WO2024256477A1 - Procédé de préparation de cellules dendritiques plasmacytoïdes - Google Patents

Procédé de préparation de cellules dendritiques plasmacytoïdes Download PDF

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WO2024256477A1
WO2024256477A1 PCT/EP2024/066260 EP2024066260W WO2024256477A1 WO 2024256477 A1 WO2024256477 A1 WO 2024256477A1 EP 2024066260 W EP2024066260 W EP 2024066260W WO 2024256477 A1 WO2024256477 A1 WO 2024256477A1
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pdcs
cells
population
medium
concentration
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Mike COLLODORO
Claude DEDRY
Joel Plumas
Sébastien MICHEL
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Pdc Line Pharma Sa
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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/56—Physical preservation processes for animal cells or human cells
    • C12N5/562—Temperature processes, e.g. following predefined temperature changes over time
    • 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
    • C12N5/56—Physical preservation processes for animal cells or human cells
    • C12N5/568—Physical preservation processes for animal cells or human cells using electromagnetic fields or radiation; using acoustic waves or corpuscular radiation
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to methods for preparing populations of plasmacytoid dendritic cells (PDCs) and their use in treating and/or preventing cancer.
  • PDCs plasmacytoid dendritic cells
  • DCs Dendritic cells
  • APCs antigen-presenting cells
  • DCs comprise four major subsets, including conventional DCs (CDCs), which further divide into type 1 (CDC1) and type 2 (CDC2); plasmacytoid DCs (PDCs); and monocyte- derived DCs (MoDC). All vary on their capacity for antigen presentation, migration, and cytokine secretion (Collin M et al, Immunology, 2018; Perez C., Nature Communications, 2019).
  • PDCs particularly, hold promise as effective anti-cancer vaccine constituents given its immuno stimulatory and tumoricidal potential (Aspord C. et al, PLoS ONE, 2010, Chaperot J.I., 2006).
  • the inventors have developed a therapeutic vaccine based on an allogeneic plasmacytoid dendritic cell line (PDC*line), which efficiently activates the CD8+ T-cell response in the context of melanoma (Aspord C. et al, JID 2019) and lung cancer (Hannani D. et al, IJMS 2023).
  • PDC*line allogeneic plasmacytoid dendritic cell line
  • a first-in-human phase Eli study with melanoma patients (clinical trial number NCT01863108) has been reported (Charles J.
  • the standard manufacturing pipeline is well known to the one skilled in the art, and includes the setting of specific cell lines as cancer- vaccine constituents.
  • This invention thus relates to an in vitro method of producing a therapeutically effective amount of a population of plasmacytoid dendritic cells (PDCs) comprising loading said PDCs with at least one antigen or fragment thereof in a first medium; loading the PDCs obtained in the step prior with at least one cryoprotectant agent in a second medium, irradiating the PDCs obtained then, and, optionally, freezing them.
  • PDCs plasmacytoid dendritic cells
  • one antigen or fragment thereof is a peptide. In some embodiments, at least one antigen or fragment thereof is a cancer or tumor antigen, or fragment thereof.
  • the first medium is a cell culture medium. In some embodiments, the first medium is a hematomasine, hematomasine, hematomasine, hematomasine, hematomasine, hematomasine, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematomasine, hematomasine, hematomasine, hematomasine, hematomasine, hematomasine, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma
  • the second medium comprises or consists of at least one cryoprotectant agent.
  • the second medium comprises DMSO as the cryoprotectant agent.
  • the second medium is CryostorTM CS5.
  • the PDCs used at the loading step are selected from the group comprising GEN2.2 PDCs, GEN3 PDCs, and any other genetically or not modified PDCs.
  • the concentration of PDCs at the loading step is from O.lxlO 6 cells/mL to lOOxlO 6 cells/mL.
  • the concentration of PDCs at loading step is from 10xl0 6 cells/mL to lOOxlO 6 cells/mL. In some embodiments, the concentration of PDCs at loading step is from 10xl0 6 cells/mL to 40xl0 6 cells/mL. In some embodiments, the concentration of PDCs at loading step is about 20xl0 6 cells/mL.
  • the concentration of at least one antigen or fragment thereof at loading step is from 0.1 pM to 1 mM.
  • the concentration of at least one antigen or fragment thereof at loading step is from 100 pM to 500 pM. In some embodiments, the concentration of at least one antigen or fragment thereof at loading step is about 200 p M.
  • PDCs are loaded with the at least one antigen or fragment thereof from 0.5 hour to 12 hours. In some embodiments, PDCs are loaded with at least one antigen or fragment thereof from 1 hour to 3 hours.
  • PDCs obtained after loading with at least one antigen or fragment thereof are contacted with at least one cryoprotectant medium from 10 mn to 5 hours.
  • PDCs in contact (or incubated) with a cryoprotectant medium are irradiated for about 10 sec to 45 mn. In some embodiments, the irradiation step is performed when the PDC cells are incubated in the cryoprotectant medium.
  • PDC cells are irradiated at a dose from 1 Gy to 120 Gy. In some embodiments, PDC cells are irradiated at a dose from 30 Gy to 90 Gy. In some embodiments, PDC cells are irradiated at a dose about 60 Gy.
  • the PDC cells are frozen at a temperature from -200°C to -50°C. In some embodiments, after irradiation the PDC cells are frozen preferably from -200°C to -80°C. In some embodiments, after irradiation the PDC cells are frozen at about -196°C.
  • a population of PDCs is obtained with the method of the invention.
  • the method of inducing and amplifying antigen specific effectors in vitro comprises obtaining purified-peripheral blood mononuclear cells (PBMCs) from an individual, said PBMCs sharing at least one major histocompatibility complex (MHC) with the population of PDCs, loading the population of PDCs according to the method herein described with said PBMCs, and culturing said loaded-PDCs with said PBMCs; and thereby inducing and amplifying antigen specific effectors in the culture in vitro.
  • MHC major histocompatibility complex
  • the method of inducing and amplifying antigen specific effectors in vitro comprises obtaining purified CD8+ T-cells, loading the population of loaded-PDCs according to the method herein described with said CD8+ T-cells, and culturing said loaded-PDCs with said CD8+ T-cells; and thereby inducing and amplifying antigen specific effectors in the culture in vitro.
  • the population of loaded-PDCs obtained according to the method of the invention it to use for treatment and/or prevention of a disease in a subject in need thereof.
  • the disease selected from the group comprising or consisting of cancer, genetic diseases and microbial infection, preferably the disease is cancer.
  • Antigen refers to a part or region of the/an antibody, which comprises fewer amino acid residues than the whole antibody.
  • An “antigen-binding fragment” binds antigen and/or competes with the whole antibody from which it was derived for antigen binding.
  • Antibody antigen-binding fragments encompasses, without any limitation, single chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)’2, Fd, defucosylated antibodies, diabodies, triabodies and tetrabodies.
  • At least one includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 103,104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015 or more.
  • cryoprotectant reagent refers to a substance used to prevent eventual cryopreservation drawbacks in either cellular or supracellular specimens. These (re)agents are generally able to prevent unwanted oxidation, as well as damaging effects of the cryopreservation process on membrane physical and chemical properties, and other cellular injuries. They act by reducing ice formation at any temperature via increasing the solutes concentration present in the system, thereby shielding membrane lipids and thus other cellular components, including cellular proteins and nucleic acids. CPAs can be divided as intracellular and extracellular cryoprotectants. The earlier are small and nonionic molecules that penetrate the cell membrane, such sulfoxides, alcohols, amides and the like. The latter corresponds to small molecules organized into polymers added to the cryoprotectant solution/medium, such as sugars.
  • cryoprotectant medium refers to a (liquid) medium formulated for freeze and storing a variety of primary cells, as well as cell lines, and shows improved cell viability and recovery after thawing. It contains at least one cryoprotective agent in its composition, and may include several other elements, such as hormones, growth factors, buffers, serum, or proteins, the presence or concentration of which is defined according to the use and cell type intended.
  • “Individual”, or “subject”, refers to an animal, preferably a mammal, more preferably a human.
  • the individual is a man.
  • the individual is a woman.
  • an individual may be a “patient”, i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of cancer.
  • the individual is an adult (for example a subject above the age of 18).
  • the individual is a child (for example a subject below the age of 18).
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, thereby forming polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labelling component.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • Polypeptide refers to both short chains, commonly referred to as “peptides”, oligopeptides or oligomers, and to longer chains, generally referred to as “proteins”. Hence, within the scope of the present invention, the terms “peptide”, “polypeptide” and “protein” are used interchangeably. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Preventing cancer is intended to mean keeping from happening at least one adverse effect or symptom of a cancer.
  • Treating cancer or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) cancer.
  • Those in need of treatment include those already with cancer as well as those prone to have cancer or those in whom cancer is to be prevented.
  • “Therapeutically effective amount” is intended to refer to the level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of cancer; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of cancer; (3) bringing about ameliorations of the symptoms of cancer; (4) reducing the severity or incidence of cancer; or (5) preventing cancer formation.
  • a therapeutically effective amount is administered prior to the onset of cancer formation, for a prophylactic or preventive action.
  • the cancer is lung cancer.
  • Vaccine refers to any preparation comprising substance or group of substances meant to cause the immune system of a subject to respond to a given antigen, in particular to a tumor.
  • Prophylactic vaccines are used to prevent a subject from ever having a particular disease, in particular cancer, or to only have a mild case of the disease, in particular cancer.
  • Therapeutic vaccines are intended to treat specific diseases in a subject, in particular cancer.
  • Anti-cancer vaccines comprise a tumor-antigen or tumor-antigens, eliciting an immune response directed against the tumor cells.
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions (preferably conservative), additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Variants should retain one or more of the biological activities of the reference polypeptide.
  • This invention relates to a method for preparing a population of plasmacytoid dendritic cells (PDCs) which comprises the following steps: a) loading said PDCs with at least one antigen or fragment thereof in a first medium; b) incubating the PDCs obtained in step (a) with at least one cryoprotectant agent in a second medium; c) irradiating the PDCs obtained in step (b); and optionally d) freezing the PDCs obtained in step (c).
  • PDCs plasmacytoid dendritic cells
  • the method according to the invention is an in vitro method.
  • Plasmacytoid dendritic cells are a subtype of DCs which play an essential role in the immune response through their ability secrete type I interferon. They also have strong antigen presentation capabilities.
  • the PDCs are a PDC line of cells.
  • the PDCs are cultured in suitable conditions. Methods to culture cells in vitro are well known in the art and are routine practice.
  • the PDCs are maintained in a controlled atmosphere, i.e., controlled temperature, pressure and composition, typically at 37°C, 1 atm, 5 % CO2.
  • the PDCs are cultured in a substrate or vessel.
  • the substrate is plastic.
  • the plastic substrate is a vessel or container suitable for cell culture, typically a plastic flask or plastic dish.
  • the PDCs are plated onto a substrate which allows adherence of cells thereto.
  • the plastic substrate is treated with at least one agent that promotes cell adherence, survival and/or proliferation; illustratively, in some embodiments, the plastic substrate is treated with poly-D-lysine, gelatin and/or collagen.
  • the agent forms a layer or a matrix at the surface of the plastic substrate.
  • the PDCs are maintained in a substrate which prevents adherence of cells thereto.
  • the PDCs are cultured in suspension.
  • the PDCs are cultured in a culture medium sustaining their further proliferation, generally a liquid culture medium, which may contain serum or may be serum-free.
  • the PDCs are growth in bioreactor in suspension in synthetic medium.
  • Culture media are known in the art.
  • the term “culture medium” or “cell culture medium” or “medium” refers to an aqueous liquid or gelatinous substance comprising nutrients which can be used for maintenance or growth of cells.
  • Cell culture media can contain serum or be serum-free.
  • the culture medium comprises a basal medium formulation as known in the art and include, without limitation, Eagle's Minimum Essential Medium (MEM), OPTI-MEM, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium 199, Waymouth's MB 752/1 X-VIVO-15, or Williams Medium E, and modifications and/or combinations thereof.
  • Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for culturing the PDC.
  • the culture medium used to cultivate the population of PDC is X-VIVO-15, or variants thereof.
  • these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g., glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodium acetate), etc. It will also be apparent that many media are available as low glucose formulations with or without sodium pyruvate.
  • physiological buffers e.g., HEPES, bicarbonate
  • nucleotides e.g., nucleosides and/or nucleic acid bases
  • ribose e.g., deoxyribose
  • amino acids e.g., amino
  • basal media are supplemented with one or more further components selected from the group comprising or consisting of transferrin, selenium salts, amino acids, sugar, and combinations thereof.
  • these components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution.
  • HBSS Hanks' Balanced Salt Solution
  • Further antioxidant supplements may be added, e.g., P- mercaptoethanol.
  • basal media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution.
  • the culture medium is further supplemented with antibiotic and/or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamycin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, zeocin, and combinations thereof.
  • the culture medium is further supplemented with fungicide compounds.
  • the culture medium is further supplemented with hormones selected from the group comprising or consisting of D-aldosterone, diethylstilbestrol (DES), dexamethasone, estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin/human growth hormone (HGH), thyrotropin, thyroxine, L- thyronine, epithelial growth factor (EGF), human recombinant epidermal growth factor, and combinations thereof.
  • the final concentration of hormones may range from 0.01 to 1000 ng/mL, preferably from 0.1 to 100 ng/mL, more preferably from 1 to 10 ng/mL.
  • the culture medium is further supplemented with lipids and lipid carriers selected from the group comprising or consisting of cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, and the like, and combinations thereof.
  • Albumin can similarly be used in fatty-acid free formulations.
  • the culture medium is further supplemented with mammalian plasma or serum.
  • the concentration of the plasma or serum in the culture medium is from 0.01% to 20%, preferably from 0.1% to 10%, more preferably from 1% to 5%. In some embodiments, the concentration of the plasma or serum in the culture medium is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.
  • serum is obtained from a sample of whole blood by first allowing clotting to take place in the sample and subsequently separating the so formed clot and cellular components of the blood sample from the liquid component (serum) by an appropriate technique, typically by centrifugation.
  • An inert catalyst e.g., glass beads or powder
  • serum can be prepared using serum- separating vessels (SST), which contain the inert catalyst to mammals. Plasma or serum often contains cellular factors and components that are necessary for viability and expansion.
  • the plasma or serum for use in the culture medium as described herein may include human plasma or serum; or plasma or serum derived from non-human animals, preferably non-human mammals, such as, e.g., non-human primates e.g., lemurs, monkeys, apes), fetal or adult bovine, horse, porcine, lamb, goat, dog, rabbit, mouse or rat serum or plasma, etc.
  • the culture medium is further supplemented with a serum replacement or analogue.
  • the cultured PDCs may be counted in order to facilitate re-seeding of the PDCs at a desired density.
  • the PDCs are cultured by re-seeding them at a density of between 10° and 10 5 cells/cm 2 , and at a splitting ratio between about 1/100 and 1/2.
  • the splitting ratio denotes the fraction of the passaged PDCs that is seeded into an empty (typically a new) culture vessel of the same surface area and/or volume as the vessel from which the PDCs were obtained.
  • the type of culture vessel, as well as of surface and/or volume allowing PDCs proliferation into the culture vessel and the cell culture media can be the same as initially used and as described hereinabove, or may be different.
  • the population of PDCs is devoid of contamination by bacteria, fungi, protozoa, archaea, or other microbial species.
  • the population of PDCs is characterized in that it is substantially pure.
  • the substantially pure population of PDCs comprises less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or less of other contaminating cell type(s).
  • the substantially pure population of PDCs comprises 0% of other contaminating cell type(s). In some embodiments, the substantially pure cell population comprises no more than one cell type.
  • the population of PDCs is of clinical grade.
  • the PDCs express major histocompatibility complex (MHC) antigen specific to humans.
  • MHC major histocompatibility complex
  • the PDCs express MHC of class I such as human leukocyte antigen (HLA).
  • HLA comprises or consists of HLA-A, HLA-B, and/or HLA-C subtypes.
  • HLA is a heterodimer and is composed of a heavy a chain and smaller P chain.
  • the a chain is encoded by a variant HLA-A gene, generating HLA-A haplotype group.
  • HLA haplotypes are selected from the list comprising or consisting of HLA-A*02:01, HLA-A*02:02, HLA- A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:ll.
  • the PDCs express HLA-A. In an embodiment, PDC express HLA-A2 subtype. In an embodiment, PDCs express HLA-A*02:01 haplotype. [0072] In some embodiments, the PDCs are not genetically modified. In some embodiments, the PDC are genetically modified.
  • genetically modified means that the genome of the PDCs comprises at least one modification consisting of a mutation and/or an insertion of one or more nucleic acid sequences.
  • Methods to genetically modify a cell include, non-imitatively, CRISPR-Cas-based techniques, Zinc finger nucleases- based techniques, TALEN and the like.
  • the PDCs are constitutively modified, i.e., the modification of the genome is transmitted to daughter cells, typically the PDCs are transduced by methods known in the art such as lenti viral vectors.
  • the PDCs are transiently modified, typically the PDCs are transfected.
  • the PDCs are genetically modified to express at least one protein of interest.
  • “at least one” means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20.
  • “genetically modified to express at least one protein of interest” means that the PDCs expresses 1.2-fold, 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold or more the at least one protein of interest, or transcript encoding thereof, compared to an unmodified PDCs. In some embodiments, the PDCs overexpress the at least one protein of interest.
  • the at least one protein of interest is a secreted protein or a surface protein.
  • the at least one protein of interest is a secreted protein.
  • the secreted protein is a cytokine.
  • the cytokine is an interleukin.
  • the interleukin is selected from the group comprising or consisting of IL-2, IL-7, IL- 12 and IL- 15. In some embodiments, the interleukin is IL- 12 or IL- 15.
  • the at least one protein of interest is IL-2.
  • IL-2 typically has the sequence as disclosed in Uniprot Ref. P60568, incorporated herein by reference.
  • IL-2 is interchangeably referred to as IL2, and interleukin 2.
  • the at least one protein of interest is IL- 12.
  • IL- 12 typically comprises 2 subunits, namely IL12A (or p35) and IL12B (or p40), typically having the sequence of Uniprot Ref. P29459 and Uniprot Ref. P29460, respectively, incorporated herein by reference.
  • IL- 12 is interchangeably referred to as IL12, interleukin 12, and p70.
  • the at least one protein of interest is IL- 15.
  • IL- 15 typically has the sequence as disclosed in Uniprot Ref. P40933, incorporated herein by reference. IL- 15 is interchangeably referred to as IL15, and interleukin 15.
  • the at least one protein of interest is IL-7.
  • IL-7 typically has the sequence as disclosed in Uniprot Ref. P13232, incorporated herein by reference. IL-7 is interchangeably referred to as IL7, and interleukin 7.
  • the at least one protein of interest is a surface protein.
  • the surface protein is of the B7 family of proteins. In some embodiments, the surface protein from the B7 family of proteins is CD80 or CD86.
  • the at least one protein of interest is CD80.
  • CD80 typically has the sequence as disclosed in Uniprot Ref. P33681 , incorporated herein by reference.
  • CD80 is interchangeably referred to as CD80, B7, B7- 1, B7.1, BB1, CD28LG, CD28LG1, and LAB7.
  • the at least one protein of interest is CD86.
  • CD86 typically has the sequence as disclosed in Uniprot Ref. P42081 , incorporated herein by reference. CD86 is interchangeably referred to as CD86, B7-2, B7.2, B70, CD28LG2, and LAB72.
  • the at least one protein of interest is mutated. In some embodiments, the at least one protein of interest comprises at least one amino acid mutation compared to the wild type protein of interest.
  • amino acid mutation comprises substitutions, deletions, insertions, inversions, and combination thereof.
  • the mutations of the at least one protein of interest do not produce any adverse effect on the PDC, i.e., the mutations of the at least one protein of interest do not alter the viability, and/or proliferation capacities of the PDC. In some embodiments, the mutations of the at least one protein of interest are not oncogenic.
  • the mutations of the at least one protein of interest increase at least 1.5-fold, 2-fold, 3-fold or more the efficiency of the PDCs as antigen presenting cells. In some embodiments, the mutations of the at least one protein of interest increase at least 1.5-fold, 2-fold, 3-fold or more the viability and/or circulating time of the PDCs.
  • the PDCs are genetically modified to reduce (knockdown) or abolish (knock-out) the expression of at least one protein or gene encoding thereof.
  • “reduce” means a decrease of expression from 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and at most 99%, of the basal expression of the at least one protein or gene encoding thereof.
  • “reduce” means a 100% decrease of expression of the basal expression of the at least one protein or gene encoding thereof.
  • PDCs are capable of activating naive T cells and memory T cells into activated, antigen- specific T cells, either by direct priming, or by cross priming (z.e., presentation of exogenously-derived antigens).
  • This second mechanism allows PDCs to be contacted, loaded, loaded, charged or the like, with any given antigen of interest or combination of antigens of interest.
  • the loaded PDCs activate T cells specific for the antigen of interest or combination of antigens of interest.
  • the population of PDCs is loaded with at least one cancer antigen or fragment thereof (step a). In some embodiments, the population of PDCs is loaded with at least two, three or four cancer antigens or fragments thereof. In some embodiments, the population of PDCs is loaded with more than four cancer antigens or fragments thereof, such as for example five, six, seven, ten or twelve cancer antigens or fragments thereof.
  • the terms “contacted”, “pulsed” and loaded” are intended to mean that the population of PDCs is incubated with one or several cancer antigens and/or fragment and/or variant thereof, resulting in the “loading” of the one or several cancer antigens and/or fragment and/or variant thereof on the population of PDCs.
  • the terms “contacted”, “loaded”, or “incubated” may be used interchangeably. This incubation itself is hereby interchangeably referred to as the “loading step”.
  • the population of PDCs is substantially devoid of free cancer antigen and/or fragment thereof. In some embodiments, the population of PDCs is substantially devoid of free cancer antigen and/or fragment thereof after a washing step.
  • the at least one cancer antigen and/or fragment thereof is not expressed in healthy tissues. In some embodiments, the at least one cancer antigen and/or fragment and/or variant thereof have low expression levels in healthy tissues.
  • the at least one cancer antigen and/or fragment thereof is a molecule selected from the group comprising or consisting of peptides, polypeptides, proteins, polysaccharides, lipids, nucleic acids, and combinations thereof.
  • the at least one cancer antigen and/or fragment thereof is a molecule selected from the group comprising or consisting of peptides, polypeptides, and proteins.
  • the at least one antigen or fragment thereof is a peptide.
  • the at least one antigen or fragment thereof is a cancer or tumor antigen, or fragment thereof. In some embodiments, the at least one antigen or fragment thereof is a cancer antigen, or fragment thereof. In some embodiments, the at least one antigen or fragment thereof is a tumor antigen, or fragment thereof.
  • the at least one antigen or fragment thereof is a cancer peptide, or fragment thereof. In some embodiments, the at least one antigen or fragment thereof is a tumor peptide, or fragment thereof.
  • the at least one antigen or fragment thereof is a microbial antigen. In some embodiments, the at least one antigen or fragment thereof is a bacterial, viral, fungal or archaeal antigen, or fragment thereof.
  • the at least one antigen or fragment thereof is a microbial peptide, or fragment thereof. In some embodiments, the at least one antigen or fragment thereof is a bacterial, viral, fungal or archaeal peptide, or fragment thereof.
  • the at least one antigen or fragment thereof is a molecule, preferably a peptide or protein, involved in a genetic disease.
  • the at least one antigen or fragment thereof is selected from the group comprising or consisting or 5T4 (TPBG), CAMEL (CTAG2), CEA, Cyclin DI (CCND1), EPCAM, GLULD1 (LGSN), GP100, HER2 (ERBB2), hTERT, IDO, LY-6K, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A9, MAGE- A10, MAGE-C2, MULTI-MAGE, MELAN-A/MART-1, MUC-1, MUC5AC, NY-BR- 1, NY-ESO-1, p53, PD-L1, PRAME, PSA, RAGE-1, SURVIVIN (BIRC5), TRP-2, Tyrosinase, Her-2/Neu, NY-ESO1, PD-L1, LY-6K, variants and fragments thereof.
  • the at least one cancer antigen and/or fragment thereof is selected from the group comprising or consisting or consisting or 5T4 (
  • the peptide comprises natural and non-natural amino acids. In a preferred embodiment, the peptide comprises or consists of natural amino acids. In another embodiment, the peptide comprises or consists of non-natural amino acids.
  • the peptide comprises at least one post-translational modification on at least one amino acid residues.
  • the peptide is glycosylated, glycated, phosphorylated, biotinylated, oxidated, nitrated, nitrosylated, acylated, alkylated, acetylated, methylated, lipidated, ubiquitinated, carboxylated, pegylated, or combinations thereof.
  • the at least one cancer antigen and/or fragment thereof is a polysaccharide. In another embodiment, the at least one cancer antigen and/or fragment and/or variant thereof is a lipid. In another embodiment, the at least one cancer antigen and/or fragment and/or variant thereof is a nucleic acid molecule.
  • the at least one cancer antigen and/or fragment thereof elicits an immune response, i.e., the at least one cancer antigen and/or fragment and/or variant thereof is immunogenic. In a preferred embodiment, the at least one cancer antigen and/or fragment thereof elicits an adaptative immune response. In a more preferred embodiment, the at least one cancer antigen and/or fragment thereof elicits T cell- mediated immune response.
  • the step a) is performed in a first medium.
  • the first medium is a cell culture medium, preferably a hematopoietic cell culture medium, more preferably a X-VIVO-15 culture medium.
  • the incubation is performed at a temperature from 32°C to 42°C, preferably at 37°C. [0120] In some embodiments, the incubation is performed for a duration from 30 minutes to 24 hours.
  • the incubation is performed for a duration from 30 minutes to 16 hours, from 30 minutes to 8 hours, from 30 minutes to 4 hours, or from 30 minutes to 2 hours.
  • the incubation is performed for a duration from 1 hour to 16 hours, from 2 hours to 8 hours, or from 2 hours to 4 hours.
  • the incubation is performed for a duration from 1 hour to 8 hours, from 1 hour to 4 hours, or from 1 hours to 3 hours.
  • the incubation is performed for about 2 hours. In a preferred embodiment, the incubation is performed for about 3 hours.
  • the cellular concentration of the population of PDCs used for the loading step is from O.lxlO 6 cells/mL to lOOxlO 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step is from O.lxlO 6 cells/mL to 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is about lxl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step is from 10xl0 6 cells/mL to lOOxlO 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is from 10xl0 6 cells/mL to 40xl0 6 cells/mL, preferably from 20xl0 6 cells/mL to 40xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is about 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step is IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 or IxlO 7 cells/mL.
  • the PDCs cellular concentration is comprised between IxlO 4 and lxl0 8 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step is at least 5xl0 6 cells/mL, at least 6xl0 6 cells/mL, at least 7xl0 6 cells/mL, at least 8xl0 6 cells/mL, at least 9xl0 6 cells/mL, at least 10xl0 6 cells/mL, at least l lxlO 6 cells/mL at least 12xl0 6 cells/mL at least 13xl0 6 cells/mL at least 14xl0 6 cells/mL at least 15xl0 6 cells/mL at least 16xl0 6 cells/mL at least 17xl0 6 cells/mL at least 18xl0 6 cells/mL at least 19xl0 6 cells/mL or at least 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step is at least 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is at least 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the incubation step is from O.lxlO 6 cells/mL to lOOxlO 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the incubation step is from O.lxlO 6 cells/mL to 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is about lxl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the incubation step is from 10xl0 6 cells/mL to lOOxlO 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is from 10xl0 6 cells/mL to 40xl0 6 cells/mL, preferably from 20x10 6 cells/mL to 40x10 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is about 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the incubation step is IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 or IxlO 7 cells/mL.
  • the PDCs cellular concentration is comprised between IxlO 4 and lxl0 8 cells/mL.
  • the cellular concentration of the population of PDCs used for the incubation step is at least 5xl0 6 cells/mL, at least 6xl0 6 cells/mL, at least 7xl0 6 cells/mL, at least 8xl0 6 cells/mL, at least 9xl0 6 cells/mL, at least 10xl0 6 cells/mL, at least l lxlO 6 cells/mL at least 12xl0 6 cells/mL at least 13xl0 6 cells/mL at least 14xl0 6 cells/mL at least 15xl0 6 cells/mL at least 16xl0 6 cells/mL at least 17xl0 6 cells/mL at least 18xl0 6 cells/mL at least 19xl0 6 cells/mL or at least 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the incubation step is at least 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is at least 20x10 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the irradiation step is from O.lxlO 6 cells/mL to lOOxlO 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the irradiation step is from O.lxlO 6 cells/mL to 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is about lxl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the irradiation step is from lOx 10 6 cells/mL to lOOx 10 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to 40xl0 6 cells/mL, preferably from 20x10 6 cells/mL to 40x10 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is about 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the irradiation step is IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 or IxlO 7 cells/mL.
  • the PDCs cellular concentration is comprised between IxlO 4 and lxl0 8 cells/mL.
  • the cellular concentration of the population of PDCs used for the irradiation step is at least 5xl0 6 cells/mL, at least 6xl0 6 cells/mL, at least 7xl0 6 cells/mL, at least 8xl0 6 cells/mL, at least 9xl0 6 cells/mL, at least 10xl0 6 cells/mL, at least l lxlO 6 cells/mL at least 12xl0 6 cells/mL at least 13xl0 6 cells/mL at least 14xl0 6 cells/mL at least 15xl0 6 cells/mL at least 16xl0 6 cells/mL at least 17xl0 6 cells/mL at least 18xl0 6 cells/mL at least 19xl0 6 cells/mL or at least 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the irradiation step is at least 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is at least 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from O.lxlO 6 cells/mL to lOOxlO 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from O.lxlO 6 cells/mL to 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is about lxl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to 100xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to 40xl0 6 cells/mL, preferably from 20xl0 6 cells/mL to 40xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is about 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 or IxlO 7 cells/mL.
  • the PDCs cellular concentration is comprised between IxlO 4 and IxlO 8 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is at least 5xl0 6 cells/mL, at least 6xl0 6 cells/mL, at least 7xl0 6 cells/mL, at least 8xl0 6 cells/mL, at least 9xl0 6 cells/mL, at least 10xl0 6 cells/mL, at least l lxlO 6 cells/mL at least 12xl0 6 cells/mL at least 13xl0 6 cells/mL at least 14xl0 6 cells/mL at least 15xl0 6 cells/mL at least 16xl0 6 cells/mL at least 17xl0 6 cells/mL at least 18xl0 6 cells/mL at least 19xl0 6 cells/mL or at least 20x10 6 cells/mL
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is at least 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is at least 20x10 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from O.lxlO 6 cells/mL to lOOxlO 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from O.lxlO 6 cells/mL to 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is about lxl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to lOOxlO 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to 40xl0 6 cells/mL, preferably from 20xl0 6 cells/mL to 40xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is about 20xl0 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 or IxlO 7 cells/mL.
  • the PDCs cellular concentration is comprised between IxlO 4 and IxlO 8 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is at least 5xl0 6 cells/mL, at least 6xl0 6 cells/mL, at least 7xl0 6 cells/mL, at least 8xl0 6 cells/mL, at least 9xl0 6 cells/mL, at least 10xl0 6 cells/mL, at least l lxlO 6 cells/mL at least 12xl0 6 cells/mL at least 13xl0 6 cells/mL at least 14xl0 6 cells/mL at least 15xl0 6 cells/mL at least 16xl0 6 cells/mL at least 17xl0 6 cells/mL at least 18xl0 6 cells/mL at least 19xl0 6 cells/mL or at least 20x10 6 cells/mL.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is at least 10xl0 6 cells/mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is at least 20x10 6 cells/mL.
  • the concentration of the cancer antigen or fragment thereof used for the loading step is from 0.1 pM to 1 mM.
  • the concentration of the cancer antigen or fragment thereof used for the loading step i.e., step a)) is from 0.1 pM to 100 pM. In some embodiments, the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 5 pM to 50 pM. In some embodiments, the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.
  • the concentration of the cancer antigen or fragment thereof used for the loading step is from 100 pM to 1 mM. In a preferred embodiment, the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 100 pM to 500 pM, preferably from 200 pM to 500 pM. In a more preferred embodiment, the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 200 pM.
  • the concentration of the cancer antigen or fragment thereof used for the loading step (z.e., step a)) is at least 50 pM, at least 75 pM, at least 100 pM, at least 125 pM, at least 150 pM, at least 175 pM, or at least 200 pM. In some embodiments, the concentration of the cancer antigen or fragment thereof used for the loading step (z.e., step a)) is at least 100 pM. In some embodiments, the concentration of the cancer antigen or fragment thereof used for the loading step (z.e., step a)) is at least 200 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from 0. IxlO 6 cells/mL to lOOxlO 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 0.1 pM to 1 mM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from O.lxlO 6 cells/mL to 10xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step i.e., step a)) is from 5 pM to 50 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is about IxlO 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to 100xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 100 pM to 1 mM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to 40xl0 6 cells/mL, preferably from 20xl0 6 cells/mL to 40xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 100 pM to 500 pM, preferably from 200 pM to 500 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is about 20xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is at least 5xl0 6 cells/mL, at least 6xl0 6 cells/mL, at least 7xl0 6 cells/mL, at least 8xl0 6 cells/mL, at least 9xl0 6 cells/mL, at least 10xl0 6 cells/mL, at least l lxlO 6 cells/mL at least 12xl0 6 cells/mL at least 13xl0 6 cells/mL at least 14xl0 6 cells/mL at least 15xl0 6 cells/mL at least 16xl0 6 cells/mL at least 17xl0 6 cells/mL at least 18xl0 6 cells/mL at least 19xl0 6 cells/mL or at least 20x10 6 cells/mL
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is at least 10xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is at least 100 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and/or irradiation step (i.e., step c)) is at least 20xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is at least 200 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from O.lxlO 6 cells/mL to lOOxlO 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 0.1 pM to 1 mM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from O.lxlO 6 cells/mL to 10xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 5 pM to 50 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is about lxl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to lOOxlO 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step i.e., step a)) is from 100 M to 1 mM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is from 10xl0 6 cells/mL to 40x10 6 cells/mL, preferably from 20x10 6 cells/mL to 40x10 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is from 100 M to 500 pM, preferably from 200 pM to 500 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is about 20x10 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is at least 5xl0 6 cells/mL, at least 6xl0 6 cells/mL, at least 7xl0 6 cells/mL, at least 8xl0 6 cells/mL, at least 9xl0 6 cells/mL, at least 10xl0 6 cells/mL, at least l lxlO 6 cells/mL at least 12xl0 6 cells/mL at least 13xl0 6 cells/mL at least 14xl0 6 cells/mL at least 15xl0 6 cells/mL at least 16xl0 6 cells/mL at least 17xl0 6 cells/mL at least 18xl0 6 cells/mL at least 19xl0 6 cells/mL or at least 20x10 6 cells/mL; and
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is at least 10xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is at least 100 pM.
  • the cellular concentration of the population of PDCs used for the loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is at least 20xl0 6 cells/mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is at least 200 pM.
  • the PDCs are washed after step a).
  • the PDCs are contacted (or incubated) with at least one cryoprotective agent i.e., a cryoprotectant) after loading them with at least one antigen or fragment thereof (step b).
  • this step b) is performed in a second medium.
  • the term “second medium” may also be referred to as “cryoprotective medium” or “preservation medium”.
  • cryoprotective media include, but are not limited to, CryoStor R/ TM CS2, CryoStor R/ TM CS5, CryoStor R/ TM CS10, mFreSRTM, MesenCultTM- ACF, STEMdiffTM, NutriFrccz R/ I M D10, and the like.
  • Cryoprotective agents i.e., a cryoprotectants
  • cryoprotective agents are part of (cryo)preservation media and are known in the art.
  • cryoprotective agents are DMSO, glycerol, propylene glycol, ethylene glycol, sucrose, threalose, serum, and the like.
  • the at least one cryoprotectant agent is selected from the group comprising or consisting of DMSO, glycerol, propylene glycol, ethylene glycol, sucrose, threalose, and serum, preferably DMSO.
  • the second medium is selected from the group comprising or consisting MesenCultTM
  • the cryprotective medium comprises from 1% to 10% of DMSO, preferably from 2% to 8% of DMSO, more preferably from 3% to 7% of DMSO. In a preferred embodiment, the cryoprotective medium comprises about 5% of DMSO. In a more preferred embodiment, the cryoprotective medium is CryoStor R/I M CS5.
  • the PDCs are contacted with at least one cryoprotective agent for a period of time from 10 min to 5 hours. In some embodiments, the PDCs are contacted with at least one cryoprotective agent for a period of time from 20 min to 4 hours, from 30 min to 3 hours, or from 45 min to 2 hours. In some embodiments, the PDCs are contacted with at least one cryoprotective agent for a period of time from 10 min to 4 hours, from 10 min to 3 hours, from 10 min to 2 hours, or from 10 min to 1 hour.
  • the PDCs are not washed after step b). In some embodiments, the PDCs remain in the same medium for the next step.
  • the PDCs are irradiated after contacting (or incubating) them with at least one cryoprotective agent (step c).
  • the PDCs are irradiated when they still are in contact (or incubated) with the cryoprotective agent.
  • the irradiation step c) is performed in a cryoprotective medium.
  • the irradiation step c) is performed in the same cryoprotective medium as for step b).
  • the PDCs are irradiated in the presence of a cryoprotectant.
  • the cryoprotectant is present and/or active during irradiation step.
  • the PDCs are irradiated at a dose from 1 to 120 Gy. In some embodiments, the PDC are irradiated at a dose from 10 to 100 Gy. In some embodiments, the PDCs are irradiated at a dose from 30 to 100 Gy. In some embodiments, the PDCs are irradiated at a dose of about 60 Gy.
  • the PDCs are irradiated for a period of time from 10 sec to 45 min. In some embodiments, the PDCs are irradiated for a period of time from 30 sec to 30 min, from 45 sec to 20 min, or from 1 min to 15 min. In some embodiments, the PDCs are irradiated for a period of time from 10 sec to 30 min, from 10 sec to 20 min, or from 10 sec to 10 min. In some embodiments, the PDCs are irradiated for a period of time from 1 sec to 45 min, from 5 min to 45 min, from 10 min to 45 min, or from 45 min to 45 min.
  • the PDCs are not washed after step c). In some embodiments, the PDCs remain in the same medium for the next step.
  • the PDCs are frozen after irradiation (step d). In some embodiments, the PDCs are frozen when they still are in contact (or incubated) with the cryoprotective agent. In other words, in some embodiments, the freezing step d) is performed in a cryoprotective medium. In some embodiments, the freezing step d) is performed in the same cryoprotective medium as for steps b) and c).
  • the PDCs are cryopreserved for storage.
  • PDCs are frozen at a temperature from -20°C to -200°C, preferably from - 80°C to -200°C.
  • the PDCs are frozen at about -196°C, typically cells are stored in liquid nitrogen.
  • PDCs are “flash-frozen” in liquid nitrogen prior to storage.
  • the method of the present invention comprises the following steps: a) loading the PDCs with at least one antigen or fragment thereof in a first culture medium; b) incubating the PDCs obtained in step (a) with at least one cryoprotectant agent in a second medium; c) irradiating the PDCs obtained in step (b); and d) optionally, freezing the PDCs obtained in step (c), wherein the cellular concentration of the population of PDCs used at steps a), b) and c) is at least 10xl0 6 cells/mL; and/or the concentration of the cancer antigen or fragment thereof used at step a) is at least 100 p M.
  • the method of the present invention comprises the following steps: a) loading the PDCs with at least one antigen or fragment thereof in a first hematopoietic cell culture medium; b) incubating the PDCs obtained in step (a) with at least one cryoprotectant agent in a second medium; c) irradiating the PDCs obtained in step (b); and d) optionally, freezing the PDCs obtained in step (c), wherein the cellular concentration of the population of PDCs used at steps a), b) and c) is at least 10xl0 6 cells/mL; and/or the concentration of the cancer antigen or fragment thereof used at step a) is at least 100 pM, and wherein the at least one cryoprotectant agent is DMSO.
  • the method of the present invention comprises the following steps: a) loading the PDCs with at least one antigen or fragment thereof in a first hematopoietic cell culture medium; b) incubating the PDCs obtained in step (a) with at least one cryoprotectant agent in a second medium; c) irradiating the PDCs obtained in step (b); and d) optionally, freezing the PDCs obtained in step (c), wherein the cellular concentration of the population of PDCs used at steps a), b) and c) is at least 20x10 6 cells/mL, wherein the concentration of the cancer antigen or fragment thereof used at step a) is at least 200 pM, and wherein the at least one cryoprotectant agent is DMSO.
  • Another object of the present invention relates to a population of PDCs obtained with the method as described hereinabove, i.e., a population of PDCs obtained with a method comprising the following steps: a) loading the PDCs with at least one antigen or fragment thereof in a first medium, preferably a culture medium, more preferably a hematopoietic cell culture medium; b) incubating the PDCs obtained in step (a) with at least one cryoprotectant agent, preferably DMSO, in a second medium; c) irradiating the PDCs obtained in step (b); and d) optionally, freezing the PDCs obtained in step (c), preferably wherein the cellular concentration of the population of PDCs used at steps a), b) and/or c) is at least 10xl0 6 cells/mL, preferably at least 20xl0 6 cells/mL, and/or the concentration of the cancer antigen or fragment thereof used at step a) is at least 100
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, PDE4B, AMIG02, FASLG, ATP1B1, SH2D2A, VGF, C15orf48, LGR4, SLX1B, SLC22A1, CHST2, GLYATL2, COL11A2, WNT1, LINC01619, FBN1, NGFR, TCF7, MT1X, CCL22, GJA1, OSR2, ELOVL7, NDRG4, SMOX, FP236383,3, FSD1L, PHLDB1, PRR5L, CCR7, ROR1, FN1, SAPCD2, SPHK1, SOCS3, DNM3, KHDRBS3, GRAMD1B, MAML3, MDK, TFPI2, NR4A1, FGFR1, EDIL3, NREP, STAT5A, TET3, MOB3B
  • altered expression means a difference of 5%, 10%, 20%, 50% or more.
  • “at least two” encompasses 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.
  • “at least three” encompasses 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.
  • “at least four” encompasses 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.
  • “at least five” encompasses 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, PDE4B, AMIGO2, FASLG, ATP1B1, SH2D2A, VGF, C15orf48, LGR4, SLX1B, SLC22A1, CHST2, GLYATL2, COL11A2, WNT1, LINC01619, FBN1, NGFR, TCF7, MT1X, CCL22, GJA1, OSR2, ELOVL7, NDRG4, SMOX, FP236383,3, FSD1L, PHLDB1, PRR5L, CCR7, ROR1, FN1, SAPCD2, SPHK1, SOCS3, DNM3, KHDRBS3, GRAMD1B, MAML3, MDK, TFPI2, NR4A1, FGFR1, EDIL3, NREP, STAT5A, TET3, MOB3B
  • “increased expression” means an increase of expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, or more, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant); or an increase of expression of at least 1.01-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of 0MA1, REPIN1-AS1, LRRN2, SAMHD1, FES, MACROD1, AL139125,1, SLC25A23, EML3, SEPTIN1, AK1, GOLGA8A, PDK2, SCARNA2, LINC-ROR, AL138756,1, TCL1B, AL022238,3, KIR3DX1, IL16, MPEG1, ST6GALNAC4, NRSN2-AS1, DNAH1, TREML2, AC073957,3, IRF8, SLC32A1, EPHB4, CHCHD10, TTC39A, APOBEC2, RNVU1-31, INPP4A, TM7SF2, AC109326,l, ZNF296, SIGLEC6, SLC46A3, PDLIM2, MLLT11, ARHGAP27, ARHGEF17, OPLAH
  • “decreased expression” means a decrease of expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, or more, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant); or a decrease of expression of at least 1.01-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, compared to PDCs irradiated in a medium that does not
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A, IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • cryoprotectant i.e., irradi
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, the regulation of cell viability and activation, and/or identity markers, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, FCGR2A, IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, and FCGR2A; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, and FCGR2A, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAME3, STAT5A, ITPR1, NCK2, PTGS2, ITPR3, DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAME3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAME3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of TCF7, IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of TCF7; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of TCF7, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased surface expression of CD83 compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • “increased surface expression” means an increase of surface expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, or more, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant); or an increase of surface expression of at least 1.01-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, compared to PDCs irradiated in a medium that does
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A, IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • cryoprotectant i.e., irradiated in the absence of at least one cryoprotectant
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to PDCs ir
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, PDE4B, AMIG02, FASLG, ATP1B1, SH2D2A, VGF, C15orf48, LGR4, SLX1B, SLC22A1, CHST2, GLYATL2, COL11A2, WNT1, LINC01619, FBN1, NGFR, TCF7, MT1X, CCL22, GJA1, OSR2, ELOVL7, NDRG4, SMOX, FP236383,3, FSD1L, PHLDB1, PRR5L, CCR7, ROR1, FN1, SAPCD2, SPHK1, SOCS3, DNM3, KHDRBS3, GRAMD1B, MAML3, MDK, TFPI2, NR4A1, FGFR1, EDIL3, NREP, STAT5A, TET3, MOB3B
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, PDE4B, AMIGO2, FASLG, ATP1B1, SH2D2A, VGF, C15orf48, LGR4, SLX1B, SLC22A1, CHST2, GLYATL2, COL11A2, WNT1, LINC01619, FBN1, NGFR, TCF7, MT1X, CCL22, GJA1, OSR2, ELOVL7, NDRG4, SMOX, FP236383,3, FSD1L, PHLDB1, PRR5L, CCR7, ROR1, FN1, SAPCD2, SPHK1, SOCS3, DNM3, KHDRBS3, GRAMD1B, MAML3, MDK, TFPI2, NR4A1, FGFR1, EDIL3, NREP, STAT5A, TET3, MOB3B
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of 0MA1, REPIN1-AS1, LRRN2, SAMHD1, FES, MACROD1, AL139125,1, SLC25A23, EML3, SEPTIN1, AK1, GOLGA8A, PDK2, SCARNA2, LINC-ROR, AL138756,1, TCL1B, AL022238,3, KIR3DX1, IL16, MPEG1, ST6GALNAC4, NRSN2-AS1, DNAH1, TREML2, AC073957,3, IRF8, SLC32A1, EPHB4, CHCHD10, TTC39A, APOBEC2, RNVU1-31, INPP4A, TM7SF2, AC109326,l, ZNF296, SIGLEC6, SLC46A3, PDLIM2, MLLT11, ARHGAP27, ARHGEF17, OPLAH
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A, IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, the regulation of cell viability and activation, and/or identity markers, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, FCGR2A, IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, and FCGR2A, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, ITPR3, DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of TCF7, IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than lOxlO 6 cells/mL.
  • the obtained PDCs have increased expression of TCF7, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, FCGR2A, IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, and FCGR2A; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IL16, MMP25, MMP11, CD40, CLEC2D, FCER1 A, NCR3LG1, and PDGFB, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, and FCGR2A, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, ITPR3, DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of TCF7, IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of TCF7 ; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of TCF7, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased surface expression of CD83 compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL. (i.e., irradiated in the absence of at least one cryoprotectant).
  • “increased surface expression” means an increase of surface expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, or more, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than lOxlO 6 cells/mL.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A, IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl0 6 cells/mL.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGEEC6, and TCE1B; and have increased surface expression of CD83, compared to PDCs ir
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, the regulation of cell viability and activation, and/or identity markers; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCE1, FASEG, NGFR, CCE22, CCR7, IE24, MMP13, TGFBR3, FCGR2A, IE16, MMP25, MMP11, CD40, CEEC2D, FCER1A, NCR3EG1, and PDGFB; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, and FCGR2A; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of TCF7, IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the obtained PDCs have increased expression of TCF7; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.
  • the present invention further relates to a population of modified PDCs having altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, PDE4B, AMIG02, FASLG, ATP1B1, SH2D2A, VGF, C15orf48, LGR4, SLX1B, SLC22A1, CHST2, GLYATL2, COL11A2, WNT1, LINC01619, FBN1, NGFR, TCF7, MT1X, CCL22, GJA1, OSR2, ELOVL7, NDRG4, SMOX, FP236383,3, FSD1L, PHLDB1, PRR5L, CCR7, ROR1, FN1, SAPCD2, SPHK1, SOCS3, DNM3, KHDRBS3, GRAMD1B, MAML3, MDK, TFPI2, NR4A1, FGFR1, EDIL3, NREP, STAT5A, TET
  • the present invention further relates to a population of modified PDCs having increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCE1, FASEG, NGFR, TCF7, CCE22, CCR7, SOCS3, MAME3, STAT5A, ITPR1, IE24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and/or having decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IE16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CEEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGEC5, IRF8, NCR3EG1, PDGFB, SERPINE2, SIGEEC6, and TCE1B, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased surface expression of CD83 compared to unmodified PDCs.
  • the present invention further relates to a population of modified PDCs having increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and/or having decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to un
  • the PDCs from the population of modified PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of CCL1, FASLG, NGFR, TCF7, CCL22, CCR7, SOCS3, MAML3, STAT5A, ITPR1, IL24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A; and have increased surface expression of CD83, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene selected from the group consisting of IL16, IGHA1, TRAV22, MMP25, MMP11, IGHA2, CD40, CLEC2D, DUSP7, EBF1, FCER1A, IFIT2, IGFBP4, IGLC5, IRF8, NCR3LG1, PDGFB, SERPINE2, SIGLEC6, and TCL1B; and have increased surface expression of CD83, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, the regulation of cell viability and activation, and/or identity markers, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, FCGR2A, IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCE1, FASEG, NGFR, CCE22, CCR7, IE24, MMP13, TGFBR3, and FCGR2A; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IE16, MMP25, MMP11, CD40, CEEC2D, FCER1A, NCR3EG1, and PDGFB, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of CCE1, FASEG, NGFR, CCE22, CCR7, IE24, MMP13, TGFBR3, and FCGR2A, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the interaction with other immune cells, preferably selected from the group consisting of IL16, MMP25, MMP11, CD40, CLEC2D, FCER1A, NCR3LG1, and PDGFB compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, ITPR3, DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in the regulation of cell viability and activation, preferably selected from the group consisting of SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have altered expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of TCF7, IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased expression of TCF7; and/or have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have increased expression of TCF7, compared to unmodified PDCs.
  • the PDCs from the population of modified PDCs have decreased expression of at least one, at least two, at least three, at least four, at least five, or at least six gene involved in identity markers, preferably selected from the group consisting of IGHA1, TRAV22, IGHA2, IGLC5, IRF8, SIGLEC6, and TCL1B, compared to unmodified PDCs.
  • the present invention thus also relates to a method for treating and/or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the population of PDCs obtained with the method of preparation of the invention, or of the population of modified PDCs as described herein.
  • the disease is selected from the group comprising or consisting of cancer, genetic diseases, and microbial infections.
  • the disease is cancer.
  • the disease is a microbial infection.
  • the disease is a genetic disease.
  • the present invention thus also relates to the population of PDCs obtained with the method of preparation of the invention, or the population of modified PDCs as described herein, for use for the manufacture of a medicament for the treatment and/or the prevention of a disease in a subject in need thereof.
  • the present invention thus also relates to the use of PDCs obtained with the method of preparation of the invention, or the population of modified PDCs as described herein, for the manufacture of a medicament for the treatment and/or the prevention of a disease in a subject in need thereof.
  • the disease is selected from the group comprising or consisting of cancer, genetic diseases, and microbial infections.
  • the disease is cancer.
  • the disease is a microbial infection.
  • the disease is a genetic disease.
  • the present invention also relates to a method of in vitro inducing and amplifying antigen specific effectors in PDCs, comprising the steps of: a. obtaining cells selected from the group comprising or consisting of purified-peripheral blood mononuclear cells (PBMCs) and CD8+ T-cells from an individual, said cells sharing at least one major histocompatibility complex (MHC) with the population of PDCs obtained with the method of preparation the invention, or the population of modified PDCs as described herein; b. loading the population of PDCs obtained with the method of preparation the invention, or the population of modified PDCs as described herein, with said cells, thereby obtaining loaded PDCs; and c. culturing said loaded PDCs with said cells; and thereby inducing and amplifying antigen specific effectors in the culture in vitro of loaded PDCs.
  • PBMCs purified-peripheral blood mononuclear cells
  • MHC major histocompatibility complex
  • the individual is a human individual.
  • Another object of the present invention relates to a population of PDCs obtained with the method of inducing and amplifying antigen specific effectors as described hereinabove.
  • Another object of the present invention relates to a kit for implementing the method as described herein, and instructions for use.
  • Figures 1A-1B are a combination of graphs showing the peptide presentation efficiency towards Jurkat Luciferase tools. For each peptide, the data of three replicates are plotted.
  • Fig. 1A shows the proportion of luminescence signal obtained in positive control condition (CD3/CD28 Dynabeads ratio 10:1) in 3 batches of tTCR Jurkat cells specific for HLA-A2/Survivin complexes.
  • Fig. IB shows the proportion of luminescence signal obtained in positive control condition (CD3/CD28 Dynabeads ratio 10:1) in 3 batches of tTCR Jurkat cells specific for HLA-A2/Melan-A complexes.
  • Figures 2A-2C are a combination of graphs and plots of the apoptosis evaluation (Annexin V + 7-AAD) for all runs.
  • Fig. 2A shows a plot of the percentage of apoptotic cells across the 24h incubation time in all batches and runs.
  • Fig. 2B shows Annexin V77- AAD staining of PDC*line cells for batches 1, 2 and 3 in all runs at T6h following irradiation. Early apoptotic cells are identified as the cells positive for Annexin V and negative for 7-AAD.
  • Fig. 2C shows a dot plot of apoptosis assays for the second run for all batches.
  • Figure 3A-3C are a combination of dot plots showing an overview of differentially expressed genes in cells at 4 hours post irradiation when comparing batch 1 and batch 2 (Fig. 3A), batch 1 and batch 3 (Fig. 3B), and batch 2 and batch 3 (Fig. 3C).
  • Figure 4 is a histogram showing CD83 expression 4 hours post irradiation in cells from batch 1, batch 2, and batch 3.
  • Example 1 Effect of cell irradiation in CS5 on the PDC*line cells features
  • Table 1 List of the selected tumor antigens.
  • PDC*line cells were a) loaded at IxlO 6 viable cells/ml in X-Vivo 15 with peptide at 10 pM; b) washed, resuspended in CryostorTM CS5 and c) frozen at 50xl0 6 total cells/ml in CryostorTM CS5. The cells were not irradiated.
  • the APA-Luciferase assay aims to evaluate the sample cells like sample potency (peptide loading efficiency, peptide presentation ability to TCR specific CD8+ T cells). It corresponds to a co-culture of the sample cells with tTCR specific Jurkat cells.
  • sample potency peptide loading efficiency, peptide presentation ability to TCR specific CD8+ T cells.
  • the more efficient the peptide presentation is (between the MHC complex of loaded PDC*line cells and the TCR complex of Jurkat TCR specific Luciferase QC tool), the more intense the Jurkat TCR specific Luciferase QC tool activation is.
  • Jurkat TCR specific Luciferase QC tool are transduced with APl/NFKb Nanoluciferase (reporter gene). Thus, the production of Nanoluciferase is directly related to cell activation.
  • Drug Product like sample potency evaluation relies on a quantification of the produced Nanoluciferase (luminescence measurement) by tTCR specific Jurkat cells
  • tTCR specific Jurkat cells correspond to TCR KO (T cell Receptor - knock out) Jurkat cells transduced with CD8aa, APl/NFKb Nanoluciferase (reporter gene and its promotor) and an engineered TCRaP (T cell receptor seqeunces) specific to one antigen. They are mimicking CD8+ cytotoxic T cells and are able to be activated through the presentation by loaded PDC*line cells of the corresponding MHC/peptide complex. tTCR specific Jurkat cells activation triggers Nanoluciferase production (reporter gene).
  • PBMC Peripheral blood mononuclear cells
  • CD8+ T cells were obtained from PBMCs through a negative selection using Miltenyi CD8+ T cell isolation kit (reference: 130-096-495). Three distinct AAA-CD8 were performed, one for each manufacturing run (Run 1, Run 2 and Run 3).
  • PBMCs The same source of PBMCs was used for each AAA-CD8 and corresponded to 21HDCYTAPH#04 sample (LabCollector ID: 2792, PBMCs collected from a Healthy Donor Cytapheresis on 03-DEC-2021, lOOxlO 6 viable HLA-A*0201 PBMCs frozen in 1.8 ml of 25% FBS - 10% DMSO freezing medium).
  • the antigen presentation assay corresponds to the co-culture of tTCR specific
  • each cell suspension was homogenized. For each cell suspension, two independent cell suspension samples of 25 pl were collected. Each sample was mixed with 25 pl of AOPI, and the resulting mix suspensions was used to load two counting chambers of one counting slide. Total cell concentration, viable cell concentration and cell viability rate of each counting chamber are evaluated though a cell counting on Nexcelom Automated Cell Counter using the “Primary cells / Cell line”. After cell counting, cell suspensions are centrifuged at 400 g for 10 minutes at room temperature. Once the centrifugation is complete, supernatants are discarded and each cell pellet is resuspended in pre-heated (37°C) complete RPMI at 1.5xl0 6 viable cells/ml.
  • Dynabead vial was vortexed for 30 seconds before collecting 1 ml (40x 10 6 beads) of bead suspension and transferring them in a 5-ml tube. Then, 2 ml of Purification Buffer were added to the beads, and the bead suspension was homogenized.
  • the Dynabead tube was uncapped and placed under a magnetic field for 1 minute using an EasySep Cell Separation Magnet. The supernatant was discarded by reversing the assembly EasySep Cell Separation Magnet / Dynabead tube. The assembly EasySep Cell Separation Magnet / Dynabead tube was replaced upside and the Dynabead tube removed from the magnet. Dynabeads were resuspended in complete RPMI at 20xl0 6 beads / ml, and the suspension diluted with complete RPMI to generate 6 distinct Dynabead dilutions as shown on Table 3.
  • Each Jurkat TCR specific Luciferase QC tool was co-cultured in 12 distinct conditions by filling one well with 100 pl of Jurkat tool cell suspension (150.000 viable cells) and 100 pl of: medium (complete RPMI), one of each tested batch (Batch 1, Batch 2, Batch 3 and Batch 4 - 150.000 total cells per well), or one of each Dynabeads diluted suspension (from 150.000 to 1.500.000 beads per well). Each co-culture condition was performed in duplicates. Co-culture plates were incubated at 37 °C - 5% CO2 for 24h.
  • the Luciferase assay corresponds to an indirect measurement of cell activation. It relies on a quantification, through a luminescence measurement, of the produced Nanoluciferase by tTCR specific Jurkat cells.
  • the Luciferase assay was performed as follows: after 24h of incubation at 37°C - 5% CO2, the co-culture plates were placed at room temperature. Each co-culture well was homogenized and a sample of 50 pl transferred to a new well of a 96 well plate - U bottom. Co-culture duplicate samples were pooled and mixed in the same well. Lor each duplicate sample mix, 50 pl were transferred to a new well of a 96 well white plate - Elat bottom.
  • IX Nano-Gio Luciferase Substrate was prepared by diluting 50X Nano-Gio Luciferase Substrate in Nano-Gio Luciferase Buffer (39 pl of 50X Nano-Gio Luciferase Substrate in 1911 pl of Nano-Gio Luciferase Buffer). 50 pl of IX Nano-Gio Luciferase Substrate were added to each well of the 96 well white plate - Elat bottom. The plate was placed on an orbital shaker for 3 minutes at 500 rpm.
  • the luminescence measurement was performed on TEC AN plate reader with the following parameters: shaking of 5 seconds with 1 mm of amplitude and on a linear mode, luminescence reading with an automatic attenuation, 1000 ms of integration time and 0 ms of settle time.
  • APA-Luciferase measurements were performed on the TECAN plate reader. For each tested condition, a luminescence signal was measured and recorded. This signal related to the amount of Nanoluciferase enzyme secreted by the tTCR specific Jurkat cells and triggered by Jurkat tool cells activation. For each Jurkat TCR specific Luciferase QC tool, two values were computed for each tested batch: a) The cell activation proportion was computed as the luminescence value ratio of the tested batch condition over the Dynabeads 10:1 beads/cell ratio condition. Indeed, at 10:1 beads/cell ratio, Jurkat tool cells reach a saturation threshold (and a Nanoluciferase production saturation threshold), and b) the batch equivalent Dynabeads/cell ratio.
  • the assay Annexin V-FITC 7-AAD kit from Beckman Coulter was used with the following modifications: i) Starting from frozen cells, the cryotubes were thawed in water bath (37°C), then 1 ml of cell suspension was transferred in 50 ml tube and washed with 9 ml of X-vivo 15, centrifuged at 400g for 10 minutes, followed by cells’ resuspension in 10 ml of X-vivo 15 and counting cells with Nexcelom. This was followed by another centrifuge cycle at 400g for 10 minutes, cell resuspension in X-vivo 15 at IM/ml and dispatching of 2M cells in a tube for each time points. ii) Incubation at 37 °C Oh, 4h, 6h and 24h
  • the Annexin-V kits contain a viability dye to evaluate membrane integrity as well as annexin-V to assess membrane asymmetry.
  • a viability dye to evaluate membrane integrity as well as annexin-V to assess membrane asymmetry.
  • three different cell populations may be observed (i) live cells that do not stain either with annexin V or with the vital dye, (ii) necrotic or late apoptotic cells that stain with both annexin V and the vital dye, and (iii) early apoptotic cells that stain with annexin V only.
  • RNA-later stabilization solution (ref: AM7020) and stored at -20 °C until extraction
  • RNA extraction samples are thawed and centrifuge 15 minutes at 8000g to remove RNA Later before resuspension in RLT buffer from RNAeasy Mini Kit
  • RNA is eluted in 30ul of Rnase-free water and samples are stored at -80°C until use
  • RNA quantification and integrity assessment is performed using Agilent RNA Nano kit
  • Cells are centrifuge (400g for 7 minutes) and washed 2x with PBS before resuspension at IM cells/ml in PBS + 2% FBS 200.000 cells are collected for cell surface staining and incubated 30 minutes at 4°C with 5 ul of PE-conjugated CD83 antibody (clone HB15a, ref: IM2218U) Cells are centrifuge at 400g for 7 minutes before resuspension in 300 ul of BD Lysis buffer and analysis using a Attune NxT Flow Cytometer
  • Figure 3A-3C shows an overview of deferentially expressed genes at 4 hours post irradiation based on the ratio of mean transcript read counts in batch 2 compared to batch 1 (Fig. 3A), batch 3 compared to batch 1 (Fig. 3B), and batch 3 compared to batch 2 (Fig. 3C); and expressed as Log2FC.
  • Y axis represents gene expression fold change (log2FC); 1 dot represents 1 gene; black dots means significantly differentially expressed genes (adjp ⁇ 0,05).
  • Table 4 Differentially expressed genes between batches 3 and 1 (4 hours post irradiation).
  • Table 5 Differentially expressed genes of interest between batches 3 and 1 (4 hours post irradiation).
  • CD83 Cluster of Differentiation 83 (CD83) cell surface expression was evaluated by flow cytometry and is shown on Figure 4 (expressed as Median Fluorescence Intensity). CD83 is significantly more expressed in batch 3 compared to batches 1 and 2, 4h post irradiation. [0295] Taken together, the results show that the PDC*line cells treated in the conditions of batch 3 show improved survival and functionality.

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Abstract

La présente invention concerne un procédé de préparation de populations in vitro de cellules dendritiques plasmacytoïdes (PDC) et leur utilisation dans le traitement et/ou la prévention du cancer.
PCT/EP2024/066260 2023-06-13 2024-06-12 Procédé de préparation de cellules dendritiques plasmacytoïdes Ceased WO2024256477A1 (fr)

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Citations (3)

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WO2023275219A1 (fr) * 2021-07-02 2023-01-05 Aarhus Universitet Production et multiplication conformes aux normes bpf de cellules dendritiques plasmacytoïdes à partir de cellules souches et progénitrices hématopoïétiques

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US20120020998A1 (en) * 2008-05-16 2012-01-26 Etablissement Francais Du Sang Plasmacytoid dendritic cell line used in active or adoptive cell therapy
WO2023275219A1 (fr) * 2021-07-02 2023-01-05 Aarhus Universitet Production et multiplication conformes aux normes bpf de cellules dendritiques plasmacytoïdes à partir de cellules souches et progénitrices hématopoïétiques

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CHARLES JULIE ET AL: "An innovative plasmacytoid dendritic cell line-based cancer vaccine primes and expands antitumor T-cells in melanoma patients in a first-in-human trial", ONCOIMMUNOLOGY, vol. 9, no. 1, 1 January 2020 (2020-01-01), XP093083278, DOI: 10.1080/2162402X.2020.1738812 *
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