WO2017214190A1 - Cellules dendritiques modifiées exprimant la 1a-hydroxylase et utilisations associées pour le traitement de maladies à médiation immunitaire - Google Patents

Cellules dendritiques modifiées exprimant la 1a-hydroxylase et utilisations associées pour le traitement de maladies à médiation immunitaire Download PDF

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WO2017214190A1
WO2017214190A1 PCT/US2017/036210 US2017036210W WO2017214190A1 WO 2017214190 A1 WO2017214190 A1 WO 2017214190A1 US 2017036210 W US2017036210 W US 2017036210W WO 2017214190 A1 WO2017214190 A1 WO 2017214190A1
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dendritic cell
cells
engineered
engineered dendritic
dcs
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Xiaolei Tang
David J. Baylink
Kin-Hing William Lau
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Loma Linda University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/0071Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/461Cellular immunotherapy characterised by the cell type used
    • A61K39/4615Dendritic cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/462Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/462Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622Antigen presenting cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/46Cellular immunotherapy
    • A61K39/464Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643Vertebrate antigens
    • A61K39/46433Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y114/00Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13013Calcidiol 1-monooxygenase (1.14.13.13), i.e. 25-hydroxyvitamin D-1-alpha-hydroxylase
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • MS Multiple sclerosis
  • CNS central nervous system
  • Certain aspects of the present disclosure are generally directed to engineered cells and methods of treating immune-mediated diseases with minimal side effects on a patient's immune defense.
  • a subset of immune cells is engineered to have a short life span and/or to synthesize de novo a therapeutic drug.
  • the engineered cells can be dendritic cells.
  • the engineered cells are dendritic cells that have been treated to inhibit or prevent cell proliferation.
  • the therapeutic drug produced by the engineered cells has the ability to induce in the immune system another immune cell subset.
  • the therapeutic drug can induce an immune cell subset that includes cells that have a long life span and/or the capacity to specifically suppress the immune attack without interfering with an individual's general immunity.
  • the therapeutic drug can induce regulatory T cells.
  • One aspect of the present disclosure is directed toward limiting the de novo synthesis of a high concentration of the therapeutic drug to the immune system, thereby preventing drug effects in other unintended tissues.
  • the short life span of the engineered cells ensures that, upon induction of the long-lived but more specific regulatory cells (e.g., regulatory T cells), the drug is removed from the patient's body to ensure safety of the therapy.
  • dendritic cells are engineered for de novo synthesis of a supra-physiological and/or therapeutic dose of the active vitamin D metabolite l,25[OH] 2 D, also referred to herein as calcitriol.
  • the de novo synthesis of a therapeutic calcitriol dose is achieved through overexpression in dendritic cells of lct- hydroxylase that physiologically converts 25[OH]D (or calcidiol), the major vitamin D form in the blood circulation and the substrate for 1 ⁇ -hydroxylase, into calcitriol.
  • the overexpression of 1 ⁇ -hydroxylase is achieved through transduction of dendritic cells with a vector that expresses cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) gene.
  • the CYP27Bl-engineered dendritic cells are loaded with one or more peptides (epitopes) that are specifically expressed in the diseased tissues and associated with pathogenesis of the disease.
  • the epitope-loaded CYP27Bl-engineered dendritic cells (CYP27Bl-engineered DCs) are irradiated or treated to prevent further proliferation (non-proliferative).
  • the epitope-loaded non-proliferative CYP27Bl-engineered DCs are intravenously injected into a mammal.
  • the CYP27Bl-engineered DCs migrate into the peripheral lymphoid tissues.
  • the CYP27Bl-engineered DCs can induce epitope-specific regulatory T cells which then specifically suppress pathogenic T cells.
  • the pathogenic T cells can be the immune cells that are associated with pathogenesis of the immune-mediated disease.
  • the accumulated epitope- specific regulatory T cells can specifically suppress pathogenic T cells.
  • the accumulated epitope-specific regulatory T cells specifically promote apoptosis of pathogenic T cells.
  • the CYP27Bl-engineered DCs spontaneously undergo apoptosis, thereby preventing prolonged generation of a high calcitriol concentration to avoid potential leaking into the systemic circulation and attending adverse side effects.
  • vitamin D and/or 25[OH]D supplementation can be an adjuvant therapy to maintain a sufficient and same level of 25[OH]D in the blood circulation.
  • the CYP27Bl-engineered DCs can be injected multiple times until the disease is completely controlled. In certain aspects, the time between the two injections can be at least two weeks.
  • an engineered cell includes a dendritic cell that has been altered to overexpress a 1 ⁇ -hydroxylase.
  • the dendritic cell has been altered by a transduction of the dendritic cell with a vector that expresses cytochrome P450 family 27 polypeptide 1.
  • the transduction may be performed using a lentivirus or an FDA-approved virus.
  • the dendritic cell is treated to achieve an inhibition of a proliferation of the dendritic cell.
  • the dendritic cell may be treated with mitomycin C to achieve the inhibition of the proliferation of the dendritic cell.
  • a method for treating an immune-mediated disease.
  • the method includes: altering a dendritic cell so that the dendritic cell overexpresses a gene of cytochrome P450 family 27 polypeptide 1, thereby producing an engineered cell; treating the engineered cell so that a proliferation of the engineered cell is inhibited, thereby producing a non-proliferative engineered cell; and immunizing a patient having said immune-mediated disease with said non-proliferative engineered cell.
  • immunizing comprises injecting into the patient having the immune-mediated disease an amount of the non-proliferative engineered cell that has been mixed with an epitope that is being targeted by the immune-mediated disease.
  • engineered dendritic cells comprising a heterologous polynucleotide that encodes a lct-hydroxylase protein are provided.
  • the engineered dendritic cell overexpresses the heterologous polynucleotide that encodes the 1 ⁇ -hydroxylase protein.
  • the heterologous polynucleotide comprises a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1) polynucleotide sequence.
  • the heterologous polynucleotide is operably linked to a promoter.
  • the promoter is a constitutively active promoter.
  • the promoter is a spleen focus-forming virus (SFFV) promoter.
  • the engineered dendritic cell comprises an expression cassette comprising a promoter operably linked to the heterologous polynucleotide that encodes the 1 ⁇ -hydroxylase protein.
  • the engineered dendritic cell comprises a vector (e.g., a viral vector) that comprises an expression cassette comprising a promoter operably linked to the heterologous polynucleotide that encodes the 1 ⁇ - hydroxylase protein.
  • the dendritic cell is a bone marrow-derived dendritic cell. In some embodiments, the dendritic cell is an activated dendritic cell.
  • the engineered dendritic cell synthesizes 1,25- dihydroxyvitamin D at an increased level, relative to a control dendritic cell lacking the heterologous polynucleotide that encodes the 1 ⁇ -hydroxylase protein, when cultured in the presence of 25-hydroxyvitamin D.
  • the engineered dendritic cell is pulsed with an antigen peptide.
  • the antigen peptide is a peptide of a myelination protein.
  • the antigen peptide is a peptide of myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG), or a combination thereof.
  • the engineered dendritic cell is treated with an anti- proliferative agent.
  • the anti-proliferative agent is mitomycin C.
  • compositions comprising a population of the engineered dendritic cell as described herein are provided.
  • expression cassettes that comprises a promoter operably linked to a heterologous polynucleotide that encodes a lct-hydroxylase protein are provided.
  • the expression cassette comprises a promoter (e.g., a constitutively active promoter, e.g., a spleen focus-forming virus (SFFV) promoter) that is operably linked to a polynucleotide comprising a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1) polynucleotide sequence such as the polynucleotide sequence of NCBI GenBank Accession No. NM_000785.3.
  • a promoter e.g., a constitutively active promoter, e.g., a spleen focus-forming virus (SFFV) promoter
  • SFFV spleen focus-forming virus
  • the expression cassette when introduced into a cell (e.g., a dendritic cell) results in the cell synthesizing 1,25-dihydroxyvitamin D at an increased level when cultured in the presence of 25-hydroxyvitamin D, relative to a control cell (e.g., control dendritic cell) lacking the expression cassette.
  • vectors comprising the expression cassettes as described herein are provided.
  • pharmaceutical compositions comprising an engineered dendritic cell or population of engineered dendritic cells as described herein and further comprising a pharmaceutically acceptable excipient are provided.
  • the method comprises contacting one or more T cells with an engineered dendritic cell, composition comprising a population of engineered dendritic cells, or pharmaceutical composition as described herein.
  • the T cells are myelin-specific T cells.
  • methods of treating a subject having an immune-mediated disease comprising administering to the subject an engineered dendritic cell, composition comprising a population of engineered dendritic cells, or pharmaceutical composition as described herein.
  • the immune-mediated disease is a demyelinating disease.
  • the demyelinating disease is multiple sclerosis.
  • FIG. 1A-1D Bone-marrow-derived dendritic cells
  • BM-DCs bone-marrow-derived dendritic cells
  • the BM-DCs were transduced with the lenti- CYP-GFP vector at days 4 and 6 during in vitro culture.
  • Lentiviral vectors used in this study included lenti-CYP-GFP that carried a CYP27B1 gene and a GFP gene. SSFV and PGK were promoters.
  • BM-DCs were transduced with no vector (Control), lenti-GFP (GFP), or lenti-CYP-GFP (hCYP27Bl). 48 hours after the last transduction, BM-DCs were then collected for analyzing expression of CYP27B1 mRNAs by real time PCR. To facilitate detection, CYP27B1 gene used in this study was human origin.
  • D BM-DCs were cultured and transduced as shown in (C). 24 hours after the last transduction, the substrate 25[OH]D was added. The cells were cultured for another 24 hours and supernatants collected for measuring l,25(OH) 2 D concentration by a radioimmunoassay.
  • FIG. 2A-2E lct-hydroxylase-overexpressing bone-marrow-derived maintain lymphoid tissue-homing capacity.
  • A BM-DCs were generated and transduced for overexpression of lct-hydroxylase as described for FIG. 1. 24 hours after the last transduction, cells were analyzed for the expression of MHC I, MHC II, CD80, and CD86 by flow cytometry.
  • B DC2.4 cells were transduced with lenti-CYP-GFP to generate a stable transfectant that constitutively overexpressed lct-hydroxylase and GFP ("DC2.4-CYP-GFP"). Subsequently, 1 x 10 6 DC2.4-CYP-GFP cells were subcutaneously injected into mice.
  • FIG. 3A-3B One intravenous injection of immunogenic but non-proliferative BM- DCs overexpressing 1 ⁇ -hydroxylase and pulsed with MOG 35-55 suppresses ongoing experimental autoimmune encephalomyelitis.
  • C57BL/6 mice were immunized with MOG 35-55 in Complete Freund Adjuvant for inducing experimental allergic encephalomyelitis (EAE).
  • EAE experimental allergic encephalomyelitis
  • EAE experimental allergic encephalomyelitis
  • mDC2.4 Mean disease severity score for animals that received no treatment (No Tx) or that received 1 x 10 6 immunogenic but non-proliferative DC2.4 (mDC2.4).
  • B Mean disease severity score for animals that received no treatment (No Tx) or that received 1 x 10 6 MOG 35-55 -pulsed immunogenic but nonproliferative DC2.4-CYP cells. Paralytic disease was monitored daily.
  • FIG. 4A-4D 1 ⁇ -hydroxylase-overexpressing BM-DCs (bone-marrow-derived DCs) enhance apoptosis of CD4 + T cells in local draining lymph nodes.
  • Animals were subcutaneously immunized with MOG 35-55 in Complete Freund Adjuvant (CFA).
  • CFA Complete Freund Adjuvant
  • animals subcutaneously received 1 x 10 s parental BM-DCs (DC) or BM-DCs that were transduced with lenti-CYP-GFP (DC-CYP).
  • DC-CYP lenti-CYP-GFP
  • mononuclear cells from local draining lymph nodes were examined for Annexin and PI staining in different T cell subsets by flow cytometry.
  • A Experimental design.
  • B Gating strategy.
  • C Representative FACS plots showing Annexin and PI staining in all, CD4 + and CD8 + T cells.
  • D Cumulative data showing the percentage of apoptotic cells among CD4 + T cells (left panel) and among CD8 + T cells (right panel). *P ⁇ 0.05.
  • FIG. 5A-5D 1 ⁇ -hydroxylase-overexpressing BM-DCs create in the peripheral lymphoid tissues a microenvironment that favors differentiation of IL-10 + Treg and Th2 cells.
  • CFA Complete Freund Adjuvant
  • C Representative plots (upper panel) and cumulative data (lower panels) showing the percentages of IL-2 + and IL-10 + CD4 + T cells.
  • D Representative plots (upper panels) and cumulative data (lower panels) showing the percentages of IL-4 + , IL-5 + , IL6 + , and IL-13 + CD4 + T cells. *P ⁇ 0.05.
  • FIG. 6 lct-hydroxylase-overexpressing BM-DCs increase secretion of IL-2, IL-10, IL- 4, and IL-6 in splenocytes.
  • CFA Complete Freund Adjuvant
  • splenocytes from the animals were stimulated with MOG 35-55 -pulsed DCs and 48 hours later supernatants collected for analyzing secretion of IL- 2 (upper left panel), IL-10 (upper right panel), IL-4 (lower left panel), and IL-6 (lower right panel) by flow cytometry. *P ⁇ 0.05.
  • FIG. 7A-7E DC-CYP stimulates the expression of Th2 and Trl cytokines in CD4 + T cells.
  • A Experimental schematic. C57BL/6 mice were subcutaneously immunized with MOG35..55 emulsified in Complete Freund Adjuvant (CFA). At days 10, 17, and 24, the animals subcutaneously received one of the following immunizations ("immu"): (1) no immunization (No immu); or (2) 1 x 10 6 DC-mCYP-GFP. Nine days after the final immunization, splenocytes from the animals were stimulated with MOG 35-55 -pulsed DC2.4.
  • CFA Complete Freund Adjuvant
  • FIG. 8A-8C DC-CYP stimulates foxp3 + Treg cells in the peripheral lymphoid tissues.
  • Immu immunizations
  • B Representative FACS plots show foxp3 + cells among CD3 + T cells.
  • FIG. 9A-9C Immunization with MOG 35-55 -pulsed DC-CYP but not parental DCs suppresses ongoing EAE.
  • C57BL/6 mice were immunized with MOG 35-55 for the induction of EAE. Ten days later, the animals intravenously received one of the following immunizations ("Immu"): (1) no immunizations ("No immu”); (2) 1 x 10 6 DC2.4-mCYP-GFP; or (3) 1 x 10 6 MOG 3 5 -S s-pulsed (100 ⁇ for 3 hours) DC2.4-mCYP-GFP. The animals were then monitored for paralytic symptoms daily. At day 18, spinal cords from the animals were analyzed by Luxol Fast Blue staining.
  • FIG. 10 Suppression of experimental autoimmune encephalomyelitis mediated by MOG35 -55 -pulsed 1 ⁇ -hydroxylase-overexpressing BM-DCs requires foxp3 + Treg cells.
  • C57BL/6 mice were immunized with MOG35-55 for the induction of EAE. The animals received either no treatment (filled triangles) or 1 x 10 6 MOG 35 -55-pulsed immunogenic but non-proliferative mDC-CYP cells (open circles and open triangles).
  • an anti-CD25 monoclonal antibody clone PC61, 500 ⁇ g/mouse/injection
  • FIG. 11A-11C Delivery of the active vitamin D metabolite to immune synapses reduces expression of CXCR3 on CD4 + T cells.
  • CFA Complete Freund Adjuvant
  • CFA Complete Freund Adjuvant
  • splenocytes from the animals were analyzed for the expression of CXCR3 on CD4 + T cells by flow cytometry.
  • A Gating strategy.
  • B Representative plots showing CXCR3 expression on CD4 + T cells.
  • FIG. 12 A model of myelin-specific immune suppression mediated by lct- hydroxylase-overexpressing dendritic cells.
  • a myelin-antigen-pulsed DC-CYP upon in vivo administration, specifically migrates into the peripheral lymphoid tissues where it interacts with myelin-specific T cells and at the same time actively de novo synthesizes a high l,25(OH) 2 D concentration at the interface of the DC-CYP and the myelin-specific T cells.
  • the DC-CYP is self-tolerized and becomes TolDC-CYP through l,25(OH) 2 D's intracrine and/or paracrine action. Additionally, the myelin-specific T cells differentiate into Treg cells through the paracrine action of l,25(OH) 2 D. Consequently, the newly induced myelin-specific Treg cells provide myelin-specific suppression of EAE.
  • compositions and methods for treating immune-mediated diseases such as but not limited to multiple sclerosis and rheumatoid arthritis
  • immune-mediated diseases such as but not limited to multiple sclerosis and rheumatoid arthritis
  • engineered dendritic cells that overexpress 1 ⁇ -hydroxylase and method of treating multiple sclerosis comprising administering the engineered dendritic cells to a subject in need thereof, are provided.
  • Multiple sclerosis (MS) is a disease characterized by chronic inflammation, demyelination, and axonal loss in the central nervous system, in which myelin sheaths around the axons of the brain and spinal cord are damaged.
  • autoimmune inflammatory attack of the myelin sheath causes demyelination.
  • the ultimate goal of MS therapy is to stop progression of the disease without interfering with global immune defense mechanisms.
  • currently available medications have not met this goal because the current therapies block in the immune system one or more components that mediate the inflammatory disorder in the CNS of MS patients.
  • these blocked immune components also participate in the general immune defense against infections and cancers. Consequently, normal immune defense mechanisms are attenuated, which leads to predictable adverse effects. For this reason, active investigations into more specific blocking of the immune attack of one's own myelin sheath are being pursued to increase efficacy of the therapy and to mitigate side effects.
  • AST antigen-specific therapy
  • AST is a major sought goal in the treatment of MS.
  • AST has a unique advantage in that it aims to specifically block immune attack of myelin sheath and hence prevent undesirable side effects which are frequently associated with the currently available therapies and caused by unintended suppression of immunity.
  • DCs programmed dendritic cells
  • Treg myelin-specific regulatory T
  • the cells, compositions, and methods disclosed herein resolve the instability issue associated with the use of DCs for augmentation of myelin-specific Treg cells in vivo.
  • a therapeutic strategy is provided that does not depend on functional status of DCs, meaning that the use of both non-immunogenic and immunogenic DCs can have therapeutic effects. If the use of immunogenic DCs is effective, then the use of non-immunogenic DCs can be expected to be effective as well. Accordingly, some embodiments herein disclosed focused on evaluating whether engineered immunogenic DCs would be able to suppress ongoing EAE. As described below in the Examples section and in FIGS. 3A-3B, although parental immunogenic DCs worsened EAE, ⁇ -hydroxylase-overexpressing immunogenic DCs effectively suppressed the progression of EAE.
  • the present disclosure relates to a novel therapy that helps resolve the DC conversion concern.
  • engineered DCs are disclosed that can de novo synthesize the active vitamin D metabolite (i.e., l,25[OH] 2 D, also referred to as calcitriol).
  • DCs are engineered for de novo production of the Treg-inducing calcitriol concentration by overexpressing 1 ⁇ -hydroxylase in the DCs.
  • the engineered DCs synthesize a supra-physiological calcitriol concentration.
  • immunization with such engineered DCs can induce Treg cells and suppress disease progression.
  • de novo synthesized calcitriol can entail several outcomes.
  • calcitriol has been shown under certain conditions to tolerize both animal and human DCs in ex vivo cultures.
  • intracrine, autocrine, and/or paracrine effects of calcitriol produced by the engineered DCs can be sufficient to suppress potential immunogenicity of in vivo converted engineered DCs, thereby circumventing the concern of conversion of the engineered DCs into pathogenic DCs.
  • calcitriol produced by the engineered DCs can program Treg cells at DC-T cell interfaces (immune synapses) during DC-T cell interaction in the peripheral lymphoid tissues, hence augmenting immune regulation and treating MS.
  • the de novo synthesis of calcitriol can ensure generation of a focal Treg- inducing calcitriol concentration in peripheral lymphoid tissues.
  • nonproliferative DCs can achieve the in vivo presence of the engineered DCs within a short period of time that is sufficient for Treg induction.
  • Both de novo calcitriol synthesis and non-proliferative status of the DCs can prevent a potential massive increase of circulating calcitriol level in systemic circulation. As a result, it can reduce side effects (e.g., hypercalcemia) in response to a high circulating level of calcitriol.
  • engineered cells are used to induce Treg cells in vivo with a safe profile.
  • DCs are engineered to overexpress 1 ⁇ -hydroxylase such that the engineered DCs can de novo synthesize a supra- physiological calcitriol concentration at DC-T cell interfaces (immune synapses) during DC-T cell interaction in the peripheral lymphoid tissues.
  • the expression of 1 ⁇ -hydroxylase in parental BM-DCs and engineered DCs confirmed functional overexpression of 1 ⁇ -hydroxylase by transducing the parental DCs with a lentiviral vector that expressed CYP27B1 gene (the gene that encodes 1 ⁇ - hydroxylase).
  • 1 ⁇ -hydroxylase refers to 25-hydroxyvitamin D-l alpha hydroxylase
  • 1 ⁇ -hydroxylase is an enzyme that catalyzes the conversion of 25- hydroxyvitamin D3 (25(OH)D) to 1,25-dihydroxyvitamin D3 (l,25(OH) 2 D).
  • the gene that encodes 1 ⁇ -hydroxylase is a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1). Sequences for human CYP27B1 mRNA and 1 ⁇ -hydroxylase protein are set forth in, e.g., NCBI GenBank Accession Nos. NM_000785.3 and NP_000776.1, respectively.
  • a dendritic cell as described herein is engineered to express a 1 ⁇ - hydroxylase protein that has at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the 1 ⁇ -hydroxylase protein set forth in NCBI GenBank Accession No. NP_000776.1.
  • identity in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity) over a specified region.
  • Methods for comparing polynucleotide or polypeptide sequences and determining percent identity are described in the art. See, e.g., Roberts et al., BMC Bioinformatics, 7:382, 2006, incorporated by reference herein.
  • nucleic acid and “polynucleotide” are used interchangeably herein and refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • the polynucleotide is DNA (e.g., genomic DNA or cDNA).
  • the polynucleotide is RNA (e.g., mRNA).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), polymorphic variants (e.g., SNPs), splice variants, and nucleic acid sequences encoding truncated forms of proteins, complementary sequences, as well as the sequence explicitly indicated.
  • protein and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins and truncated proteins.
  • a promoter refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell.
  • a promoter includes cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
  • a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
  • tissue-specific promoter initiates transcription only in one or a few particular tissue types.
  • a polynucleotide sequence is "heterologous" to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a promoter when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., the promoter is from a different gene in the same species).
  • immune-mediated disease refers to a disease that results from a dysregulation of normal immune response in a subject.
  • the immune-mediated disease is an autoimmune disease (e.g., multiple sclerosis, lupus, or rheumatoid arthritis).
  • the immune-mediated disease is a demyelinating disease.
  • demyelinating disease refers to a disease or condition of the nervous system characterized by damage to or loss of the myelin sheath of neurons.
  • a demyelinating disease can be a disease affecting the central nervous system or a disease affecting the peripheral nervous system.
  • demyelinating diseases include, but are not limited to, multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukoystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV-associated myelopathy.
  • the demyelinating disease is multiple sclerosis.
  • a "subject" is a mammal, in some embodiments, a human. Mammals can also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • farm animals e.g., cows, pigs, horses, chickens, etc.
  • sport animals e.g., pets, primates, horses, dogs, cats, mice and rats.
  • treatment refers to any indicia of success in the treatment or amelioration of an injury, disease, or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, disease, or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; and/or improving a subject's physical or mental well-being.
  • a "therapeutic amount” or a "therapeutically effective amount” of an agent is an amount of the agent that prevents, alleviates, abates, or reduces the severity of symptoms of a disease (e.g., an immune-mediated disease, e.g., MS) in a subject.
  • a therapeutically effective amount will show an increase or decrease of therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as "fold" increase or decrease.
  • a therapeutically effective amount can have at least a 1.2-fold, 1.5- fold, 2-fold, 5-fold, or more effect over a control.
  • administer refers to introducing an agent (e.g., an engineered dendritic cell, population of engineered dendritic cells, or pharmaceutical composition comprising an engineered dendritic cell as described herein) into a subject or patient, such as a human.
  • agent e.g., an engineered dendritic cell, population of engineered dendritic cells, or pharmaceutical composition comprising an engineered dendritic cell as described herein
  • the terms encompass both direct administration, (e.g., self-administration or administration to a patient by a medical professional) and indirect administration (e.g., the act of prescribing a compound or composition to a subject).
  • pharmaceutical composition refers to a composition suitable for administration to a subject.
  • a pharmaceutical composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response with the subject.
  • Pharmaceutical compositions can be designed for administration to subjects in need thereof via a number of different routes of administration, including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalational, and the like.
  • dendritic cells that have been engineered to express or overexpress a 1 ⁇ -hydroxylase are provided.
  • the dendritic cell comprises a heterologous polynucleotide that encodes a 1 ⁇ -hydroxylase protein.
  • the dendritic cell can be obtained or derived from any suitable source.
  • the dendritic cell is a bone marrow-derived dendritic cell.
  • the dendritic cell is a cord blood-derived dendritic cell.
  • the dendritic cell is a peripheral blood-derived dendritic cell.
  • the dendritic cell comprises a polynucleotide comprising a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1) polynucleotide sequence (e.g., the polynucleotide sequence of NCBI GenBank Accession No. NM_000785.3 or a variant thereof).
  • the polynucleotide encodes a human 1 ⁇ - hydroxylase protein having the sequence of the 1 ⁇ -hydroxylase protein set forth in NCBI GenBank Accession No.
  • NP_000776.1 or a variant thereof (e.g., a protein that has at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the 1 ⁇ -hydroxylase protein set forth in NCBI GenBank Accession No. NP_000776.1).
  • the engineered dendritic cell overexpresses the lct- hydroxylase protein, as compared to a dendritic cell lacking the heterologous polynucleotide.
  • the dendritic cell comprising a heterologous polynucleotide expresses the 1 ⁇ -hydroxylase protein at a level that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold higher than a dendritic cell lacking the heterologous polynucleotide.
  • Protein expression can be detected and quantified using routine techniques such as immunoassays, two-dimensional gel electrophoresis, and quantitative mass spectrometry that are known to those skilled in the art. Protein quantification techniques are generally described in "Strategies for Protein Quantitation,” Principles of Proteomics, 2nd Edition, R. Twyman, ed., Garland Science, 2013.
  • protein expression is detected by immunoassay, such as but not limited to enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); immunofluorescence (IF); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL).
  • EIA enzyme multiplied immunoassay technique
  • ELISA enzyme-linked immunosorbent assay
  • MAC ELISA IgM antibody capture ELISA
  • MEIA microparticle enzyme immunoassay
  • CEIA capillary electrophoresis immunoassays
  • RIA radioimmunoassays
  • IRMA immunoradiometric
  • protein expression is detected by quantitative mass spectrometry, for example but not limited to, spectral count MS, ion intensities MS, metabolic labeling (e.g., stable-isotope labeling with amino acids in cell culture (SILAC), enzymatic labeling, isotopic labeling (e.g., isotope-coded protein labeling (ICPL) or isotope-coded affinity tags (ICAT)), and isobaric labeling (e.g., tandem mass tag (TMT)).
  • quantitative mass spectrometry for example but not limited to, spectral count MS, ion intensities MS, metabolic labeling (e.g., stable-isotope labeling with amino acids in cell culture (SILAC), enzymatic labeling, isotopic labeling (e.g., isotope-coded protein labeling (ICPL) or isotope-coded affinity tags (ICAT)), and isobaric labeling (e.g., tandem mass tag (TMT)).
  • the heterologous polynucleotide that encodes a 1 ⁇ - hydroxylase protein is operably linked to a promoter.
  • the dendritic cell comprises a polynucleotide encoding a 1 ⁇ -hydroxylase protein (e.g., a polynucleotide comprising a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1) polynucleotide sequence such as the polynucleotide sequence of NCBI GenBank Accession No. NM_0007S5.3) that is operably linked to a promoter.
  • the promoter is a constitutively active promoter.
  • Suitable promoters include, but are not limited to, a spleen focus-forming virus (SFFV) promoter, a phosphoglycerate kinase (PGK) promoter, EFla promoter, a cytomegalovirus (CMV) promoter, a Rous sarcoma virus promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus promoter, a Moloney virus promoter, an avian leukemia virus promoter, or an Epstein-Barr virus immediate early promoter.
  • the promoter is an inducible promoter (e.g., a tetracycline-inducible promoter).
  • the promoter is a tissue-specific promoter (e.g., a dendritic cell-specific promoter, e.g., a CDllc promoter or a DC-STAMP promoter).
  • the promoter is a SFFV promoter or a PGK promoter.
  • the engineered dendritic cell comprises an expression cassette that comprises a promoter operably linked to a heterologous polynucleotide that encodes a 1 ⁇ -hydroxylase protein (e.g., a promoter operably linked to a polynucleotide comprising a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1) polynucleotide sequence such as the polynucleotide sequence of NCBI GenBank Accession No. NM_000785.3).
  • the engineered dendritic cell comprises a vector that comprises an expression cassette that comprises a promoter operably linked to a heterologous polynucleotide that encodes a 1 ⁇ -hydroxylase protein.
  • the polynucleotide that encodes a 1 ⁇ -hydroxylase protein is expressed in the dendritic cell using a virus or viral vector.
  • the virus is an adenovirus, lentivirus, adeno-associated virus, or retrovirus.
  • the virus is a lentivirus.
  • Viruses and viral vectors containing the polynucleotide that encodes a 1 ⁇ -hydroxylase protein can be introduced into the dendritic cell by methods known in the art, such as but not limited to, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, or lipofection.
  • the engineered dendritic cell is activated.
  • the dendritic cell is activated by stimulating the cell with an antigen, such as lipopolysaccharide (LPS).
  • the dendritic cell is activated by stimulating the cell with a cytokine, such as TNF- ⁇ .
  • the engineered dendritic cell is pulsed with an antigen peptide.
  • the antigen peptide is a peptide of a myelination protein.
  • the antigen peptide is a peptide of myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG), or a combination thereof.
  • the peptide comprises at least 9, 12, 15, 18, 21, 14, 17, 30, 40, 50, 60, 70, 80, 90, 100 contiguous amino acids of a MBP, PLP, or MOG protein.
  • the peptide is a MOG peptide, such as MOG peptide 35-55 (MOG 35-55 ), MOG peptide 89-113 (MOG 89- ii 3 ), or MOG peptide 92-106 (MOG 92- io6).
  • MOG peptide 35-55 MOG 35-55
  • MOG peptide 89-113 MOG 89- ii 3
  • MOG peptide 92-106 MOG 92- io6
  • the peptide is a MBP peptide, such as MBP peptide 4-14 (MBP 4- i 4 ), MBP peptide 68-86 (MBP 68-8 6), MBP peptide 84-97 (MBP 84-97 ), MBP peptide 84-105 (MBP 84-105 ), MBP peptide 87-99 ( ⁇ 87-99 ), or MBP peptide 90-106 (MBPgo-ioe)- Methods for peptide pulsing are known in the art. See, e.g., O'Neill et al., Methods Mol Med, 2005, 109:97-112.
  • the engineered dendritic cell is treated with an antiproliferative agent.
  • the anti-proliferative agent is irradiation (e.g., gamma irradiation).
  • the anti-proliferative agent is a chemical compound (e.g., mitomycin C).
  • the engineered dendritic cell is capable of de novo synthesizing 1,25-dihydroxyvitamin D when cultured in the presence of 25-hydroxyvitamin D. In some embodiments, the engineered dendritic cell synthesizes 1,25-dihydroxyvitamin D at an increased level, relative to a control dendritic cell lacking the heterologous polynucleotide that encodes the lct-hydroxylase protein, when cultured in the presence of 25-hydroxyvitamin D.
  • the engineered dendritic cell synthesizes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% more 1,25-dihydroxyvitamin D, when cultured in the presence of 25-hydroxyvitamin D, as compared to a control dendritic cell.
  • the engineered dendritic cell synthesizes 1,25-dihydroxyvitamin D at a level that is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold or more, when cultured in the presence of 25-hydroxyvitamin D, as compared to a control dendritic cell.
  • Measurement of the 1 ⁇ -hydroxylase enzymatic activity of the engineered dendritic cell can be performed according to methods known in the art. In some embodiments, measurement of 1 ⁇ -hydroxylase enzymatic activity is performed as described in the Examples section below or in Li ef al, FASEB J., 2017, doi:10.1096/fj.201601243R, incorporated by reference herein.
  • compositions and Kits [0068]
  • compositions comprising an engineered dendritic cell or population of engineered dendritic cells as described herein are provided.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • Guidance for preparing formulations for use in the present invention is found in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005.
  • a pharmaceutical composition comprises an acceptable carrier and/or excipients.
  • a pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the therapeutic agent.
  • the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration.
  • Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s).
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
  • carbohydrates such as glucose, sucrose, or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as ascorbic acid or glutathione
  • low molecular weight proteins compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
  • Other pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington: The Science and Practice of Pharmacy, supra.
  • Various pharmaceutically acceptable excipients are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipients (5 ed., Ed. Rowe et al., Pharmaceutical Press
  • the engineered dendritic cell or population of engineered dendritic cells can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, stabilizers and preservatives.
  • an aqueous solution is used, such as a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • kits comprising the engineered dendritic cells, populations of engineered dendritic cells, or pharmaceutical compositions comprising an engineered dendritic cell as described herein are provided.
  • the kit further comprises one or more additional agents, e.g., vitamin D, vitamin D3, or 25-hydroxyvitamin D3 (25(OH)D).
  • a kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention (e.g., instructions for using the kit for treating an immune-mediated disease). While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • the method comprises administering to the subject an engineered dendritic cell that expresses a heterologous polynucleotide that encodes a 1 ⁇ -hydroxylase (e.g., a heterologous polynucleotide comprising a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1) polynucleotide sequence, operably linked a heterologous promoter), or a composition comprising a population of the engineered dendritic cells or a pharmaceutical composition comprising the engineered dendritic cell.
  • a heterologous polynucleotide that encodes a 1 ⁇ -hydroxylase e.g., a heterologous polynucleotide comprising a human cytochrome P450 family 27 subfamily B member 1 (CYP27B1) polynucleotide sequence, operably linked a heterologous promoter
  • CYP27B1 human cytochrome P450 family 27 subfamily B member 1
  • the therapeutic methods comprise administering to the subject an engineered dendritic cell, population of engineered dendritic cells, or pharmaceutical composition comprising an engineered dendritic cell as described herein (e.g., as described in Section III above).
  • the engineered dendritic cell is an activated dendritic cell.
  • the engineered dendritic cell has reduced proliferative ability (e.g., due to treatment with an anti-proliferative agent).
  • the engineered dendritic cell has been pulsed with an antigen peptide (e.g., a peptide of a myelination protein, such as MOG, MBP, PLP, or a combination thereof).
  • the cells and methods herein disclosed can be used to treat immune-mediated diseases in humans.
  • DCs can be generated from peripheral blood mononuclear cells in the presence of IL-4 and GM-CSF, which are the same cytokines used in the mouse model.
  • the DCs can be engineered to overexpress the lct- hydroxylase by an FDA-approved viral vector (e.g. an adeno-associated viral vector).
  • the engineered cells can then be pulsed with one or more human myelin epitopes (equivalent to MOG 35-55 in the mouse model).
  • Such epitope-pulsed engineered DCs can be treated to prevent further proliferation by mitomycin C or by irradiation (2500 Rads).
  • These non- proliferative, epitope-pulsed, engineered DCs can be intravenously or subcutaneously injected into a patient who has the immune-mediated disease.
  • the subject to be treated is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is a juvenile.
  • the subject to be treated has an autoimmune disease.
  • the subject to be treated has a demyelinating disease.
  • the demyelinating disease is multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukodystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot- Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or human T-cell lymphotropic virus (HTLV)-associated myelopathy.
  • HTLV human T-cell lymphotropic virus
  • the subject has multiple sclerosis (MS). There are several subtypes of MS, including relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), and progressive relapsing multiple sclerosis (PRMS).
  • RRMS relapsing-remitting multiple sclerosis
  • SPMS secondary progressive multiple sclerosis
  • PPMS primary progressive multiple sclerosis
  • PRMS progressive relapsing multiple sclerosis
  • the subject has RRMS.
  • SPMS secondary progressive multiple sclerosis
  • PPMS primary progressive multiple sclerosis
  • PRMS progressive relapsing multiple sclerosis
  • the subject has RRMS.
  • SPMS secondary progressive multiple sclerosis
  • PPMS primary progressive multiple sclerosis
  • PRMS progressive relapsing multiple sclerosis
  • PRMS progressive relapsing multiple sclerosis
  • the subject has RRMS.
  • SPMS secondary progressive multiple sclerosis
  • PPMS primary progressive multiple
  • the route of administration of an engineered dendritic cell, population of engineered dendritic cells, or pharmaceutical composition comprising an engineered dendritic cell as described herein can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, inhalational, topical, intralesional, rectal, intrabronchial, intralymphatic, intradermal, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art.
  • an engineered dendritic cell, population of engineered dendritic cells, or pharmaceutical composition comprising an engineered dendritic cell as described herein is administered by intravenous injection or by subcutaneous injection. In some embodiments, an engineered dendritic cell, population of engineered dendritic cells, or pharmaceutical composition comprising an engineered dendritic cell as described herein is administered systemically. In some embodiments, an engineered dendritic cell, population of engineered dendritic cells, or pharmaceutical composition comprising an engineered dendritic cell as described herein is administered locally.
  • a therapeutically effective amount of engineered dendritic cells or composition comprising engineered dendritic cells is an amount that prevents or reverses one or more symptoms of an immune-mediated disease (e.g., MS).
  • an immune-mediated disease e.g., MS
  • a therapeutically effective amount is at least about 100, 500, 1,000, 2,500, 5,000, 10,000, 20,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, or 10,000,000 cells or more (e.g., per administration).
  • a therapeutically effective amount of the engineered dendritic cells or composition comprising engineered dendritic cells is administered about once per day, once per week, twice per week, once per month, or twice per month.
  • the engineered dendritic cells, populations of engineered dendritic cells, and compositions comprising engineered dendritic cells may be administered to a subject in need thereof for a predetermined time, an indefinite time, or until an endpoint is reached.
  • treatment is continued on a continuous daily or weekly basis for at least two to three months, six months, one year, or longer.
  • treatment is for at least 30 days, at least 60 days, at least 90 days, at least 120 days, at least 150 days, or at least 180 days.
  • treatment is continued for at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least one year.
  • treatment is continued for the rest of the patient's life or until administration is no longer effective to provide meaningful therapeutic benefit.
  • the engineered dendritic cells, populations of engineered dendritic cells, or compositions comprising engineered dendritic cells are administered in combination with one or more additional agents.
  • the engineered dendritic cells, populations of engineered dendritic cells, or compositions comprising engineered dendritic cells are administered in combination with vitamin D, vitamin D3, or 25-hydroxyvitamin D3 (25(OH]D).
  • the administration may be concurrent or sequential.
  • methods of inducing regulatory T cells are provided.
  • the method comprises contacting one or more T cells with an engineered dendritic cell, population of engineered dendritic cells, or composition comprising an engineered dendritic cell as described herein (e.g., in Section III above).
  • the T cells are myelin-specific T cells.
  • the regulatory T cells that are induced are Foxp3 + Treg cells. In some embodiments, the regulatory T cells that are induced are T h 2 cells. In some embodiments, the regulatory T cells that are induced are Trl cells.
  • the method of contacting one or more T cells with an engineered dendritic cell, population of engineered dendritic cells, or composition comprising an engineered dendritic cell as described herein is carried out in vivo (e.g., in a human subject).
  • the method of contacting one or more T cells with an engineered dendritic cell, population of engineered dendritic cells, or composition comprising an engineered dendritic cell is carried out in vitro.
  • the regulatory T cells that are induced in vitro are optionally expanded and subsequently administered to a subject.
  • the T cells are autologous to the subject to be treated.
  • the T cells are allogeneic to the subject to be treated.
  • Disclosed herein is a novel engineered dendritic cell that augments myelin-specific immune regulation and works with immunogenic DCs.
  • This engineered DC carries an overexpressed enzyme, 25-hydroxyvitamin D lct-hydroxylase ("1 ⁇ -hydroxylase") that, under physiologic conditions, synthesizes the active vitamin D metabolite 1,25-dihydroxyvitamin D ("l,25(OH) 2 D").
  • mice Female C57BL/6 mice aged 4 to 6 weeks were obtained from The Jackson Laboratory. All animals were housed in specific pathogen-free environment and were cared for in accordance with institutional and National Institutes of Health (NIH) guidelines. Animals were acclimated to new housing conditions for a minimum of 5 days before experimentation. Experiments were conducted with approval of the Institutional Animal Care and Use Committee at Loma Linda University, Loma Linda, California, USA.
  • NASH National Institutes of Health
  • DC2.4 is a DC line generated from bone marrow-derived DCs. Lentivirus production
  • the 293T cells (5 x 10 6 cells in 1 mL) were seeded into a 15 cm culture dish, which was pre-coated with the L-lysine, in standard DMEM culture medium containing 10% fetal bovine serum (FBS) and antibiotics one day before the transfection.
  • FBS fetal bovine serum
  • the cells were incubated at 37°C, 5% C0 2 overnight and then replenished with DMEM culture medium containing 4% FBS, antibiotics, and 20 mM Hepes. Supernatant was collected on the 2 nd and 3 rd days. The collected supernatants were centrifuged (4800 g) at 4°C for 24 hours. The viruses were re-suspended in PBS containing 5% glycerol, stored in -80°C, and used for transducing the 293T cells or the DCs. Generation of bone marrow-derived DCs and transduction of the DCs with the lentivirus
  • Bone marrow mononuclear cells were isolated from mouse (6-8 weeks old) tibias and femurs and cultured at a density of 1 x 10 6 cells/mL in 6-well plates in RPMI1640 medium supplemented with 10% FBS, 100 U/mL recombinant murine granulocyte/macrophage colony- stimulating factor (GM-CSF), and 10 U/ml murine interleukin-4 (IL-4) (Peprotech, Rocky Hill, NJ, USA).
  • Non-adherent cells were gently washed out after 48 hr of culture; the remaining loosely adherent clusters were cultured for another 48 hr and harvested for lentivirus transduction.
  • mice were immunized subcutaneously with 200 ug of the MOG 35-55 emulsified in Incomplete Freund Adjuvant (IFA) supplemented with 250 ⁇ g of heat-inactivated Mycobacterium tuberculosis H37Ra (Difco Laboratories, Michigan, USA).
  • IFA Incomplete Freund Adjuvant
  • Mycobacterium tuberculosis H37Ra Heat-inactivated Mycobacterium tuberculosis H37Ra
  • each mouse was administered 150 ng of pertussis toxin (Calbiochem, Germany) intraperitoneally. Animals were clinically scored and weighed on a daily basis for up to 40 days after disease induction.
  • T cells isolated from splee ns were co-cultured with MOG 35-55 pulsed DCs at a T:DC ratio of 4:1 in RPMI 1640 medium containing 100 ⁇ MOG 35-55 , 10% FBS, and antibiotics. Two days later, supernatants were collected for cytokine measurements.
  • a BD Cytometric Beads Array® (BD Biosciences, San Jose, CA, USA) kit was used for cytokine measurements according to the manufacturer's instruction.
  • the test cytokines included interleukin-2 (IL- 2), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-10 (IL-10).
  • FIG. 1A depicts a schematic representation of the lenti-CYP-GFP vector that was used to transduce parental bone- marrow-derived dendritic cells (BM-DCs), which express a low level of 1 ⁇ -hydroxylase, with CYP27B1 (cytochrome P450 family 27 subfamily B member 1) gene to overexpress 1 ⁇ - hydroxylase for de novo synthesis of a supra-physiological calcitriol concentration.
  • BM-DCs parental bone- marrow-derived dendritic cells
  • CYP27B1 cytochrome P450 family 27 subfamily B member 1
  • IB is a panel of photomicrographs that show production of green fluorescence protein (GFP) in transfected BM-DCs after 24 and 48 hours post-transfection.
  • the transduced DCs overexpressed GFP and presumably 1 ⁇ -hydroxylase as well.
  • FIG. 1C real time PCR confirmed specific overexpression of the CYP27B1 mRNA in the DCs transduced with the lenti-CYP-GFP.
  • the functional activity of the overexpressed 1 ⁇ -hydroxylase was assessed by adding 25[OH]D into the DC cultures. After overnight incubation, supernatants were collected and analyzed for the production of l,25[OH] 2 D by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • ID illustrates that DCs transduced with the lenti-CYP-GFP, as compared to control DC cells, de novo synthesized a significantly higher level of l,25[OH] 2 D in the presence of the 1 ⁇ - hydroxylase substrate.
  • Parental BM-DCs can synthesize calcitriol from 25[OH]D (the 1 ⁇ - hydroxylase substrate), suggesting that a low level of 1 ⁇ -hydroxylase activity was present.
  • FIGS. 2A-2C illustrate that the 1 ⁇ -hydroxylase-overexpressing BM-DCs maintain important DC functions, e.g. lymphoid tissue-homing capacity, such that the engineered DCs could induce Treg cells in the peripheral lymphoid tissues upon in vivo delivery.
  • FIG. 2A flow cytometry analysis indicates 1 ⁇ -hydroxylase overexpression does not alter the expression of activation markers on the cell surface of the engineered DCs.
  • the expressions of the markers MHC II, CD80, and CD86 were not significantly changed in 1 ⁇ -hydroxylase- overexpressing BM-DCs compared to controls.
  • FIG. 2B illustrates that the engineered DCs maintain lymphoid tissue-homing capacity.
  • FIG. 2C shows that the engineered DCs successfully migrated into the spleen three days after the DC injection.
  • FIGS. 3A-3B illustrate that the lymphoid-homing, engineered DC2.4 cells can a therapeutic agent for inducing myelin-specific immune regulation in mice that were immunized with MOG 35-55 for EAE, an animal model for human MS.
  • the effects of immunogenic (activated or mature) and non-proliferative (i.e., mytomycin C treated) parental DC2.4 cells (mDC2.4) on EAE disease course were examined.
  • the effects of immunogenic DCs were evaluated to determine whether the engineered DCs, when activated in a pro-inflammatory in vivo environment, would still have a therapeutic effect.
  • non-proliferative DCs were used to prevent undesirable side effects that may be caused by unnecessary presence of the engineered DC in vivo for a long period of time.
  • animals intravenously injected with the parental mDCs displayed significantly enhanced disease activity, indicating that non- modified DCs were pathogenic.
  • EAE disease course was also evaluated in animals intravenously injected with non-proliferative DC2.4 cells transduced with the lenti-CYP-GFP.
  • the engineered immunogenic and non-proliferative DC2.4 cells which were pulsed with MOG 35-55 (mDC2.4-CYP-MOG35.55), were intravenously injected into animals at the time when the paralytic symptoms just began (i.e., at day 10).
  • This pulsing of MOG 35-55 ensured that the engineered DCs would mainly interact with MOG35 -55 -specific T cells (i.e., the pathogenic T cells).
  • FIG. 3B and in Table 1 below a single injection of such engineered DCs markedly suppressed the paralytic disease. This therapeutic effect was reproduced in two subsequent independent experiments.
  • FIG. 4A depicts a schematic of the experimental protocol. Animals were immunized with MOG 35-55 emulsified in Complete Freund Adjuvant (CFA). Four days later, either parental DCs or the engineered DCs (DC-CYP) were subcutaneously injected into the EAE-bearing mice. At day 10, T cell apoptosis was examined by Annexin and PI staining using fluorescence-activated cell sorting (FACS) flow cytometry.
  • FACS fluorescence-activated cell sorting
  • FIG. 4B illustrates the gating strategy used for flow cytometry analysis.
  • FIG. 4C shows representative FACS plots showing Annexin and PI staining in all CD4 + and CD8 + T cells.
  • the DC-CYP cells significantly increased apoptosis of CD4 + (by ⁇ 30%), but not of CD8 + , T cells in local draining lymph nodes.
  • calcitriol can induce regulatory T cells. Accordingly, T cell populations were analyzed to determine whether subsets of regulatory T cells were induced by the lot-hydroxylase-overexpressing BM-DCs.
  • FIG. 5A depicts a schematic of the experimental protocol.
  • FIG. 5B illustrates the gating strategy used for flow cytometry analysis.
  • the engineered DCs compared to no treatment controls, significantly increased expression of IL-10 by almost 10-fold in T cells, suggesting that the engineered DCs induced IL-10 + Treg cells.
  • FIG. 5C illustrates the gating strategy used for flow cytometry analysis.
  • FIG. 5D shows that secretion of Th2 cytokines IL-4, IL-5, IL-6, and IL-13 were also significantly increased by 2- to 4-fold, indicating that the engineered DCs created in the peripheral lymphoid tissues a microenvironment that favors Th2 differentiation.
  • FIG. 6 illustrates that the profile of cytokine secretion by these cells (as assessed in the cell culture supernatants with a Cytometric Beads Array assay for T cell cytokine secretion) was similar to what was observed by intracellular cytokine staining.
  • FIG. 7A-7E shows that engineered DCs stimulate the expression of Th2 and Trl cytokines in CD4 + T cells. Animals were immunized as shown in FIG. 7A.
  • C57BL/6 mice were immunized with MOG 35-55 emulsified in CFA for the induction of EAE, as shown in FIG. 9A.
  • the animals received one of the following immunizations: (1) no immunization; (2) DC2.4-mCYP-GFP; or (3) MOG 35-55 -pulsed DC2.4- mCYP-GFP.
  • spinal cords were stained with Luxol Fast Blue for the analysis of demyelination and inflammation.
  • Our data showed that immunization with the MOG 35-55 - pulsed DC2.4-mCYP-GFP but not the parental DC2.4-mCYP-GFP significantly ameliorated the paralytic disease. See, FIG. 9B.
  • FIG. 10 shows the disease course in C57BL/6 mice immunized with MOG 35-55 emulsified in CFA to induce EAE.
  • the animals received either no treatment (filled triangles) or MOG 35-55 -pulsed engineered mDC2.4-CYP cells.
  • animals also received intra-peritoneal injection of an anti-CD25 monoclonal antibody (clone PC61) to deplete Foxp3 + Treg cells (open circles).
  • the mAb was administered one day before and then every four days after EAE induction until the end of the experiment.
  • immune regulation which is augmented by 1 ⁇ -hydroxylase- overexpressing DCs, may predominantly function in peripheral lymphoid tissues but not in CNS.
  • calcitriol impairs CNS homing of CD4 + T cells by suppressing expression of a CNS-homing receptor, CXCR3.
  • AST is a logical strategy to achieve this goal.
  • a significant AST-associated obstacle is the instability of ex vivo generated therapeutic cellular agents in an in vivo pro-inflammatory environment in human MS patients.
  • An example of such cellular agents is ex vivo induced myelin-specific Treg cells (iTreg). It has been shown that such myelin-specific iTreg cells can be adoptively transferred to enhance myelin-specific immune regulation in vivo.
  • TolDCs ex vivo generated tolerogenic DCs
  • MS patients are deficient in Treg cells.
  • Viglietta et al. J. Exp. Med., 199, 971-979
  • Matarese et al.. Proc. Natl. Acad. Sci. USA, 2005, 102:5150-5155
  • Carbone ef al. Nat. Med., 2014, 20:69-74.
  • the cells and methods herein disclosed provide investigative tools for elucidating the mechanisms underlying the disease suppression.
  • the engineered DCs augmented apoptosis of CD4 + T cells, induced IL-10 + Treg cells and promoted immune deviation, suggesting that disease suppression, mediated by the engineered DCs, required Treg cells.
  • VDR vitamin D receptor
  • the vitamin D and/or calcitriol supplementation may not be able to produce a Treg-inducing calcitriol concentration in the peripheral lymphoid tissues.
  • the highest possible systemic calcitriol dose which can be created by vitamin D and/or calcitriol supplementation, has to be lower than the toxicity-limiting dose and may not be sufficient to induce a strong immunoregulatory effect in the peripheral lymphoid tissues.
  • a supra-physiological (therapeutic) concentration of calcitriol has been shown, in certain contexts, to be necessary for inducing Treg cells.
  • the presently disclosed lct-hydroxylase- overexpressing DCs can de novo synthesize a focal Treg-inducing calcitriol concentration and at the same time prevent toxicity associated with a high systemic calcitriol concentration.
  • vitamin D supplementation is important to maintain a sufficient serum level of 25[OH]D that is the substrate for 1 ⁇ -hydroxylase and is therefore necessary for 1 ⁇ -hydroxylase-overexpressing DCs to function properly.
  • 25[OH]D deficiency is common among MS patients, vitamin D or 25[OH]D supplementation should be considered as an adjuvant therapy accompanying the 1 ⁇ - hydroxylase-overexpressing DC-based therapy.
  • the findings presented herein suggest that the engineered DCs disclosed herein are a promising therapy for MS with an acceptable safety profile.
  • the engineered DCs may help determine potential mechanisms underlying the disease suppression by this approach.
  • the cells, compositions, and methods of the present application can provide a promising therapeutic agent for immune-mediated diseases, such as multiple sclerosis.
  • All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and materials in connection with which the publications are cited.

Abstract

Selon un aspect, l'invention concerne des cellules dendritiques modifiées comprenant un polynucléotide hétérologue qui code pour une protéine 1α-hydroxylase. Dans certains modes de réalisation, la 1α-hydroxylase est l'élément 1 de la sous-famille B de la famille 27 du cytochrome P450 humain (CYP27B1). Selon un autre aspect, l'invention concerne des méthodes de traitement d'un sujet souffrant d'une maladie à médiation immunitaire, comprenant l'administration des cellules dendritiques modifiées.
PCT/US2017/036210 2016-06-06 2017-06-06 Cellules dendritiques modifiées exprimant la 1a-hydroxylase et utilisations associées pour le traitement de maladies à médiation immunitaire WO2017214190A1 (fr)

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WO2019241479A1 (fr) * 2018-06-14 2019-12-19 Loma Linda University Cellules souches hématopoïétiques modifiées pour le traitement de la leucémie myéloïde aiguë

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US6274359B1 (en) * 1997-07-10 2001-08-14 Kyowa Hakko Kogyo Co., Ltd. 25-hydroxyvitamin D3-1α-hydroxylase and DNA encoding the hydroxylase
US20100273748A1 (en) * 2006-09-08 2010-10-28 The Regents Of The University Of California Antimicrobial therapy
US8530153B1 (en) * 2010-03-10 2013-09-10 Technologiy Transfer Office, University of Manitoba Lentiviral transduced dendritic cells for RNAi
US20140141005A1 (en) * 2012-02-14 2014-05-22 Loma Linda University Agents and method for treating inflammation-related conditions and diseases
WO2016130845A1 (fr) * 2015-02-11 2016-08-18 Loma Linda University Procédé d'utilisation de cellules dendritiques modifiées pour induire des cellules t régulatrices migrant dans l'intestin et traiter une inflammation intestinale

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Publication number Priority date Publication date Assignee Title
US6274359B1 (en) * 1997-07-10 2001-08-14 Kyowa Hakko Kogyo Co., Ltd. 25-hydroxyvitamin D3-1α-hydroxylase and DNA encoding the hydroxylase
US20100273748A1 (en) * 2006-09-08 2010-10-28 The Regents Of The University Of California Antimicrobial therapy
US8530153B1 (en) * 2010-03-10 2013-09-10 Technologiy Transfer Office, University of Manitoba Lentiviral transduced dendritic cells for RNAi
US20140141005A1 (en) * 2012-02-14 2014-05-22 Loma Linda University Agents and method for treating inflammation-related conditions and diseases
WO2016130845A1 (fr) * 2015-02-11 2016-08-18 Loma Linda University Procédé d'utilisation de cellules dendritiques modifiées pour induire des cellules t régulatrices migrant dans l'intestin et traiter une inflammation intestinale

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019241479A1 (fr) * 2018-06-14 2019-12-19 Loma Linda University Cellules souches hématopoïétiques modifiées pour le traitement de la leucémie myéloïde aiguë
EP3806864A4 (fr) * 2018-06-14 2022-05-25 Loma Linda University Cellules souches hématopoïétiques modifiées pour le traitement de la leucémie myéloïde aiguë

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