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WO2020093003A1 - Compositions de cellules d'écotropisme de tumeur destinées à être utilisées dans des méthodes thérapeutiques - Google Patents

Compositions de cellules d'écotropisme de tumeur destinées à être utilisées dans des méthodes thérapeutiques Download PDF

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WO2020093003A1
WO2020093003A1 PCT/US2019/059532 US2019059532W WO2020093003A1 WO 2020093003 A1 WO2020093003 A1 WO 2020093003A1 US 2019059532 W US2019059532 W US 2019059532W WO 2020093003 A1 WO2020093003 A1 WO 2020093003A1
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itdc
cells
cell
tumor
therapeutic
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Shawn D. HINGTGEN
Susan B. NICHOLS
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Falcon Therapeutics Inc
University of North Carolina at Chapel Hill
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Falcon Therapeutics Inc
University of North Carolina at Chapel Hill
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector

Definitions

  • iTDC induced tumor-homing drug carrier cell
  • the iTDC is not an induced pluripotent stem cell (iPSC) or an induced neural stem cell (iNSC).
  • the therapeutic payload comprises TRAIL.
  • the therapeutic payload comprises s-TRAIL.
  • the therapeutic payload comprises thymidine kinase (TK). In some embodiments, the therapeutic payload comprises s-TRAIL and TK. In some embodiments, the method further comprises administering to the individual a therapeutically effective amount of ganciclovir or valganciclovir. In some embodiments, the method further comprises administering an additional therapeutic agent to the individual. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an alkylating agent, an anti -angiogenic agent, an intercalating agent, a thymidylate synthetase inhibitor, a topoisomerase inhibitor, and/or a PARP inhibitor.
  • the chemotherapeutic agent is melphalan, bevacizumab, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin hydrochloride, doxorubicin hydrochloride liposome, gemcitabine hydrochloride, topotecan hydrochloride, olaparib, niraparib tosylate monohydrate, niraparib tosylate monohydrate, rucaparib camsylate, paclitaxel, taxol, thiotepa, bleomycin sulfate, etoposide phosphate, and/or vinblastine.
  • the iTDC is cultured in the presence of a progenitor medium.
  • the progenitor medium is a neural progenitor medium.
  • the iTDC is transdifferentiated from a somatic cell autologous to the individual.
  • the iTDC is produced by a method comprising genetically reprogramming a somatic cell into an induced tumor-homing drug carrier cell.
  • the somatic cell is isolated from a tissue, a blood sample, a bone marrow sample or a body fluid extracted from the individual.
  • the somatic cell is a fibroblast.
  • the fibroblast is a skin fibroblast.
  • the ovarian cancer is epithelial ovarian cancer. In some embodiments, a tumor growth of the ovarian cancer is reduced or inhibited.
  • the exogenous Sox2 is encoded by a recombinant nucleic acid. In some embodiments, the recombinant nucleic acid comprises a viral vector.
  • compositions comprising: (a) an isolated and purified induced tumor-homing drug carrier cell (iTDC) expressing (i) exogenous Sox2 and (ii) a therapeutic payload, and (b) a pharmaceutically-acceptable excipient.
  • the iTDC is not an induced pluripotent stem cell (iPSC) or an induced neural stem cell (iNSC).
  • the therapeutic agent expressed by the iTDC is TRAIL, s-TRAIL, and/or thymidine kinase (TK).
  • the pharmaceutical composition comprises an additional therapeutic agent.
  • the additional therapeutic agent is not expressed by the iTDC.
  • the pharmaceutical composition comprises a cryoprotectant.
  • the exogenous Sox2 is encoded by a recombinant nucleic acid.
  • the recombinant nucleic acid comprises a viral vector.
  • iTDC induced tumor-homing drug carrier cells
  • the iTDC comprising (a) an exogenous nucleic acid sequence encoding Sox2, and (ii) an exogenous nucleic acid sequence encoding a therapeutic payload, wherein the iTDC is not a pluripotent stem cell or an induced neural stem cell.
  • the iTDC is isolated and purified.
  • the therapeutic agents is TRAIL, s-TRAIL and/or thymidine kinase (TK).
  • induced tumor-homing drug carrier cells produced by a method comprising transfecting a somatic cell with an exogenous nucleic acid sequence encoding a transdifferentiation factor, and culturing the transfected somatic cell in a progenitor medium, thereby transforming the somatic cell into an induced tumor-homing drug carrier cell, wherein the iTDC is not a pluripotent stem cell or an induced neural stem cell.
  • the method further comprises transfecting the somatic cells with an exogenous nucleic acid sequence encoding a therapeutic payload.
  • the therapeutic agents is TRAIL, s-TRAIL and/or thymidine kinase (TK).
  • the progenitor medium in a neural progenitor culture medium.
  • FIG. 1 shows fluorescence images of therapeutic iTDCs or non-therapeutic controls engineered to express green fluorescent protein (GFP) and co-cultured with human ovarian cancer cells expressing mCherry-luciferase.
  • FIG. 2 shows a summary graph of the data, plotting the surface photon emission (y-axis) of the images therapeutic iTDCs or non-therapeutic controls.
  • GFP green fluorescent protein
  • FIG. 3 shows serial bioluminescence images of mice ovaries in which firefly luciferase ovarian cancer cells were orthotopically implanted, along therapeutic iTDCs or non-therapeutic controls.
  • FIG. 4 shows summary graphs generated from the bioluminescent imaging software, plotting the tumor volume in the mice (y axis) over 20 days (x axis).
  • FIG. 5 shows white light and fluorescence photomicrographs of human fibroblasts and iTDCs grown as monolayers and neurospheres, or stained with antibodies against nestin (green).
  • GPDH signifies glyceraldehyde-3 -phosphate dehydrogenase.
  • FIG. 7 shows immunofluorescence staining images showing iTDC-GFP (green) expression of the transdifferentiation marker nestin (red) and GFAP+ astrocytes and TUJ-1+ neurons after differentiation by mitogen removal (staining shown in red). In contrast, no staining was observed for the pluripotency markers TRA-l-60 or OCT-4. Hoechst staining is shown in blue. Fluorescence images showing only the red (555 nm) secondary antibody channel are shown in the bottom row.
  • FIG. 9 shows time-lapse fluorescent images in which iTDC-mC-FL were seeded 500 pm away from mCherry (mC)-expressing human glioblastoma (GBM) cells and placed in a fluorescence incubator microscope. Time-lapse fluorescence images were captured every 20 min for 22 hours and used to construct movies that revealed the migration of iTDC to GBM in real time.
  • FIG. 9 shows summary images showing migration of iTDC-mC-FL (red) (A) or parental human fibroblasts (B) toward U87-GFP-FL (green) at 0 and 22 hours after plating.
  • FIG 9 also shows single-cell tracings depicting the paths of iTDC-mC-FL or human fibroblast-directed migration toward GBM over 22 hours.
  • FIG. 10 shows summary graphs plotting the directionality and Euclidean distance of iTDC or fibroblast migration toward GBM cells determined from the real-time motion analysis.
  • FIG. 11 shows fluorescence images of the migration of iTDC-mC-FL (red) into U87 spheroids (green) and their penetration toward the core of the tumor spheroid over time in 3D levitation culture systems.
  • FIG. 12 are bioluminescent images of iTDC-mC-FL implanted into the frontal lobes of mice taken over 3 weeks.
  • FIG. 12 also shows a summary graph generated from the
  • bioluminescent imaging software plotting the tumor volume in the mice (y axis) over 20 days (x axis).
  • FIGS. 13 A and 13B show bioluminescence images of iTDC-mC-FL implanted into the frontal lobes of mice over a period of 3 weeks.
  • FIG. 14 shows images and summary data of 3D suspension cultures showing the viability of mCherry+ human U87 GBM spheroids (red) mixed with therapeutic iTDC-sTR or control cells at a ratio of 0.5 : 1 or 1 : 1.
  • FIGS. 15A and 15B show representative BLI and summary data demonstrating the inhibition of solid U87 GBM progression by iTDC -sTR therapy compared to control -treated mice.
  • *P 0.0044 by repeated-measures ANOVA.
  • FIGS. 16 A, 16B, and 16C show representative images demonstrating the expression of cytotoxic, differentiation, and pluripotency markers in iTDC-sTR after therapy.
  • a subset of animals were sacrificed 14 days after therapy; brain sections were stained with antibodies against nestin, TRAIL, GFAP, TUJ-l, OCT-4, or TRA-l-60; and the colocalization between staining (magenta) and GFP+ iTDC -sTR (green) was visualized.
  • FIG. 17 shows two different 3D culture models modeling the antitumor effects of iTDC -TK therapy.
  • iTDC-TK red
  • the top panel shows fluorescence images of the mixed therapy, and the bottom shows fluorescence images of the established GBM4 spheroids.
  • Serial fluorescence images showed the time-dependent decrease in GBM4 spheroid volume by iTDC -TK+ GCV therapy.
  • Figure 17 also shows a summary graph demonstrating the reduction in GBM4 spheroid volume over 7 days by iTDC-TK+ GCV therapy either mixed or seeded adjacent to established spheroids.
  • FIG. 18A shows bioluminescent images of iTDC-TK therapy that was assessed in vivo by injecting iTDC-TK cells into GBM tumors established 10 days earlier in the brains of mice.
  • FIG. 18B shows Kaplan-Meier survival curves demonstrating the survival of mice bearing GBM tumors treated with iTDC-TK+ GCV therapy or control iTDC.
  • FIGS. 19A and 19B show representative whole-brain and high-magnification images showing cell nuclei (blue), GBM4 (green), and iTDC-TK (red) distribution 21 days after delivering iTDC-control (I) or iTDC-TK (J) into established GBM tumors.
  • I iTDC-control
  • J iTDC-TK
  • FIGS. 20A and 20B show fluorescence imaging of 3D suspension cultures used to assess the migration and antitumor efficacy of sECM-encapsulated iTDC against patient-derived GBM spheroids and summary data.
  • FIGS. 21 A and 21B show representative images and summary data for serial imaging demonstrating the inhibition of tumor recurrence after intracavity iTDC-TK therapy for postoperative minimal GBM8 tumors.
  • FIG. 21C shows Kaplan-Meier survival curves of mice that underwent surgical resection of diffuse patient-derived GBM tumor cells and were treated with control iTDC or iTDC-TK encapsulated in sECM and transplanted into the surgical cavity
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within 1 or more than 1 standard deviation, per the practice in the art.
  • “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value.
  • Mammals include, but are not limited to, murine (e.g., mice and rats), simians, humans, farm animals (e.g., livestock and horses), sport animals, and pets (e.g., dogs and cats).
  • Subjects may be of any age, including infant, juvenile, adolescent, adult, and geriatric subjects. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. Designation as a “subject,”“individual,”“host,”“donor,” or“patient” does not necessarily entail supervision of a medical professional.
  • the term“therapeutically effective amount” refers to an amount of an immunological cell or a pharmaceutical composition described herein that is sufficient and/or effective in achieving a desired therapeutic effect in treating a patient having a pathogenic disease. In some embodiments, a therapeutically effective amount of the iTDC will avoid adverse side effects.
  • the terms“allogeneic” or“allogenic” means the plurality of iTDCs are obtained from a genetically non-identical donor. For example, allogenic iTDCs are extracted from a donor and returned back to a different, genetically non-identical recipient.
  • the term“autologous” means the plurality of iTDCs are obtained from a genetically identical donor. For example, autologous iTDCs are extracted from a patient and returned back to the same, genetically identical patient.
  • transdifferentiation or“transdifferentiating” refer to a method in which differentiated somatic cells are directly converted to differentiated or multipotent somatic cells of a different lineage without passing through an intermediate pluripotent stem cell (iPSC) stage.
  • iPSC pluripotent stem cell
  • transdifferentiation factor refers to a protein such as a transcription factor that promotes the direct conversion of one somatic cell type to another.
  • Examples include, but are not limited to, Oct4, Sox2, Klf4, Myc, Ascll, Brn2, Mytl 1, Olig2, Zicl, or any combinations thereof.
  • the terms“treat” or“treatment” refer to any type of treatment that imparts a benefit to a patient afflicted with a disease or disorder.
  • the disease or disorder include a cancer, a neurodegenerative disorder, and a neural trauma.
  • the benefit imparted to a patient afflicted with a disease or disorder include improvement in the condition of the patient (e.g., in one or more symptoms), a delay in the progression of the disease or disorder, and a delay in an onset or a recurrence of the disease.
  • transfecting is the transfer of heterologous genetic material into a cell, often through the use of a vector (e.g., molecule used as a vehicle to carry foreign genetic material into another cell).
  • a vector e.g., molecule used as a vehicle to carry foreign genetic material into another cell.
  • Methods of transfecting eukaryotic cells are known, and may include, but are not limited to, electroporation, use of cationic liposome-based reagents, nanoparticle-based reagents, polymeric-based reagents, polymeric and liposomal-based reagents, or any combination thereof.
  • the term“transducing” is the transfer of heterologous genetic material into a cell by means of a virus.
  • viral vectors are known and may include, but are not limited to, lentiviral vectors, adenoviral vectors, retroviral vectors, or any combination thereof.
  • Neural stem cell generation can also be achieved by expressing Sox2 in fibroblasts, but this strategy requires culturing on specific feeder cells for 40 days to obtain neural stem cell expansion and passaging. However, in some instances, there is a need for alternative methods that can rapidly (e.g., in less than 40 days) provide, for example, engineered iTDCs, for use in cell-based therapies.
  • Tumor-homing iTDCs have been created by transdifferentiation, termed induced tumor-homing drug carrier cells.
  • Transdifferentiation is a method in which cells (e.g., somatic cells) are directly converted to differentiated somatic cells of a different lineage without passing through an intermediate iPSC stage. This direct conversion by transdifferentiation obviates the safety concerns associated with the iPSC state and allows faster generation of the desired therapeutic cell type.
  • Induced tumor-homing drug carrier cells are, for example, induced (e.g., derived by
  • reprogramming cells which preferentially accumulate at (e.g., home to, migrate to) tumor tissues or tumor cells and which express a therapeutic payload (e.g., thymidine kinase, TRAIL, s-TRAIL).
  • a therapeutic payload e.g., thymidine kinase, TRAIL, s-TRAIL.
  • iTDC induced tumor-homing drug carrier cell
  • the somatic cell is a fibroblast cell (e.g, a skin fibroblast cell).
  • ddifferentiated somatic cells are collected from any accessible source, such as tissue, bodily fluids (e.g., blood, urine), etc.
  • tissue e.g., tissue, bodily fluids (e.g., blood, urine), etc.
  • skin cells are collected from the border of a surgical incision, e.g., during an accompanying surgical procedure, or using a traditional skin punch as a stand-alone procedure. Skin could be collected from any area, including, but not limited to, collection from the scalp or forearm.
  • transdifferentiation is carried out by exposing the cells to one or more transdifferentiation factors and/or growing the cells in a medium that promotes
  • the transdifferentiating is carried out without the use of feeder cells, e.g., in a neural progenitor medium.
  • Feeder cells as known in the art, are additional cells grown in the same culture dish or container, often as a layer (e.g., a mouse fibroblast layer on the culture dish) to support cell growth.
  • transdifferentiation is single-factor transdifferentiation in that only one transdifferentiation factor is used.
  • factors include Oct4, Sox2, Klf4, Myc, Ascll, Bm2, Mytl 1, Olig2, Zicl, or any combinations thereof.
  • Sox2 is a member of the Sox family of transcription factors and is expressed in developing cells in the neural tube as well as in proliferating progenitor cells of the central nervous system. In some embodiments, Sox2 is used as the transdifferentiation factor in the methods taught herein. In some embodiments, Sox2 is used to carry out a single-factor transdifferentiation.
  • “Nestin” is expressed predominantly in stem cells of the central nervous system, and its expression is typically absent from differentiated central nervous cells.
  • “GFAP” or“glial fibrillary acidic protein” is an intermediate filament protein expressed by central nervous system cells, including astrocytes.
  • “Tuj-l” or“beta tubulin” is a neural marker.
  • the method comprises transducing said somatic cell with a lentiviral vector comprising said nucleic acid encoding Sox2.
  • somatic cells for example those expressing Sox2
  • iTDCs cells are cultured in a progenitor medium, such as a neural progenitor medium.
  • Progenitor medium as used herein is a medium or media, for example, incorporating supplements, small molecule inhibitors, and growth factors, that promotes the
  • the neural progenitor medium includes one or more ingredients selected from: a cell culture medium containing growth-promoting factors and/or a nutrient mixture (e.g., DMEM/F12, MEM/D-valine, neurobasal medium etc., including mixtures thereof); media supplements containing hormones, proteins, vitamins and/or amino acids (e.g., N2 supplement, B27 supplement, non-essential amino acids (NEAA), L-glutamine, Glutamax, BSA, insulin, all trans retinoic acid, etc.
  • a cell culture medium containing growth-promoting factors and/or a nutrient mixture e.g., DMEM/F12, MEM/D-valine, neurobasal medium etc., including mixtures thereof
  • media supplements containing hormones, proteins, vitamins and/or amino acids (e.g., N2 supplement, B27 supplement, non-essential amino acids (NEAA), L-glutamine, Glutamax, BSA, insulin, all trans retinoic acid, etc.
  • ingredients also include one or more of beta-mercaptoethanol, transferrin; sodium selenite; and cAMP.
  • suitable concentrations of each of these ingredients are known to those of skill in the art and/or are empirically determined. Example concentrations of ingredients is also provided in Example 5 below.
  • the neural progenitor medium is a premade medium, such as STEMdiffTM Neural Induction Medium (STEM CELL TM
  • the method further comprises transducing the iTDC with a nucleic acid encoding therapeutic payload or a reporter molecule.
  • iTDCs as taught herein are loaded with (e.g., contain) a therapeutic payload, a reporter molecule and/or a nucleic acid capable of expressing the same.
  • the therapeutic agent is a protein toxin (e.g., a bacterial endotoxin or exotoxin), an oncolytic virus (e.g., a conditionally replicative oncolytic adenovirus, reovirus, measles virus, herpes simplex virus (e.g., HSV1716), Newcastle disease 15 virus, vaccinia virus, etc.), or a pro-apoptotic agent (e.g., secretable tumor necrosis factor (TNF)-related apoptosis-inducing ligand (S-TRAIL)).
  • TRAIL is a member of the tumor necrosis factor (TNF) cytokine family.
  • TRAIL activates rapid apoptosis in ovarian tumor cells.
  • S-TRAIL a secreted form of TRAIL, in some instances exerts more potent apoptotic effects (e.g., compared to TRAIL) when delivered by the iTDCs.
  • the therapeutic payload comprises a pro-inflammatory protein such as an interleukin, cytokine, or antibody.
  • the therapeutic payload comprises an enzyme useful for enzyme/prodrug therapies (e.g., thymidine kinase (e.g., with ganciclovir prodrug),
  • enzyme/prodrug therapies e.g., thymidine kinase (e.g., with ganciclovir prodrug)
  • carboxylesterase e.g., with CTP-l l
  • cytosine deaminase etc.
  • thymidine kinase when expressed in cells, thymidine kinase enzymatically cleaves ganciclovir and subsequently transforms the ganciclovir into a cytotoxic agent
  • the therapeutic comprises an RNAi molecule such as miRNA or siRNA.
  • the iTDCs are loaded with a therapeutic payload used for the treatment of cancer.
  • the therapeutic payload used for the treatment of ovarian cancer is a chemotherapeutic agent, as described elsewhere herein.
  • the therapeutic payload used for the treatment of ovarian cancer is a diagnostic therapeutic agent.
  • the therapeutic payload used for the treatment of ovarian cancer is an imaging agent.
  • the imaging agent is 2-Deoxy-2- 18 F- fluoroglucose (FDG), Sodium 18 F-fluoride (NaF), Anti- 1 -amino-3 - 18 F-fluorocyclobutane-l- carboxcylic acid ( 18 F-fluciclovine, FACBC), 99m Tc-methoxyisobutylisonitrile ( 99m Tc-sestamibi), 3'-deoxy-3'- 18 F-fluorothymidine (FLT), l6a- 18 F-fluoro-l7P-estradiol (FES), 2l- 18 F-fluoro- l6a, l7a-[(R)-(r-a-furylmethylidene)dioxy]-l9-norpregn
  • the iTDCs are loaded with nanoparticle/drug conjugates.
  • Reporter molecules are known in the art and include, but are not limited to, Green Fluorescent Protein, f3-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase gene.
  • loading of the iTDCs with a payload is accomplished using art- known methods, such as transfecting the iTDCs with a nucleic acid capable of producing a therapeutic or reporter protein, transducing the iTDCs with a viral vector, lipid-based or polymeric loading of the cells with a therapeutic payload and/or reporter molecule, etc.
  • iTDC is allogeneic with respect to said subject.
  • iTDC is syngeneic with respect to said subject.
  • the iTDC is autologous with respect to said subject.
  • the ovarian cancer is epithelial ovarian cancer. In some embodiments, the ovarian cancer is metastatic. In some embodiments, the ovarian cancer is non- invasive. In some embodiments, the ovarian cancer is invasive. In some embodiments, the ovarian cancer is a stage I, a stage II, a stage III, or a stage IV ovarian cancer. In some embodiments, the ovarian cancer is an ovarian germ cell tumor. In some embodiments, the ovarian germ cell tumor is a teratoma, a dysgerminoma, an endodermal sinus tumor, a choriocarcinoma, or any combinations thereof.
  • the ovarian cancer is a sex cord-stromal tumor, an ovarian sarcoma, a Krukenberg tumor, an ovarian cyst that develops into an ovarian cancer, or any combination thereof.
  • the ovarian cancer is an ovarian stromal tumor.
  • the ovarian stromal tumor is a granulosa-theca tumor, a Sertoli-Leydig cell tumor, a granulosa cell tumor, a small cell carcinoma of the ovary, a primary peritoneal carcinoma, or any combinations thereof.
  • the ovarian cancer is an epithelial ovarian cancer, a germ cell tumor, a stromal cell tumor, a steroid cell tumor, or a combination thereof.
  • the ovarian cancer is a small cell ovarian carcinoma, a neuro-endocrine carcinoma, a squamous cell carcinoma rising within a dermoid cyst, a struma ovarii malignum, a psammocarcinoma, or any combinations thereof.
  • the epithelial ovarian cancer is a high-grade serous ovarian cancer, a low- grade serous ovarian cancer, a mucinous ovarian cancer, an ovarian endometrioid cancer, a clear cell ovarian cancer, an unclassified ovarian cancer, an undifferentiated ovarian cancer, a Brenner tumor, a borderline tumor, a carcinosarcoma, or any combinations thereof.
  • a high-grade serous ovarian cancer is a high-grade serous ovarian cancer, a low- grade serous ovarian cancer, a mucinous ovarian cancer, an ovarian endometrioid cancer, a clear cell ovarian cancer, an unclassified ovarian cancer, an undifferentiated ovarian cancer, a Brenner tumor, a borderline tumor, a carcinosarcoma, or any combinations thereof.
  • the germ cell tumor is a dysgerminoma, a teratoma, an ovarian yolk sac tumor, a mixed germ cell tumor, an embryonal carcinoma, a polyembryoma, or any combinations thereof.
  • the stromal cell tumor is an ovarian stromal tumor with sex cord elements, an adult type granulosa cell tumor, a juvenile type granulosa cell tumor, an
  • the steroid cell tumor is a stromal luteoma, a Leydig cell tumor, or any combinations thereof.
  • the iTDCs are obtained by any method described herein. In some embodiments, the iTDC is produced by a method comprising directly genetically
  • the somatic cell is isolated from a tissue, a blood sample, a bone marrow sample or a body fluid extracted from the individual.
  • the somatic cell is a fibroblast.
  • the fibroblast is a skin fibroblast.
  • the iTDCs are transdifferentiated ex vivo before administration to the individual. In some embodiments, the iTDCs are autologous to the individual. In some embodiments, the iTDCs are allogenic.
  • the iTDCs are fresh, i.e., not frozen or previously frozen. In some embodiments, the iTDCs are cryopreserved (frozen). In some embodiments, the iTDCs are frozen and stored for later use (for example to facilitate transport). In some embodiments, the frozen iTDCs are administered to the individual after being thawed.
  • administration of the iTDCs is performed using methods known in the art.
  • intravenous administration of the cells is performed for the treatment of an ovarian cancer.
  • intraperitoneal administration of the cells is performed for the treatment of an ovarian cancer.
  • intratumoral administration or intracavity administration is performed after surgical removal of at least a part of an ovarian tumor.
  • the cells are encapsulated by a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold, which is then be administered or implanted, e.g., intratumorally.
  • a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix)
  • iTDC isolated and purified iTDC expressing (i) exogenous Sox2 and (ii) a therapeutic payload, and (b) an exogenous therapeutic agent.
  • a method of treating a ovarian cancer in an individual in need thereof comprises: administering iTDCs produced by any method described herein, and an additional therapeutic agent.
  • the exogenous therapeutic agent is a chemotherapeutic agent.
  • the iTDCs are administered prophylactically in combination with the exogenous chemotherapeutic agent in order to treat an ovarian cancer and/or a tumor.
  • the exogenous chemotherapeutic agent is an alkylating agent, an anti -angiogenic agent, an anthracycline, a cytoskeletal disruptor, an epothilone, a histone deacetylase inhibitor, an intercalating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a thymidylate synthetase inhibitor, a PARP inhibitor, a kinase inhibitor, a nucleotide analog, a precursor analog, a peptide antibiotic, a platinum-based agent, a retinoid, or a vinca alkaloid.
  • chemotherapeutic agents include: actinomycin, albumin bound paclitaxel, altretamine, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, liposomal doxorubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,
  • compositions comprising: (a) an isolated iTDC disclosed herein; and (b) a pharmaceutically-acceptable excipient.
  • a pharmaceutical composition includes one population of iTDCs, or more than one, such as two, three, four, five, six or more populations of iTDCs.
  • the components of the pharmaceutical compositions described herein are administered either alone or in combination with pharmaceutically acceptable carriers, excipients, or diluents, in a pharmaceutical composition.
  • Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that are used pharmaceutically.
  • Pharmaceutically-acceptable excipients included in the pharmaceutical compositions will have different purposes depending, for example, on the type of iTDCs used and the mode of administration.
  • Non-limiting examples of generally used pharmaceutically- acceptable excipients include, without limitation: saline, buffered saline, dextrose, water-for- injection, glycerol, ethanol, dextran (e.g., low molecular dextran such as Dextran 40),
  • PlasmaLyte human serum albumin (HSA), and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives (such as dimethylsulfoxide (DMSO)), tonicity agents, bulking agents, and lubricating agents.
  • the formulations comprising populations of iTDCs are prepared and cultured in the absence of any non-human components, such as animal serum.
  • compositions further comprise a
  • cryoprotectant or a cryopreservative is selected from dimethylsulfoxide (DMSO), formamide, propylene glycol, ethylene glycol, glycerol, trehalose, 2-methyl-2,4-pentanediol, methanol, butanediol, or any combination thereof.
  • DMSO dimethylsulfoxide
  • formamide propylene glycol
  • ethylene glycol ethylene glycol
  • glycerol trehalose
  • 2-methyl-2,4-pentanediol methanol
  • butanediol or any combination thereof.
  • compositions comprising: (a) an isolated iTDC; and (b) a
  • any known device useful for parenteral (e.g., intraperitoneal) injection and/or infusion of the formulations is used to effect such administration.
  • compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection are presented in unit dosage form, e.g., in ampoules or in multi -dose containers, with an added preservative.
  • the compositions take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and are stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use.
  • sterile liquid carrier for example, saline or sterile pyrogen-free water
  • extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.
  • compositions for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which include suspending agents and thickening agents.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • aqueous injection suspensions contain substances which increase the viscosity of the suspension, such as sodium
  • the suspension also contains suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the compositions comprising the iTDCs and/or the combination therapies described herein are administered for prophylactic and/or therapeutic treatments of ovarian cancer.
  • the compositions are administered to a patient already suffering from ovarian cancer, in an amount sufficient to cure or at least partially arrest at least one of the symptoms of the ovarian cancer. Amounts effective for this use depend on the severity and course of the ovarian cancer, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician.
  • Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation and/or dose ranging clinical trial.
  • the pharmaceutical compositions comprising an iTDC are administered directly at a tumor site in the individual.
  • the pharmaceutical compositions comprising an iTDC are administered directly at a tumor site in the individual.
  • compositions comprising an iTDC are administered directly into a tumor, a resection margin, and/or a tumor resected area.
  • the iTDCs are administered systemically to the individual.
  • administration of the iTDCs is performed using methods known in the art.
  • intravenous administration of the cells is performed for the treatment of an ovarian cancer.
  • intraperitoneal administration of the cells is performed for the treatment of an ovarian cancer.
  • intratumoral administration or intracavity administration is performed after surgical removal of at least a part of an ovarian tumor.
  • the cells are encapsulated by a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold, which is then be administered or implanted, e.g., intratumorally.
  • a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold, which is then be administered or implanted, e.g., intratumorally.
  • the iTDCs are encapsulated by a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold.
  • a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold.
  • the scaffold is biocompatible.
  • the scaffold is biodegradable and/or bioabsorbable.
  • the scaffold is sterile.
  • the scaffold is suitable for intratumoral or intracavity administration after surgical removal of a tumor.
  • the scaffold is pliable to allow manipulation thereof prior to or during administration to conform the scaffold to the area to which the iTDCs are being delivered.
  • the scaffold is configured to line the walls of the resection cavity.
  • the average thickness of the scaffold is in the nanometer, micrometer or millimeter range.
  • the scaffold includes a polymerized and/or crosslinked material selected from polyanionic polysaccharides (e.g., hyaluronic acid (HA),
  • the scaffold comprises a bioabsorbable gelatin sponge.
  • seeding the iTDCs on the scaffold comprises: mixing a polymerizable and/or crosslinkable scaffold material with said induced drug carrier cells to form a mixture of the material and iTDCs, and polymerizing and/or crosslinking said material of said mixture, to thereby form said scaffold comprising iTDCs.
  • the intratumoral or intracavity administration of the scaffold is performed using methods known in the art.
  • iTDCs migrate away from the scaffold and towards a cancerous or damaged tissue.
  • the polymerizing and/or crosslinking are performed in situ during intracavity administration after surgical removal of a brain tumor.
  • the scaffold is administered to line the walls of a resection cavity of an ovarian tumor.
  • the scaffold has ridges, channels and/or aligned fibers to promote movement of the drug carrier cells in the direction of the cancer or damaged tissue.
  • compositions comprising the iTDC described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition.
  • a patient susceptible to or otherwise at risk of a particular disease, disorder or condition is defined to be a“prophylactically effective amount or dose.”
  • the precise amounts also depend on the state of health of the patient, the weight of the patient, and the like.
  • effective amounts for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the health status of the patient, response of the patient to the drugs, and the judgment of the treating physician.
  • prophylactic treatments include administering to an individual, who previously experienced at least one symptom of the disease being treated and is currently in remission, a pharmaceutical composition comprising an iTDC described herein, in order to prevent a return of the symptoms of the ovarian cancer.
  • an iTDC and an additional therapeutic agent described herein are administered at a dose lower than the dose at which either the induced drug carrier cell or the additional therapeutic agent are normally administered as monotherapy agents. In certain embodiments, an iTDC and an additional therapeutic agent described herein are administered at a dose lower than the dose at which either the iTDC or the additional therapeutic agent are normally administered to demonstrate efficacy. In certain embodiments, an iTDC is
  • an iTDC is administered at a dose lower than the dose at which it is normally administered to demonstrate efficacy, when administered in combination with an additional therapeutic agent described herein.
  • an additional therapeutic agent is administered at a dose lower than the dose at which it is normally administered as a monotherapy agent, when administered in combination with an iTDC.
  • an additional therapeutic agent is administered at a dose lower than the dose at which it is normally administered to demonstrate efficacy, when administered in combination with an iTDC.
  • the administration of the iTDC compositions are administered chronically, that is, for an extended period of time, including throughout the duration of the life of the patient in order to ameliorate or otherwise control or limit the symptoms of the patient’s ovarian cancer.
  • the dose of the pharmaceutical compositions being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a“drug holiday”).
  • the length of the drug holiday is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days.
  • the dose reduction during a drug holiday is, by way of example only, by 10%- 100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.
  • a maintenance dose is administered if necessary. Subsequently, in specific embodiments, the dosage or the frequency of administration, or both, is reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. In certain embodiments, however, the patient requires intermittent treatment on a long-term basis upon any recurrence of symptoms.
  • the amount of a given agent that corresponds to such an amount varies depending upon factors such as the particular compound, disease condition and its severity, the identity (e.g., weight, sex) of the individual in need of treatment, but nevertheless is determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the individual being treated.
  • the pharmaceutical compositions comprising an induced drug carrier cell are administered at a dosage in the range of about 10 3 to about 10 10 iTDC per kg of body weight iTDCs per kg of body weight, including all integer values within those ranges.
  • the desired dose is conveniently presented in a single dose or in divided doses administered simultaneously or at appropriate intervals, for example as two, three, four or more sub-doses per day.
  • the desired dose is administered as a single dose or in divided doses within about 72 hours of each other.
  • the desired dose is administered in divided does within about 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days of each other.
  • the daily dosage or the amount of active in the dosage form are lower or higher than the ranges indicated herein, based on a number of variables in regard to an individual treatment regime.
  • the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the compound used, the ovarian cancer to be treated, the mode of administration, the requirements of the individual, the severity of the ovarian cancer being treated, and the judgment of the practitioner.
  • the pharmaceutical compositions comprising an iTDC are administered at a dosage of about 10 x 10 6 cells per kilogram (kg). In some embodiments, the pharmaceutical compositions comprising an iTDC are administered at a dosage of about 12.5 x 10 6 cells/kg. In some embodiments, the pharmaceutical compositions comprising an iTDC are administered at a dosage of about 1,000 cells/kg to about 10,000,000,000 cells/kg. In some embodiments, the pharmaceutical compositions comprising an induced drug carrier cell are administered at a dosage of at least about 1,000 cells/kg. In some embodiments, the
  • compositions comprising an induced drug carrier cell are administered at a dosage of at most about 10,000,000,000 cells/kg.
  • the pharmaceutical compositions comprising induced drug carrier cell are administered at a dosage of about 1,000 cells/kg to about 10,000 cells/kg, about 1,000 cells/kg to about 100,000 cells/kg, about 1,000 cells/kg to about 1,000,000 cells/kg, about 1,000 cells/kg to about 10,000,000 cells/kg, about 1,000 cells/kg to about 100,000,000 cells/kg, about 1,000 cells/kg to about 1,000,000,000 cells/kg, about 1,000 cells/kg to about 10,000,000,000 cells/kg, about 10,000 cells/kg to about 100,000 cells/kg, about 10,000 cells/kg to about 1,000,000 cells/kg, about 10,000 cells/kg to about 10,000,000 cells/kg, about 10,000 cells/kg to about 100,000,000 cells/kg, about 10,000 cells/kg to about 1,000,000,000 cells/kg, about 10,000 cells/kg to about 10,000,000,000 cells/kg, about 100,000 cells/kg to about 1,000,000 cells/kg, about 100,000 cells/kg to about 10,000,000 cells/kg, about 100,000 cells/kg to about 100,000,000 cells/kg, about 10,000 cells
  • 1,000,000 cells/kg to about 10,000,000 cells/kg about 1,000,000 cells/kg to about 100,000,000 cells/kg, about 1,000,000 cells/kg to about 1,000,000,000 cells/kg, about 1,000,000 cells/kg to about 10,000,000,000 cells/kg, about 10,000,000 cells/kg to about 100,000,000 cells/kg, about 10,000,000 cells/kg to about 1,000,000,000 cells/kg, about 10,000,000 cells/kg to about 10,000,000,000 cells/kg, about 100,000,000 cells/kg to about 1,000,000,000 cells/kg, about 100,000,000 cells/kg to about 10,000,000,000 cells/kg, or about 1,000,000,000 cells/kg to about 10,000,000,000 cells/kg.
  • the pharmaceutical compositions comprising induced drug carrier cell are administered at a dosage of about 1,000 cells/kg, about 10,000 cells/kg, about 100,000 cells/kg, about 1,000,000 cells/kg, about 10,000,000 cells/kg, about 100,000,000 cells/kg, about 1,000,000,000 cells/kg, or about 10,000,000,000 cells/kg.
  • any of the aforementioned aspects are further embodiments in which the effective amount of the pharmaceutical compositions described herein is: (a) systemically administered to the subject; and/or (b) intravenously administered to the subject; and/or (c) administered by injection to the subject; and/or (d) administered non-systemically or locally to the subject.
  • any of the aforementioned aspects are further embodiments comprising single administrations of the effective amount of the pharmaceutical composition, including further embodiments in which (i) the pharmaceutical composition is administered once a day; or (ii) the pharmaceutical composition is administered to the individual multiple times over the span of one day.
  • any of the aforementioned aspects are further embodiments comprising multiple administrations of the effective amount of the pharmaceutical composition, including further embodiments in which (i) the pharmaceutical composition is administered continuously or intermittently: as in a single dose; (ii) the time between multiple administrations is every 6 hours; (iii) the compound is administered to the individual every 8 hours; (iv) the compound is administered to the individual every 12 hours; (v) the compound is administered to the individual every 24 hours; (vi) the compound is administered to the individual every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.
  • the method comprises a drug holiday, wherein the administration of the compound is temporarily suspended or the dose of the compound being administered is temporarily reduced; at the end of the drug holiday, dosing of the compound is resumed.
  • the length of the drug holiday varies from 2 days to 1 year.
  • the therapeutic effectiveness of one of the pharmaceutical compositions described herein is enhanced by administration of an adjuvant (i.e., by itself the adjuvant has minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced).
  • an adjuvant i.e., by itself the adjuvant has minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced.
  • the benefit experienced by a patient is increased by administering one of the pharmaceutical compositions described herein with another agent (which also includes a therapeutic regimen) that also has therapeutic benefit.
  • a pharmaceutical composition described herein is co- administered with a second therapeutic agent, wherein the pharmaceutical composition described herein, and the second therapeutic agent modulate different aspects of the disease, disorder or condition being treated, thereby providing a greater overall benefit than
  • the overall benefit experienced by the patient is additive of the two therapeutic agents or the patient experiences a synergistic benefit.
  • different dosages of the pharmaceutical composition disclosed herein are utilized in formulating pharmaceutical composition and/or in treatment regimens when the compounds disclosed herein are administered in combination with one or more additional agent, such as an additional drug, an adjuvant, or the like.
  • additional agent such as an additional drug, an adjuvant, or the like.
  • Dosages of drugs and other agents for use in combination treatment regimens are optionally determined by means similar to those set forth hereinabove for the actives themselves.
  • a combination treatment regimen encompasses treatment regimens in which administration of a pharmaceutical composition described herein, is initiated prior to, during, or after treatment with a second agent described herein, and continues until any time during treatment with the second agent or after termination of treatment with the second agent. It also includes treatments in which a pharmaceutical composition described herein, and the second agent being used in combination are administered simultaneously or at different times and/or at decreasing or increasing intervals during the treatment period. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.
  • the dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought is modified in accordance with a variety of factors (e.g. the disease, disorder or condition from which the individual suffers; the age, weight, sex, diet, and medical condition of the individual).
  • factors e.g. the disease, disorder or condition from which the individual suffers; the age, weight, sex, diet, and medical condition of the individual.
  • the dosage regimen actually employed varies and, in some embodiments, deviates from the dosage regimens set forth herein.
  • compositions vary depending on the type of co-drug employed, on the specific drug employed, on the ovarian cancer being treated and so forth.
  • the pharmaceutical compositions when co-administered with one or more other therapeutic agents, the pharmaceutical
  • composition provided herein is administered either simultaneously with the one or more other therapeutic agents, or sequentially.
  • a co-drug is administered in conjunction with the
  • the co-drug is ganciclovir or valganciclovir.
  • the dosing interval is determined by the bioavailability of the co-drug and its excretion from the body.
  • the co-drug is administered for at least 5 days, about 10 days to about 3 weeks.
  • the multiple therapeutic agents are administered in any order or even
  • the multiple therapeutic agents are, by way of example only, provided in a single, unified form, or in multiple forms (e.g., as a single pill or as two separate pills).
  • compositions described herein, or a pharmaceutically acceptable salt thereof, as well as combination therapies are administered before, during or after the occurrence of ovarian cancer, or a disease or condition associated with ovarian cancer, and the timing of administering the pharmaceutical composition containing a compound varies.
  • the pharmaceutical compositions described herein are used as a prophylactic and are administered continuously to individuals with a propensity to develop ovarian cancer in order to prevent the occurrence of ovarian cancer.
  • the pharmaceutical compositions are administered to an individual during or as soon as possible after the onset of the symptoms.
  • a pharmaceutical composition described herein is administered as soon as is practicable after the onset of a ovarian cancer is detected or suspected, and for a length of time necessary for the treatment of the disease.
  • the length required for treatment varies, and the treatment length is adjusted to suit the specific needs of each individual.
  • a compound described herein or a formulation containing the pharmaceutical composition is administered for at least 2 weeks, about 1 month to about 5 years.
  • Example 1 In vitro and in vivo experiments confirming the efficacy of iTDCs in treating ovarian cancer
  • Cell lines OVACAR human ovary cancer cells were purchased. Human fibroblast cells were provided by the University of North Carolina (UNC) School of Medicine. Cell lines were grown according to methods known in the art, particularly those described in SD Hingtgen et al ,“A novel molecule integrating therapeutic and diagnostic activities reveals multiple aspects of stem cell-based therapy,” Stem Cells 28, 832-841 (2010), and M Sena-Esteves, et al , “Optimized large-scale production of high titer lentivirus vector pseudotypes,” J. Virol. Methods 122, 131-139 (2004).
  • Lentiviral vectors Reprogramming lentiviral vectors (“LV”) encoding Sox2 were purchased from Addgene (Cambridge, MA, USA). Lentiviral vectors expressing cytotoxic agents and optical reporters were constructed by methods known in the art. The cytotoxic agents were a secreted variant of the proapoptotic molecule TRAIL (“sTR”) and thymidine kinase (“TK”). The reporters were Green Fluorescent Protein (“GFP”), mCherry (“mC”), and Firefly Luciferase (“FL”). The following vectors were constructed: LV-sTR-TK-GFP, LV-GFP, and LV-mC-FL. All lentiviral constructs were packaged as lentiviral vectors in 293T cells using a helper virus-free packaging system.
  • sTR proapoptotic molecule TRAIL
  • TK thymidine kinase
  • the reporters were Green Fluorescent Protein (“GFP”), mCherry
  • ITDCs were generated following a single-factor Sox2 and feeder-free method. Human fibroblasts were seeded in 6-well plates and transduced with reprogramming vectors in media containing protamine sulfate. Two days after infection, the media was changed to STEMdiffTM Neural Induction Medium (STEMCELL Technologies, Vancouver, Canada) containing doxycycline. Media was changed every 3 days. Neurosphere formation was induced by culturing in low-adherent flasks.
  • Co-culture viability assays The following cells were generated: (1) OVACAR cells expressing mC-FL; (2) iTDC therapeutic cells expressing sTR-TK-GFP; and (3) iTDC control cells expressing sTR-GFP. The therapeutic and control cells were seeded in separate wells. Forty-eight hours later, the OVACAR cells expressing mC-FL were seeded into both the wells containing control cells and the wells containing therapeutic cells. The cells were visualized for fluorescent protein expression by fluorescence microscopy. OVACAR viability was measured by quantitative in vitro bioluminescence imaging. Photon emission was quantified using Livingimage software (PerkinElmer).
  • OVACAR cells is significantly higher than that of the therapeutic treated OVACAR cells, reflecting that the therapeutic cells significantly reduced the viability of OVACAR cells as compared to the control.
  • OVACAR cells expressing mC-FL were implanted in the ovaries of 10 mice (2xl0 6 cells/mouse). Three days later, either therapeutic iTDCs expressing TR-TK-GFP (2xl0 6 cells/mouse) or control iTDCs expressing GFP (2xl0 6 cells/mouse) were implanted into the OVACAR implantation site of each mouse. Ganciclovir (“GCV”) was injected into each mouse daily during 20 days at a dose of 100 mg/kg.
  • Ganciclovir Ganciclovir
  • mice were given an injection of D-Luciferin and photon emission was determined 1-5 min later using an IVIS Kinetic Optical System (Perkin Elmer) with a 1-5 minute acquisition time. Images were processed and photon emission quantified using Livingimage software (PerkinElmer).
  • Results The in vivo bioluminescent imaging results reflect that iTDCs expressing sTR-TK have a significant therapeutic effect against ovarian cancer.
  • Figure 3 shows
  • FIG. 4 plots the tumor volume (normalized to 1) of the control treated mice (“control”) versus that of the therapeutic-treated mice (“therapy”). On day 0, the tumor volumes of the control treated mice and therapeutic treated mice are equal, but by day 20 the tumor volume of the control-treated mice was nearly eight times greater than that of the therapeutic treated mice. Accordingly, the in vivo bioluminescent imaging results show that the
  • iTDCs expressing TK-sTR when administered with GCV, have significant therapeutic effects against ovarian cancer in vivo.
  • Transdifferentiation of human skin cells was performed as above in Example 1, but in place of the STEMdiffTM Neural Progenitor Basal Medium was a 1 : 1 mixture ofN-2 medium and B-27 medium as follows. Chemicals were purchased from Gibco® (Invitrogen Corporation, Carlsbad, California), Sigma (Sigma-Aldrich, St. Louis, Missouri) or Selleck Chemicals (Houston, Texas) as indicated.
  • N-2 medium DMEM/F12 (Gibco®), 1 X N2 supplement (Gibco®), 5 pg/ml insulin
  • B -27 medium Neurobasal medium (Gibco®), 1 X B-27 supplement (Gibco®), and 200 mM L-glutamine (Gibco®).
  • BSA bovine serum albumin
  • This medium was supplemented with the following:
  • SB431542 (Selleck Chemicals) to a final concentration of 10 pM; LDN193189 (Selleck Chemicals) to a final concentration of 10 pM; LDN193189 (Selleck Chemicals) to a final concentration of 10 pM; LDN193189 (Selleck Chemicals) to a final concentration of 10 pM; LDN193189 (Selleck Chemicals) to a final concentration of 10 pM; LDN193189 (Selleck).
  • the medium included Insulin (25 pg/ml), Transferrin (100 pg/ml), Sodium selenite (30 nM), and/or cAMP (100 ng/ml).
  • a patient is diagnosed with ovarian cancer (e.g., epithelial ovarian cancer), and surgery is scheduled for removing the tumor soon thereafter (e.g., within 24 days, 4 weeks, or 5 weeks).
  • a skin punch is taken from the patient to obtain skin fibroblast cells.
  • the cells are transdifferentiated as disclosed herein into induced iTDCs and also loaded with a therapeutic agent and/or a reporting molecule.
  • the loaded iTDCs are administered into the resection margin and resulting cavity where the tumor had been removed.
  • the iTDCs migrate toward residual ovarian cancer cells and deliver their therapeutic agent and/or reporting molecule payload.
  • a patient is diagnosed with brain cancer (e.g., glioblastoma), and surgery is scheduled for removing the tumor soon thereafter (e.g., within one, two or three weeks).
  • a skin punch is taken from the patient to obtain skin fibroblast cells.
  • the cells are transdifferentiated as taught herein into induced iTDCs and also loaded with a therapeutic agent and/or a reporting molecule.
  • the loaded iTDCs are administered into the resulting cavity where the tumor had been removed.
  • the iTDCs migrate toward residual cancer cells and deliver their therapeutic agent/reporting molecule payload.
  • the ability to rapidly generate iTDCs from human skin enables patient-specific therapies to treat cancer.
  • the efficiency of iTDC generation is significantly higher than other cellular reprogramming strategies, suggesting large numbers of iTDCs could be generated from small amounts of skin.
  • Patient-specific derivation could avoid immune rejection to maximize tumor killing and for treatment durability.
  • Example 5 Rapid Transdifferentiation of Human Skin Cells
  • iTDCs are created in weeks, allowing for reduced turn-around time from patient sample to therapy for aggressive cancers.
  • TD-derived iTDC therapies were investigated as autologous GBM therapy for human patients. These methods are capable of converting human skin into iTDCs 6-fold faster than previous methods, which is significant because time is a priority for GBM patient therapy.
  • This strategy was used to create the first iTDCs engineered with cytotoxic agents and optical reporters.
  • a combination of real-time molecular imaging, 3-D cell culture, and multiple human GBM xenografts models were used to investigate the fate, tumor-specific homing, and efficacy of iTDC therapy against solid and surgically resected GBM.
  • Human iTDCs were generated following a single-factor Sox2 and feeder-free method. Briefly, 200,000 human fibroblasts were seeded in 6-well plates and transduced with the LV cocktail containing hTERT and Sox2 in media containing protamine sulfate (5 pg/ml, Sigma). Two days after infection, the media was changed to STEMdiffTM Neural Induction Medium (STEMCELL Technologies, Vancouver, Canada) containing doxycycline (1 0 pg/ml, Sigma, St. Louis, MO, USA). Media was changed every 3 days. Neurosphere formation was induced by culturing in low-adherent flasks.
  • Lentiviral vectors In addition to the reprogramming vectors, the following lentiviral vectors were used in this study: LV-GFP-FL, LV-GFP-RLuc, LV-mC-FL, LV-sTR, LV-diTR and LV-mRFP-hRLuc-ttk.
  • GFP-RLuc and GFP-FL were constructed by amplifying the cDNA encoding Renilla luciferase or firefly luciferase using the vectors luciferase-pcDNA3 and pAC-hRluc (Addgene), respectively.
  • the restriction sites were incorporated in the primers, the resulting fragment was digested Bglll and Sail, and ligated in frame in BgllESall digested pEGFP-Cl (Clontech, Mountain View, CA, USA).
  • the GFP-FL or GFP-RLuc fragments were digested with Agel (blunted) and Sail, and ligated into pTK402 (provided by Dr. Tal Kafri, UNC Gene Therapy Center) digested BamHI (blunted) and Xhol to create LV-GFP-FL or LV-GFP- RLuc.
  • mCFL was created by amplifying the cDNA encoding firefly luciferase from luciferase-pcDNA3, ligating into BglII/Sall digested mCherry-Cl (Clontech), and ligating the mC-FL fragment into pTK402 LV backbone using blunt/Xhol sites.
  • LV-sTR and LV- diTR the cDNA sequence encoding sTR or diTR was PCR amplified using custom-synthesized oligonucleotide templates (Invitrogen, Carlsbad, CA, USA).
  • LV -sTR and LV -diTR have 1RES-GFP (internal ribosomal entry sites-green fluorescent protein) elements in the backbone as well as CMV -driven puromycin element. All LV constructs were packaged as LV vectors in 293T cells using a helper virus-free packaging system as described previously.
  • iTDCs and GBM cells were transduced with LVs at varying multiplicity of infection (MOI) by incubating virions in a culture medium containing 5 pg/ml protamine sulfate (Sigma) and cells were visualized for fluorescent protein expression by fluorescence microscopy.
  • MOI multiplicity of infection
  • Cell viability and passage number To assess the proliferation and passage number of modified and unmodified iTDCs, iTDCs expressing GFP-FL, sTR or unmodified cells were seeded in 96-well plates. Cell viability was assessed 2, 3, 4, 5, 8, and 10 days after seeding using CellTiter-Glo® luminescent cell viability kit (Promega). Maximum passage number was assessed by monitoring the number of times iTDCs, iTDC-sTR, or WT-NTD were subcultured without alterations in morphology, growth rate, or transduction efficiency.
  • iTDCs were transduced with LV-GFP-FL or LV-sTR.
  • Engineered or unmodified cells were fixed, permeabilized, and incubated for lh with anti-nestin Polyclonal antibody (Millipore, MAB353, 1 :500, Billerica, MA, USA).
  • Cells were washed and incubated with the appropriate secondary antibody (Biotium, Hayward, CA, USA) for 1 hr. Cells were then washed, mounted, and imaged using fluorescence confocal 3Q microscopy.
  • engineered or non-transduced iTDCs were cultured for 12 days in N3 media depleted of doxycycline, EGF, and FGF. Cells were then stained with antibodies directed against nestin, glial fibrillary acidic protein (GF AP; Millipore, MAB3405, 1 :250), or Tuj-l (Sigma, T8578, 1 : 1 000) and detected with the appropriate secondary antibody (Biotium). Nuclei were counterstained with Hoechst 33342 and the results analyzed using a FV 1200 laser confocal microscope (Olympus, Center Valley, PA).
  • Three-dimensional tissue culture Three-dimensional levitation cell cultures were performed using the Bio- Assembler Kit (Nano3D Biosciences, Houston, TX). Confluent 6 well plates with GBM or iTDC were treated with a magnetic nanoparticle assembly (8 m ⁇ cm 2 of cell culture surface area or 50 m ⁇ cm 1 medium, NanoShuttle (NS), Nano3D Biosciences) for overnight incubation to allow for cell binding to the nanoparticles. NS was fabricated by mixing iron oxide and gold nanoparticles cross-linked with poly- 1 -lysine to promote cellular uptake.
  • Treated GBM and iTDC were then detached with trypsin, resuspended and mixed at different ratios (1 : 1 and 1 :0.5) in an ultra-low attachment 6 well plate with 2 ml of medium.
  • a magnetic driver of 6 neodymium magnets with field strength of 50 G designed for 6-well plates and a plastic lid insert were placed atop the well plate to levitate the cells to the air-liquid interface.
  • Media containing 4 pg/ml GCV was added to the co-culture of GBM with iTDC expressing ttk. Fluorescence images where taken over time to track the cell viability of both populations (previously labeled with different fluorescence).
  • iTDCs expressing RFP were seeded in micro-culture inserts in glass bottom microwell dishes (MatTek, Ashland, MA, EISA) using 2-chamber cell 25 culture inserts (ibidi, Verona, WI, EISA).
  • U87 glioma cells expressing GFP were plated into the adjacent well (0.5mm separation) or the well was left empty. 24 hrs after plating, cells were placed in a VivaView live cell imaging system (Olympus) and allowed to equilibrate. The insert was removed and cells were imaged at 10X magnification every 20 minutes for 36 hours in 6 locations per well (to monitor sufficient cell numbers) in three independent experiments. NIH Image was then used to generate movies and determine both the migrational velocity, total distance migrated, and the directionality of migration.
  • [00135] 3-dimensional migration iTDC migration to GBM spheroids was assessed in 3-D culture systems by creating iTDC and GBM spheroids using levitation culture as described above. iTDC and GBM spheroids were co-cultured in levitation systems. Real-time imaging was performed to visualize the penetration of GBM spheroids by iTDCs in suspension.
  • Co-culture viability assays mNTD expressing sTR or control GFP-RL (5xl0 3 ) were seeded in 96 well plates. 24 hrs later, Ei87-mC-FL, LNl8-mC-FL, or GBM8-mC-FL human GBM cells (5xl0 3 ) were seeded into the wells and GBM cell viability was measured 24 hrs later by quantitative in vitro bioluminescence imaging. GBM cells were also assessed at 18 hrs for caspase-3 l7 activity with a caged, caspase 3 l7-activatable DEVD-aminoluciferin (Caspase-Glo 317, Promega, Madison, WI, EISA).
  • Co-culture viability assays 3-D levitation culture was used in three separate in vitro cytotoxicity studies. iTDCs expressing 2 different cytotoxic agents were used to treat 1 established GBM cell line (U87) and 2 patient-derived GBM lines (GBM4, GBM8). 1) To determine the cytotoxicty of TRAIL therapy, iTDC-sTR or iTDC-mCherry spheroids were co cultured in suspension with U87-GFP-FLuc spheroids at a iNTD:GBM ratio of 1 :2 or 1 : 1. GBM spheroid viability was determined 48 hrs later by FLuc imaging. 2) To determine the
  • iTDC-TK spheroids were co-cultured in suspension with patient-derived GBM4-GFP-FLuc spheroids or mixed with GBM cells prior to sphere formation. Spheroids were cultured with or without ganciclovir (GCV) and GBM spheroid viability was determine 0, 2, 4, or 7 days after addition of the pro-drug by FLuc imaging. 3) To determine the cytotoxicity of sECM-encapsulated iNTD pro-drug/enzyme therapy, iTDC-TK were encapsulated in sECM and placed in levitation cultured with patient- derived GBM8-GFP-FLuc spheroids. Viability was determine by FLuc imaging.
  • mice were given an intraperitoneal injection of D-Luciferin (4.5 mg/mouse in 150 m ⁇ of saline) and photon emission was determined 5 minutes later using an IVIS Kinetic Optical System (PerkinElmer) with a 5 minute acquisition time. Images were processed and photon emission quantified using
  • the intracranial xenograft was identified using GFP fluorescence.
  • a small portion of the skull covering the tumor was surgically removed using a bone drill and forceps and the overlying dura was gently peeled back from the cortical surface to expose the tumor.
  • the GBM8-GFPFL tumor was surgically excised using a combination of surgical dissection and aspiration, and images of GFP were continuously captured to assess accuracy of GFP -guided surgical resection. Following tumor removal, the resulting resection cavity was copiously irrigated and the skin closed with 7-0 Vicryl suture. No procedure-related mortality was observed.
  • hyaluronic sECM hydro gels Sigma
  • Tissue processing Immediately after the last imaging session, mice were sacrificed, perfused with formalin, and brains extracted. The tissue was immediately immersed in formalin. 30 pm coronal sections were generated using a vibrating microtome (Fisher Waltham, MA, USA).
  • human fibroblasts were transduced with Sox2 and performed iTDC generation without feeder cells. Then, diagnostic iTDCs expressing optical reporters or therapeutic iTDCs expressing different cytotoxic agents were generated.
  • Figure 5 shows white light and fluorescence photomicrographs of the human fibroblasts and iTDCs grown as monolayers and neurospheres or stained with antibodies against nestin (green).
  • Figure 6 is a summary graph showing the expression of nestin over time at different days after induction of iTDC generation. Quantification showed nestin expression in iTDCs remained constant from day 2 through day 10.
  • Figure 7 shows images of the immunofluorescence staining. When induced to differentiate, the iTDCs expressed the astrocyte marker GF AP and the neural marker Tuj-l. Staining revealed the cells did not express the pluripotency makers TRA-160 or OCT4. These findings were confirmed by RT-PCR analysis.
  • Figures 8A - 8D show the RT-PCR analysis of nestin, SOX2, nanog, and OCT-4 expression in NHF, iTDC, and h-iPSC.
  • the iTDCs showed high level of nestin expression that was absent in parental fibroblasts or human iPSC (h- iPSC). Sox2 expression was high in both iTDCs and h-iPSCs because Sox2 overexpression was used to generate both cell lines.
  • iTDCs did not express high levels of the pluripotency markers Nanog or OCT3/4. Together, these data demonstrate the ability to create multi-potent iTDCs within 48 hrs using single-factor Sox2 expression.
  • iTDCs Migrate Selectively to CRM The ability to home to solid and invasive GBM deposits is one of the most beneficial characteristics of induced tumor homing cell based cancer therapies.
  • iTDC migration was compared to the parental human fibroblasts from which they were derived. It was found that iTDCs rapidly migrated towards the co-cultured GBM cells, covering the 500 pm gap in 22 hrs.
  • Figure 9 shows summary images showing migration of iTDC -mC-FL (red) or parental human fibroblasts toward U87-GFP-FL (green) at 0 and 22 hours after plating.
  • Figure 9 also shows single-cell tracings depicting the paths of iTDC -mC-FL or human fibroblast-directed migration toward GBM over 22 hours.
  • the dashed line indicates the site of GBM seeding.
  • Figure 10 shows summary graphs of the directionality and Euclidean distance of iTDC or fibroblast migration toward GBM cells determined from the real-time motion analysis.
  • FIG. 11 shows fluorescence images of the migration of iTDCs-mC-FL (red) into U87 spheroids (green) and their penetration toward the core of the tumor spheroid over time in the 3D levitation culture systems.
  • the top panel shows images of the mixed therapy, and the bottom panel shows images of the established GBM4 spheroids.
  • iTDC Persistence and In Vivo Fate We next utilized the engineered iTDCs to investigate the survival and fate of these cells in vivo in the brain.
  • a previous study of in vitro proliferation after engineering of iTDC with GFPFL and rnCFL showed no significant differences with non-engineered iTDCs.
  • iTDCs engineered with mCFL was stereotactically implanted in the brain of mice and real-time non-invasive imaging was used to monitor cell survival over time. Capturing images periodically, we found that iTDCs survive more than 20 days post implantation.
  • Figure 12 shows serial bioluminescent images taken over the course of the 20 days.
  • Post-mortem IHC revealed that approximately half of iTDC-mCFL expressed the NTD marker nestin and the other half were positive for the neuronal marker Tuj-l. No astrocyte marker GF AP was observed. Additional IHC, images of which are shown in
  • FIGS. 13A and 13B verified the transplanted iTDCs did not express the pluripotency markers Oct-4 and TDR-160.
  • iTDC-based GBM Tumoricidal iNTDs.
  • TNFa-related apoptosis- inducing ligand TRAIL; diTR
  • iNTD-diTR IRES-GFP element
  • iTDC-diTR or control iNTD-GFPRL were co-cultured at different ratios with human GBM cells expressing mCherry and firefly luciferase (mC-FL).
  • mC-FL firefly luciferase
  • iTDC Secretion of a Pro-Apoptotic Agent Reduces Solid GBM
  • Human U87 GBM cell expressing mC-FL were implanted intracranially with iNTD-sTR or 'control iNTD-GFP and tumor volumes were followed using serial bioluminescence imaging.
  • Figures 15A and 15B show representative bioluminescent images and summary data.
  • iTDC-sTR treatment induced a statistically significant reduction in tumor growth by day 3 and decreased GBM volumes 50-fold by day 24.
  • FIG. 16A, 16B, and 16C shows representative images demonstrating the expression of cytotoxic, differentiation, and pluripotency markers in iTDCTE -sTR after therapy.
  • the iTDC-sTR in the GBM were positive for the expression of the Nestin 15 and Tuj-l, and negative for GFAP and pluripotency markers Oct-4 and TRD-160.
  • iTDC prodrug/enzyme therapy for patient-derived CD133+ human GEM-initiating cells, we co-cultured GBM4 cells expressing GFP and firefly luciferase (GBM4-GFPFL) with iTDC expressing a trifunctional chimeric reporter including Rluc, RFP and thymidine kinase (TK) activities, to generate iTDC-TK.
  • GBM4-GFPFL firefly luciferase
  • iTDC expressing a trifunctional chimeric reporter including Rluc, RFP and thymidine kinase (TK) activities, to generate iTDC-TK.
  • TK thymidine kinase encoded by herpes simplex virus
  • GBM4-GFPFL and iTDC-TK were co-cultured in three-dimensional levitation system in two different models.
  • Figure 17 shows the fluorescent images taken of both models and the corresponding summary data.
  • the first model top
  • the second model the two cell types were cultured side by side to mimic the treatment of an established GBM.
  • Cell survival was monitored over time by fluorescence.
  • a significant reduction of the GBM survival was observed over time, being more significant in the mixed model.
  • FIGS 18A and 18B show the serial bioluminescence data and corresponding summary data.
  • Serial bioluminescence imaging showed that iTDC-TK treatment attenuated the progression of GBM4 tumors, reducing tumor burden by 9-fold compared to control 28 days after injection.
  • iTDC-TK therapy also led to a significant extension in survival as iTDC-TK treated animals survived an average of 67 days compared to only 37 days in control -treated mice.
  • Post-mortem IHC the images of which are shown in Figures 19A and 19B, verified the significant reduction in tumor volumes by iTDC- TK injection.
  • Figures 19A and 19B show whole-brain and high-magnification images showing cell nuclei (blue), GBM4 (green), and iTDCTE -TK (red) distribution 21 days after delivering iTDCTE -control (I) or iTDCTE -TK (J) into established GBM4 tumors.
  • I iTDCTE -control
  • J iTDCTE -TK
  • a large GBM4 tumor was present in the control iTDCTE -TK animals, and only a small GBM4 focus was detected in mice treated with iTDCTE -TK+ GCV.
  • iTDC-TK therapy has significant therapeutic effects against malignant and invasive GBM and markedly prolongs the survival of tumor-bearing mice.
  • Intracavity iTDC-TK Therapy inhibits surgically resected GBM recurrence. Surgical resection is part of the clinical standard of care for GBM patients. We previously discovered that encapsulation of stem cells is required for intracavity therapy to effectively suppress GBM recurrence.
  • sECM synthetic extracellular matrices
  • Figures 20A and 20B show the fluorescent images of the cultures and the summary data. We found that mCherry+ iTDCs migrated from the sECM matrix and populated GFP+ GBM8 spheroids within 3 days.

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Abstract

La présente invention concerne des cellules vecteurs de médicament d'écotropisme de tumeur induite destinées à être utilisées dans le traitement du cancer de l'ovaire. L'invention concerne également des compositions pharmaceutiques comprenant des cellules vecteurs de médicament d'écotropisme de tumeur induite destinées à être utilisées dans le traitement du cancer de l'ovaire.
PCT/US2019/059532 2018-11-01 2019-11-01 Compositions de cellules d'écotropisme de tumeur destinées à être utilisées dans des méthodes thérapeutiques Ceased WO2020093003A1 (fr)

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