Method for preparing plasmacytoid dendritic cells

Abstract

The present invention relates to a method for preparing in vitro populations of plasmacytoid dendritic cells (PDCs) and their use in treating and / or preventing cancer.

Description

METHOD FOR PREPARING PLASMACYTOID DENDRITIC CELLSFIELD OF INVENTION

[0001] The present invention relates to methods for preparing populations of plasmacytoid dendritic cells (PDCs) and their use in treating and / or preventing cancer.BACKGROUND OF INVENTION

[0002] Dendritic cells (DCs), first described by Ralph Steinman and Zanvil Cohn in 1973, play a pivotal role in mediating immune response and inducing adaptive immune response by various cellular mechanisms. They are recognized as the most potent antigen- presenting cells (APCs), and their superior ability to acquire, process, and present antigens to T cells made them a major immunotherapeutic target in the last two decades. 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).

[0003] 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). In fact, 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). A first-in-human phase Eli study with melanoma patients (clinical trial number NCT01863108) has been reported (Charles J. et al, Oncolmmunology 2020). The product, named “Genius Vac-Mel4”, was safe and well tolerated with positive signs of biological and clinical activity. Another example pertains the development of a PDC*line-based vaccine for lung cancer, specifically non- small-cell lung cancer (NSCLC) patients, and showed encouraging results, especially incombination with immune checkpoint inhibitor (ICI)-based (ICIs)-based therapies (Hannani D. et al, Int. Journal of Molecular Sciences, 2023). The safety allied with few adverse effects while inducing favorable immune responses lets a foreseeable growth in this field in the future. Nevertheless, there are barriers to overcome before PDC-based vaccines reach both the desired effectiveness for a treatment and its widespread clinical practice. In view of broad patient population application, one major challenge / roadblock is at the manufacturing level. Indeed, the lengthy process as carried out nowadays and yields of mature dendritic cells achieved impact the final amount and overall costs.

[0004] 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. The inventors surprisingly show a new method which improves manufacturing time and yield, both of critical relevance for wider applicability and scale-up of immunotherapies.SUMMARY

[0005] 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.

[0006] In some embodiments, 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.

[0007] In some embodiments, least one antigen or fragment thereof is selected from the group comprising 5T4 (TPBG), CAMEL (CTAG2), CEA, Cyclin DI (CCND1), EPCAM, GLULD1 (LGSN), GP100, HER2 (ERBB2), hTERT, IDO, LY-6K, MAGE- Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A10, MAGE-C2, MULTIMAGE, 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.

[0008] In some embodiments, the first medium is a cell culture medium. In some embodiments, the first medium is a hematopoietic cell culture medium. In some embodiments, the first medium is X-Vivo 15 culture medium.

[0009] In some embodiments, the second medium comprises or consists of at least one cryoprotectant agent. In some embodiments, the second medium comprises DMSO as the cryoprotectant agent. In some embodiments, the second medium is Cryostor™ CS5.

[0010] In some embodiments, 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.

[0011] In some embodiments, the concentration of PDCs at the loading step is from O.lxlO6cells / mL to lOOxlO6cells / mL.

[0012] In some embodiments, the concentration of PDCs at loading step is from 10xl06cells / mL to lOOxlO6cells / mL. In some embodiments, the concentration of PDCs at loading step is from 10xl06cells / mL to 40xl06cells / mL. In some embodiments, the concentration of PDCs at loading step is about 20xl06cells / mL.

[0013] In some embodiments, the concentration of at least one antigen or fragment thereof at loading step is from 0.1 pM to 1 mM.

[0014] In some embodiments, 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.

[0015] In some embodiments, 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.

[0016] In some embodiments, 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.

[0017] In some embodiments, 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.

[0018] In some embodiments, 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.

[0019] In some embodiments, after irradiation 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.

[0020] In some embodiments, a population of PDCs is obtained with the method of the invention.

[0021] In some embodiments, 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.

[0022] In some embodiments, 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.

[0023] In some embodiments, 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. In some embodiments, the disease selected from the group comprising orconsisting of cancer, genetic diseases and microbial infection, preferably the disease is cancer.DEFINITIONS

[0024] In the present invention, the following terms have the following meanings:

[0025] “About” when preceding a figure, means plus or less 10% of the value of said figure.

[0026] “And / Or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0027] “Antigen”, “peptide antigen”, “antigen-binding fragment” 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.

[0028] “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.

[0029] “Cryoprotectant reagent”, “cryoprotectant agent” (CPA), or “cryoprotectant” refer 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 thelike. The latter corresponds to small molecules organized into polymers added to the cryoprotectant solution / medium, such as sugars.

[0030] “Cryoprotectant medium”, “cryoprotective medium”, “cryopreservation solution” or “cryoprotectant solution” 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.

[0031] “Comprising”, “comprises” and “comprised of’ are used herein are synonymous with “including”, “includes” or “containing", “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. These terms also encompass “consisting of’.

[0032] “Individual”, or “subject”, refers to an animal, preferably a mammal, more preferably a human. In one embodiment, the individual is a man. In another embodiment, the individual is a woman. In one embodiment, 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. In one embodiment, the individual is an adult (for example a subject above the age of 18). In another embodiment, the individual is a child (for example a subject below the age of 18).

[0033] “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. As used herein the term “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.

[0034] “Preventing cancer” is intended to mean keeping from happening at least one adverse effect or symptom of a cancer.

[0035] “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.

[0036] “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. In one embodiment, a therapeutically effective amount is administered prior to the onset of cancer formation, for a prophylactic or preventive action. In certain embodiments, the cancer is lung cancer.

[0037] “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.

[0038] “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, referencepolynucleotide. 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.DETAILED DESCRIPTION

[0039] 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).

[0040] In some embodiments, the method according to the invention is an in vitro method.

[0041] Plasmacytoid dendritic cells (PDCs) are a subtype of DCs which play an essential role in the immune response through their ability secrete type I interferon. They also havestrong antigen presentation capabilities. In some embodiments, the PDCs are a PDC line of cells.

[0042] Methods to isolate, cultivate, and use PDCs have been published in Chaperot et al., Journal of immunology vol. 176,1 (2006): 248-55; Lui et al., PloS one vol. 4,9 e7111. 22 Sep. 2009; Aspord et al., The Journal of investigative dermatology vol. 132,10 (2012): 2395-2406; and Hannani et al., International Journal of Molecular Sciences, 2023, 24(3), 1897, incorporated herein by reference.

[0043] In some embodiments, the PDCs are cultured in suitable conditions. Methods to culture cells in vitro are well known in the art and are routine practice.

[0044] In some embodiments, the PDCs are maintained in a controlled atmosphere, i.e., controlled temperature, pressure and composition, typically at 37°C, 1 atm, 5 % CO2.

[0045] In some embodiments, the PDCs are cultured in a substrate or vessel. In some embodiments, the substrate is plastic. In some embodiments, the plastic substrate is a vessel or container suitable for cell culture, typically a plastic flask or plastic dish.

[0046] In one embodiment, the PDCs are plated onto a substrate which allows adherence of cells thereto. In certain embodiments, 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. In some embodiments, the agent forms a layer or a matrix at the surface of the plastic substrate.

[0047] In another embodiment, the PDCs are maintained in a substrate which prevents adherence of cells thereto. In some embodiments, the PDCs are cultured in suspension.

[0048] In some embodiments, 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. In some embodiments, the PDCs are growth in bioreactor in suspension in synthetic medium.

[0049] 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. These media, that can be further supplemented with appropriate mixture of organic or inorganic compounds may, besides providing nutrients and / or growth promoters, also promote the growth / adherence or the elimination / detachment of specific cell types.

[0050] In some embodiments, 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.

[0051] In a preferred embodiment, the culture medium used to cultivate the population of PDC is X-VIVO-15, or variants thereof.

[0052] By means of illustration and not limitation, 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.

[0053] In some embodiments, basal media are supplemented with one or more further components selected from the group comprising or consisting of transferrin, seleniumsalts, 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. Further antioxidant supplements may be added, e.g., P- mercaptoethanol. While many 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.

[0054] In some embodiments, 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. In some embodiments, the culture medium is further supplemented with fungicide compounds.

[0055] In some embodiments, 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.

[0056] In some embodiments, 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.

[0057] In some embodiments, the culture medium is further supplemented with mammalian plasma or serum. In some embodiments, 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%.

[0058] The term "serum", as conventionally defined, 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, can facilitate clotting. Advantageously, 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.

[0059] In some embodiments, 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. In some embodiments, the culture medium is further supplemented with a serum replacement or analogue.

[0060] As appreciated by those skilled in the art, the cultured PDCs may be counted in order to facilitate re-seeding of the PDCs at a desired density.

[0061] In some embodiments, the PDCs are cultured by re-seeding them at a density of between 10° and 105cells / cm2, and at a splitting ratio between about 1 / 100 and 1 / 2. As used herein, 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.

[0062] 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.

[0063] In some embodiments, the population of PDCs is devoid of contamination by bacteria, fungi, protozoa, archaea, or other microbial species.

[0064] In some embodiments, the population of PDCs is characterized in that it is substantially pure.

[0065] In some embodiments, 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).

[0066] In some embodiments, 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.

[0067] In a preferred embodiment, the population of PDCs is of clinical grade.

[0068] In some embodiments, the PDCs express major histocompatibility complex (MHC) antigen specific to humans.

[0069] In some embodiments, the PDCs express MHC of class I such as human leukocyte antigen (HLA). In some embodiments, HLA comprises or consists of HLA-A, HLA-B, and / or HLA-C subtypes.

[0070] In some embodiments, HLA is a heterodimer and is composed of a heavy a chain and smaller P chain. In some embodiments, the a chain is encoded by a variant HLA-A gene, generating HLA-A haplotype group. In some embodiments, 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.

[0071] In a certain embodiment, 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.

[0073] As used herein, “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 are known in the art and include, non-imitatively, CRISPR-Cas-based techniques, Zinc finger nucleases- based techniques, TALEN and the like. In a preferred embodiment, 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. In another, less preferred embodiment, the PDCs are transiently modified, typically the PDCs are transfected.

[0074] In some embodiments, the PDCs are genetically modified to express at least one protein of interest.

[0075] As used herein, “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.

[0076] As used herein, “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.

[0077] In some embodiments, the at least one protein of interest is a secreted protein or a surface protein.

[0078] In some embodiments, the at least one protein of interest is a secreted protein. In some embodiments, the secreted protein is a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, 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.

[0079] In some embodiments, the at least one protein of interest is IL-2.

[0080] In humans, 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.

[0081] In some embodiments, the at least one protein of interest is IL- 12.

[0082] In humans, 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.

[0083] In some embodiments, the at least one protein of interest is IL- 15.

[0084] In humans, 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.

[0085] In some embodiments, the at least one protein of interest is IL-7.

[0086] In humans, 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.

[0087] In some embodiments, the at least one protein of interest is a surface protein.

[0088] In some embodiments, 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.

[0089] In some embodiments, the at least one protein of interest is CD80.

[0090] In humans, 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.

[0091] In some embodiments, the at least one protein of interest is CD86.

[0092] In humans, 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.

[0093] In some embodiments, 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.

[0094] As used herein, “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. In some embodiments, “amino acid mutation” comprises substitutions, deletions, insertions, inversions, and combination thereof.

[0095] In some embodiments, 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.

[0096] 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 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.

[0097] In some embodiments, the PDCs are genetically modified to reduce (knockdown) or abolish (knock-out) the expression of at least one protein or gene encoding thereof.

[0098] As used herein, “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. As used herein, “reduce” means a 100% decrease of expression of the basal expression of the at least one protein or gene encoding thereof.

[0099] As antigen presenting cells, PDCs are capable of activating naive T cells and memory T cells into activated, antigen- specific T cells, either by direct priming, or bycross 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.

[0100] In some embodiments, 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.

[0101] As used herein, 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. Within the scope of the present invention, the terms “contacted”, “loaded”, or “incubated” may be used interchangeably. This incubation itself is hereby interchangeably referred to as the “loading step”.

[0102] In some embodiments, 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.

[0103] In some embodiments, 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.

[0104] In some embodiments, 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.

[0105] In some embodiments, 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.

[0106] In some embodiments, the at least one antigen or fragment thereof is a peptide.

[0107] In some embodiments, 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.

[0108] In some embodiments, 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.

[0109] In some embodiments, 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.

[0110] In some embodiments, 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.

[0111] In some embodiments, the at least one antigen or fragment thereof is a molecule, preferably a peptide or protein, involved in a genetic disease.

[0112] In some embodiments, 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.

[0113] In a preferred embodiment, the at least one cancer antigen and / or fragment thereof is a peptide or polypeptide comprising at least 3, at least 5, at least 10, at least 15, at least 20, or more, amino acid residues.

[0114] In some embodiments, 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.

[0115] In some embodiments, the peptide comprises at least one post-translational modification on at least one amino acid residues. In some embodiments, the peptide is glycosylated, glycated, phosphorylated, biotinylated, oxidated, nitrated, nitrosylated, acylated, alkylated, acetylated, methylated, lipidated, ubiquitinated, carboxylated, pegylated, or combinations thereof.

[0116] In another embodiment, 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.

[0117] In some embodiments, 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.

[0118] In some embodiments, the step a) is performed in a first medium. In some embodiments, the first medium is a cell culture medium, preferably a hematopoietic cell culture medium, more preferably a X-VIVO-15 culture medium.

[0119] In some embodiments, 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.

[0121] In some embodiments, 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.

[0122] In some embodiments, 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.

[0123] In some embodiments, 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.

[0124] In a preferred embodiment, the incubation is performed for about 2 hours. In a preferred embodiment, the incubation is performed for about 3 hours.

[0125] In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is from O.lxlO6cells / mL to lOOxlO6cells / mL.

[0126] In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is from O.lxlO6cells / mL to 10xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is about lxl06cells / mL.

[0127] In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is from 10xl06cells / mL to lOOxlO6cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is from 10xl06cells / mL to 40xl06cells / mL, preferably from 20xl06cells / mL to 40xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is about 20xl06cells / mL.

[0128] In some embodiments, the cellular concentration of the population of PDCs used for the loading step (i.e., step a)) is IxlO2, IxlO3, IxlO4, IxlO5, IxlO6or IxlO7cells / mL. In some embodiments, the PDCs cellular concentration is comprised between IxlO4and lxl08cells / mL.

[0129] In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is at least 5xl06cells / mL, at least 6xl06cells / mL, at least 7xl06cells / mL, at least 8xl06cells / mL, at least 9xl06cells / mL, at least 10xl06cells / mL, at least l lxlO6cells / mL at least 12xl06cells / mL at least 13xl06cells / mL at least 14xl06cells / mL at least 15xl06cells / mL at least 16xl06cells / mL at least 17xl06cells / mL at least 18xl06cells / mL at least 19xl06cells / mL or at least 20xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is at least 10xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the loading step (z.e., step a)) is at least 20xl06cells / mL.

[0130] In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is from O.lxlO6cells / mL to lOOxlO6cells / mL.

[0131] In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is from O.lxlO6cells / mL to 10xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is about lxl06cells / mL.

[0132] In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is from 10xl06cells / mL to lOOxlO6cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is from 10xl06cells / mL to 40xl06cells / mL, preferably from 20x106cells / mL to 40x106cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is about 20xl06cells / mL.

[0133] In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is IxlO2, IxlO3, IxlO4, IxlO5, IxlO6or IxlO7cells / mL. In some embodiments, the PDCs cellular concentration is comprised between IxlO4and lxl08cells / mL.

[0134] In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is at least 5xl06cells / mL, at least 6xl06cells / mL, atleast 7xl06cells / mL, at least 8xl06cells / mL, at least 9xl06cells / mL, at least 10xl06cells / mL, at least l lxlO6cells / mL at least 12xl06cells / mL at least 13xl06cells / mL at least 14xl06cells / mL at least 15xl06cells / mL at least 16xl06cells / mL at least 17xl06cells / mL at least 18xl06cells / mL at least 19xl06cells / mL or at least 20xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is at least 10xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the incubation step (i.e., step b)) is at least 20x106cells / mL.

[0135] In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is from O.lxlO6cells / mL to lOOxlO6cells / mL.

[0136] In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is from O.lxlO6cells / mL to 10xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is about lxl06cells / mL.

[0137] In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is from lOx 106cells / mL to lOOx 106cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is from 10xl06cells / mL to 40xl06cells / mL, preferably from 20x106cells / mL to 40x106cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is about 20xl06cells / mL.

[0138] In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is IxlO2, IxlO3, IxlO4, IxlO5, IxlO6or IxlO7cells / mL. In some embodiments, the PDCs cellular concentration is comprised between IxlO4and lxl08cells / mL.

[0139] In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is at least 5xl06cells / mL, at least 6xl06cells / mL, at least 7xl06cells / mL, at least 8xl06cells / mL, at least 9xl06cells / mL, at least 10xl06cells / mL, at least l lxlO6cells / mL at least 12xl06cells / mL at least 13xl06cells / mL atleast 14xl06cells / mL at least 15xl06cells / mL at least 16xl06cells / mL at least 17xl06cells / mL at least 18xl06cells / mL at least 19xl06cells / mL or at least 20xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is at least 10xl06cells / mL. In some embodiments, the cellular concentration of the population of PDCs used for the irradiation step (i.e., step c)) is at least 20xl06cells / mL.

[0140] 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 from O.lxlO6cells / mL to lOOxlO6cells / mL.

[0141] 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 from O.lxlO6cells / mL to 10xl06cells / 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 lxl06cells / mL.

[0142] 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 from 10xl06cells / mL to 100xl06cells / 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 from 10xl06cells / mL to 40xl06cells / mL, preferably from 20xl06cells / mL to 40xl06cells / 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 20xl06cells / mL.

[0143] 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 IxlO2, IxlO3, IxlO4, IxlO5, IxlO6or IxlO7cells / mL. In some embodiments, the PDCs cellular concentration is comprised between IxlO4and IxlO8cells / mL.

[0144] 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 5xl06cells / mL, at least 6xl06cells / mL, at least 7xl06cells / mL, at least 8xl06cells / mL, at least 9xl06cells / mL, at least 10xl06cells / mL, at least l lxlO6cells / mL at least 12xl06cells / mL at least 13xl06cells / mL at least 14xl06cells / mL at least 15xl06cells / mL at least 16xl06cells / mL at least 17xl06cells / mL at least 18xl06cells / mL at least 19xl06cells / mL or at least 20x106cells / 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 10xl06cells / 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 20x106cells / mL.

[0145] 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 from O.lxlO6cells / mL to lOOxlO6cells / mL.

[0146] 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 from O.lxlO6cells / mL to 10xl06cells / 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 lxl06cells / mL.

[0147] 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 from 10xl06cells / mL to lOOxlO6cells / 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 from 10xl06cells / mL to 40xl06cells / mL, preferably from 20xl06cells / mL to 40xl06cells / 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 20xl06cells / mL.

[0148] 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., stepc)) is IxlO2, IxlO3, IxlO4, IxlO5, IxlO6or IxlO7cells / mL. In some embodiments, the PDCs cellular concentration is comprised between IxlO4and IxlO8cells / mL.

[0149] 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 5xl06cells / mL, at least 6xl06cells / mL, at least 7xl06cells / mL, at least 8xl06cells / mL, at least 9xl06cells / mL, at least 10xl06cells / mL, at least l lxlO6cells / mL at least 12xl06cells / mL at least 13xl06cells / mL at least 14xl06cells / mL at least 15xl06cells / mL at least 16xl06cells / mL at least 17xl06cells / mL at least 18xl06cells / mL at least 19xl06cells / mL or at least 20x106cells / 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 10xl06cells / 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 20x106cells / mL.

[0150] In some embodiments, 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.

[0151] In some embodiments, 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.

[0152] In some embodiments, 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. 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.

[0153] In some embodiments, 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.

[0154] 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 from 0. IxlO6cells / mL to lOOxlO6cells / 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.

[0155] 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 from O.lxlO6cells / mL to 10xl06cells / 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. 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 IxlO6cells / mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.

[0156] 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 from 10xl06cells / mL to 100xl06cells / 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. 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 from 10xl06cells / mL to 40xl06cells / mL, preferably from 20xl06cells / mL to 40xl06cells / 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. In some embodiments, the cellular concentration of the population of PDCs used forthe loading step (i.e., step a)), loading step (i.e., step b)), and / or irradiation step (i.e., step c)) is about 20xl06cells / mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.

[0157] 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 5xl06cells / mL, at least 6xl06cells / mL, at least 7xl06cells / mL, at least 8xl06cells / mL, at least 9xl06cells / mL, at least 10xl06cells / mL, at least l lxlO6cells / mL at least 12xl06cells / mL at least 13xl06cells / mL at least 14xl06cells / mL at least 15xl06cells / mL at least 16xl06cells / mL at least 17xl06cells / mL at least 18xl06cells / mL at least 19xl06cells / mL or at least 20x106cells / mL; and the concentration of the cancer antigen or fragment thereof used for the loading step i.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 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 10xl06cells / 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. 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 20xl06cells / 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.

[0158] 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 from O.lxlO6cells / mL to lOOxlO6cells / 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.

[0159] 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 from O.lxlO6cells / mL to 10xl06cells / 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. In some embodiments, the cellular concentration of the population of PDCs used forthe loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is about lxl06cells / mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.

[0160] 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 from 10xl06cells / mL to lOOxlO6cells / 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. 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 from 10xl06cells / mL to 40x106cells / mL, preferably from 20x106cells / mL to 40x106cells / 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. 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 20x106cells / mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.e., step a)) is about 10 pM.

[0161] 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 5xl06cells / mL, at least 6xl06cells / mL, at least 7xl06cells / mL, at least 8xl06cells / mL, at least 9xl06cells / mL, at least 10xl06cells / mL, at least l lxlO6cells / mL at least 12xl06cells / mL at least 13xl06cells / mL at least 14xl06cells / mL at least 15xl06cells / mL at least 16xl06cells / mL at least 17xl06cells / mL at least 18xl06cells / mL at least 19xl06cells / mL or at least 20x106cells / mL; and the concentration of the cancer antigen or fragment thereof used for the loading step (i.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 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 10xl06cells / 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. In some embodiments, the cellular concentration of the population of PDCs used forthe loading step (i.e., step a)), loading step (i.e., step b)), and irradiation step (i.e., step c)) is at least 20xl06cells / 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.

[0162] In some embodiments, the PDCs are washed after step a).

[0163] In some embodiments, 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). In some embodiments, this step b) is performed in a second medium. As used herein, the term “second medium” may also be referred to as “cryoprotective medium” or “preservation medium”.

[0164] Preservation media, and particularly, cell-specific preservation media are formulated attending to the molecular biological aspects of cells during the cryopreservation process; intended to minimize eventual cell death, improve post-thaw cell viability and function. Examples of cryoprotective media include, but are not limited to, CryoStorR / ™ CS2, CryoStorR / ™ CS5, CryoStorR / ™ CS10, mFreSR™, MesenCult™- ACF, STEMdiff™, NutriFrcczR / I MD10, and the like. Cryoprotective agents (i.e., a cryoprotectants) are part of (cryo)preservation media and are known in the art. Examples of cryoprotective agents are DMSO, glycerol, propylene glycol, ethylene glycol, sucrose, threalose, serum, and the like.

[0165] In some embodiments, 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.

[0166] In some embodiments, the second medium is selected from the group comprising or consisting MesenCult™

[0167] In some embodiments, 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 CryoStorR / I MCS5.

[0168] In some embodiments, 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.

[0169] In some embodiments, the PDCs are not washed after step b). In some embodiments, the PDCs remain in the same medium for the next step.

[0170] In some embodiments, the PDCs are irradiated after contacting (or incubating) them with at least one cryoprotective agent (step c). In a preferred embodiment, the PDCs are irradiated when they still are in contact (or incubated) with the cryoprotective agent. In other words, in some embodiments, the irradiation step c) is performed in a cryoprotective medium. In some embodiments, the irradiation step c) is performed in the same cryoprotective medium as for step b). In some embodiments, the PDCs are irradiated in the presence of a cryoprotectant. In some embodiments, the cryoprotectant is present and / or active during irradiation step.

[0171] In some embodiments, 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.

[0172] In some embodiments, 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.

[0173] In some embodiments, the PDCs are not washed after step c). In some embodiments, the PDCs remain in the same medium for the next step.

[0174] In some embodiments, 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).

[0175] In some embodiments, the PDCs are cryopreserved for storage. In some embodiments, PDCs are frozen at a temperature from -20°C to -200°C, preferably from - 80°C to -200°C. In some embodiments, the PDCs are frozen at about -196°C, typically cells are stored in liquid nitrogen. In some embodiments, PDCs are “flash-frozen” in liquid nitrogen prior to storage.

[0176] In some embodiments, 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 10xl06cells / mL; and / or the concentration of the cancer antigen or fragment thereof used at step a) is at least 100 p M.

[0177] In some embodiments, 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 10xl06cells / 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.

[0178] In some embodiments, 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 20x106cells / 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.

[0179] 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 10xl06cells / mL, preferably at least 20xl06cells / mL, and / or theconcentration of the cancer antigen or fragment thereof used at step a) is at least 100 pM, preferably at least 200 M.

[0180] In some embodiments, 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, CPED1, PAQR4, NR4A2, FLNC, KCNMB4, DBP, IFIT2, SLC6A8, LM04, CRYBG1, SPOCK2, IGDCC4, TRERF1, EGR2, TMCC3, ITPR1, IL24, MMP13, SPON2, CAV1, AP003108,2, NCK2, EBF1, PTGS2, LRIG1, AK5, RORB, BCAT1, SATB1, H2AC8, TAF4B, IGFBP4, BLK, ROR1-AS1, SEMA3D, KLF12, CDC14A, SERPINE2, ADGRE5, CLSPN, NFIA, RALGPS2, FAM111B, GUCY1B1, CLEC2D, KIF23, COL1A2, SNTB2, TGFBR3, SOD2, LGALS3, ITPR3, PCED1B, HDAC5, ENTPD1, TOX, TCTN3, KLF5, MAP2, ADGRG1, RELN, ARRDC3, MDGA1, PCDH7, PERI, LIMS2, ADGRL2, Hl-10, CYBB, KDM7A, GLI3, FCGR2A, ERCC6L, GPAT3, NCR3LG1, MCTP1, 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, LTK, DUSP7, SEMA7A, ADSS1, SNORD3B-2, FXYD3, PDGFB, NSMF, TNK2-AS1, IGLC5, PWWP3A, ACP5, SLC20A1, MAPRE3, AL109918,l, ABCA2, ACSS2, PLEKHG3, PAK6, CU633904,l, IGHA1, TIFAB, TRAV22, PTPRE, PFKFB4, RAB39A, AC245014,3, PLOD1, SCARF1, PLCB2, MIR646HG, ZNF219, MMP25, PNMA2, AL662844,4, PNPLA7, PLXNB3, PTP4A3, ZNF385A, PPFIA4, VIPR1, CFAP251, CCDC78, NOXA1, BX571818,1, FGD2, MMP11, MYBPC2, BGLAP, P2RY14, GBGT1, PLEKHH1, AC069503,!, HSPA8, SH3TC1, IGHA2, WNT10A, LGR6, CLCN4, FCER1A,AL139020,l, SCN5A, TMEM191A, BSPRY, AD0RA1, AC104530,l, CD40, AL365181,3, ANK1, KCNJ12, DNMT3B, LGI4, PDZRN3, MYBPH, and KLHL33, 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).

[0181] As used herein, “altered expression” means a difference of 5%, 10%, 20%, 50% or more.

[0182] As used herein, “at least one” encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more. As used herein, “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. As used herein, “at least three” encompasses 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. As used herein, “at least four” encompasses 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. As used herein, “at least five” encompasses 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more. As used herein, “at least six” encompasses 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0183] In some embodiments, 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, CPED1, PAQR4, NR4A2, FLNC, KCNMB4, DBP, IFIT2, SLC6A8, LMO4, CRYBG1, SPOCK2, IGDCC4, TRERF1, EGR2, TMCC3, ITPR1, IL24, MMP13, SPON2, CAV1, AP003108,2, NCK2, EBF1, PTGS2, LRIG1, AK5, RORB, BCAT1, SATB1, H2AC8, TAF4B, IGFBP4, BLK, ROR1-AS1, SEMA3D, KLF12, CDC14A, SERPINE2, ADGRE5, CLSPN, NFIA, RALGPS2, FAM111B, GUCY1B1, CLEC2D, KIF23, COL1A2, SNTB2, TGFBR3, SOD2, LGALS3, ITPR3, PCED1B, HDAC5, ENTPD1, TOX, TCTN3, KLF5, MAP2, ADGRG1, RELN, ARRDC3, MDGA1, PCDH7, PERI, LIMS2, ADGRL2, Hl-10, CYBB, KDM7A, GLI3, FCGR2A, ERCC6L, GPAT3, NCR3LG1, and MCTP1,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).

[0184] As used herein, “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.e., irradiated in the absence of at least one cryoprotectant).

[0185] In some embodiments, 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, LTK, DUSP7, SEMA7A, ADSS1, SNORD3B-2, FXYD3, PDGFB, NSMF, TNK2-AS1, IGLC5, PWWP3A, ACP5, SLC20A1, MAPRE3, AL109918,l, ABCA2, ACSS2, PLEKHG3, PAK6, CU633904,l, IGHA1, TIFAB, TRAV22, PTPRE, PFKFB4, RAB39A, AC245014,3, PLOD1, SCARF1, PLCB2, MIR646HG, ZNF219, MMP25, PNMA2, AL662844,4, PNPLA7, PLXNB3, PTP4A3, ZNF385A, PPFIA4, VIPR1, CFAP251, CCDC78, NOXA1, BX571818,1, FGD2, MMP11, MYBPC2, BGLAP, P2RY14, GBGT1, PLEKHH1, AC069503,l, HSPA8, SH3TC1, IGHA2, WNT10A, LGR6, CLCN4, FCER1A, AL139020,l, SCN5A, TMEM191A, BSPRY, ADORA1, AC104530,l, CD40, AL365181,3, ANK1, KCNJ12, DNMT3B, LGI4, PDZRN3, MYBPH, and KLHL33, compared to PDCs irradiated in amedium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).

[0186] As used herein, “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 comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).

[0187] In some embodiments, 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).

[0188] In some embodiments, 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).

[0189] In some embodiments, 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).

[0190] In some embodiments, 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 comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).

[0191] In some embodiments, 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).

[0192] In some embodiments, 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. In some embodiments, 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 thegroup 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. In some embodiments, 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. In some embodiments, 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.

[0193] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 atleast one cryoprotectant. In some embodiments, 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.

[0194] In some embodiments, 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. In some embodiments, 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. In some embodiments, the obtained PDCs have increased expression of TCF7, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant. In some embodiments, 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.

[0195] In some embodiments, 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).

[0196] As used herein, “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 increaseof 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 not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).

[0197] In some embodiments, 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).

[0198] In some embodiments, 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).

[0199] In some embodiments, 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).

[0200] In some embodiments, 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 fromthe 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 irradiated in a medium that does not comprise at least one cryoprotectant (i.e., irradiated in the absence of at least one cryoprotectant).

[0201] In some embodiments, 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, CPED1, PAQR4, NR4A2, FLNC, KCNMB4, DBP, IFIT2, SLC6A8, LM04, CRYBG1, SPOCK2, IGDCC4, TRERF1, EGR2, TMCC3, ITPR1, IL24, MMP13, SPON2, CAV1, AP003108,2, NCK2, EBF1, PTGS2, LRIG1, AK5, RORB, BCAT1, SATB1, H2AC8, TAF4B, IGFBP4, BLK, ROR1-AS1, SEMA3D, KLF12, CDC14A, SERPINE2, ADGRE5, CLSPN, NFIA, RALGPS2, FAM111B, GUCY1B1, CLEC2D, KIF23, COL1A2, SNTB2, TGFBR3, SOD2, LGALS3, ITPR3, PCED1B, HDAC5, ENTPD1, TOX, TCTN3, KLF5, MAP2, ADGRG1, RELN, ARRDC3, MDGA1, PCDH7, PERI, LIMS2, ADGRL2, Hl-10, CYBB, KDM7A, GLI3, FCGR2A, ERCC6L, GPAT3, NCR3LG1, MCTP1, 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, LTK, DUSP7, SEMA7A, ADSS1,SNORD3B-2, FXYD3, PDGFB, NSMF, TNK2-AS1, IGLC5, PWWP3A, ACP5, SLC20A1, MAPRE3, AL1O9918,1, ABCA2, ACSS2, PLEKHG3, PAK6, CU633904,l, IGHA1, TIFAB, TRAV22, PTPRE, PFKFB4, RAB39A, AC245014,3, PL0D1, SCARF1, PLCB2, MIR646HG, ZNF219, MMP25, PNMA2, AL662844,4, PNPLA7, PLXNB3, PTP4A3, ZNF385A, PPFIA4, VIPR1, CFAP251, CCDC78, N0XA1, BX571818,1, FGD2, MMP11, MYBPC2, BGLAP, P2RY14, GBGT1, PLEKHH1, AC069503,l, HSPA8, SH3TC1, IGHA2, WNT10A, LGR6, CLCN4, FCER1A, AL139020,l, SCN5A, TMEM191A, BSPRY, ADORA1, AC104530,l, CD40, AL365181,3, ANK1, KCNJ12, DNMT3B, LGI4, PDZRN3, MYBPH, and KLHL33, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mL.

[0202] In some embodiments, 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, CPED1, PAQR4, NR4A2, FLNC, KCNMB4, DBP, IFIT2, SLC6A8, LMO4, CRYBG1, SPOCK2, IGDCC4, TRERF1, EGR2, TMCC3, ITPR1, IL24, MMP13, SPON2, CAV1, AP003108,2, NCK2, EBF1, PTGS2, LRIG1, AK5, RORB, BCAT1, SATB1, H2AC8, TAF4B, IGFBP4, BLK, ROR1-AS1, SEMA3D, KLF12, CDC14A, SERPINE2, ADGRE5, CLSPN, NFIA, RALGPS2, FAM111B, GUCY1B1, CLEC2D, KIF23, COL1A2, SNTB2, TGFBR3, SOD2, LGALS3, ITPR3, PCED1B, HDAC5, ENTPD1, TOX, TCTN3, KLF5, MAP2, ADGRG1, RELN, ARRDC3, MDGA1, PCDH7, PERI, LIMS2, ADGRL2, Hl-10, CYBB, KDM7A, GLI3, FCGR2A, ERCC6L, GPAT3, NCR3LG1, and MCTP1, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mL.

[0203] In some embodiments, 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, LTK, DUSP7, SEMA7A, ADSS1, SNORD3B-2, FXYD3, PDGFB, NSMF, TNK2-AS1, IGLC5, PWWP3A, ACP5, SLC20A1, MAPRE3, AL109918,l, ABCA2, ACSS2, PLEKHG3, PAK6, CU633904,l, IGHA1, TIFAB, TRAV22, PTPRE, PFKFB4, RAB39A, AC245014,3, PLOD1, SCARF1, PLCB2, MIR646HG, ZNF219, MMP25, PNMA2, AL662844,4, PNPLA7, PLXNB3, PTP4A3, ZNF385A, PPFIA4, VIPR1, CFAP251, CCDC78, NOXA1, BX571818,1, FGD2, MMP11, MYBPC2, BGLAP, P2RY14, GBGT1, PLEKHH1, AC069503,l, HSPA8, SH3TC1, IGHA2, WNT10A, LGR6, CLCN4, FCER1A, AL139020,l, SCN5A, TMEM191A, BSPRY, ADORA1, AC104530,l, CD40, AL365181,3, ANK1, KCNJ12, DNMT3B, LGI4, PDZRN3, MYBPH, and KLHL33, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mL.

[0204] In some embodiments, 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 10xl06cells / mL.

[0205] In some embodiments, 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 fromthe 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 10xl06cells / mL.

[0206] In some embodiments, 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 10xl06cells / mL.

[0207] In some embodiments, 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 comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mL.

[0208] In some embodiments, 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 10xl06cells / mL.

[0209] In some embodiments, 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 ofCCL1, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL.

[0210] In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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, andSERPINE2, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mL.

[0211] In some embodiments, 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 lOxlO6cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL.

[0212] In some embodiments, 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 10xl06cells / mL. In some embodiments, 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, comparedto PDCs irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL.

[0213] In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL.

[0214] In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL. In some embodiments, 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 10xl06cells / mL.

[0215] In some embodiments, the obtained PDCs have increased surface expression of CD83 compared to PDCs irradiated in a medium that does not comprise at least onecryoprotectant and at a cellular concentration of less than 10xl06cells / mL. (i.e., irradiated in the absence of at least one cryoprotectant).

[0216] As used herein, “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 lOxlO6cells / mL. (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 not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mL. (i.e., irradiated in the absence of at least one cryoprotectant).

[0217] In some embodiments, 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 10xl06cells / mL.

[0218] In some embodiments, 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 10xl06cells / mL.

[0219] In some embodiments, 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 10xl06cells / mL.

[0220] In some embodiments, 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 irradiated in a medium that does not comprise at least one cryoprotectant and at a cellular concentration of less than 10xl06cells / mE.

[0221] In some embodiments, 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).

[0222] In some embodiments, 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 thatdoes not comprise at least one cryoprotectant. In some embodiments, 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. In some embodiments, 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.

[0223] In some embodiments, 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; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant. In some embodiments, 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. In some embodiments, 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; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.

[0224] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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; and have increased surface expression of CD83, compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.

[0225] 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, TET3, MOB3B, CPED1, PAQR4, NR4A2, FLNC, KCNMB4, DBP, IFIT2, SLC6A8, LM04, CRYBG1, SPOCK2, IGDCC4, TRERF1, EGR2, TMCC3, ITPR1, IL24, MMP13, SPON2, CAV1, AP003108,2, NCK2, EBF1, PTGS2, LRIG1, AK5, RORB, BCAT1, SATB1, H2AC8, TAF4B, IGFBP4, BLK, ROR1-AS1, SEMA3D, KLF12, CDC14A, SERPINE2, ADGRE5, CLSPN, NFIA, RALGPS2, FAM111B, GUCY1B1, CLEC2D, KIF23, COL1A2, SNTB2, TGFBR3, SOD2, LGALS3, ITPR3, PCED1B, HDAC5, ENTPD1, TOX, TCTN3, KLF5, MAP2, ADGRG1, RELN, ARRDC3, MDGA1, PCDH7, PERI, LIMS2, ADGRL2, Hl- 10, CYBB, KDM7A, GLI3, FCGR2A, ERCC6L, GPAT3, NCR3LG1, MCTP1, 0MA1, REPIN1-AS1, LRRN2, SAMHD1, FES, MACROD1, AL139125,!, SLC25A23, EML3, SEPTIN1, AK1, GOLGA8A, PDK2, SCARNA2,LINC-ROR, AL138756,1, TCL1B, AL022238,3, KIR3DX1, IE16, MPEG1, ST6GALNAC4, NRSN2-AS1, DNAH1, TREML2, AC073957,3, IRF8, SLC32A1, EPHB4, CHCHD10, TTC39A, APOBEC2, RNVU1-31, INPP4A, TM7SF2, AC109326,l, ZNF296, SIGLEC6, SLC46A3, PDLIM2, MEET11, ARHGAP27, ARHGEF17, OPLAH, LTK, DUSP7, SEMA7A, ADSS1, SNORD3B-2, FXYD3, PDGFB, NSMF, TNK2-AS1, IGLC5, PWWP3A, ACP5, SLC20A1, MAPRE3, AL109918,l, ABCA2, ACSS2, PLEKHG3, PAK6, CU633904,l, IGHA1, TIFAB, TRAV22, PTPRE, PFKFB4, RAB39A, AC245014,3, PEOD1, SCARF1, PECB2, MIR646HG, ZNF219, MMP25, PNMA2, AE662844,4, PNPEA7, PEXNB3, PTP4A3, ZNF385A, PPFIA4, VIPR1, CFAP251, CCDC78, NOXA1, BX571818,1, FGD2, MMP11, MYBPC2, BGEAP, P2RY14, GBGT1, PEEKHH1, AC069503,l, HSPA8, SH3TC1, IGHA2, WNT10A, EGR6, CECN4, FCER1A, AE139020,l, SCN5A, TMEM191A, BSPRY, ADORA1, AC104530,l, CD40, AE365181,3, ANK1, KCNJ12, DNMT3B, EGI4, PDZRN3, MYBPH, and KEHE33, compared to unmodified PDCs, preferably 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 CCE1, FASEG, NGFR, TCF7, CCE22, CCR7, SOCS3, MAME3, STAT5A, ITPR1, IE24, MMP13, NCK2, PTGS2, TGFBR3, ITPR3, and FCGR2A, 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.

[0226] 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.

[0227] In some embodiments, 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.

[0228] In some embodiments, 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, compared to unmodified PDCs.

[0229] In some embodiments, the PDCs from the population of modified PDCs have increased surface expression of CD83 compared to unmodified PDCs.

[0230] 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 unmodified PDCs.

[0231] In some embodiments, 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.

[0232] In some embodiments, 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.

[0233] In some embodiments, 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.

[0234] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0235] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 regulation of cell viability and activation, preferably selected from the group consisting of DUSP7, EBF1, IFIT2, IGFBP4, and SERPINE2, compared to unmodified PDCs.

[0236] In some embodiments, 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. In some embodiments, the PDCs from the population ofmodified 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. In some embodiments, the PDCs from the population of modified PDCs have increased expression of TCF7, compared to unmodified PDCs. In some embodiments, 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.

[0237] In some embodiments, the population of PDCs obtained with the method of the invention, or the population of modified PDCs as described herein, is for use for the treatment and / or the prevention of a disease in a subject in need thereof. In some embodiments, the disease is selected from the group comprising or consisting of cancer, genetic diseases and microbial infections. In one embodiment, the disease is cancer. In another embodiment, the disease is a microbial infection. In another embodiment, the disease is a genetic disease. The present invention further relates to a pharmacological composition comprising the population of PDCs obtained with the method of the invention, or the population of modified PDCs as described herein, preferably further comprising at least one pharmaceutically acceptable excipient.

[0238] 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 as a medicament.

[0239] 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 treatment and / or the prevention of a disease in a subject in need thereof. In some embodiments, the disease is selected from the group comprising or consisting of cancer, genetic diseases, and microbial infections. In one embodiment, the disease is cancer. In another embodiment, the disease is a microbial infection. In another embodiment, the disease is a genetic disease.

[0240] 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. In some embodiments, the disease is selected from the group comprising or consisting of cancer, genetic diseases, and microbial infections. In one embodiment, the disease is cancer. In another embodiment, the disease is a microbial infection. In another embodiment, the disease is a genetic disease.

[0241] 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. In some embodiments, the disease is selected from the group comprising or consisting of cancer, genetic diseases, and microbial infections. In one embodiment, the disease is cancer. In another embodiment, the disease is a microbial infection. In another embodiment, the disease is a genetic disease.

[0242] 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; andc. culturing said loaded PDCs with said cells; and thereby inducing and amplifying antigen specific effectors in the culture in vitro of loaded PDCs.

[0243] In some embodiment, the individual is a human individual.

[0244] It will be apparent to the person skilled in the art that PBMCs and CD8+ T-cells may be obtained from an individual by a simple blood sample, which is a routine and non-invasive action commonly performed by health partitioners, that can in no way endanger the health of the individual. In some embodiments, step a) of obtaining cells selected from the group comprising or consisting of PBMCs and CD8+ T-cells from an individual does not comprise an invasive step.

[0245] 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.

[0246] Another object of the present invention relates to a kit for implementing the method as described herein, and instructions for use.BRIEF DESCRIPTION OF THE DRAWINGS

[0247] 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.

[0248] 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 followingirradiation. 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.

[0249] 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).

[0250] Figure 4 is a histogram showing CD83 expression 4 hours post irradiation in cells from batch 1, batch 2, and batch 3.EXAMPLES

[0251] The present invention is further illustrated by the following examples.Example 1: Effect of cell irradiation in CS5 on the PDC*line cells featuresMaterials and MethodsPreparation of Tumor-Peptide-Loaded PDC*Line

[0252] Sample cells correspond to three independent PDC*line cell cultures, separately loaded with two peptides (Survivin and Melan-A) which were manufactured in three manufacturing runs. Briefly, PDC*line cells (1 million / mL) were incubated in hematopoietic cell culture medium X-Vivo 15 with 10 pM of one peptide for 3 h at 37°C. The tumor peptides were purchased from SB peptide. The characteristics of the peptides are detailed in Table 1.

[0253] Table 1 : List of the selected tumor antigens.

[0254] Following the standard loading procedure, the cells underwent one of four protocols, thereby generating 4 batches produced in triplicate (3 manufacturing runs). The details of each batch are detailed on Table 2.

[0255] Table 2: Specifications of each batch.

[0256] The sequence of steps for each batch manufacturing was as follows:

[0257] In Batch 1 : PDC*line cells were a) loaded at IxlO6viable cells / ml in X-Vivo 15 with peptide at 10 p M; b) washed, resuspended in X-Vivo 15 and pooled altogether, then c) irradiated at 60 Gy at 50xl06total cells / ml in X-Vivo 15, d) washed and resuspended in Cryostor™ CS5, and finally e) frozen at 50xl06total cells / ml.

[0258] In Batch 2 : PDC*line cells were a) loaded at IxlO6viable cells / ml in X-Vivo 15 with peptide at 10 p M; b) washed, resuspended in Cryostor™ CS5 and pooled altogether, then c) irradiated at 60 Gy at 50xl06total cells / ml in Cryostor™ CS5, and d) directly frozen at 50xl06total cells / ml.

[0259] In Batch 3 : PDC*line cells were a) loaded at 20xl06viable cells / ml in X-Vivo 15 with peptide at 200 pM; b) washed, resuspended in Cryostor™ CS5 and pooled altogether, then c) irradiated at 60 Gy at 50xl06total cells / ml in Cryostor™ CS5; and d) directly frozen at 50xl06total cells / ml.

[0260] In Batch 4: PDC*line cells were a) loaded at IxlO6viable cells / ml in X-Vivo 15 with peptide at 10 pM; b) washed, resuspended in Cryostor™ CS5 and c) frozen at 50xl06total cells / ml in Cryostor™ CS5. The cells were not irradiated.Antigen Presentation Assay - Luciferase Assay (APA-Luciferase)

[0261] 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. 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 intensethe Jurkat TCR specific Luciferase QC tool activation is. Furthermore, 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. Finally, Drug Product like sample potency evaluation relies on a quantification of the produced Nanoluciferase (luminescence measurement) by tTCR specific Jurkat cells co-cultured with Drug Product like sample cells.

[0262] 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).

[0263] For the evaluation of the addition of Cryostor™ CS5 to cells before the irradiation step on the PDC*line cells, 2 distinct tTCR specific Jurkat cells were used: 1) Jurkat Survivin Luciferase with TCR specificity for Survivin antigen (LabCollector ID: 2994), 10xl06viable cells frozen in 1ml of 10% DMSO (Dimethyl Sulfoxide) as freezing medium; and 2) Jurkat Melan-A Luciferase with TCR specificity for Melan-A antigen (LabCollector ID: 2863), 10xl06viable cells frozen in 1ml of 10% DMSO as freezing medium.

[0264] Peripheral blood mononuclear cells (PBMC) were used as a source of CD8+ T cells for the achievement of Antigen Activation Assay. 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). 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, lOOxlO6viable HLA-A*0201 PBMCs frozen in 1.8 ml of 25% FBS - 10% DMSO freezing medium).

[0265] The antigen presentation assay corresponds to the co-culture of tTCR specificJurkat cells with each sample cells (Batch 1, Batch 2, Batch 3, Batch 4), medium(complete RPMI) and Dynabeads Human T-Activator CD3 / CD28 at various beads:cell ratios (1:1, 2:1; 4:1, 6:1, 8:1 and 10:1). The procedure involves the following steps.

[0266] For the Jurkat tool thawing, supplemented Thawing Medium was prepared through the addition of DNase I (10 mg / ml) in pre-heated Thawing Medium to reach a final concentration of DNase I at 30 pg / ml. Then, a cryotube of each Jurkat tool was destocked from nitrogen tank and let at room temperature for about 30 seconds, and placed in a 37°C water bath until almost complete cell suspension thawing. Thawed cell suspensions were transferred to 15-ml tubes and 1 ml of supplemented Thawing Medium was drop wise added to each cell suspension before cell incubation at 37 °C for 10 minutes. Other 5 ml of supplemented Thawing Medium were slowly added to each cell suspension before centrifugation at 400g for 10 minutes at room temperature. Supernatants were discarded and each cell pellet was resuspended in 5 ml of supplemented Thawing Medium and incubated at room temperature for Ih.

[0267] For the Jurkat tool cell counting and resuspension, after the incubation at room temperature, 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 1.5xl06viable cells / ml.

[0268] Dynabead vial was vortexed for 30 seconds before collecting 1 ml (40x 106beads) 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 20xl06beads / ml, and the suspension diluted with complete RPMI to generate 6 distinct Dynabead dilutions as shown on Table 3.

[0269] Table 3: Dynabead dilutions.

[0270] 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.

[0271] 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.

[0272] 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 wasplaced 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 Assay - Data Analysis

[0273] 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. With that view, a second order polynomial regression is done on the Dynabead conditions giving the formula luminescence = ax2 + bx + c with x the beads / cell ratio. Solving the equation luminescence of the tested batch = ax2 + bx + c provided the tested batch equivalent Dynabeads / cell ratio as the smallest of the equation roots. For APA-Luciferase Assay Kruskal- Wallis (non-parametric ANOVA) tests were performed.Apoptosis assay

[0274] 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

[0275] At the end of each timepoint the staining went as described: cells were washed in ice-cold PBS, centrifuged at 500g for 5 min at 4°C, resuspended at IM / ml in ice-cold lx binding buffer, 200 pL transferred in FACS tube, 5 l of AnnexinV-FITC and 10 pL of 7-AAD were added, followed by incubation for 15 min on ice and in the dark, then 400 pL of ice-cold lx binding buffer were added before acquisition by flow cytometry of 15000 events on the singlets gate.Apoptosis Assay - Data Analysis

[0276] In early apoptosis, cells lose membrane phospholipid asymmetry, whilst the integrity of the cell membrane is maintained. The Annexin-V kits contain a viability dye to evaluate membrane integrity as well as annexin-V to assess membrane asymmetry. When cells are double-stained with annexin V and a viability dye, 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.ResultsAPA-Luciferase Assay

[0277] It has been showed that the peptide presentation efficiency for Survivin specificity was highest for batch 3 in all runs than batch 1 (i.e., with standard loading) (Figure 1A). Similarly expressive data between batch 3 and batch 1 were obtained regarding the peptide presentation efficiency for Melan-A specificity (Figure IB).Apoptosis Assay

[0278] On assessing apoptosis at specific timepoints for the batches 1, 2 and 3 in all three runs, batch 3 displayed about 20% of early apoptosis at T6h after irradiation, i.e., the lowest amongst the batches (Figures 2A and 2B).Example 2: Analysis of differentially expressed genesMaterials and MethodsPreparation of Tumor-Peptide-Loaded PDC*Line and composition of batches

[0279] See Example 1RNA sequencing

[0280] Starting from frozen cells (batches 1 / 2 / 3 / 5):Thaw cryotubes in water bath (37 °C)Transfer of cell suspension in 50 ml tube and wash with 9 ml of X-vivo 15Centrifuge at 400g for 10 minutesResuspend cells in X-vivo 15 and count cells with NexcelomCentrifuge at 400g for 10 minutesResuspend cells in X-vivo 15 at IM / ml and incubate cells for Oh, 4h or 24h at 37°C

[0281] RNA extraction and sequencing:At each time point: cells are collected and centrifuge 400g for 7 minutesCells are washed lx in 1ml PBS and centrifuge 400g for 7 minutesCells are resuspended in 1 ml of RNA-later stabilization solution (ref: AM7020) and stored at -20 °C until extractionThe day of RNA extraction, samples are thawed and centrifuge 15 minutes at 8000g to remove RNA Later before resuspension in RLT buffer from RNAeasy Mini KitTotal mRNA are purified with RNeasy Mini Kit (ref: 74104) with a DNse step (RNase-Free DNase Set; ref: 79254) following manufacturer’s recommendations RNA is eluted in 30ul of Rnase-free water and samples are stored at -80°C until useRNA quantification and integrity assessment is performed using Agilent RNA Nano kitSequencing is performed using NovaSeq S4 Reagent Kit vl.5 (300 cycles)

[0282] Bioinformatic analysis: Fastq files were mapped to Human reference genome (GRCh38 release 103) using the nf-core maseq pipeline (version 3.0, https: / / nf- co.re / rnaseq / 3.0). The pipeline uses STAR to map the raw fastq reads to the genome, project the alignments onto the transcriptome and to perform the downstream BAM-level quantification with Salmon. We also added the options — clip_rl 1 — clip_r2 1 — three_prime_clip_rl 1 — three_prime_clip_r2 1 in order to trim 5’ and 3’ T on both reads and — trim_nextseq 10 to trim based on quality after removing poly-G tails.

[0283] Once the mapping of new data completed, we started the downstream analysis with DESeq2 (https: / / bioconductor.org / packages / release / bioc / html / DESeq2.html) by using the per gene raw count matrix. Differentially expressed gene (DE) analysis was performed between irradiation conditions in order to compare the 2 new irradiation modalities (batches 2 and 3) to batch 1.CD83 cell surface expression assessment

[0284] Starting from frozen cells (batches 1 / 2 / 3 / 5; n=3 independent experiments): Thaw cryotubes in water bath (37 °C)Transfer of cell suspension in 50 ml tube and wash with 9 ml of X-vivo 15 Centrifuge at 400g for 10 minutesResuspend cells in X-vivo 15 and count cells with NexcelomCentrifuge at 400g for 10 minutesResuspend cells in X-vivo 15 at IM / ml and incubate cells for Oh, 4h or 24h at 37°C

[0285] Cell surface staining and CD83 expression analysis by flow cytometry:At each timepoint, cells are collected and washed 2x with PBSCells are centrifuge 400g for 7 minutes and resuspended in PBS at IM / mlCells are incubated with Fixable Viability Stain 510 lul per IM cells (FVS510 ref: 564406) during 15 minutes at room temperature and in the darkCells are centrifuge (400g for 7 minutes) and washed 2x with PBS before resuspension at IM cells / ml in PBS + 2% FBS200.000 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

[0286] Statistical analysis on median fluorescence intensity values (MFI) using GraphPad Prism 10: One-way ANOVA with Tukey’s multiple comparisons test.Results

[0287] Irradiation of the loaded PDCs*line cells yields various changes in gene expression. The following results explore the effects of the specific conditions used for irradiation (presence of a cryoprotectant, cell concentration, peptide concentration during loading).

[0288] 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).

[0289] The list of upregulated genes with a fold change of at least 2 and adjP < 0,05 in batch 3 compared to batch 1 at timepoint 4 hours post irradiation was established and are shown in Table 4.

[0290] Table 4: Differentially expressed genes between batches 3 and 1 (4 hours post irradiation).

[0291] From the list above, a short list of selected genes of interest which could be linked with PDC*line cells mechanism of action was established and is shown on Table 5.

[0292] Table 5: Differentially expressed genes of interest between batches 3 and 1 (4 hours post irradiation).

[0293] As seen on Table 5, various genes involved in the interaction with other immune cells (e.g., CCL1, FASLG, NGFR, CCL22, CCR7, IL24, MMP13, TGFBR3, FCGR2A, and IL16), the regulation of cell viability and activation (e.g., SOCS3, MAML3, STAT5A, ITPR1, NCK2, PTGS2, and ITPR3), and identity markers (e.g., TCF7), are differentially expressed in batch 3 compared to batch 1.

[0294] 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.

Claims

CLAIMS1. An in vitro 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).

2. The method according to claim 1, wherein said at least one antigen or fragment thereof is a peptide.

3. The method according to claim 1 or 2, wherein said at least one antigen or fragment thereof is a cancer or tumor antigen or a fragment thereof.

4. The method according to any one of claims 1 to 3, wherein at least one antigen or fragment thereof is selected from the group consisting of 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.

5. The method according to any one of claims 1 to 4, wherein said first medium is a cell culture medium, preferably said first medium is a hematopoietic cell culture medium, more preferably said first medium is X-Vivo 15 culture medium.

6. The method according to any one of claims 1 to 5, wherein said second medium comprises or consists of at least one cryoprotectant agent, preferably at least oneDMSO-containing cryoprotectant agent, more preferably said second medium comprises or consists of Cryostor™ CS5.

7. The method according to any one of claims 1 to 6, wherein the concentration of said PDCs at step a) and / or b) is from O.lxlO6cells / mL to lOOxlO6cells / mL.

8. The method according to any one of claims 1 to 7, wherein the concentration of said PDCs at step a), b) and / or c) is about lxl06cells / mL.

9. The method according to any one of claims 1 to 7, wherein the concentration of said PDCs at step a), b) and / or c) is at least 10xl06cells / mL.

10. The method according to any one of claims 1 to 7, wherein the concentration of said PDCs at step a), b) and / or c) is at least 20xl06cells / mL.

11. The method according to any one of claims 1 to 7, wherein the concentration of said PDCs at step a), b) and / or c) is about 20xl06cells / mL.

12. The method according to any one of claims 1 to 11, wherein the concentration of said at least one antigen or fragment thereof at step a) is from 0.1 pM to 1 mM.

13. The method according to any one of claims 1 to 12, wherein the concentration of said at least one antigen or fragment thereof at step a) is about 10 p M.

14. The method according to any one of claims 1 to 12, wherein the concentration of said at least one antigen or fragment thereof at step a) is at least 100 p M.

15. The method according to any one of claims 1 to 12, wherein the concentration of said at least one antigen or fragment thereof at step a) is at least 200 p M.

16. The method according to any one of claims 1 to 12, wherein herein the concentration of said at least one antigen or fragment thereof at step a) is about 200 pM.

17. The method according to any one of claims 1 to 16, wherein the concentration of said PDCs at step a), b) and / or c) is at least 10xl06cells / mL; and wherein theconcentration of said at least one antigen or fragment thereof at step a) is at least 100 pM.

18. The method according to any one of claims 1 to 17, wherein the concentration of said PDCs at step a), b) and / or c) is at least 20xl06cells / mL; and wherein the concentration of said at least one antigen or fragment thereof at step a) is at least 200 pM.

19. A population of PDCs obtained with the method according to any one claims 1 to 18.

20. The population of PDCs according to claim 19, wherein said PDCs have increased expression of at least one 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 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.

21. The population of PDCs according to claim 19 or 20, wherein said PDCs have increased surface expression of CD83 compared to PDCs irradiated in a medium that does not comprise at least one cryoprotectant.

22. A population of modified PDCs having increased expression of at least one 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 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 unmodified PDCs.

23. The population of modified PDCs according to claim 22, wherein said PDCs have increased surface expression of CD83 compared to unmodified PDCs.

24. The population of PDCs according to any one of claims 19 to 21, or the population of modified PDCs according to claim 22 or 23, for use for treating and / or preventing a disease in a subject in need thereof.

25. The population of PDCs for use according to claim 24, wherein the disease is selected from the group consisting of cancer, genetic diseases and microbial infection, preferably cancer.

26. A method of inducing and amplifying antigen specific effectors in vitro, 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 according to any one of claims 19 to 21, or the population of modified PDCs according to claim 22 or 23; b. loading the population of PDCs according to any one of claims 19 to 21, or the population of modified PDCs according to claim 22 or 23with 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.

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