Methods for the treatment of cancer

Abstract

Combination therapies for the treatment of cancers are provided. In some aspects, a nucleic acid encoding a transdifferentiation determinant is administered in combination with a chemotherapeutic, electric fields therapy (e.g., TTField), or electromagnetic fields therapy to synergistically treat a cancer such as a glioma or glioblastoma (GBM).

Description

METHODS FOR THE TREATMENT OF CANCERREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 657,384, filed June 7, 2024, the entirety of which is incorporated herein by reference.BACKGROUND1. Field

[0002] The present disclosure relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods and compositions for treating cancer.2. Description of Related Art

[0003] Cancer continues to present a serious clinical problem. Glioblastoma (GBM) is the most common and aggressive type of primary brain tumor, resulting in the majority of the approximately 200,000 deaths related to tumors of the central nervous system worldwide each year. The current standard of care for patients with GBM consists of maximal surgical resection followed by radiotherapy and chemotherapy with temozolomide (TMZ). However, even with this combination of treatment, the median overall survival (OS) is typically only 10-16 months, with fewer than 10% of patients surviving for 5 years or more from the time of diagnosis. This prognosis has improved very little over the past four decades. Clearly, there is a need for more effective treatment strategies to improve outcomes for patients faced with cancers such as glioblastoma.SUMMARY

[0004] The present disclosure is based, in part, on the discovery that methods of transdifferentiation of cancer cells can synergistically interact with a second anticancer therapy, preferably a cytotoxic chemotherapeutic (e.g., temozolomide) and / or an electric fields therapy (e.g., TTFields therapy, EMF therapy) for the treatment of a cancer (e.g., a glioma such as GBM) in a mammalian subject. The transdifferentiation can utilize a gene therapy to induce the transformation of GBM into immune cells based on expressing one or more transdifferentiation determinants (e.g., myeloid regulator Pu.l in combination with either (i) Irf8 / Id2 / Batf3 in murine cells or (i) human Ikzf 1 in human cells) in the cancer. The transdifferentiation determinant may preferably be an antigen presenting cell transdifferentiation determinant. The transdifferentiation gene therapy can be delivered to a mammalian subject, such as a mouse or a human subject. The transdifferentiation strategy is generally outlined in FIG. 1, and it is shown that GBM cells can be reprogrammed into myeloid lineage, exhibiting either dendritic cell-like or macrophage-like features (e.g., as shown in FIGS. 2A-F) with upregulated pyroptotic machinery (FIG. 3). The transdifferentiation therapy resulted in killing relatively few cancer cells by itself, but this therapy was shown to synergize with a TTFields therapy to dramatically increase cancer cell killing (FIGS. 4A-B, FIGS. 5A-B, FIGS. 6A-B). Similarly, the transdifferentiation therapy when administered in combination with a cytotoxic chemotherapy (temozolomide) synergistically improved cancer cell killing (FIGS. 7A-B, FIG. 8). It is anticipated that administering a transdifferentiation therapy in combination with either (i) a cytotoxic chemotherapeutic such as temozolomide and / or (ii) a non-ionizing radiation therapy or an electric fields therapy such as TTFields, can synergistically work together to improve survival of a mammalian subject with a cancer (e.g., GBM). In some preferred aspects, the transdifferentiation is administered to the mammalian subject in combination with a non-ionizing radiation therapy such as a TTFields therapy or electromagnetic field (EMF) therapy to treat a cancer. Without wishing to be bound by any theory, the results presented herein support the idea that unexpected observation that the transdifferentiation therapy causes an immune fate reprogramming of cancer cells, creating a unique cellular vulnerability in cancer cells to cytotoxic injury by inducing pyroptotic cell death (a highly immunogenic cell death process). In some aspects, it is anticipated that using a non-ionizing radiation therapy (e.g., TTFieldsTTFields, EMF therapy) may be particularly beneficial, since possible longer-term adverse effects of ionizing radiation therapies may reduce the immune system reactivity of thesubject against the cancer. Nonetheless, it is anticipated that at least over the short term, the transdiffcrcntiation therapy may synergize with an ionizing radiation therapy for the treatment of the cancer. In some aspects, it is anticipated that additional therapeutic benefit for treating the cancer can be achieved by optionally administering a soluble PD-1 decoy (e.g., Sagawa et al., 2022; Liu et al. 2021; Shin et al., 2016), a decoy VEGF receptor (e.g., aflibercept, a VEGF-trap, or conbercept; for example as described in Holash et al. 2002, Wang et al. 2013 ) and / or an anti- VEGF antibody or scFv to the subject, optionally administered via intra-tumoral injection (FIG. 9). Reprogramming of human lung adenocarcinoma and renal cell carcinoma lines significantly enchance sensitivity to chemotherapeutic agents as measured by caspase- 1 cleavage (FIG. 10). Related compositions and methods are provided herein.

[0005] An aspect of the present disclosure relates to a method of treating a cancer in a mammalian subject, comprising: (i) administering to the mammalian subject a nucleic acid encoding one or more transdifferentiation determinants, and (ii) administering to the mammalian subject a cytotoxic anticancer therapy. The cancer may be a brain cancer, such as for example a glioma or anaplastic astrocytoma. The glioma may be a high-grade glioma or a glioblastoma (GBM). The transdifferentiation determinant may preferably be an antigen presenting cell transdifferentiation determinant. The cancer may be a breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, or mesothelioma, melanoma, neuroendocrine tumor (e.g., pancreatic neuroendocrine tumor or carcinoid tumor), or soft tissue sarcoma (e.g., rhabdomyosarcoma, Ewing sarcoma). The cytotoxic anticancer therapy may be a chemotherapy, an immunotherapy, a radiation therapy, or an anti-angiogenic therapy, preferably a decoy VEGF receptor, or a soluble PD-1 decoy. The decoy VEGF receptor may be aflibercept, a VEGF-trap, or conbercept. The decoy VEGF receptor may be a fusion protein comprising ligand binding domains of human VEGFR1 and human VEGFR2 (e.g., as described in Sargunas et al., 2025), preferably VEGFR1 (domain 2, aa 28-123) and VEGFR2 (domain 3, aa 130-230) in tandem. The chemotherapy may be an alkylating agent (e.g., temozolomide, carmustine, lomustine or procarbazine). The radiation therapy may be an ionizing or non-ionizing radiation therapy, and in some preferred aspects a non-ionizing radiation therapy is used. The non-ionizing radiation therapy may be an alternating electric field therapy, a TTFields therapy, or an electromagnetic field therapy. In some aspects, the cancer is a glioma or a glioblastoma, and wherein the transdifferentiation determinant is further defined as a cell fate determinant (CFD) that can resultin transdifferentiation of cells of the cancer into an antigen presenting cell (APC) or a myeloid lineage cell. The antigen presenting cell may be a macrophage, B cell, or dendritic cell. The one or more transdifferentiation determinants may be selected from the list consisting of SPI1, IKZF1, CTSZ, AEBP1, ARID3A, ATF5, ATP8B2, BASP1, BCL11B, BCL6, BID, CBFA2T3, CIITA, CIR1, CREG1, CTTN, EGR1, ELF4, ETS1, ETV5, FOXP1, FOXP4, GATA3, GM2A, GNS, GTF2IRD1, HHEX, H0XB3, HTATIP2, IRF5, IRF8, KIF21A, KLF4, KLF11, KLF12, KMT2E, LDB1, LEF1, LM02, L0XL2, MAGED1, MMP14, MREG, MXD1, MYBL1, NAB2, NC0A3, NFATC2, NFE2L1, N0TCH2, NR1H3, PAWR, PCGF2, PDLIM1, PLAGL1, PLCG1, POU6F1, PRKCB, PTPN14, RBI, RBFOX2, RBPMS, RORA, RUNX3, SALL2, SATB1, SOX13, STAT6, TCF7, TCF19, TDP2, TFEB, TFEC, TLE2, TSHZ2, USF1, USF2, ZBTB34, ZEB1, ZFP91, ZNF74, ZNF280B, ZNF366, ZNF483, ZNF507, ZNF827, AES, ARF2, AT0X1, BATF3, BCL11A, CBFA2T3, CEBPA, CEBPB, PPARD, ETV3, ETV6, GLMP, HELZ2, ID2, IKZF3, MAFB, MAZ, MYCL, POU2AF1, SPIB, TAF10, TFE3, VAV1, and ZFP384. The one or more transdifferentiation determinants may encode human full-length PU.l (Spil) or PU.l delta the PEST domain (dP). In some aspects, the subject is a human, the cancer is a brain cancer preferably a human glioblastoma, and the one or more transdifferentiation determinants are selected from the list consisting of: SPI1, IKZF1, CTSZ, AEBP1, ARID3A, ATF5, ATP8B2, BASP1, BCL11B, BCL6, BID, CBFA2T3, CIITA, CIR1, CREG1, CTTN, EGR1, ELF4, ETS1, ETV5, FOXP1, FOXP4, GATA3, GM2A, GNS, GTF2IRD1, HHEX, H0XB3, HTATIP2, IRF5, IRF8, KIF21A, KLF4, KLF11, KLF12, KMT2E, LDB1, LEF1, LM02, LOXL2, MAGED1, MMP14, MREG, MXD1, MYBL1, NAB2, NC0A3, NFATC2, NFE2L1, and N0TCH2. In some aspects, the one or more transdifferentiation determinants are SPI1 (GenBank: NM_001080547.2) and / or IKZF1 (GenBank: NM_006060.6), and the subject may preferably be a human. In some aspects, the subject a rodent, and wherein the one or more transdifferentiation determinants are Irf8, Id2, and / or Batf3. The nucleic acid may be comprised in a viral vector. The viral vector may be a lentivirus, an adeno-associated viruses (AAV), an adenovirus, a herpes simplex virus (HSV), a reovirus vector, a baculovirus, or a poliovirus. In some aspects, the viral vector is a lentivirus or an adeno- associated virus (AAV). The nucleic acid may be comprised in a virus-like particle (VLP). The nucleic acid may be comprised in a lipid composition, a liposome, or a nanoparticle. The nucleic acid nucleic acid encoding one or more transdifferentiation determinants may be comprised in a plasmid, wherein the one or more transdifferentiation determinants are operably linked to apromoter that can induce expression in cells of the cancer. The nucleic acid may further encode a soluble PD-1 decoy or a VEGF decoy. The cytotoxic anticanccr therapy may be an electric fields therapy. The electric fields therapy may be a TTFields therapy or an electromagnetic fields (EMF) therapy. The method may comprise administering the TTFields therapy or electromagnetic fields (EMF) therapy in combination with the chemotherapy. The chemotherapy may be paclitaxel. The TTFields therapy or EMF therapy may be administered before, after, or concurrently with the chemotherapeutic, preferably paclitaxel. In some aspects, the chemotherapy is temozolomide and wherein the cancer is a brain cancer, preferably a glioblastoma. The mammalian subject may be a human.

[0006] Another aspect of the present disclosure relates to use of a composition comprising a nucleic acid encoding one or more transdifferentiation determinants and a cytotoxic anticancer therapy for the treatment of cancer in a mammalian subject as described above or herein.

[0007] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0008] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0009] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0010] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0011] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.

[0012] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0013] The term “unit dose” when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e ., carrier, or vehicle.

[0014] The terms “cell,” and “cells,” and “cell population,” used interchangeably, intend one or more mammalian cells. The term includes progeny of a cell or cell population. Those skilled in the art will recognize that “cells” include progeny of a single cell, and there arc variations between the progeny and its original parent cell due to natural, accidental, or deliberate mutation or change.

[0015] The term “immunotherapy” refers to treatment of disease (e.g., cancer) by modulating an immune response to a disease antigen.

[0016] The term “cancer cell” as used herein refers to a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorageindependent growth potential, ability to promote tumor growth or development in an immunocompromised non-human animal model, or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell” or “cancerouscell” and encompasses cancer cells of a solid tumor and a liquid tumor. “Cancer” may be used interchangeably herein with “tumor”.

[0017] The term “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this application, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0018] An effective response of a patient or a patient’ s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder. Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse. For example, an effective response can be reduced tumor size or progression-free survival in a patient diagnosed with cancer.

[0019] “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0020] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the ail from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0023] FIG. 1. Computationally identified CFD combinations to re-engineer GBM cells into antigen presenting cells / dendritic cells.

[0024] FIGS. 2A-F. Computationally identified CFD combinations (mPF and hPI) successfully reprogram murine (A) and human (B) GBM cells, respectively to iMCs (CD45+MHC-II+) compared to Pu.l alone. mP: mouse Pu.l, mPF: murine Pu.l / Irf8 / Id2 / Batf3, hP: human Pu.l, hPI: human Pu.l / Ikzfl. C: Reprogramed murine GBM cells demonstrated ability to cross presentation to activate T cells. D: In vivo study plan. Only mPF group shows significant anti-tumor immunity (E) with more activated intratumoral immune activity (F). 2-way ANOVA; meaniSEM. * P <0.05; ** P <0.01; *** P<0.001.

[0025] FIG. 3. Single cell RNAseq analysis of murine iMCs from KR158 GBM cells shows upregulation of key pyroptosis genes.

[0026] FIGS. 4A-B. (A) Caspase 1 activation and GSDMD cleavage (B) in an AIM2 and TTFields dependent manner. Scr: scrambled shRNA, NT : nontreated.

[0027] FIGS. 5A-B. iMCs exhibit swelling reminiscent of pyropsis (blue arrow) and necrotic death (red arrow) in response to TTFields (A) to greater extents compared to cells expressing mP and ev control (B). 2-way ANOVA; mecm+SEM. *, p <0.05; ** p <0.01.

[0028] FIGS. 6A-B. Human iMCs are exquisitely more sensitive to TTFields-induced cytotoxicity (A) with pyroptotic characteristics: cell swelling (blue arrow) and post-rupture cell ghosts (red) (B). 2-way ANOVA; mean±SEM. *, P<0.05; ***, P <0.001.

[0029] FIGS. 7A-B. Cleaved Caspase- 1 (plO) can be detected when using TMZ to treat reprogramed cells indicating pyroptosis pathway activation (A). Time-course quantification of Caspase- 1 activation (plO band intensity normalized to total Caspase- 1) showing elevated baseline in reprogrammed iMCs and a pronounced activation peak at 6 h post-TMZ treatment (B).

[0030] FIGS. 8A-B. mPF reprogramming synergizes with TMZ to enhance survival and tumor control in a murine GBM model. A) Kaplan-Meier survival curves comparing intracranial GBM-bearing mice treated with mP alone, mPF reprogramming, TMZ alone, or mPF+TMZ combination (n=7 per group). mPF significantly prolongs survival versus mP (*P<0.05, log-rank), and addition of TMZ further extends survival (***P<0.001, log-rank). Two of seven mice in mPF+TMZ did not show detectable tumor at time of death. All other mice had visible tumors at time of death. B) Representative brain sections at endpoint showing tumor burden for each treatment group. Statistical comparisons by log-rank test.

[0031] FIGS. 9A-C. A) Schematic diagrams of the secreted PD-1 decoy (sPD-1) and negative control (NC) constructs. The NC lacks the PD-L1 -binding motif within the IgV domain. B) Representative immunoblots of His-taggcd sPD- 1 and NC proteins in conditioned media from KR158 GBM cells co-transfected with dPF and either sPD-1 or NC. Only sPD-1 is detected by the anti-PD-1 monoclonal antibody (RMP1-14), which recognizes the PD-L1 -binding motif absent in NC. C) Proposed in vivo strategy: an oncolytic virus delivers reprogramming factors to tumor cells, followed by anti-mitotic treatment. Intratumoral sPD-1 decoy counters T-cell exhaustion, while systemic anti-VEGF antibody or intratumoral VEGF Fab / scFv modulates inflammation. Tumor growth will be tracked, and immune phenotyping performed.

[0032] FIG. 10. Human lung adenocarcinoma and renal cell carcinoma lines were reprogrammed into iMCs using hPI and, upon treatment with various chemotherapeutic agents for the respective tumor types, displayed markedly increased caspase-1 cleavage (measured as higher ratios of hi / lo peaks), compared to IMCs alone or to non-reprogrammed ev cells.DETAILED DESCRIPTIONI. Definitions

[0033] " Transdifferentiation" is the conversion of one cell type into another cell type without the requirement of an intermediary step of a pluripotent state. For example, in some aspects, a cancer cell may be transdifferentiated into an antigen-presenting cell (APC) or an induced myeloid (iMC) type cell. In aspects where a cancer cell is transdifferentiated into another cell type, it is anticipated that the cancer cell may be considered to be at various stages of differentiation, including both terminally differentiated cancerous cells as well as less differentiated cancer cells such as cancer stem cells (CSC)

[0034] An "Antigen-Presenting Cell" (APC) is a cell that displays antigen complexed with major histocompatibility complex II (MHC-IT) on their surfaces. APCs can process external antigens and present them to other immune cells, such as T cells. Macrophages, B cells and dendritic cells (professional antigen presenting cells) are naturally occurring professional APCs. An APC may also express one or more co-stimulatory molecules. In some aspects, when a cancer cell is transdifferentiated into an APC (e.g.. a dendritic cell, DC-like cell), the transdifferentiated cancer cell expresses at least one or more APC cell markers that are not associated with the cancer cell prior to transdifferentiation.

[0035] " Hot" and "Cold" tumors refer to the immunological characteristics of the tumor microenvironment, particularly in terms of immune cell infiltration and activity. "Hot" tumors are characterized by a high presence of tumor-infiltrating lymphocytes (TILs), elevated expression of immune activation markers, and pro-inflammatory cytokines. These tumors often express high levels of neoantigens and are more likely to respond to immunotherapies, such as immune checkpoint inhibitors, due to an already primed immune environment. In contrast, "Cold" tumors exhibit minimal immune cell infiltration and tend to have immunosuppressive microenvironments dominated by regulatory T cells, myeloid-derived suppressor cells, or inhibitory cytokines. These tumors may lack sufficient antigen presentation or express low levels of immune recognition markers, making them less responsive to immune-based therapies. Strategies to convert "Cold" tumors into "Hot" tumors include the use of oncolytic viruses, radiation therapy, epigenetic modifiers, or combination immunotherapies aimed at enhancing antigen presentation, promotingT-cell infiltration, and reversing immune suppression. Understanding the immune phenotype of a tumor is critical for selecting and designing effective immunotherapeutic interventions.

[0036] The “tumor microenvironment (TME)” refers to the complex and dynamic network of non-cancerous cells, signaling molecules, and structural components that surround and interact with tumor cells. It includes immune cells (such as T cells, B cells, macrophages, dendritic cells, and myeloid-derived suppressor cells), stromal cells (including fibroblasts and endothelial cells), extracellular' matrix proteins, blood vessels, and a variety of cytokines and chemokines. The TME plays a critical role in tumor progression, immune evasion, and therapeutic resistance. It can be immunosuppressive, limiting the effectiveness of anti-tumor immune responses through mechanisms such as the secretion of inhibitory cytokines (e.g., TGF-0, IL- 10), expression of immune checkpoint ligands (e.g., PD-L1), and recruitment of regulatory immune cells. The TME also influences angiogenesis, metabolic reprogramming, and tissue remodeling, all of which support tumor growth and metastasis. Therapeutic strategies targeting the TME aim to reprogram the immune landscape, normalize the vasculature, and disrupt tumor- supportive stromal interactions to enhance the efficacy of conventional and immune-based therapies.

[0037] The “immune system” is a complex network of cells, tissues, and molecules that defends the body against infections, abnormal cells, and foreign substances. It comprises two main branches: the innate immune system and the adaptive immune system. The innate immune system provides immediate, non-specific defense through physical barriers (such as skin and mucosa), phagocytic cells (e.g., macrophages, neutrophils), natural killer (NK) cells, and soluble mediators like complement proteins and cytokines. The adaptive immune system provides a slower but highly specific response mediated by lymphocytes, including B cells and T cells. B cells produce antibodies that neutralize pathogens, while T cells coordinate cellular responses, including the direct killing of infected or malignant cells. The immune system relies on processes such as antigen recognition, activation, clonal expansion, and memory formation to provide lasting protection. Regulatory mechanisms maintain self-tolerance and prevent autoimmunity. Dysregulation of the immune system can contribute to diseases such as autoimmunity, immunodeficiency, and cancer. Immunotherapies aim to modulate immune responses by enhancing or suppressing specific components to treat these conditions.

[0038] Antigen-presenting cells, such as dendritic cells, B cells, and macrophages, capture and process antigens and present them on major histocompatibility complex (MHC) molecules to activate naive T cells, linking innate and adaptive immunity.

[0039] The immune response to cancer begins with the recognition and processing of tumor-associated antigens by antigen-presenting cells, leading to activation and clonal expansion of cytotoxic and helper T cells. Cytotoxic T cells induce apoptosis of tumor cells presenting antigen-MHC class I complexes, while helper T cells produce cytokines that support and enhance the immune response. B cells contribute by producing tumor- specific antibodies that facilitate antibody-dependent cellular cytotoxicity and opsonization. Other immune cells, including natural killer cells and macrophages, participate in tumor surveillance by targeting cells with altered or missing self-markers.

[0040] Tumors evade immune destruction by downregulating antigen presentation, secreting immunosuppressive cytokines such as transforming growth factor-beta (TGF-0) and interleukin- 10 (IL- 10), recruiting regulatory immune cells, and expressing immune checkpoint molecules like PD-L1 that inhibit T cell activity. Chronic exposure to tumor antigens can lead to T cell exhaustion, impairing immune effectiveness. Immunotherapies seek to overcome these barriers by enhancing antigen presentation, blocking inhibitory signals, providing engineered immune cells, and stimulating immune activation. A comprehensive understanding of the immune system and its interactions with tumors is essential for developing effective cancer treatments.

[0041] " Dendritic cells” are antigen-presenting cells having the broadest range of antigen presentation and the ability to activate naive T cells. Their main function is to process antigen material and present it on the cell surface to T cells. DCs present antigen to both helper and cytotoxic T cells. In addition to the conventional MHC class Il-mediated presentation to CD4 helper T cells and MHC class I-mediated presentation to CD8+cytotoxic T cells, dendritic cells are also capable of cross-presentation. Cross-presentation refers to the process by which exogenous antigens, typically destined for MHC class II presentation, are redirected and presented via MHC class I molecules. This mechanism is critical for the initiation of cytotoxic T cell responses against pathogens that do not directly infect antigen-presenting cells, as well as for the induction of antitumor immunity..

[0042] "Dendritic-like cells" (also termed "DC-like cells") are cells that have been transdiffcrcntiatcd to be able to act as antigen presenting cells. In some embodiments DC-likc cells express MHC-II and CDllc. In some embodiments, DC-like cells express one or more factors, including but not limited to, CD1 1c, BDCA-1, CD8, CD8a, CD103, and CD205.

[0043] "Macrophages" are a type of white blood cell of the immune system that engulf and digest cellular debris, foreign substances, microbes, cancer cells, etc. in a process called phagocytosis. The engulfed material is typically processed by the macrophage and antigens can be presented at the cell surface in the context of MHC-II.

[0044] "Macrophage-like cell" are cells that have been transdifferentiated to be able to act as antigen presenting cells. In some embodiments, macrophage-like cells express MHC-II and CD1 lb and / or CD68. In some embodiments, macrophage-like cells express one or more factors including but not limited to, CD 14, CD 16, CD64, CD71, and CCR5.

[0045] ‘ ‘B cells” are lymphoid cells that function as antigen-presenting cells and play a central role in humoral immunity. Their primary function is the production of antigen- specific antibodies following activation. B cells recognize native antigens through the B cell receptor (BCR), internalize the antigen, process it, and present peptide fragments on MHC class II molecules to helper T cells. In addition to antibody production, B cells contribute to immune regulation and memory. Upon activation, B cells can differentiate into plasma cells, which secrete large quantities of antibodies, or memory B cells, which persist long-term to confer immunological memory. B cells are also capable of presenting antigen to T cells, thereby contributing to the activation and shaping of adaptive immune responses.

[0046] ‘ ‘B cell-like cell” are cells that have been transdifferentiated to be able to act as antigen presenting cells. In some embodiments, B cell-like cells express MHC-II and CD19. In some embodiments, B cell-like cells express one or more factors including but not limited to CD20, B220, CD21, CD22, CD40, CD138 and CD27.

[0047] "Cytokines" and "Chemokines" are small secreted proteins that mediate and regulate immunity, inflammation, and hematopoises. Chemokines, a specialized subset of cytokines, primarily regulate the directed migration (chemotaxis) of immune cells to sites ofinfection, inflammation, or injury. Antigen-presenting cells, including dendritic cells, macrophages, and B cells, arc major sources of cytokines and chcmokincs during immune activation. Common cytokines produced by APCs include but are not limited to interleukin- 12 (IL- 12), which promotes differentiation of naive T cells into Thl cells; interleukin-6 (IL-6), which supports inflammation and B cell maturation; tumor necrosis factor-alpha (TNF-a), which contributes to local inflammation and leukocyte recruitment; and interleukin- 1 beta (IL- 1 P), which promotes fever and T cell activation. APCs also secrete chemokines such as CCL2 (MCP-1), CCL3 (MIP-la), and CXCL10 (IP-10), which recruit monocytes, T cells, and natural killer cells to sites of antigen presentation and inflammation. Through the production of these soluble mediators, APCs play a central role in initiating and shaping both innate and adaptive immune responses.

[0048] " Immune therapy" or "Immunotherapy" is the treatment of disease, such as cancer, by activating or suppressing the immune system. Immunotherapies can be designed to elicit or amplify an immune response. There are several types of immunotherapy, including but not limited to monoclonal antibodies, immune checkpoint inhibitors, cancer vaccines, adoptive cell transfer, and cytokine therapies. Monoclonal antibodies, such as rituximab or trastuzumab, target specific antigens on tumor cells to mediate immune destruction. Immune checkpoint inhibitors, such as anti-PD-1 (nivolumab) or anti-CTLA-4 (ipilimumab), can block inhibitory pathways in T cells, thereby enhancing anti-tumor immunity. Cancer vaccines aim to stimulate the immune system to recognize and attack tumor-associated antigens. Adoptive cell transfer generally involves the infusion of autologous or engineered immune cells, such as CAR T cells, which are modified to recognize specific tumor antigens. Cytokine therapies, such as interleukin-2 (IL-2) or interferonalpha, enhance immune cell proliferation and activation. These modalities can be used alone or in combination to improve immune-mediated control of disease.

[0049] A "nucleic acid" includes both RNA and DNA. RNA and DNA include, but are not limited to, cDNA, genomic DNA, plasmid DNA, RNA, mRNA, condensed nucleic acid, nucleic acid formulated with cationic lipids, and nucleic acid formulated with peptides or cationic polymers. Nucleic acid also includes modified RNA or DNA. The nucleic acid may comprise one or more chemical modifications or non-natural nucleotides, e.g., to slow degradation after administration to a mammalian subject.

[0050] An “expression vector” refers to a nucleic acid (e.g., RNA or DNA) encoding an expression product (c.g., polypeptide), such as a transdiffcrcntiation determinant. An expression vector may be, but is not limited to, a virus, a modified virus, a recombinant virus, an attenuated virus, a plasmid, a linear DNA molecule, or an mRNA. An expression vector is capable of expressing one or more polypeptides in a cell, such a mammalian glioblastoma or cancer cell. The expression vector may comprise one or more sequences necessary for expression of the encoded expression product. A variety of sequences can be incorporated into an expression vector to alter expression of the coding sequence. The expression vector may comprise one or more of: a 5' untranslated region (5' UTR), an enhancer, a promoter, an intron, a 3' untranslated region (3' UTR), a terminator, and a polyA signal operably linked to the DNA coding sequence. The nucleic acid encoding the transdifferentiation determinant may be operably linked to a promoter for expressing the transdifferentiation determinant in the GBM cell.

[0051] The term "plasmid" refers to a nucleic acid that includes at least one sequence encoding a polypeptide (such as a transdifferentiation determinant) that is capable of being expressed in a glioblastoma cell. A plasmid can be a closed circular DNA molecule. A variety of sequences can be incorporated into a plasmid to alter expression of the coding sequence or to facilitate replication of the plasmid in a cell. Sequences can be used that influence transcription, stability of a messenger RNA (mRNA), RNA processing, or efficiency of translation. Such sequences include, but are not limited to, 5' untranslated region (5' UTR), promoter, introns, and 3' untranslated region (3' UTR). Plasmids can be manufactured in large scale quantities and / or in high yield. Plasmids can further be manufactured using cGMP manufacturing. Plasmids can be transformed into bacteria, such as E. coli, or eukaryotic cells. In some aspects, a transdifferentiation determinant is included in a plasmid to be administered to a mammalian subject such as a human to treat a cancer.

[0052] A “promoter” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter may comprise one or more additional regions or elements that influence transcription initiation rate, including, but not limited to, enhancers. A promoter can be, but is not limited to, a constitutively active promoter, a conditional promoter, an inducible promoter, or a cell-type specific promoter. Examples of promoters can be found, forexample, in WO 2013 / 176772. The promoter can be, but is not limited to, CMV promoter, IgK promoter, mPGK, SV40 promoter, b-actin promoter (such as, but not limited to a human or chicken b-actin promoter), a-actin promoter, SRa promoter, herpes thymidine kinase promoter, herpes simplex virus (HSV) promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus maj or late promoter (Ad MLP), rous sarcoma virus (RSV) promoter, EFla promoter, and RNA promoters (including, but not limited to Hl, U6 and 7SK). The CMV promoter can be, but is not limited to, CMV immediate early promoter, human CMV promoter, mouse CNV promoter, and simian CMV promoter. The promoter can also be a hybrid promoter. Hybrid promoters include, but are not limited to, CAG promoter.

[0053] “Operably linked” refers to the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter operably linked to a coding sequence will direct RNA polymerase mediated transcription of the coding sequence into RNA, including mRNA, which may then be spliced (if it contains introns) and translated into a protein encoded by the coding sequence. A coding sequence can be “operably linked” to one or more transcriptional or translational control sequences. A terminator / polyA signal operably linked to a gene may terminate transcription of the gene into RNA and directs addition of a poly A signal onto the RNA.

[0054] A “heterologous” sequence is a sequence which is not normally present in a cell, genome or gene in the genetic context in which the sequence is currently found. For example, a heterologous sequence can be a coding sequence linked to a different promoter sequence relative to the native coding sequence. A heterologous sequence can differ from its corresponding native sequence in having one or more introns removed. A heterologous sequence can also be present in the context of an expression vector, such as, but not limited to, a plasmid or viral vector.II. Transdifferentiation determinants

[0055] A transdifferentiation determinant is a gene which when expressed either by itself or in combination with one or more additional transdifferentiation determinants in a cancer cell, such as a glioblastoma cell, can convert (transdifferentiate) the cancer cell into another cell type without the requirement of an intermediary pluripotent state of the cell. Transdifferentiationdeterminants are provided below in Table 1 . Transdifferentiation determinants that may be used include those described in WO2021133775A1. Transdiffcrcntiation determinants that can be used, e.g., to promote the transition of human glioblastoma to antigen presenting cell, are provided in Table 1. Preferably the transdifferentiation determinants human Pu.l and Ikzfl are expressed in a human cancer cell to promote transdifferentiation to an APC.* For some genes and proteins, multiple isoforms are known. The indicated NCBI Reference number represents a single isoform and is provided as an exemplary sequence. Use of any of the known isoforms is contemplated.III. Combination Therapies

[0056] In some aspects, it has been shown herein that administration of a nucleic acid encoding one or more transdifferentiation determinants in combination with a second anticancer therapy (e.g., a chemotherapy such as temozolomide, or TTFields) can additively or preferably synergistically interact to improve treatment of the cancer in a mammalian subject. Without being bound by any theory, data is provided herein that supports the idea that the different therapies can stimulate the immune system of the mammalian subject to attack the cancer. The nucleic acid encoding the one or more transdiffcrcntiation determinants can be administered before, after, or substantially at the same time as the second anticancer therapy. The administration of the different therapies may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 hours or more, or by 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3 weeks or more. Administration of one or both of the different anticancer therapies can be repeated, if desired. Preferably, the second anticancer therapy (e.g., cytotoxic therapy, chemotherapy such as temozolomide and / or a non-ionizing radiation therapy such as TTFields) are administered to the mammalian subject after the administration of the nucleic acid encoding the transdifferentiation determinant(s). The second anticancer therapy can be a chemotherapy, radiation therapy, immunotherapy, additional gene therapy, or electromagnetic therapy (e.g., TTFields). Additionally, an anticancer surgery (e.g., surgery to remove one or more primary tumor or metastases) may also be administered where appropriate.A. Chemotherapeutics

[0057] It is anticipated that a wide variety of different chemotherapeutic s can be used in aspects of the present disclosure. In some preferred aspects, temozolomide is administered in combination with a nucleic acid encoding one or more transdifferentiation determinants.

[0058] A variety of dosing regimens can be used. For example, in humans, temozolomide may be administered to a patient at a dose of 100, 150, or 200 milligrams (mg) per square meter (m[2]) of body size once a day on Days 1 to 5 of each 28-day cycle for 12 cycles starting at 4 weeks after receiving radiation treatment. TMZ can also be dosed at 75mg per square meter daily for 21 days on and 7 days off or some variations of this on-off schedule. It is anticipated that administration of a transdifferentiation to a mammalian subject may allow for reduced dosages of temozolomide to be administered to a patient to treat a cancer such as a brain cancer (e.g., GBM, anaplastic astrocytoma), melanoma, neuroendocrine tumors (e.g., pancreatic neuroendocrine tumors and carcinoid tumors), and soft tissue sarcoma (e.g., rhabdomyosarcoma, Ewing sarcoma).

[0059] The chemotherapeutic may be an alkylating agent. Alkylating agents damage cell DNA to prevent cancer cells from dividing. Nitrosoureas are a particular type of alkylating agent. Unlike other alkylating agents, nitrosoureas can travel into your brain and kill cancer cells there. Nitrosoureas can be used to treat some brain tumors. Alkylating agents damage cell DNA to prevent cancer cells from dividing. Nitrosoureas are a particular type of alkylating agent. Unlike other alkylating agents, nitrosoureas can travel into your brain and kill cancer cells there. Nitrosoureas can be used to treat brain cancers. Selected alkylating agents include: altretamine, bendamustine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazinc, ifosfamide, mechlorethamine, melphalan, oxaliplatin, procarbazine, temozolomide, thiotepa, trabectedin. Selected nitrosoureas include: carmustine, lomustine, and streptozocin.

[0060] Other chemotherapeutic s that may be used in combination with a transdifferentiation therapy include antimetabolites, topoisomerase inhibitors, mitotic inhibitors such as paclitaxel, antitumor antibiotics such as anthracyclines, and other chemotherapeutics. Antimetabolites prevent cancer cells from making the genetic material they need to create new cells (e.g., 5-fluoro uracil, hydroxyurea, etc.). Other chemotherapeutics that are contemplated for use with the present disclosure include: Platinum-based chemotherapies that may cross-link DNAstrands (e.g., cisplatin, carboplatin, oxaliplatin), proteasome inhibitors (e.g., bortezomib), and histone deacetylase inhibitors (e.g., vorinostat).B. Radiation Therapies

[0061] In some aspects the transdifferentiation therapy is administered in combination with a radiation therapy. The radiation therapy may be an ionizing radiation therapy such as an external beam radiation therapy or a brachytherapy. Preferably, the radiation therapy is a non-ionizing radiation therapy such as an electromagnetic field (EMF) therapy, an alternating electric field therapy, or a TTFields therapy. Without wishing to be bound by any theory, data presented herein support the idea that synergy of a transdifferentiation therapy and a non-ionizing therapy may result at least in part due to effects on the immune system of the subject that can more effectively attack the cancer. In some aspects, non-ionizing radiation therapies arc preferred over ionizing therapies as it is anticipated that this may further increase beneficial immune system anticancer effects in the subject resulting from the combination of the transdiffcrcntiation therapy and the non-ionizing radiation therapy.1. TTFields Therapy

[0062] Tumor-treating fields (TTFields) utilize alternating electric fields to exert biophysical force on charged and polarisable molecules known as dipoles. TTFields can be used as a complement to treat a brain cancer brain cancer such as GBM. TTFields have been used in combination with standard therapy, has been shown to extend survival for patients with newly diagnosed GBM, recurrent GBM and mesothelioma, leading to the clinical approval of this approach by the FDA. TTFields represent a non-invasive anticancer modality consisting of low- intensity (1-3 V / cm), intermediate-frequency (100-300 kHz), alternating electric fields delivered via cutaneous transducer arrays configured to provide optimal tumour-site coverage (Rominiyi et al., 2021).

[0063] Tumor-treating fields (TTFields) represent an emerging non-invasive anticancer therapeutic modality that involves the transcutaneous delivery of low-intensity (1-3 V / cm), intermediate-frequency (100-300 kHz), alternating electric fields (the approach is also known as alternating electric field therapy) that exert biophysical force on charged and polarisable molecules known as dipoles. The beneficial effects of TTFields therapy are influenced by treatment duration(with evidence that application >18 h / day improves survival; see Toms etal., 2019), electrical field intensity (where increased intensity confers greater reduction in cell proliferation) and electrical field frequency (Kirson et al., 2007), which varies between cancer types — in the case of glioma cells, TTFields are clinically delivered at an optimum frequency of 200 kHz (Kirson et al., 2004).

[0064] The frequency of the alternating field has also been shown to provide different biological effects. Low-frequency electric fields (<1 kHz) influence cell membrane polarisation and can alter the behavior of excitable tissue, such as action potential firing in neuronal cells (Moghadam et al., 2008). On the other hand, high-frequency fields (>500 kHz) cause charged and / or polar molecules inside cells to vibrate, creating friction and causing kinetic energy to transfer between molecules, which can be radiated out as thermal energy, leading to tissue heating (Cheung et al., 1984). Intermediate-frequency alternating electric fields (100-500 kHz) do not generate enough thermal energy to cause significant tissue heating and alternate too quickly to trigger action potential firing and were consequently originally thought to lack any beneficial effects (Davies et al., 2013). However, Kirson et al. demonstrated that low-intensity alternating electric fields delivered at 100-300 kHz successfully inhibited cancer cell growth, both in vitro (using cell lines derived from melanoma, glioma, lung, prostate, and breast cancer) and in vivo, by interfering with microtubule polymerisation during mitosis (Kirson et al., 2004). These findings led to the first pilot study (EF-07) in GBM patients launched in 2004 (Elion et al. 2007) and, eventually, to the development of TTFields as a strategy for treating cancer.

[0065] TTFields can be used to treat a variety of cancers of the central nervous system such as brain cancers. Preferably, TTFields are administered in combination with a nucleic acid encoding a transdifferentiation determinant to treat a brain cancer, such as a recurrent GBM, newly diagnosed GBM, brain metastases, and / or other extracranial tumors.2. Electromagnetic Fields (EMF) Therapy

[0066] The non-ionizing radiation therapy may be an EMF Therapy. The EMF therapy may be a pulsed electromagnetic field (PEMF) therapy, Pulsed electric fields (PEF), or cold atmospheric plasma (CAP) therapy (e.g., Cheng et al. 2015; Vadala et al., 2016). A PEF device typically includes a pulsed voltage source and an electrode (Xiao et al., 2013). The voltage source generates a pulsed voltage, and an electric field is generated between the two electrodes. ThePEMF device typically includes a current source and a magnetic field generating device. Due to electromagnetic induction, a pulsed magnetic field is generated. Plasma can be generated as ionized gas from high voltage. Plasma includes electrons, ions, and neutral particles. Cold atmospheric plasma (CAP) typically has a temperature in the range of about 102— 103K, and the pressure is typically close atmospheric pressure. When CAP is applied to cancer cells or tissues, physical factors such as electromagnetic fields and ultraviolet rays, and chemical factors such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) can be generated (Shashurin et al., 2015). CAP may exert additional consequences on cancer cells, contributing to cancer cell killing. PEMF may very low frequency bands, and clinical applications may utilize pulsed or sinusoidal magnetic fields below 1000 Hz with magnetic induction strengths within 100 Gs (1 Gs = 10^ T). This pulsed magnetic field typically does not result in significant thermal effects in organisms due to the short duration of action and low irradiation power. PEF and PEMF are field therapies in which the treatment target (e.g., cancer tumor) is placed in the field, while plasma therapy is a kind of contact therapy in which the ionization-generated substance in question needs to be in direct contact with the treatment target.IV. Cancers

[0067] In some aspects, the present disclosure provides methods for treating brain cancers such as gliomas including GMB. Nonetheless, it is anticipated that the methods provided herein can be used to treat a variety of cancers. Cancers that may be treated include but are not limited to cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, smalllymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoidcs; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.IV. Examples

[0068] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Results

[0069] Although immunotherapies, such as immune checkpoint inhibitors, have shown significant benefits for other solid tumors, their efficacy in glioblastoma multiformc (GBM) remains limited. The development of new treatments is further complicated by the high degree of intra- and inter-patient heterogeneity in GBM and its immunosuppressed or “cold” tumor microenvironment (TME). A gene therapy approach is used here that can re-engineer GBM cells into immune cells, specifically antigen-presenting cells. This strategy effectively "reheats" the cold TME of GBM and significantly retards tumor growth (PCT / US2020 / 066557, FIG. 1).

[0070] The viral delivery of a combination of computationally identified cell fate determinants (CFDs), anchored by the myeloid regulator Pu.l, along with species-specific functional differentiation factors (murine Irf8 / Id2 / Batf3 for mPF or human Ikzfl for hPI), successfully reprograms GBM cells into the myeloid lineage, exhibiting either dendritic cell-like or macrophage-like features (FIGS. 2A-C). Further in vivo study demonstrated that the reprogramed cells can heat up tumor and provide anti-tumor immunity (FIGS. 2D-F).

[0071] Notably, the induced myeloid cells (iMCs) (mPF) upregulate pyroptotic machinery, including gasdermin D (GSDMD, which is primarily expressed in terminal myeloid cells, and Toll-like receptors (TLRs) that activate alternative inflammasomes, to a greater extent compared to mP alone and empty vector (ev) controls (FIG. 3).

[0072] The results described herein demonstrate that Tumor Treating Fields (TTFields) induce focal nuclear envelope disruption, leading to the formation of large cytosolic micronuclei clusters in GBM cells. These clusters vigorously recruit and activate the cGAS / STING and AIM2 / Caspase 1 inflammasomes, creating a comprehensive in situ immunizing platform against GBM tumors. The data show that TTFields-activated AIM2 leads to caspase 1 stimulation, which in turn cleaves the pore-forming GSDMD, causing membrane damage and cell death (FIGS. 4A- B). This process is reminiscent of pyroptosis, a specialized type of programmed necrosis vital to innate immunity and the initiation of inflammatory responses in myeloid cells known to have robust anti-tumor immunity. Notably, only 50% of the mouse and human GBM cell lines we tested had detectable GSDMD, suggesting that other gasdermins (GSDMs) might be involved. Many cancers downregulate or mutate various GSDMs and components of the pyroptotic machinery to evade pyroptosis and immune stimulation.

[0073] It was then tested whether the combination of the two approaches could enhance pyroptosis induction. As expected, iMCs dramatically enhanced TTFields-induced cytotoxicity compared to GBM cells expressing Pu.l alone or empty vector (ev) controls. Importantly, several morphological characteristics of pyroptosis were observed in both murine (FIGS. 5A-B) and human (FIGS. 6A-B) iMCs, including cellular swelling and post-rupture cell ghosts, which are only occasionally observed in GBM cells expressing Pu.l alone.

[0074] Additionally, the use of temozolomide (TMZ) was tested. TMZ is a cytotoxic chemotherapy drug that typically induces apoptosis, which is not an immune-activating form of cell death. However, under conditions favoring programmed necrosis provided by the fate conversion regimen, classical apoptosis may also potentiate a switch to pyroptosis (FIGS. 7A-B).

[0075] The inventors evaluated reprogrammed cell therapy with and without temozolomide (TMZ) in a murine GBM model (FIGS. 8A-B). The combination of mPF reprogramming plus TMZ significantly prolonged survival and reduced tumor growth comparedto either treatment alone. Although tumors were smaller in all reprogrammed groups, two of seven mice in the mPF+TMZ cohort died without detectable tumor, suggesting potential CNS inflammation from heightened immune activation. By contrast, all other mice succumbed with measurable tumor burden. Given the heterogeneity of human GBM, the inventors anticipate that better control of reprogramming may be achieved (e.g., improved anti-mitotic therapeutic efficiency) by combining the reprogramming approach with an additional anticancer therapy such as an intratumorally administered anti-PD-1 therapeutic, a systematic anti-VEGF antibody, and / or an intratumorally administered VEGF Fab or scFv; these approaches can be tested in a humanized mouse model in case of under- or over-stimulation of immune system (FIGS. 9A-C). The investigators tested hPI-reprogrammed cell therapy, with and without chemotherapeutic agents, across multiple human cancer cell lines (FIG. 10). As in murine models, hPI reprogramming alone increased baseline caspase- 1 activation, and combining hPI with chemotherapeutic agents further augmented caspase- 1 cleavage to a significant degree.Example 2 - Methods

[0076] Generation of lentiviral CFD combination constructs for viral production. ORF expression clones for mouse and human CFDs were purchased from Genecopoeia and sequence verified. To create mF3, cDNAs encoding mouse IRF8, BATF3, and ID2 were synthesized, linked by P2A and T2A cleavage sequences, and cloned into the lentiviral vector pSF- simple (pSF-simple-T2A-mIRF8-P2A-mBATF3-E2A-mID2). For PU.l constructs, cDNAs encoding mouse and human full-length PU.l (Spil) and PU.l delta the PEST domain (dP)34were cloned into the lentiviral response plasmid pSF-Lenti that contains PGK-puro to generate pSF- Lenti-PU.l and pSF-lenti-detaPU.l. For lentiviral production, 7.5xl06HEK 293T cells were plated overnight in intact DMEM in a 10cm Poly-D lysine hydrobromide (SIGMA) coated dish, followed by transfection with a 2: 1 ratio of total DNA in PEI (pg) together with the viral packaging and envelop plasmids PSPAX2 and PMD2.G, respectively in advanced DMEM medium supplemented with 1.25%FBS, IX pyruvate, lOmM HEPES and lOmM sodium butyrate. Media were replaced at 16 hrs after transfection. Viral supernatants were collected every 24hrs and centrifuged for 20hrs at 24000g to sediment viral particles, which were resuspended in opti-MEM and viral titers measured.

[0077] Cell Culture. Mouse GBM cell lines KR158, KR 158-luc, GL261 , and human GBM cell lines U87, LN428, LN827, and lung cancer line A549, renal cancer line 786-0 were cultured in DMEM media with 10% FBS and 1% pen / strep. For lentiviral transduction, GBM cells in baseline DMEM media were incubated with indicated lentiviral constructs for 24hrs, followed by media replacement to RPMI 1640 supplemented with 10% FBS, 1% pen / strep and 3pg / p 1 puromycin for selection of transduced cells. Five days after transduction, cells were passaged into RPMI 1640 media supplemented with 10% FBS and 1% pen / strep without puromycin until analysis. TTFields were applied to cancer cell lines using the Inovitro™ system (Novocure, Israel). GBM cells were treated with TTFields at the clinically approved frequency of 200 kHz. Temozolomide was used at the concentration of lOOpM. Gemcitabine, paclitaxel, and everolimus were used at the concentration of 62.5nM, 12.5pM, and 12.5pM separately.

[0078] Western blotting. Cells were treated on ice for 20 min with RIPA buffer (150mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 25mM, PH 7.4 Tris) containing a protease inhibitor cocktail (Roche), followed by centrifugation at 13,000g at 4 °C for 20 min. Supernatants were collected and protein concentration determined using a protein assay dye reagent (Bio-Rad). Equal amounts of proteins were resolved by SDS-PAGE and transferred onto poly vinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk in TBST, then probed with indicated primary antibodies (1:1000) at 4 °C overnight, washed with TBST, and incubated with HRP-conjugated antirabbit or anti-mouse secondary antibodies (1:5000) at room temperature for 1 hr.

[0079] Caspase-1 activation assay. Caspase- 1 activation assay was performed according to the manufacturer’s protocol (Pyroptosis 660 Caspase- 1 Kit, BIO-RAD, Cat#ICT9158). FLICA is cell-permeant and will efficiently diffuse in and out of all cells. If there is an active caspase- 1 enzyme inside the cell, it will covalently bind with YVAD-FMK and retain the fluorescent signal within the cell. Unbound FLICA will diffuse out of the cell during the subsequent wash steps. Therefore, positive cells will retain a higher concentration of FLICA and fluoresce brighter than negative cells. There is no interference from pro-caspases or inactive forms of the enzymes. 6 hours after indicated treatment, adherent cells were trypsinized and washed twice in wash buffer, resuspended and incubated with FLICA at the dilution of 1:30 at 37 oC for 1 hr., washed andanalyzed by BD FACSymphony A5 SE at the channel of APC. Debris and doublets were excluded out from analysis.

[0080] In vivo experiments with reprograming and TMZ treatment. KR158 cells were infected with the indicated CFD lentiviruses for 24 hrs. prior to surgery and resuspended at a density of 3xl05cells per 3pl PBS. 3xl05cells were slowly (IpL / min) implanted into the posterior frontal lobe of the brain of 6-week-old C57BL / 6J mice (Jackson Lab) using an automated mouse stereotaxic localizer (Stoelting's) at 2mm lateral and 3.5mm deep on the right side, with the fontanelle as the reference point. Seven days post-implantation, mice received temozolomide (TMZ) treatment via intraperitoneal injection at a dosage of 25 mg / kg / day for five consecutive days. Overall survival was monitored and recorded as the study endpoint, ev + V, ev + TMZ: n =5; mPF + V, mPF + TMZ: n =7. Fog-rank test was used to compare survival rates.

[0081] Decoy construction design. The sPD-1 and Negative Control (NC) constructs were synthesized using the Genescript service. sPD-1 was constructed by ligating the synthesized cDNA encoding the codon-optimized extracellular’ domain protein of mouse wild-type PD-1 [amino acids (aa) 1 to 169] and 6 His tag into the pGenLenti vector (pGenEenti-mouse PD-1, Eot#U089YHG070-2 / J201884, Genescript). The Negative Control was constructed by ligating the synthesized cDNAs encoding the codon-optimized extracellular domain protein of sPD-1 without the PD-E1 binding motif (aa 1 -69 and 78-169) and 6 His tag into the same pGenEenti vector. Culture supernatants of KR158 mouse GBM cells transduced with sPD-1 or NC were collected and subjected to Western blotting with mouse anti-His and anti-mouse PD-1.

[0082] Building on this strategy, analogous decoy proteins targeting human PD-1 and VEGF can be constructed using a tumor- specific adeno-associated virus (AAV) delivery platform. The human PD-1 decoy will include the codon-optimized extracellular domain (amino acids 1- 167) of human PD-1 fused to a detection tag. For the VEGF decoy, a high-affinity ligand trap can be generated by fusing the ligand-binding domains of human VEGFR1 (domain 2, aa 28-123) and VEGFR2 (domain 3, aa 130-230) in tandem, followed by the Fc region of human IgG to enhance stability and systemic half-life. Both constructs will be cloned into AAV vectors driven by a tumor- selective promoter, enabling spatially restricted expression within the tumor microenvironment and minimizing off-target effects.* * *

[0083] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the ail are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.WO2021133775A1Rominiyi, O., Vanderlinden, A., Clenton, S.J. et al. Tumour treating fields therapy for glioblastoma: current advances and future directions. Br J Cancer 124, 697-709 (2021).Toms, S., Kim, C., Nicholas, G. & Ram, Z. Increased compliance with tumor treating fields therapy is prognostic for improved survival in the treatment of glioblastoma: a subgroup analysis of the EF- 14 phase III trial. J. Neuro-OncoL 141, 467-473 (2019).Kirson, E. D., Dbaly, V., Tovarys, F., Vymazal, J., Soustiel, J. F., Itzhaki, A. et al. Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors. Proc. Natl Acad. Sci. USA 104, 10152-10157 (2007).Kirson, E. D., Gurvich, Z., Schneiderman, R., Dekel, E., Itzhaki, A., Wasserman, Y. et al. Disruption of cancer cell replication by alternating electric fields. Cancer Res. 64, 3288-3295 (2004).Moghadam, M., Firoozabadi, S. & Janahmadi, M. 50 Hz alternating extremely low frequency magnetic fields affect excitability, firing and action potential shape through interaction with ionic channels in snail neurones. Environmentalist 28, 341-347 (2008).Cheung, A. Y. & Neyzari, A. Deep local hyperthermia for cancer therapy: external electromagnetic and ultrasound techniques. Cancer Res. 44, 4736s-4744s (1984).Davies, A. M., Weinberg, U. & Palti, Y. Tumor treating fields: a new frontier in cancer therapy. Ann. N. Y. Acad. Sci. 1291, 86-95 (2013).Eilon, D. K., Vladimir, D., Frantisek, T., Josef, V., Jean, F. S., Aviran, 1. et al. Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors. Proc. Natl Acad. Sci. USA 104, 10152 (2007).Toms, S., Kim, C., Nicholas, G. & Ram, Z. Increased compliance with tumor treating fields therapy is prognostic for improved survival in the treatment of glioblastoma: a subgroup analysis of the EF- 14 phase III trial. J. Neuro-Oncol. 141, 467-473 (2019).Vadala M, Morales-Medina JC, Vallelunga A, Palmieri B, Laurino C, lannitti T. Mechanisms and therapeutic effectiveness of pulsed electromagnetic field therapy in oncology. Cancer Med. 2016 Nov;5(l l):3128-3139. Epub 2016 Oct 17. PMID: 27748048; PMCID: PMC5119968.X. Cheng, K. Rajjoub, A. Shashurin, D. Yan, J. H. Sherman, K. Bian, F. Murad, M. Keidar, Bioelectromagnetics 2016, 38, 53.D. Xiao, C. Yao, H. Liu, C. Li, J. Cheng, F. Guo, L. Tang, Bioelectromagnetics 2013, 34, 512.Shashurin, M. Keidar, Phys. Plasmas 2015, 22, 122002.Sagawa et al. Soluble PD-L1 works as a decoy in lung cancer immunotherapy via alternative polyadenylation. JCI Insight. 2022 Jan 11 ;7(l):el53323.Liu, Hao, et al. "High- Affinity Decoy PD-1 Mutant Screened from an Epitope-Specific Cell Library." Engineering 7.11 (2021): 1557-1565.Shin JH, Park HB, Choi K. Enhanced Anti-tumor Reactivity of Cytotoxic T Lymphocytes Expressing PD- 1 Decoy. Immune Netw. 2016 Apr;16(2): 134-9.Holash J, et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A. 2002;99(17):11393-8.Wang Q, Li T, Wu Z, Wu Q, Ke X, Luo D, Wang H. Novel VEGF decoy receptor fusion protein conbercept targeting multiple VEGF isoforms provide remarkable anti-angiogenesis effect in vivo. PLoS One. 2013 Aug 12;8(8):e70544.Sargunas, Paul R., et al. "Bispecific receptor decoy proteins block ocular neovascularization via simultaneous blockade of vascular endothelial growth factor A and C." Molecular Therapy (2025).

Claims

WHAT IS CLAIMED IS:

1. A method of treating a cancer in a mammalian subject, comprising:(i) administering to the mammalian subject a nucleic acid encoding one or more transdifferentiation determinants, and(ii) administering to the mammalian subject a cytotoxic anticancer therapy.

2. The method of claim 1, wherein the cancer is a brain cancer.

3. The method of claim 2, wherein the brain cancer is a glioma or anaplastic astrocytoma.

4. The method of claim 2, wherein the glioma is a high-grade glioma or a glioblastoma (GBM).

5. The method of claim 1, wherein the cancer is a breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, or mesothelioma, melanoma, neuroendocrine tumor, pancreatic neuroendocrine cancer, carcinoid tumor, soft tissue sarcoma, rhabdomyosarcoma, or Ewing sarcoma.

6. The method of any one of any one of claims 1-4, wherein the cytotoxic anticancer therapy is a chemotherapy, an immunotherapy, a radiation therapy, or an anti-angiogenic therapy.

7. The method of claim 6, wherein the immunotherapy is a soluble PD-1 decoy.

8. The method of claim 6, wherein the anti- angiogenic therapy is a decoy VEGF receptor.

9. The method of claims 6 and 8, wherein the decoy VEGF receptor is aflibercept, a VEGF- trap, or conbercept.

10. The method of claim 6 wherein the decoy VEGF receptor is a fusion protein comprising ligand binding domains of human VEGFR1 and human VEGFR2, preferably VEGFR1 (domain 2, aa 28-123) and VEGFR2 (domain 3, aa 130-230) in tandem.

11. The method of claim 6, wherein the chemotherapy is an alkylating agent.

12. The method of claim 11 , wherein the alkylating agent is temozolomide, carmustine, lomustinc or procarbazine.

13. The method of claim 6, wherein the radiation therapy is a non-ionizing radiation therapy.

14. The method of claim 13, wherein the non-ionizing radiation therapy is an alternating electric field therapy, a TTFields therapy, or an electromagnetic field therapy.

15. The method of any one of claims 1-14, wherein the cancer is a glioma or a glioblastoma, and wherein the transdifferentiation determinant is further defined as a cell fate determinant (CFD) that can result in transdifferentiation of cells of the cancer into an antigen presenting cell (APC) or a myeloid lineage cell.

16. The method of claim 15, wherein the antigen presenting cell is a macrophage-like cell, B cell-like cell, or dendritic cell-like cell.

17. The method of any one of claims 1-16, wherein the one or more transdifferentiation determinants are selected from the list consisting of SPI1, IKZF1, CTSZ, AEBP1, ARID3A, ATF5, ATP8B2, BASP1, BCL11B, BCL6, BID, CBFA2T3, CIITA, CIR1, CREG1, CTTN, EGR1 , ELF4, ETS 1 , ETV5, FOXP1 , FOXP4, GATA3, GM2A, GNS, GTF2IRD1 , HHEX, H0XB3, HTATIP2, IRF5, IRF8, KIF21A, KLF4, KLF11, KLF12, KMT2E, LDB1, LEF1, LM02, L0XL2, MAGED1, MMP14, MREG, MXD1, MYBL1, NAB2, NC0A3, NFATC2, NFE2L1, N0TCH2, NR1H3, PAWR, PCGF2, PDLIM1, PLAGL1, PLCG1, POU6F1, PRKCB, PTPN14, RBI, RBF0X2, RBPMS, RORA, RUNX3, SALL2, SATB1, SOX13, STAT6, TCF7, TCF19, TDP2, TFEB, TFEC, TLE2, TSHZ2, USF1, USF2, ZBTB34, ZEB1, ZFP91, ZNF74, ZNF280B, ZNF366, ZNF483, ZNF507, ZNF827, AES, ARF2, AT0X1, BATF3, BCL11A, CBFA2T3, CEBPA, CEBPB, PPARD, ETV3, ETV6, GLMP, HELZ2, ID2, IKZF3, MAFB, MAZ, MYCL, POU2AF1, SPIB, TAF10, TFE3, VAV1, and ZFP384.

18. The method of claim 17, wherein the one or more transdifferentiation determinants encodes human full-length PU.l (Spil) or PU.l delta the PEST domain (dP).

19. The method of any one of claims 1-18, wherein the subject is a human, the cancer is a brain cancer preferably a human glioblastoma, and the one or more transdifferentiation determinants areselected from the list consisting of: SPI1 , IKZF1 , CTSZ, AEBP1 , ARID3A, ATF5, ATP8B2, BASP1, BCL11B, BCL6, BID, CBFA2T3, CIITA, CIR1, CREG1, CTTN, EGR1, ELF4, ETS1, ETV5, FOXP1, FOXP4, GATA3, GM2A, GNS, GTF2IRD1, HHEX, H0XB3, HTATIP2, IRF5, IRF8, KIF21A, KLF4, KLF11, KLF12, KMT2E, LDB1, LEF1, LM02, L0XL2, MAGED1, MMP14, MREG, MXD1, MYBL1, NAB2, NC0A3, NFATC2, NFE2L1, and N0TCH2.

20. The method of any one of claims 1-19, wherein the one or more transdifferentiation determinants are SPI1 (GenBank: NM_001080547.2) and / or IKZF1 (GenBank: NM_006060.6).

21. The method of any one of claims 1-19, wherein the subject is a rodent, and wherein the one or more transdifferentiation determinants are Irf8, Id2, and / or Batf3.

22. The method of any one of claims 1-21, wherein the nucleic acid is comprised in a viral vector.

23. The method of claim 22, wherein the viral vector is a lentivirus, an adeno-associated viruses (AAV), an adenovirus, a herpes simplex virus (HSV), a reovirus vector, a baculovirus, or a poliovirus.

24. The method of claim 23, wherein the viral vector is a lentivirus or an adeno-associated virus (AAV).

25. The method of any one of claims 1-21, wherein the nucleic acid is comprised in a viruslike particle (VLP).

26. The method of any one of claims 1-21, wherein the nucleic acid is comprised in a lipid composition, a liposome, or a nanoparticle.

27. The method of any one of claims 1-26, wherein the nucleic acid nucleic acid encoding one or more transdifferentiation determinants is comprised in a plasmid, wherein the one or more transdifferentiation determinants are operably linked to a promoter that can induce expression in cells of the cancer.

28. The method of any one of claims 1-27, wherein the nucleic acid further encodes a soluble PD-1 decoy or a VEGF decoy.

29. The method of any one of claims 1-28, wherein the cytotoxic anticancer therapy is an electric fields therapy.

30. The method of claim 29, wherein the electric fields therapy is a TTFields therapy or an electromagnetic fields (EMF) therapy.

31. The method of any one of claims 29, wherein the method comprises administering the TTFields therapy or electromagnetic fields (EMF) therapy in combination with the chemotherapy.

32. The method of claim 31, wherein the chemotherapy is paclitaxel.

33. The method of claim 32, wherein the TTFields therapy is administered before paclitaxel.

34. The method of claim 32, wherein a TTFields therapy is administered after paclitaxel.

35. The method of claim 32, wherein a TTFields therapy is administered concurrently with paclitaxel.

36. The method of claim 31, wherein the chemotherapy is temozolomide and wherein the cancer is a brain cancer, preferably a glioblastoma.

37. The method of any of claims 1-36, wherein the mammalian subject is a human.

38. Use of a composition comprising a nucleic acid encoding one or more transdifferentiation determinants and a cytotoxic anticancer therapy for the treatment of cancer in a mammalian subject as described in any one of claims 1-37.

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