WO2022187573A1 - Procédés et compositions se rapportant à l'hybridation et le camouflage de la membrane cellulaire - Google Patents

Procédés et compositions se rapportant à l'hybridation et le camouflage de la membrane cellulaire Download PDF

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WO2022187573A1
WO2022187573A1 PCT/US2022/018833 US2022018833W WO2022187573A1 WO 2022187573 A1 WO2022187573 A1 WO 2022187573A1 US 2022018833 W US2022018833 W US 2022018833W WO 2022187573 A1 WO2022187573 A1 WO 2022187573A1
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cell
engineered
cell membrane
cells
cancer
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PCT/US2022/018833
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English (en)
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Fnu KENRY
Samir Mitragotri
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration

Definitions

  • the technology described herein relates to cells in which the exterior surface of the cell composition provides improved therapeutic delivery, e.g., for cell therapy and immunotherapy applications.
  • CAR-T cell therapy has emerged as a therapeutic paradigm for a number of diseases, including cancer.
  • the therapeutic cells are themselves targeted by the patient’s immune system. This targeting canm mean that the therapeutic cells are eliminated by the patient’s body before they can provide treatment. This targeting often also results in dangerous side effects such as intolerable myeloablation, infections, cytokine release syndrome (CRS), and bleeding complications.
  • CRS cytokine release syndrome
  • This unwanted targeting of therapeutic cells is particularly common when treating diseases in which the diseased cells are of the same type or similar type as the therapeutic cells, e.g., in hematological cancers.
  • Described herein are engineered cells that are are able to avoid the unwanted immune system targeting described above.
  • the engineered cells described herein are modified with a cell membrane coating that allows them to avoid stimulating the immune system.
  • the cell membrane coating can be derived from a source cell and when applied to a recipient cell, it provides an engineered cell with biological properties and functionalities similar to the source cells from which the membrane is derived, such as homotypic targeting, cytokine binding and isolation, immune evasion, and extended in vivo residence time. This approach provides increased efficacy and safety, particularly in the treatment of hematological cancers.
  • an engineered cell comprising a cell membrane coating on the surface of the cell, the cell membrane coating comprising cell membrane components of one or more source cells.
  • the cell membrane coating comprises cell membrane components of at least two source cells. In some embodiments of any of the aspects, the cell membrane coating comprises cell membrane components of at least three source cells. In some embodiments of any of the aspects, the at least two source cells are different types of cells. In some embodiments of any of the aspects, the one or more source cells comprise a T cell, a diseased cell, and/or a macrophage. In some embodiments of any of the aspects, the T cell is a CAR-T cell. In some embodiments of any of the aspects, the diseased cell is a cancer cell. In some embodiments of any of the aspects, the cancer cell is a leukemia cell or an acute myeloid leukemia (AML) cell. In some embodiments of any of the aspects, the macrophage is a THP-1 macrophage.
  • the cell membrane components include one or more of: a lipid; a lipid bilayer; transmembrane proteins; membrane-displayed biomolecules; and cell surface proteins.
  • the cell membrane coating comprises a mixture, fusion, or hybridization of cell membrane components from two or more source cells.
  • the mixture, fusion, or hybridization of cell membrane components has an equal amount of cell membrane components from each source cell.
  • the amount of cell membrane components is the amount as measured by protein concentrations.
  • the cell membrane components obtained from each source cell further comprises a detectable label.
  • the cell membrane components obtained from each source cell each further comprise a detectable label, wherein each detectable label is distinguishable from the detectable labels comprised by the cell membrane components obtained from the other source cells.
  • the cell membrane coating further comprises a detectable label.
  • the engineered cell further comprises a detectable label.
  • the engineered cell is a T cell.
  • the T cell is a CD8+ T cell.
  • the T cell is a CAR-T cell.
  • the CAR-T cell comprises a CAR specific for or active against the diseased cell.
  • the CAR is an anti-CD 123 CAR.
  • the one or more source cells and/or the engineered cell are obtained from a subject.
  • the engineered cell is not autologous to a subject.
  • the engineered cell is autologous to a subject.
  • described herien is a method of producing an engineered cell comprising a cell membrane coating, the method comprising: a) obtaining cell membrane components from one or more source cells; b) optionally, mixing or fusing the cell membrane components of two or more source cells; and c) contacting a recipient cell with the cell membrane components to obtain an engineered cell comprising a cell membrane coating.
  • the cell membrane components are obtained by freeze-thaw of the source cells.
  • the mixing or fusing of the cell membrane components comprises sonication and/or fluid flow.
  • the method, or at least steps b) and c) is conducted in a microfluidic device.
  • a microfluididic device comprising, in the following order along a flow path(s): a) one or more initial input ports; b) a flow channel for mixing materials provided to the one or more initial input ports; c) one or more recipient cell input ports; d) a flow channel comprising sonication input, or capable of receiving sonication input; and e) one or more output ports.
  • the cancer is a hematological cancer or hematopoietic cancer.
  • the cancer is leukemia.
  • the cancer is acute myeloid leukemia.
  • the subject is at risk of cytokine release syndrome (CRS).
  • FIGs. 1A-1C depict schematics illustrating the theranostic approach described herein, e.g., to treat acunte myeloid leukemia.
  • FIG. 2 depicts an exemplary embodiment of the microfluidic devices described herein.
  • Fig. 3 depicts an exemplary microfluidic device for the formulation of cell membrane- coated nanostructures.
  • Figs. 4A-4D depict the surface morphology of macrophage membrane vesicle (MMV)- coated Au (gold) nanostructures obtained through microfluidic-mediated physical mixing.
  • White arrows indicate the macrophage membrane coatings. Scale bars represent 40 nm.
  • Fig. 5 depicts the size of different macrophage membrane vesicle-coated Au nanostructures obtained through microfluidic-mediated physical mixing.
  • Fig. 6 depicts surface charge of different macrophage membrane vesicle-coated Au nanostructures obtained through microfluidic-mediated physical mixing.
  • Fig. 7 depicts the protein expression of the bare and macrophage membrane vesicle- encapsulated Au nanostructures.
  • Lanes 1 to 7 of the polyacrylamide gel represent protein marker (1), cell lysate (2), AuNNs (3), AuNSs (4), MMVs (5), MMV-AuNNs (6), and MMV-AuNSs (7), respectively.
  • Fig. 8 depicts the variation of the hydrodynamic size of macrophage membrane vesicle- encapsulated Au nanostructures overtime.
  • Fig. 9 depicts the zeta potential variation of the macrophage membrane vesicle- encapsulated Au nanostructures overtime.
  • Figs. 10A-10C demonstrate cancer cell uptake of the macrophage membrane vesicle- encapsulated Au nanostructures.
  • FIG. 10A Representative brightfield images of the uptake of AuNNs and MMV-AuNNs by MDA-MB-231 breast cancer cells after a 12-h incubation. White arrows on the brightfield images indicate the presence of Au nanostructures. Scale bars represent 20 pm.
  • Fig. 10B The corresponding semi -quantitative estimation of the uptake of different Au nanostructures by cancer cells over time.
  • Fig. IOC Spreading areas of cancer cells after different Au nanostructure treatments over time.
  • Figs. 1 lA-11C demonstrate enhanced immune evasion of macrophage membrane vesicle- encapsulated Au nanostructures.
  • FIG. 11A Representative brightfield images of the uptake of AuNNs and MMV-AuNNs by RAW 264.7 macrophages after a 12-h incubation. White arrows on the brightfield images indicate the presence of Au nanostructures. Scale bars represent 20 pm.
  • Fig. 1 IB The corresponding semi-quantitative estimation of the uptake of different Au nanostructures by macrophages overtime.
  • Fig. 11C Spreading areas of macrophages after different Au nanostructure treatments over time.
  • Figs. 12A-12D depict the surface morphology of cancer cell membrane vesicle-coated Au nanostructures obtained through microfluidic-mediated physical mixing.
  • White arrows indicate the cancer cell membrane coatings. Scale bars represent 40 nm.
  • Fig. 13 depicts the size of different cancer cell membrane vesicle-coated Au nanostructures obtained through microfluidic-mediated physical mixing.
  • Fig. 14 depicts the surface charge of different cancer cell membrane vesicle-coated Au nanostructures obtained through microfluidic-mediated physical mixing.
  • Fig. 15 depicts the protein expression of the bare and cancer cell membrane vesicle- encapsulated Au nanostructures.
  • Lanes 1 to 7 of the polyacrylamide gel represent protein marker (1), cell lysate (2), AuNNs (3), AuNSs (4), CCMVs (5), CCMV-AuNNs (6), and CCMV-AuNSs (7), respectively.
  • Fig. 16 depicts the variation of the hydrodynamic size of cancer cell membrane vesicle- encapsulated Au nanostructures overtime.
  • Fig. 17 depicts the zeta potential variation of the cancer cell membrane vesicle- encapsulated Au nanostructures overtime.
  • coating cells with a cell membrane coating permits them to avoid stimulating the immune system and can provide further functionality such as homotypic targeting, cytokine binding and isolation, immune evasion, and extended in vivo residence time. This provides increased efficacy and safety, particularly in the treatment of hematological cancers like AML.
  • the cell membrane coatings and methods described herein also provide faster and more cost-effective functionalization than prior art methods.
  • an engineered cell comprising a cell membrane coating on the surface of the cell, the cell membrane coating comprising cell membrane components of one or more source cells.
  • cell membrane refers to a biological membrane that separates the interior of a cell from the extracellular space.
  • the cell membrane is also known in the art as the plasma membrane or cytoplasmic membrane.
  • the cell membrane includes a phospholipid bilayer with interspersed cholesterols as well as embedded or associated membrane proteins (including transmembrane, lipid-anchored, and peripheral proteins), gly colipids, glycoproteins, sterols, and carbohydrates.
  • cell membrane coating refers to a volume of material associated with a cell and which is located on or near the extracellular face of the cell’s own cell membrane.
  • the cell membrane coating can have a spherical, hemispherical, or irregular shape.
  • the cell membrane coating can have the shape of the engineered cell which it is coating.
  • the cell membrane coating can have a thickness of from 1 to 50 nm. In some embodiments of any of the aspects, the cell membrane coating can have a thickness of from 2 to 35 nm. In some embodiments of any of the aspects, the cell membrane coating can have a thickness of from 5 to 20 nm. In some embodiments of any of the aspects, the cell membrane coating can have a constant or varying thickness.
  • the cell membrane coating can form a complete coating on the engineered cell, e.g., the cell membrane coating forms a continuous surface with two faces but no edges. In some embodiments of any of the aspects, the cell membrane coating can form an incomplete coating on the engineered cell, e.g., the cell membrane coating forms a discontinuous surface with two faces and at least one edge. In some embodiments of any of the aspects, the cell membrane coating coats at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the engineered cell.
  • the cell membrane coating comprises one or more cell membrane components.
  • Cell membrane components are the molecules found in a cell membrane in nature.
  • Exemplary cell membrane components include phospholipids, cholesterols, embedded or associated membrane proteins (including transmembrane, lipid-anchored, and peripheral proteins), glycolipids, glycoproteins, sterols, and carbohydrates.
  • the cell membrane coating comprises, consists of, or consists essentially of one or more cell membrane proteins, e.g., embedded or associated membrane proteins (including transmembrane, lipid-anchored, and peripheral proteins).
  • the cell membrane coating comprises, consists of, or consists essentially of phospholipids and one or more cell membrane proteins, e.g., embedded or associated membrane proteins (including transmembrane, lipid-anchored, and peripheral proteins).
  • cell membrane proteins e.g., embedded or associated membrane proteins (including transmembrane, lipid-anchored, and peripheral proteins).
  • Individual cell membrane proteins are well known in the art and are further described, e.g, in Nano Letters 2016, 16, 9, 5895-5901; Biomaterials 2016, 76, 52-65; and ACS Nano 2018, 12, 12, 12096-12108; each of which is incorporated by reference herein in its entirety.
  • the cell membrane components include one or more of: a lipid; a lipid bilayer; transmembrane proteins; membrane-displayed biomolecules; and cell surface proteins.
  • the cell membrane components include: a. at least one lipid; at least one lipid bilayer; at least one transmembrane protein; at least one membrane-displayed biomolecule; and at least one cell surface protein; b. at least one lipid bilayer; at least one transmembrane protein; at least one membrane- displayed biomolecule; and at least one cell surface protein; c.
  • a transmembrane protein is one comprising a transmembrane domain and which positions part of the protein on one side of a phospholipid bilayer, and another part of the protein on the other side of that phospholipid bilayer. This is contrasted with a cell surface protein, which positions part or all of the protein on one side of a phospholipid bilayer and no part of the protein on the other side of that phospholipid bilayer.
  • a cell surface protein may have a part of the protein embedded in the phospholipid bilayer or no part of the protein embedded in the phospholipid bilayer.
  • a cell membrane coating and/or cell membrane components can be obtained from a source cell, e.g., by isolating or purifying the cell membrane coating and/or cell membrane components from the source cell.
  • Cell membrane coatings and/or components can be obtained from one or more source cells by any method known in the art that disrupts cellular membrane integrity or lyses a cell, e.g., contacting with detergents, freezing and then thawing the source cell(s) (i.e., freeze- thaw), bead milling, use of a blender, liquid homogenization, sonication, and the like.
  • a cell membrane coating and/or cell membrane components can be engineered and/or synthetic, e.g., provided by combining isolated or chemically synthesized/produced components.
  • the cell membrane, e.g, the source or reference cell membrane is a mammalian cell membrane.
  • the cell membrane, e.g, the source or reference cell membrane is a human cell membrane.
  • the cell membrane, e.g, the source or reference cell membrane is a T cell membrane (e.g., a CAR-T T cell membrane).
  • the cell membrane e.g. the source or reference cell membrane
  • a diseased cell membrane e.g., a cancer, leukemia, or acute myeloid leukemia (AML) cell membrane
  • AML acute myeloid leukemia
  • the cell membrane, e.g, the source or reference cell membrane is a macrophage membrane (e.g., a THP-1 macrophage membrane).
  • the cell membrane coating comprises cell membrane components of multiple source or reference cells, e.g, multiple different types of source or reference cells. In some embodiments of any of the aspects, the cell membrane coating comprises cell membrane components of at least two source or reference cells, e.g, at least two different types of source or reference cells. In some embodiments of any of the aspects, the cell membrane coating comprises cell membrane components of at least three source or reference cells, e.g, at least three different types of source or reference cells. In some embodiments of any of the aspects, the cell membrane coating comprises cell membrane components of at least four source or reference cells, e.g, at least four different types of source or reference cells.
  • the cell membrane coating comprises cell membrane components of at least five source or reference cells, e.g, at least five different types of source or reference cells.
  • the cell membrance coating comprises cell membrane components from multiple source or reference cells, any combination of the cell types described herein are contemplated herein.
  • the following table illustrates exemplary pairwise combinations of different types of source or reference cells.
  • the cell membrane coating comprises cell membrane components from a CAR-T cell, a cancer cell, and a macrophage.
  • the source cells comprise a CAR-T cell, a cancer cell, and a macrophage.
  • the cell membrane coating comprises cell membrane components from a CAR-T cell, a cancer cell, and a THP-1 macrophage.
  • the source cells comprise an CAR-T cell, a cancer cell, and a THP-1 macrophage.
  • the cell membrane coating comprises cell membrane components from a CAR-T cell, a leukemia cell, and a THP-1 macrophage.
  • the source cells comprise an CAR-T cell, a leukemia cell, and a THP- 1 macrophage.
  • the cell membrane coating comprises cell membrane components from an anti-CD 123 CAR-T cell, a AML cell, and a THP-1 macrophage.
  • the source cells comprise an anti-CD 123 CAR-T cell, a AML cell, and a THP-1 macrophage.
  • the cell membrane coating does not comprise a source cell. In some embodiments of any of the aspects, the cell membrane coating does not comprise a source cell nucleus. In some embodiments of any of the aspects, the cell membrane coating does not comprise source cell DNA.
  • the cell membrane coating when the cell membrane coating comprises cell membrane components from multiple source cells, the cell membrane coating is a mixture, fusion, or hybridization of cell membrane components from two or more source cells. In some embodiments of any of the aspects, the mixture, fusion, or hybridization of cell membrane components has an equal amount of cell membrane components from each source cell. In some embodiments of any of the aspects, the mixture, fusion, or hybridization of cell membrane components has an unequal amount of cell membrane components from each source cell.
  • the cell membrane coating when the cell membrane coating comprises cell membrane components from multiple source cells, the cell membrane coating is a mixture, fusion, or hybridization of cell membranes from two or more source cells. In some embodiments of any of the aspects, the mixture, fusion, or hybridization of cell membranes has an equal amount of cell membrane components from each source cell.
  • the amount of cell membrane components can be the amount as measured by protein concentrations or lipid concentrations.
  • the amount of cell membrane components can be the amount as measured by protein concentrations.
  • the amount of cell membrane components can be the amount as measured by lipid concentrations.
  • the cell membrane components further comprise a detectable label.
  • the cell membrane components obtained from each source cell further comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product).
  • the cell membrane components obtained from each source cell each further comprises a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product), e.g., each detectable label is distinguishable from the detectable labels comprised by the cell membrane components obtained from the other source cells.
  • the engineered cell comprises or expresses a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product) which is not present in the cell membrane coating.
  • Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents are well known in the art.
  • detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means.
  • the detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies).
  • the detectable label can be linked by covalent or non-covalent means to the reagent.
  • a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules.
  • Detectable labels can include, but are not limited to radioisotopes, biolumine scent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
  • the detectable label is a fluorescent compound.
  • a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o- phthaldehyde, fluorescamine, Cy3TM, Cy5TM, allophy cocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyesTM,
  • a detectable label can be a radiolabel including, but not limited to 3 H, 125 1, 35 S, 14 C, 32 P, and 33 P.
  • a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase.
  • An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal.
  • Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
  • a detectable label can be a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • Other detection systems can also be used, for example, a biotin-streptavidin system.
  • a first molecule is biotinylated.
  • the quantity of biotin present is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate.
  • streptavidin peroxidase detection kits are commercially available, e.g. from DAKO; Carpinteria, CA.
  • a reagent can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to a target using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the one or more cell types that may be included in the methods or compositions described herein can comprise any mammalian cell type selected from cells that make up the mammalian body, including germ cells, somatic cells, and stem cells.
  • germ cells refers to any line of cells that give rise to gametes (eggs and sperm).
  • somatic cells refers to any biological cells forming the body of a multicellular organism; any cell other than a gamete, germ cell, gametocyte or undifferentiated stem cell.
  • somatic cells include fibroblasts, chondrocytes, osteoblasts, tendon cells, mast cells, wandering cells, immune cells, pericytes, inflammatory cells, endothelial cells, myocytes (cardiac, skeletal and smooth muscle cells), adipocytes (i.e., lipocytes or fat cells), parenchyma cells (neurons and glial cells, nephron cells, hepatocytes, pancreatic cells, lung parenchyma cells) and non-parenchymal cells (e.g., sinusoidal hepatic endothelial cells, Kupffer cells and hepatic stellate cells).
  • stem cells refers to cells that have the ability to divide for indefinite periods and to give rise to virtually all of the tissues of the mammalian body, including specialized cells.
  • the stem cells include pluripotent cells, which upon undergoing further specialization become multipotent progenitor cells that can give rise to functional or somatic cells.
  • stem and progenitor cells examples include hematopoietic stem cells (adult stem cells; i.e., hemocytoblasts) from the bone marrow that give rise to red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells; epithelial stem cells (progenitor cells) that give rise to the various types of skin cells; neural stem cells and neural progenitor cells that give rise to neuronal and glial cells; and muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue.
  • hematopoietic stem cells adult stem cells; i.e., hemocytoblasts
  • mesenchymal stem cells adult stem cells
  • epithelial stem cells progenitor cells
  • neural stem cells and neural progenitor cells that give rise to neuronal and glial cells
  • muscle satellite cells progenitor cells
  • the engineered cell and/or the recipient cell e.g., the cell which is coated with the cell membrane coating to provide the engineered cell
  • the engineered cell and/or the recipient cell e.g., the cell which is coated with the cell membrane coating to provide the engineered cell
  • the engineered cell and/or the recipient cell e.g., the cell which is coated with the cell membrane coating to provide the engineered cell
  • the engineered cell and/or the recipient cell e.g., the cell which is coated with the cell membrane coating to provide the engineered cell
  • the engineered cell and/or the recipient cell are obtained from or derived from the same species of organism. In some embodiments, the engineered cell and/or the recipient cell (e.g., the cell which is coated with the cell membrane coating to provide the engineered cell) are obtained from or derived from different species.
  • the engineered cell and/or the recipient cell e.g., the cell which is coated with the cell membrane coating to provide the engineered cell
  • the engineered cell and/or the recipient cell are cell lines.
  • the engineered cell and/or the recipient cell are cells differentiated from stem cells, progenitor cells, iPSCs, or the like in vitro.
  • the engineered cell and/or the recipient cell are obtained from or derived from diseased primary cells or a subject having a disease affecting the engineered and/or receipient cell type.
  • the source cell(s) and/or reference cell(s) are mammalian cells. In some embodiments, the source cell(s) and/or reference cell(s) are murine cells. In some embodiments, the source cell(s) and/or reference cell(s) are primate cells. In some embodiments, the source cell(s) and/or reference cell(s) are human cells. In some embodiments, the source cell(s) and/or reference cell(s) are obtained from or derived from the same species of organism. In some embodiments, the source cell(s) and/or reference cell(s) are obtained from or derived from different species.
  • the source cell(s) and/or reference cell(s) are primary cells. In some embodiments, the source cell(s) and/or reference cell(s) are cell lines. In some embodiments, the source cell(s) and/or reference cell(s) are cells differentiated from stem cells, progenitor cells, iPSCs, or the like in vitro.
  • the source cell(s) and/or reference cell(s) are obtained from or derived from diseased primary cells or a subject having a disease affecting the engineered and/or receipient cell type.
  • the one or more source cells, the engineered cell comprising a cell membrane coating, and/or the recipient cell are obtained from a subject.
  • the one or more source cells, the engineered cell comprising a cell membrane coating, and/or the recipient cell are obtained from a subject and the engineered cell is administered to that same subject.
  • the one or more source cells are obtained from a subject and the engineered cell is administered to that same subject.
  • the one or more source cells, the engineered cell comprising a cell membrane coating, and/or the recipient cell are obtained from a first subject and the engineered cell is administered to a second subject.
  • the engineered cell and/or the recipient cell is not autologous to a subject. In some embodiments of any of the aspects, the engineered cell and/or the receipient cell is autologous to a subject.
  • the source cell is autologous to a subject.
  • the engineered cell, recipient cell, and/or source cell are cells from a sample.
  • sample denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample from a subject.
  • the present invention encompasses several examples of a biological sample.
  • the biological sample is cells, or tissue, or peripheral blood, or bodily fluid.
  • Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc.
  • the term also includes a mixture of the above-mentioned samples.
  • sample also includes untreated or pretreated (or pre-processed) biological samples.
  • a sample can comprise cells from a subject.
  • the sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g.
  • the sample can be an untreated sample.
  • untreated sample refers to a sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution.
  • Exemplary methods for treating a sample include, but are not limited to, centrifugation, fdtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof.
  • the sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein.
  • a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein.
  • the sample is a clarified sample, for example, by centrifugation and collection of a supernatant comprising the clarified sample.
  • a sample can be a pre-processed sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof.
  • the sample can be treated with a chemical and/or biological reagent.
  • Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing.
  • biomolecules e.g., nucleic acid and protein
  • One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing.
  • protease inhibitor is generally used to protect or maintain the stability of protein during processing.
  • the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a sample from a subject.
  • the subject can be a human subject.
  • the engineered T cell or recipient T cell is a CD8+ T cell.
  • the engineered T cell or recipient T cell is a CAR-T cell, e.g., a cell comprising or expressing a chimeric antigen receptor (CAR).
  • CAR-T-cell and related therapies relate to adoptive cell transfer of immune cells (e.g., T-cells) expressing a CAR that binds specifically to a target (e.g., CD123) to treat a subject.
  • the cells are engineered and/or genetically modified to express the CAR.
  • the structure of CARs and methods for making them and CAR-T cells are well known in the art. Further discussion of CAR-T therapies can be found, e.g., in Wei et al.
  • the CAR can be specific for or active against a diseased cell.
  • the CAR can be specific for or active against a diseased cell of the same type as from which the cell membrane components were obtained or derived from.
  • exemplary targets for CARs include but are not limited to CD123, CD19, CD20, BCMA, CLL-1, CD22, HER-2, CEA, EpCAM, TAG-72, EGFRvIII, IL13Ra2, EphA2, CD44v6, NKG2D, CD133, TNFRSF17, SDC1, MS4A1, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, ERBB2, GPC3, MSLN, Mucl, PDCD1, CD274, KDR, FOLH1, FAP, CA9, FOLR1, LI CAM, ROR1, CD23, CD44, CD174, SLAMF7, GD2, PSCA, GPNMB, CD276, CD133, CSPG4, and
  • the CAR is an anti-CD 123 CAR, e.g., it can bind specifically to CD 123.
  • CD 123 also known as Cell Differentiation 123 or Interleukin-3 receptor is a receptor for IL-3.
  • the sequences of CD123 are known in the art for a variety of species, e.g., for human (see NCBI Gene ID 3563).
  • Anti-CD 123 reagents, including CAR-T cells are known in the art and include MB-102 CAR-T cells from Mustang Bio Inc., RIVA-CAR T 123, 7G3 MAb, CSL362 (taclotuzumab) MAb, and G4723A MAb. Such reagents are further described in the art at, e.g, Testa et al. Cancers 2019 11:1358; which is incorporated by reference herein in its entirety.
  • the engineered cell present in a composition, or combination, of the disclosure exhibit an increased utility that is not exhibited when the corresponding recipient cell occurs alone or when said recipient cell is present at a naturally occurring concentration.
  • the compositions of the disclosure- comprising engineered cells as taught herein— exhibit markedly different characteristics/properties compared to their closest naturally occurring counterpart, e.g., the corresponding recipient cell. That is, the compositions of the disclosure exhibit markedly different functional and/or structural characteristics/properties, as compared to their closest naturally occurring counterpart.
  • the engineered cells of the disclosure are structurally different from a recipient cell as it naturally exists in a subject, for at least the following reasons: said engineered cell comprises a cell membranecoating, said engineered cell can be isolated and purified, such that it is not found in the milieu of the subject, said engineered cell can be present at concentrations that do not occur in the subject, and said engineered cell can be associated with acceptable carriers that do not occur in the subject.
  • the engineered cells of the disclosure are functionally different from a recipient cell as it naturally exists in a subject, for at least the following reasons: said engineered cell when applied in an isolated and purified form can lead to therapeutic effects and immune evasion, said engineered cell now has a new utility as a supplement capable of administration to a subject, wherein the recipient cell could not have such a utility in its natural state in a subject.
  • An engineered cell comprising a cell membrane coating can be prepared by a method comprising: a) obtaining cell membrane components from one or more source cells; b) optionally, mixing or fusing the cell membrane components of two or more source cells; and c) contacting a recipient cell with the cell membrane components to obtain an engineered cell comprising a cell membrane coating.
  • Cell membrane components can be obtained from one or more source cells by any method known in the art that disrupts cellular membrane integrity or lyses a cell, e.g., contacting with detergents, freezing and then thawing the source cell(s) (i.e., freeze-thaw), bead milling, use of a blender, liquid homogenization, sonication, and the like.
  • the cell membrane components are obtained by freeze-thaw of the source cell(s).
  • obtaining the cell membrane components can further comprise washing or purifying the cell membrane components, e.g., affinity purification, centriguation, size filtration, extraction, etc.
  • Cell membrane components e.g., cell membrane components from two different source cell types can be mixed or fused by physical mixing, fluid flow, sonication, or any other method of mixing known in the art.
  • the mixing or fusing of the cell membrane components comprises sonication.
  • the mixing or fusing of the cell membrane components comprises fluid flow.
  • the mixing or fusing of the cell membrane components comprises sonication and fluid flow.
  • the method of preparing an engineered cell comprising a cell membrane coating can be conducted in whole or in part in one or more fluidic (e.g., microfluidic) devices.
  • the method of preparing an engineered cell comprising a cell membrane coating can be conducted in a fluidic (e.g., microfluidic) device.
  • the steps b) and c) of a method of preparing an engineered cell comprising a cell membrane coating can be conducted in whole or in part in one or more fluidic (e.g., microfluidic) devices.
  • the steps b) and c) of a method of preparing an engineered cell comprising a cell membrane coating can be conducted in a fluidic (e.g., microfluidic) device.
  • a fluidic (e.g., microfluidic) device 100 comprising, in the following order along a flow path(s): one or more initial input ports 101; a flow channel for mixing materials provided to the one or more initial input ports 102; one or more recipient cell input ports 103; a flow channel 104 comprising sonication input 105, or capable of receiving sonication input; and one or more output ports 106.
  • a flow path for mixing materials provided to the one or more initial input ports 102; one or more recipient cell input ports 103; a flow channel 104 comprising sonication input 105, or capable of receiving sonication input; and one or more output ports 106.
  • An exemplary embodiment is depicted in Fig. 2.
  • fluidic device refers to a device of any size or orientation which comprises one or more fluid channels, e,g., flow channels, and is suitable for the culture of living cells.
  • a fluidic device can be capable of moving any amount of fluid within the fluid flow ranges described herein below, e.g. a fluidic device can be a microfluidic device or a device capable of moving larger volumes of fluid.
  • the fluidic device can be an microfluidic device.
  • microfluidic as used herein relates to components where moving fluid is constrained in or directed through one or more channels wherein one or more dimensions are 1 mm or smaller (microscale).
  • Microfluidic channels may be larger than microscale in one or more directions, though the channel(s) will be on the microscale in at least one direction.
  • the geometry of a microfluidic channel may be configured to control the fluid flow rate through the channel (e.g. increase channel height to reduce shear).
  • Microfluidic channels can be formed of various geometries to facilitate a wide range of flow rates through the channels.
  • Channels are pathways (whether straight, curved, single, multiple, in a network, etc.) through a medium (e.g., silicon) that allow for movement of liquids and gasses. Channels thus can connect other components, i.e., keep components “in communication” and more particularly, “in fluidic communication” and still more particularly, “in liquid communication.” Such components include, but are not limited to, liquid-intake ports and gas vents. Microchannels are channels with dimensions less than 1 millimeter and greater than 1 micron. Channels can include capillaries, tubes, pathways, and/or grooves deposed within or upon a medium or substrate.
  • the term “port” refers to a portion of the fluidic device described herein which provides a means for fluid and/or cells to enter and/or exit the device and/or to enter and/or exit portions of the device.
  • the port can be of a size and shape to accept and/or secure a connection with tubes, connections, or adaptors of a fluidic or microfluidic device and allow passage of fluid and/or cells when attached to a fluidic or microfluidic device.
  • Fluids can be provided in a fluid source connected to an input port 101 and/or 103.
  • a fluid source can be a reservoir or other container comprising a volume of fluid such that the fluid can be caused to move from the fluid source and through the one or more channels of the fluidic device.
  • the fluid source can be coupled to the one or more channels of the fluidic device by any means of conducting a fluid, e.g. tubing, piping, channels, or the like.
  • the fluidic device and/or the fluid source can comprise ports.
  • Either positive or negative fluid pressure, or both, can be used to cause the fluid to flow through the channels 102 and/or 104.
  • the fluid in fluid source can be pressurized and a valve can be provided between the fluid source and the channel 102 and/or 104 to control the flow of fluid into the channel.
  • a vacuum source can be connected to the outlet port of the channel to draw the fluid through the channel.
  • gravity can be used to cause the fluid to flow through the channel.
  • the fluid source can be elevated above the device and the fluid collection reservoir can be placed below the device to provide fluid pressure that causes fluid to flow through the channel.
  • a valve at the fluid source or in the fluid flow path can be used to control the rate of fluid flow.
  • one or more pumps can be used cause the fluid to flow from the fluid source through the channel.
  • the method can comprise providing fluid to one or more channels by connecting the fluid channel(s) to a fluid source, e.g, a fluid source comprised by a fluidics system.
  • a device described herein can comprise a connection of a fluid channel(s) to a fluid source, e.g, a fluid source comprised by a fluidics system.
  • the connection can be via a port in the fluidic device.
  • control of the fluid flow from the fluid source through a fluid channel can be automated.
  • a syringe pump or solenoid can be used.
  • one or more computing devices or systems may be used to control fluid flow.
  • a computing device may be coupled to a fluid source or port in order to control the flow of fluid from the fluid source. Exemplary, but non-limiting, embodiments of automated devices are described in US Patent Publication US 2014/0038279; which is incorporated by reference herein its entirety.
  • the fluid flow rate through the one or more channels of the fluidic device can be from 1 to 100 mU/h. In some embodiments of any of the aspects, the fluid flow rate through the one or more channels of the fluidic device can be from 5 to 50 mU/h. In some embodiments of any of the aspects, the fluid flow rate through the one or more channels of the fluidic device can be from 2 to 75 mU/h.
  • the structures of the fluidic devices described herein can be formed, such as by etching, 3-D printing, machining, or micro-machining. In some embodiments of any of the aspects, the device described herein is etching -free.
  • the fluidic devices described herein can be made of a biocompatible flexible material or a biocompatible non-flexible material according to the design and application requirements.
  • the fluidic devices and/or portions thereof can be made of a flexible material, including but not limited to, a biocompatible material such as polydimethyl siloxane (PDMS), polyurethane or polyimide.
  • PDMS polydimethyl siloxane
  • the fluidic devices and/or portions thereof can also be made of non-flexible materials like glass, silicon, polysulfone, hard plastic, and the like, as well as combinations of these materials.
  • a biocompatible polymer refers to materials which do not have toxic or injurious effects on biological functions. Biocompatible polymers include natural or synthetic polymers.
  • biocompatible polymers include, but are not limited to, collagen, poly(alpha esters) such as poly(lactate acid), poly(glycolic acid), polyorthoesters and polyanhydrides and their copolymers, polyglycolic acid and polyglactin, cellulose ether, cellulose, cellulosic ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether, polyester, polyestercarbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefm, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether
  • a biocompatible material can also be, for example, ceramic coatings on a metallic substrate.
  • any type of coating material and the coating can be made of different types of materials: metals, ceramics, polymers, hydrogels or a combination of any of these materials.
  • Biocompatible materials include, but are not limited to an oxide, a phosphate, a carbonate, a nitride or a carbonitride.
  • the oxide the following ones are preferred: tantalum oxide, aluminum oxide, iridium oxide, zirconium oxide or titanium oxide.
  • Substrates are made of materials such as metals, ceramics, polymers or a combination of any of these.
  • Metals such as stainless steel, Nitinol, titanium, titanium alloys, or aluminum and ceramics such as zirconia, alumina, or calcium phosphate are of particular interest.
  • engineered cells comprising a cell membrane coating as described herein provide improved safety and efficacy, e.g., by immune evasion. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating a subject, the method comprising administering an engineered cell comprising a cell membrane coating as described herein to the subject.
  • a method of administering a cell to a subject comprising administering an engineered cell comprising a cell membrane coating as described herein to the subject.
  • an engineered cell comprising a cell membrane coating for use in a method of administering a cell to a subject. In one aspect of any of the embodiments, described herein is an engineered cell comprising a cell membrane coating for use in a method of treating a subject.
  • the subject has or is in need of treatment for cancer and/or the method of treatment is a method of treating cancer.
  • the cancer is a hematological cancer or hematopoietic cancer.
  • the cancer is leukemia.
  • the cancer is acute myeloid leukemia.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems.
  • Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.
  • Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
  • Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
  • the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e.. intrusion on and destruction of adjacent tissues), and metastasis (i.e. , spread to other locations in the body via lymph or blood).
  • metastasize refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre -malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • neoplasm refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues.
  • a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma
  • Transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
  • the subject is a subject at risk of cytokine release syndrome (CRS).
  • Cytokine release syndrome is a condition that may occur after treatment with some types of immunotherapy, such as monoclonal antibodies and CAR-T cells. Cytokine release syndrome is caused by a large, rapid release of cytokines into the blood from immune cells affected by the immunotherapy. Signs and symptoms of cytokine release syndrome include fever, nausea, headache, rash, rapid heartbeat, low blood pressure, and trouble breathing. Most patients have a mild reaction, but sometimes, the reaction may be severe or life threatening. Further details on CRS and risk factors for CRS can be found in the art, e.g., in Yan et al. Front.
  • the methods described herein relate to treating a subject having or diagnosed as having cancer with a composition (e.g., an engineered cell comprising a cell membrane coating) described herein.
  • a composition e.g., an engineered cell comprising a cell membrane coating
  • Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and/or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, for example, a lump/mass/tumor, swelling, fatique, swollen lymph nodes, frequent infections, or pain. Tests that may aid in a diagnosis of, e.g.
  • cancers include, but are not limited to, x- rays, MRI, ultrasound, a biopsy, or tests for the function/activity of affected organs or systems.
  • a family history of cancer or exposure to risk factors for cancer e.g. smoke, radiation, pollutants, mutation, etc. can increase the risk of a subject having cancer.
  • compositions and methods described herein can be administered to a subject having or diagnosed as having cancer.
  • the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of a cancer.
  • "alleviating a symptom” of a cancer is ameliorating any condition or symptom associated with the cancer, e.g, a reduction in cancer cell growth or numbers. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • a variety of means for administering the compositions described herein to subjects are known to those of skill in the art.
  • Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration.
  • Administration can be local or systemic. In some embodiments of any of the apsects, the administration is subcutaneous.
  • the term “effective amount” as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the term "therapeutically effective amount” therefore refers to an amount of a composition that is sufficient to provide a particular therapeutic effect when administered to a typical subject.
  • An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e..
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for cancer cell growth, among others.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the minimal effective dose and/or maximal tolerated dose.
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a dosage range between the minimal effective dose and the maximal tolerated dose.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for cancer growth and/or size among others.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the technology described herein relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an engineered cell comprising a cell membrane coating as described herein, and optionally a pharmaceutically acceptable carrier.
  • the active ingredients of the pharmaceutical composition comprise an engineered cell comprising a cell membrane coating as described herein.
  • the active ingredients of the pharmaceutical composition consist essentially of an engineered cell comprising a cell membrane coating as described herein.
  • the active ingredients of the pharmaceutical composition consist of an engineered cell comprising a cell membrane coating as described herein.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media.
  • the use of such carriers and diluents is well known in the art.
  • Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean
  • polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non toxic compatible substances employed in pharmaceutical formulations.
  • PEG polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • agar such as a
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent, e.g. an engineered cell comprising a cell membrane coating as described herein.
  • the pharmaceutical composition as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS ® -type dosage forms and dose-dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms of compositions as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a molecule as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-re lease
  • compositions comprising an engineered cell comprising a cell membrane coating can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non- aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion.
  • Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally,
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • the a system, or cell comprising or encoding a system can be administered in a sustained release formulation.
  • Controlled-release pharmaceutical products have a common goal of improving therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled- release formulations include: 1) extended activity of the therapeutic; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total therapeutic; 5) reduction in local or systemic side effects; 6) minimization of therapeutic accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of therapeutic activity; and 10) improvement in speed of control of diseases or conditions.
  • Controlled-release formulations are designed to initially release an amount of a therapeutic (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of therapeutic to maintain this level of therapeutic or prophylactic effect over an extended period of time.
  • the therapeutic In order to maintain this constant level of therapeutic in the body, the therapeutic must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.
  • Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each of which is incorporated herein by reference.
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
  • active ingredients for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
  • OROS ® Alza Corporation, Mountain View, Calif. USA
  • the engineered cell comprising a cell membrane coating described herein is administered as a monotherapy, e.g., another treatment for the condition (e.g., cancer) is not administered to the subject.
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
  • a second agent and/or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemel
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin),
  • vinorelbine novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)
  • irinotecan including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylomithine
  • retinoids such as retinoic acid
  • capecitabine combretastatin
  • LV leucovorin
  • oxaliplatin including the oxaliplatin treatment
  • the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
  • an effective dose of an engineered cell comprising a cell membrane coating as described herein can be administered to a patient once.
  • an effective dose of a composition comprising an engineered cell comprising a cell membrane coating can be administered to a patient repeatedly.
  • subjects can be administered a therapeutic amount of a composition comprising a system, or cell comprising or encoding a system, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
  • Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. cancer cell growth by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active ingredient.
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
  • dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
  • a composition comprising a system, or cell comprising or encoding a system can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the dosage ranges for the administration an engineered cell comprising a cell membrane coating according to the methods described herein depend upon, for example, the form of an engineered cell comprising a cell membrane coating, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for cancer cell growth.
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • an engineered cell comprising a cell membrane coating in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. cancer) can be determined by the skilled clinician.
  • a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. cancer cell growth. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. a reduction in cancer cell growth). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. cancer cell growth.
  • kits for preparing an engineered cell comprising a cell membrane coating and/or treating a subject (e.g., a subject in need of cell therapy), among others.
  • kit components that can be included in one or more of the kits described herein.
  • the kit comprises an engineered cell comprising a cell membrane coating.
  • the cell can be supplied in a lyophilized form or a concentrated form that can diluted or suspended in liquid prior to use, e.g., with cells.
  • Preferred formulations include those that are non-toxic to the cells and/or does not affect growth rate or viability and can be supplied in aliquots or in unit doses.
  • the kit comprises microfluidic device as described herein.
  • kits can be provided singularly or in any combination as a kit.
  • a kit includes the components described herein, e.g., an engineered cell, a microfluidic device, a source cell, cell membrane components, and/or a recipient cell.
  • the compositions in the kit can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit.
  • a composition can be supplied in more than one container, e.g., it can be supplied in a container having sufficient reagent for a predetermined number of cell culture events, e.g., 1, 2, 3 or greater.
  • One or more components as described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the components described herein are substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • the kit optionally comprises informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of the polypeptide or system, concentration, date of expiration, batch or production site information, and so forth.
  • the informational material relates to methods for using or administering the components of the kit.
  • the kit can be provided with its various elements included in one package, e.g., a fiber- based, e.g., a cardboard, or polymeric, e.g., a Styrofoam box.
  • the enclosure can be configured so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time.
  • the present invention relates to the herein described compositions, methods, and respective componcnt(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising).
  • other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.
  • compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.
  • the absence of a given treatment or agent can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a cell or biomolecule(s) “obtained from” a specified organism refers to a cell or biomolecule(s) isolated by physical or chemical means from that organism, and the progeny of that originally isolated cell which retain the characteristics of that cell.
  • a cell or biomolecule(s) “derived from” a specified organism is descended from a cell or biomolecule(s) obtained from the specified organism but which as undergone changes ex vivo, e.g, genetic engineering, differentiation, chemical modification, or dedifferentiation. Accordingly, the cell or biomolecule(s) “derived” from a specified organism can be identified as having key genetic or phenotype characteristics of the source species but many have alterations or additions not found in the source species naturally.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition Alternatively, a subject can also be one who has not been previously diagnosed as having a condition or one or more complications related to the condition.
  • a condition in need of treatment e.g. cancer
  • a subject can also be one who has not been previously diagnosed as having a condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA.
  • a polypeptide, nucleic acid, or cell as described herein can be engineered.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be “engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.
  • progeny of an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • the engineered cell and/or the cell membrane coating described herein is exogenous. In some embodiments of any of the aspects, the engineered cell and/or the cell membrane coating described herein described herein is ectopic. In some embodiments of any of the aspects, the engineered cell and/or the cell membrane coating described herein described herein is not endogenous.
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • exogenous can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • endogenous refers to a substance that is native to the biological system or cell.
  • ectopic refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (/. e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • prophylactic refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom.
  • Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to CRS. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
  • the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine.
  • activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • statically significant or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • specific binding refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.
  • a sample is taken, obtained, or provided via minimally invasive methods and/or involves only a minor intervention.
  • a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and/or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc), fecal sampling, semen/seminal fluid sampling, or clippings (e.g., of hair or nails).
  • a secreted or expelled bodily fluid e.g., mucus, urine, sweat, etc
  • fecal sampling e.g., semen/seminal fluid sampling
  • clippings e.g., of hair or nails.
  • the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and/or hair or nail clippings.
  • the present technology may be defined in any of the following numbered paragraphs:
  • An engineered cell comprising a cell membrane coating on the surface of the cell, the cell membrane coating comprising cell membrane components of one or more source cells.
  • the one or more source cells comprise a T cell (e.g., a CAR-T cell), a diseased cell, and/or a macrophage (e.g., a THP-1 macrophage).
  • a T cell e.g., a CAR-T cell
  • a diseased cell e.g., a CAR-T cell
  • a macrophage e.g., a THP-1 macrophage
  • AML acute myeloid leukemia
  • the cell membrane components include one or more of: a lipid, a lipid bilayer, transmembrane proteins, membrane-displayed biomolecules, and cell surface proteins.
  • the cell membrane coating comprises a mixture, fusion, or hybridization of cell membrane components from two or more source cells.
  • each source cell each further comprise a detectable label, wherein each detectable label is distinguishable from the detectable labels comprised by the cell membrane components obtained from the other source cells.
  • a method of producing an engineered cell comprising a cell membrane coating comprising: a. Obtaining cell membrane components from one or more source cells; b. Optionally, mixing or fusing the cell membrane components of two or more source cells; and c. Contacting a recipient cell with the cell membrane components to obtain and engineered cell comprising a cell membrane coating. 22. The method of paragraph 21, wherein the cell membrane components are obtained by freeze-thaw of the source cells.
  • a microfluididic device comprising, in the following order along a flow path(s): a. One or more initial input ports; b. A flow channel for mixing materials provided to the one or more initial input ports; c. One or more recipient cell input ports; d. A flow channel comprising sonication input, or capable of receiving sonication input; and e. One or more output ports.
  • a method of treating a subject comprising administering an engineered cell of any of paragraphs 1-20 to the subject.
  • the present technology may be defined in any of the following numbered paragraphs:
  • An engineered cell comprising a cell membrane coating on the surface of the cell, the cell membrane coating comprising cell membrane components of one or more source cells.
  • the one or more source cells comprise a T cell, a diseased cell, and/or a macrophage.
  • the diseased cell is a cancer cell.
  • the cancer cell is a leukemia cell or an acute myeloid leukemia (AML) cell.
  • AML acute myeloid leukemia
  • the macrophage is a THP-1 macrophage.
  • the cell membrane components include one or more of: a lipid; a lipid bilayer; transmembrane proteins; membrane-displayed biomolecules; and cell surface proteins.
  • the cell membrane coating comprises a mixture, fusion, or hybridization of cell membrane components from two or more source cells.
  • the engineered cell of paragraph 11 wherein the mixture, fusion, or hybridization of cell membrane components has an equal amount of cell membrane components from each source cell.
  • the engineered cell of any of the preceding paragraphs, wherein the cell membrane components obtained from each source cell further comprises a detectable label.
  • the engineered cell of any of the preceding paragraphs, wherein the cell membrane components obtained from each source cell each further comprise a detectable label, wherein each detectable label is distinguishable from the detectable labels comprised by the cell membrane components obtained from the other source cells.
  • the engineered cell of any of the preceding paragraphs, wherein the engineered cell is a T cell.
  • the engineered cell of paragraph 18, wherein the T cell is a CD8+ T cell.
  • the engineered cell of paragraph 18, wherein the T cell is a CAR-T cell.
  • the engineered cell of paragraph 20, wherein the CAR-T cell comprises a CAR specific for or active against the diseased cell.
  • the engineered cell of any of the preceding paragraphs, wherein the one or more source cells and/or the engineered cell are obtained from a subject.
  • the engineered cell of any of the preceding paragraphs, wherein the engineered cell is not autologous to a subject. 25.
  • the engineered cell of any of the preceding paragraphs, wherein the engineered cell is autologous to a subject.
  • a method of producing an engineered cell comprising a cell membrane coating comprising: a) obtaining cell membrane components from one or more source cells; b) optionally, mixing or fusing the cell membrane components of two or more source cells; and c) contacting a recipient cell with the cell membrane components to obtain an engineered cell comprising a cell membrane coating.
  • a microfluididic device comprising, in the following order along a flow path(s): a) one or more initial input ports; b) a flow channel for mixing materials provided to the one or more initial input ports; c) one or more recipient cell input ports; d) a flow channel comprising sonication input, or capable of receiving sonication input; and e) one or more output ports.
  • a method of treating a subject comprising administering an engineered cell of any of paragraphs 1-25 to the subject.
  • AML Acute myeloid leukemia
  • CAR chimeric antigen receptor
  • CRS cytokine release syndrome
  • CRS is characterized by an abnormal elevation of serum cytokines, such as interferon-g (IFNy), interleukin-6 (IL-6), IL-8, and IL-10, and elevated inflammatory responses following CAR T cell infusion.
  • IFNy interferon-g
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • IL-10 elevated inflammatory responses following CAR T cell infusion.
  • cell membrane coating and microfluidic technologies To overcome the fundamental limitations of CAR T cell immunotherapy for AML treatment and conventional nanomedicine, described herein is the use of two emerging nanomedicine formulation approaches: cell membrane coating and microfluidic technologies. Unlike existing formulation methods that rely primarily on synthetic materials and complicated chemical processing, the cell membrane coating technique, which so far has been used to modify nanoparticles 14,15 and bacteria, 16 relies on natural materials and simple processing steps. Based on this strategy, theranostic agents are coated with natural cellular components, which endow these agents with the biological properties and functionalities similar to the source cells from which the membrane is derived, such as homotypic targeting, cytokine binding and isolation, immune evasion, and extended in vivo residence time.
  • cell membrane coating technology enables the use of cellular materials derived from patients, essentially personalizing patient treatments and minimizing excessive immune response.
  • microfluidic platforms provide a highly controlled approach to process cell membranes and construct membrane-coated theranostic agents, significantly simplifying the formulation process, shortening the formulation time, and increasing product volume, quality, and reproducibility. Therefore, it is contemplated herein that the efficacy and safety of AML therapy can be significantly improved by adopting cell membrane coating and microfluidic technologies to formulate personalized cellular theranostic agents capable of binding specifically to AML cells, destroying these malignant cells, and removing excessive serum cytokines from the body.
  • Described herein is the construction of a high-throughput microfluidic platform which permits the hybridization of, e.g., three different patient-derived cell membranes and the coating of the hybridized membrane onto cytotoxic T cells, to generate multifunctional personalized cellular agents for combinatorial AML theranostics.
  • the “one-for-all” cellular agents have several novel features: 1) hybridized membrane from CAR T cell membrane (for homing to AML cells), AML cell membrane (for highly specific homotypic binding with AML cells, essentially eliminating the need for a specific target antigen and minimizing off-target effect), and macrophage membrane (for cytokine absorption, improved tumor accumulation, and prolonged in vivo circulation lifetime) to realize triple-targeting capability, and 2) T cells camouflaged within the hybridized cell membrane, which will be labeled with a probe for realizing combinatorial immuno- and phototherapies, visualizing treatment response, and locating residual and metastatic leukemic cells.
  • the work described herein is illustrated in Figs. 1A-1C.
  • Microfluidic devices for membrane hybridization and cellular agent synthesis will be fabricated using soft lithography. Flow rates within the microfluidic devices will be simulated and optimized in COMSOL to realize a uniform mixing and fusion of different membranes.
  • CD8+ T cells will be isolated from the peripheral blood mononuclear cells.
  • CAR T cells targeting CD 123+ AML cells which are currently being actively explored for phase I/II clinical trials, will be generated through lentiviral transduction and characterized for their surface expression, specificity, and cytotoxicity.
  • the membrane of these CAR T cells, along with those of KGla AML cells and THP-1 macrophages, will be obtained through a freeze-thaw method.
  • Each membrane will be labeled with a distinct fluorescent dye (i.e., DiD, Dil, and DiO) for downstream characterization.
  • DiD DiD, Dil, and DiO
  • the different cell membranes at a ratio of 1 : 1 : 1 (based on membrane protein concentrations) will be mixed and fused within the microfluidic device at optimized flow rates under sonication to produce hybridized membrane, which will then be used to coat the CD8+ T cells within the microfluidic device.
  • theranostic agents will be synthesized to incorporate indocyanine green (ICG) dye, in place of DiD, Dil, and DiO dyes, within the hybridized membrane.
  • ICG indocyanine green
  • the FDA-approved ICG will serve as a near-infrared (NIR) fluorescence imaging probe, photothermal agent, and photosensitizer.
  • NIR near-infrared
  • the inclusion of ICG is expected to confer the cellular agents combinatorial theranostic modalities.
  • the efficiency of photothermal conversion and reactive oxygen species generation of the cellular agents over time will be characterized under NIR laser excitation.
  • the specific targeting of the cellular agents will be confirmed by using confocal fluorescence microscopy to image agent co-localization with different fluorescently-labeled co cultured cells, particularly KG la AML cells, LCL lymphoblastoid cells, BMEC-1 bone marrow endothelial cells, and THP-1 macrophages.
  • the biocompatibility and efficacy of immuno-, photothermal, and photodynamic therapies of the cellular agents will be evaluated based on cellular morphological changes, cell proliferation assay, and apoptosis/necrosis assay, in the absence (as a control) and presence of the cellular agents and NIR laser excitation.
  • the secretion of IFNy and TNFa after cell co-cultures will be characterized using flow cytometry to investigate the activation of effector pathways against AML cells.
  • Cytokine binding will be examined by incubating the cellular agents with various cytokines typically elevated in patient serum following CAR T cell administration (i.e., IFNy, IL-6, IL-8, and IL-10) over time. Each agent- cytokine mixture will then be centrifuged and the cytokine concentration in the supernatant will be quantified using ELISA. The bound cytokines will be estimated from the difference between the initial cytokine concentration and the supernatant concentration. Cytokine solutions in the absence of cellular agents will be used as a control.
  • a xenogeneic mouse model of systemic AML will first be established using a KG la leukemic cell line (KGla-ffLuc-eGFP cells) and confirmed through the detection of leukemia in the peripheral blood after several days.
  • the tumor progression will be tracked using bioluminescence and fluorescence imaging and flow cytometry of peripheral blood at various time points.
  • the circulation lifetime of the cellular agents will be estimated from the fluorescence measurement of the blood collected at different time points post agent injection.
  • the biodistribution of the cellular agents will be evaluated in vivo through fluorescence imaging and ex vivo from the fluorescence of the tissues of the main organs (i.e., heart, lung, liver, spleen, and kidney) of the AML and control mice sacrificed 48 h post agent injection.
  • the toxicity of the cellular agents will be assessed through histopathologic examination of the tissues stained with hematoxylin and eosin.
  • Personalized cellular agents will first be prepared using AML cells, T cells, and macrophages extracted from the AML mice. Following physical property characterization, subsequent study using the personalized cellular agents will be performed similarly to what is described above. To investigate the potential of the personalized cellular agents in mitigating AML recurrence and metastasis, the treated AML mice will be re-challenged with AML cells, followed by the intravenous injection of cellular agents and evaluation of their antileukemic activity. All statistical analysis in this proposal will be performed using student’s t-test.
  • the novelty of the methods and compositions described herein includes: 1) tailorable and high-throughput hybridization of three different types of cell membrane through microfluidics, 2) engineering of immune cells through membrane camouflaging, and 3) simultaneous enhanced AML targeting, minimized CRS toxicity, and AML combinatorial theranostics within a single platform. These features have not been previously demonstrated. It is expected that the establishment of the current platform, which relies entirely on patient-derived and FDA-approved materials, will catalyze a clinically and commercially translatable technology to address the huge unmet need in AML immunotherapy to improve patient treatment response and survival. Furthermore, due to its customizability and controllability, the present approach can be easily adopted for a wide range of cell types to improve the immunotherapy and combinatorial therapies of other liquid and solid malignancies.
  • Gold (Au) nanostructures (nanonuggets (NN) or nanostars (NS)) were coated with macrophage membrane vesiscles via microfluidic-mediated physical mixing (Figs. 4A-4D).
  • the size, surface charge, protein expression, hydrodynamic size, and zeta potential of the coated nanostructures were analyzed (Figs. 5-9).
  • the coated nanostructures were taken up by cancer cells (Fig. 10A-10C) and demonstrated increased immune evasion abilities (Figs. 1 lA-11C).
  • Gold (Au) nanostructures (nanonuggets (NN) or nanostars (NS)) were coated with cancer cell membrane vesiscles via microfluidic -mediated physical mixing (Figs. 12A-12D). The size, surface charge, protein expression, hydrodynamic size, and zeta potential of the coated nanostructures were analyzed (Figs. 13-17).

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Abstract

La technologie décrite ici concerne des compositions et des procédés relatifs à des cellules modifiées comprenant un revêtement de membrane cellulaire.
PCT/US2022/018833 2021-03-05 2022-03-04 Procédés et compositions se rapportant à l'hybridation et le camouflage de la membrane cellulaire WO2022187573A1 (fr)

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