Engineered plasmacytoid dendritic cells

Abstract

The present invention relates to engineered plasmacytoid dendritic cell (pDC) expressing synthetic receptors that recognise Trop2 on a target cell, and their use in methods for treating cancer.

Description

[0001] ENGINEERED PLASMACYTOI D DENDRITIC CELLS

[0002] Field of the Invention

[0003] The present invention relates to engineered plasmacytoid dendritic cells and their use in methods for treating disease, in particular cancer.

[0004] Background of the Invention

[0005] Immune cells expressing synthetic receptors represent an established and successful approach for treating various diseases, including cancer, autoimmune diseases, inflammatory diseases, and infections. An example of such a cell therapy is chimeric antigen receptor T- cell (CAR-T) therapy. Other cells have been proposed for use with CARs. Natural killer cells are a type of cytotoxic lymphocyte that naturally attack virus-infected cells and tumour cells and can be engineered with CARs to form CAR-NK cells. WO2017019848 describes use of other phagocytic cells capable of cellular cytotoxicity, in particular macrophages.

[0006] In CAR-T therapy, a T-cell population is obtained from a patient or donor and is engineered to express a chimeric antigen receptor (CAR). The extracellular domain of a typical CAR consists of the VH and VL domains - single-chain fragment variable (scFv) - from the antigen binding sites of a monoclonal antibody that recognises a tumour associated antigen. The scFv is linked to a flexible transmembrane domain followed by an intracellular signalling domain with, for example, a tyrosine-based activation motif such as that from CD3z, and optionally additional activation domains from co-stimulatory molecules such as CD28 and CD137 (41BB), which serve to activate the T-cells and enhance their survival and proliferation. CAR T-cells are administered to the patient and the CAR recognises and binds tumour cells or other disease-causing cells. Binding of cancer cells leads to activation of cytolytic mechanisms in the T-cells, which specifically kill the bound target cells. Various preclinical and early-phase clinical trials highlight the efficacy of CAR T cells to treat cancer patients with solid tumours and hematopoietic malignancies, and to treat autoimmune diseases, inflammatory diseases, and infections.

[0007] Solid tumors present a formidable challenge in cancer treatment due to their aggressive nature and resistance to traditional therapeutic modalities. Conventional treatment options, including surgery, chemotherapy, and radiation therapy, have shown varying levels of efficacy, but they often fall short in providing durable and curative outcomes for patients with advanced solid tumors and these therapies are associated with severe side effects.

[0008] The most advanced cell-based products within oncology are Chimeric Antigen Receptor (CAR) T cell therapies. These therapies are designed to detect and kill cancer cells with a specific surface tumor-antigen and the therapy has been successful in liquid tumors. However, current efforts applying CAR-T cells for targeting solid tumors have failed. For these reasons there is an unmet need to develop the next-generation of cell-based therapies that overcome the barriers seen for CAR-T cells.

[0009] Other receptors are also used in a similar manner to CARs. For example, synthetic receptors, such as synthetic intramembrane proteolysis receptors (SNIPRs), can be expressed in cells to activate a response element in a cell upon recognition of a specific antigen. Zhu et al., 2022, Cell 185, 1431-1443 describes the design of synthetic receptors from modular domains including extracellular regulatory domains, transmembrane domains, intracellular juxtamembrane domains, and transcriptional regulators.

[0010] There remains a need in the art for improved methods for treating cancer.

[0011] Summary of the Invention

[0012] The inventors have developed an immunotherapy that utilises the unique properties of plasmacytoid dendritic cells (pDCs), also referred to herein as programmed immune reactive cells (PIRCs), to target specific cells, enhance or alter immunological pathways, and treat disease. According to the invention, pDCs are engineered to express on their surface a synthetic receptor comprising an extracellular antigen recognition domain that recognises Trop2 on a target cell. The synthetic receptor allows the pDCs to specifically target disease cells and tissue to activate immune responses and treat disease in the disease microenvironment. The multifaceted nature of pDCs means that they can be used to activate varied effects on immune responses by secreting pro-inflammatory cytokines or inhibitory factors, or recruiting and activating immune cells such as Tregs or cytotoxic lymphocytes. Indeed, the powerful, multifaceted properties of pDCs including homing to tumors, direct tumor cell killing, recruitment and modulation of other immune cells in the tumor microenvironment, and most importantly the secretion of interferon a and other inflammatory cytokines differentiates pDCs from other cell therapy platforms. pDCs are also capable of antigen presentation bridging innate and adaptive immunity. pDCs thus contribute to epitope spreading and address the issue of antigen escape by tumor cells. As a consequence, pDCs are expected to exert powerful and pervasive effects, even in disease microenvironments that are refractive to the desired immune response. pDCs may also be capable of antigen presentation to immune cells, and may conduct TRAIL-mediated direct killing of target cells.

[0013] The inventors have identified that engineered pDCs may be particularly effective for targeting Trop2. Trop2 is expressed in many solid tumors that are currently hard to treat. For example, Trop2 is highly expressed in triple negative breast cancer, non-small cell lung cancer, pancreatic and ovarian cancer, which all have very low five-year survival rates. As set out above, pDCs have unique properties and a multifaceted nature that can be used to activate varied effects on immune response. Therefore, engineering pDCs to target Trop2 is expected to provide particularly potent therapeutic effects.

[0014] In certain embodiments, the synthetic receptor also comprises a transmembrane domain and an intracellular signalling domain that activates an immune response in the cell when the extracellular antigen recognition domain binds the target cell. In particular, the intracellular signalling domain may activate expression of a response element in the cell when the extracellular antigen recognition domain binds the target cell. Such constructs allow tailored immune responses to be activated upon binding of the recognition domain to the target antigen, enabling specific activation of activation of the pDCs only in the tumor microenvironment.

[0015] In preferred embodiments, the inventors have developed an engineered plasmacytoid dendritic cells (pDCs), that utilises the versatility of synthetic receptors to target specific cells that express Trop2, in combination with a potent activator of immune responses - STING (stimulator of interferon genes), to enhance or alter immunological pathways, and treat disease. According to the invention, pDC is engineered to express on its surface a synthetic receptor comprising an extracellular antigen recognition domain that recognises Trop2 on a target cell, a transmembrane domain, and an intracellular signalling domain that activates expression of a response element in the cell when the extracellular antigen recognition domain binds the target cell, wherein the response element encodes a STING protein. The extracellular antigen recognition domain allows the cells to specifically target disease cells and tissue to activate immune responses and treat disease in the disease microenvironment. The response element encoding a STING protein potently activates a robust immune response in the pDCs to treat disease. In combination, the pDCs, the synthetic receptor and the response element encoding a STING protein provide a powerful system for targeted stimulation of immune responses. The receptor binds Trop2 on target cells, initiating the synthetic receptor cascade, activating transgene transcription and producing a STING protein, optionally with gain-of-function mutations, which activation mediates a potent immune response in the pDC, including the production of pro-inflammatory cytokines and type I interferons, and recruiting and activating immune cells, such as cytotoxic lymphocytes, and the sensitization of cancer cells to immune recognition. Consequently, the cells of the invention are expected to exert powerful and pervasive effects, even in disease microenvironments that are refractory to the desired immune response.

[0016] The synthetic receptors of the invention may be synthetic intramembrane proteolysis receptors (SNIPRs), Tango receptors or modular extracellular signalling architecture (MESA) systems. In some embodiments, the synthetic receptors is a synthetic RIP family receptor, such as a synthetic Notch receptor (SynNotch receptor), a synthetic Robo receptor (SynRobo receptor), or a synthetic RoboNotch receptor (SynRobo receptor). In some embodiments, the synthetic receptor may be a chimeric antigen receptor (CAR). In a preferred embodiment, the synthetic receptor is a SynNotch receptor. The Examples demonstrate that pDCs can express SynNotch components and can specifically bind target cells. Strikingly, activation of STING via the SynNotch response element successfully induces a type I IFN and CXCL10 response in pDCs, but the pDCs maintain their unique characteristics with no effect on differentiation or function. The system is also expected to work using synthetic receptors with different transmembrane domains, e.g. Robol, as these have similar functions to Notchl.

[0017] In some embodiments, the STING response element comprises a gain-of-function mutation that causes it to be constitutively active in the absence of 2’3’-cGAMP. In some embodiments, the STING protein comprises a mutation, preferably a substitution, at one or more residues corresponding to R71, S102, V147, N154, V155, G166, C206, G207, G230, H232, R238, F279, R281, R284, R293 or Q315 of SEQ ID NO: 1. In some embodiments, the STING response element comprises a mutation at the residue corresponding to N154 or VI 55 in SEQ ID NO: 1. In preferred embodiments, the STING response element comprises an N154S mutation or a V155M mutation. These gain-of-function mutations result in constitutive activation of immune responses by STING, including production of type I interferons and CXCL10 in response to expression of the STING protein caused by binding of an antigen to the extracellular antigen recognition domain, as shown in the Examples.

[0018] In some embodiments, the intracellular signalling domain is a transcription factor.

[0019] The invention also provides a method of treating a disease in a subject comprising administering the cell of the invention.

[0020] In preferred embodiments, the cells and methods of the invention are for use in treating cancer. The ability of pDCs to secrete immunomodulatory factors and alter immune- inhibiting or inflammatory environments are expected to be particularly useful for treating cancer, where disease-causing cells reside in and are maintained by particular immune microenvironments. In addition, the potent immune response activated by the STING response element is expected to provide therapeutic effects against such cells. In some embodiments, the STING protein activates an immune response. In preferred embodiments, the immune response comprises secretion of cytokines, such as pro- inflammatory cytokines.

[0021] In further preferred embodiments, the immune response comprises recruitment or activation of host immune cells, such as cytotoxic lymphocytes.

[0022] A pDC of the invention may also perform direct TRAIL-mediated killing of target cells.

[0023] In preferred embodiments, the pDC is a stem cell-derived plasmacytoid dendritic cell (SC-pDC). Such cells can be generated ex vivo from hematopoietic stem and progenitor cells (HSPCs), which can be obtained from a patient or a donor. Moreover, HSPCs can readily be generated from induced pluripotent stem cells (iPSCs), allowing for iPSC-derived SC-pDCs. The HSPCs and iPSCs may be engineered with the synthetic receptor before differentiation to SC-pDCs.

[0024] In especially preferred embodiments, the invention provides a method of treating cancer in a subject comprising administering an engineered pDC to the subject, , wherein the cell expresses on its surface a synthetic receptor, preferably a SynNotch receptor, comprising an extracellular antigen recognition domain that recognises Trop2 on a target cell, a transmembrane domain, and an intracellular signalling domain that activates expression of a response element in the cell when the extracellular antigen recognition domain binds the target cell, wherein the response element encodes a stimulator of interferon genes (STING) protein comprising a gain-of-function mutation that causes it to be constitutively active in the absence of 2’3’-cGAMP, such as a mutation at N154 or V155, preferably an N154S mutation or a V155M mutation.

[0025] The invention also provides the engineered pDCs of the invention for use in treating disease, such as for use in treating cancer.

[0026] The invention also provides use of the engineered pDCs of the invention in the preparation of a medicament for treating disease, such as a medicament for treating cancer.

[0027] Further embodiments of the invention are provided in the numbered paragraphs below:

[0028] 1. An engineered plasmacytoid dendritic cell (pDC) that expresses on its surface a synthetic receptor comprising an extracellular antigen recognition domain that recognises Trop2 on a target cell. 2. The cell of embodiment 1, wherein the synthetic receptor further comprises a transmembrane domain and an intracellular signalling domain that activates an immune response in the cell when the extracellular antigen recognition domain binds the target cell.

[0029] 3. The cell of embodiment 2, wherein the intracellular signalling domain activates expression of a response element in the cell when the extracellular antigen recognition domain binds the target cell.

[0030] 4. The cell of embodiment 3, wherein the response element encodes a stimulator of interferon genes (STING) protein.

[0031] 5. The cell of any one of the preceding embodiments, wherein the synthetic receptor is:

[0032] (a) a synthetic intramembrane proteolysis receptor (SNIPR);

[0033] (b) a Tango receptor;

[0034] (c) a modular extracellular signalling architecture (MESA) system; or

[0035] (d) a chimeric antigen receptor.

[0036] 6. The cell of any one of the preceding embodiments, wherein the synthetic receptor is a synthetic RIP family receptor, optionally wherein the receptor is:

[0037] (a) synthetic Notch receptor;

[0038] (b) a synthetic Robo receptor; or

[0039] (c) a synthetic RoboNotch receptor.

[0040] 7. The cell of any one of the preceding embodiments, wherein the intracellular signalling domain is a transcription factor.

[0041] 8. The cell of embodiment 7, wherein the transcription factor comprises a DNA binding domain and a transcriptional activation domain.

[0042] 9. The cell of embodiment 8, wherein the DNA binding domain is selected from the group consisting of Gal4, Pax6, zinc fingers (ZFs), synthetic zinc fingers (synZFs), transcription activator like effectors (TALEs), TetR, HNF1 alpha, and vHNFl beta. 10. The cell of embodiment 8 or embodiment 9, wherein the transcription activation domain is selected from the group consisting of VP64, VP16, WWTR1, CREB3, NF-KB, p65, Rta, HSF1, and RelA.

[0043] 11. The cell of any one of the preceding embodiments, wherein the intracellular signalling domain is selected from the group consisting of Gal4-VP64, Gal4-VP16, TetR-VP64, Lacl- VP64, HNF1-WWTR1, HNF1-CREB3, Pax6-p65, ZF-p65, synZF-p65, and HNFl-p65.

[0044] 12. The cell of any one of the preceding embodiments, wherein the extracellular antigen recognition domain is an antibody-derived domain.

[0045] 13. The cell of any one of the preceding embodiments, wherein the extracellular antigen recognition domain is an scFv domain.

[0046] 14. The cell of any one of the preceding embodiments, wherein the STING protein comprises a gain-of-function mutation that causes it to be constitutively active in the absence of 2’3’-cGAMP.

[0047] 15. The cell of any one of the preceding embodiments, wherein the STING protein comprises a mutation, preferably a substitution, at one or more residues corresponding to R71, S102, V147, N154, V155, G166, C206, G207, G230, H232, R238, F279, R281, R284, R293, or Q315 of SEQ ID NO: 1.

[0048] 16. The cell of any one of the preceding embodiments, wherein the cell comprises a heterologous nucleic acid encoding the synthetic receptor.

[0049] 17. The cell of any one of the preceding embodiments, wherein the cell comprises a heterologous nucleic acid encoding the response element.

[0050] 18. The cell of any one of the preceding embodiments, wherein the cell comprises a single heterologous nucleic acid encoding the synthetic receptor and the response element.

[0051] 19. The cell of any one of embodiments 16-18, wherein the heterologous nucleic acid is selected from the group consisting of a viral construct, a plasmid, a cosmid, and an mRNA. 20. The cell of embodiment 19, wherein the viral construct is a lentiviral vector.

[0052] 21. The cell of embodiment 20, wherein the lentiviral vector comprises an expression cassette between two LTRs.

[0053] 22. The cell of embodiment 19, wherein the viral construct is an adenoviral or adeno- associated viral vector.

[0054] 23. The cell of embodiment 22, wherein the adenoviral or adeno-associated viral vector comprises an expression cassette between two ITRs.

[0055] 24. The cell of any of embodiments 16-18, wherein the heterologous nucleic acid is introduced using site-specific DNA editing, such as CRISPR / Cas.

[0056] 25. The cell of embodiment 19, wherein the mRNA is delivered via a lipid nanoparticle.

[0057] 26. The cell of any of embodiments 3d and 5-25, wherein the pDC has undergone a step of priming the pDC.

[0058] 27. The cell of embodiment 26, wherein the step of priming the pDC comprises incubating the cells with type I IFN and / or type II IFN.

[0059] 28. The cell of embodiment 26 or embodiment 27, wherein the pDC is incubated with type I IFN and / or type II IFN for 24 hours.

[0060] 29. The cell of any one of the preceding embodiments, wherein the STING protein activates an immune response.

[0061] 30. The cell of any embodiment 29, wherein the immune response comprises secretion of cytokines, optionally wherein the cytokines modulate the environment of the diseased tissue, or wherein the immune response comprises recruitment or activation of immune cells.

[0062] 31. The cell of embodiment 30, wherein the cytokines are pro-inflammatory cytokines.

[0063] 32. The cell of embodiment 30 or embodiment 31, wherein the cytokines comprise CXCL10, MCP-1, MCP-2, MIP-la and / or MIP-lb. 33. The cell of any one of embodiments 29-32, wherein the immune response comprises production of type I IFN.

[0064] 34. A method of treating cancer in a subject, comprising administering the cell of any one of the preceding embodiments.

[0065] 35. The method of embodiment 35a, wherein 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.

[0066] 36. The cell of embodiment 35a, wherein the cancer is selected from the group consisting of 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, and Waldenstrom macroglobulinemia.

[0067] 37. The cell or method of any one of the preceding embodiments, wherein the cell has been extracted from blood or bone marrow.

[0068] 38. The cell or method of any one of embodiments 3d-47, wherein the cell has been differentiated in vitro from HSPCs, optionally wherein the HSPCs have been obtained from blood or bone marrow, or wherein the HSPCs have been differentiated in vitro from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0069] 39. A pharmaceutical composition comprising the engineered pDC of any preceding embodiment and a pharmaceutically acceptable carrier.

[0070] 40. A method of producing the cell of any one of embodiments 3d-49, comprising the steps of:

[0071] (a) transfecting or transducing HSPCs with one or more vectors encoding the synthetic receptor and the response element; and

[0072] (b) differentiating the HSPCs into pDCs.

[0073] 41. A method of producing the cell of any one of embodiments 3d-49, comprising the steps of:

[0074] (a) differentiating HSPCs into pDCs; and

[0075] (b) transfecting or transducing the pDCs with one or more vectors encoding the synthetic receptor and the response element.

[0076] 42. The method of embodiment 50or embodiment 51, wherein the vector is a viral vector, optionally wherein the vector is a lentiviral vector or an adenoviral or adeno-associated viral vector.

[0077] 43. The method of embodiment 50 or embodiment 51, wherein the vector is an mRNA, optionally encapsulated in a lipid nanoparticle. 44. The method of any one of embodiments 50-53, wherein the step of differentiating the HSPCs comprises incubating said HSPCs in one or more media, which media may typically comprise one or more cytokines, growth factors, interferons (IFNs) and / or aryl hydrocarbon receptor (AHR) antagonists (such as stemregenin-1), whereby said HSPCs are differentiated into precursor-pDCs and into pDCs.

[0078] 45. The method of any one of embodiments 50-54, further comprising a step of purifying the pDCs.

[0079] 46. The method of any one of embodiments 50-55, further comprising a step of formulating the pDCs with a pharmaceutically acceptable excipient.

[0080] Brief Description of the Figures

[0081] Figure 1. Schematic illustration showing the production of SynNotch U-pDCs expressing the SynNotch elements. “U-pDC” is an alternative term that is also used throughout to refer to pDCs. HSPCs were pre-expanded for three days before being transduced with SynNotch constructs using lentiviral vectors carrying the SynNotch receptor and the response element. In some cases, the lentiviral vector encodes both the SynNotch receptor and response element in one cassette and in other cases the two elements are encoded by separate vectors. After the transduction, HSPCs were subsequently differentiated into U-pDCs over 16 days of culture.

[0082] Figure 2. Illustration of innate immune signaling of the STING VI 55M gain of function. Normal STING signaling relies on the activation of STING based on the binding of the ligand 2’3’-cGAMP. The STING VI 55M harbors a substitution of valine at position 155 to that of methionine, which enhances stability of the dimer and / or mimics the effect of ligand binding, which provides ligand-independent / constitutive activation of STING signaling. The STING N154S harbors a substitution of asparagine at position 154 to that of serine, which increases the stability of the dimer, resulting in a constitutive active protein.

[0083] Figure 3. STING signaling in normal pDCs versus the SynNotch U-pDCs carrying the STING VI 55M gain of function (GoF). STING signaling in unmodified pDCs relies on the activation of STING by cytosolic sensing of accumulated DNA. In comparison, binding of a tumor antigen to the SynNotch receptor to the SynNotch U-pDCs results in the transcription of the STING GoF variant, resulting in a STING-mediated cytokine response without the use of a STING agonist.

[0084] Figure 4. Schematic illustration showing SynNotch STING constructs (the two elements split between two LV vectors), a-b) Schematic of a SynNotch response element in a 3rd gen. lentiviral construct. The constructs contain a STING N154S (a) or STING VI 55M (b) response element under the control of an inducible miniCMV promotor (mCMV) flanked by GAL4-binding UAS sequences. Connected to the response element is an mCherry gene through an IRES domain. Downstream of this is an eGFP transgene constitutively expressed by a PGK promotor. In U-pDCs the construct will allow expression of the STING gain of function variants only when the SynNotch receptor binds to a specific tumor antigen, c) Schematic illustration of a SynNotch receptor in a 3rd gen. lentiviral construct. The construct contains a SynNotch receptor with an anti-CD19 scFv on the extracellular side fused to the GAL4-VP64 transcription factor on the intracellular side. Upstream of the anti-CD19 scFv is a c-Myc tag to allow easy validation of construct expression and activation of the receptor using anti-c-Myc beads. The SynNotch receptor is constitutively expressed by a PGK promotor. In U-pDCs the construct will allow expression of the anti-CD19 scFv SynNotch receptor, which upon binding to CD 19 on a target cell will facilitate transcription factor (GAL4-VP64) release, which induces the expression of the aforementioned response element.

[0085] Figure 5. Schematic illustration showing SynNotch STING constructs (the two elements carried by a single LV vector), a-b) Schematic of a SynNotch receptor and response element in a 3rd gen. lentiviral construct. The constructs contain a STING N154S (a) or STING V155M (b) response elements under the control of a mCMV promotor flanked by GAL4- binding UAS sequences. Downstream of this inducible transgene, a PGK promotor expresses an anti-CD19 scFv SynNotch-GAL4-VP64 receptor. Upstream of the anti-CD19 scFv is a c- Myc tag to allow easy validation of construct expression and activation of the receptor using anti-c-Myc beads. Upon transduction the construct allows a full SynNotch system to be delivered to U-pDCs by an ‘all-in-one’ lentiviral vector, which enables U-pDCs to express the STING gain of function variants upon binding to CD 19 antigen on target cells.

[0086] Figure 6. Immune responses of activated SynNotch STING GoF U-pDCs. Binding of SynNotch STING GoF U-pDCs to a specific tumor antigen will initiate transcription of the STING GoF variant. The variant will in turn initiate a broad transcriptional program in the SynNotch U-pDCs, resulting in the production of type I IFN and pro-inflammatory proteins. These will sensitize the cancer cells to immune-mediated killing, prime surrounding bystander immune cells, such as cytotoxic lymphocytes (NK cells and CD8 T cells), and recruit other immune cells to the tumor site. Overall, this will promote specific anti-tumor responses in the tumor.

[0087] Figure 7. Infusion of SynNotch STING GoF U-pDCs into a cancer patient. U-pDCs expressing the SynNotch STING GoF are injected into a patient. The U-pDCs will home to the tumor site and upon recognition of the specific tumor antigen will become activated and secrete type I IFN and pro-inflammatory cytokines. This will in turn promote recruitment of immune cells to the tumor site, such as cytotoxic lymphocytes (NK cells and CD8 T cells). Moreover, the pro-inflammatory environment will abolish the immune-inhibiting environment that the tumor cells generate, sensitizing the tumor cells, and transforming a ‘cold’ tumor into a ‘hot’ tumor to promote anti-tumor responses.

[0088] Figure 8. U-pDCs expressing the SynNotch STING variants N154S or V155M effectively respond to target cells with a STING-mediated response, a) FACS plots showing LV transduction efficiency measured 6 days post transduction using flow cytometry. Here, the two components of the SynNotch system (receptor and response element) are delivered by two separate LVs. The level of antiCD 19 SynNotch receptor and the SynNotch response elements were evaluated using an anti-FMC63 antibody and eGFP, respectively, b-c) Transduction efficiency measured 1-day after priming of U-pDCs (day3+16+l) on SynNotch U-pDCs either expressing antiCD19 SynNotch STING N154S (b) or antiCD19 SynNotch STING V155M (c). The level of antiCD19 SynNotch receptor and the SynNotch response elements were evaluated using an anti-FMC63 antibody and eGFP, respectively. Cells were gated based on viable cells, CD11c negative and CD303 positive, d) 5e5 non-primed (IL-3) or primed (IFN) SynNotch U-pDCs were seeded in 48-well plates and overlayed with 2e6 (1:4) or 4e6 (1 :8) target cells (NALM6) or non-target cells (K562). Supernatants were harvested 20 hours later and evaluated for levels of type I IFN. e) 5e5 non-primed (IL-3) SynNotch U-pDCs were seeded in 48-well plates and overlayed with 2e6 (1 :4) or 4e6 (1 :8) target cells (NALM6) or non-target cells (K562). Supernatants were harvested 20 hours later and evaluated for levels of CXCL10 using a CXCL10 ELISA. Data shown are from one donor in biological triplicates.

[0089] Figure 9. U-pDCs expressing the SynNotch STING variants N154S or V155M effectively respond to target cells with a STING-mediated type I IFN response, a) Schematic showing experimental setup. HSPCs were initially thawed and pre-expanded at low density for 3-4 days before being transduced with lentiviral vectors at an MOI of 100. This experiment uses an ‘all-in-one’ lentiviral vector carrying either the STING N154S or the STING V155M response element along an SynNotch receptor targeting CD 19. The day after transduction, medium was changed, and HSPCs were differentiated into U-pDCs. 6-days post transduction, transduction efficiency was analyzed. Cells were also sorted by labeling with antiFMC63-PE (FMC63=scFv) followed by positive immunomagnetic selection for PE. Prior to sorting, le6 HSPCs were taken out to analyze if the transduction, or the sorting of cells, affected pDC differentiation or expansion of cells. After 18-days of pDC differentiation, pDCs were taken out and primed for co-culture experiments or cryopreserved for later use. b) Transduction efficiency was measured at 6-days post transduction by flow cytometry, c) Figure illustrating immunomagnetic selection of antiCD 19 SynNotch positive cells. HSPCs were stained with anti-FMC63-PE followed by positive immunomagnetic selection of PE positive cells, d) Purity of SynNotch positive cells after selection, e-f) Proliferation of HSPCs (actual numbers) during differentiation into U-pDCs. To estimate if transduction, expression of the STING construct, or sorting affected proliferation / viability / differentiation of HSPCs into U- pDCs, a similar number of mock cells were seeded prior to and after sorting of SynNotch positive cells. For unsorted cells, le6 cells were seeded, whereas for the sorted condition 5e6 cells were seeded. Below each graph, splitting conditions are stated, g) Similar to panel e) but calculated number of total cells assuming no cells were discarded during differentiation to U- pDCs. h-j) After 18 days of pDC differentiation, bulk U-pDCs were taken out and primed for one day with IFN, or left non-primed (IL-3). U-pDCs were subsequently evaluated for pDC markers (lineage negative, CDl lc negative, CD123+, CD303+, and SynNotch receptor expression (FMC63)). The panels show primed (IFN) or non-primed (IL-3) for nontransduced (mock) U-pDCs (h), sorted SynNotch STING VI 55M U-pDCs (i), and sorted SynNotch STING N154S U-pDCs (j). k) le5 primed (IFN) or non-primed (IL-3) U-pDCs were seeded and overlayed with 8e5 target (NALM6, REH6) or non-target (K562) cells. Supernatants were collected 20 hours later, and levels of type I IFN were evaluated. Data shown are from one donor done in biological triplicates (k).

[0090] Figure 10. U-pDCs expressing the SynNotch STING variant V155M effectively respond to target cells with a STING-mediated type I IFN response. HSPCs were initially thawed and pre-expanded at low density for 3-4 days before being transduced with an MOI of 100 of lentiviral vectors carrying the STING VI 55M SynNotch cassette. An all-in-one SynNotch construct (SynNotch receptor and response element in a single construct) was used. The day after transduction, medium was changed, and HSPCs were differentiated into U-pDCs. 6- days post transduction, transduction efficiency was analyzed. SynNotch-positive cells were also selected by labeling with antiFMC63-PE followed by immunomagnetic selection for PE. Prior to sorting, le6 HSPCs were taken out to analyze if the transduction, or the sorting of cells affected pDC differentiation or expansion of cells. After 18-days of pDC differentiation, pDCs were taken out and primed for co-culture experiments, a-b) Proliferation of HSPCs (actual numbers) during differentiation into U-pDCs. To estimate if transduction, expression of the STING construct, or sorting affected proliferation / viability / differentiation of HSPCs into U-pDCs, a similar number of mock cells were seeded prior to and after sorting of SynNotch positive cells. For unsorted cells, le6 cells were seeded, whereas for the sorted condition 5e6 cells were seeded. Below each graph, splitting conditions are stated, c) Similar to panel a) but calculated number of total cells assuming no cells were thrown out during differentiation to U-pDCs. d) Percentage of cells expressing the SynNotch receptor either 6-8 days post transduction, or after a full pDC differentiation (18 days) for non-primed (IL3) or primed (IFN) U-pDCs. e) Percentage of Lin-CDl 1c- U-pDCs expressing the pDC markers CD123, CD303, and CD304. f) Schematic overview showing the co-culture setup of SynNotch U-pDCs. Ie5 SynNotch U-pDCs or mock U-pDCs were seeded in a 96-well plate and overlayed with 8e5 target cells (E:T ratio of 1 :8). 24 hours later, supernatants were collected and type I IFN levels were evaluated, g-h) Levels of type I IFN after co-culture with target cells (REH6 and NALM6) or non-target cells (K562) for unsorted SynNotch U-pDCs (g) or sorted SynNotch U-pDCs (h). Percentage of SynNotch positive U-pDCs for each donor is stated below each graph. I-h) le5 unsorted (i) or sorted (j) SynNotch U-pDCs were seeded and stimulated with the TLR7 agonist (R837, 2.5 pg / mL), or the STING agonist 2’-3’- cGAMP (4 pg / mL). Supernatants were collected 24 hours later and levels of type I IFN were evaluated using a type I IFN bioassay, k) 0.8-le6 U-pDCs were seeded in a 24-well and stimulated with 2’3’ cGAMP or anti-c-Myc magnetic beads that activate the SynNotch receptor. 24 hours later, supernatants were collected and type I IFN analyzed using a type I IFN bioassay. Data shown are from three donors performed in biological triplicates (g-k).

[0091] Figure 11. U-pDCs expressing the SynNotch-STINGV155M induce a broad range of immunostimulatory cytokines and chemokines upon recognition of cognate antigen, a) le5 primed (IFN) or non-primed (IL-3) U-pDCs were seeded out and overlayed with 8e5 target (REH) or non-target (K562) cells (effectortarget ratio of 1 :8). Supernatants were collected 20 hours later and indicated cytokines / chemokines were evaluated using Meso-scale multiplex ELISA. Heat-map showing the compiled cytokine measurements defined in the range of pg / mL. Black squares indicate values above the maximum range detection, b) le6 primed (IFN) or non-primed (IL-3) U-pDCs were seeded out and stimulated with anti-c-Myc magnetic beads (15 pL per le6 cells), cGAMP (4 pg / mL), or left unstimulated (UT). Supernatants were collected 20 hours later and indicated cytokines / chemokines were evaluated using Meso-scale multiplex ELISA. Data are mean value + / - SEM of three donors.

[0092] Figure 12. RNA-seq profile of U-pDCs with activated SynNotch-STINGV155M shows upregulation of a broad immune response, a) Schematic showing the setup. 0.5-le6 primed (IFN) or non-primed (IL-3) U-pDCs were seeded out and stimulated with anti-c-Myc magnetic beads (15 pL per le6 cells), cGAMP (4 pg / mL), or left unstimulated (UT). Following 20 hours of stimulation, RNA was extracted and subjected to qPCR and RNA-seq. b-c) Levels of CXCL10 (b) and IFNal / 13 (c) measured using qPCR. d) Volcano plot showing differentially expressed genes of Mock versus SynNotch STINGV155M U-pDCs stimulated with anti-cMyc magnetic beads. Arrows indicate different known inflammatory genes, e) Gene ontology bubble chart displaying the 20 most enriched biological processes for the differentially expressed genes in activated SynNotch STINGV155M U-pDCs. The x- axis shows the ratio between the number of differentially expressed genes within the biological process and the number of total genes annotated in that process (rich ratio). The size of the bubble represents the number of differentially expressed genes within the process. Data are mean value + / - SEM of three donors (d-e) using technical triplicates (b-c).

[0093] Figure 13. U-pDCs expressing the SynNotch-STINGV155M are only activated upon recognition of cognate antigen. A total of le5 Mock or SynNotch-STINGV155M U-pDCs were co-cultured with Raji cells, or Raji cells with CRISPR-Cas9 knockout of CD19 (Raji CD 19 KO). Following 20-hours of stimulation supernatants were harvested and evaluated by IFNa ELISA. Data are mean value + / - SEM of three donors.

[0094] Figure 14. U-pDCs expressing the SynNotch-STINGV155M activated by cognate antigen primes NK cells, a) Schematic showing the setup. NK cells were isolated from PBMCs using negative selection. Following selection, NK cells were primed for 20 hours in conditioned medium from Mock or SynNotch U-pDCs that had prior been stimulated with 2’3’-cGAMP or anti-c-Myc magnetic beads. Following priming, NK cells were analyzed for known NK activation markers (CD314, CD253, and CD69). Next a co-culture with primed NK cells and target cancer cells (genetically engineered to express eGFP and nanoluciferase) were established and lysis of target cells was estimated using flow cytometry (eGFP levels) or release of nanoluciferase in the supernatant, b) Expression of NK priming markers following culture of NK cells in conditioned medium from Mock or SynNotch U-pDCs. As a control DC medium alone (UT) or IFNa or IFNb was used (500 U / mL). Cells were gated based on viable, CD3 negative, and CD56 positive cells. Data are from one representative donor, c-e) Percentage expression of the NK priming markers CD69 (c), CD314 (d), and CD253 following culture in conditioned medium from Mock or SynNotch-STINGV155M U-pDCs. f-g) Following priming of NK cells, NK cells were overlayed with target cells K562 (f) or REH (g) expressing nanoluciferase at an E:T ratio of 2: 1. Specific lysis of target cells was analyzed 20 hours later by assessing release of nanoluciferase in the supernatant. Percentage lysis is normalized to target cells lysed with tergitol. Target cell death without the effect of NK cells has been subtracted to get the specific lysis of cells. Data are mean value + / - SEM of three NK donors run in biological triplicates.

[0095] Figure 15. Activated SynNotch-STINGV155M U-pDCs directly kill cancer cells expressing cognate antigen, a) Schematic illustration of setup. In a co-culture setup U-pDCs or NK cells were either co-cultured together or alone with target cancer cells (REH expressing eGFP- nanoluciferase). An E:T ratio of 1 :2 or 2: 1 was used. To promote the activity of pDCs, cells were stimulated with cGAMP, or left unstimulated. Specific lysis of target cells was analyzed 24 hours or 48 hours later by counting viable target cells using flow cytometry. Percentage lysis is normalized to target cells cultured alone, b-c) Lysis of cancer cells following coculture of NK cells and target cells with or without the addition of Mock or SynNotch STINGV155M U-pDCs. Lysis of target cells was determined after 24 hours (b) or 48 hours (c). d) Following 24 hours and 48 hours of co-culture of NK cells and U-pDCs numbers of U- pDCs were analyzed using flow cytometry to evaluate if the U-pDCs are killed by NK cells, e-f) Co-culture of NK cells, Mock U-pDCs, or SynNotch-STINGV155M U-pDCs with target cells (REH expressing eGFP-NL). Following 24-hours (e), or 48-hours (f) lysis of target cells was evaluated by counting numbers of viable target cells, g) REH cells were overlayed with conditioned medium from Mock or SynNotch-STINGV155M U-pDCs that had been stimulated for 20 hours with either cGAMP, anti-cMyc magnetic beads (which activates the SynNotch receptor), or left unstimulated. Specific lysis of REH were evaluated 20 hours later by assissing release of nanoluciferase in the supernatant. Data are mean value + / - SEM of three U-pDC and NK donors done in biological dublicates.

[0096] Figure 16. Killing of target cells and viability of U-pDCs during prolonged co-culture. U- pDCs were co-cultured with REH or NALM6 at an E:T ratio of 2: 1. During co-culture cells were continuously taken out, and numbers of U-pDCs or target cells were evaluated using flow cytometry, a-b) Accumulated killing of REH cells (a), or NALM6 cells at an E:T ratio of 2: 1 (b) during 6-days of co-culture with Mock or SynNotch-STINGV155M U-pDCs. Data are mean value + / - SEM of three U-pDC donors in biological dublicates.

[0097] Figure 17. Kinetic response of SynNotch STINGV155M activation in U-pDCs. a) Schematic illustration of setup. SynNotch-STINGV155M U-pDCs were seeded out and stimulated with anti-cMyc magnetic beads. Following 6, 12, 24, 48, 72 and 96 hours of stimulation RNA or supernatant was harvested. In a second setup, anti-cMyc magnetic beads were removed from cells, and cells were left for 24-hours before supernatant and RNA were harvested, b) Levels of STINGV155M, IFNa2, IFNb, fFITl and CXCL10 following stimulation or rest-phase, c) Levels of type I IFN during culture. Data are mean value + / - SEM of two U-pDC donors run in technical triplicates.

[0098] Figure 18. SynNotch-STINGV155M U-pDCs induce immune responses and tumor regression in an in vivo model, a) Schematic illustration of setup. NXG mice (Janvier) were injected subcutaneously (s.c.) with 5e6 katushka-positive NALM6 cells. When mice had developed a mean tumor size of 120 mm3 ± 50 mm3, le7 Mock or SynNotch-STINGV155M U-pDCs were injected intravenously (i.v.) by the tail vein or intratumorally (i.t.). b-d) 48- hours after i.v. or i.t. administration of Mock or SynNotch-STINGV155M U-pDCs, spleen and tumor tissue was harvested and processed, and the proportion of U-pDCs were evaluated by flow cytometry, b) shows a gating strategy to evaluate infiltration of U-pDCs. Cells were initially gated based on FSC and SSC. Next, red blood cells (Teri 19 positive), and katushka positive NALM6 tumor cells were excluded. Cells double negative for Katushka and Teri 19 were next gated on HLA-ABC, and cells expressing CD123 and CD86 were evaluated, c-d) Proportion of U-pDCs from two mice evaluated from the spleen (c) or the tumor (d) 48-hours following injection, e) The tumor growth volume (n=3 mice per group) measured 48-hours post i.v. or i.t. injection with Mock or SynNotch-STINGV155M U-pDCs. Data are presented as mean + / - SEM. Comparison of groups (e) was done by One-way ANOVA followed by the Bonferroni multiple comparisons test.

[0099] Figure 19. Expression of the next-generation SNIPR System in U-pDCs. a) Schematic illustration showing the anti-Her2 SynNotch and anti-Her2 SNIPR harboring the STINGV155M response element. The constructs contain the STING VI 55M response element under the control of an inducible miniCMV promotor (mCMV) flanked by GAL4- binding UAS seuqences. A PGK controls the expression of the anti-Her2 SynNotch, or anti- Her2 SNIPR respectively, which are connected to a Gal4-VP64. Upstream of the anti-Her2 scFv is a c-Myc tag to allow for easy validation of construct expression and activation using anti-cMyc magnetic beads, b) Expression of the anti-Her2 SynNotch or anti-Her2 SNIPR in pDCs. c) A total of le5 Mock, anti-Her2 SynNotch, or anti-Her2 SNIPR pDCs were cocultured with K562, K562 expressing a truncated variant of Her2 (K562-Her2), or the breast cancer cell line Her2 at an E:T ratio of 1 :4. As a control cells were activated with anti-cMyc magnetic beads. Following 20-hours of stimulation supernatants were harvested and evaluated by IFNa ELISA., d) Mock, anti-Her2 SynNotch, or anti-Her2 SNIPR pDCs were co-cultured with JIMT1 cells expressing eGFP-NL at a E:T of 2: 1. Viability of JIMT1 cells were evaluated 48-hours later by release of nanoluciferase. Data are mean value + / - SEM of two donors.

[0100] Figure 20. Fold expansion of U-pDC differentiated from cord blood CD34+ cells and their phenotype before and after freezing: (A) U-pDCs were produced over 17 days using N=10 different CD34+ cord blood donors and expansion fold was calculated. (B) Representative donor analyzed by flow cytometry for lack of lineage markers and Cdl 1c as well as expression of classical pDC markers: CD123, Cd303 and CD304. (C) U-pDCs were produced over 17 days using N=5 different CD34+ cord blood donors. At day 17, bulk populations were cryopreserved and thawed afterwards to assess lineage specific and pDC specific marker before and after freezing. Each UpDC donor was assessed by flow cytometry for lack of lineage markers and CD11c, as well as expression of classical pDC markers CD 123, CD303, and CD304 fresh before freezing and cryo-thawed after freezing.

[0101] Figure 21. Engineering of U-pDCs: (A) Description of the SWITCH system (B) vector gene of interest schematic (C) schematic of SWITCH U-pDC differentiation process (D) SWITCH expression during differentiation process: Expression of SWITCH during pDC differentiation for non-primed (IL-3) or primed (IFN) U-pDCs at day 18 post transduction. The column diagram shows transduction efficiency for both sorted or unsorted cells. Data are + / - SEM of three donors. (D) Percentage of cells expressing the transgene either 6-8 days post transduction, or after a full pDC differentiation (18 days) for non-primed (IL3) or primed (IFN) U-pDCs. (E) and (F) a SWITCH construct comprising an anti-Trop2 scFv, in circular and linearized form.

[0102] Figure 22. SWITCH U-pDC characterization: phenotype U-pDCs expressing the SWITCH system display similar phenotype as non-transduced (MOCK) U-pDCs: (A) Expression of the SWITCH system is shown before and after positive selection of transduced cells. FACS plot are representative for one donor, and bargraphs are + / - SEM of N=3.

[0103] Figure 23. Type I IFN release by U-pDCs: (A) activated U-pDCs and SWITCH U-pDCs with c-myc beads were assessed for IFNa release; (B) U-pDCs and SWITCH U-pDCs were co-cultured with tumor antigen positive and negative cell line and assessed for IFNa release.

[0104] Figure 24. Activation and migration of a broad immune response induced by SWITCH U- pDC: (A) Mock or SWITCH U-pDCs were activated with c-myc for 24 hrs before a transwell containing 1.25e6 PBMC were added on top. Following 2 and 5 hours of incubation 200 uL of medium was removed from the bottom well. Counting beads were added to the medium before staining and a fixed volume was analyzed for each sample. (B) PBMCs were activated with conditioned medium from cultures with U-pDCs or SWITCH U-pDCs and B cells were stained for CD69 activation marker (C) Lysis of target cells by primed NK cells. Following priming of NK cells, NK cells were overlayed with target positive cells expressing nanoluciferase at an E:T ratio of 2: 1. Specific lysis of target cells was analyzed 20 hours later by assessing release of nanoluciferase in the supernatant. Percentage lysis is normalized to target cells lysed with tergitol. Target cell death without the effect of NK cells has been subtracted to get the specific lysis of cells. Data are + / - SEM of three NK donors. Figure 25. SWITCH U-pDCs kill tumor cells in vitro: U-pDCs or NK cells were added at an E:T ratio of 2: 1 together with REH6 (nanoluciiferase expressing). To promote pDC activity, cells were stimulated with cGAMP, anti-cMyc, R837, or left unstimulated (UT). Specific lysis of target cells was analyzed 24 or 48 hours later by assessing release of nanoluciferase in the supernatant or by counting viable target cells using flow cytometry. Percentage lysis is normalized to target cells lysed with tergitol (for NLuc assay), or from target cells cultured alone. Target cell death without the effect of NK cells has been subtracted to get the specific lysis of cells. Data are + / - SEM of three pDC donors, using the same NK donor (B) U-pDCs or SWITCH U-pDCs cells were added at an E:T ratio of 1 :2 together with tumor antigen positive cells (nanoluciiferase expressing). Specific lysis of target cells was analyzed 48 hours later by assessing release of nanoluciferase in the supernatant. Percentage lysis is normalized to target cells lysed with tergitol (for NLuc assay), or from target cells cultured alone. Data are + / - SEM of three donors. (C) U-pDCs were thawed and primed for 24 hours. Following priming, a co-culture setup was performed with the target positive cells expressing GFP. 25.000 target cells were seeded out, and overlayed with 50.000 U-pDCs or SWITCH U- pDCs corresponding to an E:T ratio of 2: 1.

[0105] Figure 26. SWITCH U-pDCs kill tumors in vivo: NXG mice implanted with tumor antigen positive cell line were followed for tumor growth for 15 days. U-pDCs or SWITCH U-pDCs were infused i.v. to the respective groups and mice were sacrificed 48h later and assess for tumor growth inhibition. Tumor were also analyzed for SWITCH U-pDC infiltration by flow cytometry staining for CD123 and CD86 markers specific to U-pDCs

[0106] Figure 27. SWITCH U-pDCs are tumor specific and have no basal activity: (A) le5 pDCs were seeded out per well (96-well), and subsequently overlayed with indicated target cell lines: TA+ and TA-. A pDGtarget ratio of 1 :4 or 1 :8 was used (4e5 or 8e5 target cells, respectively). Supernatants were harvested and evaluated for levels of IFNa using ELISA

[0107] Figure 28. SWITCH U-pDC construct characterization: (A) U-pDCs and SWITCH UpDCs were thawed and primed for 24 hours. Following priming, a co-culture setup was performed with the tumor antigen positive target cells. 25.000 target cells were seeded out, and overlayed with 50.000 U-pDCs or SWITCH U-pDCs corresponding to an pDC:T ratio of 2: 1. As a control, cells were stimulate with the TLR9 agonist CpG-A (2.5 pg / mL). Following 48 hours of co-culture the morphology of cells were checked. (B) HSPCs transduced with SWITCH constructs with different promotors (MND, MSCV and PGK) were stained to assess transgene expression 6-days post transduction. (C) Indicated cell lines were stained with a commercially-available antigen specific antibody, Black histograms indicate stained cells, whereas gray bars are either unstained cells for commercially Ab. (D) U-pDCs and SWITCH U-pDCS generated over 16-days of culture were seeded out (le5 cells per well), and co-cultured with target positive and negative cells at indicated pDC: target ratios. Target cells were trypsinized and seeded out on the same day of co-culture. Following 20-hours of stimulation, supernatants were harvested and levels of IFNa evaluated by IFNa ELISA. Data are from one donor run in biological duplicates as an initial pilot study.

[0108] Figure 29. Flow cytometry of LentiX cells based on cMyc detection. FACS plots compare transgene expression in cells (A) given a mock transfection treatment, or transfected with p089 (B), p090 (C), or pl 08 (D) constructs.

[0109] Figure 30. Flow cytometry of LentiX cells based on cMyc detection. (A) Histograms comparing transgene expression in LentiX cells given a mock transfection treatment, or transfected with p089, p090, or pl 08 constructs. (B) A bar chart comparing transfection efficiency of p089, p090, and pl 08 constructs into LentiX cells 48 hours post transfection.

[0110] Figure 31. Histograms comparing expression of (A) p089, (B) p090, and (C) pl 08 constructs in K562 cells using flow cytometry based on cMyc detection.

[0111] Figure 32. Bar chart comparing lentivirus production in K562 cells transduced with p089, p090, and pl 08 constructs.

[0112] Figure 33. FACS plots comparing transgene expression 8 days post transduction using flow cytometry based on cMyc detection in HSPCs either given a mock transduction treatment, or transduced with p089, p090, or pl 08.

[0113] Figure 34. Bar chart comparing transgene expression in HSPCs transduced with p089, p090, or pl 08 8 days post transduction. Figure 35. Comparison of fold expansion accumulation in pDCs transduced with PL108 vs mock-transduced pDCs.

[0114] Figure 36. Bar chart comparison of IFN-a production in pDCs transduced with PL108 and mock-transduced pDCs, when co-cultured with cell lines (A549, 0VCAR3, or HCC1937) at a pDC: target cell ratio of 1 : 1 or 1 :4, or stimulated with an antigen (60 nM CpG-A, 300 nM diaBZI, TROP2 protein, or anti-cMyc magnetic beads).

[0115] Figure 37. Flow cytometry analysis of pDCs based on cMyc detection. Left - FACS plots confirming expression of markers (CD123 and HLA-DR) associated with pDCs in mock- transduced pDCs and PL108-transduced pDCs. pDC populations were successively gated. Right - FACS plot comparing TROP2 expression in PL108-transduced cells and mock- transduced cells.

[0116] Figure 38. Line graph comparing fold expansion over 21 days of culture in pDCs transduced with p090 or pl 08, or given a mock-transduction treatment.

[0117] Figure 39. Bar chart comparison of IFN-a production in pDCs transduced with p090 or pl 08, or given a mock-transduced treatment, when co-cultured with OVCAR-3 cells at a pDC: OVCAR-3 ratio of 1 :4, or stimulated with anti-cMyc magnetic beads.

[0118] Figure 40. Flow cytometry analysis of pDCs based on cMyc detection. Top - FACS plot confirming expression of HLA-DR and CD 123 markers associated with pDCs in mock- transduced pDCs and pDCs transduced with pl08 or p090. Bottom - FACS plots comparing transgene expression in pl08-transduced pDCs with p090-transduced and mock-transduced pDCs.

[0119] Figure 41. Line graph comparing fold expansion over 21 days of culture in pDCs transduced with pl 14, or given a mock-transduction treatment.

[0120] Figure 42. Bar chart comparison of IFN-a production in pDCs transduced with pl 14, or given a mock-transduced treatment, when co-cultured with OVCAR3, JIMT-1, HCC1937, or A549 cell lines. Mock-transduced DI 17 and DI 19 cells, as well as cancer cells, were used as controls. Figure 43. Flow cytometry analysis of pDCs based on cMyc detection. Top - FACS plot confirming expression of HLA-DR and CD 123 markers associated with pDCs in mock- transduced pDCs and pDCs transduced with pl 14. Bottom - FACS plots comparing transgene expression in pl 14-transduced pDCs and mock -transduced pDCs.

[0121] Figure 44. Flow cytometry analysis of LentiX cells based on cMyc detection. The histogram compares expression of various scFv SNIPR constructs. Mock-transfected cells were used as a control.

[0122] Detailed Description of the Invention

[0123] Synthetic Receptors

[0124] The invention provides engineered plasmacytoid dendritic cells (pDCs)that express on their surface a synthetic receptor comprising an extracellular antigen recognition domain that recognises Trop2 on a target cell. Preferably, the synthetic receptor also comprises a transmembrane domain and an intracellular signalling domain that activates expression of a response element in the cell when the extracellular antigen recognition domain binds the target cell, wherein the response element preferably encodes a STING protein. In such embodiments, the intracellular signalling domain may be intracellularly cleaved upon binding of the antigen recognition domain, releasing the intracellular signalling domain. The synthetic receptors allow the cells to recognise and bind specific target cells and to activate immune responses upon binding.

[0125] The inventors have identified that engineered pDCs may be particularly effective for targeting Trop2. Trop2 overexpression is associated with decreased patient survival as well as increased tumor aggressiveness and metastasis in many cancers (Shvarstur and Bonavida, Genes Cancer. 2015 Mar;6(3-4):84-105). Trop2 is also associated with tumor progression via immune and nonimmune mechanisms (Italiano et al., Journal of Clinical Oncology 41, no. 16_suppl (June 01, 2023) 9040 9040. Trop2 protein expression is also highly restricted in healthy adult tissue and overexpressed in cancers. Therefore, treatment of cancer with pDCs of the invention is expected to provide therapeutic effects.

[0126] In some embodiments, the synthetic receptor is a synthetic intramembrane proteolysis receptor (SNIPR). SNIPRs comprise an extracellular antigen recognition domain, which may be an scFv with specificity for a Trop2, a transmembrane domain, which controls proteolysis and cleavage, and an intracellular signalling domain, which may be a transcription factor. SNIPRs comprise a transmembrane domain from the regulated intracellular proteolysis (RIP) family of proteins. For example, a SNIPR may comprise a transmembrane domain selected from the group consisting of Notchl, Notch2 and Robol. In preferred embodiments, the SNIPR is a SynNotch receptor. Binding of the antigen recognition domain releases the transcription factor, which can target a response element that encodes STING either wildtype or with a selective mutation, such as a gain-of-function mutation. Recognition elements for the transcription factor drive expression of STING protein, thereby supporting constitutive activation of an immune response. As shown in the Examples, binding of a pDC of the invention to its target cell activates expression of STING, which induces a targeted and potent immune response useful for treating disease, including production of type I and III IFNs, CXCL10, MCP-1, MCP-2, MIP-la, MIP-lb. STING and preferred STING proteins are discussed in the subsequent section. In some embodiments, the SNIPR may further comprise a juxtamembrane domain, such as a human N0TCH2 juxtamembrane domain.

[0127] In some embodiments, the pDCs comprise a heterologous nucleic acid encoding the synthetic receptor. In some embodiments, the pDCs comprise a heterologous nucleic acid encoding the response element. In some embodiments the synthetic receptor and response element are encoded by a single heterologous nucleic acid. In some embodiments, the heterologous nucleic acid or acids is or are selected from the group consisting of a viral construct, a plasmid, a cosmid, an mRNA. In some embodiments, the viral construct is an AAV construct, an adenoviral construct, a lentiviral construct, or a retroviral construct.

[0128] In some embodiments, the heterologous nucleic acid is integrated into the genome of the engineered cell. In some embodiments, the heterologous nucleic acid is not integrated into the genome of the engineered cell. In some embodiments, the heterologous nucleic acid is introduced by a transposase, retrotransposase, episomal plasmid, mRNA, or random integration. Preferably, the heterologous nucleic acid is introduced with a gene editing system such as TALEN, zinc finger or, most preferably, CRISPR / Cas9.

[0129] The term heterologous as used herein has its normal meaning. In particular, the heterologous nucleic acid is a nucleic acid that has been introduced into the cell and that is not present in an unmodified cell in the same configuration or location. The heterologous nucleic acid may be constructed using endogenous (preferably human) sequences. Each domain may be heterogeneous, that is, comprised of sequences derived from different protein chains.

[0130] The extracellular antigen recognition domain recognises Trop2 on a target cell and can be designed or selected according to the desired therapeutic use or target cell. In some embodiments, the antigen recognition domain is an antibody or fragment thereof, e.g., a Fab, Fab’, Fv, F(ab’)2, dAb, or preferably, one or more single-domain antibodies (“nanobodies”) or one or more single chain antibody fragments (“scFv”). A scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. A single-domain antibody, also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. scFvs and nanobodies are useful in synthetic receptors because they may be engineered to be expressed as part of a single chain along with the other synthetic receptor components.

[0131] The extracellular antigen recognition domain recognises Trop2 on the cell surface of a target cell. In certain embodiments, the domain specifically binds Trop2 and does not exhibit significant binding to any other antigens, or exhibits significantly stronger binding to Trop2 than any other antigen. In preferred embodiments, expression of the response element only occurs when the extracellular antigen recognition domain binds Trop2. Accordingly, the antigen recognition domain will bind Trop2, preferably specifically bind. Binding will bring the pDC and target cell into close proximity, such that the pDC may exert its anti-cancer effects. Additionally, binding of Trop2 by the antigen recognition domain may activate immune responses in the pDC, specifically targeting the cell expressing Trop2, in the tumour microenvironment, bind Trop2

[0132] In certain embodiments, the extracellular antigen recognition is bispecific or multispecific, with specificity to more than one target of interest.

[0133] The intracellular signalling domain activates expression of a response element which activates expression of STING in the cell when the extracellular antigen recognition domain binds the target cell. The expression of STING activates an inflammatory immune response. In some embodiments, the expression of STING results in the induction of Interferon Stimulated Genes (ISGs) in the cell. In some embodiments, the ISGs induced following the expression of STING comprise IFIT1, IFIT2, MX2, CXCL11, CXCL10, IFNL1, IFNL2, IFNL3, IFNB1, IFNA8 and IFNA10.

[0134] As shown in the examples, induction of STING expression and resulting expression of ISGs is rapid. In some embodiments, STING expression is induced within 12 hours of binding the antigen on the target cell, such as within 10 hours, 8 hours, 6 hours or 5 hours. Preferably, STING expression is induced within 6 hours. In preferred embodiments, maximal STING expression is induced within 12 hours, such as within 10 hours, 8 hours, 6 hours or 5 hours. Preferably, maximal STING expression is induced within 6 hours. In some embodiments, expression of ISGs is induced within 12 hours of binding the antigen on the target cell, such as within 10 hours, 8 hours, 6 hours or 5 hours. Preferably, expression of ISGs is induced within 6 hours. In preferred embodiments, maximal expression of ISGs is induced within 12 hours, such as within 10 hours, 8 hours, 6 hours or 5 hours. Preferably, maximal expression of ISGs is induced within 6 hours.

[0135] In preferred embodiments, the immune response activated in the cell is the secretion of cytokines. The cytokines may include pro-inflammatory cytokines. For example, pro- inflammatory cytokines may promote an inflammatory response, which may be useful in cancer therapy. Examples of pro-inflammatory cytokines include, but are not limited to, IL- la, IL-lb, IL-6, IL-13, IL-17a, tumour necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase-proteins include, but are not limited to, C -reactive protein (CRP) and serum amyloid A (SAA). Preferred cytokines expressed following activation of the intracellular signalling domain and expression of STING include IFNa, IFN , IFN , TNFa, IL-6, CXCL9, CXCL10, CCL2, CCL3, CCL4, CCL8 and / or CXCL11. Most preferred cytokines are CXCL10 and type-I interferons. Further preferred cytokines are MCP-1, MCP-2, MIP-la, MIP-lb.

[0136] As shown in the examples, secretion of cytokines is rapid. In some embodiments, secretion of cytokines occurs within 12 hours of binding the antigen on the target cell, such as within 10 hours, 8 hours, 6 hours or 5 hours. Preferably, secretion of cytokines occurs within 6 hours. In preferred embodiments, maximal secretion of cytokines occurs within 12 hours, such as within 10 hours, 8 hours, 6 hours or 5 hours. Preferably, maximal secretion of cytokines occurs within 6 hours.

[0137] The response element may encode a STING protein. In some embodiments, the STING protein activates an immune response. In some embodiments, the immune response comprises secretion of cytokines or comprises recruitment or activation of host immune cells, such cytotoxic lymphocytes (e.g. NK cells and CD8 T cells). In some embodiments, the cytokines are pro-inflammatory cytokines. In some embodiments, the cytokines comprise CXCL10, MCP-1, MCP-2, MIP-la and / or MIP-lb. In some embodiments, the immune response comprises production of Type I IFN.

[0138] In some embodiments, the immune response comprises the activation of NK cells. Activation may induce CD69 and TRAIL on the NK cells. In some embodiments, activation of the NK cells increases their cytotoxic activity. In preferred embodiments, activation of the NK cells stimulates the NK cells to lyse target cells expressing the antigen.

[0139] In certain embodiments, the immune response that is activated by STING is the recruitment of immune cells, such as or cytotoxic lymphocytes (e.g. NK cells and CD8 T cells).

[0140] In some embodiments, the immune response is the direct lysis of target cells by the pDC.

[0141] The intracellular signalling domain is preferably a transcription factor. The transcription factor may comprise a DNA binding domain and a transcriptional activation domain. The transcription factor is generally released by intracellular proteolysis following binding to the target. The DNA binding domain may be selected from the group consisting of Gal4, Pax6, zinc fingers (ZFs), synthetic zinc fingers (synZFs), transcription activator like effectors (TALEs), TetR, HNF1 alpha, vHNFl beta. The response element will contain the cognate recognition element for the DNA binding domain. Appropriate recognition elements are well established, including UAS elements, which are bound by Gal4, and HNF1 -binding site, which is bound by HNF1 alpha, etc.

[0142] The DNA binding domain may be derived from the group consisting of Gal4, Pax6, zinc fingers (ZFs), synthetic zinc fingers (synZFs), transcription activator like effectors (TALEs), TetR, HNF1 alpha, vHNFl beta. The transcription activation domain may be selected from the group consisting of VP64, VP16, WWTR1, CREB3, NF-KB, p65, Rta, HSF1, and RelA. The transcription activation domain may be derived from the group consisting of VP64, VP16, WWTR1, CREB3, NF-KB, p65, Rta, HSF1, and RelA. The intracellular signalling domain may be selected from the group consisting of Gal4-VP64, Gal4-VP16, TetR-VP64, LacI-VP64, HNF1-WWTR1, HNF1-CREB3, Pax6-p65, ZF-p65, synZF-p65, or HNFl-p65. The response element comprises a promoter that is activated by the intracellular signalling domain. Preferably, the response element comprises an inducible promoter that is activated by the intracellular signalling domain. For example, a suitable promoter is an inducible miniCMV promoter, optionally comprising UAS sequences that are bound by GAL-4. The promoter is operably linked to a gene that encodes a STING protein. The response element and STING gene may be integrated in the genome or present on a vector such as a plasmid. The response element and gene may be introduced by any appropriate technique, such as by transposase, retrotransposase, episomal plasmid or random integration. Preferably, the response element is introduced with a gene editing system such as TALEN, zinc finger or, most preferably, CRISPR / Cas9. Preferred synthetic receptors comprise extracellular antigen recognition domains described above. Preferred synthetic receptors comprise intracellular signalling domains, such as transcription factors, that activate expression of STING.

[0143] In some embodiments, the synthetic receptor comprises a Notch domain encoded by a Notch gene. A preferred Notch gene is N0TCH1. Alternative Notch genes include N0TCH2, N0TCH3 and N0TCH4. In other embodiments, the synthetic receptor comprises a Robol domain.

[0144] In certain embodiments, the synthetic receptor system is multiplexed with multiple transcription factor domains and / or response elements. In certain embodiments, the pDC expresses on its surface a synthetic receptor, optionally a SynNotch receptor, comprising an extracellular antigen recognition domain that recognises an antigen on a target cell, a transmembrane domain, and two or more intracellular signalling domains that activate expression of a two or more response elements in the cell when the extracellular antigen recognition domain binds the target cell, wherein at least one response element encodes a stimulator of interferon genes (STING) protein, and wherein at least one response element encodes an additional gene, preferably a gene that activates an immune response. In certain embodiments, the pDC expresses on its surface two or more synthetic receptors, optionally SynNotch receptors, wherein at least one activates expression of STING, and at least one activates expression of an additional gene, preferably a gene that activates an immune response. In certain such embodiments, the additional gene encodes TRAIL, a checkpoint inhibitor, such as PDL1, IL-12 or Type I UN. In preferred embodiments, expression of PD- L1 or IL10 is activated. In preferred embodiments, expression of IL-12 is activated.

[0145] The synthetic receptor comprises a transmembrane domain linking the extracellular antigen recognition domain and the intracellular signalling domain. The transmembrane domain may be derived from the transmembrane domain of 4-1BB / CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a gamma chain of a T cell receptor, a delta chain of a T cell receptor, CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD34, CD37, CD45, CD64, CD80, CD86, CD 134, CD 137, CD 154, or a zeta chain of a T cell receptor, or any combination thereof. The transmembrane domain may be derived from a member of the regulated intramembrane proteolysis (RIP) family, for example the transmembrane domain may be derived from Notchl, Notch2, Notch3, Notch4, Robol. In a preferred embodiment, the transmembrane domain is derived from Notchl.

[0146] In certain embodiments, the synthetic receptor comprises a hinge or spacer, in particular to allow free movement of the extracellular antigen recognition domain. Suitable hinge or spacers include regions of IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM, or a fragment thereof. In some embodiments, the hinge or spacer is from CD8 alpha. Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the construct. In some embodiments, the linker may be derived from repeats of glycine-glycine-glycine-glycine-serine. The linkers may also be used as a peptide tag. The linker peptide sequence may be of any appropriate length to connect one or more proteins of interest and is preferably designed to be sufficiently flexible so as to allow the proper folding and / or function and / or activity of one or both of the peptides it connects. a)

[0147] In preferred embodiments, the pDC, comprises a single nucleic acid which encodes both the SNIPR and response element, wherein the nucleic acid encodes:

[0148] (a) a promoter, responsive to the transcription factor in the SNIPR, such as the minimal CMV promoter preceded by 5x UAS elements, which are target sequences for Gal4, and

[0149] (b) STING, preferably comprising a gain-of-function mutation that causes it to be constitutively active in the absence of 2’3’-cGAMP

[0150] (c) a second promoter, such as PGK,

[0151] (d) an extracellular antigen recognition domain, such as an anti-Trop2 scFv,

[0152] (e) a CD8 hinge region,

[0153] (f) a transmembrane domain, preferably a human NOTCH 1 transmembrane domain

[0154] (hNl TMD),

[0155] (g) a Gal4-VP64 transcription factor, and

[0156] (h) optionally a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE).

[0157] Constructs illustrating the expected effects of such constructs are set forth in the Examples.

[0158] The Examples demonstrate that such constructs can be expressed by pDCs, which maintain their functionality and type I IFN response, and can be used to successfully recognise target cells, bind and subsequently become activated to produce IFN-alpha and pro- inflammatory factors. In certain embodiments the synthetic receptor and the response element are encoded by separate heterologous nucleic acids. In certain embodiments, the synthetic receptor and the response element are encoded by the same heterologous nucleic acid, which can simplify manufacture of the engineered immune cell, as demonstrated in the Examples.

[0159] In preferred embodiments, in a composition comprising the pDCs of the invention, at least 5%, such as at least 10%, at least 20%, at least 40%, at least 50%, at least 55% or most preferably at least 60%, of the cells express the synthetic receptor.

[0160] STING protein

[0161] The examples demonstrate that the stimulation of interferon genes (STING) protein is effective to activate a potent and targeted immune response in engineered pDCs in combination with a synthetic receptor system.

[0162] The response element may encode a stimulator of interferon genes (STING) protein. In such embodiments the sequence encoding STING will be heterologous to the cell. STING is an ER-associated membrane protein that is critical for innate immune sensing of pathogens and STING is a key immune response regulator. STING-mediated activation of the IFN-I pathway through the TBK1 / IRF3 signaling axis involves both cyclic-dinucleotide binding and its translocation from the ER to vesicles. Activation of STING is known to induce production of type I interferons and inflammatory cytokines. By activating STING on binding to a target cell, the pDCs of the invention may induce a targeted and potent immune response to treat disease. For example, activation of STING upon binding to a target cell may result in cytotoxic T cell activation and or NK cell activation, which may mediate target cell apoptosis, in particular tumor cell apoptosis. Upon activation of STING, the pDC of the invention may recruit cytotoxic T cell and NK cells to the target cell and the disease microenvironment. Upon activation of STING, the cell of the invention may also mediate direct killing of the target cell, for example via cytotoxic activity or TRAIL-mediated killing.

[0163] An exemplary STING protein sequence is SEQ ID NO: 1 (Uniprot Q86WV6)

[0164] 10 20 30 40 50

[0165] MPHSSLHPS I PCPRGHGAQK AALVLLSACL VTLWGLGE PP EHTLRYLVLH 60 70 80 90 100

[0166] LASLQLGLLL NGVCSLAEEL RHIHSRYRGS YWRTVRACLG CPLRRGALLL 110 120 130 140 150

[0167] LS IYFYYSLP NAVGPPFTWM LALLGLSQAL NI LLGLKGLA PAE I SAVCEK 160 170 180 190 200

[0168] GNFNVAHGLA WSYYI GYLRL I LPELQARIR TYNQHYNNLL RGAVSQRLYI 210 220 230 240 250 LLPLDCGVPD NLSMADPNIR FLDKLPQQTG DHAGIKDRVY SNSIYELLEN 260 270 280 290 300

[0169] GQRAGTCVLE YATPLQTLFA MSQYSQAGFS REDRLEQAKL FCRTLEDILA 310 320 330 340 350

[0170] DAPESQNNCR LIAYQEPADD SSFSLSQEVL RHLRQEEKEE VTVGSLKTSA 360 370

[0171] VPSTSTMSQE PELLISGMEK PLPLRTDFS

[0172] An exemplary nucleotide sequence encoding a STING protein sequence is SEQ ID NO:2 (NM_198282.4)

[0173] 1 gttcattttt cactcctccc tcctaggtca cacttttcag aaaaagaatc tgcatcctgg

[0174] 61 aaaccagaag aaaaatatga gacggggaat catcgtgtga tgtgtgtgct gcctttggct

[0175] 121 gagtgtgtgg agtcctgctc aggtgttagg tacagtgtgt ttgatcgtgg tggcttgagg

[0176] 181 ggaacccgct gttcagagct gtgactgcgg ctgcactcag agaagctgcc cttggctgct

[0177] 241 cgtagcgccg ggccttctct cctcgtcatc atccagagca gccagtgtcc gggaggcaga

[0178] 301 agatgcccca ctccagcctg catccatcca tcccgtgtcc caggggtcac ggggcccaga

[0179] 361 aggcagcctt ggttctgctg agtgcctgcc tggtgaccct ttgggggcta ggagagccac

[0180] 421 cagagcacac tctccggtac ctggtgctcc acctagcctc cctgcagctg ggactgctgt

[0181] 481 taaacggggt ctgcagcctg gctgaggagc tgcgccacat ccactccagg taccggggca

[0182] 541 gctactggag gactgtgcgg gcctgcctgg gctgccccct ccgccgtggg gccctgttgc

[0183] 601 tgctgtccat ctatttctac tactccctcc caaatgcggt cggcccgccc ttcacttgga

[0184] 661 tgcttgccct cctgggcctc tcgcaggcac tgaacatcct cctgggcctc aagggcctgg

[0185] 721 ccccagctga gatctctgca gtgtgtgaaa aagggaattt caacgtggcc catgggctgg

[0186] 781 catggtcata ttacatcgga tatctgcggc tgatcctgcc agagctccag gcccggattc

[0187] 841 gaacttacaa tcagcattac aacaacctgc tacggggtgc agtgagccag cggctgtata

[0188] 901 ttctcctccc attggactgt ggggtgcctg ataacctgag tatggctgac cccaacattc

[0189] 961 gcttcctgga taaactgccc cagcagaccg gtgaccatgc tggcatcaag gatcgggttt

[0190] 1021 acagcaacag catctatgag cttctggaga acgggcagcg ggcgggcacc tgtgtcctgg

[0191] 1081 agtacgccac ccccttgcag actttgtttg ccatgtcaca atacagtcaa gctggcttta

[0192] 1141 gccgggagga taggcttgag caggccaaac tcttctgccg gacacttgag gacatcctgg

[0193] 1201 cagatgcccc tgagtctcag aacaactgcc gcctcattgc ctaccaggaa cctgcagatg

[0194] 1261 acagcagctt ctcgctgtcc caggaggttc tccggcacct gcggcaggag gaaaaggaag

[0195] 1321 aggttactgt gggcagcttg aagacctcag cggtgcccag tacctccacg atgtcccaag

[0196] 1381 agcctgagct cctcatcagt ggaatggaaa agcccctccc tctccgcacg gatttctctt

[0197] 1441 gagacccagg gtcaccaggc cagagcctcc agtggtctcc aagcctctgg actgggggct

[0198] 1501 ctcttcagtg gctgaatgtc cagcagagct atttccttcc acagggggcc ttgcagggaa

[0199] 1561 gggtccagga cttgacatct taagatgcgt cttgtcccct tgggccagtc atttcccctc

[0200] 1621 tctgagcctc ggtgtcttca acctgtgaaa tgggatcata atcactgcct tacctccctc

[0201] 1681 acggttgttg tgaggactga gtgtgtggaa gtttttcata aactttggat gctagtgtac

[0202] 1741 ttagggggtg tgccaggtgt ctttcatggg gccttccaga cccactcccc acccttctcc

[0203] 1801 ccttcctttg cccggggacg ccgaactctc tcaatggtat caacaggctc cttcgccctc

[0204] 1861 tggctcctgg tcatgttcca ttattgggga gccccagcag aagaatggag aggaggagga

[0205] 1921 ggctgagttt ggggtattga atcccccggc tcccaccctg cagcatcaag gttgctatgg

[0206] 1981 actctcctgc cgggcaactc ttgcgtaatc atgactatct ctaggattct ggcaccactt

[0207] 2041 ccttccctgg ccccttaagc ctagctgtgt atcggcaccc ccaccccact agagtactcc

[0208] 2101 ctctcacttg cggtttcctt atactccacc cctttctcaa cggtcctttt ttaaagcaca

[0209] 2161 tctcagatta

[0210] In preferred embodiments, the STING response element comprises a gain-of-function mutation that causes it to be constitutively active in the absence of 2’3’-cGAMP. As demonstrated in the Examples, use of such a mutant STING protein provides robust activation of immune responses. In preferred embodiments, the STING protein comprises a mutation, preferably a substitution, at one or more residues corresponding to R71, SI 02, V147, N154, V155, G166, C206, G207, G230, H232, R238, F279, R281, R284, R293, or Q315 of SEQ ID NO: 1. Proteins with mutations at these residues may be constitutively active (as described in W02020028743 Tse el al. Mol Ther. 2021 Jul 7;29(7):2227-2238) and so may be particularly useful in pDCs of the present invention. Preferred mutations at these residues are R71H, V147L, N154S, V155M, V155R, G166E, G230A, H232R, R293Q, R281M, R284M, R238M, and R293M. In more preferred embodiments, the STING proteins comprises a mutation at one or more residues selected from V147, N154 and V155. In most preferred embodiments, the STING protein comprises a mutation at residue N154 and / or V155. The preferred mutation at N154 is N154S. The preferred mutations at V155 are V155R and, more preferably V155M.

[0211] The STING protein encoded by the response element may comprise a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, across the length of SEQ ID NO: 1, optionally with one or more of the mutations described in the preceding paragraph. In preferred embodiments, the STING protein comprises SEQ ID NO: 1, preferably with one or more of the mutations described in the preceding paragraph. The STING protein may be truncated.

[0212] The response element encoding the STING protein may comprise a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2, across the length of SEQ ID NO:2, optionally with one or more substitutions to introduce the mutations described in the paragraph above. In preferred embodiments, the response element encoding the STING protein comprises SEQ ID NO:2, preferably with one or more substitutions to introduce the mutations described in the paragraph above. The response element encoding the STING protein may be codon-optimised.

[0213] An exemplary sequence encoding STING N154 is provided as SEQ ID NO:3

[0214] Sequence for STING N154S:

[0215] Atgccccacagctctctgcaccccagcatcccttgccccagaggccacggcgcccagaaggccgccctggtgctgctgagcgcttg tctggtcacactgtggggactcggagaacctcctgagcacaccctgcggtacctggtgctgcatctggcatctctgcagctgggcctg ctgctgaatggcgtgtgcagcctggccgaggaactgcggcacatccactctagatatagaggctcttattggagaaccgtgcgggcct gcctgggatgtcctctgcggagaggcgctctgctgctgctctctatctacttctactactccctgcccaatgccgtcggccctccattcac ctggatgctggctctgctgggcctgagccaggccctgaacatcctgctgggcctcaagggtctggctcctgctgaaatcagcgccgtg tgcgagaagggcaacttcagcgtggcccacggcctggcctggtcctactacatcggctacctgaggctgattctgcctgagctgcaag ccagaatccggacatacaaccagcactacaacaatctgctgcggggagctgtgtcccagcgcctgtacatcctgctgcctctggattg cggcgttcctgacaacctgagcatggccgatcctaacatcagattcctggacaagctgccccagcagacaggcgaccacgccggca tcaaggacagagtgtacagcaacagcatctacgagctgctggaaaacggccagagggccggcacctgtgtgctggaatacgccac acctctgcagaccctgtttgccatgtcccaatacagccaagccggatttagcagagaggatagactggaacaggccaagctgttctgc cggaccctggaggacatccttgctgacgcccctgagagccagaacaactgcagactgatcgcctaccaggagccagccgacgaca gcagcttcagcctgtctcaggaggtgctgagacacctgagacaggaagagaaagaggaagtgaccgtgggcagcctgaagacctc tgccgtgccctccaccagcaccatgagccaggagcccgagctgcttattagcggcatggaaaaacctctgccactgagaacagatttc agctga

[0216] An exemplary sequence encoding STING V155M is provided as SEQ ID NO:4

[0217] Sequence STING V155M atgccccacagctcccttcatcctagcatcccctgccccagaggccacggcgcccagaaggccgccctggtcctgctgtctgcctgc ctggtgaccctgtggggcctgggcgagcctccagagcacaccctgcggtacctggtgctgcacctggcatctcttcagctgggcctgt tgcttaatggcgtgtgcagcctggccgaggaactgagacacatccactctagataccggggcagctactggcggacagttagagctt gtctgggctgccctctgcgcagaggcgccctgctgctcctgagcatctacttctactacagcctgccaaatgccgtgggacctcctttca cctggatgctggccctgctgggactgagccaggctctgaatatcctgctcggcctgaagggcctggctcctgctgagatcagcgccgt gtgtgaaaaaggcaacttcaacatggcccacggcctggcctggtcctactatatcggatatctgcggctgattctgcccgagctgcaag ccagaatccggacctacaaccagcactacaacaacctgctgagaggagctgtgtcccagcggctgtacatcctgctgcctctggactg cggcgtgcccgacaacctgagcatggccgatcctaacatcagattcctggataagctgcctcagcagacaggcgaccacgccggca tcaaggacagagtgtacagcaacagcatctacgagctgctggaaaacggtcagagagccggaacatgcgtgctggagtacgccac acctctgcagaccctgttcgccatgagccaatactctcaggccggctttagccgggaagatagactggaacaggccaagctgttttgta gaaccctcgaggacatcctggctgatgcccctgagagccagaacaactgcagactgatcgcctaccaggagcctgctgacgacagc tcattcagcctgagccaagaggtgctgaggcacctgagacaggaggagaaggaagaagtgacagtcggctctctgaagaccagcg ccgtgccatctaccagtaccatgtcccaggagcccgagctgctgatcagcggcatggaaaaacctctgcccctgcggaccgacttca gctga

[0218] The response element encoding the STING protein may comprise a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3 or 4, across the length of SEQ ID NO:3 or 4. In preferred embodiments, the response element encoding the STING protein comprises SEQ ID NO:3 or 4. In certain embodiments, the response element comprises a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 or 4, across the length of SEQ ID NO: 3 or 4, which encodes a STING protein that additionally comprises one or more substitutions to introduce the mutations described in the paragraphs above and / or that does not comprise the N154S or V155M mutations. The codon optimisation exemplified in to SEQ ID NO:3 and 4 will be also useful for sequences encoding other substitutions. Plasmacytoid dendritic cells (pDCs) for treating disease

[0219] The examples demonstrate that engineered pDCs are particularly effective to activate a potent and targeted immune response, in particular in combination with a synthetic receptor system. 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.

[0220] The engineered cells for use in the invention are plasmacytoid dendritic cells (pDCs). Plasmacytoid dendritic cells (pDCs) have a multifaceted role in the immune system, which makes them extremely adaptable for the targeted treatments of the invention. 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 hyperactivation of pDCs has been associated with the pathogenesis of several diseases, including viral infections, autoimmune diseases and turn ouri genesis (Swiecki and Colonna, Nat Rev Immunol, 2015. 15(8); and Tang and Cattral, Cell Mol Life Sci, 2016).

[0221] Preferably, the engineered pDCs express TRAIL. In another embodiment said pDCs express CD123, CD303, CD304, CD4 and / or HLA-DR. In yet another embodiment said pDCs express IFN type I, IFN type III and / or proinflammatory cytokines. In a further embodiment said pDCs express IRF7, TLR7 and / or TLR9. In one embodiment said pDCs express CD40, CD80, CD83 and / or CD86. For example, said pDCs express TRAIL, CD123, CD303, CD304, CD4, HLA-DR, IFN type I, IFN type III, IRF7, TLR7 and / or TLR9. In preferred embodiments, the pDCs of the invention express CD123, CD303 and CD304 and are negative for lineage markers and CD11c, as shown in the Examples.

[0222] In one preferred embodiment of the present invention, the pDCs express TNF-related apoptosis-inducing ligand (TRAIL). TRAIL, which is also designated CD253, is a ligand involved in killing cancer cells by interacting with TRAIL receptors (death receptor 5, DR5) on the surface of cancer cells and thereby triggering apoptosis.

[0223] The engineered pDCs of the invention are preferably matured cells having a surface phenotype that strongly resembles blood pDCs. In a preferred embodiment, said pDCs express CD 123, CD303, CD304, CD4 and / or HLA-DR.

[0224] A key attribute of the cells of the invention for use in therapeutic compositions may be the phenotype CD123+ and HLA-DR+. In preferred embodiments, in a composition comprising the pDCs of the invention, at least 10%, such as at least 20%, at least 40%, at least 50%, at least 55% or most preferably at least 60%, of the cells have the phenotype CD123+ and HLA-DR+.

[0225] In another preferred embodiment said pDCs express IFN type I, IFN type III and / or proinflammatory cytokines.

[0226] The pDCs may in one preferred embodiment express Toll-like receptors, such as for example Toll-like receptor 7 (TLR7) and / or Toll-like receptor 9 (TLR9).

[0227] In yet another preferred embodiment said pDCs express Interferon regulatory factor 7 (IRF7).

[0228] In a preferred embodiment said pDCs secretes IL-6.

[0229] In preferred embodiments, the engineered plasmacytoid dendritic cell is capable of a type I IFN response.

[0230] In another preferred embodiment said pDCs express Cluster of differentiation 80 (CD80), which is a protein found on Dendritic cells, activated B cells and monocytes that provides a costimulatory signal necessary for T cell activation and survival. The pDCs may in a preferred embodiment also express proteins characteristic for antigen presenting cells such as for example Cluster of Differentiation 86 (CD86) and / or Cluster of Differentiation 40 (CD40). CD86 is a protein expressed on antigen-presenting cells that provides costimulatory signals necessary for T cell activation and survival, whereas CD40 is a costimulatory protein found on antigen presenting cells and is required for their activation.

[0231] Preferably, said pDCs express CD40, CD80, CD83 and / or CD86. In another preferred embodiment said pDCs express interleukin 6 (IL-6).

[0232] In preferred embodiments, the engineered pDCs of the invention are stem cell-derived plasmacytoid dendritic cell.

[0233] In certain embodiments, pDCs are autologous. Such treatments may minimise any risk of rejection of the transferred cells. In alternative 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.

[0234] 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 innate lymphocytes or interferon-producing cells and methods of treatment using such cells.

[0235] Methods for treating cancer

[0236] The invention provides a method of treating cancer in a subject comprising administering an engineered pDC of the invention to the subject. Therefore, the invention provides 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, albeit using different transferred immune cells with different immune-regulating effects and activities.

[0237] The inventors have identified that engineered pDCs may be particularly effective for targeting Trop2 (Trophoblast cell surface antigen 2). Trop2 overexpression is associated with decreased patient survival as well as increased tumor aggressiveness and metastasis in many cancers (Shvarstur and Bonavida, Genes Cancer. 2015 Mar;6(3-4):84-105). Trop2 is also associated with tumor progression via immune and nonimmune mechanisms (Italiano et al., Journal of Clinical Oncology 41, no. 16_suppl (June 01, 2023) 9040 9040. Trop2 protein expression is also highly restricted in healthy adult tissue and overexpressed in cancers. Therefore, treatment of cancer with pDCs of the invention is expected to provide therapeutic effects.

[0238] In preferred embodiments, the cancer to be treated expresses Trop2. Trop2 expression may be assessed by immunohistochemistry (H4C) or in situ hybridization (ISH). In preferred embodiment, the cancer to be treated is Trop2+.

[0239] TROP2 is known to be upregulated in a range of cancers, including bladder urothelial carcinoma, head and neck squamous cell carcinoma, lung squamous cell carcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, lung adenocarcinoma, prostate adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, thyroid carcinoma, pancreatic cancer and uterine corpus endometrial carcinoma. Sacituzumab govitecan is a Trop2 antibody conjugate and is currently progressing in clinical trials for treatment of epithelial cancers, triple negative breast cancer, non-small cell lung cancer, urothelial carcinoma, castration-resistant prostate cancer, HR+ / HER2- metastatic breast cancer, head and neck squamous cell carcinoma, endometrial cancer, ovarian cancer, glioblastoma, brain tumors and endometrial carcinoma (Wen et al. Ann TranslMed 2022; 10(24): 1403). Accordingly, in preferred embodiments the cells and methods of the invention are for use in treating one of the cancers listed above, preferably a Trop2-positive cancer. In preferred embodiments, the cancer to be treated in accordance with the invention is metastatic.

[0240] In preferred embodiments, the cells of the invention are for use in treatment of triple negative breast cancer, non-small cell lung cancer, pancreatic or ovarian cancer. Trop2 is highly expressed in triple negative breast cancer, non-small cell lung cancer, pancreatic and ovarian cancer, which all have very low five-year survival rates, and so the cells of the invention are expected to provide particular benefits.

[0241] In preferred embodiments, the cells of the invention are for use in treatment of triple negative breast cancer. pDCs are known to infiltrate triple negative breast cancer tumours and be associated with better survival (Oshi et al Cancers (Basel). 2020 Nov 12; 12(11):3342), and triple negative breast cancer is known to express Trop2, so the therapy of the invention is expected to be particular effective against triple negative breast cancer.

[0242] In preferred embodiments, the cells of the invention are for use in treatment of colon cancer. The presence and density of tumour-infiltrating pDCs is known to be correlated with survival in colon cancer (Kiessler, J Immunother Cancer. 2021 Mar;9(3):e001813)., and colon cancer is known to express Trop2, so the therapy of the invention is expected to be particular effective against colon cancer.

[0243] In preferred embodiments, the patient to be treated has relapsed following a previous treatment. In preferred embodiments, the cancer to be treated as been characterised as refractory to treatment, for example because at least one treatment has been tried and has failed.

[0244] In certain embodiments of the invention, the methods of treatment may comprise (i) collecting autologous hematopoietic stem progenitor cells (HSPCs), either from the subject to be treated or a healthy donor; (ii) preparing engineered pDCs, for example using a method discussed below; (iii) optionally administering to the subject lymphodepleting chemotherapy; and (iv) administering to the subject the engineered pDCs.

[0245] In certain embodiments, the methods of the invention may comprise administering cells expressing more than one exogenous construct. Individual cells may express more than one construct, or the population of cells administered may comprise a plurality of different cells.

[0246] In certain embodiments, the engineered cells are further modified to express immune- modulatory proteins, such as cytokines (e.g., IL-2, IL-12 or IL-15), which may stimulate T- cell activation and recruitment, and may thus aid in combating the tumour microenvironment. Thus, the cells may comprise a population of cells expressing the exogenous construct and further expressing an immune modulatory protein such as, for example, IL-2, IL- 12, or IL-15.

[0247] In certain embodiments, the engineered cells may be isolated from a subject and used fresh, or frozen for later use, in conjunction with (e.g., before, simultaneously or following) lymphodepletion.

[0248] In certain embodiments, the engineered cells 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.

[0249] 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.

[0250] 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.

[0251] Preferred doses are between 5xlOE6 pDCs and 50xl0E6 pDCs, such as between 5xlOE6 pDCs and 35xlOE6 pDCs, or between 10xl0E6 pDCs and 50xl0E6 pDCs or between 10xl0E6 pDCs and 35xlOE6 pDCs, or between 20xl0E6 pDCs and 50xl0E6 pDCs, or between 5xlOE6 pDCs and 15xlOE6 pDCs, or between 20xl0E6 pDCs and 30xl0E6 pDCs, or between 40xl0E6 pDCs and 50xl0E6 pDCs. Most preferred are 10 x 10E6, 25 x 10E6, and 50 x 10E6 pDCs.

[0252] In certain embodiments of the invention, the compositions comprising the cells of the invention are administered once a month. In certain embodiments, at least 3 doses are administered.

[0253] 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 engineered cells, or subsequent to. In certain embodiments, lymphodepletion is performed both before and after administration of the engineered cells.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] The engineered cells of the invention may be administered by any appropriate route. Generally, the cells will be administered by intravenous infusion. Preferably, the cells will be suspended from vials in a patient-specific infusion bag. Preferably, the cells will be stored in liquid nitrogen and thawed before use. Preferably, the cells will be stored in liquid nitrogen in a liquid nitrogen storage infusion bag for direct intravenous infusion after thawing.

[0258] The methods and cells of the invention are expected to be particularly effective for treating cancer. The ability of pDCs to secrete immunomodulatory factors and alter immune- inhibiting tumour microenvironments, and the potent effects of STING, are expected to be particularly useful for treating cancer. Also, cancer cells present Trop2 that can be recognised by the extracellular antigen recognition domain to provide targeted therapy at tumour sites, avoiding healthy cells.

[0259] In preferred embodiments, the cancer is a solid tumour. The cells of the invention may be particular effective at targeting and altering the microenvironment 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.

[0260] In certain embodiments, the method and cells 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.

[0261] 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. In preferred embodiments, the method or cell of the invention is for use in treating cancer and the cell is an engineered stem cell-derived plasmacytoid dendritic cell that expresses a synthetic receptor comprising an extracellular antigen recognition domain that recognises an antigen on a target cell, a transmembrane domain, and an intracellular signaling domain that activates expression of a response element in the cell when the extracellular antigen recognition domain binds the target cell, wherein the response element encodes a stimulator of interferon genes (STING) protein.

[0262] In the therapeutic methods of the invention, engineered cells 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.

[0263] Methods for generating cells of the invention

[0264] The engineered cells may be generated by any appropriate method. Exemplary methods for generating pDCs in significant amounts are provided in WO2018 / 206577. The invention also provides methods of generating engineered plasmacytoid dendritic cells.

[0265] The methods of the invention may further comprise a step of producing the pDCs.

[0266] In certain embodiments, the method for producing an engineered plasmacytoid dendritic cell (pDCs) comprises:

[0267] - providing hematopoietic stem progenitor cells (HSPCs)

[0268] - transfecting or transducing said HSPCs with a vector encoding a synthetic receptor as described herein and a vector encoding a response element as described herein, optionally wherein a single vector encodes both elements,

[0269] - incubating said HSPCs in one or more media, which media may typically comprise one or more cytokines, growth factors, interferons (IFNs) and / or aryl hydrocarbon receptor (AHR) antagonists (such as stemregenin-1), whereby said HSPCs are differentiated into precursor-pDCs and into pDCs.

[0270] In certain embodiments, the method for producing an engineered plasmacytoid dendritic cell (pDCs) comprises:

[0271] - providing hematopoietic stem progenitor cells (HSPCs), - incubating said HSPCs in one or more media, which media may typically comprise one or more cytokines, growth factors, interferons (IFNs) and / or aryl hydrocarbon receptor (AHR) antagonists (such as stemregenin-1), whereby said HSPCs are differentiated into precursor-pDCs and into pDCs,

[0272] - transfecting or transducing said pDCs with a vector encoding a synthetic receptor as described herein and a vector encoding a response element as described herein, optionally wherein a single vector encodes both elements.

[0273] Accordingly, in certain embodiments, the method for producing an engineered plasmacytoid dendritic cell (pDCs) comprises:

[0274] - providing hematopoietic stem progenitor cells (HSPCs),

[0275] - incubating said HSPCs in one or more media, which media may typically comprise one or more cytokines, growth factors, interferons (IFNs) and / or aryl hydrocarbon receptor (AHR) antagonists (such as stemregenin-1), whereby said HSPCs are differentiated into precursor-pDCs and into pDCs, and

[0276] - transfecting or transducing said HSPCs prior to differentiation, or transfecting or transducing said pDCs subsequent to differentiation, with a vector encoding a synthetic receptor as described herein and a vector encoding a response element as described herein, optionally wherein a single vector encodes both elements.

[0277] In preferred embodiments, the method provides producing pDCs from pluripotent stem cells, wherein the method comprises:

[0278] (a) transfecting or transducing pluripotent stem cells with a vector encoding a synthetic receptor as described herein, and optionally a response element as described herein on the same or a different vector;

[0279] (b) differentiating the pluripotent stem cells into Hematopoietic Stem and Progenitor cells (HSPCs);

[0280] (c) optionally isolating the HSPCs;

[0281] (d) differentiating the HSPCs into pDCs; and

[0282] (e) optionally isolating the pDCs.

[0283] In preferred embodiments, the method provides producing pDCs from pluripotent stem cells, wherein the method comprises: (a) transfecting or transducing pluripotent stem cells with a vector encoding a synthetic receptor as described herein, and optionally a response element as described herein on the same or a different vector;

[0284] (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;

[0285] (c) optionally isolating the HSPCs;

[0286] (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

[0287] (e) optionally isolating the pDCs.

[0288] Transfecting or transducing the cells can be achieved by any appropriate technique.

[0289] 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.

[0290] 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, or the extracellular domain of the synNotch may contain an epitope, to aid sorting of pDCs with the construct. 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 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.

[0291] 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. In certain embodiments, the heterologous nucleic acid is introduced using a sitespecific DNA editing such as TALEN, zinc finger or CRISPR / Cas.

[0292] In preferred embodiments, CD34+ HSPC are transfected or transduced and then differentiated into pDCs.

[0293] In certain embodiments, the method for producing an engineered plasmacytoid dendritic cell (pDCs) comprises:

[0294] - providing hematopoietic stem progenitor cells (HSPCs)

[0295] - transfecting or transducing said HSPCs with a vector encoding a synthetic receptor as described herein and a vector encoding a response element as described herein, optionally wherein a single vector encodes both elements,

[0296] - incubating said HSPCs in a first medium comprising cytokines and growth factor whereby said HSPCs are differentiated into precursor-pDCs

[0297] - adding interferons (IFNs) to said first medium to obtain a second medium whereby said precursor- pDCs are differentiated into pDCs

[0298] In certain embodiments, the method for producing an engineered plasmacytoid dendritic cell (pDCs) comprises:

[0299] - providing hematopoietic stem progenitor cells (HSPCs)

[0300] - transfecting or transducing said HSPCs with a vector encoding a synthetic receptor as described herein and a vector encoding a response element as described herein, optionally wherein a single vector encodes both elements,

[0301] - incubating said HSPCs in a first medium comprising cytokines and growth factor whereby said HSPCs are differentiated into precursor-pDCs

[0302] - adding stem cell factor (SCF) and an aryl hydrocarbon receptor antagonist (such as stemreginin- 1) in a first medium to obtain high yield of pre-cursor pDCs

[0303] - providing a second medium comprising interferons (IFNs)

[0304] - adding interferons (IFNs) to said a second medium to said first medium comprising pre-cursor pDCs, whereby said precursor- pDCs are transformed into to obtain a high yield of fully activated and differentiated pDCs a second medium whereby said precursor- pDCs are differentiated into pDCs

[0305] In some embodiments, the invention uses pDCs extracted from blood or bone marrow. Any appropriate technique may be used for isolating pDCs from blood or bone marrow. Such cells may be transfected with a vector comprising an expression cassette comprising a transgene encoding the at least one antigen or receptor using any appropriate method.

[0306] In some embodiments, the invention uses pDCs derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Any appropriate technique may be used for differentiating iPSCs and ESCs into HSPCs and into pDCs. Exemplary protocols are provided herein for differentiating HSPCs into pDCs. The production of DCs from ESCs and iPSCs are disclosed in, for example, Li et al. World J Stem Cells. 2014 Jan 26; 6(1): 1-10 and the production of HSPCs from ESCs and iPSCs are disclosed in, for example, Tan et al., Proc Natl Acad Sci USA. 2018;l 15(9):2180-2185, Tursky c / a / . Stem Cell Reports. 2020;15(3):735-748 and Demirci et al. Stem Cell Res Ther. 2020;l 1(1):493. Similar processes may be used to generate pDCs.

[0307] In some embodiments of the invention, the pDCs comprise one or more heterologous nucleic acids encoding the synthetic receptor and the response element. In some embodiments, the heterologous nucleic acid is integrated into the genome of the engineered cell. In some embodiments, the heterologous nucleic acid is not integrated into the genome of the engineered cell. In some embodiments, the heterologous nucleic acid is introduced by a transposase, retrotransposase, episomal plasmid, mRNA, or random integration. In certain embodiments, the heterologous nucleic acid is introduced with a gene editing system such as TALEN, zinc finger or CRISPR / Cas9.

[0308] In preferred embodiments said second medium comprises IFN-y and / or IFN-p. In another embodiment said second medium further comprises IL-3. Preferably, said second medium comprises IL-3, IFN-y and IFN-p.

[0309] The precursor-pDCs may for example be incubated for at least 24 hours in said second medium. Said precursor-pDCs may be incubated for 24 to 72 hours in said second medium. Preferably, said precursor pDCs are incubated for around 24 hours, such as 20-28, 22-26 or 24 hours.

[0310] In one embodiment said first medium comprises Flt3 ligand, thrombopoietin and / or interleukin-3. In another embodiment said first medium further comprises stem cell factor and StemRegenin 1. In another embodiment said first medium further comprises stem cell factor and UM 171. In another embodiment said first medium further comprises RPMI medium supplemented with fetal calf serum (FCS). In another embodiment said first medium comprises serum-free medium (SFEM). Preferably, said first medium comprises Flt3 ligand, thrombopoietin, SCF, interleukin-3 and StemRegenin 1. The HSPCs may for example be incubated for 21 days in said first medium.

[0311] In one embodiment the method as described herein, further comprises a step of immunomagnetic negative selection to enrich for differentiated pDCs.

[0312] Before differentiation of HSCs into precursor-pDCs, the HSCs may be cultured in a culture medium not comprising differentiation factors. 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.

[0313] “Hematopoietic stem cells” (HSCs) 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.

[0314] In a preferred embodiment HSCs are Hematopoietic stem and progenitor cells (HSPCs) that are positive for the marker CD34. HSCs or HSPCs are found in the bone marrow of humans, such as in the pelvis, femur, and sternum. They are also found in umbilical cord blood and in peripheral blood.

[0315] Stem and progenitor cells can be taken from the pelvis, at the iliac crest, using a needle and syringe. The cells can be removed as liquid for example to perform a smear to look at the cell morphology or they can be removed via a core biopsy for example to maintain the architecture or relationship of the cells to each other and to the bone.

[0316] The HSCs or HSPCs may also be harvested from peripheral blood. To harvest HSCs or HSPCs from the circulating peripheral blood, blood donors can be injected with a cytokine that induces cells to leave the bone marrow and circulate in the blood vessels. The cytokine may for example be selected from the group consisting of granulocyte-colony stimulating factor (G-CSF), GM-CSF granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclophosphamide. They are usually given as an injection into the fatty tissue under the skin every day for about 4-6 days.

[0317] The HSCs or HSPCs may also be harvested or purified from bone marrow. Stem cells are 10-100 times more concentrated in bone marrow than in peripheral blood. The hip (pelvic) bone contains the largest amount of active marrow in the body and large numbers of stem cells. Harvesting stem cells from the bone marrow is usually done in the operating room.

[0318] HSCs or HSPCs may also be purified from human umbilical cord blood (UCB). In this method, blood is collected from the umbilical cord shortly after a baby is born.

[0319] The first medium is a differentiation medium, wherein HSCs are differentiated into precursor-pDCs. Thus, the first medium comprises differentiation factors.

[0320] Before differentiation of HSCs into precursor-pDCs, the HSCs may be cultured in a culture medium not comprising differentiation factors. 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.

[0321] To initiate differentiation of HSCs into precursor-pDCs, differentiation factors, such as Flt3 ligand, thrombopoietin and / or at least one interleukin selected from interleukin-3, IFN-b and PGE2 are added to the medium. SCF and / or an aryl hydrocarbon receptor antagonist (such as stemreginin- 1) can also be used.

[0322] Thus, in a preferred embodiment said first medium comprises Flt3 ligand, thrombopoietin and / or at least one interleukin selected from interleukin-3, IFN-b and PGE2. More preferably, said first medium comprises Flt3 ligand, thrombopoietin and / or interleukin- 3. In another preferred embodiment, the first medium comprises SCF and / or an aryl hydrocarbon receptor antagonist (such as stemreginin- 1).

[0323] Appropriate culture media can be prepared by the skilled person, for example using the guidance in WO 2018 / 206577.

[0324] The HSPCs are incubated in the first medium under conditions that are typical for human cell cultures and well known to the skilled person. Typical conditions for incubation of cell cultures are for example a temperature of 37 °C, 95% humidity and 5% CO2.

[0325] 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 in said first medium. In a more preferred embodiment the culture is incubated for at least 16 days, such as at least 18 days, at least 20 days or such as for example at least 21 days in said first medium. In a further preferred embodiment, the culture is incubated for at least 7 days in said first medium. In a further preferred embodiment, the culture is incubated for at least 1 day in said first medium.

[0326] The HSCs may for example be incubated for 1 week, 2 weeks, 3 weeks or 4 weeks in said first medium. In a preferred embodiment said HSPCs are incubated for 21 days in said first medium.

[0327] In one embodiment the first medium is refreshed during the incubation period. The medium may for example be refreshed every second day, every third day or every fourth day during the incubation period. The first medium is preferably refreshed with medium containing one or more components of the first medium as described herein and above. Preferably the medium is refreshed with medium comprising the cytokines.

[0328] After incubation of HSPCs in the first medium, wherein HSCs are differentiated into precursor-pDCs, IFNs are added to the first medium thereby obtaining a second medium.

[0329] Alternatively, a second medium is provided, which comprises IFNs, such as IFN type I, IFN type II and / or IFN type III.

[0330] In one embodiment said second medium comprises IFN-a, IFN-y and / or IFN-p.

[0331] In one preferred embodiment said second medium comprises IFN-y and / or IFN-p. Preferably, said second medium comprises IFN-y and IFN-p.

[0332] In another preferred embodiment said second medium comprises interleukin-3 (IL-3). In the embodiment, wherein the first medium comprises IL-3, IL-3 may be added to the medium again, for example together with the interferons. It is understood that the three components can be added in any order. In a particular preferred embodiment said second medium comprises IFN-y, IFN-P and IL-3.

[0333] The precursor-pDCs are incubated in the second medium under conditions that are typical for human cell cultures and well known to the skilled person. Typical conditions for incubation of cell cultures are for example a temperature of 37 °C, 95% humidity and 5% CO2.

[0334] In one embodiment said precursor-pDCs are incubated in said second medium for at least 1 hour, such as at least 5 hours, such as for example at least 10 hours, such as at least 15 hours or such as at least 20 hours in said second medium. In one preferred embodiment precursor-pDCs are incubated for at least 24 hours in said second medium.

[0335] In another embodiment said precursor-pDCs are incubated in said second medium for at least 1 day, at least two days, at least three days or at least 4 days. In some embodiments, the methods of the invention 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.

[0336] Construct sequences

[0337] The present invention provides cells transfected or transduced with the following constructs: UTXp090 (SEQ ID NO: 5); UTXp0108 (SEQ ID NO: 6); UTXpllS (SEQ ID NO: 7); UTXplOS (SEQ ID NO: 8); UTXpll4 (SEQ ID NO: 9); UTXpll5 (SEQ ID NO: 10); UTXpll6 (SEQ ID NO: 11); UTXpll7 (SEQ ID NO: 12); UTXpll8 (SEQ ID NO: 13); UTXpll9 (SEQ ID NO: 14); UTXpl43 (SEQ ID NO: 15); UTXpl44 (SEQ ID NO: 16); UTXpl45 (SEQ ID NO: 17); and UTXpl46 (SEQ ID NO: 18).

[0338] General

[0339] It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0340] In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes “polypeptides”, and the like.

[0341] Unless specifically prohibited, the steps of a method disclosed herein may be performed in any appropriate order and the order in which the steps are listed should not be considered limiting.

[0342] All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Examples

[0343] Example 1

[0344] Hematopoietic stem and progenitor cells (HSPCs) were transduced with two lentiviral vectors, each encoding one of the SynNotch components; one construct encoding the anti- CD19 scFv SynNotch receptor, and one encoding the STING gain-of-function (STING N154S or STING VI 55M) response element (Figure 4a-c). Subsequently, cells were differentiated into U-pDCs. Figure 8a shows representative FACS plots, showing the expression of the anti-CD19 scFv SynNotch receptor (FMC63) and the STING N154S SynNotch response element, or the STING VI 55M SynNotch response element (eGFP) 6- days post transduction (Mock = cells that did not receive any of the two lentiviral vectors). At the end of pDC differentiation, U-pDCs were primed for one day, and a pDC phenotyping was performed on cells. Figure 8b-c presents representative FACS plots showing the phenotype of primed (IFN) or non-primed (IL-3) U-pDCs transduced with the antiCD19 SynNotch receptor and SynNotch STING N154S (b), or SynNotch STING VI 55M (c) response element. U-pDCs were found to display a pDC phenotype, with no expression of the conventional dendritic cell marker CD11c (data not shown), and high levels of the pDC marker CD303 upon priming. U-pDCs were found to still express the SynNotch components, with -20% of the pDCs being double positive for both constructs. This example shows that it is possible to express the SynNotch components in U-pDCs.

[0345] Example 2

[0346] Primed (IFN) or non-primed (IL-3) U-pDCs, where 20% of the cells express the antiCD19 SynNotch and STINGN154S response element, or STING V155M response element, or U- pDCs not expressing the construct (mock), were co-cultured with CD 19 positive target cells (NALM6) or CD19 negative non-target cells (K562) at an effectortarget ratio of 1 :4 or 1 :8. Supernatants were subsequently collected 24 hours later, and bioactive type I IFN (Figure 8d) or CXCL10 (Figure 8e) were evaluated using a commercially-available type I IFN bioassay (HEK-blue Type I IFN reporter bioassay), or a CXCL10 ELISA, respectively. The data in Figure 8d-e show that the SynNotch STING U-pDCs specifically recognize and respond to the target cell line, and subsequently become activated and produce type I IFN and CXCL10. Data are representative of one donor. This example shows that expression of the SynNotch system components in U-pDCs allows the U-pDCs to recognize target cells, resulting in a type I IFN and CXCL10 response. Example 3

[0347] Hematopoietic stem and progenitor cells (HSPCs) were transduced with one ‘all-in-one’ lentiviral vector encoding both the SynNotch receptor and the STING N154S response element, or STING VI 55M response element in (Figure 5a-b). Next, HSPCs were differentiated into U-pDCs. During pDC differentiation, the population was split in two where one population was purified for SynNotch positive cells by positive immunomagnetic selection and the other population was left unsorted. After pDC differentiation, U-pDCs were either cryopreserved or primed for experiments (Figure 9a). 6-days post transduction (before splitting the culture in to two populations) 50.2% and 55.1% of cells were found to express the SynNotch components for STING N154S and STING VI 55M, respectively (Figure 9b). Next, the populations were split in to two, and in one population SynNotch-positive cells were purified by staining with anti-FMC63-PE, followed by positive immunomagnetic selection for PE (Figure 9c). After the selection, 88.0% and 88.9% of cells were positive for the SynNotch components for STING N154S and STING VI 55M, respectively (Figure 9d). This example shows that it is possible to transduce HSPCs with an ‘all-in-one’ lentiviral vector that encodes both the SynNotch receptor and the SynNotch STING response element, allowing expression of both components of the SynNotch system in cells. This further allows purification of SynNotch positive cells using positive selection.

[0348] Example 4

[0349] Subsequent to the experiments outlined in Example 3, lentiviral vector-transduced HSPCs were differentiated into pDCs. A similar proliferation was observed for cells transduced with the STING VI 55M construct as non-transduced cells (mock) (Figure 9e and Figure 9g). This was also evident for cells that had been immunomagnetically selected for FMC63 expression, showing that neither expression of STING VI 55M nor sorting of cells affected proliferation of cells during pDC differentiation (Figure 9e-g). Proliferation of cells expressing the SynNotch STING N154S was slightly reduced (Figure 9e-f). After the pDC differentiation, pDCs were primed (IFN) or left non-primed (IL-3) for 24 hrs. Next, pDCs were phenotyped based on the expression of lineage markers, CD11c, CD 123, CD303, and FMC63. SynNotch STING VI 55M U-pDCs were found to show a similar phenotype as non-transduced U-pDCs (mock), being negative for lineage markers and CD11c, and expressing CD 123 and CD303 upon IFN priming (Figure 9h-i). SynNotch STING N154S U-pDCs were found to display an affected pDC phenotype with high expression of lineage and CD11c, and low expression of CD123 and CD303 (Figure 9j). Next, SynNotch U-pDCs were co-cultured with CD19 positive target cells (NALM6 or REH6), or CD 19 negative non-target cells (K562) at an effector :target ratio of 1 :8. Supernatants were collected 24 hours later and type I IFN was evaluated using a commercially available type I IFN bioassay (HEK-blue Type I IFN reporter bioassay). The data show in figure 9k that the U-pDCs expressing the ‘all-in-one’ construct with both SynNotch system components specifically recognize the target cell lines NALM6 and REH6, and subsequently become activated and produce type I IFN (Figure 9k).

[0350] Example 5

[0351] Hematopoietic stem and progenitor cells (HSPCs) were transduced with one ‘all-in-one’ lentiviral vector encoding the SynNotch receptor and the STING VI 55M response element (Figure 5b). Next, HSPCs were differentiated into U-pDCs. During pDC differentiation SynNotch positive cells were purified by immunomagnetic positive selection. After pDC differentiation, U-pDCs were either cryopreserved or primed for experiments. The setup was conducted using three individual donors. Transduction or sorting of SynNotch positive cells did not affect proliferation of HSPCs or differentiation into U-pDCs (Figure lOa-c). An average transduction efficiency of 54% was obtained, and after purification a purity of 93.9% SynNotch positive cells was achieved (Figure lOd). The level of SynNotch receptor expression was found to be retained during differentiation and priming of U-pDCs. Next, SynNotch U-pDCs or non-transduced U-pDCs (mock) were primed with IFN (IFN) or left non-primed (IL-3). Phenotypic analysis of the U-pDCs showed a similar phenotype, with U- pDCs being negative for lineage markers and CD11c and expressing the pDC markers CD123, CD303 and CD304 at high levels (Figure lOe).

[0352] Example 6

[0353] Primed (IFN) or non-primed (IL-3) U-pDCs expressing the ‘all-in-one’ anti-CD19 SynNotch with STING VI 55M response element, or U-pDCs not expressing the construct (mock), were next co-cultured with CD 19 positive target cells (NALM6 or REH6), or CD 19 negative nontarget cells (K562) at an effectortarget ratio of 1 :8. Supernatants were collected 24 hours later and levels of type I IFN was evaluated using a commercially available type I IFN bioassay (HEK-blue Type I IFN reporter bioassay) (Figure 1 Of). The data in Figure lOg-h show that SynNotch STING VI 55M U-pDCs specifically recognize the target cell lines NALM6 and REH6, and subsequently become activated to produce type I IFN. The response is found to be higher in U-pDCs that have undergone sorting for the SynNotch receptor during pDC differentiation versus unsorted cells (Figure lOg-h). The production of type I IFN in U-pDCs upon SynNotch activation occurs independent of priming of the U-pDCs.

[0354] Example 7

[0355] Anti-CD19 SynNotch STING V155M U-pDCs, or U-pDCs not expressing the construct (mock), were activated with the TLR7 agonist R837, or the STING agonist 2’3’-cGAMP. 24 hours later, supernatants were collected, and type I IFN was analyzed using a commercially available type I IFN bioassay (HEK-blue Type I IFN reporter bioassay). The data in Figure lOi-j show that SynNotch STING V155M U-pDCs display a similar induction of type I IFN as the mock U-pDCs upon agonist stimulation, indicating expression of the SynNotch components in U-pDCs does not affect the differentiation or function of U-pDCs. A similar production of type I IFN is observed for SynNotch U-pDCs that have been sorted for the SynNotch receptor during U-pDC differentiation versus unsorted cells, indicating that the sorting process do not affect U-pDC differentiation, or function of U-pDCs.

[0356] Example 8

[0357] Anti-CD19 SynNotch STING V155M U-pDCs, or U-pDCs not expressing the construct (mock), were activated with 2’3’-cGAMP or anti-c-Myc magnetic beads. 24 hours later, supernatants were collected, and type I IFN was analyzed using a commercially available type I IFN bioassay (HEK-blue Type I IFN reporter bioassay). The data in Figure 10k show that SynNotch STING VI 55M U-pDCs can be activated using anti-c-Myc magnetic beads, which bind the SynNotch receptor, allowing activation of the receptor without being cocultured with target cells carrying the cognate antigen.

[0358] Example 9

[0359] Anti-CD19 SynNotch-STINGvl55MU-pDCs, or U-pDCs not expressing the construct (mock), were primed (IFN) or not primed (IL-3), and then activated by exposure to target cells expressing CD19, with 2’3’-cGAMP or anti-c-Myc magnetic beads. The data in Figurel 1 show SynNotch STING VI 55M pDCs induce a broad cytokine response in response to activation by target cells expressing cognate antigen, by cGAMP or by c-Myc. The cytokines induced include type I and III IFNs, as well as various chemokines, including MCP-2, CXCL10 (IP-10), MCP-1, MIP-la, and MIP-lb (Figure l la-b).

[0360] The broad immune response of SynNotch-STINGvl55MU-pDCs activated by cGAMP or c- Myc is also reflected on the RNA level, as measured by RNA-seq (Figure 12a-e). This is reflected in the induction of multiple interferon-induced genes (ISGs), and different IFN subtypes, including IFIT1, IFIT2, MX2, CXCL11, CXCL10, IFNL1, IFNL2, IFNL3, IFNB1, IFNA1, IFNA8, and IFNA10 (Figure 12d). Overall, the resulting response of activation of the SynNotch STING-gain-of-function correlates with a potent induction of an immune response, which is linked with anti-viral defences, type I IFN signalling, induction of cytokine- mediated signalling pathways, and activation of various immune cells, including NK cells and B cells (Figure 12e).

[0361] Example 10

[0362] The response of SynNotch-STINGvl55MU-pDCs is dependent on recognizing cognate antigen. Secretion of IFNa was induced in anti-CD19 SynNotch STINGV155MpDCs upon exposure to CD19-positive target cells (Raji cells). CD19-knock-out Raji cells did not induce a response, suggesting that no ligand-independent activation occurred (Figure 13)

[0363] Example 11

[0364] Activated SynNotch-STINGvl55MU-pDCs produce factors that promote the activity of NK cells (Figure 14a-f). Conditioned medium from activated SynNotch STING gain of function pDCs induces activation markers on NK cells, including CD69 and CD253 (TRAIL) (Figure 14b-e). The capacity of NK cells to lyse the cancer cell lines K562 and REH is increased (Figure 14f-g) following priming with conditioned medium from SynNotch pDCs . The killing function of the NK cells can also be recapitulated with conditioned medium from cGAMP stimulated Mock U-pDCs, in addition to SynNotch pDCs (Figure 14f-g).

[0365] SynNotch-STINGvl55MU-pDCs also cooperate with NK cells in killing cancer cells in a triple culture system, but also show killing capacity on their own (Figure 15). In a triple culture system comprising target cells, NK cells and pDCs, killing is highly induced for the co-culture of NK cells with the SynNotch U-pDCs, with up to 60% killing achieved after 48- hours (Figure 15b-c). This effect can also be replicated for Mock U-pDCs when stimulated with cGAMP (Figure 15b-c). Of note, the pDCs do not seem to be killed by the NK cells during the co-culture (Figure 15d). Importantly, co-culture of SynNotch U-pDCs alone with target cells (without the presence of NK cells) leads to potent killing of the target cells with up to 40% killing of the target cells during 48-hours of culture, whereas less than 10% of target cells are killed with NK cells alone. This indicates the SynNotch- STINGV155MU-pDCs are more potent at killing the target cells than NK cells (Figure 15e-f). Conditioned medium from TAA-activated SynNotch-STINGvl55MU-pDCs do not lead to target cell death, indicating the killing is mediated by cell-to-cell contact (Figure 15g).

[0366] Example 12

[0367] The killing function of the SynNotch-STINGvl55MU-pDCs is rapid with killing already being observed after 1-day of culture, but also increases during 6-days of co-culture with the target cells REH and NALM6, with accumulated killing reaching up to 60% (Figure 16a-b).

[0368] The induction of the STING-gain-of-function variant occurs rapidly with mRNA expression topping during 6-hours after activation (Figure 17a-c). Accordingly, type I IFNs (IFNa2 and IFNb), and various ISGs (IFIT1 and CXCL10) are rapidly induced during the first 6-hours of stimulation (Figure 17b). Similarly, type I IFN is rapidly secreted from SynNotch activated pDCs, topping after 24-hours post stimulation (Figure 17c). The response seems to be rapidly turned off as the activation agent (anti-cMyc magnetic beads) is taken up by the cells, or removed from the culture using magnetic depletion (Figure 17b and c).

[0369] Example 13

[0370] NXG mice (Janvier) were injected subcutaneously (s.c.) with 5e6 katushka-positive NALM6 cells. When mice had developed a mean tumor size of 120 mm3± 50 mm3, le7 Mock or SynNotch-STINGvl55MU-pDCs were injected intravenously (i.v.) by the tail vein or intratumorally (i.t.) (Figure 18a). 48-hours after i.v. or i.t. administration of Mock or SynNotch-STINGvl55MU-pDCs, the SynNotch pDCs homed to the tumor (Figure 18c) and induce rapid regression of the tumor following 48-hours after injection (Figure 18e).

[0371] Example 14

[0372] A next-generation Synthetic Intramembrane Proteolysis Receptor (SNIPR) expressing the STING gain-of-function component can be expressed and is functional in pDCs (Figure 19a- d). The SNIPR system seems to be more efficient versus the conventional SynNotch, both when it comes to expression levels in cells (Figure 19b), response to target cells (Figure 19c), and killing capacity (Figure 19d).

[0373] Example 15 - Expansion of CD34+ derived cells and effect of freezing

[0374] The protocol described in WO 2018 / 206577 (see particularly Figure 1 of WO 2018 / 206577) was used to generate CD34+-derived pDCs (henceforth describes as U-pDCs), with the inclusion of SCF and SRI in the cytokine enriched medium to promote expansion. Using this method, 3400-fold expansion was achieved (Figure 20A).

[0375] Cells expanded through the protocol exhibited the same phenotype (Figure 20B) as peripheral blood-derived plasmacytoid dendritic cells (pDCs). Notably, in contradistinction to pDCs isolated from peripheral blood, the protocol described herein enabled the derivation of unique pDCs from CD34+ progenitor cells that could be subjected to cryopreservation and subsequent thawing (Figure 20C) This pivotal attribute enhances the scalability of production, rendering these unique CD34+-derived plasmacytoid dendritic cells (U-pDCs) amenable to large-scale manufacturing for application in cell therapy modalities. The capability to freeze and thaw U-pDCs not only facilitates logistical aspects of production but advances the feasibility and practicality of utilizing these cells in therapeutic interventions on a broader scale.

[0376] Example 16 SWITCH construct

[0377] As illustrated schematically in Figure 21 A and B, the constructs of the invention, which are herein below named “SWITCH” constructs, comprise an scFv which may be directed against a specific antigen. As will be evident, the SWITCH construct illustrated in Figure 21(B) also comprises a STING gain of function mutation and a SynNotch / SNIPR construct.

[0378] Figures 2 IE and 2 IF provide schematics for an exemplary construct for use in the invention.

[0379] As shown in Figure 2 ID and 22, positive selection for the SynNotch receptor increased the purity of engineered cells. U-pDCs were subsequently evaluated for pDC markers (lineage negative, CDl lc negative, CD123+, CD303+, CD304+, and SWITCH expression). These results further confirm that cells transduced with a SWITCH construct maintain expression of CD 123, CD303 and CD304 and therefore their pDC phenotype.

[0380] Example 17 - Type I IFN release by U-pDCs

[0381] HSPC derived SWITCH U-pDCs were primed for 72 hr with IFN, after which RNA was extracted and subjected to RNA-seq. The differential gene expression identified is as shown in Figure 12d, and replicates the differential gene expression shown for SynNotch STING pDCs. As shown in this figure, IFN priming of U-pDCs promoted expression of many IFNa isoforms, type III IFN lambda, and chemokines such as CXCL10 and CXCL11. Activated U- pDCs and SWITCH U-pDCs with c-myc beads were assessed for IFNa release (Figure 23 A). U-pDCs transduced with a SWITCH construct demonstrated a greater Type I IFN response than U-pDCs not transduced with the construct. U-pDCs and SWITCH U-pDCs were cocultured with tumor antigen positive and negative cell line and assessed for IFNa release (Figure 23B). As shown in Figure 23C, U-pDCs transduced with a SWITCH construct demonstrated a significant Type I IFN response when exposed to cells expressing the tumour antigen bound by their scFv. This increase in secreted IFN is significant. The autocrine and paracrine effects of secreted IFN (by binding to IFNAR) also support upregulation of hundreds of IFN-stimulated genes (supported by the RNA-seq data), in various tumour microenvironment residual cells. The effect of this can be repolarization and reprogramming of immune cells, as well as increasing cancer cells sensitization toward the immune system. The discerned upregulation of numerous genes intricately linked to the functional repertoire of pDCs provides compelling evidence of the heightened functional potential and immune- activating attributes inherent to U-pDCs, thus elucidating their potential utility as a potent cellular resource in therapeutic applications.

[0382] Example 18 - Activated SWITCH U-pDCs release proinflammatory cytokines responsible for migration of other immune cell types at the tumor site:

[0383] 0.5-le6 IFN primed U-pDCs and SWITCH U-pDCS were seeded out and stimulated with anti-c-Myc magnetic beads (15 pL per le6cells), or cGAMP (4 pg / mL). RNA was collected 20 hours later, and levels of CXCL10 and IFNal / 13 were estimated using qPCR. The effect on expression of CXCL10 and IFNal / 13 were consistent with those shown in Figures 12b and 12c. le5primed (IFN) or non-primed (IL-3) U-pDCs were seeded out and overlayed with 8e5 target (REH6) or non-target (K562) cells (effector: target ratio of 1 :8). Supernatants were collected 20 hours later and indicated cytokines / chemokines were evaluated using Meso-scale multiplex ELISA. The results of the analysis are shown in the heatmap in Figure I la. Increased concentrations of chemokines associated with the migration of immune cells was observed (IP-10, MCP-1, MIP-la, MIP-lb and TNF-a).

[0384] Example 19 - Activation and migration of a broad immune response induced by SWITCH U-pDC.

[0385] The ability of U-pDC and SWITCH U-pDCs to recruit PBMCs, activate B cells and to promote lysis when provided in combination with NK cells. As demonstrated in Figures 24A- C, SWITCH transduced U-pDCs had a much greater ability to promote PBMC migration, activation of B cells and lysis by NK cells. Therefore, transduction with a SWITCH construct produced U-pDCs with increased ability to recruit other cell types, and promote both innate and adaptive responses, including antibody responses. This data suggests that a SWITCH construct enhances the ability of the pDC to act as an antigen-presenting cell.

[0386] Example 20 - SWITCH U-pDCs kill tumor cells in vitro

[0387] The ability of U-pDCs to promote lysis of target cells (REH6) in vitro was further tested as shown in Figure 25. The ability of the SWITCH U-pDCs to promote lysis was compared to that of NK cells alone, and U-pDCs not engineered with a SWITCH construct, with and without chemical activators, at an effector cell: target cell ratio of 2: 1. SWITCH U-pDCs were able to lyse target cells even in the absence of further stimulation, and demonstrated the greatest ability to promote lysis under every condition (Figure 25A). However, chemical activation enhances the ability of SWITCH U-pDCs to promote lysis. When the effector: target cell ratio was reversed, (effector: target ratio of 1 :2) the SWITCH U-pDCs were still able to lyse target cells (Figure 25B). A visualisation of the lysis of target cells expressing GFP is shown in Figure 25C.

[0388] Example 21 - SWITCH U-pDCs kill tumor cells in vivo

[0389] The ability of SWITCH U-pDCs to promote lysis of target cells was also tested, as shown in Figure 26. SWITCH U-pDCs were found to be present in tumour, indicating that the U-pDCs successfully homed to the tumour. Moreover, tumour size was reduced in mice which received SWITCH U-pDCs, and the difference between the delta-tumour growth in mice given SWITCH U-pDCs and non-transduced U-pDCs, and the difference between SWITCH U-pDCs and mice given the PBS control, was significant.

[0390] Example 22 - SWITCH U-pDCs are tumor specific and have no basal activity

[0391] To ascertain the safety profile of our SWITCH U-pDCs, the inventors have generated data illustrating their responsiveness exclusively in the presence of target antigen (TA)-positive tumor cell lines, as delineated in Figure 28A. Notably, when co-cultured with TA-negative cell lines, these cells do not release IFNa, underscoring their discriminative and antigendependent activation mechanism. Furthermore, to gauge the controllability of our SWITCH system in regulating the initiation and cessation of IFNa release and release of STING GoF gene, the inventors employed c-myc beads as a potent inducer of system activation, resulting in a 100% activation rate. Our observations revealed a prompt surge in IFNa levels and STING (Stimulator of Interferon Genes) gain-of-function gene expression upon system activation, as illustrated in Figure 17B and described in the context of the SynNotch construct. Equally noteworthy is the attenuation of this response upon the removal of c-myc beads from the culture milieu. These findings underscore the precision and reversibility of our SWITCH system, reinforcing its potential as a finely tunable tool for modulating the immune response in a controlled and target-specific manner.

[0392] The inventors also performed the analysis and characterization of different vectors to choose a clinical candidate. The selection process involved the identification of a single-chain variable fragment (scFv) with a proven safety profile in clinical trials, derived from the established and approved drug Sacituzumab (Trop2 ADC directed drug). Subsequently, the vector construct underwent optimization through a meticulous examination of various promoters to elucidate their differential impact on the expression levels of our SWITCH system, as depicted in Figure 28A. The diverse iterations of our SWITCH systems underwent comprehensive studies for expression dynamics, culminating in the selection of the variant exhibiting the highest expression, thereby augmenting the therapeutic product's efficacy, as illustrated in Figure 28B.

[0393] Further, the inventors conducted a thorough characterization of distinct tumor cell lines, distinguishing between those positive and negative for the target antigen. Furthermore, cell lines expressing varying levels of the antigen were characterized, as delineated in Figure 28C. To refine our vector construct selection, a series of co-culture assays encompassing the aforementioned conditions were executed. The assessment focused on the quantification of interferon-alpha (IFNa) release, and the optimal construct was discerned based on its maximal IFNa release in conjunction with an absence of activation when co-cultured with antigen-negative cell lines. This comprehensive selection process ensures the judicious choice of the vector construct that optimally aligns with therapeutic objectives and safety considerations.

[0394] Example 23 - Confirmation of functionality for Trop2 SWITCH construct The data discussed above and presented in Figures 1-27 demonstrate the functionality of pDCs expressing SWITCH constructs of the invention. In these experiments, the SWITCH construct targeted CD 19. The same functionality and effects are expected for pDCs expressing a construct targeting Trop2. Indeed, Figure 28, discussed above, provides data demonstrating the effectiveness of pDCs expressing a Trop2 SWITCH construct. In addition, the following observations have been made confirming the functionality for a Trop2 SWITCH construct:

[0395] Objective: Differentiate Trop2 SWITCH UpDCs from CD34+ cells Model: research grade material, N=3 donors differentiated 21 days

[0396] We observed an up to 6000 fold expansion of these cells in research grade process

[0397] Objective: Transduction efficiency for Trop2 SWITCH construct

[0398] Model: research grade material N=3

[0399] We could transduce and differentiate Trop2 SWITCH U-pDCs and obtained an average of 40% transduction efficiency

[0400] Objective: Antigen specific activation of Trop2 SWITCH construct Model: research grade material

[0401] N=3

[0402] Trop2 SWITCH U-pDCs cells released type I IFNa when co-cultured with 2 out of 2 cell lines (OVCAR3 and JIMT-1) tested

[0403] Objective: No antigen independent activity of Trop2 SWITCH construct Validation article: Therapeutic candidate

[0404] Model: research grade material N=3

[0405] Trop2 SWITCH U-pDCs cells did not get activated by antigen negative cell lines: 2 out of 2 (PAI and BT549) tested and did not release type I IFNa

[0406] Example 24 - Comparing transgene expression and transfection efficiency in LentiX cells transduced with anti-CD19 and anti-TROP2 constructs

[0407] Transgene expression and transfection efficiency in LentiX cells transfected with p089 (an anti-CD19 SNIPR), p090 (an anti-TROP2 SNIPR), or pl 08 (a codon optimized anti-TROP2 SNIPR) constructs was compared using flow cytometry based on cMyc detection. LentiX cells were transfected in a pl 5 dish using PeiMax and a transfer vector at a dose of 3.1 pg per well. Cells were transfected with p089, p090, or pl08.

[0408] 48 hours after transfection, cells were stained with the Zombie Violet viability dye, blocked with TruStain FxC, then stained using anti-cMyc (APC). Mock-transfected LentiX cells were used as controls.

[0409] Transgene expression is shown in Figures 29 A to D and Figure 30A. Minimal transgene expression was observed for mock-transformed cell lines (Figure 29A). However, as shown in Figures 29B to 29D and 30A, strong transgene expression was observed p089, p090 and p0108, with the codon optimisation of pl 08 providing higher expression than p090.

[0410] Transfection efficiency in transformed LentiX lines was also compared (Figure 30B). p089, p90 and pl 08 showed high transfection efficiencies and the codon optimisation of pl 08 provided higher transfection efficiency than p090.

[0411] Example 25 - Transgene expression in K562 cells transduced with p089, p090, or pl 08 constructs

[0412] Transgene expression in K562 cells transduced with p089, p090, or pl 08 constructs using lentiviral vectors was compared using flow cytometry based on cMyc detection. Lentivirus titers for transduced lines was also compared.

[0413] 50,000 K562 cells were seeded out and transduced with lentiviral vectors. Cells were transduced with p089 (an anti-CD19 SNIPR), p090 (an anti-TROP2 SNIPR), or pl 08 (a codon optimized anti-TROP2 SNIPR).

[0414] 5 days after transduction, cells were stained with Zombie Violet viability dye, blocked with TruStain FxC, then stained with anti-cMyc (APC). For some samples, anti-IgG2a isotype control (APC) was evaluated.

[0415] Transgene expression was visualised for each cell line using FACS plots (Figures 31 A to C). Figures 31 A to C demonstrate that K562 cells transduced with p089, p090, or pl08 constructs all showed high levels of transgene expression.

[0416] Figure 32 shows the analysis of lentiviral titers in transduced cells. Lentivirus titer was greatest for cells transduced with p089 (1.3 x 109TU / mL). Increased levels of lentivirus production were observed for cells transduced with pl 08 (titer of 0.8 x 109TU / mL) relative to cells transduced with p090 (titer of 0.2 x 109TU / mL).

[0417] Example 26 - Transgene expression in HSPCs transduced with p089, p090, or pl08 constructs

[0418] Transgene expression in HSPCs transduced with p089, p090, or pl 08 constructs was compared using flow cytometry based on cMyc detection. HSPCs obtained from donor 117 (DI 17) were transduced with p089 (an anti-CD19 SNIPR), p090 (an anti-TROP2 SNIPR), or pl 08 (a codon optimized anti-TROP2 SNIPR).

[0419] 8 days post transduction, cells were stained with Zombie Violet viability dye, blocked with TruStain FxC, then stained with anti-cMyc (APC). Mock-transduced HSPCs were used as controls (DI 17 Mock).

[0420] Transgene expression was visualised using FACS plots (Figure 33) and a bar chart (Figure 34). Minimal transgene expression was observed for mock-transformed cell lines (Figure 33, far left panel). Successful expression of both the anti-CD19 and anti-TROP2 constructs was observed, with codon optimisation providing higher transgene expression.

[0421] Example 27 - Functional analysis of PL 108 in pDCs pDCs derived from HSPCs obtained from donor 95 (D095) and expressing PL108 (also referred to as pl08 herein) were stained with Zombie Violate Fixable viability stain (423113, BioLegend) for 10 minutes. A full antibody cocktail comprising: anti -human CD1 Ic-FITC (301604, BioLegend), anti-human CD123-PE (130-115-264, Miltenyi), anti-human HLA- DR-PerCPVio700 (130-111-793, Miltenyi), andanti-cMyc-AF647 (2233s, Cell Signaling Technologies), or an FMO-cocktail was added. Cells were then stained for an additional 30 minutes, washed twice, then resuspended in a fixation buffer (containing PBS and 0.9% formaldehyde). Mock-transduced HSPCs were used as controls (D095 Mock).

[0422] Cells were analysed phenotypically after <48 hours of fixation. Flow cytometry analysis was performed to confirm mock-transduced and PL108-transduced cells exhibited markers associated with pDCs. Gating was performed: first to isolate pDCs, then to isolate single cells, then to isolate lives expressing markers associated with pDCs (HLA-DR and CD123), but not CD11c (Figure 37, left side). Due to low cell numbers throughout the production, the phenotype was not evaluated on days 7 or 14.

[0423] No difference was observed in fold expansion when comparing mock-transduced cells with PL108-transduced cells after 21 days (Figure 35).

[0424] Mock-transduced and PL108-transduced pDCs were also co-cultured with cell lines (A549, OVCAR3, or HCC1937) at a pDC: target cell ratio of either 1 : 1 or 1 :4, or stimulated with an antigen (60 nM CpG-A, 300 nM diaBZI, TROP2 protein, or anti-cMyc magnetic beads). Non-stimulated cells were used as a control. Cells were stimulated for 20 hours before supernatants were harvested and IFN-a levels were analysed using IFN-a ELISA. Cells transduced with PL108 showed increased levels of IFN-a production compared to mock- transfected cells when stimulated with CpG-A, anti-cMyc magnetic beads, or TROP2 protein, or when co-cultured with OVCAR3, or HCC1937 (Figure 36).

[0425] Expression of TROP2 protein was compared between mock-transduced and PL-108- transduced cells. TROP2 protein expression was observed in PL-108-transduced cells, but not mock-transduced cells (Figure 37, right side).

[0426] These data demonstrate that expression of the anti-TROP2 construct does not affect expansion or phenotype of the pDCs, and they retain high functional performance. Example 28 - Functional comparison of p090 and pl 08 in pDCs derived from mobilized HSPCs pDCs derived from mobilized HSPCs (moHSPCs) obtained from donor 111 (Di l l) and transduced with either pl 08 or p090 were stained with Zombie Violate Fixable viability stain (423113, BioLegend) for 10 minutes. A full antibody cocktail comprising: anti-human CDl lc-FITC (301604, BioLegend), anti-human CD123-PE (130-115-264, Miltenyi), antihuman HLA-DR-PerCPVio700 (130-111-793, Miltenyi), andanti-cMyc-AF647 (2233s, Cell Signaling Technologies), or an FMO-cocktail was added. Cells were then stained for an additional 30 minutes, washed twice, then resuspended in a fixation buffer (containing PBS and 0.9% formaldehyde). Mock-transduced HSPCs were used as controls (Dl l 1 Mock).

[0427] Cells were analysed phenotypically after <48 hours of fixation. Flow cytometry analysis was performed to confirm mock-transduced and pl08-transduced cells exhibited markers associated with pDCs (HLA-DR and CD123) (Figure 40, top).

[0428] Due to low cell numbers throughout the production, the phenotype was not evaluated on days 7 or 14. Similar fold expansion was observed for mock-transduced, p090-transduced, and pl08-transduced cells (Figure 38).

[0429] Mock-transduced, pl08-transduced, and p090-transduced pDCs were also co-cultured with OVCAR-3 cells at a pDC: OVCAR-3 ratio of 1 :4, or stimulated with anti-cMyc magnetic beads. Cells were stimulated for 20 hours before supernatants were harvested and IFN-a levels were analysed using IFN-a ELISA. Untreated cells were used as a control. Cells transduced with pl 08 showed much greater IFN- a production when co-cultured with OVCAR-3 or stimulated with anti-cMyc magnetic beads than p090-transduced and mock transduced cells (Figure 39).

[0430] Flow cytometry also demonstrated that expression of the transgene was greater in cells transduced with pl08 than cells transduced with p090 (Figure 40. bottom).

[0431] These data demonstrate that both p090 and pl 08 cells successfully differentiate to pDCs. The cells with the codon optimised construct pl 08 exhibit higher transgene expression and functional performance.

[0432] Example 29 - Functional analysis of pl 14 in pDCs

[0433] HSPCs obtained from either donor 117 (DI 17) or donor 119 (DI 19) were transduced with pl 14 (an anti-TROP2 fully human SNIPR)

[0434] Flow cytometry analysis was performed to confirm mock-transduced and pl 14-transduced cells exhibited markers associated with pDCs (HLA-DR and CD123) (Figure 43, top)

[0435] No difference was observed in fold expansion when comparing mock-transduced cells with pl 14 transduced cells after 20 days (Figure 41). pl 14-transduced DI 17 and DI 19 pDCs were also co-cultured with OVCAR3, JIMT-1, HCC1937, and A549 cell lines. Mock-transduced DI 17 and DI 19 cells, as well as cancer cells, were used as controls. Cells were co-cultured for 20 hours before supernatants were harvested and IFN-a levels were analysed using IFN-a ELISA. DI 17 and DI 19 cells transduced with pl 14 showed much greater IFN- a production when co-cultured with OVCAR-3, JIMT-1, and HCC1937 compared to mock-transduced cells and cancer cells alone (Figure 42).

[0436] Flow cytometry also demonstrated that expression of the transgene was observed in cells transduced with pl 14, but not in mock-transduced cells (Figure 43, bottom).

[0437] Example 29 - Lentivirus production for different constructs

[0438] Expression of various scFv SNIPR constructs was compared in LentiX cells.

[0439] LextiX cells were transfection with p089, pl08, pl 13, pl 14, pl 15, pl 17, pl 18, pl 19, pl44, or pl46. Mock-transfected cells were used as a control. 48 hours after transfection cells were stained with Zombie Violet viability dye, blocked with TruStain FxC, then stained with anti- cMyc (APC).

[0440] Flow cytometry based on cMyc detection was performed to evaluate expression of the different constructs. Comparisons are shown in Figure 44, which demonstrates that all transfected cell lines expressed transgenes efficiently.

Claims

CLAIMS1. An engineered plasmacytoid dendritic cell (pDC) that expresses on its surface a synthetic receptor comprising an extracellular antigen recognition domain that recognises Trop2 on a target cell.

2. The cell of claim 1, wherein the synthetic receptor further comprises a transmembrane domain and an intracellular signalling domain that activates an immune response in the cell when the extracellular antigen recognition domain binds the target cell.

3. The cell of claim 2, wherein the intracellular signalling domain activates expression of a response element in the cell when the extracellular antigen recognition domain binds the target cell.

4. The cell of claim 3, wherein the response element encodes a stimulator of interferon genes (STING) protein.

5. The cell of any one of the preceding claims, wherein the synthetic receptor is:(a) a synthetic intramembrane proteolysis receptor (SNIPR), such as wherein the synthetic receptor is a synthetic RIP family receptor, optionally wherein the receptor is:(i) synthetic Notch receptor;(ii) a synthetic Robo receptor; or(iii) a synthetic RoboNotch receptor;(b) a Tango receptor;(c) a modular extracellular signalling architecture (MESA) system; or(d) a chimeric antigen receptor.

6. The cell of any one of claims 2-5, wherein the intracellular signalling domain is a transcription factor, optionally wherein the transcription factor comprises a DNA binding domain and a transcriptional activation domain, such as wherein:(a) the DNA binding domain is selected from the group consisting of Gal4, Pax6, zinc fingers (ZFs), synthetic zinc fingers (synZFs), transcription activator like effectors (TALEs), TetR, HNF1 alpha, and vHNFl beta; and / or(b) the transcription activation domain is selected from the group consisting of VP64, VP16, WWTR1, CREB3, NF-KB, p65, Rta, HSF1, and RelA; and / or(c) the intracellular signalling domain is selected from the group consisting of Gal4- VP64, Gal4-VP16, TetR-VP64, LacI-VP64, HNF1-WWTR1, HNF1-CREB3, Pax6-p65, ZF- p65, synZF-p65, and HNFl-p65.

7. The cell of any one of the preceding claims, wherein the extracellular antigen recognition domain is an antibody-derived domain, such as wherein the extracellular antigen recognition domain is an scFv domain.

8. The cell of any one of the preceding claims, wherein the STING protein comprises:(a) a gain-of-function mutation that causes it to be constitutively active in the absence of 2’3’-cGAMP; and / or(b) a mutation, preferably a substitution, at one or more residues corresponding to R71, S102, V147, N154, V155, G166, C206, G207, G230, H232, R238, F279, R281, R284, R293, or Q315 of SEQ ID NO: 1.

9. The cell of any one of claims 3-8, wherein:(a) the cell comprises a heterologous nucleic acid encoding the synthetic receptor; and / or(b) the cell comprises a heterologous nucleic acid encoding the response element; and / or(c) the cell comprises a single heterologous nucleic acid encoding the synthetic receptor and the response element.

10. The cell of claim 9, wherein the heterologous nucleic acid is selected from the group consisting of a viral construct, a plasmid, a cosmid, and an mRNA, optionally wherein:(a) the viral construct is a lentiviral vector, optionally wherein the lentiviral vector comprises an expression cassette between two LTRs; or(b) the viral construct is an adenoviral or adeno-associated viral vector, optionally wherein the adenoviral or adeno-associated viral vector comprises an expression cassette between two LTRs; or(c) wherein the heterologous nucleic acid is introduced using site-specific DNA editing, such as CRISPR / Cas.

11. The cell of claim 10, wherein the mRNA is delivered via a lipid nanoparticle.

12. The cell of any of any preceding claim, wherein the pDC has undergone a step of priming the pDC, optionally wherein the step of priming the pDC comprises incubating the cells with type I IFN and / or type II IFN, such as wherein the pDC is incubated with type I IFN and / or type II IFN for 24 hours.

13. The cell of any one of claims 4-12, wherein the STING protein activates an immune response, such as wherein:(a) the immune response comprises secretion of cytokines, optionally wherein the cytokines modulate the environment of the diseased tissue, or wherein the immune response comprises recruitment or activation of immune cells, such as wherein the cytokines are pro- inflammatory cytokines, optionally wherein the cytokines comprise CXCL10, MCP-1, MCP- 2, MIP-la and / or MIP-lb; and / or(b) the immune response comprises production of type I IFN.

14. A method of treating cancer in a subject, comprising administering the cell of any one of the preceding claims.

15. The method of claim 14, wherein:(a) 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 orureter, 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; or(b) the cancer is selected from the group consisting of 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, and Waldenstrom macroglobulinemia.

16. The method of claim 14, wherein the cancer is selected from the group consisting of urothelial carcinoma, head and neck squamous cell carcinoma, lung squamous cell carcinoma, cholangiocarcinoma, colon cancer, such as colon adenocarcinoma, esophageal carcinoma, lung adenocarcinoma, prostate adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, thyroid carcinoma, and uterine corpus endometrial carcinoma, epithelial cancer, triple negative breast cancer, non-small cell lung cancer, castration-resistant prostate cancer, HR+ / HER2- metastatic breast cancer, endometrial cancer, ovarian cancer, glioblastoma, brain tumors and endometrial carcinoma.

17. The method of any of claims 14-16, wherein the cancer expresses Trop2.

18. The method of any of claims 14-17, wherein the cancer is metastatic or refractory to at least one further treatment.

19. The cell or method of any one of the preceding claims, wherein the cell has been extracted from blood or bone marrow.

20. The cell or method of any of claims 1-18, wherein the cell has been differentiated in vitro from HSPCs, optionally wherein the HSPCs have been obtained from blood or bone marrow, or wherein the HSPCs have been differentiated in vitro from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

21. A pharmaceutical composition comprising the engineered pDC of any preceding claim and a pharmaceutically acceptable carrier.

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