Cancer vaccines based on induced pluripotent stem cells

Abstract

In one embodiment, the present application discloses a mammalian autologous or allogeneic vaccine comprising an effective amount of mammalian induced pluripotent stem cells (iPSCs) obtained by reprogramming of somatic cells from a patient, the mammalian autologous or allogeneic vaccine expressing a gene selected from the group consisting of ASTE1, BIRC5, CDCA1, CDKN2A, DEPDC1, EGER, ERBB2, FOXM1, GPC3, HJURP, HSPA8, HSP90B1, IDH1, IDO1, IGF2BP3, IMP3, KIF20A, KIF20B, MEEK, MGAT5, NUF2, PMEL, RAS, TAF1B, TOMM34, TTK, TP53, VEGFR1 and VEGFR2, and inducing an immune response in the patient for the treatment of cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Patent Application No. 63 / 195,609, filed June 1, 2021, and U.S. Provisional Patent Application No. 63 / 233,141, filed August 13, 2021. [Background technology]

[0002] Yamanaka and colleagues (reviewed in Yamanaka S. et al. Cell 126:663-76, 2006; Shi Y. et al. Nat Rev Drug Discov. 16:115-130, 2017) have developed methods to revert mature cells to an embryonic state ("reprogramming"). Such induced pluripotent stem cells (iPSCs) can be generated by introducing four transcription factors into mature somatic cells, which transform the transcriptional and epigenetic state to a pluripotent state that closely resembles embryonic stem cells (ESCs).

[0003] Kooreman et al. (US Patent Application Publication No. 2019 / 0290697, incorporated herein by reference in its entirety) observed many similarities in gene expression between iPSCs and cancer cells and developed a vaccine using iPSCs to prevent the growth of many cancers. These authors determined that the best efficacy seen with this vaccine was to obtain cells from an individual, reprogram such cells into iPSCs, and use the individual's iPSCs as a vaccine for the individual along with an adjuvant ("autologous" vaccination). These researchers also identified a particular type of adjuvant composed of oligodeoxynucleotides based on short synthetic unmethylated CpG motifs ("CpG") that was most effective in these models.

[0004] In particular, the patent publication discloses a method for the treatment of cancer in a patient, comprising vaccinating a patient with a vaccine, the vaccine comprising an effective amount of mammalian pluripotent stem cells obtained by reprogramming somatic cells from the patient, the vaccine further comprising an adjuvant that is an immunological preparation that boosts the immune response to the vaccine, and the vaccination comprises administering the mammalian pluripotent stem cells to a patient in need thereof. In one embodiment of this method, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). In another embodiment of this method, the mammalian pluripotent stem cells are undifferentiated pluripotent stem cells. In another embodiment of this method, the pluripotent stem cells are generated using a mini-intron plasmid that includes four reprogramming factors, including Oct4, c-Myc, KLF-4 and Sox2, with the possibility of shRNAp53 addition. In another embodiment, the stem cells obtained by reprogramming somatic cells are selected from the group consisting of fibroblasts, keratinocytes, peripheral blood cells and kidney epithelial cells. In another embodiment, the vaccine is administered according to at least one of the following methods: a) as a stand-alone vaccination, b) as an adjuvant therapy before tumor resection, c) as an adjuvant therapy after tumor resection, d) in a metastatic setting, e) in a prophylactic setting in the absence of tumor or cancer, and f) in combination with chemotherapy, immunotherapy, targeted therapy using biologics, small molecule drugs, nanoparticles containing biologics or small molecule drugs, or combinations thereof. In another embodiment of this method, the cancer is selected from the group consisting of breast cancer, melanoma, and mesothelioma. In another aspect of this method, the cancer is selected from the group consisting of leukemia, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoma, myeloproliferative disorder, squamous cell carcinoma, adenocarcinoma, sarcoma, neuroendocrine carcinoma, bladder cancer, skin cancer, brain and spinal cord cancer, head and neck cancer, thyroid, bone cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastrointestinal cancer, (hypopharynx) cancer, esophageal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, eye cancer, renal cell carcinoma, kidney, liver cancer, ovarian cancer, gastric cancer, testicular cancer, thyroid and thymic cancer.

[0005] In another embodiment, a patent publication discloses a method for vaccination of a mammal with a pluripotent stem cell cancer vaccine, the method comprising: introducing mammalian pluripotent stem cells obtained by reprogramming somatic cells from a recipient, the vaccine further comprising an adjuvant, an immunological preparation that boosts the immune response to the vaccine, and providing the vaccine to the recipient. In one aspect of the method, the mammalian cells are undifferentiated pluripotent cells. In another aspect, the pluripotent stem cells are generated using a mini-intron plasmid that includes four reprogramming factors, including Oct4, c-Myc, KLF-4, and Sox2. In yet another aspect, the pluripotent stem cells are obtained by reprogramming somatic cells selected from the group consisting of fibroblasts, keratinocytes, peripheral blood cells, and kidney epithelial cells. In yet another aspect, the vaccine is irradiated prior to vaccination.

[0006] Another embodiment of the patent publication provides a thermostable vaccine composition comprising an effective amount of mammalian pluripotent stem cells obtained by reprogramming somatic cells from a mammal, and an adjuvant or immunological preparation that boosts the immune response to the vaccine. In one aspect of the vaccine composition, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). In another aspect, the mammalian pluripotent stem cells are undifferentiated pluripotent stem cells. In yet another aspect, the stem cells obtained by reprogramming somatic cells are obtained by reprogramming somatic cells selected from the group consisting of fibroblasts, keratinocytes, peripheral blood cells, and kidney epithelial cells. In further embodiments, the adjuvant is selected from the group consisting of CpG, QS21, poly(di(carboxylatophenoxy)phosphazene; derivatives of lipopolysaccharide, e.g., monophosphoryl lipid A, muramyl dipeptide (MDP; Ribi), threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174; cholera toxin (CT), and Leishmania elongation factor. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have found that there is a need to develop iPSCs that can present additional antigens to enhance individual responses to cancer.There is also a need to develop a readily available formulation that can use iPSCs from an individual to treat genetically unrelated individuals ("allogeneic vaccination"), as well as to identify other adjuvants and targeting strategies that can generate stronger or more effective immune responses.There is also a need to develop standards for judging the effectiveness of iPSC cancer vaccines during their production ("release criteria") and after administration to individuals ("biomarkers"). [Means for solving the problem]

[0008] In one embodiment, the present application provides compositions and methods for the production of cancer vaccines that prophylactically or therapeutically target multiple types of cancer. In one aspect, the vaccine comprises an adjuvant and iPSCs or mini-intron plasmid-generated iPSCs (MIP-iPSCs). In one variation, the adjuvant is mixed with the iPSCs. The adjuvant can be loaded onto the iPSCs by incubating the cells with the adjuvant. In one variation, the adjuvant is genetically encoded in the iPSCs.

[0009] In one embodiment, the cancer vaccine is autologous (or self-generated). As used herein, autologous or autologous vaccine refers to a vaccine that can be prepared by reprogramming cells from an individual and used to confer immunity to the same individual.

[0010] In one embodiment, the cancer vaccine is a readily available allogeneic formulation.

[0011] In one embodiment, the method and vaccination treatment plan for autologous and allogeneic cancer vaccine production are provided.The inventors have surprisingly found that in one particular aspect, allogeneic iPSC vaccine is not as effective as autologous vaccine, and have developed a method to improve the effectiveness of both autologous and allogeneic vaccination.

[0012] In one aspect, the cancer vaccine is genetically modified to improve the effectiveness of antigen presentation.

[0013] In another embodiment of the composition and method, the autologous or allogeneic vaccine is genetically modified to express an antibody or an antibody fragment. The antibody may be a monoclonal antibody, a humanized antibody, a chimeric antibody, a single chain antibody, an antibody fragment, or a combination thereof. The antibody may be secreted by the iPS cell, or they may be expressed as a transmembrane protein on the cell surface. The antibody may be reactive to, for example, a cancer antigen, a cytokine, a growth factor or its receptor, or a protein expressed on the surface of a T cell. The term "antibody" may be used to define any endogenous cell surface protein capable of binding to a ligand, including, but not limited to, immunoglobulins composed of heavy and light chains, single variable fragments (scFv) that are fusion proteins of immunoglobulin heavy (VH) and light chain (VL) variable regions, camelid single domain antibodies, nanobodies, or other variations known in the art.

[0014] In one variation, the iPS cells are genetically modified to express anti-CD28 and anti-CD80 antibodies on the cell surface.

[0015] In one variation, the autologous or allogeneic vaccine is genetically modified to express a cell surface ligand selected from the list consisting of ICAM1, LFA-1, LFA-3, CD80, CD81, CD28, ICOS, 4-1BB, anti-DEC-205 antibody, anti-CLEC9A (DNGR) antibody, anti-DCIR-2 antibody, anti-DECTIN antibody, anti-ASGPR antibody, anti-mannose receptor antibody and anti-CLEC12 (DCAL-2) antibody. In another variation, the autologous or allogeneic vaccine is genetically modified to express connexin 43. In another variation, the autologous or allogeneic vaccine is genetically modified to secrete a protein selected from the group consisting of XCR1, CCL3, CCL4, CCL5, CCL20, CCL25 and FLT3L or a combination thereof. In one variation, the allogeneic vaccine is genetically modified to express CCL3 and FLT3L. In another variation, the iPSCs are genetically modified to secrete a protein selected from the group consisting of GM-CSF, INF alpha, INF beta, IL-2, IL-12, IL-15 and IL-21, or a combination thereof. In another variation, the allogeneic vaccine expresses IL-15 on the cell surface. In another variation, the iPSCs are genetically modified to express a protein selected from the group consisting of gp96, hsp90, hsp70, CD91, calreticulin and LOX-1. In a particular variation, the iPSCs are genetically modified to express hsp70. In another variation, the autologous or allogeneic vaccine is genetically modified to express a cell surface protein selected from the group consisting of B7, OX40, CD28, CD40L, TLR4, CD70, MHC class I, MHC class II and OX40L. In another variation, the vaccine is modified to express OX40.

[0016] In one embodiment, the autologous or allogeneic vaccine is genetically modified to include an inhibitory RNA. In one variation, the inhibitory RNA is selected from the group consisting of antisense RNA, siRNA, shRNA, miRNA, lncRNA, pri-miRNA, antisense oligonucleotide, and pre-miRNA. In one variation, the inhibitory RNA inhibits expression of a gene selected from the group consisting of MHC class I, MHC class II, beta 2 microglobulin, and LAMP. In another variation, the inhibitory RNA inhibits beta 2 microglobulin. In one variation, the inhibitory RNA inhibits expression of a gene selected from the group consisting of PD-1, PDL-1, PDL-2, Nodal, cytokine signaling 1 (SOCS1), IL-10, IL-10R, TGF-β, and TGF-β. In one variation, the inhibitory RNA inhibits expression of TGF-β. In one variation, the inhibitory RNA is expressed at a concentration high enough to inhibit expression of one or more of such genes in antigen-presenting cells that take up the iPSC vaccine. Those skilled in the art will understand that methods other than inhibitory RNA, such as those involving CRISPR, can be used to inhibit the expression of specific genes in autologous or allogeneic vaccines.See Li, HL et al. Methods 101:27-35, 2015; Terns MP Mol. Cell 72:404-412.

[0017] In another aspect, autologous or allogeneic vaccine is administered in combination with small molecule drug.In one embodiment, this drug is selected from the group consisting of HDAC inhibitor, bromodomain inhibitor, Vps34 kinase inhibitor, PRMT5 inhibitor, autophagy inhibitor, angiogenesis inhibitor, vascular disruptor, STING pathway activator and Toll receptor pathway activator.In another variant, the small molecule drug is STING activator and Toll receptor pathway activator.

[0018] In one variation, the HDAC inhibitor is selected from the group consisting of valproic acid, gibinostat, belinostat, entinostat, mocetinostat, practinostat, chidamide, xinostat, abexinostat, vorinostat, romidepsin, panobinostat, and belinostat. In another variation, the HDAC inhibitor is vorinostat.

[0019] In one variation, the BET inhibitor is selected from the group consisting of I-BET151 (GSK1210151A), I-BET762 (GSK525762), OTX-015, TEN-010, CPI-203 and CPI-0610. In one variation, the BET inhibitor is CPI-0610. In another variation, the Vps34 kinase inhibitor is selected from the group consisting of SB02024 and SAR405. In yet another variation, the PRMT5 inhibitor is GSK3326595.

[0020] In one variation, the autophagy inhibitor is selected from the group consisting of 3-methyladenine, bafilomycin A1, chloroquine, hydroxychloroquine, N-2--(1H-benzimidazol-6-yl)-N-4--(5-cyclobutyl-1H-pyrazol-3-yl)quinazoline-2,4-diamine, MRT68921, MRT67307, SBI-0206965, ULK100, ULK101, and SB02024. In another variation, the autophagy inhibitor is SB02024.

[0021] In one variation, the angiogenesis inhibitor is selected from the group consisting of axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and ramucirumab. In another variation, the angiogenesis inhibitor is sorafenib.

[0022] In one variation, the vascular disrupting agent is selected from the group consisting of combretastatin, AVE8062, ZD6126, ABT-571, MN-029, CKD516, OXi8006, 5,6-dimethylxanthenone 4-acetic acid, combretastatin A-4 phosphate, ZD6126, Oxi4503, DMXAA, and the dolatastatin derivative TZT-1027. In another variation, the vascular disrupting agent is DMXAA.

[0023] In one variation, the STING activator is selected from the group consisting of ADU-S100, MK-1454, MK-2118, BMS-986301, SR-717, GSK3745417, SB-11285, IMSA-101, c-di-GMP, c-di-AMP and cGAMP. In another variation, the STING activator is SR-717.

[0024] In one variation, the Toll pathway activator is selected from the group consisting of diacyl lipopeptide, triacyl lipopeptide, flagellin, poly I:C, hexa-acetylated lipid A, monophosphoryl lipid A, gardikimod, imiquimod, and R848. In another variation, the Toll pathway activator is imiquimod.

[0025] In another variation, the autologous or allogeneic vaccine is co-administered with an antibody selected from the group consisting of anti-CD47, rituximab, cetuximab, daratumumab, trastuzumab, trastuzumab emtansine, pertuzumab, panitumumab, ramucirumab, necitumumab, and blinatumomab. In another variation, the autologous or allogeneic vaccine is co-administered with blinatumomab. In another variation, the autologous or allogeneic vaccine is co-administered with a small molecule drug selected from the group consisting of ibrutinib, acalabrutinib, and galuniseritib. In another variation, the autologous or allogeneic vaccine is co-administered with ibrutinib.

[0026] In another aspect, pluripotent stem cells are induced to undergo a specific type of cell death.The inventors have surprisingly discovered that pluripotent stem cells do not need to be viable at the time of injection to induce the desired anti-cancer immunity.In one embodiment, iPSCs that are killed by in vitro administration of oxaliplatin or doxorubicin and induced to express calreticulin, then mixed with adjuvant, are particularly potent.

[0027] In one variation, the cell death inducer is glutamic acid, sorafenib, ML162, FIN56, FINO2, erastin, sulfazine, RSL3, Ki8751, SGX-523, AZD7762, KW-2449, NVP-TAE684, AZD4547, TG-101348, bleomycin, axitinib, cytochalasin B, dasatinib. , SNX-2112, semagacestat, CHIR-99021, B02, olaparib, silmitasertib, tanespimycin, nintedanib, ML031, canertinib, SMER-3, BCL-LZH-4, SN-38, tamatinib, ML334 diastereomers, analogs, salts or derivatives thereof. In another variation, the cell death inducer is bleomycin.

[0028] The formulation can be administered to a patient after freezing and thawing without loss of activity. In one variation, the iPSCs are mixed with an adjuvant and then frozen. In one variation, the adjuvant is a saponin formulation, a virosome, a virus-like particle, a non-toxic derivative of enterobacterial lipopolysaccharide (LPS), an immunostimulatory oligonucleotide (e.g., an immunostimulatory oligonucleotide containing a CpG motif), a mineral-containing composition, an oil-based emulsion, a polymer, a micelle-forming adjuvant (e.g., liposome), an immunostimulatory complex matrix (e.g., ISCOMATRIX), a particle, squalene, phosphate, a cationic liposome-DNA complex (CL DC), DDA, DNA adjuvants, gamma-insulin, ADP-ribosylating toxins, detoxified derivatives of ADP-ribosylating toxins, Freund's complete adjuvant, Freund's incomplete adjuvant, muramyl dipeptide, monophosphoryl lipid A (MPL), poly IC, CpG oligodeoxynucleotide (ODN), imiquimod, QS21, AS101, adjuvant system AS0, adjuvant system AS02, adjuvant system AS03, MF59®, polydi(carboxylatophenoxy)phosphazene; derivatives of lipopolysaccharides, such as monophosphoryl lipid A, muramyl dipeptide (MDP; Ribi), threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174; cholera toxin (CT), and Leishmania elongation factor, and aluminum or aluminum salts (e.g., alum, aluminum phosphate, aluminum hydroxide). In another variation, the adjuvant is AS101.

[0029] Release assays can be used to qualify a particular lot of autologous or allogeneic iPSCs as having suitable properties for use in a vaccine formulation, while qualification assays can be used to validate a particular manufacturing process. Release or qualification assays can be based on the expression of appropriate genes by measuring mRNA, protein or carbohydrate in iPSCs according to methods known in the art, including but not limited to PCR, RNAseq, flow cytometry, ELISA and immunohistochemical staining. In addition to previously published criteria for iPSCs for regenerative medicine (Sullivan et al. 2018), the inventors have identified genes whose expression in iPSCs makes them particularly useful as vaccines for certain cancers. Such genes are listed in Table 1.

[0030] Table 1 - Genes useful as release or qualification assays for the production of iPSCs for cancer vaccines: Astrocytoma IDH1 Bladder DEPDC1 KIF20B Breast BIRC5 CDCA1 DEPDC1 ERBB2 KIF20A KIF20B Cervical FOXM1 HJURP MELK Colorectal ASTEl IGF2BP3 TAF1B TOMM34 VEGFRl VEGFR2 Esophagus CDCA1 IGF2BP3 IMP3 TTK Stomach ERRB2 Glioblastoma EGFR HSP90B1 Head and neck CDCAl CDKN2A IMP3 Liver GPC3 HSPA8 Melanoma HSP90B1 MGAT5 PMEL NSCLC ERBB2 HSP90Bl IDO1 IMP3 NUF2 TTK TP53 VEGFRl VEGFR2 Ovarian BIRC5 ERBB2 FOXM1 HJURP MELK VEGFRl VEGFR2 Pancreas ERBB2 HSPA8 KIF20A RAS TP53 VEGFRl VEGFR2 Prostate BIRC5 ERBB2

[0031] One or more genes selected from Table 1 can be used to qualify a manufacturing lot of iPSCs for use in an autologous vaccine for patients with a cancer listed in the table. In another embodiment, one or more genes selected from Table 1 can be used to qualify a manufacturing lot of iPSCs.

[0032] In one embodiment, one or more genes selected from Table 1 can be used to track a patient's immune response to a vaccine. In one variation, antibody responses to one or more genes selected from Table 1 can be tracked using assays including, but not limited to, ELISA or Luminex. In one variation, cellular immune responses to one or more genes selected from Table 1 can be tracked using assays including, but not limited to, ELISPOT or cytotoxic T cell killing assays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Compositions and methods are provided for generating pluripotent vectors, generating iPSCs harboring the vectors, establishing cancer vaccines, and prophylactic and therapeutic vaccination of subjects.

[0034] A cancer vaccine, as used herein, uses a host's pluripotent stem cells in combination with an adjuvant to stimulate the same host's immune system in targeting cancer cells.

[0035] The host is generally a mammal, including, but not limited to, humans, dogs, cats, or horses. Laboratory animals, such as rodents, are subject to cancer selection studies, epitope screening, and mechanistic studies. Larger animal studies, such as pigs and monkeys, are subject to safety studies.

[0036] For purposes of the present invention, pluripotent cells can be autologous, allogeneic and xenogeneic to the recipient.

[0037] "Treatment" refers to both therapeutic and prophylactic or preventative treatment. Those in need of treatment include those who already have the disorder as well as those who are to prevent the disorder. In another embodiment, "treating" or "treatment" of any condition or disorder refers to the amelioration of the condition or disorder present in a subject in certain embodiments, including prophylactic amelioration. In another embodiment, "treatment" includes the amelioration of at least one physical parameter that may be indiscernible by the subject. The term "treatment" includes either physical (e.g., stabilization of discernible symptoms) or physiological (e.g., stabilization of physical parameters) or both of the modulation of the condition or disorder. The term "treatment" includes the delay of onset of the condition or disorder. In addition, the term "treatment" includes the reduction or elimination of any of the symptoms (e.g., pain) or one or more symptoms (e.g., pain) of the condition (e.g., cancer), or the delay of the progression of the condition or one or more symptoms of the condition, or the reduction of the severity of the condition or one or more symptoms of the condition. In one variation, "treatment" includes prophylactic administration of a vaccine described herein.

[0038] As used herein, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, etc. In one aspect, the mammal is a human.

[0039] As used herein, "pluripotency" and "pluripotent" stem cells refer to the ability of such cells to differentiate into any type of cell in an adult organism. The term "induced pluripotent stem cells" encompasses pluripotent cells derived from differentiated somatic cells that, like embryonic stem cells (ESCs), can be cultured for extended periods while maintaining the ability to differentiate into any type of cell in an organism, but unlike ESCs (derived from the inner cell mass of a blastocyst), i.e., cells whose potential is narrower and more defined and which cannot be said to generate any type of cell in an organism in the absence of experimental manipulation. "Having the potential to become iPSCs" refers to differentiated somatic cells that can be induced to become iPSCs, i.e., reprogrammed to become iPSCs. That is, somatic cells can be induced to differentiate to establish cells with the morphological characteristics, proliferation capacity, and pluripotency of pluripotent cells. iPSCs have a human ESC-like morphology and grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders, and prominent nucleoli. In addition, iPSCs express one or more key pluripotency markers known to those skilled in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT and zfp42.In addition, pluripotent cells can form teratomas.In addition, they can form or contribute to ectodermal, mesodermal or endodermal tissues in living organisms.

[0040] The term "viable", for example, as used in the section "...pluripotent stem cells do not need to be viable...", means that the cells do not need to have intact membranes or be metabolically active as assessed by commonly used viability stains, e.g., trypan blue, propidium iodide, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) and similar assays (reviewed in Kamiloglu et al., 2020, Food Frontiers 1:332-349).

[0041] Somatic cells can be differentiated / reprogrammed into a state not clearly distinguishable from embryonic stem cells (ESCs) by combining three, four, five, six or more factors. Such reprogrammed cells are called "induced pluripotent stem cells" (iPSCs, iPCs, iPSCs) and can be generated from a variety of tissues (Shi Y. et al., Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 2017 Feb; 16(2):115-130).

[0042] The vaccine may also include an adjuvant. Adjuvants useful in vaccines are well known to those skilled in the art, and thus the selection of an appropriate adjuvant can be routinely performed by those skilled in the art upon review of this application. Examples of useful adjuvants include, but are not limited to, complete and non-complete Freund's mineral gels, such as aluminum hydroxide, surfactants, such as lysolecithin, pluronic polyols, polyanions, peptides, and oil emulsions. Particularly useful adjuvants are those that stimulate cell-mediated immunity, including, but are not limited to, CG-enriched oligodeoxynucleotides (CpG), Bacillus Calmette-Guerin (BCG), activators of the cGAS-STING pathway, MPL (3-O-desacyl-4'-monophosphoryl lipid A), AS04 adjuvant, and mycobacterial cell wall peptidoglycan. In some embodiments, the vaccine is a sterile, pyrogen-free, isotonic formulation, particulate-free injectable composition. The purity standards required for injectable compositions are well known, as are the manufacturing and purification methods used to prepare injectable compositions. The vaccine may be administered by any means known in the art. Injectable pharmaceutical compositions may be administered parenterally, i.e., intravenously, subcutaneously, and intramuscularly. In some embodiments, pharmaceutical vaccine compositions may be administered intranasally or to tissues in the oral cavity, for example, sublingually or by administration to buccal tissue.

[0043] The term "stem cell" refers to a non-specialized cell that can replicate or self-renew itself and develop into specialized cells of various cell types. The product of stem cells undergoing division is at least one additional cell with the same ability as the original cell. Pluripotent stem cells are cells derived from any type of tissue (usually embryonic tissue, e.g., fetal or pre-embryonic tissue), which have the property that under appropriate conditions they can generate progeny of various cell types that are derivatives of all three germ layers (endoderm, mesoderm, and ectoderm). Induced pluripotent stem cells (iPSCs) are generated by exogenously high expression of pluripotency markers (OCT4, SOX2, c-MYC, NANOG, and KLF4) using viral or non-viral vectors, thereby inducing pluripotency into transfected cell lines.

[0044] Pluripotent stem cells are considered to be undifferentiated when they are not committed to a specific lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated iPSCs are easily recognized by those skilled in the art, and typically can be seen by two-dimensional microscopic observation of cell colonies, with high nucleus / cytoplasm ratio and prominent nucleoli. Undifferentiated iPSCs express genes that can be used as markers to detect the presence of undifferentiated cells, and the polypeptide products can be used as negative selection markers.

[0045] The term "dendritic cells" as used herein refers to antigen-presenting cells of the mammalian immune system. Their main function is to process antigenic material and present it on the cell surface to T cells of the immune system. Dendritic cells are believed to be the only immune cells capable of activating naive T cell responses.

[0046] The term "treatment" refers to the reduction, reversal, remission, alleviation, inhibition, or prevention of the progression of a disease or condition, such as cancer. In another embodiment, the term also encompasses prophylaxis, therapy, and cure. The subject or patient undergoing "treatment" or "treatment" is any mammal in need of such treatment for cancer, including primates, and humans, as well as other mammals, such as horses, cows, pigs, and sheep; and domesticated mammals and pets.

[0047] The term "reprogramming" refers to the induction of a pluripotent state on a differentiated somatic cell and as used in the art.

[0048] Somatic cells of interest include, but are not limited to, fibroblasts, blood cells, urinary cells, and the like.

[0049] As used herein, an "adjuvant" is an immunological preparation that boosts the immunological response of the recipient's immune system to target pluripotent stem cells. Adjuvants include those disclosed in this application and those known in the art for boosting the immunological response of the recipient's immune system to target pluripotent stem cells. The term "adjuvant" refers to any substance or agent that can stimulate an immune response. Some adjuvants can cause activation of cells of the immune system. For example, adjuvants can cause immune cells to produce and secrete cytokines. Examples of adjuvants that may result in activation of cells of the immune system include the nanoemulsion formulations described herein, CG-enriched oligodeoxynucleotides (CpG), Bacillus Calmette-Guerin (BCG), activators of the STING pathway, MPL (3-O-desacyl-4'-monophosphoryl lipid A), AS04 adjuvant, and mycobacterial cell wall peptidoglycan, saponins purified from the bark of the Q saponaria tree, e.g., QS21, polydi(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides, e.g., monophosphoryl lipid A (MPL; RibiImmunoChemResearch, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (glucosamine disaccharide associated with lipid A; OM PharmaSA, Meyrin, Switzerland); cholera toxin (CT), and Leishmania elongation factor (purified Leishmania protein; Corixa Corporation, Seattle, Wash.); or mixtures thereof. Other adjuvants known in the art may include, for example, aluminum phosphate or hydroxide salts. In some embodiments, the pluripotent stem cells are administered with one or more adjuvants.In some embodiments, the adjuvants utilized are described in U.S. Patent Publication No. 2005158329, U.S. Patent Publication No. 2009010964, U.S. Patent Publication No. 2004047882, or U.S. Patent No. 6,262,029, and adjuvants for cancer vaccines are reviewed in William S. Bowen et al., 2018, Current challenges for cancer vaccine adjuvant development, Expert Review of Vaccines, 17:3, 207-215.

[0050] As used herein, the term "an amount effective to boost (or induce) an immune response" (e.g., a composition for inducing or boosting an immune response) refers to the dosage level or amount (e.g., when administered to a mammal) required to stimulate, generate and / or induce an immune response in a mammal. An effective amount can be administered in one or multiple administrations over various time periods (e.g., via the same or different routes) as disclosed herein. The application or administration is not intended to be limited to a particular formulation or route of administration or time period.

[0051] Tumor-associated antigens (TAA) or tumor-specific antigens (TSA), as used herein, refer to known as well as unknown antigens / epitopes present on cancer cells.

[0052] The optimal immune response from a cancer vaccine is to stimulate the host's immune system to target such TAAs and TSAs present on pluripotent cells and confer immunity against cancer types that express the TAAs and TSAs. Known TAAs and TSAs include, but are not limited to, EPCAM, CEACAM, TERT, WNK2, and survivin.

[0053] Method of vaccination: Pluripotent stem cells can be obtained from any mammalian species, particularly human cells, including, for example, humans, primates, horses, cows, pigs, etc., as a source of cancer vaccines. Induced pluripotent cells can use multiple starting tissues or cells, including, but not limited to, blood cells, skin cells, fibroblasts, and epithelial cells.

[0054] Pluripotent stem cells are generated and expanded using standard methods known in the art, preferably under feeder cell-free conditions until a stable pluripotent stem cell population is formed (Sun N., Panetta NJ, Gupta DM, Wilson KD, Lee A, Jia F, Hu S, Cherry AM, Robbins RC, Longaker MT, Wu JC. Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad Sci USA. 2009 Sep 15;106(37):15720-5;Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, Panetta NJ, Chen ZY, Robbins RC, Kay MA, Longaker MT, Wu JC. A nonviral minicircle vector for deriving human iPS cells Nat Methods. 2010 Mar; 7(3):197-9;K. Turksen et al. (Eds) Induced Pluripotent Stem (iPS) Cells: Methods and Protocols. 2016. Humana Press. ISBN 978-1-4939-3054-8). This population contains a pure pluripotent stem cell percentage of >90% as assessed by pluripotent stem cell sorting using magnetic antibody sorting (MACS) or fluorescent antibody sorting (FACS).

[0055] In one embodiment of the disclosed vaccine composition, the amount of CpG provided per dose of the vaccine composition is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In another embodiment of the vaccine composition, the number of iPS cells provided per dose is about 10 million, 25 million, 50 million, 100 million, 200 million, 400 million, 800 million, or 2 billion cells.

[0056] In another embodiment, the amount of CpG per dose may be 1 mg and is given or administered with a multiplicity of iPS cells of about 10 million, 25 million, 100 million, 200 million, 400 million, 800 million or 2 billion cells. The cell dose used for the cancer vaccine (1×10 cells) 6 ~1×10 9 The vaccine efficacy ranges from 2×10 cells per dose to 1×10 cells per dose, which may need to be adjusted for the mammal in which the vaccine is used. In small rodents, vaccine efficacy is estimated at 2×10 cells per dose. 6 The cells were set to pluripotent stem cells.

[0057] In clinical applications as cancer vaccines, dosage is expressed in terms of cells and milligrams of CpG per dose. As used herein, the term "CpG" refers to synthetic immunomodulatory oligonucleotides having an unmethylated deoxycytidylyl deoxyguanosine dinucleotide motif (reviewed in Kayraklioglu et al. in Angela Sousa (ed.), DNA Vaccines: Methods and Protocols, Methods in Molecular Biology, vol. 2197, https: / / doi.org / 10.1007 / 978-1- 0716-0872-2_4, Springer Science+Business Media, LLC, part of Springer Nature 2021; Feher K. Protein Pept Sci 20:1060-1068, 2019. and Campbell JD Methods Mol. Biol. 1494:15-27, 2017; each of which is incorporated herein by reference in its entirety). Many such CpG oligonucleotides have been described in the literature.

[0058] In one embodiment, use effective amount of CpG.The term "effective amount" is defined herein and also refers to the amount of CpG that is required together with iPS cells to induce immune response against said iPS cells in mammals.It can be understood that the determination of effective amount is empirical and depends on the species of mammals treated, the number of iPS cells co-administered and the route and schedule of administration.

[0059] In one embodiment, the CpG is selected from the group consisting of CpG1018, CpG7909, SD-101, CpG10104, CpG55.2, CpG10101, CpG52364, MGN1703 and DV281. In one embodiment, the CpG used is CpG1018.

[0060] In another embodiment, the amount of CpG given per dose is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg. In a particular embodiment, the amount per dose is 3 mg. In one embodiment, the number of iPS cells given per dose is about 10 million, 25 million, 50 million, 100 million, 200 million, 400 million, 800 million or 2 billion cells. In a particular embodiment, the number of iPS cells given per dose is 100 million cells.

[0061] In one embodiment, iPS cells and CpG are administered in a single dosage form.In one embodiment, iPS cells and CpG are administered separately or sequentially.In one embodiment, iPS cells and CpG are administered in a pharmaceutically acceptable solution, which may contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and other therapeutic ingredients as appropriate.

[0062] In one embodiment, the iPS cells and CpG are administered as parenteral injection (subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intrathecal, etc.) or mucosal administration (intranasal, intratracheal, inhalation, and rectal, intravaginal, etc.). The injection can be by bolus or continuous infusion. The iPS cells and CpG can be microencapsulated, co-created, coated on fine gold particles, contained in liposomes, misted, aerosolized, pelleted for skin implantation, or dried on a sharp object to scratch the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with delayed release of active compounds, and such formulation excipients and additives and / or auxiliary substances, such as disintegrants, binding substances, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizing agents, are customarily used as described above. Pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of current methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.

[0063] In certain embodiments, the iPS cells and CpG are delivered as a subcutaneous bolus injection.

[0064] Treatment of patients may require different doses depending on the activity of the compound, the method of administration, the purpose of immunization (i.e., preventive or therapeutic), the nature and severity of the disorder, and the age and weight of the patient. The administration of a given dose can be carried out both by single administration of individual dosage unit forms or by administration of several small dosage unit forms. Multiple administration of doses at specific intervals of several weeks or months apart is common for boosting antigen-specific responses.

[0065] In a specific embodiment, the iPS cells and CpG are delivered as a course of four weekly injections every four months.

[0066] In one variation, the method comprises in vitro generation and vaccination of iPSC-based vaccine, for example, subcutaneous vaccination of recipient for several weeks, including several consecutive weeks, for example, 4 consecutive weeks.In one variation, vaccination is carried out once a week for at least 2 consecutive weeks, 3 consecutive weeks, 4 consecutive weeks, 5 consecutive weeks, or at least 6 consecutive weeks.In another variation, the vaccine comprises the use of iPSC with adjuvant, where the adjuvant is an immunological preparation, for example, an antibody, peptide, or small molecule, that boosts or enhances the immune response to the vaccine.

[0067] In one embodiment, the adjuvant is a saponin formulation, a virosome, a virus-like particle, a non-toxic derivative of enterobacterial lipopolysaccharide (LPS), an immunostimulatory oligonucleotide (e.g., an immunostimulatory oligonucleotide containing a CpG motif), a mineral-containing composition, an oil-based emulsion, a polymer, a micelle-forming adjuvant (e.g., a liposome), an immunostimulatory complex matrix (e.g., ISCOMATRIX), a particle, squalene, phosphate, a cationic liposome-DNA complex (CL DC), DDA, DNA adjuvants, gamma-insulin, ADP-ribosylating toxins, detoxified derivatives of ADP-ribosylating toxins, Freund's complete adjuvant, Freund's incomplete adjuvant, muramyl dipeptide, monophosphoryl lipid A (MPL), poly IC, CpG oligodeoxynucleotide (ODN), imiquimod, QS21, adjuvant system AS0, adjuvant system AS02, adjuvant system AS03, MF59®, polydi(carboxylatophenoxy)phosphazene; derivatives of lipopolysaccharides, such as monophosphoryl lipid A, muramyl dipeptide (MDP; Ribi), threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174; cholera toxin (CT), and Leishmania elongation factor, and aluminum or aluminum salts (e.g., alum, aluminum phosphate, aluminum hydroxide). In one variation, the adjuvant is AS 101. Other suitable adjuvants include TLR agonists, NOD agonists and lipid DNA agonist complexes.

[0068] In one embodiment, the adjuvant is mixed with the iPSC cells. In one embodiment, the adjuvant is simply injected with the iPSC cells. In another embodiment, the adjuvant is incubated with the iPSC cells for a period of time to allow the adjuvant to be taken up by the iPSC cells by either a non-specific mechanism, such as pinocytosis or a receptor-mediated mechanism. In one variation, the incubation is carried out for at least 1 hour, 2 hours, 6 hours, 12 hours or 24 hours. In one embodiment, the iPSC cells are emulsified in the adjuvant. In one variation, the emulsifier used is selected from the group consisting of oil-in-water, saponin, squalene, QS21, adjuvant system AS0, adjuvant system AS02 and adjuvant system AS03. In another embodiment, the adjuvant and the iPSC cells are incorporated into a delivery system. In one variation, the delivery system is an absorbent matrix. In one variation, the absorbent matrix is ​​selected from the group consisting of hydrogel, collagen gel, alginate, poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA) and polycaprolactone. In one embodiment, the adjuvant is covalently attached to the cells. In one embodiment, the adjuvant is included in the nanoparticles. In one variation, the nanoparticles are selected from the group consisting of gold, poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA) and polycaprolactone nanoparticles. In one variation, the nanoparticles are simply injected with the iPSC cells. In another variation, the adjuvant is incubated with the iPSC cells for a period of time to allow the nanoparticles to be taken up by the iPSC cells by either a non-specific mechanism, such as pinocytosis or a receptor-mediated mechanism. In one variation, the incubation is performed for at least 1 hour, 2 hours, 6 hours, 12 hours or 24 hours. In one variation, the nanoparticles are covalently attached to the iPSCs.

[0069] In one embodiment, the pluripotent stem cells are not genetically modified. In one embodiment, the pluripotent stem cells are genetically modified using standard methods known in the art. Delivery of genes to pluripotent stem cells can be achieved by, for example, viral vectors, including but not limited to, lentivirus, adenovirus, adeno-associated virus and Sendai virus vectors. Delivery of genes to pluripotent stem cells can also be achieved by delivery of DNA plasmids, DNA minicircles or RNA to cells. This delivery method can be achieved by electroporation, nanoparticle delivery or the use of lipids, as well as other means known in the art.

[0070] In one embodiment, the pluripotent stem cells express one or more immune cell binding proteins (e.g., ICAM1, LFA-1, LFA-3, CD80, CD81, CD28, ICOS, 4-1BB, anti-DEC-205 antibody, anti-CLEC9A (DNGR) antibody, anti-DCIR-2 antibody, anti-DECTIN antibody, anti-ASGPR antibody, anti-mannose receptor antibody, anti-CLEC12 (DCAL-2) antibody, or a combination thereof) without being genetically modified. In another embodiment, the pluripotent stem cells are genetically modified to express one or more immune cell binding proteins (e.g., ICAM1, LFA-1, LFA-3, CD80, CD81, CD28, ICOS, 4-1BB, anti-DEC-205 antibody, anti-CLEC9A (DNGR) antibody, anti-DCIR-2 antibody, anti-DECTIN antibody, anti-ASGPR antibody, anti-mannose receptor antibody, anti-CLEC12 (DCAL-2) antibody, or a combination thereof). In one variation, the pluripotent stem cells are genetically modified to express CD28 and CD80.

[0071] In another embodiment, the immune cell binding protein enhances antigen cross-presentation in antigen-presenting cells. The level of cross-presentation in antigen-presenting cells can be determined by methods known in the art, including but not limited to, measuring the expression of peptides on MHC proteins on dendritic cells by flow cytometry, measuring the level of T cells activated by cross-presenting dendritic cells in vitro, and measuring cytotoxic T cell activation in vivo.

[0072] In one embodiment, the pluripotent stem cells express one or more cytokines (e.g., XCR1, CCL3, CCL4, CCL5, CCL20, CCL25 and FLT3L, or a combination thereof) without genetic modification. In another embodiment, the pluripotent stem cells are modified to express CCL3 and FLT3L. In another variation, the iPSCs are genetically modified to secrete a protein selected from the group including GM-CSF, INF alpha, INF beta, IL-2, IL-12, IL-15 and IL-21, or a combination thereof. In another embodiment, the allogeneic vaccine expresses IL-15. In another variation, the iPSCs are genetically modified to express a protein selected from the group including gp96, hsp90, hsp70, CD91, calreticulin and LOX-1. In one embodiment, the iPSCs are genetically modified to express hsp70. In another variation, the autologous or allogeneic vaccine is genetically modified to express a cell surface protein selected from the group including B7, OX40, CD28, CD40L, TLR4, CD70, MHC class I, MHC class II and OX40L.In another embodiment, the iPSC is modified to express OX40.In another embodiment, the iPSC is modified to express connexin 43.

[0073] In one embodiment, the expression level of the genetically modified protein is adjusted to demonstrate chemotaxis or activation of immune cells. In one variation, the strength of the promoter used is adjusted to obtain the desired expression level. In another variation, the copy number of the gene to be expressed is adjusted to obtain the desired expression level. In another variation, the number of transfected pluripotent stem cells in the vaccine is adjusted to obtain the desired expression level. Chemotaxis of immune cells can be measured by methods known in the art, including but not limited to in vitro assays using Boyden chambers and in vivo assays examining immune infiltrates at the vaccination site. Activation of immune cells can be measured by methods known in the art, including but not limited to measuring antigen levels on cells using flow cytometry or immunohistochemical staining, measuring the number of activated immune cells by ELISpot, measuring the number of activated immune cells by cytotoxicity assays, or other means known in the art.

[0074] In another variation, the autologous or allogeneic vaccine is genetically modified to include an inhibitory RNA. In one variation, the inhibitory RNA is selected from the group consisting of antisense RNA, siRNA, shRNA, miRNA, lncRNA, pri-miRNA, antisense oligonucleotide, and pre-miRNA. In one variation, the inhibitory RNA inhibits expression of a gene selected from the group consisting of MHC class I, MHC class II, beta 2 microglobulin, and LAMP. In one embodiment, the inhibitory RNA inhibits beta 2 microglobulin. In one variation, the inhibitory RNA inhibits expression of a gene selected from the group consisting of PD-1, PDL-1, PDL-2, Nodal, cytokine signaling 1 (SOCS1), IL-10, IL-10R, TGF-β, and TGF-β. In another variation, the inhibitory RNA inhibits expression of TGF-β. In one variation, the inhibitory RNA is expressed at a concentration high enough to inhibit expression of one or more of such genes in antigen-presenting cells that take up the iPSC vaccine. One of skill in the art will understand that methods other than inhibitory RNA, for example, including CRISPR, can be used to inhibit expression of specific genes in autologous or allogeneic vaccines.

[0075] In one embodiment, allogeneic vaccine is developed by using CRISPR to delete the genes of beta 2 microglobulin and HLA-DR.The inventors have surprisingly found that the presence of such genes directs immune system to attack allogeneic antigens first, instead of focusing on the cancer-related genes of interest.Without intending to be bound by a particular theory, the inventors have found that deleting beta 2 microglobulin and HLA-DR forces the recipient's immune system to respond to cross-presented antigens first, thereby generating an allogeneic product that is effective in inducing immune response against cancer.

[0076] In another aspect, autologous or allogeneic vaccine is administered in combination with small molecule drug.In one embodiment, this drug is selected from the group consisting of HDAC inhibitor, bromodomain inhibitor, Vps34 kinase inhibitor, PRMT5 inhibitor, autophagy inhibitor, angiogenesis inhibitor, vascular disruptor, STING pathway activator and Toll receptor pathway activator.In another variant, the small molecule drug is STING activator and Toll receptor pathway activator.

[0077] In one variation, the HDAC inhibitor is selected from the group consisting of valproic acid, gibinostat, belinostat, entinostat, mocetinostat, practinostat, cidamide, xinostat, abexinostat, vorinostat, romidepsin, panobinostat, and belinostat. In another variation, the HDAC inhibitor is vorinostat.

[0078] In one variation, the BET inhibitor is selected from the group consisting of I-BET151 (GSK1210151A), I-BET762 (GSK525762), OTX-015, TEN-010, CPI-203 and CPI-0610. In one variation, the BET inhibitor is CPI-0610.

[0079] In one variation, the Vps34 kinase inhibitor is selected from the group consisting of SB02024 and SAR405. In another variation, the PRMT5 inhibitor is GSK3326595. In one variation, the autophagy inhibitor is selected from the group consisting of 3-methyladenine, bafilomycin A1, chloroquine, hydroxychloroquine, N-2--(1H-benzimidazol-6-yl)-N-4--(5-cyclobutyl-1H-pyrazol-3-yl)quinazoline-2,4-diamine, MRT68921, MRT67307, SBI-0206965, ULKlOO, ULK101 and SB02024. In one variation, the autophagy inhibitor is SB02024.

[0080] In one variation, the angiogenesis inhibitor is selected from the group consisting of axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and ramucirumab. In another variation, the angiogenesis inhibitor is sorafenib.

[0081] In one variation, the vascular disrupting agent is selected from the group consisting of combretastatin, AVE8062, ZD6126, ABT-571, MN-029, CKD516, OXi8006, 5,6-dimethylxanthenone 4-acetic acid, combretastatin A-4 phosphate, ZD6126, Oxi4503 and the dolatastatin derivative TZT-1027.

[0082] In one variation, the STING activator is selected from the group consisting of ADU-S100, MK-1454, MK-2118, BMS-986301, SR-717, GSK3745417, SB-11285, IMSA-101, c-di-GMP, c-di-AMP and cGAMP. In one variation, the STING activator is SR-717.

[0083] In one variation, the Toll pathway activator is selected from the group consisting of diacyl lipopeptide, triacyl lipopeptide, flagellin, poly I:C, hexa-acetylated lipid A, monophosphoryl lipid A, gardikimod, imiquimod, and R848. In another variation, the Toll pathway activator is imiquimod.

[0084] In another variation, the autologous or allogeneic vaccine is co-administered with an antibody selected from the group consisting of anti-CD47, rituximab, cetuximab, daratumumab, trastuzumab, trastuzumab emtansine, pertuzumab, panitumumab, ramucirumab, necitumumab, and blinatumomab. In one variation, the autologous or allogeneic vaccine is co-administered with blinatumomab.

[0085] In another variation, the autologous or allogeneic vaccine is co-administered with a small molecule drug selected from the group consisting of ibrutinib, acalabrutinib, and galuniseritib. In another variation, the autologous or allogeneic vaccine is co-administered with ibrutinib.

[0086] In one variation, the small molecule drug and iPSC cells are incorporated into a delivery system. In one variation, the delivery system is an absorbent matrix. In one variation, the absorbent matrix is ​​selected from the group consisting of hydrogel, collagen gel, alginate, poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA) and polycaprolactone. In one embodiment, the small molecule drug is contained in a nanoparticle. In one variation, the nanoparticle is selected from the group consisting of gold, poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA) and polycaprolactone nanoparticles. In one variation, the nanoparticle is injected with the iPSC cells. In another variation, the adjuvant is incubated with the iPSC cells for a period of time to allow the nanoparticles to be taken up by the iPSC cells by either a non-specific mechanism, such as pinocytosis or a receptor-mediated mechanism. In one variation, the incubation is performed for at least 1 hour, 2 hours, 6 hours, 12 hours or 24 hours. In one variation, the nanoparticles are covalently attached to the iPSCs.

[0087] In one variation, small molecule drug is given orally, parenterally or intravenously together with iPSC vaccine.In another variation, small molecule drug is given 4 days before vaccine, 3 days before vaccine, 2 days before vaccine, 1 day before vaccine, at the same time as vaccine, 1 day after vaccine, 3 days after vaccine, 4 days after vaccine or any combination of days before, during and after vaccine administration.

[0088] In one aspect, pluripotent stem cells are induced to undergo a specific type of cell death. The inventors surprisingly discovered that pluripotent stem cells do not need to be viable at the time of injection to induce the desired anti-cancer immunity.

[0089] In one embodiment, iPCs are killed to induce expression of calreticulin on the cell surface.

[0090] In one embodiment, iPSCs that are killed by administration of oxaliplatin or doxorubicin in vitro and induced to express calreticulin, and then mixed with an adjuvant, are particularly potent.

[0091] In one variation, the cell death inducer is selected from the group including glutamic acid, sorafenib, ML162, FIN56, FINO2, erastin, sulfazine, RSL3, Ki8751, SGX-523, AZD7762, KW-2449, NVP-TAE684, AZD4547, TG-101348, bleomycin, axitinib, cytochalasin B, dasatinib, SNX-2112, semagacestat, CHIR-99021, B02, olaparib, silmitasertib, tanespimycin, nintedanib, ML031, canertinib, SMER-3, BCL-LZH-4, SN-38, tamatinib, ML334 diastereomers, analogs, salts or derivatives thereof. In another variation, the cell death inducer is bleomycin.

[0092] In one variation, the cell death inducer is incubated with iPSC cells for 1 hour, 2 hours, 6 hours, 12 hours or 24 hours. In another variation, the small molecule drug is incubated with iPSC cells for 2 hours, and the cells are harvested and frozen. In one variation, the cell death inducer is administered orally, parenterally or intravenously with the iPSC vaccine. In one variation, the cell death inducer is administered 4 days before the vaccine, 3 days before the vaccine, 2 days before the vaccine, 1 day before the vaccine, simultaneously with the vaccine, 1 day after the vaccine, 3 days after the vaccine, 4 days after the vaccine or any combination of days before, during and after the vaccine administration.

[0093] In another embodiment, the vaccine is comprised of dendritic cells, which are obtained from the patient to be treated, expanded in tissue culture, pulsed with antigens derived from pluripotent stem cells, and then delivered back to the patient. The dendritic cells can be pulsed with whole pluripotent stem cells, extracts of pluripotent stem cells, mRNA from pluripotent stem cells, cDNA from pluripotent stem cells, or proteins or peptides from pluripotent stem cells.

[0094] In another embodiment, a method is provided for vaccination of a mammal with a pluripotent stem cell cancer vaccine, the method comprising: 1) introducing a mammalian pluripotent stem cell from an embryonic source, or 2) by reprogramming a somatic cell from a recipient, and providing the recipient with the pluripotent stem cell. In another aspect, the mammalian cell is an undifferentiated pluripotent cell.

[0095] In another aspect of this method, pluripotent stem cells are genetically modified from the patient's tumor cells. Any cell type derived from the tumor can be used, but the stem cell population can be used from the tumor. The stem cell population derived from the tumor can be isolated by many methods known in the art.

[0096] In another embodiment, pluripotent stem cells are genetically modified from normal cells to fuse with the patient's tumor cells. Cell fusion can be achieved by methods known in the art, such as electrical cell fusion, polyethylene glycol cell fusion, Sendai virus-induced cell fusion, and optionally, thermally controlled plasmonics.

[0097] In one embodiment, the vaccine is irradiated prior to vaccination to prevent iPSC proliferation. In one variation, proliferation is prevented by exposing iPSC to a DNA crosslinking agent instead of irradiation. In one variation, the crosslinking agent is selected from the group including nitrogen mustard, cisplatin, BCNU, psoralen and mitomycin C. The amount of crosslinking agent used is sufficient to prevent iPSC from undergoing cell division. Additional DNA crosslinking agents can be used provided that they inhibit cell division and do not interfere with the vaccine properties of iPSC. In one variation of this method, the vaccine is injected subcutaneously for a period of 4 weeks or less, for example, 3 weeks, 2 weeks or about 1 week. In another variation, vaccination is performed once a week. In another variation, vaccination may be performed daily, several times a week, for example, 2 or 3 times a week, or every 2 weeks, and the period can be 2, 3, 4, 5, 6, 7, or 8 weeks or more.

[0098] A therapeutically effective amount of the vaccine can boost or enhance an in vivo immune response against cancer by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90% or more relative to the effect of not administering the vaccine of the present application. Assays used to measure T cell responses include, but are not limited to, delayed type hypersensitivity tests, flow cytometry using major histocompatibility complex tetramer peptides, lymphocyte proliferation assays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunospot assays (ELISPOT), cytokine flow cytometry, cytotoxic T lymphocyte (CTL) assays, CTL precursor frequency assays, T cell proliferation assays, carboxyfluorescein diacetate succinimidyl ester assays, polyfunctional T cell assays, measurement of cytokine mRNA by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), RNAseq and limiting dilution analysis.

[0099] Analysis of tumor biopsy can be used to monitor immune response to cancer vaccine.This biopsy can evaluate the infiltration of immune cells into tumor.Immune cells can be evaluated by immunohistochemical staining, flow cytometry, Q-TOF, ELISPOT, RNAseq, or any of the methods known in the art or described above.

[0100] Responses to cancer vaccines can also be assessed by the expression of cell surface antigens on immune cells. Increased expression of antigens such as CD69, Ox40, HLA-DR and CD154 and / or decreased expression of antigens such as CD25, PD-L1 and TIGIT can be used to measure activation of immune cell responses either within the tumor or in the circulation.

[0101] Other assays to assess immune responses include, but are not limited to, gene expression profiling, protein microarrays to assess antibody responses to multiple antigens at once, luciferase immunoprecipitation, phosphoflow to measure multiple intracellular signaling molecules in the immune system at the single cell level for lymphocyte immune surveillance, and surface plasmon resonance biosensors to monitor antibody immunity in serum.

[0102] Clinical signs of efficacy include, but are not limited to, reduction in tumor size by imaging techniques including, but not limited to, MRI or PET scan, biomarkers including, but not limited to, PSA, AFP, CA19-9, CA-125, CEA, circulating tumor cells, circulating miRNA, and clinical parameters including, but not limited to, morbidity and fat percentage.

[0103] In one variation of this method, vaccination causes the destruction of tumor cells.Those tumor cells release small molecules, peptides, proteins or genetic materials into circulation that can be detected and act as biomarkers of vaccine activity.When vaccine induces an immune response that kills tumor cells, biomarkers can rise in blood, plasma, serum, urine, fecal matter or other body fluids.Also, biomarkers can decrease over time as the amount of tumor cells that produce them decreases.

[0104] In one embodiment, the biomarkers are detected by methods known in the art, including but not limited to, PCR, ELISA, flow cytometry, or analytical chemistry.

[0105] In one embodiment, the protein biomarker is selected from the group including lactate dehydrogenase, alpha 1-acid glycoprotein, fucosylated alpha 1-acid glycoprotein, PSA, PSA-3, CEA, CA19-9, CA15-3, CA27.29, NMP-22, calcitonin, thyroglobulin, HCG-β, alpha-fetoprotein, HER-2, MAGEA3, NY-ESO-1, PMEL, and IGFBP2. In one embodiment, the biomarker is cell-free DNA. In one embodiment, the biomarker is lncRNA or miRNA. In one embodiment, the miRNA is selected from the group including mi-24, mi-320a, mi-423-5q, miR-21-5p, miR-20a-5p, miR-141-3p, miR-145-5p, miR-155-5p, as well as miR-223-3p, miR-23a-3p, miR-27a-3p, miR-142-5p, miR-376c-3p, miR-642b-3p, miR-1202-5p, miR-1207-5p, miR-1225-5p, miR-4270-5p, miR-1825-3p and miR-4281-3p.

[0106] In another embodiment, the biomarker is expressed in the tumor. In one variation, the biomarker is a measure of tumor mutation burden (TMB). In another variation, the biomarker is tumor expression of one or more proteins selected from the group including PDL-1, PDL-2, TGF beta, VEGF, CXCL12, CCL18, ARG1, iNOS, IL-10, IL-35 and Galectin 1.

[0107] In another variation of this method, the vaccine is administered prophylactically to patients with a family history or genetic abnormalities (including but not limited to mutations in BRCA1, BRCA2, APC, FAP, HNCC, TP53, P16 and PTEN) prior to the onset of cancer. In another variation of this method, the vaccine is administered prophylactically to healthy individuals prior to the onset of cancer.

[0108] In another embodiment, a heat stable vaccine composition is provided that comprises an effective amount of mammalian pluripotent stem cells obtained from an embryonic source or obtained by reprogramming somatic cells derived from a mammal, and optionally an adjuvant or immunological preparation that boosts the immune response to the vaccine.

[0109] In one variation, iPSCs that are killed by in vitro administration of oxaliplatin or doxorubicin and induced to express calreticulin prior to formation of a heat-stable vaccine and then mixed with an adjuvant are particularly potent. In one variation, the cell death inducer is selected from the group comprising glutamic acid, sorafenib, ML162, FIN56, FIN02, erastin, sulfazine, RSL3, Ki8751, SGX-523, AZD7762, KW-2449, NVP-TAE684, AZD4547, TG-101348, bleomycin A2, axitinib, cytochalasin B, dasatinib, SNX-2112, semagacestat, CHIR-99021, B02, olaparib, silmitasertib, tanespimycin, nintedanib, ML031, canertinib, SMER-3, BCL-LZH-4, SN-38, tamatinib, ML334 diastereomers, analogs, salts or derivatives thereof.

[0110] In another embodiment of the vaccine composition, the pluripotent stem cells are induced pluripotent cells (iPSCs). In another embodiment of the vaccine composition, the mammalian pluripotent stem cells are undifferentiated pluripotent stem cells. In another embodiment of the vaccine composition, the pluripotent stem cells are produced from the group consisting of fibroblasts, keratinocytes, peripheral blood cells and kidney epithelial cells. In another embodiment of the vaccine composition, the pluripotent stem cells are killed by exposure to oxaliplatin or doxorubicin at optimal concentrations and duration to induce expression of calreticulin. In one variation, the cell death inducer is selected from the group comprising glutamic acid, sorafenib, ML162, FIN56, FINO2, erastin, sulfazine, RSL3, Ki8751, SGX-523, AZD7762, KW-2449, NVP-TAE684, AZD4547, TG-101348, bleomycin A2, axitinib, cytochalasin B, dasatinib, SNX-2112, semagacestat, CHIR-99021, B02, olaparib, silmitasertib, tanespimycin, nintedanib, ML031, canertinib, SMER-3, BCL-LZH-4, SN-38, tamatinib, ML334 diastereomers, analogs, salts or derivatives thereof.

[0111] In one variation, the present application discloses a formulation for use in treating cancer in a patient comprising administering to the patient via a vaccine, the vaccine comprising an effective amount of mammalian pluripotent stem cells obtained from an embryonic source or obtained by reprogramming somatic cells from the patient, and vaccination comprising administering to a patient in need thereof the mammalian pluripotent stem cells.

[0112] In one embodiment, a vaccination for use in treating cancer in a patient is provided, the vaccine comprising an effective amount of mammalian pluripotent stem cells, or fragments thereof, obtained from an embryonic source or by reprogramming somatic cells from a patient, and the vaccination comprises administering the mammalian pluripotent stem cells to a patient in need thereof.In another variation, the vaccine is a thermostable vaccine composition comprising an effective amount of mammalian pluripotent stem cells, obtained from an embryonic source or by reprogramming somatic cells from a mammal, and an adjuvant or immunological preparation that boosts the immune response to the vaccine.In another aspect, the vaccine is a combination of mammalian pluripotent stem cells, obtained from an embryonic source or by reprogramming somatic cells from a mammal, and mammalian pluripotent stem cells that are killed by exposure to optimal concentrations and duration of oxaliplatin or doxorubicin to induce expression of calreticulin, and an adjuvant or immunological preparation that boosts the immune response.

[0113] Working Example The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to utilize the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to be all or only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation must be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (° C.), and pressure is at or near atmospheric.

[0114] The present invention has been described with respect to specific embodiments discovered or proposed by the inventor, including preferred methods for carrying out the invention. In light of this disclosure, those skilled in the art will understand that numerous modifications and changes can be made in the specific embodiments illustrated without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalence considerations, changes can be made in the protein structure without affecting the biological action by type or amount. All such modifications are intended to be included within the scope of the appended claims.

[0115] For further elaboration of the general techniques useful in carrying out the methods of the present application, the practitioner may refer to standard textbooks and reviews in cell biology, tissue culture and embryology. With regard to tissue culture and ESCs, see Teratocarcinomas and embryonic stem cells: A practical approach (EJ Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (PM Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (MV Wiles, Meth. Enzymol. 225:900, 1993);Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (PD Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

[0116] General methods in molecular and cellular biochemistry can be found in standard texts such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial suppliers, such as BioRad, TakaRa, Thermofisher, Sigma-Aldrich, and Qiagen. [Example 1]

[0117] Allogeneic iPSC vaccine using shRNA In a particular embodiment, the present inventors have found that autologous iPSC vaccine is more potent than allogeneic iPSC vaccine.The main difference between autologous and iPSC vaccine is the difference in major histocompatibility locus (MHC class I and MHC class II).MHC mismatch can induce strong immune response.For example, in mice, skin grafts with matching MHC locus are acutely rejected within about one week, while skin grafts with mismatching major histocompatibility locus take much longer to be rejected and can remain intact for more than one month.

[0118] In the case of iPSC vaccines, the overwhelming immune response to MHC differences precludes the generation of an immune response to the cancer-associated antigen of interest, and therefore, in this case, it is desirable to remove MHC antigens in the iPCs used for the allogeneic vaccine.

[0119] In one method, siRNA or shRNA is used to suppress the expression of beta2-microglobulin. Lentiviral particles containing beta-2-microglobulin shRNA plasmid are obtained from a commercial source, SantaCruzBiotechnology, Inc. (SantaCruz, CA), and used according to the manufacturer's instructions. Briefly, 24 hours prior to viral infection, iPC cells are seeded into 12-well tissue culture plates at a concentration that results in approximately 50% confluence on the day of infection (day 2). On day 2, iPC medium is prepared with Polybrene® (sc-134220) at a final concentration of approximately 5 μg / ml (depending on the lot of polybrene, each lot was tested for optimal concentration that does not induce toxicity). The medium in the iPC culture plate is removed and replaced with 1 ml of polybrene / medium mixture per well (for 12-well plates). Lentiviral particles are thawed and added to the cells at a concentration that depends on the lot of lentivirus (each lot was tested for optimal particle concentration that results in maximum gene inhibition before incubation). The particles are incubated with the cells overnight, then washed and fresh medium is added to the plates.

[0120] To obtain stable expression of the shRNA, puromycin selection is used. Puromycin is added to the culture at the highest concentration that does not kill non-transfected cells, usually 2-10 μg / mL. Culture is continued in the presence of puromycin, and puromycin-resistant clones are selected and expanded in the presence of puromycin to the desired cell number to be used for the vaccine.

[0121] The effectiveness of shRNA can be evaluated by PCR of beta 2 microglobulin or FACS analysis of cells using antibodies of HLA class I using standard methods known in the art.Knockout of beta 2 microglobulin is sufficient to inhibit the expression of HLA class I.Similar methods can also be used to inhibit the expression of HLA class II using shRNA directed against individual class II members such as HLA-DR. [Example 2]

[0122] Allogeneic iPSC vaccines using CRISPR An alternative to using shRNA is to use CRISPR technology. Two gene-specific gRNAs designed around the 5' end of the coding sequence of beta 2 microglobulin are obtained from commercial suppliers, such as Origene (Rockville, MD), as part of their CRISPR knockout kit, and used according to the manufacturer's instructions.

[0123] Briefly, approximately 18-24 hours prior to transfection, plate approximately 3 x 10 cells. 5Seed adherent iPSC cells in 2 ml of culture medium in each well of a 6-well plate, adjusted to the exact concentration to obtain 50-70% confluence the next day. Dilute 1 μg of each of the gRNA vectors in 250 μL of Opti-MEM I (LifeTechnologies) and vortex gently. Then, dilute 1 μg of donor DNA in the same 250 μL of Opti-MEM I and vortex gently. Add 6 μL of Turbofectin 8.0 to the diluted DNA (not in the reverse order), then mix thoroughly by gently pipetting dropwise (perform an initial titration to see if a ratio of 3:1 Turbofectin 8.0 to DNA is optimal, or if a different ratio improves transfection efficiency). The mixture is then incubated at room temperature for 15 min, after which it is added dropwise to the cells without changing the medium. Gently rock the plate back and forth and side to side to evenly distribute the compounds, and incubate the cells in a 5% CO incubator.

[0124] 48 h after transfection, split the cells 1:10 and grow for an additional 3 days. Split the cells 1:10 again and repeat the process to split the cells 2-4 times in total.

[0125] To select cells with successful CRISPR knockout, puromycin selection is used. Puromycin is added to the culture at the highest concentration that does not kill non-transfected cells, usually 2-10 μg / mL. Culture is continued in the presence of puromycin to select for puromycin-resistant clones. Such clones can then be grown in the absence of puromycin if desired.

[0126] The effectiveness of CRISPR knockout can be evaluated by PCR of beta 2 microglobulin or FACS analysis of cells using antibodies of HLA class I using standard methods known in the art.Knockout of beta 2 microglobulin is enough to inhibit the expression of HLA class I.Similar methods can also be used to inhibit the expression of HLA class II using vectors with gRNA directed against individual class II members such as HLA-DR. [Example 3]

[0127] Boosted allogeneic vaccines iPSC cell lines are genetically modified to not express beta2 microglobulin or class II HLA antigens as in Example 2. The cells are then infected with adeno-associated virus particles (VectorBioLabs, Malvern PA) containing CD28 and CD80 expression cassettes according to the manufacturer's instructions. Briefly, virus-containing medium is prepared by thawing a virus stock and adding the desired amount of virus to growth medium to achieve the desired multiplicity of infection (MOI). This is calculated using the following formula: AAV GC particles used = MOI (multiplicity of infection) * number of cells to be infected. The optimal MOI is determined using green fluorescent protein (GFP)-expressing AAV from the same supplier with MOIs ranging from 2,000 to 500,000, probing for optimal GFP expression by flow cytometry. To infect AAV viruses containing CD28 and CD80 expression cassettes, the cell culture medium is removed and AAV-containing medium is added to the cell culture and incubated for 6 hours. The levels of CD28 and CD80 on the iPS cells are monitored using flow cytometry. [Example 4]

[0128] Fusion of cancer cells and iPS cells iPSC vaccines present many embryonic antigens in common with cancer cells. However, cancer cells may also contain antigens that arise from somatic mutations that are unique to each individual cancer. To expand the antigen presentation of iPSCs, the inventors discovered that fusing an individual's cancer cells with iPSCs allows for further expression of such antigens.

[0129] Tissue from tumor biopsies is dissociated using collagenase by means known in the art. Briefly, tumor tissue from surgical resection is placed in tissue culture medium and minced with a scalpel into approximately 1-3 mm pieces. 3 Depending on the amount of tissue, transfer the minced tissue into a 15 mL or 50 mL conical tube and centrifuge the tube at 100 x g for 5 min at room temperature. Add a solution containing collagenase II to reach a final concentration of 1 mg / mL and a solution of DNase I to reach a final concentration of 100 Kunitz / mL (adjust the final concentration depending on the lot of enzyme).

[0130] The mixture is placed in a flask, which is placed on a vibrating platform and rocked at room temperature until the tissue is visibly dissociated.

[0131] The resulting cell suspension is placed in a 15 mL or 50 mL conical tube and washed three times with serum-free tissue culture medium. The cells are resuspended in serum-free tissue culture medium and counted. The cell suspension of iPS cells in serum-free tissue culture medium is added at a ratio of 2 cancer cells for every 1 iPS cell. The cells are centrifuged at 100×g for 5 minutes at room temperature in serum-free tissue culture medium. The supernatant is removed and the cell pellet is gently resuspended by tapping the bottom of the tube. Fusion is performed by slowly adding 1 ml of 50% PEG1500 pre-warmed to 37° C. to the pellet over 1 minute using a 1 ml pipette and constantly mixing the cells by gentle shaking for an additional 2 minutes. 3 ml of medium pre-warmed to 37° C. is slowly added over 3 minutes while the cells are continuously agitated by gentle shaking. Then 10 ml of medium pre-warmed to 37° C. is gently added and the cells are incubated for an additional 10 minutes and then centrifuged at 100×g for 5 minutes. The cells are then washed again to remove residual PEG and are either prepared for injection or frozen using standard cryopreservation techniques.

[0132] All references cited in this specification, including publications, books, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. References [Table 1]

[0133] Although the invention has been described in conjunction with specific embodiments and examples, it will be apparent to those of skill in the art in light of the teachings and this disclosure that equivalents of the specifically disclosed materials and methods may also be applied to the present invention, and such equivalents are intended to be encompassed within the scope of the following claims.

Claims

1. 1. A mammalian autologous or allogeneic vaccine comprising an effective amount of mammalian induced pluripotent stem cells (iPSCs) obtained by reprogramming somatic cells from a patient, comprising: expressing a gene selected from the group consisting of ASTE1, BIRC5, CDCA1, CDKN2A, DEPDC1, EGFR, ERBB2, FOXM1, GPC3, HJURP, HSPA8, HSP90B1, IDH1, IDO1, IGF2BP3, IMP3, KIF20A, KIF20B, MELK, MGAT5, NUF2, PMEL, RAS, TAF1B, TOMM34, TTK, TP53, VEGFR1 and VEGFR2; Optionally, it may contain an adjuvant, Autologous or allogeneic vaccines to induce an immune response in patients for the treatment of cancer.

2. The selected gene or genes are Astrocytoma IDH1; Bladder DEPDC1 KIF20B; Breast BIRC5 CDCA1 DEPDC1 ERBB2 KIF20A KIF20B; Cervical FOXM1 HJURP MELK; Colorectal ASTEl IGF2BP3 TAF1B TOMM34 VEGFRl VEGFR2; Esophagus CDCA1 IGF2BP3 IMP3 TTK; Stomach ERRB2; Glioblastoma EGFR HSP90B1; Head and neck CDCAl CDKN2A IMP3; Liver GPC3 HSPA8; Melanoma HSP90B1 MGAT5 PMEL; NSCLC ERBB2 HSP90Bl IDO1 IMP3 NUF2 TTK TP53 VEGFRl VEGFR2; Ovarian BIRC5 ERBB2 FOXM1 HJURP MELK VEGFRl VEGFR2; Pancreatic ERBB2 HSPA8 KIF20A RAS TP53 VEGFRl VEGFR2; and Prostate BIRC5 ERBB2 The vaccine of claim 1, wherein the type of cancer in the patient is selected from the group consisting of:

3. 1. A mammalian autologous or allogeneic vaccine comprising an effective amount of mammalian induced pluripotent stem cells (iPSCs) obtained by reprogramming somatic cells from a patient, comprising: genetically engineered to contain an inhibitory RNA that is not involved in the reprogramming process; Autologous or allogeneic vaccines to induce an immune response in patients for the treatment of cancer.

4. 4. The vaccine of claim 3, wherein the inhibitory RNA is selected from the group consisting of antisense RNA, siRNA, shRNA, miRNA, lncRNA, pri-miRNA, antisense oligonucleotides and pre-miRNA.

5. 5. The vaccine of claim 3 or 4, wherein the inhibitory RNA inhibits expression of a gene selected from the group consisting of MHC class I, MHC class II, beta 2 microglobulin and LAMP.

6. 6. The vaccine of claim 5, wherein the inhibitory RNA inhibits expression of a gene selected from the group including PD-1, PDL-1, PDL-2, Nodal, cytokine signaling 1 (SOCS1), IL-10, IL-10R, TGF-β, and TGF-βR.

7. 1. A mammalian autologous or allogeneic vaccine comprising an effective amount of mammalian induced pluripotent stem cells (iPSCs) obtained by reprogramming somatic cells from a patient, comprising: Genetically engineered to express genes that are not involved in the reprogramming process; Autologous or allogeneic vaccines to induce an immune response in patients for the treatment of cancer.

8. The vaccine of claim 7, wherein the gene is selected from the group consisting of ICAM1, LFA-1, LFA-3, CD80, CD81, CD28, ICOS, 4-lBB, anti-DEC-205 antibody, anti-CLEC9A (DNGR) antibody, anti-DCIR-2 antibody, anti-DECTIN antibody, anti-ASGPR antibody, anti-mannose receptor antibody and anti-CLEC12 (DCAL-2) antibody.

9. 8. The vaccine of claim 7, wherein the gene is selected from the group consisting of XCR1, CCL3, CCL4, CCL5, CCL20, CCL25 and FLT3L.

10. 8. The vaccine of claim 7, wherein the gene is selected from the group consisting of GM-CSF, INF alpha, INF beta, IL-2, IL-12, IL-15 and IL-21.

9.

11. 8. The vaccine of claim 7, wherein the gene is selected from the group consisting of gp96, hsp90, hsp70, CD91, calreticulin and LOX-1.

12. 8. The vaccine of claim 7, wherein the gene is selected from the group consisting of B7, OX40, CD28, CD40L, TLR4, CD70, MHC class I, MHC class II and OX40L.

13. 2. The mammalian autologous or allogeneic vaccine of claim 1, wherein the mammalian induced pluripotent stem cells are non-viable and express a protein selected from the group consisting of calreticulin, Hsp70 and HSP90 on the cell surface.

14. 14. The mammalian autologous or allogeneic vaccine of claim 13, wherein the mammalian induced pluripotent stem cells are killed by in vitro administration of a chemotherapeutic agent.

15. 15. The mammalian autologous or allogeneic vaccine of claim 14, wherein the chemotherapeutic agent is selected from the group consisting of oxaliplatin or doxorubicin.

16. 2. The mammalian autologous or allogeneic vaccine of claim 1, wherein the vaccine further comprises dendritic cells, the dendritic cells being obtained from the patient.

17. 17. The mammalian autologous or allogeneic vaccine of claim 16, wherein the dendritic cells are pulsed with iPS cells reprogrammed from mature cells obtained from the patient.

18. 17. The mammalian autologous or allogeneic vaccine of claim 16, wherein the dendritic cells are expanded in tissue culture and pulsed with induced pluripotent stem cell-derived antigens prior to delivery back to the patient.

19. 20. The mammalian autologous or allogeneic vaccine of claim 18, wherein the dendritic cells are selected from the group of dendritic cells pulsed with total pluripotent stem cells, extracts of pluripotent stem cells, mRNA from pluripotent stem cells, cDNA from pluripotent stem cells, or proteins or peptides from pluripotent stem cells.

20. 2. The mammalian autologous or allogeneic vaccine of claim 1, wherein the cancer is selected from the group consisting of leukemia, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoma, myeloproliferative disorder, squamous cell carcinoma, adenocarcinoma, sarcoma, neuroendocrine carcinoma, bladder cancer, skin cancer, brain and spinal cord cancer, head and neck cancer, thyroid, bone cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastrointestinal cancer, (hypopharynx) cancer, esophageal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, eye cancer, renal cell carcinoma, kidney, liver cancer, ovarian cancer, stomach cancer, testicular cancer, thyroid and thymic cancer.

21. 21. The mammalian autovaccine or allogeneic vaccine composition of claim 20, wherein the adjuvant is CpG and the amount of CpG per dose is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg.

22. 22. The mammalian autologous or allogeneic vaccine composition of claim 21, wherein the number of iPS cells per dose is about 10 million, 25 million, 50 million, 100 million, 200 million, 400 million, 800 million or 2 billion cells.