtained by:

a. differentiating the population of pluripotent cells into a pathway associated with a particular cancer in the patient, wherein the pluripotent cells have optionally been expanded in the presence of a mutagen,

b. the cells thus differentiated are expanded and,

c. optionally exposing to a mutagen during amplification to induce mutagenesis of the gene in cells of the population,

d. verifying that at least 70% of the cells of the population express a fetal marker,

e. optionally verifying whether the cells of the population express at least one cancer or neoantigen present in the cancer cells of the subject,

f. inactivating said cells such that the cells lose their ability to divide.

The use of a population of foetal cells according to the invention is of particular interest. In fact, these cells contain a variety of fetal antigens that are readily expressed by cancer cells.

The invention also relates to methods of developing and producing cell populations intended for use in treating cancer in a patient.

The method comprises the following steps:

a) optionally, a biopsy of the cancer is performed,

b) analyzing cells recovered from a cancer biopsy of a patient to identify fetal markers and cancer markers expressed by the cancer cells,

c) differentiating the population of pluripotent cells by a pathway associated with a particular cancer in the patient (e.g., if the patient has renal cancer, differentiation of the renal pathway will be induced),

d) optionally introducing mutations in the differentiated cell population: this step is optional, but is preferably performed. It is intended to increase the diversity of antigens expressed by cells of a population, to improve the ability to control cancer cells upon exposure to the cellular immune system, even if there are mutations of their cells. The mutation rate can be controlled by examining the sequence of one or more genes of a population of cells. The presence of a mutated sequence of a given gene in a population can be identified and quantified, e.g., compared to the gene sequences in the population. For example, a mutation rate of 0.1% of a given gene indicates that 0.1% of the sequences identified for that gene in the population exhibit a mutation. The mutation rate of a given gene is calculated by sequencing the DNA of that gene and calculating the percentage of copies containing the mutation relative to the native sequence, which is the basic and predominant sequence (since the predominant sequence is a native "wild-type" sequence).

e) Optionally verifying that the cells of said population express at least one cancer or neoantigen present in the cancer cells of said subject,

f) inactivating said cells such that the cells lose their ability to divide. This is to avoid proliferation of cells in vivo after administration of all or part of the cell population to the patient.

Once the cell population is obtained, it can be administered to an animal (preferably a mammal, more preferably a human) in whole or in part in the presence of HDACi or a compound that stimulates MHC-I expression and a TLR3 agonist. As described above, in all methods, the inactivated fetal cell population or extract thereof, or T lymphocytes or antigen presenting cells primed with the population or extract thereof, may be administered.

In a specific embodiment, the pluripotent cells of step c) are iPS cells (induced pluripotent stem cells) that have developed from patient cells. This may reduce the risk of cross-immunity when administering fetal cells to a patient. In fact, non-fetal antigens should not be recognized by the immune system, while fetal antigens (present on both population cells and cancer cells) should be recognized.

Alternatively, the present invention relates to a method for treating a patient, comprising the steps of:

a) optionally, a cancer biopsy is performed, and,

b) analyzing cells recovered from a cancer biopsy of a patient to identify fetal and cancer markers expressed by the cancer cells,

c) selecting a population of inactivated and optionally mutagenized fetal cells involved in a differentiation pathway associated with a particular cancer of the patient,

d) administering to the patient cells, HDACi or a compound that stimulates or increases MHC-I expression, and a TLR3 agonist.

In particular embodiments, the fetal cells are involved in a lung differentiation pathway. Thus, they will express the above markers for the lung. These cells are particularly suitable for the treatment of lung cancer.

In particular embodiments, the fetal cells are involved in a thyroid differentiation pathway. Thus, they will express the above-described markers for thyroid. These cells are particularly suitable for the treatment of thyroid cancer.

In particular embodiments, the fetal cells are involved in a renal differentiation pathway. Therefore, they will express the above markers for kidney. These cells are particularly suitable for the treatment of kidney cancer.

In particular embodiments, the fetal cells are involved in hematopoietic differentiation pathways. Thus, they will express the above-described markers for hematopoietic cells. These cells are particularly suitable for the treatment of blood cancers (leukemia).

In particular embodiments, the fetal cells are involved in an intestinal differentiation pathway. Thus, they will express the above markers for the intestine. These cells are particularly suitable for the treatment of gastrointestinal cancer.

In particular embodiments, the fetal cells are involved in a pancreatic differentiation pathway. Therefore, they will express the above markers for pancreas. These cells are particularly suitable for treating pancreatic cancer.

In particular embodiments, the fetal cells are involved in neural differentiation pathways. Thus, they will express the above-described markers for neurons or brain. These cells are particularly suitable for the treatment of brain cancer (in particular glioblastoma).

In particular embodiments, the fetal cells are involved in a bone differentiation pathway. Thus, they will express the above markers for bone. These cells are particularly suitable for the treatment of bone cancer.

This ingredient is disclosed in WO 2019101956.

Pluripotent cell population

The pluripotent stem cells may be Embryonic Stem Cells (ESC) or Induced Pluripotent Stem Cells (iPSC), preferably of human origin. However, when the subject is from a species other than human, the pluripotent cells are from the subject's species.

As used herein, the term "human embryonic stem cell (hESC)" refers to an isolated cell from a pre-blastocyst stage embryo. In another embodiment, hES cells are prepared by de-differentiation of at least partially differentiated cells (e.g., pluripotent cells), and are totipotent in practice. Methods of making hescs are well known and are taught, for example, in U.S. Pat. nos. 5,843,780, 6,200,806, 7,029,913, 5,453,357, 5,690,926, 6,642,048, 6,800,480, 5,166,065, 6,090,622, 6,562,619, 6,921,632, and 5,914,268, U.S. published application No. 2005/0176707, and international application No. WO 2001085917. In the context of the present invention, human embryonic stem cells (hescs) are generated without destroying the embryo according to the technique described in Chung et al 2008.

As used herein, the term "human-induced pluripotent stem cells (hipscs)" refers to pluripotent stem cells that are artificially derived from non-pluripotent cells by reprogramming procedures using methods known in the art and by the methods originally disclosed by Yamanaka (in particular WO2012/060473, PCT/JP2006/324881, PCT/JP02/05350, US 9,499,797, US 9,637,732, US8,158,766, US8,129,187, US8,058,065, US8,278,104). Briefly, somatic cells were reprogrammed to induce 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 a specific embodiment, the induced pluripotent stem cells are derived from a mammal, particularly (but not limited to) rodents, pigs, cats, dogs, non-human primates, and humans.

Preparation of cell populations

In a specific embodiment, the cell membrane is retained (thereby antigen presentation via the MHC-I pathway). In a specific embodiment, the cells are inactivated as described above or below. In a specific embodiment, the cell is a human embryonic stem cell (hESC), a human induced pluripotent stem cell (hiPSC), a fetal stem cell, a cancer stem cell, a virus-infected cell, or a bacterial cell as described below. In another embodiment, the immunogenic component is a cellular composition comprising Antigen Presenting Cells (APCs) that have been primed (prime) in vitro by an antigen of interest. The composition is an antigen presenting cell vaccine, and is made from an antigen and Antigen Presenting Cells (APCs). Antigen presenting cells are cells that display an antigen complexed with a Major Histocompatibility Complex (MHC) on their surface. In the context of the present invention, reference may be made to Dendritic Cells (DCs) which are preferred because they are capable of presenting antigens to helper cells and cytotoxic T cells, macrophages or B cells. These APCs may be native cells or engineered cells. In particular, Eggermont et al (Trends in Biotechnology,2014,32,9,456-465) can be cited, which review the progress in the development of artificial antigen-presenting cells. Methods for developing anti-cancer vaccines using APC have been widely proposed in the art and are known to those skilled in the art.

In another embodiment, the immunogenic component consists of a composition of T cell lymphocytes primed in vitro against the antigen of interest (e.g., by exposure to antigen presenting cells presenting the antigen of interest). Thus, the composition is capable of generating an immune response in vivo against the antigen of interest. This strategy may be referred to as "adoptive transfer of T cells," and such adoptively transferred T cells are known to persist in vivo for long periods of time and to readily migrate between the lymphatic and vascular compartments (Bear et al, J Biomed Biotechnol.2011; 2011: 417403; Melief et al, J Clin invest.2015; 125(9): 3401-.

In some embodiments, HDACi is administered in combination with a vaccine composition containing an immunogenic component. The administration may be simultaneous, separate or sequential, as in the embodiments disclosed below, wherein the immunogenic component is a composition of a population of i) human embryonic stem cells (hescs), ii) human induced pluripotent stem cells (hipscs), or iii) fetal stem cells.

As mentioned above, the present specification highlights that HDAC inhibitors (in particular valproic acid) are combined with populations of i) human embryonic stem cells (hescs), ii) human induced pluripotent stem cells (hipscs) or iii) fetal stem cells, as these cells express antigens and neo-antigens that are also present in very aggressive cancers. Thus, regardless of the immunogenic component, as described above and below, it is preferred that the antigen of interest is an antigen expressed by a cancer cell or a neoantigen.

In a specific embodiment, the immunogenic component is a cell composition, wherein a population of i) human embryonic stem cells (hESC), ii) human induced pluripotent stem cells (hiPSC) or iii) a fetal stem cell composition has been obtained from the inactivation of pluripotent stem cells and fetal stem cells, as disclosed in further detail below.

In a particular embodiment, the immunogenic component is a cellular composition, wherein the cellular composition has been obtained by differentiating pluripotent stem cells (ESC and iPSC) or fetal stem cell compositions in vitro. More particularly, the immunogenic component is a population of i) a human embryonic stem cell (hESC) composition, ii) a human induced pluripotent stem cell (hiPSC) composition or iii) a fetal stem cell composition. Typically, methods for generating pluripotent or fetal stem cell populations are described in the following patent applications, respectively: WO2017/202949 and EP 17306635.8.

Typically, the method for generating hescs or hipscs comprises the steps of: i) expanding the pluripotent cells in the presence of an agent that induces MHC-I presentation of the antigen in the population during the inducing step under conditions that maintain pluripotency of the cells; ii) exposing the expanded cells to an inactivating agent which inactivates the cells, iii) recovering and conditioning the expanded inactivated cells.

Cell expansion is performed under conditions to maintain the pluripotency (medium, temperature) of the cells. Such culture conditions are known in the art. Maintaining the pluripotency of the cell will ensure that such cells will express (and thus present) all embryonic antigens, thereby increasing the ability of the cell to present such antigens on its surface via the MHC I pathway.

The more embryonic antigens that are presented on the surface of pluripotent cells, the higher the likelihood that at least one of these antigens will also be presented on the surface of cancer cells, which will then be recognized and targeted by the immune system that has been primed by the vaccine composition of the present invention.

Thus, maintenance of the pluripotency of the cells of the composition according to the invention obtained by the methods disclosed herein results in the presentation of a variety of embryonic antigens and thus in the general efficacy of the vaccine composition of the invention in the treatment methods disclosed herein.

It is known in the art to expand cells under conditions such as maintenance of pluripotency. In particular, this is described in all iPSC amplification protocols described so far (Shi Y and al, Nat Rev Drug Discovery 2017; Chen KG and al Cell Stem Cell. 2014). The following conditions are preferably used:

the population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), or fetal stem cells are mutagenized cells.

E8 medium or all clinical grade ESC/iPSC medium was used, and optionally supplemented with VPA and/or mutagens (such as ENU, see below).

The temperature is 37 ℃ (under the condition of oxygen deficiency or no oxygen deficiency)

-changing the medium daily using the same medium supplemented with VPA (from 0.1mM to 5mM) and/or ENU (0.1. mu.g/ml to 100. mu.g/ml) and/or a p53 inhibitor and/or a compound that enhances cell survival such as Y-27632Rock inhibitor.

Cells are usually cultured for 8 weeks, maintaining 90% of the optimal density by routine passaging once a week using an enzymatic method (collagenase, trypsin).

In a specific embodiment, the pluripotent cells and fetal stem cells used in the methods of treatment disclosed herein are inactivated. The term "inactivated" and grammatical variants thereof is used herein to refer to cells (e.g., pluripotent cells or fetal stem cells) that are viable but have been unable to proliferate (i.e., mitotically inactivated). One skilled in the art can use techniques known in the art, including but not limited to exposure to chemical agents, radiation, and/or lyophilization. The pluripotent cells and fetal stem cells may be inactivated such that the pluripotent cells are unable to divide when administered to a subject and therefore unable to form a teratoma in the subject. It will be appreciated that in the case of a plurality of cells, not every cell need be incapable of proliferation. Thus, as used herein, the phrase "inactivated to an extent sufficient to prevent teratoma formation in a subject" refers to the degree of inactivation in the entire population such that teratomas do not form after administration to the subject, as the irradiated pluripotent stem cells or fetal stem cells no longer divide, as demonstrated by in vitro culture. It should be noted that even if one or more of the plurality of cells are actually capable of proliferating in a subject, it may be assumed that the host's immune system will destroy those cells prior to the formation of a teratoma. This inability to proliferate and teratoma formation can be demonstrated by testing in mice with functional and non-functional immune systems.

In some embodiments, an "inactivated" cell is a killed cell, and in some embodiments, an inactivated cell is a whole cell lysate, a pluripotent stem cell or fetal stem cell-derived exosome, an enriched cancer stem cell neoantigen, a fully purified cancer stem cell neoantigen, a DNA RNA and protein extract, a lyophilized whole cell suspension, a cell lysate fraction (such as a membrane fraction, a cytoplasmic fraction), or a combination thereof.

When mice were vaccinated with hescs or hipscs in combination with valproic acid or other HDACi, the inactivated pluripotent or fetal stem cells were still able to stimulate the immune response. This vaccination was able to induce an effective immune and anti-tumor response against 4T1 breast cancer without signs of side effects and autoimmune disease.

Typically, to inactivate stem cells, they are exposed to a lethal dose of radiation (e.g., a single 5-100 Gy). The precise dose and length of dose delivered to the cells is not critical as long as the cells are rendered non-viable.

The recovery step of the method comprises one (or more) steps of washing the cell culture and resuspending the cells in any suitable medium, such as X-Vivo/Stemflex medium or any other clinical grade cell culture medium.

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

It is reminded that pluripotent cells or fetal stem cells are genetically very stable cells. Indeed, since they are present very early in the embryonic development process and they must be propagated for embryonic development, it is important that these cells are less prone to mutations in order to have homogeneity in the embryo. Thus, the cells present in a pluripotent or fetal stem cell population are typically very homogeneous (i.e., greater than 95% of the cells in the population have the same genetic background) when considered for their genetic content.

When iPSC or fetal stem cells were prepared, some selective advantage of the cells appeared during multiple passages, which resulted in clonal populations of ipscs exhibiting specific mutations but with sequences of the cell genome close to 100% in late passages.

However, ipscs are as stable as hescs over several passages (hussei SM and al, Nature 2011). Culture-induced (adaptive) mutations with few genetic changes will be obtained after long-term culture (hussei SM and al, Bioessays, 2012).

However, it would be advantageous to be able to induce mutations in cells to increase the variability of fetal/embryonic neoantigens on treated cellular material found in aggressive cancers. In this way, the likelihood that the immune system will produce T cells against these mutant cells and be able to fight against cancer cells as well as those that will undergo subsequent variation during tumor growth will be increased.

This will help fight against cancers caused by the accumulation of genetic variation resulting from DNA replication errors and/or environmental damage during cancer stem cell proliferation. These mutations include cancer-driving mutations and genome-destabilizing mutations that cause carcinogenesis. This increased genomic instability leads to clonal evolution, which in turn leads to the selection of more aggressive clones with increased resistance.

Thus, in a particular embodiment, the cells are expanded under conditions that will induce genetic mutation of said cells.

Thus, cells can be exposed to mutagens, i.e., physical or chemical agents that alter the genetic material (usually DNA) of an organism, thereby increasing the frequency of mutations above the natural background level.

The mutagen can be selected from physical mutagens and chemical mutagens.

Among the physical mutagens, mention may be made of:

ionizing radiation, such as X-rays, gamma rays and alpha particles, which can cause DNA fragmentation and other damage. Mention may in particular be made of the radiation from cobalt-60 and cesium-137. The level of radiation should be much lower than that used for cell inactivation and can be designed by one skilled in the art;

uv radiation with wavelengths above 260nm, which, if not corrected, may lead to copying errors;

or radioactive decay, such as 14C in DNA.

Among chemical mutagens, mention may be made of:

reactive Oxygen Species (ROS), such as superoxide, hydroxyl radical, hydrogen peroxide;

deaminating agents such as nitrous acid, which can cause conversion mutations by converting cytosine to uracil;

-Polycyclic Aromatic Hydrocarbons (PAHs) that bind to DNA when activated as diol-epoxides;

alkylating agents, such as ethyl nitrosourea (ENU, CAS No. 759-73-9), mustard gas or vinyl chloride;

aromatic amines and amides, such as 2-acetamidofluorene;

alkaloids from plants, such as alkaloids from the species vinca rosea;

bromine and some bromine-containing compounds;

-sodium azide;

-bleomycin;

-psoralen in combination with ultraviolet radiation;

-benzene;

-base analogues that can substitute DNA bases during replication and cause transition mutations;

-intercalating agents, such as ethidium bromide, proflavine, daunorubicin;

metals, such as arsenic, cadmium, chromium, nickel and compounds thereof which may be mutagenic.

The inventors have shown that culture conditions can be designed such that DNA replication errors can be induced in pluripotent stem cells or fetal stem cells without triggering DNA damage-dependent apoptosis.

This is particularly unexpected because, as mentioned above, pluripotent cells or fetal stem cells are naturally very stable and therefore should introduce as few mutations as possible early in embryogenesis. It follows that DNA repair mechanisms are very effective in these cells, correcting most defects and/or inducing apoptosis in cases where it is not possible to correct them.

In one embodiment, a starting population of pluripotent cells (such as ESCs or IPSCs) is expanded and maintained in a medium that allows for pluripotency (as known in the art) to preserve the pluripotency phase during iterative passaging. In these cases, a small number of exome mutations (5-10 mutations per exome) are typically observed.

The pluripotent or fetal cells are then cultured in vitro using mutagenic compounds to induce and increase genomic instability in pluripotent stem cells or fetal cells (such as those listed above), for example. As a marker for Double Strand Breaks (DSBs), γ H2AX phosphorylation confirmed DNA damage. Both the proportion of γ H2AX positive cells and the frequency of γ H2AX foci in ESC or IPSC increased, and the number of micronuclei as markers of genomic instability was higher.

In a particular embodiment, the agent is bleomycin, ENU, an alkylating agent, actinomycin D, ROS modulator, UV, H2O2, ionizing radiation (gamma rays, X rays), all of which allow to induce and enhance the mutation rate of pluripotent stem cells or foetal cells accumulated during culture.

In a specific embodiment, N-ethyl-N-nitrosourea (ENU) has been shown to produce new mutations and enhance neoantigen levels in treated pluripotent or fetal stem cells during long term culture at doses <50 μ g/ml for at least 7-60 days. These mutations are similar to those reported in cancer.

In a particular embodiment, the method of the invention, wherein the mutagen is selected from the group consisting of: chemical mutagens and radiation mutagens (X-rays, UV radiation), wherein the mutagens are selected from the group consisting of: ENU, reactive oxygen species, deaminating agent, polycyclic aromatic hydrocarbon, aromatic amine, and sodium azide. In this embodiment, a population of pluripotent or fetal stem cells will be obtained, wherein the cells have random mutations (often cell-specific, resulting in a heterogeneous population), particularly in cancer-associated neoantigens.

Thus, it is possible to accumulate multiple mutations during pluripotent or fetal cell proliferation in response to DNA damage at high mutation rates from a selective advantage in long term culture, while maintaining the pluripotency of the cells, particularly when the cells are cultured with HDACi in a culture medium. The presence of HDACi in culture can maintain increased epigenetic signatures of active histones (H3K4me3 and H3K9ac) and pluripotency in response to induced DNA damage and replication and proliferation rates during passaging.

In another embodiment of the compositions and methods described herein, mutations are induced in pluripotent or fetal stem cells by genetically modifying the cells with genes that promote high levels of genomic instability. In particular, appropriate inhibitors such as NER/BER/DSBR/MMR inhibitors may be used to delete or reduce the activity of genes or signaling pathways involved in DNA repair and replication. These methods of inducing genomic instability associated with increased DNA damage can be performed by using "vectors" or "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 are DNApkC, Ku70, Rad51, Brca1, or Brca 2.

In other embodiments, the pluripotent or fetal stem cell is modified to inhibit apoptosis-related genes such as p53 by genetic modification or chemistry of p53 (e.g., Pifithrin-mu, Nutlin-3, nestin), or by using compounds that enhance cell survival such as Y-27632, a selective inhibitor of p160-Rho associated frizzled kinase (ROCK).

In a specific embodiment, the population of pluripotent cells consists of induced pluripotent stem cells (ipscs) produced from somatic cells (e.g., cells isolated from a patient that has contained genomic alterations associated with:

i) DNA repair disorders including, for example, ataxia telangiectasia, bloom syndrome, Cockayne syndrome, fanconi anemia, Werner syndrome, xeroderma pigmentosum, nemenre's breakdown syndrome;

ii) hereditary family cancer syndromes with genomic instability, such as lingeri syndrome (hereditary nonpolyposis colorectal cancer with mutations in the MMR gene (including MLH1, MSH2, MSH6, PMS1 and PMS2), Li-Fraumeni with mutations in the TP53 gene or CHEK2, Hereditary Breast and Ovarian Cancer (HBOC) syndrome with deletions or mutations in the BRC1/2 gene, Familial Adenomatous Polyposis (FAP) with mutations in the APC gene;

iii) somatic cells that induce genomic instability, such as translocation in CML (T9; 22).

In a specific embodiment, the population of mutated pluripotent cells consists of induced pluripotent stem cells and is produced by somatic cells containing genomic alterations associated with a disease. Typically, the genomic alteration may be a translocation (T9:22), a deletion (BRCA1/2) or a mutation (BRCA, RET).

In a specific embodiment, the population of pluripotent stem cells consists of ipscs generated from cancer cell lines or patient-specific cancer cells.

In another embodiment, the ESC, IPSC or fetal stem cell population is genetically modified by use of a "vector" to overexpress a plurality of non-random cancer stem associated neoantigens. In particular embodiments, the ESC, IPSC, or fetal stem cell population is genetically modified by "genome editing" techniques to express multiple mutations and cancer stem cell specific neoantigens (at least 5) in pluripotent stem cells or fetal stem cells. The present invention provides compositions and methods for providing ESCs, IPSCs or fetal stem cells by introducing multiple neoantigens thereof through RNA-guided multiple genome editing, modification, expression suppression and other RNA-based techniques.

The term "genome editing" as used herein refers to RNA-mediated genetic manipulation and specifically includes guide RNAs for cas 9-mediated genome editing. The guide rna (grna) was transfected with endonuclease cas 9. 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 by using the criprpr-Cas 9 system according to standard methods in the art. Compositions and methods of making and using a criprpr-Cas system are known in the art and are described, inter alia, in u.s.8,697,359.

In a specific embodiment, a population of pluripotent fetal stem 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 the neoantigen, providing excellent immune responses by increasing oligoclonal expansion of TILs and Th1/Th2 cellular immunity.

In the present invention, the alkylating agent is selected from the following: nitrogen mustards, nitrosoureas, alkyl sulfonates, triazines, ethyleneimines, and combinations thereof. Non-limiting examples of nitrogen mustards include nitrogen mustard hydrochloride (Lundbeck), chlorambucil (GlaxoSmithKline), cyclophosphamide (Mead Johnson Co.), bendamustine (Astellas), ifosfamide (Baxter International), melphalan (Ligand), melphalan fluoropiperazine (Oncopeptides), and pharmaceutically acceptable salts thereof. Non-limiting examples of nitrosoureas include streptozocin (Teva), carmustine (Eisai), lomustine (Sanofi), and pharmaceutically acceptable salts thereof. Non-limiting examples of alkyl sulfonates include busulfan (Jazz Pharmaceuticals) and pharmaceutically acceptable salts thereof. Non-limiting examples of triazines include dacarbazine (Bayer), temozolomide (Cancer Research Technology), and pharmaceutically acceptable salts thereof. Non-limiting examples of ethyleneimine include thiotepa (Bedford Laboratories), altretamine (MGI Pharma), and pharmaceutically acceptable salts thereof. Other alkylating agents include ProLindac (Access), Ac-225 BC-8(Actinium Pharmaceuticals), ALF-2111(Alfact Innovation), Trifosfamide (Baxter International), MDX-1203(Bristol-Myers Squibb), Thiourobutyronitrile (CellCeutix), dibromonitrobenzene (Chinoin), dibromodulcitol (Chinoin), nimustine (Daiichi Sankyo), glufosfamide (Eleison Pharmaceuticals), HuMax-TAC and PBD ADC combinations (Genmab), BP-C1(Meabco), Suxian (Messac), nifurtimox (Methonomx), Thioflavist (Mitsubishi tan), ramustine (Mitsubishi), Pharmata-01 (Metapur), Mariotriene P (Mitsubishi), Natraline (Mitsubishi), Nartisin (Na 6332), Norfectine (Na-P), Nartisin (Na-P), Nartin-P (S-P) (SG5), Nartin-P (S), Nartin-S (S-P) (Nartin) combinations (S6332), and Nartin (S) and (Nartin (S) P, Fotemustine (Servier), nedaplatin (Shionogi), heptaplatin (Sk Holdings), apigenin (spectra Pharmaceuticals), SG-2000(Spirogen), TLK-58747(Telik), lamotrigine (Vion Pharmaceuticals), procarbazine (Alkem Laboratories Ltd.) and pharmaceutically acceptable salts thereof. In another embodiment, the alkylating agent is selected from the group consisting of nitrogen mustard hydrochloride (Lundbeck), chlorambucil (GlaxoSmithKline), cyclophosphamide (Mead Johnson Co.), streptozocin (Teva), dacarbazine (Bayer), thiotepa (Bedford Laboratories), hexamethylmelamine (MGI Pharma), pharmaceutically acceptable salts thereof, and combinations thereof. In another embodiment, the alkylating agent is selected from ProLindac (Access), Ac-225 BC-8(Actinium Pharmaceuticals), ALF-2111(Alfact Innovation), bendamustine (Astelans), ifosfamide (Baxter International), Trifosfamide (Baxter International), MDX-1203(Bristol-Myers Squibb), temozolomide (Cancer Research Technology), thionuronitrile (CellCeutix), dibromonitrobenzene (Chinoin), dibromodulcitol (Chinoin), nimustine (Daiichi Sankyo), carmustine (Eisai), phosphoramide (Eleisen Pharmaceuticals), HuMax-TAC and PBD ADC (combined), leucoderma (Jazzz Pharmaceu BP (American BP), Melissubne-D (Melothiofur C-1), Melothioxane (Melothioxane), Melanolide (Melothiofur Bione), Melanolide (Melanolide), Melanolide (C-1), Melanolide (Melanolide), Melanolide (C-D ADC (Melanolide), Melanolide (C-891), Melanolide (Melanolide), Melanolide (C-891 (Melanolide), Melanolide (Melanolide), 22P1G cells in combination with ifosfamide (Nuvilex), melphalan fluoropiperazine (Oncopeptides), estramustine phosphate (Pfizer), prednimustine (Pfizer), lurkinetin (Pharmamar), trabectedin (Pharmamar), altretamine (Sanofi), lomustine (Sanofi), SGN-CD33A (Seattle Genetics), fotemustine (Service), nedaplatin (Shionogi), heptoplatin (Sk Holding), apigenin (spectra Pharmaceuticals), SG-2000(Spirogen), TLK-58747(Telik), lamustine (Vion Pharmaceuticals), carbohydrazide (Alkem Laboratories Ltd.), pharmaceutically acceptable salts thereof, and combinations thereof.

In a particular embodiment, a population of pluripotent cells or fetal stem cells is treated with N-ethyl N-nitrosourea (ENU, CAS number 759-73-9). ENU has the following chemical formula C₃H₇N₃O₂It is a highly efficient mutagen by transferring an ethyl group to a nucleobase in a nucleic acid.

As mentioned above, the purpose of the mutagen is to introduce random mutations in the genes of pluripotent or fetal cells during amplification (mutations introduced during replication and division of the cells). Populations of pluripotent stem cells or fetal stem cells acquire mutations that may provide growth advantages and are selected to promote culture adaptation. Passage of ESC, IPSC or fetal stem cells is subject to high levels of selection pressure and, upon expansion, populations of multiple clonal mutations can be advantageously selected.

It is noted that since pluripotent cells are very stable, administration of the mutagen may have to be performed for a long time. Illustratively, when ENU is used, it may be administered 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, more preferably at least 50 days or even at least 60 days. After administration of the mutagen, the cells are washed (if the mutagen is a chemical agent) and may be further amplified in the presence of an agent that favors MHC-I expression (especially HDACI). The agent is preferably also present during the administration of the mutagen.

Thus, it can be observed and examined that mutagens will induce mutations (i.e., non-synonymous, nonsense, frameshift, StopGain, splice variants, CNV, SNV) in certain embryogenic genes expressed by pluripotent cells, thereby increasing the diversity of these antigens (new neoantigens throughout the genome). This would therefore increase the likelihood that the vaccine composition will have enhanced immunogenicity, being able to stimulate a broad immune response against aggressive cancers where there are rapid and frequent mutations.

It may indeed be difficult to obtain an effective immune response for certain cancers in which clonal expansion of cancer cells occurs with mutation of an antigen expressed by the tumor cells. Thus, the immune response will depend on the mutational load of the cancer. Thus, the generation of random mutations in pluripotent cell populations or fetal stem cells through the use of mutagens will result in the expression of mutated embryonic antigens and increase the diversity of antigens presented to the immune system after vaccination.

Thus, there have been primed T cells against mutant antigens that will appear in cancer cells during their division, which will accelerate and improve the immune response against these cells.

In a specific embodiment, the pluripotent stem cell or fetal stem cell population is genetically modified to overexpress a compound that stimulates an immune response by gene integration within the genome of the pluripotent cell or fetal stem cell. Typically, in a first step, a population of pluripotent stem cells or fetal stem cells is isolated and expanded. In a second step, the gene of interest is packaged into an integrating viral vector (e.g., a retrovirus or lentivirus). In the third step, the integrated viral vector containing the target gene is transferred into a pluripotent stem cell population and differentiated into fetal stem cells.

In a specific embodiment, the pluripotent cell or fetal stem cell population is genetically modified with a protein that stimulates MHC expression and/or an immune response. These compounds are selected from the group consisting of interferon alpha (IFN-alpha), interferon gamma (IFN-gamma), interleukin 2(IL-2), interleukin 4(IL-4), interleukin 6(IL-6), interleukin 12(IL-12), Tumor Necrosis Factor (TNF), and granulocyte-macrophage colony stimulating factor (GM-CSF), functional fragments thereof, and combinations thereof.

Interferons (IFNs) contemplated by the present invention include the common IFN types, IFN- α, IFN- β, and IFN- γ. IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior, and/or increasing their antigen production, thereby making cancer cells more readily recognized and destroyed by the immune system. IFNs may also act indirectly on cancer cells, for example, by slowing angiogenesis, enhancing the immune system, and/or stimulating Natural Killer (NK) cells, T cells, and macrophages. Recombinant IFN- α is commercially available as Roferon (Roche pharmaceuticals) and Intron A (Schering Corporation).

Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include(IL-2; Chiron Corporation) and(IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc (Seattle, Wash) is currently testing recombinant forms of IL-21, which are also contemplated for use in the combinations of the present invention.

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 sargrastim), and erythropoietin (alfa-ibetin, dabigatran). Treatment with one or more growth factors may help stimulate the production of new blood cells in subjects undergoing traditional chemotherapy. Thus, treatment with CSF may help reduce side effects associated with chemotherapy and may allow the use of higher doses of chemotherapeutic agents. Various recombinant colony stimulating factors are commercially available, such as Neupogen (G-CSF; Amgen), Neulasta (pelfilistim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), and Arnesp (erythropoietin).

As used herein, the term "adjuvant" refers to any compound capable of enhancing an immune response against an immunogenic antigen and capable of increasing recruitment of antigen presenting cells. Such adjuvants are capable of activating APCs and enhancing antigen presentation, inducing expression of inflammatory cytokines and pro-inflammatory environment, priming natural T cells to drive acquired immunity, modulating adaptive immune response, acting as immunopotentiators by exhibiting immunostimulatory effects during antigen presentation and by inducing expression of co-stimulatory molecules on APCs, improving the quality of downstream T helper cytokine profiles and differentiation of antigen-specific T helper cell populations, thereby generating needles from B cellsAntibodies specific for these antigens. Typically, the adjuvant is selected from, but not limited to, the following group: i) agonists of toll-like receptors (TLRs): TLR1, TLR2, TLR3, TLR4 (e.g. GLA, MPLA), TLR5, TLR6, TLR7, TLR8(egVTX-2337), TLR9 (egIC-31; IM-2125, SD-101, ODN2395, ODN 1826), TLR10 TLR11, C-type lectin-like receptor; retinoic acid inducible gene-like receptors, nucleotide-binding oligodomain-like receptors, proteins containing a nucleotide-binding domain and a leucine-rich repeat (LRR) (NLR), RIG-I-like receptors (RLR); ii) GM-CSF, IL2, IFN alpha-2 a, IFN alpha-2 b; iii) aluminum salts, aluminum hydroxide (alum), IFA,QS21 (e.g. QS21STIMULON), QS21 saponin, QS21 isoergosin, MPL + AlumLiposomes, trehalose-6, 6', dibehenate (TDB), Complete Freund's Adjuvant (CFA), Lipopolysaccharide (LPS), Pam3CysSerLys4(Pam3CSK 4). In a particular embodiment, the adjuvant is a TLR3 agonist. As used herein, the term "TLR 3" refers to Toll-like receptor 3. TLR3 agonists are well known to those skilled in the art. It refers to an affinity agent (i.e., a molecule that binds to a target molecule) that is capable of activating a TLR3 polypeptide to induce all or part of a receptor-mediated response. For example, TLR3 agonists directly or indirectly induce TLR3 dimerization/oligomerization and trigger TLR 3-mediated signaling. A TLR3 agonist as used herein may, but need not, bind to a TLR3 polypeptide and may or may not interact directly with a TLR3 polypeptide. They include double-stranded ribonucleic acids (dsRNA), such as: poly (A: U) for Poly (A) -Poly (uridylic acid), Poly (I: C) for Poly (inosine-polycytidylic acid), Poly (ICLC)PolyI：PolyC12UOr RGIC dsRNA, such as RGIC100.1

For example, such TLR3 agonists are described in patent US8409813 (particularly in the ninth to twenty-second columns), patent EP2281043, patent application WO2015/091578 and patent application WO 2008/109083. In particular, RGIC100.1 is described in examples 1 and 2 of WO 2015/091578.

In a specific embodiment, the TLR3 agonist is poly (a: U).

As used herein, the term "administering" refers to the act of injecting or otherwise physically delivering a substance (e.g., a combined preparation) present outside the body to a subject, e.g., by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art. When a disease or symptom thereof is being treated, the substance is typically administered after the onset of the disease or symptom thereof. When preventing a disease or symptoms thereof, the substance is generally administered prior to the onset of the disease or symptoms thereof. The term "simultaneous administration" as used herein refers to the administration of 2 active ingredients by the same route and simultaneously or substantially simultaneously. The term "separately administering" means that the 2 active ingredients are administered simultaneously or substantially simultaneously by different routes. The term "sequential administration" means that the 2 active ingredients are administered at different times, the routes of administration being the same or different.

In a specific embodiment, the vaccine composition (pluripotent stem cells or fetal stem cells + agent stimulating MHC presentation + adjuvant) is injected subcutaneously. The injections may be performed simultaneously, sequentially, separately, at the same injection point or at different injection points, in the same syringe or in separate syringes.

In a particular embodiment, the follow-up treatment (administration of a compound that stimulates MHC I and/or the immune system, e.g. HDACi, in particular VPA) is administered by the oral route.

By "therapeutically effective amount" is meant the minimum amount of active agent necessary to confer a therapeutic benefit to a subject. For example, a "therapeutically effective amount" for a subject is an amount that induces, alleviates, or causes amelioration of a pathological symptom, disease progression, or physiological condition associated with a disorder or resistance to a disease. It will be appreciated that the total daily amount of a compound of the 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 upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular compound used; the particular composition used, the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the particular compound employed; the duration of the treatment; drugs used in combination or coincidental with the particular compound employed; and factors well known in the medical arts. For example, it is known to those skilled in the art to start doses of the compounds at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved. However, the daily dosage of the product may vary within a wide range of 0.01-1,000mg per adult per day. Typically, the compositions contain 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 500mg of the active ingredient for symptomatic adjustment of the dose to the subject to be treated. The medicament will generally contain from about 0.01mg to about 500mg of active ingredient, preferably from 1mg to about 100mg of active ingredient. The effective amount of the drug is typically provided at a dosage level of from 0.0002mg/kg to about 20mg/kg body weight per day, particularly from about 0.001mg/kg to 7mg/kg body weight per day.

Vaccine compositions include an immunogenic component intended to generate an immune response in a subject against one or more antigens of interest. The antigen of interest is any antigen for which an immune response is desired, including any peptide, protein, e.g., bacterial, viral or parasitic protein, from self (e.g., antigens from cancer cells) or foreign, other kinds of antigens, e.g., nucleic acids, sugars, lipopolysaccharides, and the like.

The methods described herein also include the step of administering HDACi several days after administration of the vaccine composition. Such continuous administration of HDACi can be used to maintain the change in microenvironment long enough to allow immune cells to "take over" the tumor. Typically, such further continuous administration of HDACi consists in administering a sufficient dose of HDACi daily for at least three days, and up to one month, after administration of the vaccine. However, it is preferred that HDACi is further administered for at least one week, more preferably at least or about two weeks.

The method according to the invention, wherein the treatment is a prophylactic treatment.

Pharmaceutical and vaccine compositions

The population of pluripotent stem cells or fetal stem cells as described above may be used in a vaccine composition. Thus, in another aspect, the invention relates to a vaccine composition comprising i) a population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells, ii) an agent that induces MHC-1 presentation of an antigen (e.g. HDACi) and iii) an adjuvant.

In particular, such population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells is inactivated and optionally mutated to inhibit their proliferative capacity and optionally a cell extract is obtained.

In a specific embodiment, the vaccine composition according to the invention, wherein the population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells is mutagenized cells.

In a particular embodiment, the agent that stimulates an immune response is HDACi (used at a dose of 0.2mM-4 mM). In another embodiment, an adjuvant in the context of the present invention is a TLR3 agonist.

In a particular embodiment, the HDACi is VPA.

In a specific embodiment, the TLR3 agonist is Poly (a: U).

The invention also relates to devices (such as syringes) containing the vaccine compositions, which can be used to administer the HDACi compound, adjuvant and cell composition simultaneously.

In a specific embodiment, the vaccine composition of the invention is used to treat a subject suffering from cancer.

Such vaccine compositions may be used as therapeutic vaccines against cancer cells (cancer cells expressing immunogenic neo-antigens, driver or passenger mutations; progenitor cells as epigenetic de-differentiated cells, tumor initiating cells expressing fetal and embryonic genes) to cure patients, or as prophylactic vaccines to prevent such cancers from occurring, especially in patients predisposed to such cancers. For example, a Susceptibility gene is (see also Lindor et al,2008Journal of the National Cancer Institute monograms, No.38, convention Handbook of family Cancer Suminor Syndromes, Second Edition):

breast/ovary: BRCA1, BRCA2, PALB2, RAD 51;

the forest syndrome: MLH1, MSH2, MSH6, PMS2, EPCAM;

hereditary papillary renal cell carcinoma: FH. MET;

and (3) Kaimen disease: PTEN, PIK3 CA;

fanconi disease: FANC;

von Hippel-Lindau disease: VHL;

malignant melanoma: CDKN2A, MITF, BAP1, CDK 4;

endocrine tumors: MEN1, RET, CDKN 1B;

neurofibromatosis: NF1, NF2, LZTR1, SMARCB1, SPRED 1;

hereditary pheochromocytoma paraganglioma: SDH, TMEM127, MAX, EPAS 1;

familial adenomatous polyposis: APC, MUTYH;

retinoblastoma: RB 1;

birt-hogg-dube syndrome: FLCN;

bloom syndrome: BLM;

carrey syndrome: PRKAR 1A;

gorlin syndrome: PTCH 1;

Li-Fraumeni syndrome: TP53, CHEK 2;

nemehne syndrome: NBN;

Peutz-Jeghers syndrome: STK 11;

familial juvenile polyposis: BMPR1A, SMAD 4;

xeroderma pigmentosum is XP.

This list is non-limiting.

In certain embodiments, the cancer stem cell vaccine product comprises a mixture of lyophilized cell lysates from human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells, enriched multi-cancer stem neoantigens, purified cancer stem neoantigens, exosomes derived from fetal stem cells, DNA, RNA, proteins or a mixture of multiple polypeptides. These are immunogenic agents as described above, which are formulated in the presence of HDACi.

In another embodiment, the cancer stem cell vaccine product is mixed with GMP medium, supernatant from engineered irradiated populations of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hipscs), or fetal stem cells, which are used as adjuvant effectors.

In a specific embodiment, the derived population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells fetal cells in the composition are inactivated (i.e., unable to repopulate).

The compositions of the derived populations of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells of the invention are readily obtained by any of the methods described above.

It should be noted that the derived population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells in the composition is genetically heterogeneous, carrying specific somatic mutations when mutagens are used, and thus is different from the derived population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cell compositions that are produced according to methods known in the art and are genetically more homogeneous.

In particular embodiments, both HDACi and DNA methyltransferase inhibitors are used simultaneously. Indeed, it has been shown that the combined use of VPA and 5-azacytidine (an analogue of nucleoside cytosine, which can be incorporated into DNA and RNA) results in a synergistic effect of the re-expression of novel anti-embryonic antigens.

HDACi is administered in a therapeutically effective amount. For VPA, it may be 10-15 mg/kg/day, up to 60 mg/kg/day. Plasma levels of VPA should preferably be within the generally accepted therapeutic range (50-100. mu.g/mL).

In a specific embodiment, the method according to the invention further comprises one or more of radiotherapy, targeted therapy, immunotherapy or chemotherapy. Generally, 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 an immune response, as a combined preparation, as well as radiation therapy, targeted therapy, immunotherapy or chemotherapy.

In some embodiments, a subject is administered i) a population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells, ii) a compound selected from the group that activates MHC expression and/or an immune response, and iii) an adjuvant, and a chemotherapeutic agent, as a combined preparation.

The term "chemotherapeutic agent" refers to a chemical compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, metodepa, and uredepa; ethyleneimine and methylmelamine, including hexamethylmelamine, tritamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethamine; polyacetyl (especially bullatacin and bullatacin ketone); camptothecin (including the synthetic analog topotecan); bryostatins; a colistin; CC-1065 (including its aldorexin, kazelaixin, and bizelaixin synthetic analogs); nostoc cyclopeptides (specifically nostoc cyclopeptide 1 and nostoc cyclopeptide 8); dolastatin; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM 1); (ii) soft coral alcohol; (ii) coprinus atramentarius alkali; alcohol of coral tree; spongistatin; nitrogen mustards, such as chlorambucil, mechlorethamine, ifosfamide, mechlorethamine oxide hydrochloride, melphalan, neomustard, cholesteryl-p-phenylacetic acid mechlorethamine (phenesterine), prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorouramicin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin 11 and calicheamicin 211, see, e.g., Agnew Chem Intl.Ed. Engl.33: 183-) (1994)); daptomycin, including daptomycin a; an esperamicin; and novel oncostatin chromophores and related chromoprotein enediyne antibioticsChromophore), aclacinomycin, actinomycin, anthranomycin, azaserine, bleomycin, actinomycin C, carrubicin, carminomycin, pheochromocin, tryptomycin, dactinomycin, daunomycin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin, isorubicin, idarubicin, sisomicin, mitomycin, mycophenolic acid, nogomycin, olivomycin, pelomycin, epirubicin, puromycin, quinomycin, roxobicin, streptonigromycin, streptozotocin, tubercidin, ubenimex, sethoxystatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dinotefuran, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiaguanine, thioguanine; pyrimidine analogs, such as, for example, Cytidine, azacitidine, 6-azauridine, carmofur, Cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, fluorouridine, 5-FU; androgens such as karitesterone, drotandrosterone propionate, epitioandrostanol, meindrotane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trostane; folic acid replenisher such as folinic acid; acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; amsacrine; besubbs; a bisantrene group; edatrexae; desphosphamide; colchicine; diazaquinone; (ii) nilotinib; ammonium etiolate; an epothilone; ethydine; gallium nitrate; a hydroxyurea; (ii) mushroom polysaccharides; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanol; diamine nitracridine; gustatostatin; methionine; pirarubicin; podophyllinic acid; 2-acethydrazide; (ii) procarbazine;lezoxan; rhizomycin); a texaphyrin; a germanium spiroamine; (ii) zonecanoic acid; a tri-imine quinone; 2,2', 2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, myxomycin A, bacillocin A and serpentine); uratan; changchun wineDigoxin; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; adding the star of tussingo; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (A)Bristol-Myers SquibbOncology, Princeton, N.J.) and docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine (navelbine); norfloxacin (novantrone); (ii) teniposide; daunorubicin; aminopterin; (xiloda); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included within this definition are anti-hormonal agents, such as anti-estrogenic agents, for modulating or inhibiting the effects of hormones on tumors, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, 4-hydroxytamoxifene, trovaxifene, raloxifene hydrochloride (keoxifene, LY117018, onapristone, and toremifene (Fareston), and anti-androgenic agents, such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

In some embodiments, a subject is administered i) a population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells as a combined preparation, ii) a compound selected from the group that activates MHC expression and/or an immune response, and iii) an adjuvant, and targeted cancer therapies.

Targeted cancer therapies are drugs or other substances that block cancer growth and spread by interfering with specific molecules ("molecular targets") involved in cancer growth, progression, and spread. Targeted cancer therapies are sometimes referred to as "molecular targeted drugs," "molecular targeted therapies," "precision drugs," or similar names. In some embodiments, the targeted therapy consists in administering to the subject a tyrosine kinase inhibitor. The term "tyrosine kinase inhibitor" refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and are described in U.S. patent publication 2007/0254295, which is incorporated by reference herein in its entirety. It will be appreciated by those skilled in the art that compounds related to tyrosine kinase inhibitors will reproduce the effect of tyrosine kinase inhibitors, e.g. the related compounds will act on different members of the tyrosine kinase signalling pathway to produce the same effect as tyrosine kinase inhibitors of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in the methods of the present embodiments include, but are not limited to: dasatinib (BMS-354825), PP2, BEZ235, saratinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW 572016; GW2016), cantinib (CI 1033), semazenil (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; 6474), bevacizumab (avastin), MK-2206(8- [ 4-aminocyclobutyl) phenyl ] -9-phenyl-1, 2, 4-triazolo [3,4-f ] [1,6] naphthyridin-3 (2H) -one hydrochloride) derivatives, analogs thereof, and combinations thereof. Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are described, for example, in U.S. patent publication 2007/0254295, U.S. patent nos. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated by reference herein in their entirety. In certain embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been administered orally 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, most preferably approved by the FDA for at least one hematologic or oncology indication. Examples of such inhibitors include, but are not limited to, gefitinib, erlotinib, lapatinib, canertinib, BMS-599626(AC-480), lenatinib, KRN-633, CEP-11981, imatinib, nilotinib, dasatinib, AZM-475271, CP-724714, TAK-165, sunitinib, vatalanib, CP-547632, vandetanib, bosutinib, lestatinib, tandutinib, midostaurin, Enzastaurin (Enzastaurin), AEE-788, pazopanib, axitinib, motasinib, OSI-930, cedanib, KRN-951, dolisib, Sericicliib, SNS-032, PD-0332991, MKC-1 (Ro-317453; R-440), sorafenib, ABT-869, brib (BMS-582664), SU-14813, lapatinib-6668, SU-3568, OSI-668, and others, (TSU-68), L-21649, MLN-8054, AEW-541 and PD-0325901.

In some embodiments, a subject is administered i) a population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells as a combined agent, ii) a compound selected from the group that activates MHC expression and/or an immune response, and iii) an adjuvant (particularly a TLR3 agonist), and an immune checkpoint inhibitor.

As used herein, the term "immune checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with or modulates, in whole or in part, one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD 86; and PD1 and its ligands PDL1 and PDL2(Pardol, Nature Reviews Cancer 12: 252-264,2012). These proteins are responsible for either 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: anti-CTLA 4 antibodies (e.g., ipilimumab), anti-PD 1 antibodies (e.g., nivolumab, pembrolizumab), anti-PDL 1 antibodies, anti-TIM 3 antibodies, anti-LAG 3 antibodies, anti-B7H 3 antibodies, anti-B7H 4 antibodies, anti-BTLA antibodies, and anti-B7H 6 antibodies. Examples of anti-CTLA-4 antibodies are described in U.S. patent nos. 5,811,097, 5811097, 5855887, 6051227, 6207157, 6,682,736, 6,984,720, and 7,605,238. An anti-CTLA-4 antibody is tremelimumab (tremelimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also referred to as 10D1, MDX-D010), which is a fully human monoclonal IgG antibody that binds 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, 7943743, 8008449, 8168757, 8,217,149 and PCT published patent application nos: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400 and WO 2011161699. In some embodiments, the PD-1 blocking agent comprises an anti-PD-L1 antibody. In certain other embodiments, PD-1 blockers include anti-PD-1 antibodies and similar binding proteins, such as nivolumab (MDX 1106, BMS 936558, ONO 4538), which is a fully human IgG4 antibody that binds to and blocks PD-1 activation by its ligands PD-L1 and PD-L2; langlizumab (MK-3475 or SCH 900475), humanized monoclonal IgG4 antibody against PD-1; CT-011, a humanized antibody that binds PD-1; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559(MDX-1105-01) for PD-L1(B7-H1) blockade. Other immune checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al, 2007, J.Immunol.179: 4202-4211). Other immune checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271(Loo et al, 2012, clin. Also included are TIM3(T cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al, 2010, j.exp.med.207:2175-86 and Sakuishi et al, 2010, j.exp.med.207: 2187-94). In some embodiments, the immunotherapeutic treatment consists of Adoptive immunotherapy, as described by Nicholas p.restifo, Mark e.dudley and Steven a.rosenberg ("adaptive immunization for cancer: harnessing the T cell response, Nature Reviews Immunology, Volume 12, April 2012). In adoptive immunotherapy, circulating lymphocytes or tumor-infiltrating lymphocytes of a patient are isolated in vitro, activated by lymphokines such as IL-2 and re-administered (Rosenberg et al, 1988; 1989). Activated lymphocytes are most preferably patient's own cells that have been isolated from a blood sample at an early stage and activated (or "expanded") in vitro. This embodiment is particularly preferred because HDACi can increase the expression of PDL1 on the surface of cancer cells.

In some embodiments, a subject is administered i) a population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells, ii) a compound selected from the group that activates MHC expression and/or an immune response, and iii) an adjuvant (particularly a TLR3 agonist), and a radiotherapeutic agent, as a combined preparation.

As used herein, the term "radiotherapeutic agent" means, without limitation, any radiotherapeutic agent known to those of skill in the art to be effective in treating or ameliorating cancer. For example, the radiotherapeutic agent may be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods may optionally further comprise administering one or more additional cancer therapies, such as, but not limited to, chemotherapy and/or another radiation therapy.

In another embodiment, a compound that activates MHC expression and/or an immune response, as described above; the adjuvant and population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells can be combined with a pharmaceutically acceptable excipient and optionally a sustained release matrix, such as a biodegradable polymer, to form a pharmaceutical composition.

The present invention further comprises: i) a population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells; ii) a compound selected from the group activating MHC expression and/or an immune response, and iii) an adjuvant.

The pharmaceutical composition of the invention is useful in therapy.

The pharmaceutical composition of the invention is used for treating cancer.

The pharmaceutical composition of the invention, wherein the compound selected from the group activating MHC expression and/or immune response is HDACi.

The pharmaceutical composition of the invention, wherein the adjuvant is a TLR3 agonist.

In a specific embodiment, the vaccine composition according to the invention comprises: i) a population of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hipSCs) or fetal stem cells, ii) VPA and iii) Poly (A: U).

In a specific embodiment, the pharmaceutical composition according to the invention comprises: i) a population of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hipSCs) or fetal stem cells, ii) VPA and iii) Poly (A: U).

"pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce deleterious, allergic, or other untoward effects when properly administered to a mammal, particularly a human. Pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids of any type. The pharmaceutical compositions of the present invention are intended for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, and the active ingredient (alone or in combination with another active ingredient) may be administered in unit administration form as a mixture with conventional pharmaceutical supports to animals and humans. Suitable unit administration forms include oral route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical composition contains a pharmaceutically acceptable carrier for the formulation to be injected. These may be in particular isotonic sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures of these salts), or dry, in particular freeze-dried compositions which, when added in sterile or physiological saline (depending on the case), allow the constitution of injectable solutions. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations include sesame oil, peanut oil, or propylene glycol aqueous solutions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injection is possible. It must be stable under the conditions of preparation and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Solutions comprising the compounds of the present invention as the free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding it) may be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids (e.g., hydrochloric or phosphoric acids) or organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. A sterile injectable solution is prepared by: the desired amount of active polypeptide is added to an appropriate solvent containing several other ingredients as listed above (as required), followed by filter sterilization. Typically, the dispersion is prepared by: the various sterilized active ingredients are added to the sterile vehicle which contains the base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Once formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as the types of injectable solutions described above, but drug-releasing capsules and the like may also be used. For example, for parenteral administration in aqueous solution, the solution should be suitably buffered if necessary, and the liquid diluent first 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 in accordance with the present disclosure are known to those skilled in the art. For example, one dose can be dissolved in 1ml of isotonic NaCl solution and added to 1000ml of subcutaneous infusion or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.

More particularly, a population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells and a compound that activates MHC expression and/or an immune response are formulated into a vaccine composition. Accordingly, the present invention relates to a vaccine composition comprising: i) a population of human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC) or fetal stem cells, ii) a compound selected from the group activating MHC expression and/or an immune response and iii) an adjuvant.

In a specific embodiment, the vaccine composition according to the invention comprises: i) a population of human embryonic stem cells (hescs), human induced pluripotent stem cells (hipscs), or fetal stem cells; ii) valproic acid and iii) a TLR3 agonist.

In a specific embodiment, the vaccine composition according to the invention comprises: i) expressing a neoantigen, in particular a population of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hipSCs) or fetal stem cells enhanced by a mutagen or genetic modification, ii) valproic acid and iii) Poly (A: U).

The composition may also comprise 5 azacytidine.

Furthermore, as described above, the vaccine composition of the present invention may be used in a subject suffering from cancer.

The vaccine composition according to the invention can be formulated in the same way as the immunogenic composition, together with the physiological excipients indicated above. For example, pharmaceutically acceptable carriers include, but are not limited to, phosphate buffered saline solutions, distilled water, emulsions such as oil/water emulsions, sterile solutions of various types of wetting agents, and the like. Adjuvants such as muramyl peptides such as MDP, IL-12, aluminum phosphate, aluminum hydroxide, alum and/or montanide (r) may be used in the vaccine.

The vaccine composition according to the invention may be administered by subcutaneous (s.c), intradermal (i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, oral administration and intranasal administration or inhalation. The vaccine administered is typically a single dose. Alternatively, the administration of the vaccine of the invention is a first (primary vaccination) followed by 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 85, 86, 87, 85, 84, 87, 85, or more of the same population of stem cells, compounds that stimulate the immune system, or a combination thereof and/or further radiotherapy, or a further chemotherapy, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 boosts (recals) (subsequent administration).

Vaccine compositions are also provided in kits. The kit comprises a vaccine composition according to the invention and a leaflet providing instructions for immunization. The kit also includes all materials used to administer the product.

In summary, the inventors demonstrate that TLR-3 agonists can be used to enhance the anti-cancer activity of vaccines comprising antigenic compositions administered together with HDAC inhibitors (and possibly checkpoint inhibitors as well).

The antigenic composition comprises inactivated cells (pluripotent or foetal cells, which have preferably been inactivated after mutagenesis). Using these types of cells, either ubiquitous vaccines (when using pluripotent cells) or more specific vaccines (when using fetal cells involved in the differentiation pathway of the cancer to be treated) can be produced, expressing the neofetal antigens present on the surface of the cancer cells. The presence of HDAC inhibitors (and their ability to increase MHC-1 expression) and TLR-3 agonists enhances cytotoxic T cell responses. This response may also be improved by the use of checkpoint inhibitors which may further increase the response, in particular as HDAC inhibitors which may increase the expression of PDL-1 (programmed death ligand 1 or CD274), thereby limiting the immune response. However, as shown in the examples, despite this effect of HDACi, the combination disclosed herein is able to control the proliferation of cancer even in the absence of checkpoint inhibitors.

The invention will be further illustrated by the following figures and examples. These examples and drawings, however, should not be construed as limiting the scope of the invention in any way.

Drawings

FIG. 1: hESC with valproic acid (VPA), Poly (A: U), ODN2395 orAdjuvant in combination with vaccination studies on a 4T1 mouse model. hESC with valproic acid (VPA), Poly (A: U), ODN2395 orAdjuvant in combination with vaccination studies on 4T1 mouse model. Blab/c mice were boosted twice with vaccines containing 3 different adjuvants on the day of tumor challenge. Tumor growth was assessed up to 26 days using calipers allowing measurement of longitudinal (a, mm) and transverse (b, mm) diameters to follow tumor size. Tumor volumes were calculated and plotted using the calculation formula 1/6 Π (a + b/2)3mm 3.

FIG. 2: valproic acid treatment increases MHC I expression in breast cancer cells. Changes in MHC I expression during VPA treatment at 2mM on 4T1 adherent cells and 4T1 derived mammospheres.

FIG. 3: HDACi upregulates MHC1 in lung cancer cell lines. The effect of VPA, levetiracetam, vorinostat and entinostat on the immunogenicity of LLC 1. MHCI and MHCII expression were quantified by flow cytometry analysis measuring Relative Fluorescence Intensity (RFI). LLC1 was treated with valproic acid (A), entinostat (B), levetiracetam (C) and vorinostat (D) at doses corresponding to IC50 for 48 hours.

Detailed description of the preferred embodiments

Example 1

The experiment was carried out as follows: with 3 different adjuvants (500 μ g TLR3, 50 μ g TLR9 agonist or 50 μ g/ml)Saponin vaccine adjuvant) mixed 2x10⁶Five mice per group received two booster vaccinations, 7 days and 14 days, from each irradiated hESC cell. After 14 days, 5X10⁴Individual 4T1 cells were injected into the mammary fat pad of mice and valproic acid was added to the drinking water at a dose of 4 mg/ml. Five untreated mice were used as controls.

Adjuvants used in vaccine products: three different adjuvants were tested:

1/TLR 3-based adjuvant: poly (A: U) (ref # tlrl-pau, InvivoGen) Poly A-polyuridylic acid (Poly (A: U)) is a synthetic double stranded RNA molecule that can be specifically signaled through TLR 3. Poly (A: U) is known to induce dendritic cell and T lymphocyte activation to promote antigen-specific Th1 immune responses and to boost antibody production. Poly (A: U) has been developed for potent adjuvant activity and is approved for the treatment of TLR3 expressing breast cancer (convention R. et al, 2010. manipulating effects of toll-like receptor (TLR3) signaling in tumor of therapeutic approach to inhibition of the anticancer effect of TLR3 ligands. cancer Res.70(2): 490-500).

2/TLR 9-based adjuvant: ODN2395 VacciGrade^TMCpG ODN, type C (ref # vac-2395-1.InvivoGen) ODN2395 is a type C CpG ODN consisting of synthetic Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN) that are predominantly present in bacterial DNA. CpG ODN is recognized by murine TLR9, which is expressed only on human B cells and dendritic cells, thereby inducing a Th 1-predominant immune response. ODN2395, which specifically activates mouse TLR9, is a strong IFN- α inducer from dendritic cells and a strong B cell activator.

Saponin vaccine adjuvant (ref # vac-quil InvivoGen).The adjuvant is a saponin adjuvant comprising a saponin from a water extractable fraction of Quillaja saponaria Molina of the south american tree. Saponins induce strong adjuvant responses to T-dependent and T-dependent antigens and induce a strong cytotoxic CD8+ lymphocyte response. In combination with cholesterol and phospholipids to form an immunostimulatory complex,adjuvants can activate both cell-mediated and antibody-mediated immune responses against a variety of tumor antigens.

Results

We found that this is in contrast to unvaccinated mice, as opposed to using a TLR9 agonist orSaponin vaccine adjuvant mice vaccinated with hESC in combination with TLR3 agonist gave the greatest reduction in breast tumor volume (p)<0.001) (fig. 1).

Table 1 shows the reduction rate of tumor growth, where there was a statistical difference in mean tumor size.

Table 1: percentage of tumor reduction at day 26 and statistical test (Student test) compared to control group receiving PBS only.

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

Example 2: ability of compounds to increase MHC I

The ability of a compound to increase MHC I on a tumor cell can be assessed (and is therefore suitable in the context of the present method).

Valproic acid increases MHC I on breast cancer cells

4T1 cells were a triple negative breast cancer cell line incubated with increasing doses (0.2 and 2mM) of valproic acid (VPA). After 4 days of treatment, MHC I surface markers were quantified by flow cytometry analysis. VPA has the property of increasing MHC I levels in a dose-dependent manner, which highlights that VPA can enhance anti-tumor immune responses by improving T cell tumor recognition. Furthermore, the Relative Fluorescence Intensity (RFI) measured by flow cytometry analysis showed that MHC I expression increased 2.1-fold and 2.7-fold in 4T1 and mammosphere-derived 4T1 cells, respectively, when treated with 2mM VPA (fig. 2). These results indicate that VPA treatment can effectively enhance MHC I expression at baseline and in "stem cell-like" situations.

Furthermore, we also observed a 2.1-fold increase in PDL1 expression when 4T1 cells were treated with 2mM VPA, whereas PDL2 expression was only weakly upregulated after VDL treatment (fig. 2).

HDAC inhibitors increase MHC1 expression in lung tumor cells

Non-small cell lung cancer (NSCLC) cell lines (lewis lung cancer, LLC) expressing a particularly aggressive metastatic phenotype were used. Four HDAC inhibitor molecules were evaluated using flow cytometry analysis: entinostat, levetiracetam, vorinostatAnd valproic acid (VPA)Expression of Major Histocompatibility Complex (MHC) types I and II.

The half maximal inhibitory concentration (IC50) of each HDACi was first determined using the MTT cell proliferation assay. LLC1 was treated with increasing doses of HDCAi and proliferation was assessed by measuring absorbance of cells after incorporation of MMT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) substrate. Vorinostat, entinostat and levetiracetam have an IC50 (level) of 2-4 μ M and valproic acid of about 2 mM.

For all HDACi tested, LLC1 treated with 4 HDACi at a dose corresponding to IC50 showed altered MHC class I expression. Vorinostat, levetiracetam, entinostat and VPA have the property of significantly increasing MHC1 expression on the cell membrane by a factor of 2, in contrast to MHC type 2 expression which is not regulated with dose, compared to untreated cells (figure 3). VPA proved to overexpress MHC 110-fold compared to untreated LLC1 cells (fig. 3A).

It should also be noted that some HDACi increased PDL-1 on the cell surface (data not shown).

Example 3:treatment of 4T1 cells with valproic acid (VPA) induced up-regulation of the immune response

To determine the effect of VPA on 4T1 cells, transcriptome analysis was performed on cells treated with 0.5mM VPA for 10 days and compared to cells not treated with VPA. These analyses allow the identification of genomes associated with TNF- α signaling and IFN- α and IFN- γ responses. Furthermore, the use of the SAM algorithm allowed for the differentiation of 44 immune-related genes between VPA-treated and control samples and was verified by principal component analysis (p-value 3.3x 10)^-4). Among the 44 immune-related genes, CD74, CCL2, and TNFRSF9 were found to be overexpressed with fold changes greater than 2. These three molecules are known to contribute to clonal expansion, survival and development of T cells, and to modulate CD28 costimulation to promote Th1 cell responses. Thus, the HDACi improves the immune response against cancer cells.