WO2024145168A1 - Compositions et méthodes pour traiter le cancer - Google Patents

Compositions et méthodes pour traiter le cancer Download PDF

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WO2024145168A1
WO2024145168A1 PCT/US2023/085419 US2023085419W WO2024145168A1 WO 2024145168 A1 WO2024145168 A1 WO 2024145168A1 US 2023085419 W US2023085419 W US 2023085419W WO 2024145168 A1 WO2024145168 A1 WO 2024145168A1
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amount
fold increase
increase over
therapeutic agent
active agent
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PCT/US2023/085419
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Remo MOOMIAIE
Richard Klemke
Huawei Wang
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Cytonus Therapeutics, Inc.
The Regents Of The University Of California
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Publication of WO2024145168A1 publication Critical patent/WO2024145168A1/fr

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  • the IL-12 polypeptide comprises a membrane-bound IL-12 polypeptide. In some embodiments, the IL-12 polypeptide is complexed to the cell surface of the enucleated cell. In some embodiments, the enucleated cell induces an endogenous immune response at a site associated with the disease or condition of the subject. In some embodiments, the method further comprises treating the WSGR Docket No.53712-720.601 disease or condition of the subject, at least in part, by inducing an endogenous immune response in the subject. In some embodiments, the site associated with the disease or condition comprises a microenvironment. In some embodiments, the microenvironment is a tumor microenvironment.
  • a method for inducing endogenous immune response in a subject comprising: administering to the subject an enucleated cell comprising (a) an exogenous mRNA encoding a cytokine polypeptide; and (b) one or more organelles for expressing the cytokine polypeptide on a cell surface of the enucleated cell, wherein the cytokine polypeptide induces the endogenous immune response in the subject.
  • the cytokine polypeptide comprises a membrane-bound cytokine polypeptide.
  • the cytokine polypeptide is complexed to the cell surface of the enucleated cell.
  • the enucleated cell induces the endogenous immune response at a microenvironment.
  • the microenvironment is a tumor microenvironment.
  • the microenvironment is a lymphatic microenvironment, a hepatic microenvironment, a spleen microenvironment, a pancreatic WSGR Docket No.53712-720.601 microenvironment, an epidermis microenvironment, or a combination thereof.
  • the enucleated cell induces the endogenous immune response in proximity of a cell.
  • the cell is a cancer cell.
  • the cell is an immune cell.
  • the immune cell is a leukocyte.
  • the cytokine polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours. In some embodiments, the cytokine polypeptide is complexed with the enucleated cell for the at least 24 hours. In some embodiments, the cytokine polypeptide is not secreted by the enucleated cell. In some embodiments, the cytokine polypeptide is not delivered to the microenvironment. In some embodiments, the cytokine polypeptide is not delivered to the cell. In some embodiments, the cytokine polypeptide comprises a full-length cytokine or antigen binding fragment thereof of the cytokine.
  • the cytokine polypeptide encoded by the exogenous mRNA results in a decreased toxicity in the subject compared to a toxicity caused by direct delivery of the cytokine polypeptide.
  • the cytokine polypeptide comprises an interleukin polypeptide.
  • the interleukin polypeptide is an IL-12 polypeptide.
  • the cytokine polypeptide comprises an interleukin antibody or antigen binding fragment thereof.
  • the cytokine polypeptide comprises IL-12 or antigen binding fragment thereof.
  • the endogenous immune response comprises an innate immune response or adaptive immune response.
  • the enucleated cell further comprises an immune checkpoint inhibitor.
  • the enucleated cell expresses an abundance of the cytokine polypeptide that is at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 1.0 fold, at least 2.0 fold, or at least 5.0 fold higher than the enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the abundance of the cytokine polypeptide and the abundance of the comparable naturally-occurring cytokine polypeptide is determined by FACS.
  • the cytokine polypeptide is not secreted by the enucleated cell.
  • the cytokine polypeptide comprises an interleukin polypeptide.
  • the interleukin polypeptide is IL-12 polypeptide.
  • the enucleated cell further comprises an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.
  • the immune checkpoint inhibitor comprises the PD-1 inhibitor.
  • the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell.
  • the enucleated cell is depleted of an immune recognition molecule.
  • the immune recognition molecule comprises HLA antigen, proteoglycan, sugar moiety, embryonic antigen, or any combination thereof.
  • the enucleated cell comprises a targeting moiety.
  • the targeting moiety comprises CXCR2, CCR2, PSGL- 1, or a combination thereof.
  • the composition further comprises an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.
  • the immune checkpoint inhibitor comprises the PD- 1 inhibitor. In some embodiments, the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell. In some embodiments, the immune checkpoint inhibitor is encoded by a second enucleated cell.
  • Fig.1B illustrates mesenchymal stem cells (MSCs), MSC-derived enucleated cells transfected with IL-12 mRNA (MSC IL-12 and Enucleated cell IL-12 respectively), or conditioned medium (CM) collected at the indicated time points.
  • Fig.1C illustrates CM from cells/enucleated cells as shown in Fig.1B assessed in mouse splenocytes for the activation of the phosphorylated/activated form of Stat4 (p-Stat4) by western blot.
  • Fig.1D illustrates a schematic for using the enucleated cell to treat a triple negative breast cancer (TNBC) mouse model.
  • TNBC triple negative breast cancer
  • Fig.1E illustrates measurements of IL-12 secreted by the enucleated cell within tumor microenvironment.
  • Fig.1F illustrates serum IL-12 measurement.
  • Fig.1G illustrates measurements of IL-12 mediated inflammatory biomarkers.
  • Fig.1H illustrates mice bearing SQ E0771 tumors injected with a total of three doses of either PBS, MSCs secreting IL-12, or CA-IL-12.
  • Fig.1I illustrates FACS analysis for detecting leukocyte activation.
  • Fig.2A illustrates a schematic of enucleated cell that secreted IL-12 alone or in combination with the checkpoint inhibitor anti-PD-1 antibody for treating cancer mouse model.
  • Fig.2B illustrates a survival curve for animals injected as described in Fig.2A.
  • Fig.2C illustrates a graph of tumor size over time post-injection.
  • Fig.2D illustrates a graph of fold change in animal weight during the treatment phase of the survival experiments shown in Fig.2B.
  • Fig.3B illustrates Boyden Chamber migration assay showing mesenchymal stem cells or enucleated cells engineered for expressing CCR2, CXCR4, and leukocyte adhesion molecule (PSGL-1; termed CCP MSCs or CCP enucleated cells respectively) for robustly homing towards CCL2, SDF-1a, and EO771 conditioned medium relative to a no-chemoattractant negative control.
  • PSGL-1 leukocyte adhesion molecule
  • Fig.4B illustrates homing of the enucleated cells to tumors.
  • Fig.4C illustrates FACS for determining the abundance of enucleated cells homed to tumors.
  • Fig.5A illustrate infiltration of the enucleated cell infiltration.
  • Fig.5B illustrates optical sectioning and 3D reconstruction of the center of macrometastasis revealed that clusters of CCP enucleated cells.
  • Fig.5C illustrates single channel acquisition of DAPI stained sections showing normal lung architecture and no tumor mass.
  • Fig.8A illustrates an exemplary design of the enucleated cell expressing cell membrane tethered IL-12.
  • Fig.8B illustrates non-limiting example of a vector map of a vector encoding IL-12.
  • Fig.8C illustrates engineering and screening for the enucleated cell expressing membrane-bound scIL-12.
  • WSGR Docket No.53712-720.601 illustrates an exemplary mechanism of immune cell activation by CA-scIL-12.
  • Fig.9A illustrates MSCs or enucleated cells engineered with or without scIL-12 that were co-cultured with primary murine splenocytes and concanavalin A for 48 hours.
  • Fig.9B illustrates enucleated cells (1 x10 6 ) engineered to secrete IL-12 (CA-IL-12).
  • Fig.11A illustrates a schematic for engineering the hTERT-MSC for expression both scIL-12 and IL-15R ⁇ and the subsequent enucleation of the hTERT-MSC.
  • Fig.11B illustrates hTERT MSCs infected with two lentiviruses encoding constructs for either scIL-12 or IL-15R ⁇ . Following drug selection, cells were FACS sorted for single cell clones. Clone 4 was enucleated to generate enucleated cells expressing both scIL-12 and IL- 15R ⁇ , and receptor expression was analyzed by FACS at the indicated time points.
  • Fig.11C illustrates functionality of expressing membrane-bound IL-12, IL-15, or a combination of both IL-12 and IL-15.
  • Fig.12A illustrates biodistribution of the enucleated cells by bioluminescence.
  • Fig.12B illustrates quantification of biodistribution of the enucleated cells of Fig.12A.
  • Fig.13A illustrates increased homing of the enucleated cell described herein compared to nucleated mesenchymal stem cell (MSC).
  • MSC nucleated mesenchymal stem cell
  • Fig.13B illustrates quantification of homing of the enucleated cell of Fig.13A.
  • Fig.14A illustrates homing of the enucleated cell based on increased chemoattractants in the TNBC tumor metastatic sites.
  • Fig.14B illustrates dot plots of expression of Cxcl (Top) and Ccl (Bottom) chemokines within Vcam1-positive cells in Cluster 1 confirming Cxcl12 (encoding SDF-1 ⁇ ) and Ccl2 were the highest expressed Cxcl and Ccl chemokines, respectively.
  • the active agent may be a protein.
  • the active agent may be an antibody, antigen, or a fragment of said antibody or antigen.
  • cells comprising mRNA to express a protein, an antibody, an antigen, or portion thereof, and compositions containing such cells.
  • the cells are enucleated.
  • the protein, antibody, antigen, or portion thereof is a therapeutic agent.
  • the enucleated cell is obtained or derived from a nucleated cell (e.g., a parent cell). In some aspects, the enucleated cell comprises a transmembrane moiety. In some embodiments, the enucleated cells comprises a targeting moiety. In some embodiments, the enucleated cell comprises a therapeutic agent. In some embodiments, the enucleated cell comprises a protein. In some embodiments, the enucleated cell comprises a cytokine. In some WSGR Docket No.53712-720.601 embodiments, the enucleated cell comprises an antibody or antigen binding fragment thereof or a single-domain antibody or an antigen binding fragment thereof.
  • an enucleated cell may be derived from an adult stem cell, a mesenchymal stromal cell (MSC), a natural killer (NK) cell, a macrophage, a myoblast, a neutrophil, endothelial cell, endothelial precursor cell, and/or a fibroblast.
  • an enucleated cell is derived from a mesenchymal stromal cell.
  • the enucleated cell is derived from an inducible pluripotent stem cell (iPSC).
  • the parent cell is derived from a cell is immortalized using suitable methods.
  • parent cell is immortalized by expressing human telomerase reverse transcriptase (hTERT), an oncogene, or a viral gene such as simian virus 40 (SV40).
  • hTERT human telomerase reverse transcriptase
  • SV40 viral gene
  • the cytoplast is derived from a parent cell using suitable methods provided in United States Patent No.10,927,349, which is hereby incorporated by reference in its entirety.
  • the enucleated cell retains one or more intracellular organelles for synthesis of an exogenous protein.
  • the enucleated cell retains one or more intracellular organelles for synthesis of an exogenous cytokine.
  • the enucleated cell does not require differentiation of the parent cell.
  • the parent cell containing a nucleus is engineered to express the single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein; and subsequently, the nucleus of the parent cell is removed.
  • the parent cell containing the nucleus is enucleated, and the enucleated cell is engineered to express single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein.
  • the parent cell is engineered to express one or more of the biomolecules above (e.g., immune-evading moiety and/or targeting moiety), and the resulting enucleated cell (e.g., already expressing the immune- evading moiety and/or targeting moiety) is further engineered to express a second of the biomolecules above (e.g., a therapeutic agent).
  • the biomolecules above e.g., immune-evading moiety and/or targeting moiety
  • a second of the biomolecules above e.g., a therapeutic agent
  • the enucleated cells of the present WSGR Docket No.53712-720.601 disclosure can be extensively engineered prior to enucleation, stored for long periods of time as needed (through for e.g., lyophilization, cryohibernation, cryopreservation), and quickly engineered to express a therapeutic agent closer to the time of need.
  • the cell can originate from any organism having one or more cells.
  • Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g.
  • algal cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g.
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g. fruit fly, cnidarian, echinoderm, nematode, etc.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).
  • the cell is a somatic cell.
  • the cell is a stem cell or a progenitor cell.
  • the cell is a mesenchymal stem or progenitor cell.
  • the cell is a hematopoietic stem or progenitor cell.
  • the cell is a muscle cell, a skin cell, a blood cell, or an immune cell.
  • lymphoid cells such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell), Natural killer cell, cytokine induced killer (CIK) cells; myeloid cells such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil), Monocyte/Macrophage, Red blood cell (Reticulocyte), Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell), parathyroid (Parathyroid chief cell, Oxyphil cell), adrenal (Chromaffin cell), pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia), Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope
  • the nucleated cell is an immune cell (e.g., a lymphocyte (e.g., a T cell, a B cell), a macrophage, a natural killer cell, a neutrophil, a mast cell, a basophil, a dendritic cell, a monocyte, a myeloid- derived suppressor cell, an eosinophil).
  • the nucleated cell is a phagocyte or a leukocyte.
  • the nucleated cell is a stem cell (e.g., an adult stem cell (e.g., a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, mesenchymal stem cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a testicular cell), an embryonic stem cell, an inducible pluripotent stem cell (iPS)).
  • the nucleated cell is a progenitor cell.
  • the nucleated cell is from a cell line.
  • the nucleated cell is a suspension cell.
  • the nucleated cell is an adherent cell.
  • the nucleated cell is a cell that has been immortalized by expression of an oncogene.
  • the nucleated cell is immortalized by the expression of human telomerase reverse transcriptase (hTERT) or any oncogene.
  • the nucleated cell is immortalized by the expression of a viral gene such as simian virus 40 (SV40).
  • the nucleated cell is a patient or subject derived cell (e.g., an autologous patient-derived cell, or an allogenic patient-derived cell).
  • the enucleated cell is derived from an immune cell.
  • the enucleated cell is derived from a natural killer (NK) cell, a neutrophil, a macrophage, a lymphocyte, a fibroblast, an adult stem cell (e.g., hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchymal stem cell, a mesenchymal stromal cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a skin stem cell, or a testicular cell), a mast cell, a basophil, an eosinophil, an endothelial cell, an endothelial cell precursor cell, or an inducible pluripotent stem cell.
  • NK natural killer
  • neutrophil e.g., hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchy
  • the parent cell is not an erythrocyte or erythroid precursor cell. In some embodiments, the parent cell is a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial precursor cell. In some embodiments, the enucleated cell is not an erythrocyte or erythroid precursor cell. In some embodiments, the enucleated cell is not a platelet cell. In some embodiments, the enucleated cell is not an endothelial cell. In some embodiments, the enucleated cell is not an endothelial precursor cell.
  • the enucleated cell does not express complement receptor one (CR1). In some embodiments, the enucleated cell does not express CD44. In some embodiments, the enucleated cell does not express VLA-4. In some embodiments, the enucleated cell does not express BCAM. In some embodiments, the enucleated cell does not express ICAM. In some embodiments, the enucleated cell does not express a receptor for collagen. In some embodiments, the enucleated cell does not express a receptor for thrombopoietin. In some embodiments, the enucleated cell does not express a receptor for collagen.
  • CR1 complement receptor one
  • the enucleated cell does not express CD44. In some embodiments, the enucleated cell does not express VLA-4. In some embodiments, the enucleated cell does not express BCAM. In some embodiments, the enucleated cell does not express ICAM. In some embodiments, the enucleated cell does not express
  • the enucleated cell does not express a receptor for von Willebrand factor (VWF). In some embodiments, the enucleated cell does not express a receptor for fibrinogen. In some embodiments, the enucleated cell does not express GP1b-IX-V receptor. In some embodiments, the enucleated cell does not express GPIIb/IIIa receptor. In some embodiments, the enucleated cell does not express prostanoid receptor. In some embodiments, the enucleated cell does not express purinergic receptor. In some embodiments, the enucleated cell does not express thromboxane receptor.
  • VWF von Willebrand factor
  • the parent cell is not an endothelial precursor cell.
  • a parent cell may be treated with cytochalasin to soften the cortical actin cytoskeleton.
  • the nucleus may then physically extracted from the cell body by high-speed centrifugation in gradients of Ficoll to generate an enucleated cell. Because enucleate cells and intact nucleated cells sediment to different layers in the Ficoll gradient, enucleated cells may be easily isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated).
  • the enucleation process is clinically scalable to process tens of millions of cells.
  • enucleated cells may be used as a disease-homing vehicle to express clinically relevant cargos/payloads to treat various diseases.
  • the enucleated cells described herein retain one or more intracellular organelles that are endogenous to the parent cell. In some embodiments, all of the one or more intracellular organelles are retained. In some embodiments, fewer than all of the one or more intracellular organelles are retained. In some embodiments, the Golgi apparatus and/or the endoplasmic reticulum are retained, which are involved in protein synthesis and secretion.
  • Enucleated cells may be smaller than their nucleated counterparts (e.g., the nucleated parent cells), and for this reason may migrate better through small openings in the vasculature and tissue parenchyma.
  • removing the large dense nucleus alleviates a major physical barrier allowing the cell to move freely through small openings in the vessels and tissue parenchyma.
  • an enucleated cell has improved bio-distribution in the body and movement into target tissues.
  • an enucleated cell comprises at least 1 ⁇ m in diameter. In some embodiments, an enucleated cell is greater than 1 ⁇ m in diameter.
  • the amount of the active agent is about 2.00 to about 10.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 2.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 35.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 15.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 115.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 15.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 45.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 25.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 120.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 50.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 85.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 50.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 95.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 110.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 60.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 100.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 70.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 95.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 70.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 105.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 70.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 135.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 75.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 120.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 75.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the WSGR Docket No.53712-720.601 active agent is about 75.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 145.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 75.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 105.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 80.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 130.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 80.00 to about 135.00. In some embodiments, the amount of the active agent is about 80.00 to about 140.00.In some embodiments, the amount of the active agent is about 80.00 to about 145.00.In some embodiments, the amount of the active agent is about 80.00 to about 150.00.In some embodiments, the amount of the active agent is about 85.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 100.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 85.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 120.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 85.00 to about 125.00. In some embodiments, the amount of the active agent is about 85.00 WSGR Docket No.53712-720.601 to about 130.00.In some embodiments, the amount of the active agent is about 85.00 to about 135.00.In some embodiments, the amount of the active agent is about 85.00 to about 140.00.In some embodiments, the amount of the active agent is about 85.00 to about 145.00.In some embodiments, the amount of the active agent is about 85.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 95.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 90.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 125.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 95.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 130.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 95.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is WSGR Docket No.53712-720.601 about 95.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 105.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 100.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 135.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 100.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 120.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 105.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 150.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 110.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 120.00 fold increase over WSGR Docket No.53712-720.601 endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 135.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 110.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 130.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 115.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 140.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 115.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 130.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 125.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 145.00 fold increase over endogenous amount of the active agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the active agent is about 125.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 140.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 130.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 140.00 to about 145.00 fold increase over endogenous amount of the active agent.
  • the amount of the active agent is about 140.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 145.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 11
  • the amount of the active agent is greater than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell.
  • the amount of the active agent is less than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell.
  • the WSGR Docket No.53712-720.601 amount of the active agent and the amount of the comparable endogenous active agent is determined by flow cytometry (FACS).
  • the active agent may be a cytokine.
  • the cytokine may be expressed by the mRNA in the enucleated cell.
  • the cytokine may be expressed by the enucleated cell on the surface of the enucleated cell.
  • the cytokine polypeptide encoded by the exogenous mRNA has decreased toxicity in the subject as compared to direct delivery of the cytokine polypeptide.
  • the cytokine may comprise a transmembrane domain.
  • the mRNA may encode the cytokine and the transmembrane domain.
  • the cytokine may be attached to the cell surface of the enucleated cells.
  • the cytokine may be attached, complexed, tethered, connected, coupled, linked, or any of the like to the cell surface of the enucleated cell.
  • the cytokine may be attached to the cell surface of an enucleated cell exogenously.
  • the cytokine is not delivered to a target tissue or microenvironment by an enucleated cell.
  • the cytokine is not secreted to a target tissue or microenvironment by an enucleated cell.
  • the mRNA encodes a cytokine or an antigen-binding fragment of a cytokine.
  • the cytokine is an antigen-binding fragment of said cytokine.
  • the cytokine is a full-length cytokine.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.1 fold to at least 5.0 fold higher than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.1 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.3 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.4 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.5 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.7 fold than an enucleated cell expressing an abundance of a WSGR Docket No.53712-720.601 comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.8 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.9 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine WSGR Docket No.53712-720.601 polypeptide that is at least 3.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
  • the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 5.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the abundance of the cytokine polypeptide and the abundance of the comparable naturally- occurring cytokine polypeptide is determined by flow cytometry (FACS).
  • FACS flow cytometry
  • the cytokine polypeptide is coupled to the enucleated cell surface for a certain length of time. In some embodiments, the length of time is following delivery to a subject (e.g., administration) disclosed herein. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 36 hours.
  • a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 72 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 24 hours.
  • a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 24 hours. In some embodiments, a cytokine polypeptide can be expressed on a cell surface of an enucleated cell for at least 36 hours. In some embodiments, a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide can be expressed on a cell surface of an enucleated cell for at least 72 hours. [0082]
  • the enucleated cell disclosed herein may comprise an mRNA encoding a polypeptide.
  • the polypeptide may be a cytokine.
  • the cytokine may comprise an interleukin.
  • the interleukin may comprise IL-12.
  • the IL-12 may comprise IL-12 ⁇ .
  • IL-12 ⁇ comprises SEQ ID NO: 35.
  • IL-12 ⁇ comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.
  • the IL-12 may comprise IL-12 ⁇ .
  • IL-12 ⁇ comprises SEQ ID NO: 36.
  • IL-12 ⁇ comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36.
  • the IL-12 may comprise IL-12 ⁇ and IL-12 ⁇ .
  • the IL-12 ⁇ and IL-12 ⁇ interact with a disulfide bond connecting the IL-12 ⁇ and IL-12 ⁇ .
  • the IL-12 may comprise a transmembrane domain, such as a transmembrane from a B7 cell surface ligand.
  • the transmembrane from the B7 cell surface ligand is from a B7-2 cell surface ligand.
  • B7-2 cell surface ligand comprises SEQ ID NO: 37. In some embodiments, B7-2 cell surface ligand comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37.
  • the IL-12 comprises IL-12 ⁇ , IL-12 ⁇ , and a transmembrane B7-2 domain. In some embodiments, IL-12 ⁇ , IL-12 ⁇ , and the transmembrane B7-2 domain comprises SEQ ID NO: 33.
  • IL-12 ⁇ , IL-12 ⁇ , and the transmembrane B7-2 domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33.
  • the transmembrane domain e.g., transmembrane B7-2, can allow the IL-12 to be tethered to the cell surface.
  • the interleukin may be IL-15.
  • IL-15 comprises SEQ ID NO: 38.
  • IL-15 comprises an amino acid WSGR Docket No.53712-720.601 sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38.
  • the IL-15 may further comprise IL-15 receptor alpha (RA).
  • IL-15 RA comprises SEQ ID NO: 40.
  • IL-15 RA comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 40.
  • the IL-15 RA may comprise a sushi domain.
  • the sushi domain comprises SEQ ID NO: 41.
  • sushi domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41.
  • the sushi domain can allow the IL-15RA to interact with the IL-15.
  • the sushi domain can allow the IL-15RA to bind to the IL-15.
  • the IL-15 RA may comprise a transmembrane domain In some embodiments, the transmembrane domain comprises SEQ ID NO: 42.
  • transmembrane domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42.
  • the IL-15 RA may comprise a sushi domain and a transmembrane domain.
  • the sushi domain comprises cysteine residues that form disulfide bonds, such as cysteine residues at position 3, 29, 45, and 63 of SEQ ID NO: 41.
  • the IL-15 and IL-15 RA may be connected.
  • the IL-15 and IL-15 RA may be connected by a linker.
  • the linker comprises SEQ ID NO: 39.
  • linker comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39.
  • the polypeptide may comprise the IL-15, the IL-15 RA, and the linker.
  • the IL-15, the IL-15 RA, and the linker comprises SEQ ID NO: 34.
  • the IL- 15, the IL-15 RA, and the linker comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34.
  • the sequences for these regions may be found in Table 9.
  • Table 9 Amino acid sequences for IL-12 and IL-15 complexes WSGR Docket No.53712-720.601 [0083]
  • the enucleated cell disclosed herein may comprise more than one mRNAs encoding more than one polypeptides.
  • the polypeptides may comprise cytokines.
  • the cytokines may comprise interleukins.
  • the interleukins may comprise IL-12 and IL-15.
  • IL-12 and IL-15 are expressed on the surface of the enucleated cell.
  • the polypeptides comprise a cytokine and an immune checkpoint inhibitor.
  • the enucleated cell disclosed herein may comprise an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor may comprise a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.
  • the immune checkpoint inhibitor may be encoded by a second exogenous mRNA in the enucleated cell.
  • the cells of the present disclosure comprise at least one therapeutic agent.
  • the cells are enucleated, such as with the methods of enucleation disclosed herein.
  • the therapeutic agent is or comprises an active agent of the present disclosure.
  • an active agent comprises at least one of a DNA molecule, a RNA molecule, a protein (e.g., an enzyme, an antibody, an antigen, a toxin, cytokine, a protein hormone, a growth factor, a cell surface receptor, or a vaccine), a peptide (e.g., a peptide hormone or an antigen), a small molecule (e.g., a steroid, a polyketide, an alkaloid, a toxin, an antibiotic, an antiviral, a colchicine, a taxol, a mitomycin, or emtansine), a gene editing factor, a nanoparticle, or another active agent (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses (e.g., oncolytic viruses), exosomes, lipids, or ions).
  • a protein e.g., an enzyme, an antibody, an antigen, a toxin
  • the amount of the therapeutic agent is about 3.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 4.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 5.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 5.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 35.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 45.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 45.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 55.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 60.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 60.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 65.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 65.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 70.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 70.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 70.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 70.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 75.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 75.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 75.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 85.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 85.00 to about 125.00. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 130.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 135.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 140.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 145.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 90.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 90.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 145.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 95.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 95.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 100.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 145.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 105.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 105.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 110.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 115.00 to about 130.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 115.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 120.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 125.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 130.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 135.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 135.00 to about 145.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 135.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 140.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 140.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 145.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
  • the amount of the therapeutic agent is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or a nucleated parent cell.
  • the amount of the therapeutic agent is greater than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or nucleated parent cell.
  • the amount of the therapeutic agent is less than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or nucleated parent cell. In some embodiments, the amount of the therapeutic agent and the amount of the
  • the therapeutic agent is recombinantly expressed by the enucleated cell or parent cell thereof.
  • the parent cell from which the enucleated cell is derived or obtained is engineered to produce or express the therapeutic agent.
  • expression of the therapeutic agent is stable (e.g., permanent).
  • the expression of the therapeutic agent by the parent cell is transient (e.g., non- permanent).
  • the parent cell is enucleated prior to engineering the WSGR Docket No.53712-720.601 enucleated cell to recombinantly express the therapeutic agent.
  • the parent cell is engineered to recombinantly express the therapeutic agent prior to enucleation.
  • the therapeutic agent is not naturally expressed (e.g., in the absence of engineering) in the cell from which the enucleated cell was derived or obtained (e.g., the therapeutic agent is exogenous to the parent cell). In some embodiments, the therapeutic agent is not naturally expressed in the subject (e.g., the therapeutic agent is exogenous to the subject).
  • the therapeutic agent is not naturally expressed in the subject at the intended site of therapy (e.g., a tumor, or a particular tissue such as the brain, the intestine, the lungs, the heart, the liver, the spleen, the pancreas, muscles, eyes, and the like) (e.g., the therapeutic agent is exogenous to the intended site of therapy).
  • the level of the therapeutic agent is not naturally occurring in the enucleated cell of the parent cell, such as over expression or under expression of the therapeutic agent.
  • the therapeutic agent is derived from a synthetic cell and loaded into the enucleated cell.
  • the therapeutic agent may be endocytosed into the cell prior to or after enucleation of the cell.
  • the therapeutic agent may be synthesized by the cell.
  • the enucleated cell comprises a plurality of therapeutic agents.
  • the enucleated cell comprises at least 1 (e.g., at least 1, 2, 3, 4, 5, or more) different DNA molecules, RNA molecules, proteins, peptides, small molecule active agents, or gene-editing factors, in any combination.
  • a therapeutic agent comprises a DNA molecule and a small molecule active agent.
  • the therapeutic agent comprises two different small molecule active agents.
  • the therapeutic agent comprises a chemokine receptor (e.g., for targeting) and a small molecule active agent.
  • the therapeutic agent comprises a polypeptide.
  • the polypeptide is exogenous.
  • the polypeptide is encoded by an exogenous polynucleotide delivered into the parent cell or the enucleated cell.
  • the polypeptide is synthesized by at least one intracellular organelle of the enucleated cell.
  • the polypeptide is tethered to the enucleated cell.
  • the polypeptide is expressed on the cell surface or the enucleated cell.
  • the enucleated cell expresses the polypeptide in a target environment.
  • the target environment is a microenvironment.
  • the microenvironment is a tumor microenvironment.
  • the enucleated cell expresses the polypeptide at a target cell.
  • the target cell is a cancer cell.
  • the cancer cell expresses the cancer biomarker of any cancer described WSGR Docket No.53712-720.601 herein.
  • the target cell is an endothelial cell.
  • the endothelial cell expresses an endothelial biomarker described herein.
  • the endothelial cell is a blood vessel cell. In some embodiments, the endothelial cell is a lymphatic vessel cell.
  • the exogenous polypeptide comprises a cytokine.
  • An “exogenous” polypeptide provided herein may refer to a presence of the polypeptide that is exogenous to the cell or enucleated cell, or an amount or level of expression of the polypeptide that exogenous to the cell or enucleated cell.
  • the cytokine comprises a membrane binding domain. In some embodiments, the exogenous polypeptide comprises a soluble cytokine.
  • the cytokine comprises interferons. In some embodiments, the cytokine comprises a lymphokine. In some embodiments, the cytokine comprises a tumor necrosis factor. In some embodiments, the cytokine comprises a monokine. In some embodiments, the cytokine comprises a colony-stimulating factor. In some embodiments, the cytokine comprises a transforming growth factor.
  • Non-limiting examples of cytokines include bFGF, TNF- ⁇ , IL-10, IL-12(p70), IL-1 ⁇ , IL-2, IL-6, GM-CSF, IL-13, IFN- ⁇ , TGF- ⁇ 1, TGF-02, TGF- ⁇ 3, and IL-15.
  • the exogenous polypeptide comprises an interleukin. In some embodiments, the exogenous polypeptide comprises more than one interleukin.
  • interleukins include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36.
  • a cytokine of the composition is an interleukin or a mutant thereof, including, but not limited to, wild-type and mutant forms of IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8. (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, Il29, IL30, IL31, IL32, IL33, IL35, or IL36.
  • the cytokine comprises IL-12.
  • the IL-12 comprises an IL-12 complex.
  • the IL-12 complex comprises SEQ ID NO: 33.
  • the IL-12 complex comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33.
  • the IL*12 complex comprises IL-12 ⁇ , IL-12 ⁇ , and a transmembrane domain.
  • the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 33. In some embodiments, the exogenous polypeptide comprises IL-12 ⁇ or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 35. In some embodiments, the exogenous polypeptide comprises IL-12 ⁇ or a catalytically active fragment thereof.
  • the IL-15 complex comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34.
  • the IL-15 complex is formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor ⁇ (SuIL-15 RA).
  • the complex is a fusion protein, e.g., with IgG4 Fc.
  • the IL-15 complex comprises IL-15 RA with a sushi domain and a transmembrane domain connected to IL-15 by a linker.
  • the cytokine comprises IL-15 and IL-12 in the same enucleated cell.
  • the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 34.
  • the exogenous polypeptide comprises IL-15 or a catalytically active fragment thereof.
  • the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 38.
  • the exogenous polypeptide comprises a linker.
  • the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 39.
  • the exogenous WSGR Docket No.53712-720.601 polypeptide comprises IL-15 RA or a catalytically active fragment thereof.
  • the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 40.
  • the exogenous polypeptide comprises a sushi domain of IL-15 RA or a catalytically active fragment thereof.
  • the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 41. In some embodiments, the exogenous polypeptide comprises a transmembrane domain of IL-15 RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 42. In some embodiments, the exogenous polypeptide comprises a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof.
  • TNF tumor necrosis factor
  • TNF superfamily member polypeptide examples include Lymphotoxin alpha (TNF ⁇ ), Tumor necrosis factor (TNF ⁇ ), Lymphotoxin beta (TNF ⁇ ), OX40 ligand (CD252, Gp34, or CD134L), CD40 ligand (CD154, TRAP, Gp39, or T-BAM), Fas ligand (CD178, APTL, or CD95L), CD27 ligand (CD70), CD30 ligand (CD153), CD137 ligand (4-1 BBL), TNF-related apoptosis-inducing ligand (CD253 or APO-2L), Receptor activator of nuclear factor kappa- ⁇ ligand (CD254, OPGL, TRANCE, or ODF), TNF-related weak inducer of apoptosis (APO-3L or DR3L), a proliferation-inducing ligand (CD256, TALL-2, or TRDL1), B-cell activating factor (CD257,
  • the therapeutic agent comprises any one of the immune checkpoint molecule described herein or an immune checkpoint molecule inhibitor for inhibiting any one of the immune checkpoint molecule described herein.
  • the immune checkpoint molecule include PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), B7-H3 (also called CD276), A2AR, CD27, LAG3, TIM-3, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD73, NKG2A, PVRIG, PVRL2, CEACAM1, CEACAM5, CEACAM6, FAK, CCR-2, CCL-2, LIF, CD47, SIRP ⁇ , M-CSF, CSF-1R, IL-3, IL-1RAP, IL-8, SEMA4D, Angiopoietin-2, CLEVER-1, Axl, phosphatidylserine or a fragment thereof.
  • the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 5% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 10% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 20% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety.
  • the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 90% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 100% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. [0101] In some embodiments, the targeting moiety comprises an exogenous antibody or an exogenous antigen-binding fragment for targeting a biomarker described herein.
  • the cells are enucleated, such as with the methods of enucleation disclosed herein.
  • transmembrane moiety is coupled to a polypeptide.
  • the polypeptide is a cytokine.
  • the polypeptide is an interleukin.
  • the polypeptide is a single-domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed here, or a combination thereof.
  • the transmembrane moiety is coupled by way of a covalent bond.
  • the transmembrane moiety is a fusion protein comprising the single-domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed here, or a combination thereof.
  • the exogenous polypeptide is complexed to the transmembrane moiety.
  • the transmembrane moiety comprises a full length protein or a variation thereof or a fragment thereof.
  • the transmembrane moiety is endogenous to the parent cell that is being enucleated for obtaining the enucleated cell.
  • the transmembrane moiety may be an exogenous transmembrane moiety to the parent cell or to the enucleated cell.
  • the enucleated cell comprises a cytokine.
  • the cytokine comprises an interleukin.
  • the cytokine comprises an IL-12 (interleukin 12).
  • the cytokine comprises an IL-15 (interleukin 15).
  • the exogenous cytokine or fragment thereof is complexed with a transmembrane moiety.
  • the transmembrane moiety comprises a WSGR Docket No.53712-720.601 transmembrane polypeptide.
  • the exogenous cytokine or fragment thereof is complexed with N-terminus of the transmembrane polypeptide.
  • IL-12 is coupled to a cell surface protein ligand.
  • the cell surface protein ligand is from the B7 family of cell surface ligand proteins.
  • B7 family members includes but is not limited to B7.1, B7.2, B7-H4, and B7-H3.
  • Il-12 is coupled to a portion of a cell surface protein ligand.
  • IL-12 is coupled to only a transmembrane portion of a cell surface protein ligand.
  • IL-12 is coupled to the transmembrane portion of a B7 family member.
  • IL-12 is coupled to the transmembrane portion of B7.1.
  • IL-12 comprises IL-12 ⁇ .
  • IL-12 comprises IL-12 ⁇ and IL-12 ⁇ .
  • IL-12 ⁇ comprises a signal peptide and an IL-12 subunit alpha chain.
  • IL-12 ⁇ comprises an IL-12 subunit alpha chain.
  • IL-15 may be coupled to IL-15 R ⁇ to the exoplasmic side of the cell membrane of the enucleated cell. As depicted in FIG.10A, IL-15 R ⁇ spans the cell membrane of an enucleated cell. The portion of IL-15 R ⁇ on the exoplasmic surface is coupled to IL-15 by a linker. On the opposite end of IL-15 from the linker connecting to IL-15 R ⁇ is a signal peptide. In some embodiments, IL-15 R ⁇ comprises a Sushi domain and a transmembrane region.
  • the enucleated cells may be engineered with immune evading moieties (e.g., CD34+) to avoid an antigenic response in the host.
  • Enucleated cells may also be engineered to express cell-surface receptors (e.g, adhesion molecules, chemokine receptors) used for cellular homing, chemokine sensing, and other biological functions that are essential to targeting damaged tissue.
  • An immune-evading moiety may comprise a signaling peptide, or portion thereof, that reduces cellular phagocytosis through its interaction with a signal receptor protein expressed by phagocytic cells such as macrophages and dendritic cells.
  • the immune-evading moiety blocks immune cell recognition or immune cell activation.
  • the compositions, methods and systems of the present disclosure utilize an enucleated cell platform developed by the inventors of the present disclosure and initial described in United States Patent Application No.10,927,349, which is hereby incorporated by reference in its entirety.
  • additional utility and advantages of the enucleated cells disclosed herein are discussed in U.S. Patent Application No.18/176,259, filed February 28, 2023; U.S. Patent Application No.17/885,867, filed August 11, 2022; and U.S. Patent Application No. 18/190,838, filed March 27, 2023, each of which is hereby incorporated by reference in its entirety.
  • the IL-12 B7TM coding region is preceded by a human eukaryotic translation elongation factor 1 ⁇ 1 promoter (EF1A) and a signal peptide or Kozak.
  • the IL-12 B7TM coding region comprises SEQ ID NO: 10.
  • the IL-12 B7TM coding region comprises a nucleotide sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.
  • IL-15 is encoded by a vector.
  • IL-15 and IL-15 R ⁇ are encoded by a vector.
  • IL-15, IL-15 R ⁇ , and a linker are encoded by a vector.
  • IL-15, IL-15 R ⁇ , a linker, and a signal peptide are encoded by a vector.
  • the vector is a lentiviral vector.
  • the vector is a mammalian expression vector. As depicted in FIG.10B, IL-15 and IL-15 R ⁇ may be inserted in a vector such as the vector depicted (vector ID: VB190220- WSGR Docket No.53712-720.601 1118ayw).
  • compositions described herein comprising the enucleated cells or the compositions described herein.
  • the pharmaceutical formulations further comprise a pharmaceutically acceptable: carrier, excipient, diluent, or nebulized inhalant.
  • the pharmaceutical formulations include two or more active agents, or two or more therapeutic agents as disclosed herein.
  • the two or more active agents are contained in a single dosage unit such as, for example, when the enucleated cell comprises two or more therapeutic agents.
  • the two or more active agents are contained in separate dosage units such as when the enucleated cell is administered separately from an additional therapeutic agent or adjuvant.
  • the active agents that may be, in some embodiments, the additional therapeutic agent include a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic agent, cardio protectant, and/or checkpoint inhibitor.
  • Non-limiting checkpoint inhibitor includes IMP321/Eftilagimod alpha (Immutep), Relatlimab BMS-986016, Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy), LAG525, MK-4280, Irinotecan, Oxaliplatin, REGN3767, TSR-033, BI754111, Sym022, FS118 (a bi-specific anti-LAG3/PD-L1 antagonistic mAb), MGD013 (a bi-specific anti-LAG3/PD-1 antagonistic mAb), TSR-022, Niraparib, Bevacizumab, MBG453, Decitabine, Spartalizumab, Sym
  • Non-limiting examples of active agents that may be, in some embodiments, the additional therapeutic agent include CPI- WSGR Docket No.53712-720.601 006 (for inhibiting CD73 and allowing T cell and APC activation); Monalizumab (for inhibiting NKG2A); COM701 (for inhibiting PVRIG/PVRL2 and activating T cell); CM24 (for inhibiting CEACAM1 and allowing T and NK cells activation); NEO-201 (for inhibiting CEACAM5 and CEACAM6 which allows T cell activation while interfering with tumor cell growth); Defactinib (for inhibiting FAK and interfering with tumor growth); PF-04136309 (for inhibiting CCR-2 and CCL-2 and allowing T cell recruitment and activation); MSC-1 (for inhibiting LIF and allowing T cell and APC activation while interfering with cancer growth); Hu5F9-G4 (5F9), ALX148,
  • composition described herein may include, but not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.
  • the pharmaceutical formulations including a therapeutic agent may be manufactured in a conventional manner such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
  • the pharmaceutical formulations may include at least an exogenous therapeutic agent as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form.
  • the methods and compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity.
  • therapeutic agents exist in WSGR Docket No.53712-720.601 unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the therapeutic agents are also considered to be disclosed herein.
  • pharmaceutical formulations provided herein include one or more preservatives to inhibit microbial activity.
  • stabilizing agents include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, I about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v.
  • polysorbate 20 (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.
  • aqueous oral dispersions liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.
  • a therapeutic agent as discussed herein e.g., therapeutic agent is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection.
  • formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions.
  • suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene- glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, WSGR Docket No.53712-720.601 emulsifying, and dispensing agents.
  • Prevention of the growth of microorganisms may be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • it is desirable to include isotonic agents such as sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption such as aluminum monostearate and gelatin.
  • a pharmaceutical formulations described herein is formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.
  • Parenteral injections may involve bolus injection or continuous infusion.
  • Pharmaceutical formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative.
  • the composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a therapeutic agent is formulated for use as an aerosol, a mist or a powder.
  • WSGR Docket No.53712-720.601 other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present.
  • the nasal dosage form should be isotonic with nasal secretions.
  • Pharmaceutical preparations for oral use are obtained by mixing one or more solid excipient with one or more of the compositions described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents are added such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose,
  • the pharmaceutical formulations of the exogenous therapeutic agents are in the form of a capsules, including push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push fit capsules contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active therapeutic agent is dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers are added.
  • a capsule may be prepared, for example, by placing the bulk blend of the formulation of the therapeutic agent inside of a capsule.
  • the formulations non-aqueous suspensions and solutions
  • the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC.
  • the formulation is placed in a sprinkle capsule, wherein the capsule is swallowed whole or the capsule is opened and the contents sprinkled on food prior to eating.
  • compositions for oral administration are in dosages suitable for such administration.
  • solid oral dosage forms are prepared by mixing a composition with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.
  • the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt WSGR Docket No.53712-720.601 tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules.
  • the composition is in the form of a powder.
  • Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above.
  • the pharmaceutical formulations described herein are self- emulsifying drug delivery systems (SEDDS).
  • SEDDS self- emulsifying drug delivery systems
  • Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets.
  • emulsions are created by vigorous mechanical dispersion.
  • SEDDS as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation.
  • An advantage of SEDDS is that only gentle mixing is required to distribute the WSGR Docket No.53712-720.601 droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient.
  • a pharmaceutical formulation is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.
  • Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative.
  • compositions include, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion.
  • Other methods include, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.
  • the compositions are provided that include particles of a therapeutic agent and at least one dispersing agent or suspending agent for oral administration to a subject.
  • the pharmaceutical formulations optionally include one or more salts in an amount required to bring osmolality of the composition into an acceptable range.
  • salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
  • Other the pharmaceutical formulations optionally include one or more preservatives to inhibit microbial activity.
  • An aerosol formulation for nasal administration is generally an aqueous solution designed to be administered to the nasal passages in drops or sprays.
  • Nasal solutions may be similar to nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values outside of this range may additionally be used.
  • Antimicrobial agents or preservatives may also be included in the formulation.
  • An aerosol formulation for inhalations and inhalants may be designed so that the agent or combination of agents is carried into the respiratory tree of the subject when administered by the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer.
  • Inhalations or insufflations comprising finely powdered or liquid drugs, may be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the agent or combination of agents in a propellant, e.g., to aid in disbursement.
  • Propellants may be liquefied gases, including halocarbons, for example, fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.
  • Halocarbon propellants may include fluorocarbon propellants in which all hydrogens are replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen- containing chlorofluorocarbon propellants.
  • Hydrocarbon propellants useful include, for example, propane, isobutane, n-butane, pentane, isopentane and neopentane.
  • a blend of hydrocarbons may also be used as a propellant.
  • Ether propellants include, for example, dimethyl ether as well as the ethers.
  • An aerosol formulation may also comprise more than one propellant.
  • the aerosol formulation comprises more than one propellant from the same class such as two or more fluorocarbons; or more than one, more than two, more than three propellants from different classes such as a fluorohydrocarbon and a hydrocarbon.
  • the compositions of the present disclosure may also be dispensed with a compressed gas, e.g., an inert gas such as carbon dioxide, nitrous oxide or nitrogen.
  • Aerosol formulations may also include other components, for example, ethanol, isopropanol, propylene glycol, as well as surfactants or other components such as oils and detergents. These components may serve to stabilize the formulation and/or lubricate valve components.
  • the aerosol formulation may be packaged under pressure and may be formulated as an aerosol using solutions, suspensions, emulsions, powders and semisolid preparations.
  • a solution aerosol formulation comprises a solution of an agent such as a transporter, carrier, or ion channel inhibitor in (substantially) pure propellant or as a mixture of propellant and solvent.
  • the solvent may be used to dissolve the agent and/or retard the evaporation of the propellant.
  • Solvents may include, for example, water, ethanol and glycols. Any combination of suitable solvents may be use, optionally combined with preservatives, antioxidants, and/or other aerosol components.
  • An aerosol formulation may be a dispersion or suspension.
  • a suspension aerosol formulation comprises a suspension of an agent or combination of agents, e.g., a transporter, carrier, or ion channel inhibitor, and a dispersing agent.
  • Dispersing agents may include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin and corn oil.
  • a suspension aerosol formulation may also include lubricants, preservatives, antioxidant, and/or other aerosol components.
  • An aerosol formulation may similarly be formulated as an emulsion.
  • the methods comprise treating a disease or a condition of a subject by administering the enucleated cells to the subject.
  • the method comprises administering to the subject having the disease or the condition an enucleated cell, wherein the enucleated cell comprises an active agent (e.g., exogenous polypeptide or a catalytically active fragment thereof) coupled to a surface of the enucleated cell.
  • the active agent is expressed by the enucleated cell in the absence of the nucleus at the surface of the enucleated cell.
  • the active agent is or comprises a cytokine polypeptide, or a catalytically active fragment thereof.
  • the resulting enucleated cell is engineered to express and, in some cases, secrete the therapeutic agent such as, for example, an antibody or an antigen- binding fragment thereof (e.g., single-domain antibody).
  • the enucleated WSGR Docket No.53712-720.601 cell may be administered to a subject in need thereof to treat a disease or a condition in the subject.
  • the enucleated cell of the present disclosure may be prepared in advance and cryopreserved for a length of time. This means, the enucleated cell of the present disclosure (e.g., engineered to express the homing receptors, immune activators, etc.) may be rapidly deployed. Such technical aspect is particular important when the enucleated cell is used to treat a disease or a condition stemmed from an outbreak of pathogen exposure or infection.
  • removal of the nucleus involves mechanically removing the nucleus.
  • the parent cell may be treated with cytochalasin to soften the cortical actin cytoskeleton.
  • enucleated cells are then physically extracted from the cell body by high-speed centrifugation in gradients of Ficoll to generate an enucleated cell. Because enucleate cells and intact nucleated cells sediment to different layers in the Ficoll gradient, enucleated cells may be easily isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated). The enucleation process is clinically scalable to process tens of millions of cells. In some embodiments, enucleated cells may be used as a disease-homing vehicle to deliver clinically relevant cargos/payloads to treat various diseases.
  • a biomolecule e.g., the therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein.
  • Non-limiting examples of methods that may be used to introduce a biomolecule into the parent cell or the enucleated cell include: liposome mediated transfer, an adenovirus, an adeno-associated virus, a herpes virus, a retroviral based vector, a lentiviral vector, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetofection, nanoparticle transfection, or combinations thereof.
  • a therapeutic agent, a virus, an antibody, or a nanoparticle may be introduced into the enucleated cells.
  • the enucleated cell is preserved via cryopreservation, cryohibernation, or lyophilization.
  • Cryopreservation comprises freezing the enucleated cell
  • cryohibernation comprises storing the enucleated cell at a temperature that is below room WSGR Docket No.53712-720.601 temperature but without freezing the enucleated cell.
  • the enucleated cell is lyophilized.
  • the lyophilization of the enucleated cell comprises the use of lyoprotectants for retaining cell viability and biologic function.
  • Lyoprotectant comprises addition of reagents, salts, or additives that protects cell during the desiccation process.
  • Common lyoprotectants include trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Additionally, methods for increasing the transport of lyoprotectants inside the cells in suspension can be utilized as a way of improving the viability and function of cells after lyophilization.
  • Inflammation cytokine measurements [0199] Finally, in a bilateral E0771 TNBC model, tumors were injected unilaterally with 3 doses of intertumoral enucleated cells that secreted IL-12 or PBS (control), and bilateral tumor size measured over time. Unilateral injection of intertumoral enucleated cells that secreted IL-12 reduced tumor growth in both tumors compared to controls, indicating that injection at a single site of enucleated cells that secreted IL-12 induced a systemic antitumor response to limit tumor growth at a distant site (contralateral tumor).
  • RNA-seq datasets generated from human TNBC primary tumors TME, and well-established mouse models of TNBC (EO771 and 4T1) were interrogated.
  • Fig.3A shows scRNA-seq datasets generated from two human TNBC primary tumors were integrated with eight normal mammary datasets.
  • VCAM1-positive vascular endothelial cells from clusters 0 and 4 Top panel of Fig.
  • EO771 tumors showed significantly upregulated CCL2, SDF-1a1ct and P- /E-selectin by qPCR relative to untreated contralateral tumor that peaked on day 4 post RT (Fig. 4B).
  • CCP enucleated cells (1x10 6 labeled with DiD) dye
  • the animals were sacrificed 24 hours later. Both tumors were then resected, and processed for FACS quantification of enucleated cells.
  • Tables 3-5 illustrate: vector summary of the vector encoding IL-12; vector components of the vector encoding IL-12; and a nucleic acid sequence (SEQ ID NO: 1) of the vector encoding IL-12.
  • Table 3. Vector summary of the vector encoding IL-12 Table 4.
  • Vector components of the vector encoding IL-12 WSGR Docket No.53712-720.601
  • WSGR Docket No.53712-720.601 Table 5.
  • Fig.8D shows that CCP MSCs were infected with lentiviruses encoding scIL-12 and a stable resistant cell line generated by drug selection, then enriched by fluorescence- activated cell sorting (FACS) using anti-IL-12 monoclonal antibody (mAb) to select the top 1% cells expressing the highest amounts of scIL-12, designated CA-scIL-12 MSC parent.
  • FACS fluorescence- activated cell sorting
  • mAb anti-IL-12 monoclonal antibody
  • CA-IL-12 readily homed to and integrated into micro- (93 ⁇ 11%) and macrometastasis (67% ⁇ 15%) in the lungs, which was similar to CCP enucleated cells (97 ⁇ 11% and 59 ⁇ 9%). Changes were not noted in integrated enucleated cells as in Fig.5. Strikingly, 93 ⁇ 11% and 97 ⁇ 11% of metastases observed showed infiltrated CA-IL-12 and CCP enucleated cells, respectively.
  • CA-IL-12 product has significant potential to clear the lungs of micrometastatic disease, which has implications as an adjuvant therapy for TNBC as well as late-stage metastatic cancers.
  • a single i.v. dose of CA-IL-12 did not impact the number of macrometastasis indicating that additional doses of CA-IL-12 are not necessary to impact large metastatic nodules.
  • CA-scIL-12 with similar findings.
  • Fig.10A illustrates an exemplary design of the enucleated cell expressing cell membrane tethered IL-15.
  • the IL-15 is connected to the IL-15 receptor alpha by a linker.
  • a signal peptide can also be connected to the IL-15.
  • the fusion of IL-15 receptor alpha can increase IL-15 activity and response.
  • Fig.10B illustrates a non-limiting example of a vector map for a vector encoding IL-15.
  • Fig.11A illustrates a schematic for engineering the hTERT-MSC for expression both WSGR Docket No.53712-720.601 scIL-12 and IL-15R ⁇ and the subsequent enucleation of the hTERT-MSC.
  • Fig.11B illustrates hTERT MSCs infected with two lentiviruses encoding constructs for either scIL-12 or IL-15R ⁇ . Following drug selection, cells were FACS sorted for single cell clones. Clone 4 was enucleated to generate enucleated cells expressing both scIL-12 and IL-15R ⁇ , and receptor expression was analyzed by FACS at the indicated time points.
  • Fig.11C illustrates functionality of expressing membrane-bound IL-12, IL-15, or a combination of both IL-12 and IL-15.
  • Pan-T cells were isolated from C57BL/6 mice.1.4x10 ⁇ 6 Pan-T cells/2 ml were co-cultured with hTERT MSCs expressing surface IL-12 a+b, hTERT MSCs expressing surface IL-15 R ⁇ , or hTERT MSCs expressing surface IL-12 a+b & IL-15 R ⁇ .
  • 1.4x10 ⁇ 6 Pan-T cells/ml were co-cultured with hTERT MSCs without transfection or with IL-12 a+b transfection for 1 hour. The T cell only was taken as a negative control.
  • CA-IL-12 and CA-scIL-12 Comparing the potential of CA-IL-12 and CA-scIL-12 to induce antitumor immunity against primary orthotopic TNBC tumors as well as secondary tumors in lungs and liver. Changes can be measured in a panel of key protein, gene, and immune cell biomarkers that report immune activation in the TME using established preclinical murine models of TNBC. The ability of CA-IL-12 medicines to transform the TME from relatively immunologically “cold” to “hot” tumors based on established biomarkers of antitumor immunity can be assessed. Changes in tumor regression and metastatic burden of CA-IL-12L medicine in combination with immune checkpoint blockade (ICB) therapy can be detailed.
  • IRB immune checkpoint blockade
  • the biodistribution of engineered MSCs and enucleated cells were monitored by bioluminescence.
  • the mouse ear dermis was treated with WSGR Docket No.53712-720.601 LPS and i.v.-injected with 3D cultured CCP (CXCR4, CCR2, and PSGL-1) MSC or CCP enucleated cells each transfected with firefly luciferase mRNA.
  • Mice were IP injected with D- luciferin at indicated time points after i.v. injection and mouse organs were subjected to bioluminescence imaging with IVIS Lumina Series III. Representative images were shown from two independent experiments in Fig.12A.
  • significant numbers of 3D CCP enucleated cells rapidly home to SDF-1 ⁇ , CCL2, and P-selectin produced within inflamed tissues within 2 hours post injection (Fig.13A and Fig.13B).
  • Fig.13A and Fig.13B show that CCP enucleated cells home better to the inflamed mouse ear than MSCs.
  • Primary tumors can be formed by inoculating 4T1 or E0771.LMB TNBC cells (50,000 in 50 ⁇ L) orthotopically into the inguinal mammary fat pad (mfp), and tumor growth can be monitored using digital calipers and by whole body luminescence. Under these conditions, primary tumors measuring 100 mm 3 form reproducibly (100% of mice) between 14-18 days.
  • Pulmonary metastases can be formed by seeding TNBC cells by tail vein injection (200 ⁇ L).
  • liver specific metastases can be established by standard hemi spleen injection. These transplantation models generate secondary tumors in nearly 100% of mice within 14 days and secondary tumor growth can be monitored using whole body luminescence BLI.
  • enucleated cells can be labelled with Vybrant DiO dye (3,3'- Dioctadecyloxacarbocyanine perchlorate ( ⁇ ex 484 nm/ ⁇ em 501 nm) for direct fluorescence imaging (Figs.5-7) or PCR using human mitochondrial DNA loci.
  • Vybrant DiO dye (3,3'- Dioctadecyloxacarbocyanine perchlorate ( ⁇ ex 484 nm/ ⁇ em 501 nm) for direct fluorescence imaging (Figs.5-7) or PCR using human mitochondrial DNA loci.
  • Animal weight and tumor size can be recorded by BLI and analyzed semi-quantitatively by luminoscore and compared across groups by two-way ANOVA. Growth of primary tumors can be terminated when tumors reach 2,000 mm 3 or animals show signs of physical distress.
  • mice euthanized and mammary fat pad (mfp), lungs, liver, heart, kidney, spleen as well as inguinal and popliteal lymph nodes excised and profiled for changes in health and immune biomarkers.
  • mfp mammary fat pad
  • Portions of each tissue can be prepared for fluorescent microscopy, IHC, nanostring analyses, or disaggregated into single cell suspensions for flow cytometry (FACS).
  • FACS flow cytometry
  • Serum and tissue samples from tumor bearing mice can be used for measurements of IL-12 and its effector IFN- ⁇ .
  • tissue and serum concentrations of IL-12 and IFN- ⁇ can be measured by ELISA and by measuring changes in IL-12-induced biomarkers IFN- ⁇ , PD- 1/PD-L1, CXCL9, and CXCL10 by RT-PCR.
  • Host-derived vs enucleated cell-derived IL-12 mRNA expression in breast, spleen, and liver by RT-PCR using oligonucleotide primers that distinguish innate host and engineered forms of IL-12 mRNAs can be measured.
  • Tissues can also be examined for changes in 200 nCounter® Inflammation Biomarkers using a standard NanoString nCounter Analysis.
  • Tissues and tumors can be collected for FACS analysis of key infiltrating leukocyte populations, including: CD8+ T cells (CD45.2+CD8+CD3+CD4-), CD4+ T cells (CD45.2+CD8- CD3+CD4+); T regulatory cells (CD45.2+CD4+CD25+FoxP3+); M1 macrophages (CD45+Ly6c-F4/80+MHC II+); M2 macrophages (CD45+Ly6c-F4/80+MHC II-); dendritic cells (CD45+ and Btla+Flt3+ or Ly-6C+TLR7+TLR9+), activated NK cells (CD45+CD3-NK1.1+ and CD107A, Granzyme B+ IFN- IFN-y+), and mature memory effector T cells (CD45ROne
  • the remaining tumors can be split in half for analysis by ELISA and IHC.
  • One set of halves can be analyzed by ELISA for IL-12 and IFN- ⁇ levels.
  • the other half can be used for IHC staining with anti-CD8, anti-CD4, anti-FoxP3, and PD-1 and PD-L1 antibodies.
  • the intratumor location and number of infiltrating leukocytes of each type can be quantified by counting immunopositive cells in 10 high-powered (400X) within the tumor and tumor/stroma interface. For FACS and IHC, the relative number of each leukocyte type can be compared between groups using two-way ANOVA.
  • CA-IL-12 or CA-scIL-12 treatment can also increase the number of activated cytotoxic effector cells locally in the tumor containing tissues, peripherally in inguinal, and popliteal lymph nodes as measured by IFN- ⁇ and Granzyme B-secreting (or expressing) cells.
  • activated cytotoxic effectors cells cannot be present in peripheral non-tumor bearing tissues.
  • no adverse health, weight loss, or organ pathology can be noted in IL-12 treated animals.
  • CA-IL-12 and CA-scIL-12 can induce adaptive and innate immune responses that can reprogram the immunologically “cold” TME into a “hot” TME in primary tumors and secondary tumors in lungs and liver without overt animal toxicity.
  • the proposed dosing regimen of CA-IL-12 and CA-scIL-12 can slow tumor growth and metastatic progression, and this response is likely be attenuated by IL-12/ IFN- ⁇ induced immune cell exhaustion due to upregulation of PD-1/PD-L1 signaling, leading to reduction in primary and secondary tumor burden. Therefore, local delivery of IL-12 medicines in combination with anti-PD-1 therapy can result in more robust and sustained antitumor and antimetastatic activity.
  • Example 1 Data discussed in Example 1 and Example 6 indicates that IT administration of CA-IL- 12 ignites a favorable shift in the composition of infiltrating immune cells in the TME without adverse health events, including: a) five-fold increase in CD8+ cells, b) two-fold increase in the M1/M2 ratio and c) two-fold decrease in CD25+ T regulatory cells, d) no or minimal changes in animal health as indicated by body weight, serum IFN-IL-12/IFN-y levels, and pathology analyses of the liver and other vital organs. Similar changes in immune cell composition in the TME and health measures in mice receiving CA-IL-12L medicines injected i.v. can be considered as criterion for success of the experiments described in this example.
  • CA-IL-12 activates innate and adaptive immunity in tumor tissue better than CA-scIL- 12.
  • CA-IL-12 and CA-sc-IL-12 are equally competent with regards to their ability to home to and interdigitate within secondary TNBC tumors. It is expected that CA-scIL-12 can show the most favorable toxicity profile since membrane tethered IL-12 has limited access to the circulation relative to secreted forms of extracellular IL-12; however, its efficacy may be inferior compared to CA-IL-12. If this is the case, CA-IL-12 development can be continued, but only if it shows a favorable toxicity profile based on the following criteria.
  • IFN- ⁇ is the prime mediator of both the beneficial and adverse effects of high dose rIL-12 immunotherapy. This relationship is further complicated by the observations that: 1) humans are significantly more sensitive to IL-12/ IFN- ⁇ toxicity than mice, 2) direct measurements of IFN- ⁇ in tissue in humans have not been determined although tissue IFN- ⁇ has been linked to toxicity. The concentrations of serum IFN- ⁇ can be determined and examined to see if they remain lower than concentrations known to be toxic in humans. For reasons stated above, tissue IFN- ⁇ can also be measured and its association with toxicity can be determined.
  • a threshold for IFN- ⁇ toxicity can be set at 5 ng/mL, and if CA-IL-12 produces serum concentrations of IFN- ⁇ ⁇ 5 ng/mL than this modality of localized IL-12 delivery can be dismissed. Leveraging the genetic versatility of the enucleated cell platform, two next generation enucleated cell products can be developed with improved benefit/risk ratios.
  • CA-scIL-12 can be engineered to co-express known immune cell surface anchored co-stimulatory proteins 4- 1BBL and/or CD40L, which is expected to synergistically drive potent immune activation in combination with scIL-12 within the TME and maintain a safe profile.
  • CA-IL-12 can be engineered to secrete IL-12 that is conditionally activated by the high proteolytic activity in the TME.
  • IL-12 can be engineered with a peptide mask with a protease linker that prevents binding to the IL-12 receptor using a similar approach for masking cytokines and antibodies.
  • the linker sequence was selected using cellular libraries of peptide substrates (CLiPS) for its responsiveness to multiple tumor-associated proteases, including urokinase-type plasminogen activator (uPA), membrane-type serine protease 1 (MT-SP1/matriptase), and legumain, all of which are strongly upregulated in the tumor microenvironment of most human tumors.
  • uPA urokinase-type plasminogen activator
  • MT-SP1/matriptase membrane-type serine protease 1
  • legumain all of which are strongly upregulated in the tumor microenvironment of most human tumors.
  • the mask contains specific protease consensus cleavage sites that upon exposure to high protease activity in the TME removes the mask and restores IL-12 function. Because proteolytic activity is largely absent in blood and normal tissues IL-12 bioactivity is retained within the TME.
  • Criteria for selecting CA-IL-12 medicine can be based on detecting a favorable shift in the composition of infiltrating immune cells in the TME without adverse health events while the experiments detailed for reducing metastatic burden, improving overall animal survival, and inducing durable antitumor immunity can be conducted using; a) CA-IL-12, b) CA-scIL-12, c) WSGR Docket No.53712-720.601 CA-scIL-12 engineered to contain additional cell surface immune activators 4-1BBL/CD40L or d) CA-IL-12 secreting protease activated IL-12. The medicine developed can then be referred to as CA-IL-12L.
  • CA-IL-12L can then be further evaluated for efficacy.
  • Example 9 Determination of the ability of IL-12 medicine (CA-IL-12L) in combination with ICB therapy to reduce metastatic burden, improve overall animal survival, and induce durable antitumor immunity [0227] Coupling the potent adaptive and innate immune activity of IL-12 with ICB is a powerful combinatorial therapy to overcome ICB resistance and drive antitumor immunity. While IL-12 monotherapy can reduce tumor progression, it is not sufficient to drive durable antitumor responses.
  • ICB therapy (Atezolizumab) have been achieved in the adjuvant setting and tumor-free and overall survival in mice whose primary tumors are resected prior to CA-IL-12L can be evaluated, and anti-PD-1 therapy as a clinical trial correlate.
  • 4T1 and EO771 tumors can be used, which are refractory to ICB therapy as is the case with most human TNBCs.
  • Primary tumors can be formed following orthotopic transplantation of 4T1 or E0771.LMB TNBC cells as described previously. At day 14, primary tumors can be excised, and spontaneous metastases can be monitored by bioluminescence imaging (BLI). Under these conditions, tumor weights at resection show a low coefficient of variation ( ⁇ 15% CV).
  • Animal weight and tumor size can be recorded by BLI and analyzed semi-quantitatively by luminoscore once a week and compared across groups by two-way ANOVA. Metastases can be measured by marking the whole body minus the 4th inguinal mfp as a ROI. Survival rates can be determined from Kaplan-Meier plots and the log-rank test. The durability of the antitumor response can be evaluated by rechallenging surviving mice (from group 10) with tumor cells. In these experiments, in mice with no measurable primary tumors 60 days after treatment, tumor cells can be reintroduced into the 4th mfp and tumor growth can again be monitored by BLI. These experiments can be terminated when tumors reach 2,000 mm 3 or show signs of labored respiration.
  • CA-IL- 12L medicine in combination with ICB therapy can yield an 80% overall response rate with ⁇ 50% reduction in metastatic burden, and an improved overall animal survival of at least 20 days with a ⁇ 40% complete response rate based on Kaplan–Meier plots, the log rank test, and the Cox proportional hazards test.
  • CA-IL-12L in combination with ICB therapy can generate durable antitumor immunity ( ⁇ 120 days disease free) and prevent tumor regrowth following tumor rechallenge in 100% of the surviving animals.
  • CA-IL-12L can be present in tumor tissue but fail to induce immune cell infiltration robust enough to drive anticancer immunity. If this is the case, CA-IL-12L may be engineered to secrete potent NK and cytotoxic T cell chemoattractants (CXCL9 or CXCL10). On the other hand, if CA-IL-12L does promote immune cell infiltration, activate IL-12 biomarkers, and prevent immune exhaustion markers as expected, but fails to impact overall survival an in-depth analyses of the infiltrated immune cell phenotypes and immune cytokines can be performed to identify potential new targets and immunomodulators that could be targeted by bioengineered enucleated cells alone or in combination with ICB therapy.
  • CXCL9 or CXCL10 cytotoxic T cell chemoattractants
  • the PyMT mouse is ideally suited as a model for testing ICB in combination with CA-IL-12L because the primary tumors are ICB refractory and substantial work supports a role for innate and adaptive immune systems in metastasis regulation.
  • MMTV-PyMT tumor vasculature overexpresses CCP enucleated cell ligands.
  • CD31-positive endothelial cells within MMTV-PyMT tumors were FACS sorted and utilized for 10x Genomics scRNA-seq (Fig.14A and Fig.14B).
  • mice can be euthanized on the day following the last treatment (day 90 when >90% of mice have metastatic disease).
  • Animal health and biomarker analyses of blood and tissues can be performed as described previously with minor modifications.
  • the presence of primary and secondary tumors in mfp, lungs, liver, and brain can be evaluated by gross inspection at the time of necropsy and by H&E and IHC using specific PyMT (Novus, NB100-2749), CK18 (Abcam, EPR17347) and EpCam (Abcam, EPR20533-63) antibodies.

Abstract

Sont décrites des compositions et des méthodes pour traiter le cancer par administration d'une cellule énucléée, la cellule énucléée comprenant un ARN messager exogène (ARNm) codant pour un polypeptide de cytokine et des organelles pour exprimer le polypeptide de cytokine à partir de l'ARNm exogène, le polypeptide de cytokine étant exprimé par la cellule énucléée sur une surface de cellule. La cellule énucléée peut également comprendre un ARNm exogène codant pour une autre cytokine ou un autre inhibiteur de point de contrôle immunitaire.
PCT/US2023/085419 2022-12-28 2023-12-21 Compositions et méthodes pour traiter le cancer WO2024145168A1 (fr)

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