Method of producing plasmacytoid dendritic cells

Abstract

The present invention relates to a method of producing plasmacytoid dendritic cells (pDCs) from pluripotent stem cells. The invention also relates to the pDCs produced by said method, and the use of said pDCs in methods of therapy, in particular, methods of treating a cancer, an autoimmune disease, or an inflammatory disease, wherein the method comprises administering a therapeutically effective amount of the pDCs to the patient in need thereof.

Description

[0001] METHOD OF PRODUCING PLASMACYTOID DENDRITIC CELLS

[0002] Field of the Invention

[0003] The present invention relates to a method of producing plasmacytoid dendritic cells from pluripotent stem cells and to plasmacytoid dendritic cells for use in methods for treating disease, in particular a cancer, an autoimmune disease, or an inflammatory disease. of the Invention

[0004] Plasmacytoid dendritic cells (pDCs) are a rare type of immune cell that initiate immune responses and link the innate and adaptive immune systems. pDCs also induce tolerance to exogenous and endogenous antigens (Swiecki, and Colonna, Nat Rev Immunol, 2015. 15(8)). pDCs express high levels of interferon regulatory factor 7, primarily sense pathogens through pattern recognition receptors including toll-like receptor (TLR) 7 and 9, and produce and secrete high levels of type I interferon (IFN) (Swiecki and Colonna, Nat Rev Immunol, 2015. 15(8); and Tangand Cattral, Cell Mol Life Sci, 2016). In particular, pDCs produce type I IFNs in response to a viral infection. pDCs also present antigens and activate T-cells in the lymph nodes. Thus, activated pDCs link the innate and adaptive immune system together via cytokine production combined with antigen-presenting cell (APC) activity. Taken together, pDCs are key effectors in cellular immunity and have a multifaceted role in the immune system, which makes them extremely adaptable for targeted treatments. For example, pDCs have been proposed for use in vaccines where they present antigens and are delivered to lymph nodes to activate T-cells (Charles et al., 2020, Oncology, 9(1); Thordardottir, S., et al., 2017, Oncoimmunology, 6(3): p. el285991). pDCs are also used as immunotherapies. WO2022 / 063818 provides engineered pDCs for use in immunotherapy to target diseased cells and activate immune responses for the treatment of disease. Furthermore, the antiviral state during infections that pDC functionality achieves provides vital adjuvant activity in the context of vaccination, and promotes immunogenic anti-tumour responses upon activation (Swiecki, and Colonna, Nat Rev Immunol, 2015. 15(8); Tovey, et al. Biol Chem, 2008. 389(5); and Rajagopal, et al. Blood, 2010. 115(10): p. 1949-57).

[0005] As pDCs are a rare type of immune cell in blood, methods of producing pDCs from Haemopoietic Stem and Progenitor Cells (HSPCs) have been developed in order to obtain large numbers of pDCs for use in immunotherapies. For example, WO2018 / 206577 provides methods for generating populations of pDCs from HSPCs. CGMP compliant methods to produce pDCs from HSPCs have also been described in WO 2023 / 275219. However, these methods still require obtaining HSPCs from patients, which is laborious, inconvenient, expensive and reduces the utility of the resulting pDCs for therapeutic applications.

[0006] A need in the art remains for improved methods of producing pDCs.

[0007] Summary of the Invention

[0008] The invention provides a method of producing plasmacytoid dendritic cells (pDCs) from pluripotent stem cells. As demonstrated in the examples, the inventors have successfully developed a method of producing pDCs from an accessible source of cells, that is, pluripotent stem cells. Accordingly, the invention provides a method of producing plasmacytoid dendritic cells (pDCs) from pluripotent stem cells wherein the method comprises:

[0009] (a) differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs);

[0010] (b) optionally isolating the HSPCs;

[0011] (c) differentiating the HSPCs into pDCs; and

[0012] (d) optionally isolating the pDCs. pDCs are a rare population of cells existing in blood. Contrastingly, pluripotent stem cells can be “off-the-shelf’ or produced from skin in large numbers, so called “induced pluripotent stem cells”. By developing a first method for producing pDCs from pluripotent stem cells, the invention provides a more efficient, cost-effective and versatile process for generating pDCs that does not require obtaining HSPCs. Therefore, the inventors provide a means of improved pDC production for use in adoptive cell therapy in the treatment of disease.

[0013] Strikingly, the Examples also demonstrate that using a culture medium comprising one or more agents associated with pDC differentiation to differentiate pluripotent stem cells into HSPCs improves the capacity of the HSPCs to be further differentiated into pDCs in a subsequent step. Accordingly, in preferred embodiments, step (a) comprises differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising one or more agents associated with pDC differentiation. The one or more agents associated with pDC differentiation may be selected from the list consisting of a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, an aryl hydrocarbon receptor (AhR) antagonist, a thrombopoietin (TPO) receptor agonist, IL-3, UM171 and ascorbic acid.

[0014] The Examples demonstrate that, for example, using a culture medium comprising a c- Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist is particularly effective for differentiating pluripotent stem cells into HSPCs, and for generating HSPCs that can be effectively differentiated into pDCs.

[0015] The Examples also demonstrate that, for example, using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid is effective for differentiating HSPCs, that are obtained from pluripotent stem cells, into pDCs.

[0016] In certain embodiments, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[0017] In certain embodiments, the medium used in step (a) is an embryoid body formation medium supplemented with c-Kit agonist, an FLT3 agonist, and an aryl hydrocarbon receptor (AhR) antagonist. In certain embodiments of the invention, the medium used in step (a) further comprises IL-3, or IL-3 and UM171. In certain embodiments of the invention, the AhR antagonist is StemRegenin 1. In certain embodiments of the invention, the c-Kit agonist is Stem Cell Factor (SCF). In certain embodiments of the invention, the FLT3 agonist is Fms-related tyrosine kinase 3 ligand (Flt3-L). In certain embodiments of the invention, the TPO receptor agonist is TPO. In certain embodiments of the invention, the ascorbic acid is L-ascorbic acid. The Examples demonstrate that these factors are particularly effective.

[0018] In certain embodiments of the invention, the pluripotent stem cells are genetically modified. The inventors have developed a method to produce pDCs that may be genetically modified. Thus, the pDCs of the invention may be genetically modified to enhance pDC functionality and / or reduce side effects of pDC for use in a method of treatment of human disease. This may lead to more effective treatments of said human disease.

[0019] In certain embodiments of the invention, the method comprises a step of genetically modifying the pluripotent stem cells. In certain embodiments of the invention, the step of genetically modifying the pluripotent stem cells comprises introducing a heterologous nucleic acid, optionally wherein the heterologous nucleic acid encodes a chimeric antigen receptor (CAR), or a synthetic RIP family receptor, optionally wherein the synthetic RIP family receptor is a synthetic Notch receptor (SynNotch receptor), a synthetic Robo receptor (SynRobo receptor), or a synthetic RoboNotch receptor, or synthetic intramembrane proteolysis receptor (SNIPR), or a Tango receptor, or a modular extracellular signalling architecture (MESA) system. In certain embodiments of the invention, the heterologous nucleic acid is introduced into the cell by transfection or transduction with a vector, optionally wherein the vector is a viral construct, optionally wherein the viral construct is an adenoviral construct, a lentiviral construct, or a retroviral construct, or an mRNA, optionally wherein the mRNA is delivered to the cell using a lipid nanoparticle (LNP), or a plasmid, or a cosmid. In certain embodiments of the invention, the heterologous nucleic acid is introduced into the cell by using site-specific DNA editing, optionally wherein the site-specific DNA editing is CRISPR / Cas, or TALEN, or zinc finger. Use of such genetic modifications in combination with the method of the invention for producing pDCs will provide particularly potent therapeutic products.

[0020] In certain embodiments of the invention, the HSPCs express CD34, and / or CD45, and / or CD31, and / or CD43, and / or CD117, and / or CD 123. In certain embodiments of the invention, the pDCs express CD304, and / or CD123, and / or CD303, and / or HLA-DR.

[0021] In certain embodiments, the HSPCs generated in step (a) and / or the pDCs generated in step (c) are isolated, in steps (b) and / or (d). Such isolation may be useful for generating purified cells and removing undifferentiated cells. However, such isolation may not be necessary, if for example steps (a) and (c) generate cell populations of the required purity.

[0022] In certain embodiments of the invention, step (d) comprises enriching by immunomagnetic separation. In certain embodiments of the invention, the enrichment by immunomagnetic separation is performed using HLA-DR, CD123, CD304 or CD303 as the target protein.

[0023] In certain embodiments of the invention, step (c) comprises isolating by enzymatic dissociation. In certain embodiments of the invention, step (c) comprises isolating by enzymatic dissociation using a protease enzyme, optionally wherein the protease is collagenase, trypLE express, trypsin, and / or accutase. In certain embodiments of the invention, the isolating by enzymatic dissociation comprises isolating the cells from the adherent layer, and / or suspension layer.

[0024] In certain embodiments of the invention, the pDCs respond to TLR-7 agonists, and / or TLR-9 agonists, and / or STING agonists. In certain embodiments of the invention, the pDC response is type I interferon (IFN) expression, optionally wherein the type I IFN is IFN-a. In certain embodiments of the invention, the method further comprises formulating the pDCs in a pharmaceutical composition. pDCs obtained from pluripotent stem cells in accordance with the invention are particularly suitable for use in pharmaceutical compositions.

[0025] The invention also provides a pDC obtained by the method of the invention.

[0026] The invention also provides a pDC obtained by the method of the invention for use in a method of treatment of a cancer, an autoimmune disease, or an inflammatory disease, wherein the method comprises administering a therapeutically effective amount of the pDC to the patient in need thereof.

[0027] In certain embodiments of the invention, the method further comprises priming step pDCs, optionally wherein the priming medium comprises type I IFNs, optionally wherein the type I IFN is IFN-0 and / or IFN-a, and / or type II IFNs, optionally wherein the type II IFN is IFN-y. In certain embodiments of the invention, the method does not further comprise a priming step the pDCs.

[0028] The pDCs of the invention are able to make responses to TLR agonists and / or STING agonists with or without prior priming using type I and / or type II IFN. Thus, the inventors have produced pDCs with enhanced functionality compared with previous methods. The absence of such need for type I and / or type II IFN priming enables said pDCs for use in a method of treatment of disease. Furthermore, unprimed pDCs for use in a method of treatment of disease may reduce the risk of hyper-activation of pDCs, which has been associated with pathogenesis.

[0029] In certain embodiments of the invention, the pDC obtained by the method is cryopreserved, optionally wherein the cryopreservation uses CryoStorlO, and / or is at -20°C or -80°C.

[0030] The inventors have developed a method of producing pDCs suitable for cryopreservation. Thus, the pDCs may be cryopreserved prior to pDCs for use in a method of treatment of a disease. This is beneficial for a patient in need thererof, without having to wait for the production of said pDCs. Furthermore, pDCs of the invention could be produced by the method prophylactically, for use in a method of treatment of a disease later in life when said patient may be in need thereof. Brief Description of the Figures

[0031] Figure 1. Exemplary illustration showing the generation of iPSC-derived pDCs for treatment of cancer. iPSCs or ES are cultured under specific conditions to first differentiate into mesoderm, followed specification into hematopoietic progenitor cells. Once hematopoietic cells are formed these cells are differentiated into pDCs. The pDCs can be cryopreserved as an off-the-shelf therapy for patients, in example a patient with a tumor.

[0032] Figure 2. Lymphoid progenitor specification of hematopoietic progenitors fails to produce cells expressing CD123 and CD303. a) Schematic overview showing overall setup. Briefly, iPSCs were seeded out as single-cells in Aggrewells in EB formation medium A and B (StemCell Technologies). Following 12 days of culture, setup is divided into two setups; for setup 1, cells are transferred to pDC differentiation following hematopoietic specification, whereas for setup 2, cells first undergo a lymphoid progenitor differentiation (using a lymphoid specification kit, StemCell Technologies) prior to differentiation into pDCs. b) Expression of the hematopoietic markers CD34 versus CD45 following hematopoietic specification (12-days). c) Numbers of cells during culture in pDC medium following 12- days of hematopoietic specification (setup 1). d) Expression of the pDC markers CD123 and CD303 during culture in pDC medium for cells derived from hematopoietic specification (post day 12) (setupl). e) Expression of the hematopoietic markers CD34 and CD45, as well as the lymphoid progenitor markers CD5 and CD7 following lymphoid progenitor specification (26-days) (setup 2). f) expansion of cells during lymphoid specification (post 12-days of hematopoietic specification) (setup 2). g) numbers of cells during culture in pDC medium following lymphoid progenitor specification (post day 26) (setup 2). h) Expression of the pDC markers CD 123 and CD303 during culture in pDC medium for cells derived from lymphoid progenitor specification (post day 26). Data shown is from one setup (setup 2).

[0033] Figure 3. Addition of cytokines and small-molecule inhibitors using a commercially available hematopoietic specification kit promotes expansion of iPSC-derived hematopoietic progenitors, and generation of cells expressing the pDC markers CD 123, CD303, CD304. a) Schematic overview showing overall setup. Briefly, iPSCs were seeded out as single-cells in Aggrewells in EB formation medium A and B (StemCell Technologies). Following 5-days of EB formation, medium was supplemented with the indicated cytokines and small-molecule inhibitors depicted as strategies F-K. After 14-days of hematopoietic specification, EBs were harvested, and cells were transferred to pDC medium on or tissue -treated (TC) culture plates. Cells were differentiated to pDCs over 21 -days of culture. During the pDC differentiation cells were taken out continuously to evaluate expression of pDC markers using flow cytometry, b) Numbers of cells after harvesting EBs following 14-days of hematopoietic specification for strategy F-K. c) Expression of CD45 versus CD34 on harvested EBs derived from strategy F-K. d-e) Fold expansion (d) and viability (e) through pDC differentiation of iPSC-derived HSPCs. f-g) Cells expressing pDC markers also show minor expression of CD56. (f) shows an example of a FACS plot of CD123 versus CD56 for strategy F at day 9 of pDC differentiation, whereas (g) shows percentage expression of CD3, CD14, CD19, CD56, and CD 123 for strategy F-K at day 9 of pDC differentiation, h) Example of gating strategy for strategy F at 12-days of pDC differentiation using spectral flow cytometry with a large panel consisting of 27 markers. Cells were initially gated based on FSC and SSC to exclude debris. Next, doublets were excluded in regards to FSC and SSC, and viable cells were evaluated for expression of CD 123 and CD66b. Cells expressing CD 123 or CD 123 (high) were next evaluated for expression of CD45RA and CD4, followed by CD56 / CD86. i) Expression of markers within the viable cell gate (all cells) shown in panel h. j) Expression of markers within the CD123(h) gate shown in panel h. k-m) During pDC differentiation expression of pDC markers were evaluated, (k) shows an example of a FACS plot showing expression of the pDC markers CD123 versus CD303 for viable cells at day 5, 9, 14 and 21 of pDC differentiation. Of note, cells within the CD123+ and CD303+ gate (Q6) were also found to be positive for CD304. 1-m) Graphs showing percentage expression of the CD123+CD303+CD304 (Q6) (1), or cells with high expression of CD123+CD303+CD304+ (+++) for cells taken out at different days during culture in pDC medium, n) At day 16 of pDC differentiation cells were taken out and primed for one day or left unprimed. Next, 2e5 cells were stimulated with the TLR7 agonist R837 (2.5 pg / mL), CpG-A (2.5 pg / mL), cGAMP (5 pg / mL), or left unstimulated. After 20 hours of stimulation, supernatants were harvested and levels of IFNa was evaluated by an IFNa ELISA. Data shown are + / - SEM of biological duplicates (n) for one setup.

[0034] Figure 4. Generation of iPSC-derived pDCs using a modified commercially available kit for hematopoietic specification of iPSCs. a) Schematic overview showing overall setup. Briefly, iPSCs were seeded out as single-cells in Aggrewells in EB formation medium A and B (StemCell Technologies). Following 5-days of EB formation, medium was supplemented with the indicated cytokines and small-molecule inhibitors depicted as strategy F and K. After 14-days of hematopoietic specification, EBs were harvested, and cells were transferred to pDC medium on tissue-coated plates. Cells were differentiated to pDCs over 17-days of culture. During the pDC differentiation cells were taken out continuously to evaluate expression of pDC markers using flow cytometry, b) Analysis of the expression of the hematopoietic markers CD31, CD34, CD43, CD45, CD117 and CD 123 following 14-days of hematopoietic specification for strategy F and K. c) Percentage expression of the hematopoietic markers for strategy F and K following 14-days of hematopoietic specification, d-f) Cell numbers (d), fold expansion (e), and viability (f) of cells through pDC differentiation for strategy F and K. g-h) Gating example for cells taken out at day 11 of pDC differentiation for strategy F (g) and strategy K (h). Cells were gated based on FSC and SSC to exclude debris. Next, doublets were excluded for FSC and SSC, respectively. Viable cells were analyzed for the expression of CD123, CD303, CD304, and HLA-DR. Of note, antibodies with modified Fc-part (REAfflnity) were used to exclude background staining, i) At day 17 of culture in pDC medium cells were taken out and sorted for CD304 using positive selection (Miltenyi). FACS plot shows the evaluation of pDC marker expression before sorting and after sorting for strategy F. j) Percentage expression of CD123+CD303+ population on live cells taken out during culture in pDC differentiation medium for strategy F. k) Cells were taken out during culture in pDC medium from strategy F and primed for 20 hours. Next, cells were stimulated with a TLR7 agonist (R837), TLR9 agonist (CpG-A), or left unstimulated. After 20 hours supernatants were harvested and levels of IFNa were evaluated by an IFNa ELISA. Data are + / - SEM of biological duplicates. I-m) Following positive selection for CD304 (described above), cells were primed for 20 hours. Next cells were stimulated with a TLR7 agonist (R837), TLR7 / 8 agonist (R848), a TLR9 agonist (CpG- A), or left unstimulated (UT) (1) or cells were stimulated with cGAMP or unstimulated (UT) (m). 20-hours later supernatants were harvested and levels of IFNa were evaluated by an IFNa ELISA. Data are + / - SEM of biological duplicates. Data is shown from two different experiments.

[0035] Figure 5. Generation of iPSC-derived pDCs using an optimized protocol for hematopoietic specification, a) Schematic illustration showing the generation of iPSC-derived pDC using an in-house specified protocol. iPSCs are initially seeded as single cells in aggrewells. During hematopoietic specification medium is changed at the indicated timepoints to reflect the stage of commitment. After 14-days of hematopoietic specification, cell suspension and adherent cells are harvested and resuspended in pDC medium to promote the differentiation of progenitors into pDCs. b-c) Analysis of the expression of the hematopoietic markers CD31, CD34, CD43, CD45, CD117 and CD 123 following 14-days of hematopoietic specification for suspension cells or adherent cells differentiated in medium either without SRI (b) or containing SRI (c). d) Percentage expression of the hematopoietic markers following 14-days of hematopoietic specification, e-g) Total live cell numbers (e), fold expansion (f) and viability (g) during culture of cells post hematopoietic specification in pDC differentiation medium, h-i) Flow plots of cells taken out at day 7 (h) or day 11 (i) of pDC differentiation. Cells were gated based on FSC and SSC to exclude debris. Next, doublets were excluded for FSC and SSC, respectively. Viable, CDllc negative or viable, CDllc positive cells were analyzed for the expression of CD123, CD303, CD304, and HLA-DR. Of note, antibodies with modified Fc-part (REAffinity) were used to exclude background staining. Data shown are from one experiment, j) At day 16 of culture in pDC medium cells harvested from the adherent layer were taken out and sorted for CD303 using positive selection (Miltenyi). Hereafter suspension cells and CD303 sorted adherent cells were primed for 20 hours. Next cells were stimulated with a TLR7 agonist (R837), a TLR7 / 8 agonist (R848), a TLR9 agonist (CpG-A), or left unstimulated (UT). 20-hours later supernatants were harvested and levels of IFNa were evaluated by an IFNa ELISA. Data are + / - SEM of biological duplicates.

[0036] Detailed Description of the Invention

[0037] Plasmacytoid dendritic cells (pDCs)

[0038] Plasmacytoid dendritic cells (pDCs) are a rare type of immune cell that are considered to be key in linking the innate and adaptive immune systems. They are known to secrete large quantities of type 1 interferon (IFNs) in response to a viral infection and are key effectors in cellular immunity with the ability to not only initiate immune responses but also to induce tolerance to exogenous and endogenous antigens. Plasmacytoid dendritic cells have been proposed for use in vaccines, where they present antigens and are delivered to lymph nodes to activate T-cells (Charles et al., 2020, Oncology, 9(1)). WO2018 / 206577 provides methods for generating populations of pDCs. WO2022 / 063818 describes engineered pDCs that express CAR or SynNotch constructs that activate particular immune responses in the treatment of disease. pDCs have a multifaceted role in the immune system, which makes them extremely adaptable for targeted treatments. pDCs are key effectors in cellular immunity with the ability to not only initiate immune responses but also to induce tolerance to exogenous and endogenous antigens (Swiecki, and Colonna, Nat Rev Immunol, 2015. 15(8)). pDCs are distinct from conventional DCs as their final stage of development occurs within the bone marrow; their antigens are taken up by receptor-mediated endocytosis; they express high levels of interferon regulatory factor 7; and they primarily sense pathogens through toll-like receptor (TLR) 7 and 9 (Swiecki and Colonna, Nat Rev Immunol, 2015. 15(8); and Tangand Cattral, Cell Mol Life Sci, 2016). Through these pattern-recognition receptors, pathogen nucleic acids can activate pDCs to produce high levels of type I interferon (IFN). Thus, activated pDCs link the innate and adaptive immune system together via cytokine production combined with antigen-presenting cell (APC) activity. Furthermore, pDC functionality is also essential to achieve an antiviral state during infections, provide vital adjuvant activity in the context of vaccination, and for promoting immunogenic anti-tumour responses upon activation (Swiecki, and Colonna, Nat Rev Immunol, 2015. 15(8); Tovey, et al. Biol Chem, 2008. 389(5); and Rajagopal, et al. Blood, 2010. 115(10): p. 1949-57). A delicate balance must be maintained, however, as hyper-activation of pDCs has been associated with the pathogenesis of several diseases, including viral infections, autoimmune diseases and tumourigenesis (Swiecki and Colonna, Nat Rev Immunol, 2015. 15(8); and Tang and Cattral, Cell Mol Life Sci, 2016).

[0039] In some embodiments, the pDCs produced according to the invention express CD304, CD123, CD303, CD4 and / or HLA-DR. Preferably, the pDCs produced according to the invention express IFN-a. In yet another embodiment said pDCs express type I IFN. pDCs also express type III IFN and / or pro inflammatory cytokines. In a further embodiment said pDCs express Toll-like receptor 7 (TLR-7), Toll-like receptor 9 (TLR-9), and / or STING protein. In yet another embodiment said pDCs respond to TLR-7 agonists, TLR-9 agonists, and / or STING agonists. Said pDCs express CD40, CD80, CD83 and / or CD86. For example, said pDCs express TRAIL, CD123, CD303, CD304, CD4, HLA-DR, type I IFN, and / or type III IFN, IRF7, TLR-7 and / or TLR-9.

[0040] The pDCs produced according to the invention are preferably matured cells having a surface phenotype that strongly resembles blood pDCs. In a preferred embodiment, said pDCs express CD123, CD303, CD304, CD4 and / or HLA-DR.

[0041] In another preferred embodiment said pDCs express type I IFN, type III IFN and / or proinflammatory cytokines.

[0042] The pDCs may in one preferred embodiment express Toll-like receptors, such as for example TLR-7 and / or TLR-9.

[0043] The pDCs may in one preferred embodiment express STING protein. In some embodiments, the pDCs produced according to the invention express CD73, CD28, CD69, TIM3, CD16, CD45RA, CD38, CD25, PD-1, CD45, CD123, CD27, CDllb, TIGIT, CD86, CD163, CD19, CD66b, CD4, CD3, ICOS, CD14, CD8, CDl lc, TCRgd, PD- Ll, or CD56. In some embodiments, the pDCs produced according to the invention express at least 2, 5, 10, 15, 20, or 25 markers selected from the list consisting of CD73, CD28, CD69, TIM3, CD16, CD45RA, CD38, CD25, PD-1, CD45, CD123, CD27, CDllb, TIGIT, CD86, CD163, CD19, CD66b, CD4, CD3, ICOS, CD14, CD8, CDllc, TCRgd, PD-L1 and CD56. In preferred embodiments, the pDCs produced according to the invention express, CD28, CD69, TIM3, CD16, CD45RA, CD38, CD25, PD-1, CD45, CD123, CD27, CDllb, TIGIT, CD86, CD163, CD19, CD66b, CD4, CD3, ICOS, CD14, CD8, CDl lc, TCRgd, PD- L1 and CD56.

[0044] In some embodiments, the pDCs of the invention are not primed. In other embodiments, the pDCs of the invention are primed by pro-inflammatory cytokine(s) to induce an activated state. The pro -inflammatory cytokine may be IFN. In one embodiment, the priming medium comprises type I IFN or type II IFN. The type I IFN may be IFN-0 and / or IFN-a. The type II IFNs may be IFN-y. pDCs produced by the invention can be for use in methods of treatment of diseases, wherein the unprimed pDCs can become activated after administration of the pDCs to the patent in need thereof.

[0045] As set out in the preceding paragraphs, pDCs are a rare cell type but their characteristics and functionality are well-characterized. pDCs have also been described as “innate lymphocytes” or “interferon-producing cells” (Ziegler-Heitbrock et al. , Nat Rev Immunol, 2023. 23, 1-2; and Hornero and Idoyaga, Molecular Immunol, 2023. 159, 38-45). Accordingly, any aspect or embodiment of the invention may relate to a method of generating innate lymphocytes or interferon-producing cells from pluripotent cells.

[0046] Methods for generating pDCs

[0047] The invention provides methods of generating plasmacytoid dendritic cells from pluripotent stem cells. The method comprises:

[0048] (a) differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs);

[0049] (b) optionally isolating the HSPCs;

[0050] (c) differentiating the HSPCs into pDCs; and (d) optionally isolating the pDCs.

[0051] In preferred embodiments, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[0052] In certain embodiments, step (a) comprises differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising one or more agents associated with pDC differentiation. Preferably, the one or more agents associated with pDC differentiation are selected from the list consisting of a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, an aryl hydrocarbon receptor (AhR) antagonist, a thrombopoietin (TPO) receptor agonist, IL-3, UM171 and ascorbic acid. In certain embodiments, at least 2, at least 3, at least 4, at least 5, at least 6 or such agents are used. Preferably, one agent from at least 2, at least 3, at least 4, at least 5, at least 6, or all 7 classes of agents are used.

[0053] In certain embodiments, step (a) comprises differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c- Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist.

[0054] In certain embodiments, step (c) comprises differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid.

[0055] In certain embodiments, the method of producing pDCs from pluripotent stem cells comprises:

[0056] (a) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist;

[0057] (b) optionally isolating the HSPCs;

[0058] (c) differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid; and

[0059] (d) optionally isolating the pDCs.

[0060] In certain embodiments, the medium to differentiate the pluripotent stem cells into HSPCs comprises embryoid body formation medium supplemented with c-Kit agonist, an FLT3 agonist, and an aryl hydrocarbon receptor (AhR) antagonist. An exemplary embryoid body formation medium for supplementation is EB formation medium, EB medium A and / or EB medium B from StemCell Technologies.

[0061] Accordingly, in certain embodiments, the method of producing pDCs from pluripotent stem cells comprises:

[0062] (a) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a embryoid body formation medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist;

[0063] (b) optionally isolating the HSPCs;

[0064] (c) differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid; and

[0065] (d) optionally isolating the pDCs.

[0066] The medium to differentiate the pluripotent stem cells into HSPCs may further comprise IL-3, or IL-3 and UM171.

[0067] Accordingly, in certain embodiments, the method of producing pDCs from pluripotent stem cells comprises:

[0068] (a) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, an aryl hydrocarbon receptor (AhR) antagonist, and IL-3;

[0069] (b) optionally isolating the HSPCs;

[0070] (c) differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid; and

[0071] (d) optionally isolating the pDCs.

[0072] In certain embodiments, the method provides producing pDCs from pluripotent stem cells, wherein the method comprises: (a) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, an aryl hydrocarbon receptor (AhR) antagonist, IL-3 and UM171;

[0073] (b) optionally isolating the HSPCs;

[0074] (c) differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid; and

[0075] (d) optionally isolating the pDCs.

[0076] In preferred embodiments, the method provides producing pDCs from pluripotent stem cells, wherein the method comprises:

[0077] (a) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising SCF, Flt3-L, SRI, and IL-3;

[0078] (b) optionally isolating the HSPCs;

[0079] (c) differentiating the HSPCs into pDCs using a culture medium comprising one or more of SCF, Flt3-L, SRI, TPO, IL-3 and L-ascorbic acid; and

[0080] (d) optionally isolating the pDCs.

[0081] In preferred embodiments, the AhR antagonist is StemRegenin 1. In other embodiments, the AhR antagonist is CH-223191. In other embodiments, the AhR antagonist is GNF351. In other embodiments, the AhR antagonist is PD98059. In other embodiments, the AhR antagonist is BAY 2416964. In other embodiments, the AhR antagonist is BAY- 218. In other embodiments, the AhR antagonist is PDM2.

[0082] In preferred embodiments, the c-Kit agonist used in the method is Stem Cell Factor (SCF).

[0083] In preferred embodiments, the FLT3 agonist is Fms-related tyrosine kinase 3 ligand (Flt3-L).

[0084] In preferred embodiments, the TPO receptor agonist is TPO. In other embodiments, the TPO receptor agonist is AMG-531. In other embodiments, the TPO receptor agonist is SB-497115. In preferred embodiments, the ascorbic acid is L-ascorbic acid. In other embodiments, the ascorbic acid is D-ascorbic acid.

[0085] In any method of the invention, the differentiation factors are used at a concentration effective to promote differentiation to HSPCs or to pDCs.

[0086] The method of the invention may comprise differentiating the pluripotent stem cells into mesoderm cells prior to differentiation into HSPCs.

[0087] The method of the invention may not comprise differentiating the pluripotent stem into lymphoid progenitor cells prior to differentiation into HSPCs.

[0088] The methods of the invention may further comprise a step of enriching the pDCs using immunomagenetic separation.

[0089] Thus in some embodiments, the method of producing pDCs from pluripotent stem cells comprises:

[0090] (a) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist;

[0091] (b) isolating the HSPCs;

[0092] (c) differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid;

[0093] (d) isolating the pDCs; and

[0094] (e) enriching by immunomagnetic separation.

[0095] In preferred embodiments, the step of immunomagnetic separation may be performed using CD304 as the target protein. In other preferred embodiments, the step of immunomagnetic separation may be performed using CD303, CD 123 or HLA-DR as the target protein.

[0096] The methods of the invention may further comprise a step of isolating the HSPCs using enzymatic dissociation.

[0097] Thus in some embodiments, the method of producing pDCs from pluripotent stem cells comprises: (a) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist;

[0098] (b) isolating the HSPCs by enzymatic dissociation;

[0099] (c) differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid; and

[0100] (d) isolating the pDCs.

[0101] In preferred embodiments, the step of enzymatic dissociation may be performed using a protease enzyme. In preferred embodiments, the step of enzymatic dissociation may be performed using collagenase and / or trypLE express. In other embodiments, the step of enzymatic dissociation may be performed using trypsin, and / or accutase. In preferred embodiments, the step of enzymatic dissociation may be performed by isolating the cells from the adherent layer. In other preferred embodiments, the step of enzymatic dissociation may be performed by isolating the cells from the suspension layer.

[0102] In certain embodiments, pluripotent stem cells are differentiated into HSPCs in bulk. Alternatively, a single pluripotent stem cell or a single clone population are differentiated.

[0103] Any appropriate culture format may be used. For scaled-up manufacturing, alternative approaches may be used. For example, in certain embodiments, steps (a) and / or (c) comprise suspension culture, for example, dynamic suspension culture. Optional bioreactors for use in step (a) and / or step (c) include wave bioreactors, rotating wall bioreactors, stirred tank bioreactors and rotaing Erlenmeyer flasks. Potential culture strategies include microcarrier and aggregates. Bioreactor systems and dynamic suspension culture are useful in order to reduce environmental inhomogeneity. The rotating wall bioreactor (RWB) is a cylindrical culture vessel that maintains continuous rotation along its longitudinal axis and horizontal axis. Depends on the rotational speed, the immersed cells can be dynamically suspended in quasi-periodic circular motion. The stirred tank bioreactor (STBR) consists of a glass or plastic vessel with an internal impeller and enables keeping cells in suspension. The culture environment, including pH and temperature can be detected by integrated probes. Scale-up culture may also be improved using cell immobilisation, for example on microcarriers, or matrix-free cell aggregation. In preferred embodiments, the pluripotent stem cells are hypoimmune stem cells, in particular hypoimmune iPSCs. Use of such cells may allow the generation of an allogenic pDCs product. The generation of hypoimmune stem cells and particularly hypoimmune iPSCs is known in the art. Exemplary methods include depleting expression of major histocompatibility complex (MHC) genes, optionally with expression of CD47 (as described in, for example, Deuse et al., Nat Biotechnol. 2019 Mar;37(3):252-258). Genes that may be targeted for disruption, for example with Cas9 nuclease and guides include the B2m and Ciita genes. Similarly, HLA genes or expression may be ablated. Any appropriate technique may be used.

[0104] The extent of differentiation of the cell population during each step may be determined by monitoring and / or detecting the expression of one or more cell markers in the population of differentiating cells. For example, an increase in the expression of markers characteristic of the more differentiated cell type or a decrease in the expression of markers characteristic of the less differentiated cell type may be determined. The expression of cell markers may be determined by any suitable technique, including immunocytochemistry, immunofluorescence, RT-PCR, immunoblotting, fluorescence activated cell sorting (FACS), and enzymatic analysis. In preferred embodiments, a cell may be said to express a marker if the marker is detectable on the cell surface. For example, a cell which is stated herein not to express a marker may display active transcription and intracellular expression of the marker gene, but detectable levels of the marker may not be present on the surface of the cell.

[0105] A population of partially differentiated cells that is produced by a step in the methods described herein may be cultured, maintained or expanded before the next differentiation step. Partially differentiated cells may be expanded by any convenient technique.

[0106] After each step, the population of partially differentiated cells which is produced by that step may contain 1% or more, 5% or more, 10% or more or 15% or more partially differentiated cells, following culture in the medium. If required, the population of partially differentiated cells may be purified by any convenient technique, such as MACs or FACS.

[0107] Suitable techniques for cell culture are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451 ; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293, Ho WY et al J Immunol Methods. (2006) 310:40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430) Basic Cell Culture Protocols’ by J. Pollard and J. M. Walker (1997), ‘Mammalian Cell Culture: Essential Techniques’ by A. Doyle and J. B. Griffiths (1997), ‘Human Embryonic Stem Cells’ by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside’ by A. Bongso (2005), Peterson & Loring (2012)Human Stem Cell Manual: A Laboratory Guide Academic Press and ‘Human Embryonic Stem Cell Protocols’ by K. Turksen (2006). Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be employed for the above culture steps, for example 37°C, 5% or 21% Oxygen, 5% Carbon Dioxide. Media is preferably changed every two days and cells allowed to settle by gravity. Cells may be cultured in a culture vessel. Suitable cell culture vessels are well-known in the art and include culture plates, dishes, flasks, bioreactors, and multi-well plates, for example 6-well, 12-well or 96-well plates.

[0108] The methods of the invention may comprise differentiating pluripotent stem cells that are genetically modified. Preferred forms of genetic modification are discussed in the following paragraphs.

[0109] The methods of the invention may further comprise a step of genetically modifying the pDCs.

[0110] Accordingly, in certain embodiments, the method provides producing pDCs from pluripotent stem cells, wherein the method comprises:

[0111] (a) genetically modifying pluripotent stem cells;

[0112] (b) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs);

[0113] (c) optionally isolating the HSPCs;

[0114] (d) differentiating the HSPCs into pDCs; and

[0115] (e) optionally isolating the pDCs.

[0116] In preferred embodiments, the method provides producing pDCs from pluripotent stem cells, wherein the method comprises:

[0117] (a) genetically modifying pluripotent stem cells; (b) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist;

[0118] (c) optionally isolating the HSPCs;

[0119] (d) differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, an FLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid; and

[0120] (e) optionally isolating the pDCs.

[0121] The step of genetically modifying the pluripotent stem cells may include introducing a heterologous nucleic acid into the cell. Introducing a heterologous nucleic acid may be performed by transfecting or transducing with a vector said pluripotent stem cells prior to differentiation. The transfecting or transducing may be performed in the HPSCs subsequent to differentiation from pluripotent stem cells to HSPCs, or the transfecting or transducing may be performed in the pDCs subsequent to differentiation from HSPCs to pDCs. In some embodiments, the heterologous nucleic acid encodes a chimeric antigen receptor (CAR). In some embodiments, the heterologous nucleic acid encodes a synthetic RIP family receptor, optionally wherein the synthetic RIP family receptor is a synthetic Notch receptor (SynNotch receptor), a synthetic Robo receptor (SynRobo receptor), or a synthetic RoboNotch receptor. In some embodiments, the heterologous nucleic acid encodes a synthetic intramembrane proteolysis receptor (SNIPR). In some embodiments, the heterologous nucleic acid encodes a Tango receptor. In some embodiments, the heterologous nucleic acid encodes a modular extracellular signalling architecture (MESA) system. In preferred embodiments, any such receptor comprises an extracellular antigen recognition domain that recognises a tumour- associated antigen. In certain embodiments, the tumour antigen is selected from the group consisting of CD19 , CD20, CD22, CD30, CD33, CD38, CD123, CD138, CS-1, B-cell maturation antigen (BCMA), MAGEA3, MAGEA3 / A6, KRAS, CLL1, MUC-1, HER2, EpCam, GD2, GPA7, PSCA, EGFR, EGFRvIII, R0R1, mesothelin, CD33 / IL3Ra, c-Met, CD37, PSMA, Glycolipid F77, GD-2, gplOO, NY-ESO-1 TCR, FRalpha, CD24, CD44, CD133, CD166, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6, or a combination thereof. In some embodiments, the heterologous nucleic acid is introduced into the cell by transfection or transduction with a vector encoding a synthetic receptor as described herein.

[0122] Transfecting or transducing the cells can be achieved by any appropriate technique.

[0123] The vector may be an appropriate vector, such as selected from the group consisting of a viral construct, an mRNA, a plasmid or a cosmid.

[0124] In some embodiments the vector is a viral construct. In some embodiments, the viral construct is an AAV construct, an adenoviral construct, a lentiviral construct, or a retroviral construct. The construct may comprise a reporter gene such as GFP, mCherry, truncated EGFR, or truncated tNGFR. In some embodiments, the viral construct is a lentiviral construct and transduction is performed using retronectin-coated plates or using lentiboost and protamine sulfate. In preferred embodiments of the invention, the method includes the step of transducing the pluripotent stem cells with a viral construct before the step of differentiating the pluripotent stem cells into HSPCs. In other embodiments of the invention, the method includes the step of transducing the HSPCs with a viral construct before the step of differentiating the HSPCs into pDCs. pDCs differentiated from HSPCs transduced with a viral construct encoding an antigen may stably express the antigen.

[0125] In some embodiments, the vector is an mRNA encoding an antigen. The mRNA may be delivered to the cell using any appropriate technique, such as electroporation or using a lipid nanoparticle (LNP). In a preferred embodiment, the step of transfecting the pDCs with an LNP comprising an mRNA encoding an antigen occurs after the step of differentiating the HSPCs into pDCs. pDCs transfected with an LNP comprising an mRNA encoding an antigen may transiently express the antigen.

[0126] In certain embodiments, the heterologous nucleic acid is introduced using a sitespecific DNA editing such as TALEN, zinc finger or CRISPR / Cas. In some embodiments the site-specific DNA editing is CRISPR / Cas. In some embodiments the site-specific DNA editing is TALEN. In some embodiments the site-specific DNA editing is zinc finger nuclease.

[0127] In preferred embodiments, pluripotent stem cells are transfected or transduced and then differentiated into HSPCs followed by pDCs.

[0128] In some embodiments, CD34+ HSPC derived from pluripotent stem cells are transfected or transduced and then differentiated into pDCs. In some embodiments, the produced pDCs of the invention are cryopreserved.

[0129] In some embodiments, the produced pDCs of the invention are cryopreserved before enrichment by immunomagnetic separation of the pDCs. In some embodiments, the produced pDCs of the invention are cryopreserved after enrichment by immunomagnetic separation of the pDCs.

[0130] In some embodiments, the pDC produced by the invention is cryopreserved using uses CryoStorlO.

[0131] In some embodiments, the pDC produced by the invention is cryopreserved at -20°C. In other embodiments, the pDC produced by the invention is cryopreserved at -80°C.

[0132] In some embodiments, the invention uses pDCs derived from pluripotent stem cells. In preferred embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0133] “Pluripotent stem cells” as used herein are a type of cell that are undifferentiated and have self-renewing capacity. Thus, pluripotent stem cells can divide indefinitely. Pluripotent stem cells also have the capacity to develop into any cell type. Therefore, pluripotent stem cells can be used to induce differentiation into other cell types including immune cells, such as pDCs. Pluripotent stem cells can be separated into two types, that are, induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs). ESCs may be derived from parthenogenetically activated human oocytes. In preferred embodiments, ESCs are not derived from human embryos. iPSCs can be obtained by reprogramming somatic cells. iPSCs can be generated from adult fibroblast cultures. For example, iPSCs were generated from adult fibroblast cultures by introducing the four factors Oct3 / 4, Sox2, c-Myc, and Klf4 in Takahashi and Kazutoshi. Cell. 2006; 126(4): 663-676.

[0134] HSPCs can be generated from iPSCs, allowing for iPSC-derived pDCs. HSPCs can also be obtained from a patient or a donor. “Hematopoietic stem and progenitor cells” (HSPCs) as used herein are multipotent stem cells that are capable of giving rise to all blood cell types including myeloid lineages and lymphoid lineages. Myeloid lineages may for example include monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets and dendritic cells, whereas lymphoid lineages may include T-cells, B-cells and NK-cells.

[0135] In a preferred embodiment HSPCs are positive for the marker CD34. In another preferred embodiment HSPCs are positive for the marker CD45.

[0136] In another preferred embodiment HSPCs are positive for the marker CD31.

[0137] In another preferred embodiment HSPCs are positive for the marker CD43.

[0138] In another preferred embodiment HSPCs are positive for the marker CD 117.

[0139] In another preferred embodiment HSPCs are positive for the marker CD 123.

[0140] Thus in a preferred embodiment, the HSPCs are positive for the markers CD34, CD45, CD31, CD43, CD 117, and / or CD 123.

[0141] In some embodiments, the HSPCs are isolated from the adherent layer following differentiation in culture from pluripotent stem cells.

[0142] In some embodiments, the HSPCs are isolated from the suspension layer following differentiation in culture from pluripotent stem cells.

[0143] The pluripotent stem cells are incubated in the medium under conditions that are typical for human cell cultures and well known to the skilled person, in combination with the additional factors identified by the inventors. The HSPCs are incubated in the medium under conditions that are typical for human cell cultures and well known to the skilled person, in combination with the additional factors identified by the inventors. Typical conditions for incubation of cell cultures are for example a temperature of 37 °C, 95% humidity and 5% CO2.

[0144] The culture medium may be supplemented with conventional cell culture components such as serum, such as for example fetal calf serum, b-mercaptoethanol, antibiotics, such as penicillin and / or streptomycin, nutrients, and / or nonessential amino acids. Conventional cell culture components can also be substituted for conventional serum-free medium supplemented with conventional penicillin and / or streptomycin.

[0145] In one embodiment the pluripotent stem cells are incubated for at least 1 day, such as at least 2 days, at least 3 days, such as for example at least 4 days, such as at least 5 days, at least 6 days, such as for example at least 7 days, such as at least 8 days, at least 9 days, such as for example at least 10 days, such as at least 12 days, at least 14 days in the culture medium.

[0146] In one embodiment the HSPCs are incubated for at least 1 day, such as at least 2 days, at least 3 days, such as for example at least 4 days, such as at least 5 days, at least 6 days, such as for example at least 7 days, such as at least 8 days, at least 9 days, such as for example at least 10 days, such as at least 12 days, at least 14 days, such as at least 15 days, at least 16 days, such as for example at least 17 days, such as at least 18, at least 19 days, at least 20 days, such as for example at least 21 days in the culture medium.

[0147] In one embodiment the culture medium is refreshed during the incubation period. The culture medium may for example be refreshed every second day, every third day or every fourth day during the incubation period. Preferably the culture medium is refreshed with medium comprising the cytokines and / or small molecule inhibitors.

[0148] In some embodiments, the methods may include a step of priming the pDCs before administration. In some embodiments, the step of priming the pDCs comprises incubating the pDCs with type I IFN and / or type II IFN. The pDCs may be incubated with the type I IFN and / or type II IFN for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours or at least 24 hours. In a preferred embodiment, the pDCs are incubated with the type I IFN and / or type II IFN for 24 hours. The step of priming the pDCs may increase the level of IFNa expressed by the pDCs. This may improve the ability of the pDCs to activate and / or pre-condition an adoptive cell transfer immunotherapy.

[0149] Methods for treating disease

[0150] The invention provides a pDC for use in a method of treating a disease in a subject comprising administering the pDC to the subject. Therefore, the invention provides a pDC for use in a new adoptive cell therapy. Adoptive cell therapy is the transfer of ex vivo grown cells, most commonly immune-derived cells, into a host with the goal of transferring the immunologic functionality and characteristics of the transferred cells. Adoptive cell therapy is well established for treating cancer and autoimmune, inflammatory and infectious diseases, albeit using different transferred immune cells with different immune -regulating effects and activities. In certain embodiments of the invention is a pDC obtained by the invention for use in a method of treatment, the method comprising: (i) providing pluripotent stem cells; (ii) producing pDCs, for example using a method discussed herein; (iii) optionally administering to the subject lymphodepleting chemotherapy; and (iv) administering to the subject the pDCs. In some embodiments of the invention is a pDC obtained by the invention further comprising formulating the pDCs in a pharmaceutical composition for use in a method of treatment, the method comprising: (i) providing pluripotent stem cells; (ii) producing pDCs, for example using a method discussed herein; (iii) optionally administering to the subject lymphodepleting chemotherapy; and (iv) administering to the subject the pDCs.

[0151] In certain embodiments, the pDCs may be isolated and used fresh, or frozen for later use, in conjunction with (e.g., before, simultaneously or following) lymphodepletion.

[0152] In certain embodiments, the pDCs may be administered to the subject by dose fractionation, wherein a first percentage of a total dose is administered on a first day of treatment, a second percentage of the total dose is administered on a subsequent day of treatment, and optionally, a third percentage of the total dose is administered on a yet subsequent day of treatment.

[0153] An exemplary total dose comprises 103to 1011cells / kg body weight of the subject, such as 103to IO10cells / kg body weight, or 103to 109cells / kg body weight of the subject, or 103to 108cells / kg body weight of the subject, or 103to 107cells / kg body weight of the subject, or 103to 106cells / kg body weight of the subject, or 103to 105cells / kg body weight of the subject. Moreover, an exemplary total dose comprises 104to 1011cells / kg body weight of the subject, such as 105to 1011cells / kg body weight, or 106to 1011cells / kg body weight of the subject, or 107to 1011cells / kg body weight of the subject.

[0154] An exemplary total dose may be administered based on a patient body surface area rather than the body weight. As such, the total dose may include 103to 1013cells per m2.

[0155] In certain embodiments of the invention, the methods comprise lymphodepletion. Lymphodepletion may be achieved by any appropriate means. Lymphodepletion may be performed prior to administration of the pDCs, or subsequent to. In certain embodiments, lymphodepletion is performed both before and after administration of the pDCs.

[0156] In certain embodiments of the invention, the methods may comprise administration of one or more additional therapeutic agents. Exemplary therapeutic agents include a chemotherapeutic agent, an anti-inflammatory agent, an immunosuppressive, an immunomodulatory agent, or a combination thereof.

[0157] Therapeutic agents may be administered according to any standard dose regime known in the field. Exemplary chemotherapeutic agents include anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine. Exemplary chemotherapeutic agents include a topoisomerase inhibitor, such as topotecan. Exemplary chemotherapeutic agents include a growth factor inhibitor, a tyrosine kinase inhibitor, a histone deacetylase inhibitor, a P38a MAP kinase inhibitor, inhibitors of angiogenesis, neovascularization, and / or other vascularization, a colony stimulating factor, an erythropoietic agent, an anti-anergic agents, an immunosuppressive and / or immunomodulatory agent, a virus, viral proteins, immune checkpoint inhibitors, BCR inhibitors (e.g., BTK, P13K, etc.), immune -metabolic agents (e.g., IDO, arginase, glutaminase inhibitors, etc.), and the like. According to certain aspects of the present invention, the one or more therapeutic agents may comprise an antimyeloma agent. Exemplary antimyeloma agents include dexamethasone, melphalan, doxorubicin, bortezomib, lenalidomide, prednisone, carmustine, etoposide, cisplatin, vincristine, cyclophosphamide, and thalidomide, several of which are indicated above as chemotherapeutic agents, anti-inflammatory agents, or immunosuppressive agents.

[0158] Treatment of a disease, according to the invention, refers to a biological effect that may present as a decrease in disease burden, disease incidence or disease severity. For example, in relation to cancer treatment, this may manifest as a reduction in tumour volume, a decrease in the number of tumour cells, a decrease in tumour cell proliferation, a decrease in the number of metastases, an increase in overall or progression- free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumour.

[0159] The pDCs of the invention may be administered by any appropriate route. Generally, the cells will be administered by intravenous infusion.

[0160] In certain embodiments, pDCs are autologous. Such treatments may minimise any risk of rejection of the transferred cells. In preferred embodiments, the cells are allogenic, such as isolated from healthy donors. Such treatments can potentially be prepared more quickly and offered “off the shelf’. In certain embodiments, the cells are or have been cryopreserved. Moreover, the cells may be xenogeneic.

[0161] Methods for treating cancer

[0162] In preferred embodiments of the invention, the pDCs of the invention are for use in treating cancer. The ability of pDCs to secrete immunomodulatory factors and alter immune- inhibiting tumour microenvironments, are expected to be particularly useful for treating cancer. Also, cancer cells present specific antigens that can be recognised by extracellular antigen recognition domains to provide targeted therapy at tumour sites, avoiding healthy cells.

[0163] In preferred embodiments, the cancer is a solid tumour. The pDCs of the invention may be particular effective at targeting and altering the micro environment of solid tumours. Accordingly, in certain embodiments, the cancer is a solid cancer, such as a cancer selected from the group consisting of: bone cancer, breast cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, cancer of the anal region, colon cancer, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, pediatric tumors, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, glioblastoma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer and squamous cell cancer.

[0164] In certain embodiments, the pDCs of the invention are for use in treating acute lymphoblastic leukemia (ALL) (including non T cell ALL), acute myeloid leukemia, B cell prolymphocytic leukemia, B-cell acute lymphoid leukemia (“BALL”), blastic plasmacytoid dendritic cell neoplasm, Burkitt s lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's Disease, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, monoclonal gammapathy of undetermined significance (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorder (including asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytomas (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B cell lymphoma (PMBC), small cell- or a large cell-follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia (“TALL”), T-cell lymphoma, transformed follicular lymphoma, or Waldenstrom macroglobulinemia, or a combination thereof. In some embodiments, the cancer is a myeloma. In one particular embodiment, the cancer is multiple myeloma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is acute myeloid leukemia. In some embodiments, the cancer is relapsed or refractory large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, high grade B- cell lymphoma, or DLBCL arising from follicular lymphoma.

[0165] In some embodiments, the methods further comprise administering a chemotherapeutic. In certain embodiments, the chemotherapeutic selected is a lymphodepleting (preconditioning) chemotherapeutic, and is preferably administered before the cells of the invention. Such administration of a chemotherapeutic may improve survival of the transplanted cells.

[0166] In preferred embodiments, the pDC of the invention is for use in treating cancer.

[0167] In the invention, the pDC are administered to a subject already suffering from cancer, in an amount sufficient to cure, alleviate or partially arrest the cancer or one or more of its symptoms. Such therapeutic treatment may result in remission, stabilisation, reduction in metastasis or elimination of the cancer. An amount adequate to accomplish this is defined as "therapeutically effective amount". The subject may have been identified as suffering from cancer and being suitable for an adoptive cell transfer immunotherapy by any suitable means.

[0168] Methods for treating autoimmune and inflammatory diseases

[0169] In further embodiments of the invention, the methods and cells of the invention are for use in treating an autoimmune or inflammatory disease. The ability of pDCs to secrete immunomodulatory factors and alter inflammatory tissues or sites of autoimmune attack are expected to be particularly useful for treating autoimmune and inflammatory diseases. Also, autoimmune diseases are often triggered by specific autoantigens that are targeted by the patient’s immune system and these autoantigens can be recognised by extracellular antigen recognition domains to provide targeted therapy to sites of inflammation. Similarly, specific tissues that are inflamed, or that have been transplanted, can be targeted using the extracellular antigen recognition domain in the cells of the invention.

[0170] Targeting engineered pDCs to inflamed tissue and sites of inflammatory disease is expected to be effective because depletion of pDCs (in a murine asthma model) leads to T cell-mediated hyper-responsiveness, resulting in breakdown of tolerance (see Jan de Heer, 2004, J Exp Med, 200(l):89-98). pDCs are also important for long-term graft survival after allogeneic stem cell transplantation (see Peric et al. Biol Blood Marrow Transplant, 2015, 21(8), Goncalves et al. Biol Blood Marrow Transplant, 21(7), and Waller et al. J Clin Oncol, 2014, 32(22)). Also, high expression of PD-L1 / CD86 in pDCs correlate with increased T- regs in liver transplant tolerance (see Tokita et al., Transplantation. 2008, 85(3):369-77). Also, adoptive transfer (pre-operative) of donor pDCs highly promotes heart transplant survival (see PMID: Bjorck et al. J Heart Lung Transplant, 2005, 24(8) and Abe et al., Am J Transplant, 2005, 5(8)).

[0171] In certain embodiments, the autoimmune disease is selected from the list consisting of: type 1 diabetes, thyroid autoimmune diseases (e.g. Hashimoto’s and Graves’), Addison’s adrenal insufficiency, oophoritis, orchitis, lymphocytic hypophysitis, autoimmune hypoparathyroidism, autoimmune hypoparathyroidism, Goodpasture’s disease, autoimmune myocarditis, membranous nephropathy, autoimmune hepatitis, ulcerative colitis, Crohn’s disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica.

[0172] In certain embodiments, the inflammatory disease is selected from the list consisting of: cystic fibrosis, chronic inflammatory intestinal diseases like, for example, ulcerative colitis or Crohn's disease, vasculitis, in particular Kawasaki disease, chronic bronchitis, inflammatory arthritis diseases like, for example, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, and systemic onset juvenile rheumatoid arthritis (SOJRA, Still's disease), graft- versus-host disease, asthma, psoriasis, systemic lupus erythematosus and allograft rejection.

[0173] In certain embodiments, the autoimmune or inflammatory disease is transplant rejection or graft- versus-host-disease (GVHD). Exemplary organ transplant to be treated according to the invention include: kidney, heart, lung, liver, intestine, pancreas and islet of Langerhans.

[0174] The extracellular antigen recognition domain in the exogenous construct is selected to target the autoantigen associated with the disease to be treated. Exemplary antigens to be targeted include (with the associated disease to be treated in parenthesis): GAD65 (type 1 diabetes), insulinoma associated protein 2 - IA-2 (type 1 diabetes), thyroid peroxidase - TPO (thyroid autoimmune diseases, Hashimoto’s and Graves’), thyrotropin receptor - TSHR (thyroid autoimmune diseases, Hashimoto’s and Graves’), adrenocorticotropic hormone receptor - ACTHR (Addison’s adrenal insufficiency), 21 -hydroxylase (Addison’s adrenal insufficiency), 17-alpha-hydroxylase (oophoritis), P450 side-chain cleavage enzyme

[0175] (oophoritis), sperm antigens (orchitis), pituitary cytosolic protein (lymphocytic hypophysitis), calcium sensing receptor - CaSR (autoimmune hypoparathyroidism), NACHT LRR and PYD domains-containing protein 5 - NALP5 (autoimmune hypoparathyroidism), alpha 3 chain of type IV collagen (Goodpasture’s disease), myosin (Autoimmune myocarditis), Type-M phospholipase A2 receptor - PLA2R (membranous nephropathy), Cytochrome P450 1 A2 (autoimmune hepatitis), tropomyosin isoform 5 - hTM5 (ulcerative colitis), major zymogen granule membrane glycoprotein 2 -GP2 (Crohn’s disease), myelin basic protein - MBP (multiple sclerosis), nicotinic acetylcholine receptor - AChR (myasthenia gravis), aquaporin- 4 - AQP4 (neuromyelitis optica).

[0176] When used to treat transplant rejection, the extracellular antigen recognition domain in the exogenous construct is selected to target the appropriate organ. Exemplary antigens to be targeted include (with the associated organ in parenthesis): GAD65 (pancreas or islet of Langerhans), insulinoma associated protein 2 - IA-2, (pancreas or islet of Langerhans), type- M phospholipase A2 receptor - PLA2R (kidney), myosin (heart), alpha 3 chain of type IV collagen (lung), cytochrome P450 1A2 (liver), major zymogen granule membrane glycoprotein 2 - GP2 (intestine), tropomyosin isoform 5 - hTM5 (intestine).

[0177] In preferred embodiments, the method or cell of the invention is for use in treating an autoimmune or inflammatory disease and the cell is an engineered stem cell-derived plasmacytoid dendritic cell that expresses an exogenous chimeric antigen receptor or synNotch receptor comprising an extracellular antigen recognition domain that recognises an autoantigen or a tissue specific antigen, a transmembrane domain, and an intracellular signalling domain that activates an anti-inflammatory immune response in the cell when the extracellular antigen recognition domain binds the target cell.

[0178] In the therapeutic methods of the invention, engineered cells are administered to a subject already suffering from an autoimmune disease, in an amount sufficient to cure, alleviate or reduce the frequency of one or more symptoms. An amount adequate to accomplish this is defined as "therapeutically effective amount". The subject may have been identified as suffering from an autoimmune disease and being suitable for an adoptive cell transfer immunotherapy by any suitable means.

[0179] Examples

[0180] Example 1 - pDCs can be produced from iPSCs pDCs are generated from iPSCs using a setup without lymphoid progenitor differentiation. Figure 2a illustrates two different setups: setup 1 consisting of a 33 day culture period; and setup 2 consisting of a 40 day culture period. In both setup 1 and setup 2, iPSCs were seeded in Aggrewell plates as single-cells in EB formation medium A and B (StemCell Technologies) for 12 days. At day 12, cells were transferred to pDC differentiation following hematopoietic specification (setup 1), or cells first undergo a lymphoid progenitor differentiation (using a lymphoid specification kit, StemCell Technologies) prior to the differentiation into pDCs (setup 2). Flow cytometry phenotypic assessment of HSPC marker expression after 12 days of culture was then performed. Flow cytometry plots of the haematopoietic markers CD34 and CD45 showed that a large proportion of cells expressed CD34, and a proportion of these cells also expressed CD45 (Figure 2b). Using setup 1, the number of cells were calculated over 21 days in culture of pDC medium post-haematopoietic specification (post day 12) and demonstrated that the cells did not expand during pDC differentiation (Figure 2c). Flow cytometry phenotypic assessment of pDC marker expression post day 12 during culture in pDC medium for cells derived from hematopoietic specification (setup 1) was then performed. Flow cytometry plots of the pDC markers CD 123 and CD303 showed that the cells expressed CD123 and CD303 at day 7, 9, 12, 14, 16, 19, 21, and day 23 (Figure 2d). Flow cytometry phenotypic assessment of HSPC cell marker expression and lymphoid progenitor cell marker expression at day 26 (setup 2) was then performed. Flow cytometry plots of the haematopoietic markers CD34 and CD45 and the lymphoid progenitor cell markers CD5 and CD7 showed that the cells expressed these markers and differentiated into the lymphoid progenitor cells (Figure 2e). Using setup 2, the number of cells were calculated at at day 0 and day 14 of lymphoid specification (post day 12 of hematopoietic specification, Figure 2f), and during culture in pDC medium at day 0, 2, 5, 7, and day 9 following lymphoid progenitor specification (post day 26, Figure 2g). The results demonstrated that the cells expanded during lymphoid progenitor differentiation but not during the pDC differentiation. Flow cytometry phenotypic assessment of pDC marker expression during culture in pDC medium for cells derived from lymphoid progenitor cells (setup 2) was then performed. Flow cytometry plots of the pDC markers CD 123 and CD303 showed that the cells did not express both CD123 and CD303 at day 2, 7, 9, and day 12 (Figure 2h). Example 1 shows that CD123+ CD303+ pDCs can be generated from iPSCs using a setup without lymphoid progenitor differentiation.

[0181] Example 2 - Functional pDCs can be produced from iPSCs using medium supplemented with cytokines and small molecule inhibitors

[0182] Functional pDCs are generated from iPSCs using medium supplemented with cytokines and small molecule inhibitors at day 5 of culture. Figure 3a illustrates the overall setup of producing pDCs from iPSCs over a total culture period of 30 days (from day -14 to day 16). iPSCs were seeded in Aggrewell plates as single-cells in EB formation medium A and B (StemCell Technologies) or DC medium for 14 days (day -14 to day 0). Following 5 days of EB formation (day -9), different cytokines and small molecule inhibitors were added to the medium according to different strategies (Strategy F - K):

[0183] At day 0, After 14-days of hematopoietic specification (day 0), EBs were harvested and cells were transferred to pDC medium on or tissue-treated (TC) culture plates. Cells were differentiated to pDCs over 21 -days of culture. During the pDC differentiation cells were taken out continuously to evaluate expression of pDC markers using flow cytometry. The number of cells were calculated after harvesting EBs following 14 days of hematopoietic specification and demonstrated that the total number of cells differed between strategy F-K (Figure 3b). Flow cytometry phenotypic assessment of HSPC cell marker expression after harvesting EBs following 14 days of hematopoietic specification was then performed. Flow cytometry plots of the haematopoietic markers CD34 and CD45 demonstrated that the cells expressed differing levels of CD34 and CD45 expression between strategy F-K, and that strategy F, G and J generated iPSC-derived hematopoietic cells expressing both CD34+ and CD45+ (Figure 3c). The fold expansion (Figure 3d) and the percentage of viable cells (Figure 3e) were then calculated at days 0, 2, 7, 9, 12, 14, and day 16 of pDC differentiation of the iPSC-derived HSPCs using strategy F-K. The line graphs illustrate that strategy F and G led to the greatest fold expansion (Figure 3d). The percentage of viable cells were comparable for strategy F, G, H and J. At day 9 of pDC differentiation, flow cytometry phenotypic assessment of pDC marker expression CD 123, as well as other cell lineage markers CD3, CD14, CD19, and CD56 was performed. A flow cytometry plot of the markers CD123 and CD56 using Strategy F demonstrated that the CD 123+ cells also show minor expression of CD56 (Figure 3f). The percentage of CD4, CD14, CD19 and CD123 for strategy F-K also demonstrated some CD5 expression (Figure 3g). Flow cytometry phenotypic assessment using a large panel consisting of 27 markers was then performed at 12-days of pDC differentiation using Strategy F (Figures 3h-i). Figure 3h shows flow cytometry plots of a representative gating strategy: 1) cells were initially gated based on forward scatter (FSC) and side scatter (SSC) to exclude debris; 2) doublets were excluded via FSC and SSC; 3) viable cells were evaluated using expression of CD 123 and CD66b; and 4) cells expressing CD123 or CD123(high) were next evaluated for expression of CD45RA and CD4, followed by CD56 and CD86 (Figure 3h). Flow cytometry plots illustrate the eexpression of markers (CD73, CD28, CD69, TIM3, CD16, CD45RA, CD38, CD25, PD-1, CD45, CD123, CD27, CDl lb, TIGIT, CD86, CD163, CD19, CD66b, CD4, CD3, ICOS, CD14, CD8, CDl lc, TCRgd, PD-L1, and CD56) within either the viable cell gate (all cells) shown in Figure 3h (Figure 3i) or within the CD123(high) gate shown in Figure 3h (Figure 3j). Flow cytometry phenotypic assessment of pDC marker expression at day 5, 9, 14 and day 21 during culture in pDC medium was then performed. Example flow cytometry plots of the pDC markers CD 123 and CD303 showed that the cells expressed CD123 and CD303 at day 5, 9, 14 and day 21 (Figure 3k). Of note, cells within the CD123+ and CD303+ gate (Q6) were also found to be positive for CD304. Bar graphs showing the percentage expression of the CD123+CD303+CD304 (Q6) cell population (Figure 31), or cells with high expression of CD123+CD303+CD304+ (+++) (Figure 3m), for cells taken out at day 0, 2, 5, 7, 9, 12, 14, 16, 19, or day 21 during culture in pDC medium for strategy F-K showed that the expression varied depending on the strategy used. To assess the pDCs functionality and their response to agonists, cells cultured with strategy F or strategy G were taken out at day 16 of pDC differentiation and primed for one day with IFN or left unprimed. 2e5 cells were then stimulated with the TLR7 agonist R837 (2.5 pg / mL), CpG-A (2.5 pg / mL), cGAMP (5 pg / mL), or left unstimulated. Following 20 hours of stimulation, cell supernatants were harvested and the level of IFN-a was calculated using a IFN-a Enzyme Linked Immunosorbent Assay (ELISA). Data shown are + / - SEM of biological duplicates (n) for one setup. Results showed that functional active iPSC-derived pDCs are generated using all of the tested strategies. Strategy F and G were most effective (Figure 3n). Example 2 shows that functional active CD123+ CD303+ CD304+ pDCs can be generated from iPSCs, in particular using a setup containing medium supplemented with cytokines and small molecule inhibitors.

[0184] Example 3 - CD304-based immunomagnetic enrichment of pDCs results in increased functional responses Increased functional pDCs are generated from iPSCs with an additional step of CD304+ pDC enrichment by immunomagnetic separation. Figure 4a illustrates the overall setup of producing pDCs from iPSCs over a total culture period of 31 days (from day -14 to day 17). iPSCs were seeded in Aggrewell plates as single-cells in EB formation medium A and B (StemCell Technologies) or DC medium for 14 days (day -14 to day 0). Following 5 days of EB formation (day -9), cytokines and small molecule inhibitors in accordance with Strategy F or Strategy K were added to the medium:

[0185] At day 0, After 14-days of hematopoietic specification (day 0), EBs were harvested and cells were transferred to pDC medium on or tissue-treated (TC) culture plates. Cells were differentiated to pDCs over 17-days of culture. During the pDC differentiation cells were taken out continuously to evaluate expression of pDC markers using flow cytometry. Flow cytometry phenotypic assessment of HSPC cell marker expression after harvesting EBs following 14 days of hematopoietic specification was then performed. Flow cytometry plots of the haematopoietic markers CD31, CD34, CD43, CD45, CD117 and CD 123 between strategy F and K (Figure 4b). The percentage of cells expressing the hematopoietic markers (CD31+, CD34+, CD43+, CD45+, CD117+ , CD123+, CD34+CD43+ and CD43+CD45+) for strategy F and K following 14-days of hematopoietic specification was then calculated (Figure 4c). Figures 4b-c demonstrated comparable expression of haematopoietic markers between strategy F and K. Next, the number of cells (Figure 4d), the fold expansion (Figure 4e), and the percentage of viable cells (Figure 4f) were calculated for cells throughout pDC differentiation (day -14, 0, 3, 5, 7, 11, 13 and day 17) for strategy F and K, indicating that strategy F was most effective for generating pDC that are expanding during pDC differentiation. Flow cytometry phenotypic assessment of HSPC cell marker expression after harvesting EBs following 14 days of hematopoietic specification was then performed. Flow cytometry plots of the haematopoietic markers CD34 and CD45 demonstrated that the cells expressed differing levels of CD34 and CD45 expression between strategy F-K, and that strategy F, G and J generated iPSC-derived hematopoietic cells expressing both CD34+ and CD45+ (Figure 3c). Flow cytometry phenotypic assessment of pDC markers was then performed at day 11 of pDC differentiation using Strategy F or Strategy K (Figures 4g-h). The flow cytometry plots show a representative gating strategy: 1) cells were initially gated based on FSC and SSC to exclude debris; 2) doublets were excluded via FSC and SSC; 3) viable cells were gated on; and 4) cells expressing CD123, CD303, CD304, and HLA-DR were next evaluated. Results indicated CD 123, CD303, CD304 and HLA-DR expression (Figure 4g-h). Of note, antibodies with modified Fc-part (REAffinity) were used to exclude background staining. Next, at day 17 of culture in pDC medium using Strategy F, cells were taken out and sorted for CD304 using positive immunomagnetic selection (Miltenyi), and flow cytometry phenotypic assessment of pDC markers was then performed on the unsorted and sorted cells (Figures 4i-j). Flow cytometry plots showing CD123 and CD303 expression (Figure 4i) and the bar graph showing the percentage of live CD123+CD303+ expressing cells (Figure 4j) showed that CD304+ sorted cells had increased expression of the pDC markers CD123+ and CD303+. To assess the functionality of the pDCs, cells were taken out during culture in pDC medium from strategy F and primed for 20 hours, followed by stimulation with a TLR-7 agonist (R837), TLR-9 agonist (CpG-A), or left unstimulated. After 20 hours supernatants were harvested and levels of IFN-a were evaluated by an IFN-a ELISA, indicating both TLR-7 and TLR-9 responses (Figure 4k). Data are + / - SEM of biological duplicates. Following enrichment of the pDC population by immunomagnetic separation (positive selection for CD304 as described above), cells were primed for 20 hours and then stimulated with a TLR-7 agonist (R837), TLR-7 / 8 agonist (R848), a TLR-9 agonist (CpG-A), or left unstimulated (UT) (Figure 41), or were stimulated with a STING agonist (cGAMP) or unstimulated (UT) (Figure 4m). After 20 hours supernatants were harvested and levels of IFN-a were evaluated by an IFN-a ELISA, indicating both TLR-7 / 8, TLR-9 and STING responses. Data are + / - SEM of biological duplicates. Data is shown from two different experiments. Example 3 shows that enhanced functional pDCs can be generated from iPSCs after CD304+ enrichment of the CD123+ CD303+ pDCs. Example 4 - Isolation ofHSPCs within adherent or suspension layer results in functional pDCs

[0186] Functional pDCs are generated from iPSCs using both isolation of the adherent or suspension cells after haematopoetic specification. Figure 5 a illustrates the overall setup of producing pDCs from iPSCs over a total culture period of 30 days (from day -14 to day 16). iPSCs were seeded in Aggrewell plates as single-cells and cultured for 14 days (day -14 to day 0). After 14-days of hematopoietic specification, cell suspension and adherent cells were harvested and resuspended in pDC medium to promote the differentiation of progenitors into pDCs. Flow cytometry phenotypic assessment of hematopoietic marker expression after harvesting suspension cells or adherent cells differentiated in medium either without SRI (Figure 5b) or containing SRI (Figure 5c) was performed. Flow cytometry plots of the haematopoietic markers CD31, CD34, CD43, CD45, CD117 and CD123 demonstrated that the cells expressed differing levels of CD31, CD34, CD43, CD45, CD117 and CD 123 depending on whether the cells were harvested from the suspension or adherent layer, and whether they are differentiate in medium with or without SRI. The percentage of live cells expressing the hematopoietic markers following 14-days of hematopoietic specification as a bar chart (Figure 5d) illustrated that iPSCs are differentiated into hematopoietic cells in medium containing SRI . <90% cells harvested from the suspension are positive for CD43+CD45+CD43+. Next, the number of cells (Figure 5e), the fold expansion (Figure 5f), and the percentage of viable cells (Figure 5g) were calculated for cells throughout the pDC differentiation (day 0, 2, 4, 7, 9, 11 , 14, and day 16 post hematopoietic specification) indicated that increased cells were harvested from the adherent layer, but the fold expansion during pDC differentiation is comparable between adherent and suspension cells until day 14. Flow cytometry phenotypic assessment of pDC marker expression of cells taken out at day 7 (Figure 5h) or day 11 (Figure 5i) of pDC differentiation after harvesting suspension cells or adherent cells was performed. The flow cytometry plots show a representative gating strategy: 1) cells were initially gated based on FSC and SSC to exclude debris; 2) doublets were excluded via FSC and SSC; and 3) Viable, CDllc negative or viable, CDllc positive cells were analysed for the expression of CD 123, CD303, CD304, and HLA-DR. Of note, antibodies with modified Fc-part (REAffinity) were used to exclude background staining. Data shown are from one experiment. Results indicated expression of the pDC markers at both day 7 and day 11. At day 16 of culture in pDC medium, cells harvested from the adherent layer were sorted for CD303 using positive selection (Miltenyi). Suspension cells and CD303 sorted adherent cells were then primed for 20 hours followed by stimulation with a TLR-7 agonist (R837), a TLR-7 / 8 agonist (R848), a TLR-9 agonist (CpG-A), or left unstimulated (UT). 20-hours later supernatants were harvested and levels of IFN-a were evaluated by an IFN-a ELISA, indicating that functional responses can be observed in both suspension cells and CD303 sorted adherent cells (Figure 5j). Data are + / - SEM of biological duplicates. Example 4 shows that functional pDCs can be generated from iPSCs after isolation of either the adherent or suspension cells after haematopoetic specification.

Claims

CLAIMS1. A method of producing plasmacytoid dendritic cells (pDCs) from pluripotent stem cells, wherein the method comprises:(a) differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs);(b) optionally isolating the HSPCs;(c) differentiating the HSPCs into pDCs; and(d) optionally isolating the pDCs.

2. The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

3. The method of claim 1 or claim 2, wherein step (a) comprises differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising one or more agents associated with pDC differentiation.

4. The method of claim 3, wherein the one or more agents associated with pDC differentiation are selected from the list consisting of a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, an aryl hydrocarbon receptor (AhR) antagonist, a thrombopoietin (TPO) receptor agonist, IL-3, UM171 and ascorbic acid.

5. The method of any preceding claim, wherein step (a) comprises differentiating pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs) using a culture medium comprising a c-Kit agonist, an Fms-related tyrosine kinase 3 (FLT3) agonist, and an aryl hydrocarbon receptor (AhR) antagonist.

6. The method of any preceding claim, wherein step (c) comprises differentiating the HSPCs into pDCs using a culture medium comprising one or more of a c-Kit agonist, anFLT3 agonist, an AhR antagonist, a thrombopoietin (TPO) receptor agonist, IL-3 and ascorbic acid.

7. The method of any preceding claim, wherein the medium used in step (a) further comprises:(a) IL-3; or(b) IL-3 and UM171.

8. The method of any preceding claim, wherein:(i) the AhR antagonist is StemRegenin 1 , CH-223191, GNF351 , PD98059, BAY 2416964, BAY-218, or PDM2;(ii) the c-Kit agonist is Stem Cell Factor (SCF);(iii) the FLT3 agonist is Fms-related tyrosine kinase 3 ligand (Flt3-L);(iv) the TPO receptor agonist is TPO, AMG-531 or SB-497115; and / or(v) the ascorbic acid is L-ascorbic acid or D-ascorbic acid.

9. The method of any preceding claim, wherein the pluripotent stem cells are genetically modified, optionally wherein the pluripotent stem cells comprise a heterologous nucleic acid.

10. The method of claims 1 to 8, wherein the method comprises a step of genetically modifying the pluripotent stem cells.

11. The method of claim 10, wherein the step of genetically modifying the pluripotent stem cells comprises introducing a heterologous nucleic acid, optionally wherein the heterologous nucleic acid encodes a:(a) chimeric antigen receptor (CAR); or(b) synthetic RIP family receptor, optionally wherein the synthetic RIP family receptor is a synthetic Notch receptor (SynNotch receptor), a synthetic Robo receptor (SynRobo receptor), or a synthetic RoboNotch receptor; or(c) synthetic intramembrane proteolysis receptor (SNIPR); or(d) Tango receptor; or(e) modular extracellular signalling architecture (MESA) system.

12. The method of claim 10 or claim 11, wherein the heterologous nucleic acid is introduced into the cell by:(a) transfection or transduction with a vector, optionally wherein the vector is:(i) a viral construct, optionally wherein the viral construct is an adenoviral construct, a lentiviral construct, or a retroviral construct; or(ii) an mRNA, optionally wherein the mRNA is delivered to the cell using a lipid nanoparticle (LNP); or(iii) a plasmid; or(iv) a cosmid; or(b) using site-specific DNA editing, optionally wherein the site-specific DNA editing is:(i) CRISPR / Cas; or(ii) TALEN; or(iii) zinc finger nuclease.

13. The method of any preceding claim, wherein the HSPCs express:(a) CD34;(b) CD45;(c) CD31;(d) CD43;(e) CD 117; and / or(f) CD123.

14. The method of any preceding claim, wherein the pDCs express:(a) CD304;(b) CD 123;(c) CD303; and / or(d) HLA-DR.

15. The method of any preceding claim, wherein step (d) is performed and comprises enriching by immunomagnetic separation.

16. The method of claim 15, wherein the enrichment by immunomagnetic separation is performed using HLA-DR, CD123, CD304 or CD303 as the target protein.

17. The method of any preceding claim, wherein step (c) is performed and comprises isolating by enzymatic dissociation.

18. The method of claim 17, wherein step (c) comprises isolating by enzymatic dissociation using a protease enzyme, optionally wherein the protease is collagenase, trypLE express, trypsin, and / or accutase.

19. The method of claim 17 or claim 18, wherein the isolating by enzymatic dissociation comprises isolating the cells from the:(a) adherent layer; and / or(b) suspension layer.

20. The method of any preceding claim, wherein the pDCs respond to:(a) TLR-7 agonists;(b) TLR-9 agonists; and / or(c) STING agonists.

21. The method of claim 23, wherein the pDC response is type I interferon (IFN) expression, optionally wherein the type I IFN is IFN-a.

22. The method of any preceding claim, further comprising formulating the pDCs in a pharmaceutical composition.

23. The method of any preceding claim, wherein the method further comprises priming the pDCs, optionally wherein the priming medium comprises:(a) type I IFNs, optionally wherein the type I IFN is IFN-0 and / or IFN-a; and / or(b) type II IFNs, optionally wherein the type II IFN is IFN-y.

24. The method of any of claims 1-22, wherein the method does not further comprise a step of priming the pDCs.

25. The method of any preceding claim, where the method further comprises cryopreserving the pDCs, optionally wherein the cryopreservation is at -20°C or -80°C.

26. A pDC obtained by the method of any one of the preceding claims.

27. A pDC according to claim 26 for use in a method of therapy, such as treatment of a cancer, an autoimmune disease, or an inflammatory disease, wherein the method comprises administering a therapeutically effective amount of the pDC to the patient in need thereof.

28. The pDC according to claim 26 or the pDC for use according to claim 27, wherein the pDC is cryopreserved, optionally wherein the cryopreservation is at -20°C or -80°C.

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