[go: up one dir, main page]

WO2016082034A1 - Cellules dendritiques non monocytaires, précurseurs de celles-ci, et procédés associés - Google Patents

Cellules dendritiques non monocytaires, précurseurs de celles-ci, et procédés associés Download PDF

Info

Publication number
WO2016082034A1
WO2016082034A1 PCT/CA2015/051226 CA2015051226W WO2016082034A1 WO 2016082034 A1 WO2016082034 A1 WO 2016082034A1 CA 2015051226 W CA2015051226 W CA 2015051226W WO 2016082034 A1 WO2016082034 A1 WO 2016082034A1
Authority
WO
WIPO (PCT)
Prior art keywords
monocytic
cells
cell
dcs
precursor cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CA2015/051226
Other languages
English (en)
Inventor
Mickie Bhatia
Rahul KUSHWAH
Jong-Hee Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO2016082034A1 publication Critical patent/WO2016082034A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • A61K40/4272—Melan-A/MART
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/45—Artificially induced pluripotent stem cells

Definitions

  • TITLE NON-MONOCYTIC DENDRITIC CELLS, PRECURSORS THEREOF,
  • This application relates to immunotherapy and more specifically to methods and products for producing non-monocytic dendritic cells useful for immunotherapy.
  • DCs Dendritic cells
  • DCs are currently classified based on origin of cells that give rise to subsets of functional DCs in the mouse, and can be broadly divided into monocytic and non-monocytic derived DC subsets (Kushwah and Hu, 201 1 ; Shortman and Liu, 2002).
  • monocytes differentiate into DCs under highly specific conditions such as lipopolysaccharide (LPS) stimulation or stimulation by gram-negative bacteria (Cheong et al., 2010).
  • LPS lipopolysaccharide
  • monocytic DCs have been associated with Th2 immune responses (Plantinga et al., 2013) and have also been associated with tumor growth (Augier et al., 2010).
  • mouse non-monocytic DCs In contrast to monocytic DCs, mouse non-monocytic DCs have been shown to have the inherent flexibility in immunomodulation and have been shown to drive antitumor immune response both in vitro and in vivo (Fields et al., 1998; Mayordomo et al., 1995).
  • plasmacytoid DCs pDCs
  • pDCs plasmacytoid DCs
  • the inventors have identified and isolated human non-monocytic DC precursors from adult hematopoietic tissue, as well as from renewable induced pluripotent stem cells (iPSCs) reprogrammed from blood (hBiPS) that uniquely give rise to non-monocytic DCs including plasmacytoid DCs both in clonal assays along with in vivo human-mice xenografts.
  • hBiPS cells are a potential renewable source for autologous DC vaccine development.
  • an isolated non-monocytic Dendritic Cell (DC) precursor cell In one embodiment, the non-monocytic DC precursor cell is obtained from a population of CD34+CD38+CD45RA+ cells. Optionally, the non-monocytic DC precursor cell is isolated from peripheral blood or from a population of human blood iPSCs. In one embodiment, the non-monocytic DC precursor cell expresses CD1 15. In one embodiment, the non-monocytic DC precursor cell expresses CD1 15, Flt3 and HLA-DR. In one embodiment, the DC precursor cell does not express CD14 or CX3CR1 .
  • DC Dendritic Cell
  • the non-monocytic DC precursor cell does not secrete IL-6 or TNF-a following stimulation with lipopolysaccharide (LPS). Also provided are cell lines and cell cultures comprising the non-monocytic DC precursor cells as described herein.
  • LPS lipopolysaccharide
  • cell lines and cell cultures comprising the non-monocytic DC precursor cells as described herein.
  • an isolated non-monocytic dendritic cell DC.
  • the non-monocytic DC is obtained from a non-monocytic DC precursor cell as described herein.
  • the non-monocytic DC is obtained by differentiating a non- monocytic DC precursor cell as described herein.
  • the monocytic DC is obtained by differentiating, or expanding and differentiating, a non-monocytic DC precursor cell in the presence of one or more of GM- CSF, IL-4, Flt3 Ligand (Flt3L) and bone marrow stromal cells (BMSCs).
  • GM- CSF GM- CSF
  • IL-4 Flt3 Ligand
  • BMSCs bone marrow stromal cells
  • the non-monocytic DCs described herein exhibit an increased Th1 response relative to DCs derived from Cd14+ monocytic cells. In one embodiment, the non-monocytic DC exhibits a preferential Th1 response relative to Th2. In one embodiment, the increased Th1 response is characterized by increased IFN- ⁇ production. In one embodiment, the Th2 response is characterized by I L-4 production.
  • the non-monocytic DC exhibits an antitumor response.
  • the non-monocytic DC exhibits an increased anti-tumor response relative to CD14+ monocytic DCs.
  • a pharmaceutical composition comprising one or more non-monocytic DC precursor cells and/or non-monocytic DCs as described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an anti-cancer therapeutic and/or a cell culture media.
  • Another aspect of the disclosure includes the use of a non- monocytic DC precursor cell or a non-monocytic DC as described herein for immunotherapy, autologous therapy, autoimmune immunotherapy, vaccination or development of vaccine adjuvants. Also provided are methods for providing immunotherapy, autologous therapy, autoimmune immunotherapy or vaccination to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a non- monocytic DC precursor cell or a non-monocytic DC as described herein.
  • the immunotherapy, autologous therapy or autoimmune immunotherapy if for the treatment of cancer.
  • the cancer is myeloma, melanoma or prostate cancer.
  • the non- monocytic DC precursor cells or a non-monocytic DCs described herein are for use in the treatment of cancer in a subject in need thereof and the cells are autologous cells from the subject.
  • a method for isolating a non-monocytic DC precursor cell from a population of cells comprising obtaining a population of CD34+CD38+CD45RA+ cells, culturing the cells and separating one or more cells that express CD1 15 from the population of cells.
  • the method further comprises separating cells that express Flt3 and/or HLA-DR from the population of CD34+CD38+CD45RA+ cells wherein cells that express CD1 15, Flt3 and/or HLA-DR are non- monocytic DC precursor cells.
  • the methods described herein further comprise differentiating, or expanding and differentiating, the non-monocytic DC precursor cells into non-monocytic dendritic cells.
  • the differentiating, or expanding and differentiating, the non-monocytic DC precursor cells comprises culturing the cells in the presence of one or more of GM-CSF, IL-4, Flt3 Ligand (Flt3L) and bone marrow stromal cells (BMSCs).
  • GM-CSF GM-CSF
  • IL-4 Flt3 Ligand
  • BMSCs bone marrow stromal cells
  • FIG. 1 shows that human CD1 15 + Flt3 + HLA-DR + cells represent a distinct population of non-monocytic cells isolatable from adult blood with DC generation capacity.
  • A Established CD34 + CD38 + CD45RA + progenitors were cultured in presence of GM-CSF and I L-4 or Flt3L alone, and screened for expression of markers expressed on murine precursors of the DC lineage.
  • B Of mouse DC precursors markers examined (CD1 15, CD135, HLA-DR and CX3CR1 ), human CD1 15 showed a distinct transition expression pattern at day 2 of culture with DC differentiation inducing cytokine conditions.
  • CD1 15 + and CD1 15 " sub-fractions were isolated by FACS. Representative kwik-diff stained images of isolated CD1 15 + and CD1 15 " subfractions from cultured CD34 + CD38 + CD45RA + progenitors are shown.
  • D Differential marker expression of CD1 15 + and CD1 15 " cells isolated from CD34 + CD38 + CD45RA + cells.
  • E Gating strategy for de novo isolation of CD1 15 + Flt3 + HLA-DR + cells derived from adult human blood.
  • CD14 and CX3CR1 expression was used to isolate CD14 X3CR1 " subset that was further subdivided for CD1 15, HLA-DR and Flt3 expression to allow pure isolation of CD1 15 + Flt3 + HLA-DR + cells.
  • Representative Kwik-Diff stained image of de novo isolated CD1 15 + Flt3 + HLA-DR + cells is shown.
  • Non- monocytic CD1 15 + Flt3 + HLA-DR + cells isolated from adult human blood lack expression of HSC, DC/Monocyte and T/B cell lineage markers, including markers expressed on monocytic CD14 + cells.
  • Non-monocytic CD1 15 + Flt3 + HLA-DR + cells isolated from human blood fail to produce IL-6 and TNF-a, cytokines produced by monocytic CD14 + cells following LPS stimulation (ND-not detectable).
  • H Non-monocytic CD1 15 + Flt3 + HLA-DR + cells isolated from human blood fail to produce IL-12, which is produced by differentiated human DCs derived from monocytic CD14 + cells (ND-not detectable).
  • Non-monocytic CD1 15 + Flt3 + HLA-DR + cells were de novo isolated from adult human blood and underwent expansion following 6 days of Flt3L culture. Shown is a histogram comparing absolute number of cells following Flt3L stimulation. Representative images of Kwik-Diff stained differentiated cells are shown.
  • N CD4+ T cell proliferation induced by DCs derived from monocytic CD14 + cells versus non-monocytic CD1 15 + Flt3 + HLA-DR + cells.
  • Figure 2 shows that human non-monocytic CD1 15 + Flt3 + HLA-
  • DR + cells represent clonal non-monocytic precursors of human DCs.
  • FIG. 1 Representative flow plots showing generation of non-monocytic CD1 15 + Flt3 + HLA-DR + cells from xenografts transplanted with CD34 + CD38 + CD45RA + progenitors.
  • E Gating strategy for identification of clonal DC generation in vitro. DCs were characterized by CD1 1 c, HLA-DR, CD83 and CD1 a expression.
  • pDCs were characterized by expression of BDCA-2 and CD123 with a lack of CD1 a expression.
  • F IFN-a production by BDCA-2+CD123+ cells derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells demonstrating generation of functional pDCs.
  • G Monocytic CD14 + cells isolated from human blood exclusively generated non pDCs, with a complete absence of pDC generation as shown and tabulated.
  • H de novo isolated CD1 15 + Flt3 + HLA-DR + cells co-cultured with BMSCs in presence of 2 differentiation cytokine cocktails.
  • FIG. 3 shows that human non-monocytic CD1 15 + Flt3 + HLA-
  • DR + cells generate plasmacytoid DCs in vivo.
  • A NSG mice were transplanted with monocytic CD14 + cells and non-monocytic CD1 15 + Flt3 + HLA-DR + cells isolated from adult human blood followed by assessment of DC differentiation in vivo.
  • B Gating strategy to identify DC generation in vivo. Gating of hCD45 + cells was carried out to identify human cells and further focused on human hematopoeitic cells expressing the myeloid marker CD33.
  • Figure 4 shows that DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells are superior to DCs derived from bulk CD34 + cells as well as monocytic CD14 + cells in survival and induction of Th1 response in vitro.
  • A Schema for in vitro assessment of survival and function of DCs generated from non-monocytic CD1 15+Flt3+HLA-DR+ cells versus bulk CD34+ and monocytic CD14+ cells.
  • B Proportions of surviving DCs, assessed by propidium iodide staining following overnight treatment with LPS.
  • C Representative flow plots of BDCA-2 and CD123 expression on DCs generated from bulk CD34+ cells under different conditions, identifying lack of pDC generation.
  • D-F To assess in vitro capacity of DCs to drive T cell activation in vitro, DCs were stimulated overnight with LPS and cultured with CD4 + T cells for 5 days.
  • D IFN- ⁇ and IL-4 production by CD4 + T cells.
  • E Proportions of I FN- ⁇ producing CD4 + T cells upon culture with DCs.
  • F Proportions of IL-4 producing CD4 + T cells upon culture with DCs.
  • Figure 5 shows that human blood induced pluripotent stem cells (hBiPS) give rise to non-monocytic CD1 15 + Flt3 + HLA-DR + cells, which generate non-monocytic DCs both in vivo and in vitro.
  • hBiPS human blood induced pluripotent stem cells
  • D-E DCs were derived from ES cells (hES derived DCs), hFib-iPS (hFib-iPS derived DCs), from monocytic CD14+ cells derived from hB-iPS (hB-iPS DCs from monocytic DCs) and from non-monocytic CD1 15 + Flt3 + HLA-DR + cells derived from hB-iPS (hB-iPS DCs from non-monocytic CD1 15 + Flt3 + HLA-DR + cells).
  • DCs were matured overnight with LPS and cultured with CD4+ T cells for 5 days. Representative flow plots of IFN- ⁇ and I L-4 production by CD4+ T cells are shown. Also shown are histograms comparing proportions of I FN- ⁇ producing CD4+ T cells and IL-4 producing CD4+ T cells upon culture with DCs.
  • E DCs were also treated with Th2 inducing cytokine and cultured with CD4+ T cells for 5 days. Representative flow plots of IFN- ⁇ and I L-4 production by CD4+ T cells are shown. Also shown are histograms comparing proportions of IFN- ⁇ producing CD4+ T cells and IL-4 producing CD4+ T cells upon culture with DCs.
  • FIG. 6 shows that DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells are superior to DCs derived from monocytic CD14 + cells in homing and inducing Th1 response in vivo.
  • DCs were derived from monocytic CD14 + cells, and from non-monocytic CD1 15 + Flt3 + HLA-DR + cells.
  • A Schematic for in vivo assays in NSG mice to test the function of in vitro generated human DCs to drive Th response. Following antigen challenge with ovalbumin, T cell response(s) were assessed by human CD4 + T cells from the spleen.
  • C IL-17, I FN- ⁇ and I L-4 production by human CD4+ T cells.
  • D Proportions of IL-17 producing (Th17), IFN- ⁇ producing (Th1 ) and IL-4 producing (Th2) CD4 + T cells.
  • FIG. 7 shows that DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells are superior to DCs derived from monocytic CD14 + cells in inducing anti-tumor response both in vitro and in vivo.
  • DCs were derived from monocytic CD14 + cells, and from non-monocytic CD1 15 + Flt3 + HLA-DR + cells.
  • A-B DCs were pulsed with Melan-A and cultured with T cells.
  • A Shown are representative flow cytometry plots of Melan-A tetramer + CD8 + T cells. Non-pulsed DCs were cultured with T cells as a control.
  • C Schematic for in vivo assays in NSG mice to test the function of in vitro generated human DCs to drive Melan-A specific CD8 + T cell response.
  • D Gating strategy for identification of human CD8 + T cells in the spleens of NSG recipients.
  • E Shown are representative flow cytometry plots of Melan-A tetramer + CD8 + T cells in the spleens of NSG recipients. Non-pulsed DCs were used as control.
  • Figure 8 shows the isolation of CD34 + CD38 + CD45RA + progenitors, transcriptional profile analysis of CD1 15 + Flt3 + HLA-DR + cells compared to mouse non-monocytic DC precursor and analysis of DC differentiation in comparison with human monocytes.
  • A Gating strategy for FACS isolation of CD34 + CD38 + CD45RA + progenitors from human blood.
  • B Representative images of CD34 + CD38 + CD45RA + progenitors following isolation by FACS.
  • C Heat map comparing gene expression profiles of non- monocytic CD1 15 + Flt3 + HLA-DR + cells with human common myeloid progenitor (CMP), human granulocyte and macrophage progenitor (hGMP), mouse common DC progenitor (mCDP), mouse macrophage and DC progenitor (mMDP), mouse common myeloid progenitor (mCMP) and mouse non-monocytic DC precursor (mPreDC) is shown.
  • CMP Human common myeloid progenitor
  • hGMP human granulocyte and macrophage progenitor
  • mCDP mouse common DC progenitor
  • mMDP mouse macrophage and DC progenitor
  • mPreDC mouse non-monocytic DC precursor
  • E Expression profiles of T cell stimulatory markers: CD83, CD86 and HLA-DR on DCs differentiated from monocytic CD14 + cells versus non-monocytic CD1 15 + Flt3 + HLA-DR + cells. Representative of 8 independent experiments. *p ⁇ 0.05. Error bars, s.d.
  • CFU Colony forming unit
  • DCs derived from non- monocytic CD1 15 + Flt3 + HLA-DR + precursors demonstrated superior CD4 + T cell proliferation potential.
  • DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + precursors demonstrated superior CD8 + T cell proliferation potential.
  • DCs derived from non-monocytic CD1 15 + Flt3 + HLA- DR + precursors demonstrated significantly increased levels of Th1 inducing cytokines IL12p35, IL12p40 and I L- 1 ⁇ in comparison to DCs derived from monocytic CD14 + cell origin.
  • Figure 9 shows the clonal expansion of non-monocytic CD1 15 + Flt3 + HLA-DR + cells on BMSCs with Flt3L, related to Figure 2.
  • Non- monocytic CD1 15 + Flt3 + HLA-DR + cells from adult blood or NSG recipients were sorted as single cells on a 96 well plate containing a monolayer of human bone marrow stromal cells (BMSCs). After 5-6 days of culture with/without Flt3L, expansion was observed.
  • A Shown is a histogram comparing average cell count/well indicating expansion from a single non- monocytic CD1 15 + Flt3 + HLA-DR + cell.
  • Figure 10 shows phenotypical and functional characterization of DCs generated from human pluripotent stem cells (hPSCs), related to Figure 5.
  • A Representative expression profile of DC markers (CD1 1 c, CD14, CD1 a, CD86, HLA-DR) on DCs generated from different pluripotent stem cell sources are shown.
  • B CD4+ T cell proliferation induced by DCs from different pluripotent stem cell source(s).
  • C CD8+ T cell proliferation induced by DCs from different pluripotent stem cell source(s).
  • D Schema for in vitro assessment of survival and function of DCs generated from various pluripotent stem cell sources.
  • OVA ovalbumin
  • DCs Dendritic cells
  • the inventors have identified clonally derived non-monocytic DC precursors from adult humans as well as renewable induced pluripotent stem cells (iPSCs) when reprogrammed from blood (hBiPS).
  • hBiPS are a potentially renewable source for autologous DC vaccines.
  • Human non-monocytic DC precursor cells hNM-DCPs were identified to generate unique DC subtypes compared to monocytic DCs and are capable of generating a complete repertoire of DC subtypes including plasmacytoid DCs.
  • DCs generated from hNM-DCPs are capable of superior Th1 anti-tumor response compared to sources previously utilized clinically.
  • an isolated non-monocytic Dendritic Cell (DC) precursor cell as described herein.
  • the DC precursor cell expresses CD1 15.
  • the DC precursor cell expresses CD1 15, Flt3 and HLA-DR.
  • the DC precursor cell is a human cell.
  • the non-monocytic DC precursor cell is obtained from blood.
  • the non-monocytic DC precursor cell is obtained from a pluripotent stem cell such as blood derived iPSCs.
  • the non-monocytic DC precursor cell is obtained from a population of CD34+CD38+CD45RA+ cells.
  • the DC precursor cell transiently expresses CD1 15 in culture.
  • the non-monocytic DC precursor cells express CD1 15 around day 2 of culture.
  • the non-monocytic DC precursor cells express CD1 15 from about day 0.5 to about day 4 after culturing the cells.
  • the non-monocytic DC precursor cell does not express the monocytic marker CD14.
  • the non-monocytic DC precursor cell does not express CX3CR1 .
  • the non-monocytic DC precursor cell described herein also exhibit certain functional characteristics that distinguish them from other cells.
  • the DC precursor cells do not secrete IL-6 or TNF-a following stimulation with lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • an isolated non-monocytic Dendritic Cell as described herein.
  • the non- monocytic DC is obtained from a non-monocytic DC precursor cell as described herein.
  • the non-monocytic DC is a plasmacytoid cell.
  • the cell is obtained from a population of pluripotent stem cells, optionally human blood pluripotent stem cells.
  • the non-monocytic DC may be obtained by differentiating and/or expanding a precursor cell as described herein in the presence of GM-CSF and/or IL-4.
  • non-monocytic DC may be obtained by differentiating and/or expanding the precursor cells in the presence of Flt3 Ligand (Flt3L). In one embodiment, non-monocytic DC may be obtained by differentiating and/or expanding the precursor cell in the presence of Flt3L and bone marrow stromal cells (BMSCs). In one embodiment, the non-monocytic DC may be obtained by differentiating and/or expanding the precursor cell in the presence of bone marrow stromal cells (BMSCs), Flt3L, Stem Cell Factor (SCF), GM-CSF and IL-4.
  • BMSCs bone marrow stromal cells
  • SCF Stem Cell Factor
  • the non-monocytic DCs described herein also exhibit characteristics that distinguish them from other cells such as monocytic DCs.
  • the non-monocytic DCs described herein exhibit an increased Th1 response relative to DCs derived from CD14+ monocytic cells.
  • an increased Th1 response is characterized by increased IFN- ⁇ production.
  • the non-monocytic DCs described herein exhibit a preferential Th1 response relative to Th2.
  • the non-monocytic DCs described herein exhibit a preferential Th1 response relative to Th2 compared to DCs derived from CD14+ monocytic cells.
  • the Th2 response may be characterized by determining IL-4 production.
  • the non-monocytic DCs described herein exhibit superior CD4+ and CD8+ T cell proliferation relative to monocytic CD14+ cells (see for example Figures 8H and 8I).
  • the non-monocytic DCs described herein also show increased levels of Th1 inducing cytokines.
  • the non- monocytic DC expresses increased levels of IL12p35 and/or IL12p40 relative to DC derived from monocytic CD14+ cells (see, for example, Figure 8J).
  • a cell culture or cell line comprising non-monocytic DC precursor cells or non monocytic DC cells as described herein.
  • the term "cell culture” refers to one or more cells grown under controlled conditions and optionally includes a cell line.
  • the term "cell line” refers to a plurality of cells that are the product of a single group of parent cells.
  • the cell culture or cell line is a clonal cell culture or cell line derived from a single cell.
  • the cells and compositions described herein are useful for immunotherapy and for eliciting and immune response in vitro or in vivo.
  • the non-monocytic DC precursor cells and/or the non-monocytic DCs of described herein are for use in immunotherapy, autologous therapy, autoimmune immunotherapy, vaccination or development of vaccine adjuvants.
  • the cells and compositions described herein are useful for the treatment of cancer in a subject in need thereof.
  • cancer refers to one of a group of diseases caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body.
  • the subject has a cancer suitable for treatment by immunotherapy.
  • the subject has myeloma, melanoma or prostate cancer.
  • treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease (e.g. maintaining a patient in remission), preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treating” and “treatment” as used herein also include prophylactic treatment.
  • treatment methods comprise administering to a subject a therapeutically effective amount of the non-monocytic DC precursor cells or non-monocytic DCs as described herein and optionally consists of a single administration, or alternatively comprises a series of administrations.
  • the cells described herein are prepared or formulated for administration to a subject in need thereof as known in the art. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • the non-monocytic DCs described herein are also useful for inducing a preferential Th1 response and/or a preferential Th17 response in vitro or in vivo in a subject in need thereof.
  • the subject has cancer, optionally myeloma, melanoma or prostate cancer.
  • methods for inducing a preferential Th1 response and/or a preferential Th17 response in vivo in a subject in need thereof comprising administering to the subject the non-monocytic DC precursor cells or non-monocytic DCs as described herein.
  • the methods and uses described herein involve cells that are autologous, such as non-monocytic DC from a subject with cancer or suspected of having cancer, or allogenic, such as non- monocytic cells from a healthy donor without cancer.
  • the methods described herein include obtaining a sample of peripheral blood from a subject in order to obtain autologous non-monocytic DC precursor cells or non-monocytic DCs using the methods described herein.
  • a non-monocytic Dendritic Cell (DC) precursor cell from a population of cells.
  • a non-monocytic DC precursor cell may be isolated by separating cells based on the expression of one or more biomarkers described herein to be associated with monocytic DC precursor cells.
  • the method comprises obtaining a population of cells and separating one or more cells that express CD1 15 from the population of cells.
  • the method comprises separating cells that express Flt3 and/or HLA-DR from the population of cells.
  • cells that express CD1 15, FLT3 and/or HLA-DR are non- monocytic DC precursor cells.
  • a method of isolating a non-monocytic Dendritic Cell (DC) precursor cell comprising:
  • the method further comprises separating cells that express Flt3 and/or HLA-DR from the population of cells, wherein cells that express CD1 15, FLT3 and/or HLA-DR are non-monocytic DC precursor cells.
  • the cultured CD34+CD38+CD45RA+ cells transiently express CD1 15 between 0.5 and 5 days, between 0.5 and 4 days, or between 1 and 3 days after culturing the cells.
  • the cultured CD34+CD38+CD45RA+ cells transiently express CD1 15 about 2 days after culturing the cells.
  • the CD34+CD38+CD45RA+ cells may be obtained from different sources.
  • the CD34+CD38+CD45RA+ cells are obtained from peripheral blood cells.
  • the CD34+CD38+CD45RA+ cells are obtained from differentiating pluhpotent stem cells.
  • the pluhpotent stem cells are human blood induced pluhpotent stem cells (hB-iPSCs).
  • differentiating the hB-iPSCs comprises culturing the cells in the presence of SCF, Flt3L, II-3, II-6, GM-CSF and BMP4.
  • the characteristics of the non-monocytic DC precursor cells and/or the non- monocytic DC cells described herein can be used in methods to identify and/or isolate non-monocytic DC precursor cells and/or non-monocytic DC cells.
  • the non-monocytic DC precursor cells and non-monocytic DC cells exhibit different patterns of gene expression relative to other cells such as monocytic DC cells. Accordingly, the present disclosure includes methods of identifying and/or isolating non-monocytic DC precursor cells and/or non-monocytic DC cells based on gene expression.
  • non-monocytic DC precursor cells and/or non-monocytic DC cells include those based on morphological and/or functional characteristics.
  • the non- monocytic DC precursor cells described herein do not exhibit monocytic morphological features such as cytoplasmic vacuoles or horseshoe shaped nuclei.
  • the non-monocytic DC cells exhibit a distinct cytokine response profile from CD14+ monocytic cells.
  • the non-monocytic DC cells described herein expand and differentiate in response to Flt3L.
  • the non-monocytic DC cells described herein exhibit increased levels of Th1 inducing cytokines such as I L12p35, IL12P40 and I L-1 ⁇ in comparison to DCs derived from monocytic CD14+ cells.
  • the levels of biomarkers may be determined using fluorescent activated cell sorting (FACS).
  • FACS fluorescent activated cell sorting
  • the methods described herein comprise differentiating and/or expanding a population of non-monocytic DC precursor cells to form a population of non-monocytic DCs. Also provided are methods for expanding a population of non-monocytic DCs to form an expanded population of non-monocytic DCs.
  • differentiation refers to the process by which a less specialized cell, such as a precursor cell, becomes a more specialized cell type, such that it is committed to a specific lineage.
  • precursor cell refers to cell with a limited replicative capability that shows signs of differentiation towards a target cell, such as the non-monocytic DC precursor cells described herein.
  • the non-monocytic DC precursor cells may be expanded by culturing the cells in the presence of Flt3 Ligand (Flt3L) and bone marrow stromal cells (BMSCs).
  • Flt3L Flt3 Ligand
  • BMSCs bone marrow stromal cells
  • the non-monocytic DC precursor cells may be differentiated and/or expanded by culturing the cells in the presence of one or more of GM-CSF, IL-4, Flt3 Ligand (Flt3L), Stem Cell Factor (SCF) and bone marrow stromal cells (BMSCs).
  • GM-CSF GM-CSF
  • IL-4 Flt3 Ligand
  • Flt3L Flt3 Ligand
  • SCF Stem Cell Factor
  • BMSCs bone marrow stromal cells
  • the DC precursor cells may be differentiated and/or expanded by culturing the cells in the presence of GM- CSF, IL-4, Flt3 Ligand (Flt3L), Stem Cell Factor (SCF) and bone marrow stromal cells (BMSCs).
  • GM- CSF GM- CSF
  • IL-4 Flt3 Ligand
  • Flt3L Flt3 Ligand
  • SCF Stem Cell Factor
  • BMSCs bone marrow stromal cells
  • Example 1 Identification and characterization of non-monocytic precursors capable of generating dendritic cells for immunotherapy
  • Tested cells (CD14+, NMDCPs) were then added with a mixture of serum-free media (StemSpan, Stem Cell technologies) containing different cytokine combinations (Miltenyi) of SCF (20ng/ul) GM-CSF (50 ng/ul), I L-4 (10ng/ul), Flt3L (100ng/ul). All cytokines were purchased from Miltenyi.
  • Human T cells were isolated by flow cytometry using CD4 and CD45RA for naive T cells and CD8, CD45RO, CCR7 for naive CD8 + T cells.
  • Human CD14 + monocytic cells were isolated from human MNCs using flow cytometry.
  • T cell response in humanized NSG mice engrafted with human MNCs followed by vaccination with OVA pulsed DCs was assessed by culturing splenocytes with OVA for 24 hours followed by assessment of T cell producing cytokines using Human Th1 Th2 and Th17 kits (BD Biosciences, Burlington, ON).
  • Anti-Melan-A specific CD8+ T cell response was assessed in humanized mice engrafted with human MNCs followed by vaccination with Melan-A pulsed DCs.
  • CD8+ T cell response specific to Melan-A was assessed by using Human HLA tetramers specific for Melan-A to identify Melan-A+ CD8+ T cells in the spleens of the humanized mice (BD Biosciences, Burlington, ON).
  • hESCs and hiPSCs were performed as previously described(Risueno et al. , 2012).
  • Human cord blood derived induced pluripotent stem cells were used as a source of human blood iPSCs.
  • Day 15 embryoid bodies (EBs) were dissociated into single cells and CD34+CD45+ cells were isolated by flow cytometry on a BD FACAria, which were subsequently cultured in the presence of either GM-CSF along with TNF-a or GM-CSF along with IL-4 to drive DC generation.
  • total cellular RNA was isolated using the RNeasy Plus Mini kit according to the manufacturer's instructions (Qiagen, Mississauga, ON) and cDNA was synthesized using qScript SuperMix (Quanta Biosciences, Gaithersburg, MD).
  • qScript SuperMix Quanta Biosciences, Gaithersburg, MD
  • Output data was normalized using Robust Multichip Averaging (RMA) algorithm and baseline transformed to the median of all samples using GeneSpring 12.5 software (Agilent Technologies).
  • Mouse microarray data was used from publicly available GEO source (GSE15907, GSE1 1430, GSE37566, and GSE37029). Common gene entities between mouse and human data were identified and hierarchical clustering using Pearson distance metric and centroid linkage rule was done on normalized expression values using dChip software(Li and Wong, 2001 ). Multiple hypotheses testing, such as Benjamini- Hochberg false discovery rate P-value correction was performed for comparison.
  • CD34 + CD38 + CD45RA + human progenitors have been shown to possess pDCs potential (Chicha et al., 2004). The inventors therefore hypothesized that precursors of non-monocytic DCs may emerge from this parent CD34 + CD38 + CD45RA + population upon differentiation induction.
  • CD34 + CD38 + CD45RA + progenitors were isolated ( Figure 8A) and cultured to screen for acquisition of markers expressed by murine non- monocytic DC precursors, including CX3CR1 , CD1 15 and CD135 (Onai et al., 2007) (Sasmono et al., 2003) ( Figure 1A).
  • CD1 15 showed a transient but distinct expression pattern that was maximal at day 2 of differentiation culture ( Figure 1 B).
  • Human CD1 15 + subset isolated from differentiated CD34 + CD38 + CD45RA + progenitors had distinct features from monocyte DCs used clinically for immunotherapy.
  • CD1 15 + cells lacked monocytic morphological features ( Figure 1 C).
  • Figure 1 C and 1 D we examined whether CD1 15 + Flt3 + HLA-DR + cells could be found in the circulation of human adult peripheral blood (PB).
  • the phoenotype of this subset was in sharp contrast to human monocytic CD14 + cells that express CD14, CD1 1 b and CD62L ( Figure 1 F) (Geissmann et al., 2003) and are devoid of CD1 15, Flt3 or HLA-DR ( Figure 1 G) and give rise to monocytic DCs. Beyond cellular morphology and phenotypic expression, de novo isolated non-monocytic CD1 15 + Flt3 + HLA-DR + cells failed to secrete IL-6 and TNF-a following LPS stimulation (de Waal Malefyt et al., 1991 ) ( Figure 1 G), as well as IL-12 (Ebner et al.
  • Figure 1 H produced by DCs derived from CD14 + monocytes.
  • Global gene expression profiling demonstrated that human CD1 15 + Flt3 + HLA- DR + cells demonstrated similar signatures to established mouse non- monocytic DC precursors (Figure 8B) (Liu et al., 2009; Onai et al., 2007) while displaying a distinct molecular profile from human monocytic CD14 + cells ( Figure 1 1).
  • CD1 15 + Flt3 + HLA-DR + cells represent a unique subset of cells of non-monocytic origin.
  • Flt3 Ligand shown to expand non-monocytic DC precursors in the mouse (Onai et al., 2007), inhibited survival of monocytic CD14 + cells while promoting expansion and subsequent DC differentiation of non-monocytic CD1 15 + Flt3 + HLA-DR + cells ( Figure 1 L and 1 M).
  • GM-CSF and I L-4 known to drive direct differentiation of monocytic CD14 + cells into DCs without expansion
  • non-monocytic CD1 15 + Flt3 + HLA-DR + cells induced significant expansion/differentiation of non-monocytic CD1 15 + Flt3 + HLA-DR + cells into DCs with increased expression of T cell stimulatory markers (CD83, CD86) (Dissanayake et al., 201 1 ) ( Figure 8C and 8D).
  • T cell stimulatory markers CD83, CD86
  • Figure 8C and 8D T cell stimulatory markers
  • CD1 15 + Flt3 + HLA-DR + cells therefore appear to represent candidate non- monocytic DC precursors in the human (hNM-DCPs).
  • hNM-DCPs human
  • GM-CSF and IL4 Compared to the solely presence of GM-CSF and IL4 (GMI), the combination of Flt3L, Stem Cell Factor (SCF), GM-CSF and IL4 (FSGMI) demonstrated better overall human cells input (CD45) and significant frequencies of differentiation into mature DCs (phenotypically identified as CD1 a + " CD1 1 c + HLA-DR + CD83 + ) and pDCs (CD1 a " CD123 + BDCA-2 + ) ( Figure 2I), thereby delineating a robust read-out assay for hNMDCPs differentiation.
  • SCF Stem Cell Factor
  • FSGMI GM-CSF and IL4
  • CD1 15 + Flt3 + HLA-DR + cells may be a heterogeneous population, these cells were clonally examined in vitro to quantitate the DC developmental capacity of this unique subset.
  • Two sources of human non- monocytic CD1 15 + Flt3 + HLA-DR + cells were used in clonal analysis as depicted in Figure 2A: 1 ) De novo isolated directly from adult human blood, and 2) Purified from human-mouse xenograft recipients transplanted with CD34 + CD38 + CD45RA + progenitors.
  • Non-monocytic CD1 15 + Flt3 + HLA-DR + cells from either source were clonally deposited on human BMSCs to drive clonal expansion in presence of Flt3L followed by DC differentiation inducing conditions also in presence of Flt3L or GM-CSF and IL-4 (Figure 2B). All xenograft recipients transplanted with CD34 + CD38 + CD45RA + progenitors generated non-monocytic CD1 15 + Flt3 + HLA-DR + cells in vivo ( Figure 2C), where GM-CSF and IL-4 treatment significantly increased non-monocytic
  • CD1 15 + Flt3 + HLA-DR + reconstitution compared to untreated transplanted cells Figure 2D.
  • In vitro monocytic CD14 + cells generated DCs that were phenotypically identified as CD1 a + " CD1 1 c + HLA-DR + CD83 + ( Figure 2E) at 100% frequency, and were completely devoid of CD1 a " CD123 + BDCA-2 + cells representing human pDC (Dzionek et al., 2001 ) ( Figure 2E and 2G).
  • CD1 15 + Flt3 + HLA-DR + cells clonally generated either DCs or pDCs exclusively, or a mixture of DCs and pDCs (Figure 2E) at a frequency of 43, 33 and 24% respectively ( Figure 2G).
  • Plasmacytoid DCs derived from non-monocytic precursors was correlated with IFN-a production, confirming functional responsiveness of pDCs generated (Dzionek et al., 2001 ) ( Figure 2F).
  • Non-monocytic DC precursors exclusively give rise to human plasmacytoid DCs in vivo
  • CD1 15 + Flt3 + HLA-DR + cells were able to generate CD123 + BDCA-2 + pDCs in vivo ( Figure 3B). These results were consistent in all independent experiments and recipient mice ( Figure 3C and 3D).
  • CD1 15 + Flt3 + HLA-DR + cells represent a non-monocytic precursor that gives rise to a unique repertoire of DCs that includes generation of pDCs in vivo.
  • Preferential Th1 vs Th2 in vitro response can be induced by human non- monocytic DCs
  • Th1 response (characterized by IFN- ⁇ production) is required to promote antitumor immune capacity, whereas Th2 response (characterized by I L-4 production) is suppressive (Cerundolo et al., 2004; Fields et al., 1998; Plantinga et al., 2013).
  • IFN- ⁇ production characterized by IFN- ⁇ production
  • Th2 response characterized by I L-4 production
  • DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells demonstrated a preferential tolerance and enhanced survival in response to bacterial infectious LPS induced cell death as compared to monocytic CD14+ and bulk CD34+ cells (Figure 4B).
  • Analysis of DC populations derived from bulk CD34+ cells in presence of both GM-CSF and IL-4 or FLt3L identified a lack of pDC generation (Figure 4C), a property unique to non-monocytic CD1 15 + Flt3 + HLA- DR + cells ( Figure 2G) and highlighting an absence of this population in clinically utilized DC regimens for immunotherapy.
  • DCs derived from non- monocytic CD1 15 + Flt3 + HLA-DR + cells generated significantly higher proportions of I FN- ⁇ producing Th1 T cells ( Figure 4D and 4E) along with a significant reduction in generation of IL-4 producing Th2 cells when compared with DCs derived from monocytic CD14 + cells as well as bulk CD34+ cells ( Figure 4D and 4F).
  • TSLP thymic stromal lymphopoietin
  • TSLP thymic stromal lymphopoietin
  • DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + precursors preferentially induced IFN- ⁇ production of T cells over IL-4 production with a five-fold higher frequency in Th1 cells than Th2 cells compared to DCs derived from monocytic CD14 + and bulk CD34+ cells, which preferentially drove the expected Th2 over a Th1 response ( Figure 4G, 4H and 4I).
  • CCR7 C-C chemokine receptor type 7
  • hB-iPS derived non-monocytic CD1 15+Flt3+HLA-DR+ cells gave rise to pDCs, confirmed by expression of BDCA-2 and CD123 ( Figure 5B) and functionally validated by production of IFN-a ( Figure 5C), with a complete absence of pDC generation from other hPSC sources.
  • DCs generated from hB-iPS derived CD1 15+Flt3+HLA-DR+ cells also showed a significantly greater potential for both CD4+ and CD8+ T cell proliferation compared to DCs derived from other hPSC sources ( Figure 10B and 10C).
  • DCs generated from hB-iPSCs derived non-monocytic CD1 15+Flt3+HLA-DR+ cells also generated higher proportions of IFN- ⁇ producing Th1 T cells than DC subsets generated from other hPSC sources, along with significantly reduced generation of IL-4 producing Th2 cells (Figure 5D).
  • DCs generated from hB- iPSCs derived non-monocytic CD1 15+Flt3+HLA-DR+ cells preferentially induced IFN- ⁇ production in T cells with five-fold higher frequency of Th1 cells vs. Th2 cells ( Figure 5E).
  • hB-iPS derived non-monocytic CD1 15 + Flt3 + HLA-DR + cells showed exclusive generation of BDCA-2+ pDCs ( Figure 5F and 10F).
  • hB-iPSCs offer an abundantly available resource for non- monocytic DC-based vaccine development and related applications.
  • Non-monocytic DC precursors are capable of superior migration and Th1 induction in vivo
  • DCs derived from monocytic CD14 + cells selectively gave rise to a preferential Th2 response over Thl with 6-7 fold higher IL-4 + CD4 + T cells compared to IFN-y + CD4 + T cells ( Figure 6C and 6D).
  • DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells led to a preferential induction of Th1 over Th2 response with 2-3 fold higher proportions of IFN- ⁇ secreting T cells than I L-4 producing T cells ( Figure 6C and 6D).
  • Th1 associated transcription factor T-bet (Zhu et al., 2010) was several fold higher than that of the Th2 associated transcription factor GATA-3 (Zhu et al., 2010) observed in spleens of recipients transplanted with DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells compared to monocytic CD14 + cells ( Figure 6E).
  • Th17 another subset of T cells characterized by IL-17 production, termed Th17, has recently been identified (Yang et al., 2008) to have potent tumor eradication capacity (Martin-Orozco et al. , 2009).
  • Th17 induction was examined using novel non-monocytic DC precursors.
  • DCs derived from monocytic CD14 + cells showed poor induction of Th17 response in vivo
  • DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells generated a significantly higher splenic Th17 response along with increased expression of Th17 associated transcription factor, RORyT (Yang et al., 2008) ( Figures 6C, 6D and 6E).
  • Non-monocytic human DCs drive superior anti-tumor immune response in vivo
  • Melan-A is an antigen widely expressed in melanoma, where CD8 + T cell response against Melan-A is indicative of an anti-myeloma immune response (Boon and van der Bruggen, 1996) and is used as a surrogate for measuring the levels of protective immune response in melanoma patients (Romero et al., 2002).
  • DCs derived from non-monocytic CD1 15 + Flt3 + HLA-DR + cells possessed nearly 10-fold higher capacity to drive induction of Melan-A specific CD8 + T cells compared to DCs derived from monocytic CD14 + cells in vitro ( Figure 7A and 7B).
  • human DCs derived from novel non-monocytic precursors demonstrated a > 10 fold enhanced ability to drive Melan-A specific CD8+ T cell response in vivo compared to clinically utilized monocytic DCs, further supporting unique applications and suitability for cancer therapy.
  • Mouse DC generation proceeds from macrophage/dendritic cell precursor (MDP) (Fogg et al., 2006), which differentiates into common dendritic cell precursor (CDP) (Onai et al., 2007), giving rise to non-monocytic DCs(Liu et al., 2009).
  • MDP macrophage/dendritic cell precursor
  • CDP common dendritic cell precursor
  • MDP and CDP are phenotypically overlapping populations within the mouse BM with both populations identified by CX3CR1 expression (Auffray et al., 2009) (Fogg et al., 2006; Onai et al., 2007).
  • CD1 15 + Flt3 + HLA-DR + cells lack CX3CR1 expression, highlighting unique differences between mouse and human hematopoiesis in the context of DC biology. Given that the current understanding of human DC biology largely comes from studies in the mouse (Ardavin, 2003; Geissmann et al., 2010), these differences are critical in informing and revisiting clinical applications.
  • DCs have also been explored as vaccines for viral infections such as HCV (Zhou et al., 2012) and HIV (Garcia et al., 2013) and hB-iPS derived non-monocytic CD1 15+Flt3+HLA-DR+ cells offers a renewable source for autologous vaccine development.
  • HCV Zhou et al., 2012
  • HIV HIV
  • hB-iPS derived non-monocytic CD1 15+Flt3+HLA-DR+ cells offers a renewable source for autologous vaccine development.
  • preclinical work with hB-iPS derived non-monocytic CD1 15+Flt3+HLA-DR+ cells provides validation for their utility in vaccine development due to their propensity for enhanced survival, T cell proliferation and Th1 differentiation, further optimization has been limited by the complexities of generating functional T cells from pluripotent stem cell sources.
  • the methodology described herein can be applied with ease in a translational setting for the development of clinical grade DC vaccines for immunotherapy as well as use in anti-tumor DC therapies. Additionally, identification of human non-monocytic DC precursors allows for these cells to be used in screening platforms for identification of human vaccine adjuvants, which has been limited with murine DC populations. Moreover, identification of human non-monocytic DC precursors also allows for investigation of DC biology in human autoimmune disorders, which have previously relied on DCs derived from monocytes.
  • non-monocytic precursors comprise a rare population in blood ( ⁇ 1 %)
  • identification of human precursors capable of clonal expansion to several hundred-fold in the presence of Flt3L provides a method to apply to patient immunotherapy, in addition to generation from patient specific iPSCs generated from blood cells.
  • standard clinical conditions to expand DCs using GM-CSF do not support human non-monocytic DC precursors, suggesting that these same non-monocytic DCs have not participated in clinical therapies used to date.
  • the present Example provides a proof of principle for generation and utility of unique human DC precursors for immunotherapy as compared to current clinically used sources of DCs (Banchereau and Steinman, 1998; Cerundolo et al., 2004; Palucka and Banchereau, 2012).
  • HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nature medicine 6, 435-442.
  • Interleukin 10(l L-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of I L-10 produced by monocytes.
  • Nuclear factor-kappaBI controls the functional maturation of dendritic cells and prevents the activation of autoreactive T cells. Nature medicine 17, 1663- 1667.
  • Figdor, C.G. de Vries, I. J., Lesterhuis, W.J., and Melief, C.J. (2004). Dendritic cell immunotherapy: mapping the way. Nature medicine 10, 475- 480. Fogg, D.K., Sibon, C, Miled, C, Jung, S., Aucoutuher, P., Littman, D.R., Cumano, A. , and Geissmann, F. (2006). A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 31 1 , 83-87.
  • Th2 polarization by Der p 1 -pulsed monocyte-derived dendritic cells is due to the allergic status of the donors. Blood 98, 1 135-1 141 .
  • Monocyte-derived dendritic cells induce a house dust mite-specific Th2 allergic inflammation in the lung of humanized SCID mice: involvement of CCR7. Journal of immunology 169, 1524-1534.
  • Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat Biotechnol 29, 1 1 17-1 1 19.
  • T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity 31 , 787-798.
  • Dendritic cells On the move from bench to bedside. Nature medicine 7, 761 -765.
  • Tumour immunology TSLP drives human tumour progression. Nature reviews Immunology 1 1 , 235.
  • a macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101 , 1 155-1 163.
  • Circulating human basophils lack the features of professional antigen presenting cells. Sci Rep 3, 1 188.
  • Dendritic cells genetically modified with an adenovirus vector encoding the cDNA for a model antigen induce protective and therapeutic antitumor immunity.
  • T-cell tolerance may reflect selective activation of lymphokine synthesis.
  • Noncanonical Wnt signaling orchestrates early developmental events toward hematopoietic cell fate from human embryonic stem cells.
  • T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28, 29-39.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des cellules précurseurs de cellules dendritiques non monocytaires capables de générer des cellules dendritiques, des cellules dendritiques non monocytaires, des procédés associés et leurs utilisations. Les cellules dendritiques non monocytaires sont utiles pour susciter une réponse immunitaire in vivo ou in vitro, telle qu'une réponse de type Th1, et/ou en immunothérapie comme en immunothérapie anticancéreuse.
PCT/CA2015/051226 2014-11-24 2015-11-24 Cellules dendritiques non monocytaires, précurseurs de celles-ci, et procédés associés Ceased WO2016082034A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462083730P 2014-11-24 2014-11-24
US62/083,730 2014-11-24

Publications (1)

Publication Number Publication Date
WO2016082034A1 true WO2016082034A1 (fr) 2016-06-02

Family

ID=56073250

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2015/051226 Ceased WO2016082034A1 (fr) 2014-11-24 2015-11-24 Cellules dendritiques non monocytaires, précurseurs de celles-ci, et procédés associés

Country Status (1)

Country Link
WO (1) WO2016082034A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7341870B2 (en) * 2002-12-16 2008-03-11 Etablissement Francais Du Sang Dendritic cell line

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7341870B2 (en) * 2002-12-16 2008-03-11 Etablissement Francais Du Sang Dendritic cell line

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ASPORD C ET AL.: "A Novel Cancer Vaccine Strategy Based on HLA-A*0201 Matched Allogeneic Plasmacytoid Dendritic Cells", PLOS ONE, vol. 5, no. 5, 1 May 2010 (2010-05-01), ISSN: 19326203 *
BRETON G ET AL., THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 212, no. 3, 9 March 2015 (2015-03-09), pages 401 - 413, ISSN: 1540-9538 *
BUENO C ET AL.: "NG2 Antigen Is Expressed In CD 34+ HPCS and Plasmacytoid Dendritic Cell Precursors: Is NG2 Expression in Leukemia Dependent on the Target Cell Where Leukemogenesis is Triggered?", LEUKEMIA, vol. 22, no. 8, August 2008 (2008-08-01), pages 1475 - 1478, ISSN: 08876924 *
LEE J ET AL.: "Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 212, no. 3, 1 January 2015 (2015-01-01), pages 385 - 399, ISSN: 1540-9538 *
MERAD M ET AL.: "The Dendritic Cell Lineage: Ontogeny and Function of Dendritic Cells and Their Subsets in the Steady State and the Inflamed Setting", ANNUAL REVIEW OF IMMUNOLOGY., vol. 563, 6 December 2013 (2013-12-06) *

Similar Documents

Publication Publication Date Title
TWI382843B (zh) 成熟樹突細胞組合物及培養彼等之方法
US20090297541A1 (en) Maturation of dendritic cells
TWI572718B (zh) 免疫抑制細胞及其製造方法和組成物
Karsunky et al. Developmental origin of interferon-α–producing dendritic cells from hematopoietic precursors
US20150166955A1 (en) Method for loading of dendritic cells with class i antigens
CN110914411A (zh) 干扰素引发的浆细胞样树突细胞
US20240141294A1 (en) Cgmp compliant production and expansion of plasmacytoid dendritic cells from hematopoietic stem and progenitor cells
US20200017831A1 (en) Methods of preparing an isolated population of dendritic cells and methods of treating cancer using same
JP6334810B2 (ja) Ｉｆｎを用いた非接着培養による樹状細胞の調製方法
US20220249567A1 (en) Low density cell culture
Decker et al. Dendritic cell immunotherapy for the treatment of neoplastic disease
US20230149459A1 (en) V delta1+ t cells for the treatment of myeloid malignancies
Baek et al. Dendritic cell (DC) vaccine in mouse lung cancer minimal residual model; comparison of monocyte-derived DC vs. hematopoietic stem cell derived-DC
US20250352570A1 (en) Methods for treating myeloid malignancies
US20170267974A1 (en) Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells
CN116322714A (zh) 使用血小板裂解物制备树突状细胞的方法
Hutten et al. Ex Vivo Generation of Interstitial and Langerhans Cell-like Dendritic Cell Subset–based Vaccines for Hematological Malignancies
WO2016082034A1 (fr) Cellules dendritiques non monocytaires, précurseurs de celles-ci, et procédés associés
US20210284963A1 (en) Methods of obtaining a mixed population of human xcr1+ and plasmacytoid dendritic cells from hematopoietic stem cells
US20240400992A1 (en) Compositions and methods for antigen-specific t cell expansion
Han et al. Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells from hematopoietic stem cells
JP2023153286A (ja) Ｎｋ細胞を含む細胞集団の製造方法
Lin In vitro generation of hematopoietic progenitors and functional T cells from pluripotent stem cells
van Lent Lymphoid development; a dynamic interplay of timing and dosing

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15862740

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15862740

Country of ref document: EP

Kind code of ref document: A1