WO2015165869A1 - Méthode de génération de cellules suppressives - Google Patents

Méthode de génération de cellules suppressives Download PDF

Info

Publication number
WO2015165869A1
WO2015165869A1 PCT/EP2015/059134 EP2015059134W WO2015165869A1 WO 2015165869 A1 WO2015165869 A1 WO 2015165869A1 EP 2015059134 W EP2015059134 W EP 2015059134W WO 2015165869 A1 WO2015165869 A1 WO 2015165869A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
mdscs
host cell
csf
Prior art date
Application number
PCT/EP2015/059134
Other languages
English (en)
Inventor
David Escors Murugarren
Therese Maria LIECHTENSTEIN
Original Assignee
Fundación Pública Miguel Servet
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 Fundación Pública Miguel Servet filed Critical Fundación Pública Miguel Servet
Publication of WO2015165869A1 publication Critical patent/WO2015165869A1/fr

Links

Classifications

    • 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
    • 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
    • C12N5/064—Immunosuppressive dendritic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to methods for the production of myeloid derived suppressor cells (MDSCs).
  • MDSCs myeloid derived suppressor cells
  • This cell population can be used as a research tool for screening of new therapies for the treatment of several diseases, especially cancer.
  • the invention can also have multiple applications in applied immunology and in the development of new immunotherapies.
  • Cancer cell immunotherapy aims at stimulating the patient's immune system to destroy the cancer cells of a growing tumor. It is now widely recognized that a malignant tumor develops, among other reasons, because there is a failure of the immune system to destroy the cancerous cells. It is believed that the tumor cells develop a series of strategies such as downregulating tumor antigen presentation (APC) or promoting immune tolerance that ultimately lead to escape from immune surveillance.
  • APC tumor antigen presentation
  • cell immunotherapy was just a promise only a few years ago, the field is now becoming a reality thanks to the emergence of the first success stories, such as that of Sipuleucel-T (trade name Provenge), which is a therapeutic vaccine for treating prostate cancer. It is the first cell immunotherapy approved by the FDA. This is fuelling increasing interest in the field.
  • DCs cultured dendritic cells
  • APCs antigen presenting cells
  • DCs are used to evaluate different treatments as they are the most potent antigen presenting cells (APCs), and can be cultured and expanded ex vivo in large numbers from bone marrow and peripheral blood monocytes.
  • APCs antigen presenting cells
  • many of the treatments found in this way fail to raise effective anti-cancer responses, as DCs are dysfunctional in advanced stages of disease.
  • pre-clinical evaluation of new therapies should complement DC-based tests with MDSC-based tests.
  • WO2010062990 discloses methods of isolating, culturing and differentiating myeloid derived suppressor cells (MDSCs) from genetically modified embryonic stem cells (ES) and hematopoietic cells using a combination of factors including macrophage colony-stimulating factor (M-CSF).
  • MDSCs myeloid derived suppressor cells
  • ES embryonic stem cells
  • M-CSF macrophage colony-stimulating factor
  • the methods described comprise contacting the ES with effective amounts of M-CSF and additional factors such as Kit Ligand, VEGF, TPO and others, and culturing these cells under conditions suitable for their propagation.
  • Bayne et.al (“Tumor-derived granulocyte-macrophage colony-stimulating factor regulates myeloid inflammation and T cell immunity in pancreatic cancer” Cancer cell 2012, vol. 21 , pp. 821 -835) discloses that granulocyte- colony stimulating factor (GM-CSF) produced and secreted to the tumor environment by the cancer cells plays an important role in the development of MDSCs. It also discloses that MDSC in vitro differentiation can be achieved by the addition of recombinant GM-CSF to the culture medium of purified haematopoietic precursor cells. Likewise, the efficiency of production disclosed leaves room for improvement.
  • GM-CSF granulocyte- colony stimulating factor
  • MDSCs myeloid derived suppressor cells
  • the invention provides means to easily produce ex vivo (in vitro) large numbers of MDSCs whose behavior mimics that of the in vivo tumor infiltrating myeloid derived suppressor cells responsible for the suppression of T-cell responses. This allows to set up in vitro tests for easily screening any therapies that seek to modulate the central role played by the myeloid derived suppressor cells in many ailments, such as cancer and autoimmune diseases.
  • a first aspect of the present invention is an in vitro method for the production of a myeloid derived suppressor cell (MDSC) comprising the step of transducing the granulocyte-macrophage colony stimulating factor (GM- CSF) gene with a lentiviral vector to an animal host cell.
  • MDSC myeloid derived suppressor cell
  • GM- CSF granulocyte-macrophage colony stimulating factor
  • a second aspect of the present invention is the myeloid derived suppressor cell obtainable by the method according to the first aspect of the invention.
  • a third aspect of the invention is a method for screening a substance for therapy comprising maintaining a cell as defined in the second aspect of the invention in the presence of the substance, wherein the substance is selected from the group consisting of organic molecules, peptides, proteins, vaccines and mixtures thereof.
  • FIG.1 Ex vivo cancer-specific MDSC differentiation system.
  • A Bar graph on the left shows number of myeloid cells in millions (yield of BM-derived myeloid cells) after a 5 day incubation of mouse bone marrow with the percentages of conditioning media (CM) from 293T or B16 cells as indicated.
  • CM conditioning media
  • Phenotype of myeloid cells as a function of the percentage of 293T-CM.
  • B Phenotype of myeloid cells at day 8. Bar graph on the left represents the percentage of expression of the indicated markers (bottom) after 8 days differentiation with DC medium or 293T-CM as indicated. Statistical comparisons between DC and MDSC markers are shown. Histogram on the right shows CD62L expression on conventional DCs, 293T-MDSCs (bold continuous line), and B16-MDSCs (dashed line). Percentages of expression are shown within the graph. The horizontal dotted line represents the gate in which 95% of the control unstained cells (c) are excluded. Percentages of expression are shown within the histogram.
  • (C) Dot plot of Ly6C-Ly6G marker profiles from conventional DCs, or MDSCs differentiated in 293T- or B16-CM as indicated. Monocyte-(M) or granulocytic-(G) MDSCs are gated within the graphs with their corresponding percentages. M-MDSCs and G-MDSCs, monocytic and granulocytic MDSCs.
  • FIG.2 Ex vivo MDSCs conserve their plasticity of differentiation.
  • A On the left, the Ly6C-Ly6G profile of day 5 293T-MDSCs is shown as a density-plot. If grown in MDSC-CM, most of the cells become Ly6G positive (granulocytic MDSCs, top density plot) without significant CD1 1 c expression (upper right density plot, Ly6G-CD1 1 c profile). If 293T-MDSCs are grown in conventional DC differentiation medium (recombinant GM-CSF), Ly6G expression is lost
  • Ex vivo melanoma-specific MDSCs resemble tumor-infiltrating MDSCs and are strongly suppressive.
  • CD62L/Ly6G co-expressing cells are shown. Quadrants were established using unstained cells as controls.
  • B Immunoblot of iNOS and GADPH expression (as a loading control) in DC, 293T-MDSCs and B16-MDSCs as indicated on top. Only B16-MDSCs expressed iNOS.
  • C Phenotype profiling of M-MDSC and G-MDSC populations from different sources (tumor-infiltrating MDSCs - (T), spleen MDSCs from tumor-bearing mice - (Spl), and ex vivo B16-MDSCs) are shown, as bar graphs with percentage of expression of the indicated markers on top of each graph.
  • the left bar graphs shows the percentage of CD8 T cells in an MLR using the indicated C57BL/6-derived DCs/MDSCs as APCs and purified Balb-c CD8 T cells as allogenic effectors.
  • the right graph represents the corresponding percentage of Ki67-positive CD4 and CD8 T cells. This graph shows data from one of two experiments with similar results. Statistical comparisons between MDSCs and DCs are shown within the graphs. No, no MDSCs or DCs in reactions.
  • the left bar graph represents the percentage of suppression of T cell proliferation by bulk or purified B16 M-MDSCs or G-MDSCs. CFSE-labelled autologous CD8 T cells activated with anti-CD3/anti-CD28 beads were used as targets.
  • the bar graph on the right represents the fold increase/decrease in secreted IFN- ⁇ from the cultures shown on the left. Different APC/T cell ratios were used for the experiments as indicated in the graphs. Appropriate statistical
  • FIG.4. BT459-MDSCs exhibit particular phenotypic characteristics, but retain their T cell suppressive activities.
  • A Bar graph representing the number of MDSCs from samples taken from cultures on day 5 and day 8, as indicated, using the indicated cells (bottom legend).
  • B Bar graph representing the percentage of surface expression of the indicated makers (bottom legend) on DCs, 293T-, B16- and BT549-MDSCs as indicated (bottom legend). The cells were left untreated or treated with ILI beta or IFN-gamma as shown in the figure.
  • C Bar graph with error bars representing the percentage of Ki67+ balb-c CD4 or CD8 T cells as shown (cells under proliferation) in mixed lymphocyte reactions (MLRs) using as "stimulators" DCs, 293T-, B16- and BT549-MDSC cells as indicated (bottom legend). ** , *** , very and highly significant differences between the shown groups and DCs.
  • FIG.5 The structure of an HIV-1 derived pCSGW vector (top), a typical vector that could be used for carrying out the invention, and the structure of the pDUAL lentiviral vector developed by the inventors (bottom) and used in the present invention, that features the WPRE element upstream the ubiquitin promoter, the RRE and CPPT element upstream the SFFV promoter. It also contains a 3' U3-deleted long terminal repeat (LTR) to make it self-inactivating after integration (SIN). ⁇ , HIV-1 packaging signal.
  • FIG.6 Flow cytometry density plot of MDSCs generated by direct transduction of bone marrow cells with the lentivector pDUAL-GMCSF-Puro. The Ly6C- Ly6G expression profiles demonstrate the differentiation of both monocytic and granulocytic MDSC populations. The relative percentage of each population is indicated within the graph.
  • transduction refers to the process whereby heterologous (foreign) DNA is introduced into a host cell via a viral vector.
  • the aim of this process is for the piece of DNA to be integrated in the genome of the host cell so that the protein or RNA that the foreign DNA codes for is stably expressed in the host cell.
  • the transduction of the GM-CSF gene is carried out with the aim of having the host cell stably express the GM-CSF protein.
  • transfection refers to the process whereby heterologous (foreign) DNA is introduced into a host cell via any non-viral method, such as electroporation, or a chemical-based method. Transfection usually involves opening transient holes or pores in the cell membrane, to allow the uptake of the genetic material.
  • lentivirus refers to a genus of viruses of the
  • Retroviridae family characterized by a long incubation period. They are the only retroviruses that are able to infect non-dividing cells. Among them, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline Immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) are found.
  • HIV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • FIV feline Immunodeficiency virus
  • EIAV equine infectious anemia virus
  • lentiviral vector refers to a genetically modified lentivirus that is used to deliver genetic material into a host cell.
  • the lentiviral vector is engineered so that it delivers the GM-CSF gene to the host cell.
  • Examples of lentiviral vectors that could be used for the present invention are pCSGW (Ikeda Y. et.al.
  • the term "host cell” as used herein refers to an isolated cell from any animal organism that is selected to be genetically modified.
  • the genetic modification consists on the transduction of a GM-CSF gene, so that the host cell stably expresses the GM-CSF gene products. Once transduced successfully, the host cell is propagated and cultured in vitro. This transduced host cell can further be used as a research tool, for instance for screening of new therapies based on the modulation of any process mediated by myeloid suppressor cells.
  • host cells examples include mammalian cells, bone marrow cells, cells derived from a tumor, non-human embryonic stem cells, human somatic stem cells and human embryonic stem cells derived from established cell lines or generated without the destruction of human embryos, and mammal (including human) induced pluripotent stem cells (iPSCs) obtained from adult cells and reprogrammed with reprogramming factors.
  • iPSCs induced pluripotent stem cells
  • the host cell is a bone marrow cell
  • the host cell itself differentiates into MDSCs in an autocrine fashion when transduced the GM-CSF with a lentivector.
  • the "host cell” is for instance a tumour derived host cell
  • this transduced cell does not differentiate into MDSCs, but generates a CM that, when contacted to a second "myeloid precursor cell", drives the differentiation of the latter cell into MDSCs.
  • stem cell refers to somatic stem cells, non-human embryonic stem cells and human embryonic stem cells which have been generated without the destruction of human embryos.
  • human embryonic stem cell sources include commercial embryonic cell lines which do not derive from the destruction of a human embryo, and human embryonic stem cell derived form a blastocyst by the technique disclosed in Chung et.al. "Human embryonic stem cell lines generated without embryo destruction” Cell Stem Cell 2008, vol. 2, pp. 1 13-1 17. This technique allows the production of human embryonic stem cell lines generated without the destruction of the human embryo.
  • myeloid precursor cell refers to a cell that is exposed to the conditioning medium (CM) produced by the lentivirally-GM- CSF transduced "host cell".
  • CM conditioning medium
  • the exposure of the "precursor cell” to this CM drives its differentiation into MDSCs.
  • tumor-derived host cell refers to any host cell that is derived from a tumor, either from an established immortalized cell line or from tumor cells derived from a particular patient that can later on be treated with the therapies that are screened with the use of the MDSCs generated by the method of the invention, or even with the generated MDSCs themselves (MDSCs can be re-infused to the patient).
  • screening refers to the use of the myeloid derived suppressor cell obtained by the method of the invention for testing organic molecules, peptides, proteins, therapeutic vaccines or any cell-based immunotherapy for the treatment of any pathology where MDSC plays a relevant role, such as cancer.
  • lentiviral vector refers to a lentiviral vector devised by the inventors, described in Escors D. et al. "Targeting dendritic cell signaling to regulate the response to immunization" Blood 2008, vol. 1 1 1 , pp. 3050-3061 , comprising two promoters (spleen focus forming virus promoter, and ubiquitin promoter), driving expression of the GM-CSF gene and the puromycin resistance gene. It includes the Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE) upstream the ubiquitin promoter, the RRE and CPPT element upstream the SFFV promoter.
  • WPRE Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element
  • DMEM Dulbecco/Vogt modified Eagle's minimal essential medium. It is a cell culture medium. DMEM is commercial and differs from the original MEM in that it contains approximately 4 times as much of the vitamins and amino acids present in the original formula, and some (2 to 4 fold) more glucose. Additionally, it contains iron and phenol red. DMEM is suitable for most types of cells.
  • RPMI Roswell Park Memorial Institute medium. It is a cell and tissue culture medium. It is very rich in phosphate and is formulated for use in a 5% carbon dioxide atmosphere. RPMI has
  • IMDM is an abbreviation for Iscove's modified Dulbecco's medium). This medium is a modification of Dulbecco's Modified Eagle's Medium (DME) containing selenium, additional amino acids and vitamins, sodium pyruvate, HEPES buffer, and potassium nitrate instead of ferric nitrate.
  • DME Dulbecco's Modified Eagle's Medium
  • HEK cell refers to the Human Embryonic Kidney 293 cell which is a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. HEK 293 cells are very easy to grow, transfect and transduce, and have been widely used in cell biology research for many years.
  • GM-CSF Human granulocyte-Macrophage Colony Stimulating Factor
  • GM-CSF Human granulocyte-Macrophage Colony Stimulating Factor
  • This protein is coded by the CSF2 gene, and can be found in the Uniprot database with the entry P04141 (last modified January 22 nd , 2014).
  • the sequence of human GM-CSF used in the present invention is (SEQ ID NO 1 ):
  • GM-CSF encompasses any GM-CSF coded by the GM-CSF gene whatever the origin of the gene is.
  • the term does also include any GM-CSF variant coming as a result of mutations or truncations, and any other structural variant that still displays the same biological activity as the wild type GM-CSF.
  • the biological activity of a GM-CSF variant can be tested by methods such as enzyme-linked immuno assay (ELISA) and functional assays such as GM-CSF-dependent proliferation of the FDCP1 cell line.
  • the first aspect of the invention is an in vitro method for the production of a myeloid derived suppressor cell (MDSC) comprising the step of transducing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene with a lentiviral vector to an animal host cell.
  • MDSC myeloid derived suppressor cell
  • the method of the invention can be carried out with two different strategies, depending on the nature of the host cell.
  • host cell types such as bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, stem cells, and peripheral blood mononuclear cells
  • the transduction of the GM-CSF gene with a lentiviral vector allows their direct differentiation into MDSCs.
  • the method can involve the lentiviral-based transduction of the GM-CSF gene into a different type of host cell such as a tumour derived host cell.
  • the host cell produces a conditioning medium (CM) which, when put into contact with a second cell type (a precursor cell such as a bone marrow cell) induces its differentiation into MDSCs.
  • CM conditioning medium
  • the method of the invention can involve the use of a host cell which itself differentiates into MDSCs when lentivirally transduced, or can involve the use of a host cell which, when lentivirally transduced, produces a conditioning medium which, when put into contact with a second precursor cell drives the differentiation of the latter into MDSCs.
  • the in vitro method for the production of a myeloid derived suppressor cell comprises the step of transducing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene with a lentiviral vector to an animal host cell, wherein: i) the animal host cell is selected from the group consisting of bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, peripheral blood mononuclear cells, and stem cells or alternatively ii) the animal host cell is a tumour-derived host cell which generates a conditioning medium, said conditioning medium differentiating bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, peripheral blood mononuclear cells and stem cells into MDSCs, with the proviso that if the stem cell is a human embryonic stem cell, said human embryonic stem cell has been generated without the destruction of human embryos.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • a particular embodiment of the first aspect of the invention is the in vitro method for the production of a myeloid derived suppressor cell (MDSC) comprising the step of transducing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene with a lentiviral vector to an animal host cell, wherein the animal host cell is selected from the group consisting of bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, peripheral blood mononuclear cells and stem cells; with the proviso that if the stem cell is a human embryonic stem cell, said human embryonic stem cell has been generated without the destruction of human embryos.
  • MDSC myeloid derived suppressor cell
  • a particular embodiment of the first aspect of the invention is the in vitro method for the production of a myeloid derived suppressor cell (MDSC) comprising the step of transducing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene with a lentiviral vector to an animal host cell, wherein the animal host cell is a tumour-derived host cell which generates a conditioning medium, said conditioning medium differentiating bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, peripheral blood mononuclear cells and stem cells into MDSCs; with the proviso that if the stem cell is a human embryonic stem cell, said human embryonic stem cell has been generated without the destruction of human embryos.
  • MDSC myeloid derived suppressor cell
  • the in vitro method for the production of a myeloid derived suppressor cell comprises the step of transducing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene with a lentiviral vector to an animal host cell, wherein: i) the animal host cell is selected from the group consisting of bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, and peripheral blood mononuclear cells, or alternatively ii) the animal host cell is a tumour-derived host cell which generates a conditioning medium, said conditioning medium differentiating bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, and peripheral blood mononuclear cells into MDSCs,
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the in vitro method for the production of a myeloid derived suppressor cell comprises the step of transducing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene with a lentiviral vector to an animal host cell, wherein: i) the animal host cell is selected from the group consisting of bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, peripheral blood mononuclear cells, and stem cells, said stem cells selected from the group consisting of non-human embryonic stem cells, human somatic stem cells, and mammal (including human) induced pluripotent stem cells (iPSCs) or alternatively ii) the animal host cell is a tumour-derived host cell which generates a conditioning medium, said conditioning medium differentiating bone marrow cells, spleen myeloid precursor cells, lymph node precursor cells, peripheral blood mononuclear cells and stem cells into MDSCs, said stem cells selected from the group
  • GM-CSF granulocyte-macro
  • the animal host cell is a mammalian host cell.
  • the mammalian host cell is a human host cell.
  • the GM-CSF gene is a mammalian GM-CSF gene.
  • the mammalian GM-CSF gene is a murine GM- CSF gene.
  • the mammalian GM-CSF gene is a human GM- CSF gene.
  • the in vitro method is that where the host cell is a tumor-derived cell.
  • the in vitro method is that where the tumor-derived cell is a melanoma B16 cell.
  • the in vitro method is that where the host cell is an embryonic stem cell, provided that if the embryonic stem cell is a human embryonic stem cell, this is derived from established cell lines or from human embryonic cells generated without the destruction of a human embryo.
  • the in vitro method is that where the embryonic cell is a human embryonic kidney (HEK) cell.
  • the in vitro method is that where the lentiviral vector is a pDUAL lentiviral vector.
  • the in vitro method is that where the MDSC cell expresses at least one of the surface markers selected from the group consisting of CD1 1 b, Gr1 , CD62L and does not express CD1 1 c.
  • the in vitro method further comprises cultuhng the MDSC in a medium of DMEM or RPMI, and 10% fetal calf serum.
  • a second aspect of the present invention is the myeloid derived suppressor cell obtainable by the method according to the first aspect of the invention.
  • This aspect can be reformulated as a myeloid derived suppressor cell obtainable by the method comprising the step of transducing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene with a lentiviral vector to an animal host cell.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the cell obtained by the method of the invention is new, since there are no references in the prior art disclosing any host cell transduced via a lentivector with the GM-CSF gene, and therefore the cell obtained the method comprises a new genome where the foreign genetic material is integrated by means of the lentiviral vector. It is well established that the integration patterns of lentivectors in the target genome are distinct from those of other vectors, including retrovirus vectors. (Beard B.C. et.al. "Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, or foamy virus". Hum. Gene Ther. 2007, vol. 18, pp.
  • a third aspect of the invention is a method for screening a substance for therapy comprising maintaining a cell as defined in the second aspect of the invention in the presence of the substance, wherein the substance is selected from the group consisting of organic molecules, peptides, proteins, vaccines and mixtures thereof.
  • the method for screening a substance for therapy is in vitro. In a particular embodiment, the method for screening a substance for therapy is in vivo. In another particular embodiment, the method is for screening a cancer therapy.
  • This last aspect may also be formulated as the use of the second aspect of the present invention for screening a substance for therapy wherein the substance is selected from the group consisting of organic molecules, peptides, proteins, vaccines and mixtures thereof.
  • the screening is in vitro screening.
  • the screening is in vivo screening.
  • the screening is for novel cancer treatments.
  • 293T-GM-CSF, B16F0-GM-CSF and BT549- GMCSF were generated by transduction using lentivectors co-expressing mouse GM-CSF and puromycin resistance and selected with 3 g/ml puromycin.
  • MDSCs were expanded from BM cells extracted from femurs/tibias of C57BL/6 mice, by incubation for five to ten days with 75% conditioning medium (CM) from the GM-CSF-producing cells in IMDM supplemented with 10% FCS, penicillin/streptomycin and gentamycin.
  • CM conditioning medium
  • concentrations of recombinant mouse GM-CSF 5000 to 50 ng/ml as concentration standards. Proliferation by CFSE dilution was evaluated by flow cytometry.
  • Immunoblot was performed as described in (Escors D. et.al. "Targeting dendritic cell signalling to regulate the response to immunisation”. Blood 2008 vol. 1 1 1 , pp. 3050-61 ), using mouse anti-iNOS antibodies (from Cell
  • Peroxidase-conjugated anti- mouse and anti-rabbit antibodies were purchased from DAKO. Plasmids, lentivector production, titration and cell transduction
  • the pDUAL-GM-CSF-Puro lentivector was a pDUAL plasmid described before, in which the mouse GM-CSF gene was cloned by reverse
  • BioLegend FITC-Ly6C, PE-CD3, PE-CD86, APC-CD80, APC-I-A/I-F, Biotin- ICAM I, Biotin-H2kb, FITC-IFN- ⁇ , Biotin-PD-L1 , PE-Cy7-Ly6G, PE-IL6, PE- CD34, PE-CD14, PE-CD3, PE-F4/80, Biotin-IL12/IL13, Biotin-IL15, PE-IL17A, PE-Cy7-streptavidin, APC-streptatividin; From eBioscience: PE-CD4, AF700- Foxp3, APC-CD1 1 c, FITC-IA/IF.
  • FITC-IL17A APC-IL10; From Raybiotech: FITC-class I H-2Kb; From BD bioscience pharmigen: v500-CD4, PE- CD8alpha, APC-CD1 1 b, Biotin-IL12, ACP-Ki67; from Southernbiotech: PE- Cy7-CD25; from LSBio: PE-IL4; from Invitrogen: PE-streptavidin, FITC- streptavidin, APC-granzyme B; from AbD Serotec: PE-CD62L; from R&D Systems: anti-human/mouse PE-Arginase 1 .
  • Tumor and spleen cells were stained and analysed by flow cytometry on a LSR Fortessa (BD). CD45+ CD1 1 b+ populations were gated and analyzed.
  • T lymphocytes were isolated from the spleen of a C57BL/6 or Balb-c mice (Harlan) using CD8a and CD4 T cell Isolation Kits II (Miltenyi Biotec, Germany). If required, CD8 T lymphocytes were labeled with CFSE. Cells were plated at 10 5 cells/100 ⁇ /96 well. Subsequently, the cells were either left unstimulated or were stimulated with a 1/800 dilution of anti- CD3/anti-CD28 coated beads (Invitrogen).
  • MDSCs were either used in bulk or sorted using the MDSC Isolation Kit (Miltenyi Biotec, Germany). When indicated, supernatants containing IL12 were added at 250 pg per well while monoclonal antibodies against CTLA-4, PD1 , and control hamster IgG
  • ELISPOT data and mean fluorescence intensities from surface or intracellular staining from multiple groups were analysed by one-way ANOVA followed by a Tukey ' s a posteriori test as described in ibid. Escors et.al. Blood 2008 and Arce F. et. al. Arthritis Rheum. 201 1 .
  • Percentages of T cells in MLRs were normally distributed and analysed with one way ANOVA followed by Tukey ' s test. Three independent reactions per group were used for these experiments.
  • CM MDSC-conditioning medium
  • Ly6G l0W neg Ly6C + , Ly6G high MDSC populations
  • Fig 1 C granulocytic MDSC populations
  • Profiles on day 10 suggested that G-MDSCs in our system derived from M-MDSCs as recently shown in vivo in melanoma. Accordingly, purified day 5 Ly6G-negative 293T-MDSCs quickly up-regulated Ly6G and some CD1 1 c (Fig 1 D).
  • MDSCs Day 5 MDSCs showed a high degree of plasticity of differentiation. They re- differentiated to DCs depending on culture conditions by losing Ly6G and re- expressing CD1 1 c (Fig. 2). MDSC proliferation slowed down after day 5, losing viability from day 10 and suggesting that G-MDSCs were the terminal differentiation stage (Fig. 2). 293T-, B16-, and BT549-MDSCs are phenotypicallv distinct, but functionally suppressive.
  • melanoma B16-MDSCs showed a higher Ly6G/CD62L co-expression compared to 293T-MDSC controls (Fig 3A).
  • B16-MDSCs expressed inducible nitric oxide synthase (iNOS) and arginase-1 , a signature of tumor-infiltrating MDSCs (Figs 3B and 3C).
  • B16-MDSCs generated ex vivo were equivalent to tumor-infiltrating MDSCs according to expression of CD62L, PD- L1 , arginase-1 , CD86 and CD80, with lower surface levels of MHC II, as expected for non-activated MDSCs (Fig 3C).
  • Fig 3C phenotypically resembled tumor-infiltrating rather than spleen MDSCs from melanoma- bearing mice.
  • both 293T- and B16-MDSCs strongly inhibited T cells.
  • MLR mixed lymphocyte reaction
  • MDSCs were differentiated using CM from BT549-GMCSF, and compared to 293T- and B16-generated MDSCS (Fig 4).
  • BT549-MDSCs exhibited a higher proliferation capacity than the other MDSCs, while it was also phenotypically distinct, with a higher expression of MHC II and CD1 1 c compared to 293T- and B16-MDSCS (Fig. 4B).
  • BT549-MDSC strongly inhibited T cell proliferation (Fig 4C).
  • MDSCs can be easily differentiated from any cell type, they were characteristic of the type of tumor cell from which they derived.
  • the method of the invention allows to quickly and efficiently produce MDSCs from several animal and in particular human host cell types, both from two different tumor types (melanoma and breast cancer) and from human embryonic stem cell lines.
  • the MDSCs produced by the method of the invention induce a strong inhibition of T-cell responses, and can thus be used in the search of novel therapies aiming at regulating the role played by the myeloid derived suppressor cells.
  • MDSC obtainable by the method of the invention may also serve as cell biology experimental research tools.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode in vitro de production d'une cellule suppressive issue de myéloïde (MDSC) comprenant l'étape consistant à convertir le gène du facteur de stimulation de colonies de granulocytes-macrophages (GM-CSF) avec un vecteur lentiviral vers une cellule hôte animale. La méthode permet de générer de manière très efficace des cellules MDSC, ouvrant ainsi la voie à des modes plus pratiques d'essai et de criblage de nouveaux traitements cherchant à moduler la population desdites cellules suppressives.
PCT/EP2015/059134 2014-04-28 2015-04-28 Méthode de génération de cellules suppressives WO2015165869A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14166221.3 2014-04-28
EP14166221 2014-04-28

Publications (1)

Publication Number Publication Date
WO2015165869A1 true WO2015165869A1 (fr) 2015-11-05

Family

ID=50679855

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/059134 WO2015165869A1 (fr) 2014-04-28 2015-04-28 Méthode de génération de cellules suppressives

Country Status (1)

Country Link
WO (1) WO2015165869A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111197029A (zh) * 2020-01-09 2020-05-26 广东省第二人民医院（广东省卫生应急医院） 一种尿酸钠诱导产生髓系抑制性细胞的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DAVID ESCORS ET AL: "Assessing T-cell responses in anticancer immunotherapy: Dendritic cells or myeloid-derived suppressor cells?", ONCOIMMUNOLOGY, vol. 2, no. 10, October 2013 (2013-10-01), pages e26148, XP055141483, ISSN: 2162-4011, DOI: 10.4161/onci.26148 *
LAURENJ BAYNE ET AL: "Tumor-Derived Granulocyte-Macrophage Colony-Stimulating Factor Regulates Myeloid Inflammation and T Cell Immunity in Pancreatic Cancer", CANCER CELL, CELL PRESS, US, vol. 21, no. 6, 9 April 2012 (2012-04-09), pages 822 - 835, XP028490389, ISSN: 1535-6108, [retrieved on 20120425], DOI: 10.1016/J.CCR.2012.04.025 *
MELISSA G LECHNER ET AL: "Functional characterization of human Cd33+ And Cd11b+ myeloid-derived suppressor cell subsets induced from peripheral blood mononuclear cells co-cultured with a diverse set of human tumor cell lines", JOURNAL OF TRANSLATIONAL MEDICINE, BIOMED CENTRAL, LONDON, GB, vol. 9, no. 1, 9 June 2011 (2011-06-09), pages 90, XP021103820, ISSN: 1479-5876, DOI: 10.1186/1479-5876-9-90 *
SERAFINI PAOLO ET AL: "High-dose granulocyte-macrophage colony-stimulating factor-producing vaccines impair the immune response through the recruitment of myeloid suppressor cells", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 64, no. 17, 1 September 2004 (2004-09-01), pages 6337 - 6343, XP002553177, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-04-0757 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111197029A (zh) * 2020-01-09 2020-05-26 广东省第二人民医院（广东省卫生应急医院） 一种尿酸钠诱导产生髓系抑制性细胞的方法

Similar Documents

Publication Publication Date Title
KR102575976B1 (ko) 자연살해 세포의 증식방법
JP2023016809A (ja) 腫瘍浸潤リンパ球拡大培養用の改変人工抗原提示細胞
US11413307B2 (en) Induced dendritic cells and uses thereof
JP2019523001A (ja) 改変されたナチュラルキラー細胞を生成する方法および使用方法
EP3475413B1 (fr) Lymphocytes t génétiquement modifiés
JP2020506713A (ja) 多能性幹細胞から模倣自然免疫細胞を生成する方法及びキット
CN107075484A (zh) 从多能性干细胞诱导细胞免疫治疗用t细胞的方法
US9670459B2 (en) Production method for cell populations
Kitchen et al. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice
WO2019106163A1 (fr) Reprogrammation de cellules immunitaires primaires génétiquement modifiées
EA010434B1 (ru) Способ получения цитотоксических лимфоцитов
Vimond et al. Genetic engineering of human and mouse CD4+ and CD8+ Tregs using lentiviral vectors encoding chimeric antigen receptors
JP5911810B2 (ja) 制御性ｔ細胞の製造方法
WO2015165869A1 (fr) Méthode de génération de cellules suppressives
JP5485139B2 (ja) 遺伝子導入細胞の製造方法
CN113913458A (zh) 非病毒方法制备稳定高表达嵌合受体的nk细胞
RU2803178C1 (ru) Рекомбинантная клеточная линия TMDK562-15, проявляющая способность к активации и пролиферации ЕК клеток человека
US20220228164A1 (en) Engineered antigen presenting cells
Valizadeh et al. MARCH-I: A negative regulator of dendritic cell maturation
Panchal Developing novel therapies for X-linked lymphoproliferative disease type 1
CN116410931A (zh) 一种通用型car-t细胞的制备技术及其应用
CN116410930A (zh) 一种通用型car-t细胞的制备技术及其应用
CN116410932A (zh) 一种通用型car-t细胞的制备方法及其应用
Santos Towards GMP Production of γðTCR Engineered T Cells
Friedman Augmenting the effector phase of adoptive T cell immunotherapy of cancer

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: 15717923

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: 15717923

Country of ref document: EP

Kind code of ref document: A1