WO2021003425A1 - Procédés d'induction de l'hématopoïèse - Google Patents

Procédés d'induction de l'hématopoïèse Download PDF

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WO2021003425A1
WO2021003425A1 PCT/US2020/040746 US2020040746W WO2021003425A1 WO 2021003425 A1 WO2021003425 A1 WO 2021003425A1 US 2020040746 W US2020040746 W US 2020040746W WO 2021003425 A1 WO2021003425 A1 WO 2021003425A1
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seq
aspects
scaffold
protein
cell
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PCT/US2020/040746
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Shelly Ann MARTIN
Christine MCCOY
Ke Xu
Nuruddeen LEWIS
Raymond MONIZ
Sriram Sathyanarayanan
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Codiak Biosciences, Inc.
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Publication of WO2021003425A1 publication Critical patent/WO2021003425A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes

Definitions

  • the present disclosure relates to modified extracellular vesicles, e.g. , exosomes, (e.g, comprising an FMS-like tyrosine kinase 3 ligand (FLT3L) polypeptide) that can be used to induce hematopoiesis in a subject.
  • exosomes e.g., comprising an FMS-like tyrosine kinase 3 ligand (FLT3L) polypeptide
  • FLT3L FMS-like tyrosine kinase 3 ligand
  • FLT3/FLT3L signaling stimulates dendritic cell (DC) proliferation and differentiation further strengthening an immune response.
  • DC dendritic cell
  • traditional means of administering recombinant FLT3L require extensive and frequent dosing due to the short half-life of recombinant FLT3L.
  • EVs, exosomes are important mediators of intercellular communication. They are also important biomarkers in the diagnosis and prognosis of many diseases.
  • drug delivery vehicles e.g ., exosomes offer many advantages over traditional drug delivery methods (e.g. , peptide immunization, DNA vaccines) as a new treatment modality in many therapeutic areas.
  • Certain aspects of the present disclosure are directed to a method of inducing hematopoiesis in a subject in need thereof comprising administering an extracellular vesicle (EV) comprising an FMS-like tyrosine kinase 3 ligand (FLT3L) polypeptide.
  • EV extracellular vesicle
  • FLT3L FMS-like tyrosine kinase 3 ligand
  • the subject exhibits increased proliferation of one or more myeloid progenitor cells or one or more lymphoid progenitor cells relative to the proliferation of the one or more myeloid progenitor cells or the one or more lymphoid progenitor cells prior to the administration.
  • the subject exhibits increased proliferation of one or more myeloid cells selected from a megakaryocyte, an erythrocyte, a mast cell, a myeloblast cell, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, and any combination thereof relative to the proliferation of the one or more myeloid cells prior to the administration.
  • myeloid cells selected from a megakaryocyte, an erythrocyte, a mast cell, a myeloblast cell, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, and any combination thereof relative to the proliferation of the one or more myeloid cells prior to the administration.
  • the subject exhibits increased macrophage proliferation relative to the macrophage proliferation prior to the administration.
  • the subject exhibits increased proliferation of one or more lymphoid cells selected from a natural killer cell, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasma cell, and any combination thereof relative to the proliferation of the one or more lymphoid cells prior to the administration.
  • the subject exhibits increased dendritic cell proliferation relative to the dendritic cell proliferation prior to the administration.
  • the subject exhibits increased differentiation of one or more myeloid progenitor cells or one or more lymphoid progenitor cells relative to the differentiation of the one or more myeloid progenitor cells or the one or more lymphoid progenitor cells prior to the administration.
  • the subject exhibits increased differentiation of one or more myeloid cells selected from a megakaryocyte, an erythrocyte, a mast cell, a myeloblast cell, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, and any combination thereof relative to the differentiation of the one or more myeloid cells prior to the administration.
  • the subject exhibits increased macrophage differentiation relative to the macrophage differentiation prior to the administration.
  • the subject exhibits increased differentiation of one or more lymphoid cells selected from a natural killer cell, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasma cell, and any combination thereof relative to the differentiation of the one or more lymphoid cells prior to the administration.
  • the subject exhibits increased dendritic cell differentiation relative to the dendritic cell differentiation prior to the administration.
  • the subject has a tumor comprising one or more immune cells, wherein the subject exhibits an increase in the number of the one or more immune cells in the tumor after the administration relative to the number of the one or more immune cells in the tumor prior to the administration.
  • Certain aspects of the present disclosure are directed to an extracellular vesicle (EV) comprising an exogenous FMS-like tyrosine kinase 3 ligand (FLT3L) polypeptide.
  • EV extracellular vesicle
  • FLT3L FMS-like tyrosine kinase 3 ligand
  • the EV induces hematopoiesis in a subject in need thereof.
  • the EV induces proliferation of one or more myeloid progenitor cells or one or more lymphoid progenitor cells.
  • the EV induces proliferation of one or more myeloid cells selected from a megakaryocyte, an erythrocyte, a mast cell, a myeloblast cell, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, and any combination. In some aspects, the EV induces macrophage proliferation.
  • the EV induces proliferation of one or more lymphoid cells selected from a natural killer cell, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasma cell, and any combination thereof. In some aspects, the EV induces dendritic cell proliferation.
  • the EV induces differentiation of one or more myeloid progenitor cells or one or more lymphoid progenitor cells. In some aspects, the EV induces differentiation of one or more myeloid cells selected from a megakaryocyte, an erythrocyte, a mast cell, a myeloblast cell, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, and any combination thereof. In some aspects, the EV induces macrophage differentiation.
  • the EV induces differentiation of one or more lymphoid cells selected from a natural killer cell, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasma cell, and any combination thereof. In some aspects, the EV induces dendritic cell differentiation.
  • the EV further comprises a scaffold moiety.
  • the FLT3L polypeptide is linked to the scaffold moiety.
  • the scaffold moiety is a Scaffold X.
  • the scaffold moiety is a Scaffold Y.
  • the Scaffold X is a scaffold protein that is capable of anchoring the FLT3L polypeptide on the luminal surface of the EV and/or on the exterior surface of the EV.
  • the Scaffold X is selected from the group consisting of prostaglandin F2 receptor negative regulator (the PTGFRN protein); basigin (the BSG protein); immunoglobulin superfamily member 2 (the IGSF2 protein); immunoglobulin superfamily member 3 (the IGSF3 protein); immunoglobulin superfamily member 8 (the IGSF8 protein); integrin beta-1 (the ITGB1 protein); integrin alpha-4 (the ITGA4 protein); 4F2 cell-surface antigen heavy chain (the SLC3A2 protein); a class of ATP transporter proteins (the ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3, ATP2B1, ATP2B2, ATP2B3, ATP2B4 proteins); a functional fragment thereof; and any combination thereof.
  • the scaffold moiety is PTGFRN protein or a functional fragment thereof.
  • the scaffold moiety comprises an amino acid sequence as set forth in SEQ ID NO: 33.
  • the scaffold moiety comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% identical to SEQ ID NO: 1.
  • the Scaffold Y is a scaffold protein that is capable of anchoring the FLT3L polypeptide on the luminal surface of the EV and/or on the exterior surface of the EV.
  • the Scaffold Y is selected from the group consisting of myristoylated alanine rich Protein Kinase C substrate (the MARCKS protein), myristoylated alanine rich Protein Kinase C substrate like 1 (the MARCKSLl protein), brain acid soluble protein 1 (the BASP1 protein), a functional fragment thereof, and any combination thereof.
  • the Scaffold Y is a BASP1 protein or a functional fragment thereof.
  • the Scaffold Y comprises an N terminus domain (ND) and an effector domain (ED), wherein the ND and/or the ED are associated with the luminal surface of the EV.
  • the ND is associated with the luminal surface of the exosome via myristoylation.
  • the ED is associated with the luminal surface of the exosome by an ionic interaction.
  • the ED comprises (i) a basic amino acid or (ii) two or more basic amino acids in sequence, wherein the basic amino acid is selected from the group consisting of Lys, Arg, His, and any combination thereof.
  • the basic amino acid is (Lys)n, wherein n is an integer between 1 and 10.
  • the ED comprises Lys (K), KK, KKK, KKKK (SEQ ID NO: 205), KKKKK (SEQ ID NO: 206), Arg (R), RR, RRR, RRRR (SEQ ID NO: 207); RRRRR (SEQ ID NO: 208), KR, RK, KKR, KRK, RKK, KRR, RRK, (K/R)(K/R)(K/R) (SEQ ID NO: 209), (K/R)(K/R)(K/R)(K/R)(K/R) (SEQ ID NO: 210), or any combination thereof.
  • the ND comprises the amino acid sequence as set forth in G:X2:X3:X4:X5:X6, wherein G represents Gly; wherein represents a peptide bond, wherein each of the X2 to the X6 is independently an amino acid, and wherein the X6 comprises a basic amino acid.
  • the X2 is selected from the group consisting of Pro, Gly, Ala, and Ser;
  • the X4 is selected from the group consisting of Pro, Gly, Ala, Ser, Val, lie, Leu, Phe, Trp, Tyr, Gin and Met;
  • the X5 is selected from the group consisting of Pro, Gly, Ala, and Ser;
  • the X6 is selected from the group consisting of Lys, Arg, and His; or (v) any combination of (i)-(iv).
  • the ND comprises the amino acid sequence of
  • the ND and the ED are joined by a linker.
  • the linker comprises one or more amino acids.
  • the ND comprises an amino acid sequence selected from the group consisting of (i) GGKLSKK (SEQ ID NO: 211), (ii) GAKLSKK (SEQ ID NO: 212), (iii) GGKQSKK (SEQ ID NO: 213), (iv) GGKLAKK (SEQ ID NO: 214), (v) GGKLSKK (SEQ ID NO: 211), or (vi) any combination thereof.
  • the ND comprises an amino acid sequence selected from the group consisting of (i) GGKLSKKK (SEQ ID NO: 238), (ii) GGKLSKK S (SEQ ID NO: 239), (iii) GAKLSKKK (SEQ ID NO: 240), (iv) GAKLSKK S (SEQ ID NO: 241), (v) GGKQSKKK (SEQ ID NO: 242), (vi) GGKQSKK S (SEQ ID NO: 243), (vii) GGKLAKKK (SEQ ID NO: 244), (viii) GGKLAKK S (SEQ ID NO: 245), (ix) GGKLSKKK (SEQ ID NO: 238), (x) GGKLSKKS (SEQ ID NO: 239), and (xi) any combination thereof.
  • the ND comprises the amino acid sequence GGKLSKK (SEQ ID NO: 211).
  • the Scaffold Y is at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, 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 100, at least about 105, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 amino acids in length.
  • the Scaffold Y comprises (i) GGKLSKKKKGYNVN (SEQ ID NO: 246), (ii) GAKL SKKKKGYNVN (SEQ ID NO: 247), (iii) GGKQ SKKKKGYNVN (SEQ ID NO: 248), (iv) GGKLAKKKKGYNVN (SEQ ID NO: 249), (v) GGKL SKKKKGY S GG (SEQ ID NO: 250), (vi) GGKLSKKKKGSGGS (SEQ ID NO: 251), (vii) GGKL SKKKK S GGS G (SEQ ID NO: 252), (viii) GGKLSKKKSGGSGG (SEQ ID NO: 253), (ix)
  • GGKL SKK S GGS GGS (SEQ ID NO: 254), (x) GGKL SKS GGS GGS V (SEQ ID NO: 255), or (xi) GAKK SKKRF SFKK S (SEQ ID NO: 256).
  • the Scaffold Y consists of (i) GGKLSKKKKGYNVN (SEQ ID NO: 247), (ii) GAKL SKKKKGYNVN (SEQ ID NO: 247), (iii) GGKQ SKKKKGYNVN (SEQ ID NO: 248), (iv) GGKLAKKKKGYNVN (SEQ ID NO: 249), (v) GGKL SKKKKGY S GG (SEQ ID NO: 250), (vi) GGKLSKKKKGSGGS (SEQ ID NO: 251), (vii) GGKL SKKKK S GGS G (SEQ ID NO: 252), (viii) GGKLSKKKSGGSGG (SEQ ID NO: 253), (ix)
  • GGKL SKK S GGS GGS (SEQ ID NO: 254), (x) GGKL SKS GGS GGS V (SEQ ID NO: 255), or (xi) GAKK SKKRF SFKK S (SEQ ID NO: 256).
  • the Scaffold Y does not comprise Met at the N terminus. In some aspects, the Scaffold Y comprises a myristoylated amino acid residue at the N terminus of the scaffold protein. In some aspects, the amino acid residue at the N terminus of the Scaffold Y is Gly.
  • the FLT3L polypeptide is linked to the scaffold moiety on the exterior surface of the EV. In some aspects, the FLT3L polypeptide is linked to a scaffold moiety on the luminal surface of the EV. In some aspects, the FLT3L polypeptide is linked to the scaffold moiety by a linker. In some aspects, the linker is a polypeptide. In some aspects, the linker is a non-polypeptide moiety. [0032] In some aspects, the EV is an exosome.
  • Certain aspects of the present disclosure are directed to a pharmaceutical composition
  • a pharmaceutical composition comprising an EV disclosed herein and a pharmaceutically acceptable carrier.
  • Certain aspects of the present disclosure are directed to a cell that produces an EV disclosed herein.
  • Certain aspects of the present disclosure are directed to a cell comprising one or more vectors, wherein the one or more vectors comprise a nucleic acid sequence encoding a FLT3L polypeptide disclosed herein.
  • kits comprising an EV disclosed herein and instructions for use.
  • Certain aspects of the present disclosure are directed to a method of making EVs comprising culturing a cell disclosed herein under a suitable condition and obtaining the EVs.
  • Certain aspects of the present disclosure are directed to a method of inducing hematopoiesis in a subject in need thereof comprising administering an EV disclosed herein to the subject.
  • the EV is administered parenterally, orally, intravenously, intramuscularly, intra-tumorally, intranasally, subcutaneously, or intraperitoneally.
  • the method comprises administering an additional therapeutic agent.
  • FIGs. 1A-1B are drawings of various FLT3L fusion constructs.
  • FIG. 1A shows the pCB-1001 and pCB-1082 protein constructs.
  • pCB-1001 comprises a FLT3L monomer of fused to full length protein X (PrX)
  • pCB-1082 comprises a forced dimer of FLT3L fused to full length PrX.
  • FIG. IB shows two engineered FLT3LG fusion constructs (1251 and 1252) with CD81, which preserve FLT3LG's natural configuration.
  • FIGs. 2A-2B shows western blot confirmation of the expected monomer and dimer sizes of the various constructs illustrated in FIGs. 1 A-1B.
  • FIG. 3 shows an ELISA quantitation of the amount (ng) of hFLT3L per 1 x 10 10 exosomes in a tested preparation.
  • FIGs. 4A-4B are graphical representation of two in-vitro analyses of phosphorylated ERK (pERK) when treated with the indicated engineered FLT3L constructs, using unstimulated cells and free rhFLT3L as controls.
  • FIG. 5 is a graphical representation of FLT3L receptor binding distance, as measured by various sensors, of the engineered exosome constructs using recombinant human Flt-3/Flk-2 Fc chimera protein on protein A biosensors, with 1 x 10 11 particles per well.
  • FIGs. 6A-6B are graphical representations of the results of normalized phosphorylated ERK (pERK, normalized to the rhFLT3L values) as measured via pERK/THP-1 assay.
  • FIGs. 6C-6D are graphical representations of the results of CD45 + IA/IE + % Parent (Live CD45 + IA/IE + F4/80 ) count as measured via the XCR1 + CD1 lc + assay.
  • FIGs. 7A-7B show the 50% effective concentration (EC50) for both measured values in both the pERK/THP-1 assay and the XCR1 + CD1 lc + (FIG. 7A) and the signal to noise ratio as measured using the pERK/THP-1 assay and the XCR1 + CD1 lc + assays (FIG. 7B).
  • FIGs. 8A-8D are graphical representations of the results of an XCR1 + CDl lc + assay of the 1001 and 1082 FLT3L constructs as compared to unstimulated cells, native exosomes, and recombinant human FLT3L, as indicated.
  • FIGs. 8A-8B show the Live CD45+ Cell values as measured by the assay with respect to rhFLT3L concentration (ng/mL)
  • FIGs. 8C-8D show the F4/80 + values as measured by the assay with respect to particle concentration (particles/mL).
  • the present disclosure is directed to methods of inducing hematopoiesis in a subject in need thereof, comprising administering an EV, e.g ., exosome, comprising a FLT3L polypeptide, to the subject.
  • an EV e.g ., exosome
  • the FLT3L polypeptide is attached (or linked) to one or more scaffold moieties on the surface of the EV, e.g. , exosome, or on the luminal surface of the EV, e.g. , exosome.
  • a or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the exosomes described herein comprise exogenous FLT3L.
  • FLT3L refers to a single pass type-1 transmembrane domain or soluble fragment thereof that binds and activates the tyrosine kinase receptor (FLT3, CD135).
  • FLT3 is expressed on hematopoietic stem cells (HSC), early progenitor cells (e.g ., hematopoietic stem cells (HSCs) and hematopoietic progenitor cells), immature thymocytes, and steady state dendritic cells (DCs).
  • HSC hematopoietic stem cells
  • HSCs early progenitor cells
  • DCs steady state dendritic cells
  • FLT3L Activation of FLT3 by FLT3L results in proliferation, differentiation, development, and mobilization of these cells in the bone marrow, peripheral blood, and lymphoid organs.
  • administration of FLT3L to a subject can enhance an immune response by promoting the stability and expansion of immune cells.
  • Recombinant FLT3L proteins are available, including CDX-301, a soluble human recombinant FLT3L protein marketed by CELLDEX THERAPEUTICS. See Anandasabapathy et al., Bone Marrow Transplantation 50:924-930 (2015).
  • recombinant FLT3L administration has been shown to increase sustained levels of hematopoietic progenitor cells (CD34+ and colon-forming cells) when administered as part of an autologous hematopoietic stem cell (HSC) transplantation therapy.
  • HSC autologous hematopoietic stem cell
  • extracellular vesicle refers to a cell-derived vesicle comprising a membrane that encloses an internal space.
  • Extracellular vesicles comprise all membrane-bound vesicles (e.g., exosomes, nanovesicles) that have a smaller diameter than the cell from which they are derived.
  • extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular payload either within the internal space (i.e., lumen), displayed on the external surface of the extracellular vesicle, and/or spanning the membrane.
  • the payload can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof.
  • an extracellular vehicle comprises a scaffold moiety.
  • extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g, by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells ( e.g ., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane).
  • Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, prokaryotic or eukaryotic cells, and/or cultured cells. In some aspects, the extracellular vesicles are produced by cells that express one or more transgene products.
  • exosome refers to an extracellular vesicle with a diameter between 20-300 nm (e.g., between 40-200 nm). Exosomes comprise a membrane that encloses an internal space (i.e., lumen), and, in some aspects, can be generated from a cell (e.g, producer cell) by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. In certain aspects, an exosome comprises a scaffold moiety. As described infra, exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. In some aspects, the EVs, e.g, exosomes, of the present disclosure are produced by cells that express one or more transgene products.
  • the term "nanovesicle” refers to an extracellular vesicle with a diameter between 20-250 nm (e.g, between 30-150 nm) and is generated from a cell (e.g, producer cell) by direct or indirect manipulation such that the nanovesicle would not be produced by the cell without the manipulation.
  • Appropriate manipulations of the cell to produce the nanovesicles include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. In some aspects, production of nanovesicles can result in the destruction of the producer cell.
  • population of nanovesicles described herein are substantially free of vesicles that are derived from cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane.
  • a nanovesicle comprises a scaffold moiety. Nanovesicles, once derived from a producer cell, can be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.
  • surface-engineered EVs e.g, exosomes
  • e.g, Scaffold X-engineered EVs, e.g, exosomes refers to an EV, e.g, exosome, with the membrane or the surface of the EV, e.g, exosome, modified in its composition so that the surface of the engineered EV, e.g, exosome, is different from that of the EV, e.g, exosome, prior to the modification or of the naturally occurring EV, e.g, exosome.
  • the engineering can be on the surface of the EV, e.g, exosome, or in the membrane of the EV, e.g., exosome, so that the surface of the EV, e.g, exosome, is changed.
  • the membrane is modified in its composition of a protein, a lipid, a small molecule, a carbohydrate, etc.
  • the composition can be changed by a chemical, a physical, or a biological method or by being produced from a cell previously or concurrently modified by a chemical, a physical, or a biological method.
  • the composition can be changed by a genetic engineering or by being produced from a cell previously modified by genetic engineering.
  • a surface-engineered EV e.g.
  • exosome comprises an exogenous protein (i.e., a protein that the EV, e.g. , exosome, does not naturally express) or a fragment or variant thereof that can be exposed to the surface of the EV, e.g. , exosome, or can be an anchoring point (attachment) for a moiety exposed on the surface of the EV, e.g. , exosome.
  • exogenous protein i.e., a protein that the EV, e.g. , exosome, does not naturally express
  • a fragment or variant thereof that can be exposed to the surface of the EV, e.g. , exosome, or can be an anchoring point (attachment) for a moiety exposed on the surface of the EV, e.g. , exosome.
  • a surface-engineered EV e.g.
  • exosome comprises a higher expression (e.g, higher number) of a natural exosome protein (e.g., Scaffold X) or a fragment or variant thereof that can be exposed to the surface of the EV, e.g, exosome, or can be an anchoring point (attachment) for a moiety exposed on the surface of the EV, e.g, exosome.
  • a natural exosome protein e.g., Scaffold X
  • a fragment or variant thereof that can be exposed to the surface of the EV, e.g, exosome, or can be an anchoring point (attachment) for a moiety exposed on the surface of the EV, e.g, exosome.
  • lumen-engineered exosome refers to an EV, e.g, exosome, with the membrane or the lumen of the EV, e.g, exosome, modified in its composition so that the lumen of the engineered EV, e.g, exosome, is different from that of the EV, e.g, exosome, prior to the modification or of the naturally occurring EV, e.g., exosome.
  • the engineering can be directly in the lumen or in the membrane of the EV, e.g, exosome so that the lumen of the EV, e.g, exosome is changed.
  • the membrane is modified in its composition of a protein, a lipid, a small molecule, a carbohydrate, etc. so that the lumen of the EV, e.g, exosome is modified.
  • the composition can be changed by a chemical, a physical, or a biological method or by being produced from a cell previously modified by a chemical, a physical, or a biological method.
  • the composition can be changed by a genetic engineering or by being produced from a cell previously modified by genetic engineering.
  • a lumen-engineered exosome comprises an exogenous protein (i.e., a protein that the EV, e.g, exosome does not naturally express) or a fragment or variant thereof that can be exposed in the lumen of the EV, e.g, exosome or can be an anchoring point (attachment) for a moiety exposed on the inner layer of the EV, e.g, exosome.
  • exogenous protein i.e., a protein that the EV, e.g, exosome does not naturally express
  • a fragment or variant thereof that can be exposed in the lumen of the EV, e.g, exosome or can be an anchoring point (attachment) for a moiety exposed on the inner layer of the EV, e.g, exosome.
  • a lumen-engineered EV e.g, exosome
  • a lumen-engineered EV comprises a higher expression of a natural exosome protein (e.g, Scaffold X or Scaffold Y) or a fragment or variant thereof that can be exposed to the lumen of the exosome or can be an anchoring point (attachment) for a moiety exposed in the lumen of the exosome.
  • a natural exosome protein e.g, Scaffold X or Scaffold Y
  • an anchoring point (attachment) for a moiety exposed in the lumen of the exosome.
  • modified when used in the context of EVs, e.g ., exosomes described herein, refers to an alteration or engineering of an EV, e.g. , exosome and/or its producer cell, such that the modified EV, e.g. , exosome is different from a naturally-occurring EV, e.g. , exosome.
  • a modified EV, e.g. , exosome described herein comprises a membrane that differs in composition of a protein, a lipid, a small molecular, a carbohydrate, etc. compared to the membrane of a naturally-occurring EV, e.g.
  • exosome e.g, membrane comprises higher density or number of natural exosome proteins and/or membrane comprises proteins that are not naturally found in exosomes ( e.g a FLT3L polypeptide).
  • modifications to the membrane changes the exterior surface of the EV, e.g. , exosome (e.g, surface-engineered EVs, e.g, exosomes described herein).
  • such modifications to the membrane changes the lumen of the EV, e.g, exosome (e.g, lumen-engineered EVs, e.g, exosomes described herein).
  • a scaffold moiety refers to a molecule that can be used to anchor a payload or any other compound of interest (e.g, a FLT3L polypeptide) to the EV, e.g, exosome either on the luminal surface or on the exterior surface of the EV, e.g, exosome.
  • a scaffold moiety comprises a synthetic molecule.
  • a scaffold moiety comprises a non-polypeptide moiety.
  • a scaffold moiety comprises a lipid, carbohydrate, or protein that naturally exists in the EV, e.g, exosome.
  • a scaffold moiety comprises a lipid, carbohydrate, or protein that does not naturally exist in the EV, e.g, exosome.
  • a scaffold moiety is Scaffold X.
  • a scaffold moiety is Scaffold Y.
  • a scaffold moiety comprises both Scaffold X and Scaffold Y.
  • Non-limiting examples of other scaffold moieties that can be used with the present disclosure include: aminopeptidase N (CD 13); Neprilysin, AKA membrane metalloendopeptidase (MME); ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1); Neuropilin-1 (NRP1); CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin (MFGE8), LAMP2, and LAMP2B.
  • CD 13 aminopeptidase N
  • MME AKA membrane metalloendopeptidase
  • ENPP1 ectonucleotide pyrophosphatase/phosphodiesterase family member 1
  • NBP1 Neuropilin-1
  • CD9 CD63
  • CD81 CD81
  • PDGFR GPI anchor proteins
  • lactadherin lactadherin
  • LAMP2B lactadherin
  • Scaffold X refers to exosome proteins that have recently been identified on the surface of exosomes. See, e.g, U.S. Pat. No. 10,195,290, which is incorporated herein by reference in its entirety.
  • Non-limiting examples of Scaffold X proteins include: prostaglandin F2 receptor negative regulator ("the PTGFRN protein”); basigin (“the BSG protein”); immunoglobulin superfamily member 2 (“the IGSF2 protein”); immunoglobulin superfamily member 3 (“the IGSF3 protein”); immunoglobulin superfamily member 8 (“the IGSF8 protein”); integrin beta-1 ("the ITGB1 protein); integrin alpha-4 (“the ITGA4 protein”); 4F2 cell-surface antigen heavy chain (“the SLC3 A2 protein”); a class of ATP transporter proteins ("the ATP1A1 protein,” “the ATP1A2 protein,” “the ATP1A3 protein,” “the ATP1A4 protein,” “the ATP1B3 protein,” “the ATP2B1 protein,” “the ATP2B2 protein,” “the ATP2B3 protein,” “the ATP2B protein”); and a functional fragment thereof.
  • the PTGFRN protein prostaglandin F2 receptor negative regulator
  • a Scaffold X protein can be a whole protein or a fragment thereof (e.g ., functional fragment, e.g. , the smallest fragment that is capable of anchoring another moiety on the exterior surface or on the luminal surface of the EV, e.g., exosome).
  • a Scaffold X can anchor a moiety (e.g, a FLT3L polypeptide) to the external surface or the luminal surface of the exosome.
  • Scaffold Y refers to exosome proteins that were newly identified within the lumen of exosomes. See, e.g, International Appl. No. PCT/US2018/061679, which is incorporated herein by reference in its entirety.
  • Non-limiting examples of Scaffold Y proteins include: myristoylated alanine rich Protein Kinase C substrate ("the MARCKS protein”); myristoylated alanine rich Protein Kinase C substrate like 1 (“the MARCKSL1 protein”); and brain acid soluble protein 1 (“the BASP1 protein”).
  • a Scaffold Y protein can be a whole protein or a fragment thereof (e.g, functional fragment, e.g, the smallest fragment that is capable of anchoring a moiety to the luminal surface of the exosome).
  • a Scaffold Y can anchor a moiety (e.g, a FLT3L polypeptide) to the luminal surface of the EV, e.g, exosome.
  • fragment of a protein refers to an amino acid sequence of a protein that is shorter than the naturally- occurring sequence, N- and/or C-terminally deleted or any part of the protein deleted in comparison to the naturally occurring protein.
  • functional fragment refers to a protein fragment that retains protein function. Accordingly, in some aspects, a functional fragment of a Scaffold X protein retains the ability to anchor a moiety on the luminal surface or on the exterior surface of the EV, e.g, exosome.
  • a functional fragment of a Scaffold Y protein retains the ability to anchor a moiety on the luminal surface of the EV, e.g, exosome. Whether a fragment is a functional fragment can be assessed by any art known methods to determine the protein content of EVs, e.g, exosomes including Western Blots, FACS analysis and fusions of the fragments with autofluorescent proteins like, e.g, GFP.
  • a functional fragment of a Scaffold X protein retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% of the ability, e.g.
  • a functional fragment of a Scaffold Y protein retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% of the ability, e.g. , an ability to anchor another molecule, of the naturally occurring Scaffold Y protein.
  • variant of a molecule refers to a molecule that shares certain structural and functional identities with another molecule upon comparison by a method known in the art.
  • a variant of a protein can include a substitution, insertion, deletion, frameshift or rearrangement in another protein.
  • a variant of a Scaffold X comprises a variant having at least about 70% identity to the full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter proteins or a fragment (e.g, functional fragment) of the PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, or ATP transporter proteins.
  • variants or variants of fragments of PTGFRN share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with PTGFRN according to SEQ ID NO: 1 or with a functional fragment thereof.
  • variants or variants of fragments of BSG share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with BSG according to SEQ ID NO: 9 or with a functional fragment thereof.
  • variants or variants of fragments of IGSF2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with IGSF2 or with a functional fragment thereof.
  • variants or variants of fragments of IGSF3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with IGSF3 or with a functional fragment thereof.
  • variants or variants of fragments of IGSF8 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with IGSF8 according to SEQ ID NO: 14 or with a functional fragment thereof.
  • variants or variants of fragments of ITGB1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ITGB1 according to SEQ ID NO: 21 or with a functional fragment thereof.
  • variants or variants of fragments of ITGA4 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ITGA4 according to SEQ ID NO: 22 or with a functional fragment thereof.
  • variants or variants of fragments of SLC3A2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SLC3A2 according to SEQ ID NO: 23 or with a functional fragment thereof.
  • variants or variants of fragments of ATP1A1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1A1 or with a functional fragment thereof.
  • variants or variants of fragments of ATP1A2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1 A2 or with a functional fragment thereof.
  • variants or variants of fragments of ATP1 A3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP 1 A3 or with a functional fragment thereof.
  • variants or variants of fragments of ATP1A4 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1A4 or with a functional fragment thereof.
  • variants or variants of fragments of ATP1B3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP1B3 or with a functional fragment thereof.
  • variants or variants of fragments of ATP2B1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B1 or with a functional fragment thereof.
  • variants or variants of fragments of ATP2B2 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B2 or with a functional fragment thereof.
  • variants or variants of fragments of ATP2B3 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B3 or with a functional fragment thereof.
  • variants or variants of fragments of ATP2B4 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with ATP2B4 or with a functional fragment thereof.
  • the variant or variant of a fragment of Scaffold X protein disclosed herein retains the ability to be specifically targeted to EVs, e.g ., exosomes.
  • the Scaffold X includes one or more mutations, for example, conservative amino acid substitutions.
  • a variant of a Scaffold Y comprises a variant having at least 70% identity to MARCKS, MARCKSL1, BASP1, or a fragment of MARCKS, MARCKSLl, or BASP1.
  • variants or variants of fragments of MARCKS share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with MARCKS according to SEQ ID NO: 47 or with a functional fragment thereof.
  • variants or variants of fragments of MARCKSLl share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with MARCKSLl according to SEQ ID NO: 48 or with a functional fragment thereof.
  • variants or variants of fragments of BASP1 share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with BASP1 according to SEQ ID NO: 49 or with a functional fragment thereof.
  • the variant or variant of a fragment of Scaffold Y protein retains the ability to be specifically targeted to the luminal surface of EVs, e.g. , exosomes.
  • the Scaffold Y includes one or more mutations, e.g. , conservative amino acid substitutions.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g ., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g
  • a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • percent sequence identity or “percent identity” between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences.
  • a matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.
  • the percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the comparison of sequences and determination of percent sequence identity between two sequences may be accomplished using readily available software both for online use and for download. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S.
  • B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • Other suitable programs are, e.g, Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.
  • Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • sequence alignments are not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments.
  • One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org.
  • Another suitable program is MUSCLE, available from www.drive5.com/muscle/.
  • ClustalW2 and MUSCLE are alternatively available, e.g ., from the EBI.
  • sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g, crystallographic protein structures), functional data (e.g, location of mutations), or phylogenetic data.
  • a suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T- Coffee, available at www.tcoffee.org, and alternatively available, e.g, from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity may be curated either automatically or manually.
  • the polynucleotide variants can contain alterations in the coding regions, non coding regions, or both.
  • the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
  • nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • Polynucleotide variants can be produced for a variety of reasons, e.g, to optimize codon expression for a particular host (change codons in the human mRNA to others, e.g, a bacterial host such as E. coll).
  • Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present disclosure.
  • non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.
  • variants can be generated to improve or alter the characteristics of the polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.
  • interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al, ./. Biotechnology 7: 199- 216 (1988), incorporated herein by reference in its entirety.)
  • polypeptide variants include, e.g. , modified polypeptides.
  • Modifications include, e.g. , acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethyl ati on, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:210-19 (2010), which is incorporated herein by reference in its
  • the term "linked to” or “conjugated to” are used interchangeably and refer to a covalent or non-covalent bond formed between a first moiety and a second moiety, e.g ., Scaffold X and a FLT3L polypeptide, respectively, e.g. , a scaffold moiety expressed in or on the extracellular vesicle and a FLT3L polypeptide, e.g. , Scaffold X (e.g, a PTGFRN protein), respectively, in the luminal surface of or on the external surface of the extracellular vesicle.
  • a first moiety and a second moiety e.g ., Scaffold X and a FLT3L polypeptide, respectively, e.g. , a scaffold moiety expressed in or on the extracellular vesicle and a FLT3L polypeptide, e.g. , Scaffold X (e.g, a PTGFRN protein), respectively, in
  • encapsulated refers to a status or process of having a first moiety (e.g, a FLT3L polypeptide) inside a second moiety (e.g, an EV, e.g, exosome) without chemically or physically linking the two moieties.
  • a first moiety e.g, a FLT3L polypeptide
  • a second moiety e.g, an EV, e.g, exosome
  • the term “encapsulated” can be used interchangeably with “in the lumen of.”
  • Non-limiting examples of encapsulating a first moiety (e.g, a FLT3L polypeptide) into a second moiety are disclosed elsewhere herein.
  • the term "producer cell” refers to a cell used for generating an EV, e.g, exosome.
  • a producer cell can be a cell cultured in vitro, or a cell in vivo.
  • a producer cell includes, but not limited to, a cell known to be effective in generating EVs, e.g, exosomes, e.g, HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblast cells, fHDF fibroblast cells, AGE.HN ® neuronal precursor cells, CAP ® amniocyte cells, adipose mesenchymal stem cells, RPTEC/TERT1 cells.
  • a cell known to be effective in generating EVs e.g, exosomes, e.g, HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSC
  • a producer cell is not an antigen-presenting cell. In some aspects, a producer cell is not a dendritic cell, a B cell, a mast cell, a macrophage, a neutrophil, Kupffer-Browicz cell, cell derived from any of these cells, or any combination thereof.
  • the EVs e.g, exosomes useful in the present disclosure do not carry an antigen on MHC class I or class II molecule exposed on the surface of the EV, e.g, exosome, but instead can carry an antigen in the lumen of the EV, e.g, exosome or on the surface of the EV, e.g, exosome by attachment to Scaffold X and/or Scaffold Y.
  • isolating or purifying is the process of removing, partially removing (e.g, a fraction) of the EVs from a sample containing producer cells.
  • an isolated EV composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other aspects, an isolated EV composition has an amount and/or concentration of desired EVs at or above an acceptable amount and/or concentration. In other aspects, the isolated EV composition is enriched as compared to the starting material (e.g, producer cell preparations) from which the composition is obtained. This enrichment can be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material.
  • the starting material e.g, producer cell preparations
  • isolated EV preparations are substantially free of residual biological products.
  • the isolated EV preparations are 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free, or 90% free of any contaminating biological matter.
  • Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.
  • Substantially free of residual biological products can also mean that the EV composition contains no detectable producer cells and that only EVs are detectable.
  • the term "payload” refers to an agent that acts on a target (e.g, a target cell) that is contacted with the EV.
  • a target e.g, a target cell
  • Payloads that can be introduced into an EV, e.g, exosome, and/or a producer cell include agents such as, nucleotides (e.g, nucleotides comprising a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g, DNA or mRNA molecules that encode a polypeptide such as an enzyme, or RNA molecules that have regulatory function such as miRNA, dsDNA, IncRNA, and siRNA), amino acids (e.g, amino acids comprising a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g, enzymes), lipids, carbohydrates, and small molecules (e.g, small molecule drugs and toxins).
  • nucleotides e.g, nucleotides comprising a detectable moiety or a toxin or that disrupt transcription
  • nucleic acids e.g, DNA or mRNA molecules that encode a polypeptide such as an enzyme, or RNA molecules that have regulatory function such
  • a payload comprises a FLT3L polypeptide.
  • the term “antibody” encompasses an immunoglobulin whether natural or partly or wholly synthetically produced, and fragments thereof. The term also covers any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibody” further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the term “antigen” refers to any agent that when introduced into a subject elicits an immune response (cellular or humoral) to itself.
  • antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, e.g ., scFv, (scFv)2, Fab, Fab', and F(ab')2, F(abl)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides.
  • Antibody includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.
  • the terms "individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • the compositions and methods described herein are applicable to both human therapy and veterinary applications.
  • the subject is a mammal, and in other aspects the subject is a human.
  • a “mammalian subject” includes all mammals, including without limitation, humans, domestic animals (e.g, dogs, cats and the like), farm animals (e.g, cows, sheep, pigs, horses and the like) and laboratory animals (e.g, monkey, rats, mice, rabbits, guinea pigs and the like).
  • the term "substantially free” means that the sample comprising EVs, e.g, exosomes, comprise less than 10% of macromolecules by mass/volume (m/v) percentage concentration. Some fractions may contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% (m/v) of macromolecules.
  • macromolecule means nucleic acids, contaminant proteins, lipids, carbohydrates, metabolites, or a combination thereof.
  • the term "conventional exosome protein” means a protein previously known to be enriched in exosomes, including but is not limited to CD9, CD63, CD81, PDGFR, GPI anchor proteins, lactadherin (MFGE8), LAMP2, and LAMP2B, a fragment thereof, or a peptide that binds thereto.
  • "Administering,” as used herein, means to give a composition comprising an EV, e.g ., exosome, disclosed herein to a subject via a pharmaceutically acceptable route. Routes of administration can be intravenous, e.g. , intravenous injection and intravenous infusion. Additional routes of administration include, e.g. , subcutaneous, intramuscular, oral, nasal, and pulmonary administration. EVs, e.g. , exosomes can be administered as part of a pharmaceutical composition comprising at least one excipient.
  • Hematopoiesis refers to the formation of all blood cellular components. Hematopoiesis occurs both during embryonic development and throughout adulthood. In humans, hematopoiesis occurs primarily in the bone marrow, liver, and spleen. Hematopoiesis includes the differentiation and proliferation of erythrocytes and leukocytes, including lymphocytes (T cells and B cells), neutrophils, eosinophils, basophils, macrophages, and dendritic cells (DCs).
  • T cells and B cells lymphocytes
  • neutrophils neutrophils
  • eosinophils neutrophils
  • basophils basophils
  • macrophages macrophages
  • DCs dendritic cells
  • an "immune response,” as used herein, refers to a biological response within a vertebrate against foreign agents or abnormal, e.g. , infected cells, which response protects the organism against these agents and diseases caused by them.
  • An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • An immune reaction includes, e.g. , activation or inhibition of a T cell, e.g. , an effector T cell, a Th cell, a CD4+ cell, a CD8+ T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g. , NK cell.
  • an immune response can comprise a humoral immune response (e.g, mediated by B-cells), cellular immune response (e.g, mediated by T cells), or both humoral and cellular immune responses.
  • an immune response is an "inhibitory" immune response.
  • An inhibitory immune response is an immune response that blocks or diminishes the effects of a stimulus (e.g., antigen).
  • the inhibitory immune response comprises the production of inhibitory antibodies against the stimulus.
  • the inhibitory response comprises the production of antibodies against the FLT3L polypeptide.
  • an immune response is a "stimulatory" immune response.
  • a stimulatory immune response is an immune response that results in the generation of effectors cells (e.g ., cytotoxic T lymphocytes).
  • Treating refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition.
  • the term also includes prophylaxis or prevention of a disease or condition or its symptoms thereof.
  • the "treating" or “treatment” includes inducing hematopoiesis in a subject in need thereof.
  • the disease or condition is associated with a hematopoiesis or a deficiency thereof.
  • the disease or condition is a cancer.
  • the treating enhances hematopoiesis in a subject having a cancer, wherein the enhanced hematopoiesis comprises increased proliferation and/or differentiation of one or more immune cell in the subject
  • Prevent refers to decreasing or reducing the occurrence or severity of a particular outcome. In some aspects, preventing an outcome is achieved through prophylactic treatment.
  • an EV e.g, an exosome, comprising a FLT3L polypeptide, described herein, is administered to a subject prophylactically.
  • the subject is at risk of developing cancer. In some aspects, the subject is at risk of developing a hematopoietic disorder.
  • Certain aspects of the present disclosure are directed to methods of inducing hematopoiesis in a subject in need thereof, comprising administering an EV, e.g, an exosome, comprising a FLT3L polypeptide to the subject.
  • an EV e.g, an exosome
  • the induced hematopoiesis is characterized by an increase in the proliferation of one or more blood cells, relative to the level of proliferation of the one or more blood cells prior to the administration of the EV, e.g, an exosome, comprising a FLT3L polypeptide.
  • the induced hematopoiesis is characterized by an increase in the differentiation of one or more blood cells, relative to the level of differentiation of the one or more cells prior to the administration of the EV, e.g, an exosome, comprising a FLT3L polypeptide.
  • the one or more blood cells can be selected from any cell that is a decedent of a hematopoietic stem cell.
  • the one or more blood cells comprise an immune cell.
  • the one or more cells of the blood are selected from a myeloid progenitor cell, a lymphoid progenitor cell, a megakaryocyte, an erythrocyte, a mast cell, a myeloblast, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, a natural killer cell, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasma cell, and any combination thereof.
  • the subject exhibits increased proliferation of one or more myeloid progenitor cells relative to the proliferation of the one or more myeloid progenitor cells prior to the administration. In some aspects, the subject exhibits increased differentiation of one or more myeloid progenitor cells relative to the differentiation of the one or more myeloid progenitor cells prior to the administration. In some aspects, the subject exhibits increased proliferation of one or more myeloid cells, relative to the proliferation of the one or more myeloid cells prior to the administration. In some aspects, the subject increased differentiation of one or more myeloid cells, relative to the differentiation of the one or more myeloid cells prior to the administration.
  • the one or more myeloid cells are selected from a megakaryocyte, an erythrocyte, a mast cell, a myeloblast, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, and any combination thereof.
  • the myeloid cell is a macrophage.
  • the subject exhibits increased macrophage proliferation relative to the macrophage proliferation prior to the administration.
  • the subject exhibits increased macrophage differentiation relative to the macrophage differentiation prior to the administration.
  • the subject exhibits increased monocyte proliferation relative to the monocyte proliferation prior to the administration.
  • the subject exhibits increased monocyte differentiation relative to the monocyte differentiation prior to the administration.
  • the subject exhibits increased myeloblast proliferation relative to the myeloblast proliferation prior to the administration.
  • the subject exhibits increased myeloblast differentiation relative to the myeloblast differentiation prior to the administration.
  • the subject exhibits increased proliferation of one or more lymphoid progenitor cells relative to the proliferation of the one or more lymphoid progenitor cells prior to the administration. In some aspects, the subject exhibits increased differentiation of one or more lymphoid progenitor cells relative to the differentiation of the one or more lymphoid progenitor cells prior to the administration. In some aspects, the subject exhibits increased proliferation of one or more lymphoid cells, relative to the proliferation of the one or more lymphoid cells prior to the administration. In some aspects, the subject increased differentiation of one or more lymphoid cells, relative to the differentiation of the one or more lymphoid cells prior to the administration.
  • the one or more lymphoid cells are selected from a natural killer cell, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasma cell, and any combination thereof.
  • the lymphoid cell is a dendritic cell.
  • the subject exhibits increased dendritic cell proliferation relative to the dendritic cell proliferation prior to the administration.
  • the subject exhibits increased dendritic cell differentiation relative to the dendritic cell differentiation prior to the administration.
  • the lymphoid cell is a dendritic cell.
  • the subject exhibits increased mature dendritic cell proliferation relative to the mature dendritic cell proliferation prior to the administration.
  • the subject exhibits increased mature dendritic cell differentiation relative to the mature dendritic cell differentiation prior to the administration.
  • the subject exhibits increased immature dendritic cell proliferation relative to the immature dendritic cell proliferation prior to the administration.
  • the subject exhibits increased immature dendritic cell differentiation relative to the immature dendritic cell differentiation prior to the administration.
  • the subject has a tumor, wherein the tumor comprises one or more immune cell, e.g ., one or more tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • the subject exhibits an increase in the number of the one or more immune cells in the tumor after the administration relative to the number of the one or more immune cells in the tumor prior to the administration.
  • the EVs are administered intravenously to the circulatory system of the subject. In some aspects, the EVs are infused in suitable liquid and administered into a vein of the subject.
  • the EVs are administered intra-arterialy to the circulatory system of the subject. In some aspects, the EVs are infused in suitable liquid and administered into an artery of the subject.
  • the EVs are administered to the subject by intrathecal administration. In some aspects, the EVs are administered via an injection into the spinal canal, or into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). [0103] In some aspects, the EVs are administered to the subject by intratracheal inhalation and/or intratracheal instillation.
  • CSF cerebrospinal fluid
  • the EVs are administered to the subject by intranasal administration.
  • the EVs can be insufflated through the nose in a form of either topical administration or systemic administration.
  • the EVs are administered as nasal spray.
  • the EVs are administered to the subject by intraperitoneal administration.
  • the EVs are infused in suitable liquid and injected into the peritoneum of the subject.
  • the intraperitoneal administration results in distribution of the EVs to the lymphatics.
  • the intraperitoneal administration results in distribution of the EVs to the thymus, spleen, and/or bone marrow.
  • the intraperitoneal administration results in distribution of the EVs to one or more lymph nodes.
  • the intraperitoneal administration results in distribution of the EVs to one or more of the cervical lymph node, the inguinal lymph node, the mediastinal lymph node, or the sternal lymph node. In some aspects, the intraperitoneal administration results in distribution of the EVs to the pancreas.
  • the EVs are administered to the subject by periocular administration.
  • the EVs, e.g. , exosomes are injected into the periocular tissues.
  • Periocular drug administration includes the routes of subconjunctival, anterior sub-Tenon’s, posterior sub-Tenon’s, and retrobulbar administration.
  • Extracellular Vesicles e.g., Exosomes
  • EVs e.g. , exosomes
  • the EVs, e.g. , exosomes, useful in the present disclosure have been engineered to produce a FLT3L polypeptide.
  • an EV, e.g. , exosome comprises a FLT3L polypeptide.
  • EVs, e.g. , exosomes, described herein are extracellular vesicles with a diameter between about 20-300 nm.
  • an EV, e.g. , exosome, of the present disclosure has a diameter between about 20-290 nm, 20-280 nm, 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220 nm, 20-210 nm, 20-200 nm, 20-190 nm, 20-180 nm, 20-170 nm, 20-160 nm, 20-150 nm, 20-140 nm, 20-130 nm, 20-120 nm, 20-110 nm, 20-100 nm, 20-90 nm, 20-80 nm, 20-70 nm, 20-60 nm, 20-50 nm, 20-40 n
  • an EV, e.g. , exosome, of the present disclosure comprises a bi lipid membrane ("EV, e.g. , exosome, membrane”), comprising an interior (luminal) surface and an exterior surface.
  • the interior (luminal) surface faces the inner core (i.e., lumen) of the EV, e.g. , exosome.
  • the exterior surface can be in contact with the endosome, the multi vesicular bodies, or the membrane/cytoplasm of a producer cell or a target cell [0110]
  • the EV, e.g, exosome, membrane comprises lipids and fatty acids.
  • the EV, e.g. , exosome, membrane comprises phospholipids, gly colipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterols, and phosphatidylserines.
  • the EV, e.g. , exosome, membrane comprises an inner leaflet and an outer leaflet.
  • the composition of the inner and outer leaflet can be determined by transbilayer distribution assays known in the art, see, e.g., Kuypers et al, Biohim Biophys Acta 1985 819: 170.
  • the composition of the outer leaflet is between approximately 70-90% choline phospholipids, between approximately 0-15% acidic phospholipids, and between approximately 5-30% phosphatidylethanolamine.
  • the composition of the inner leaflet is between approximately 15-40% choline phospholipids, between approximately 10-50% acidic phospholipids, and between approximately 30-60% phosphatidylethanolamine.
  • the EV, e.g, exosome, membrane comprises one or more polysaccharide, such as glycan.
  • the EV e.g, exosome
  • the EV comprises a FLT3L polypeptide, wherein the FLT3L polypeptide is linked to the EV via a scaffold moiety, either on the exterior surface of the EV or on the luminal surface of the EV.
  • the EV, e.g, exosome, of the present disclosure comprises a FLT3L polypeptide in the lumen of the EV.
  • the EV, e.g, exosome, of the present disclosure comprises a FLT3L polypeptide on the exterior surface of the EV.
  • the EV comprises a FLT3L polypeptide on the exterior surface of the EV, optionally linked via a scaffold moiety (e.g, Scaffold X), and a FLT3L polypeptide on the luminal surface of the EV, optionally linked via the scaffold moiety (e.g, Scaffold X).
  • the first scaffold moiety and the second scaffold moiety are the same.
  • the first scaffold moiety and the second scaffold moiety are different.
  • the EV comprises a FLT3L polypeptide on the exterior surface of the EV, optionally linked via a Scaffold X, and a FLT3L polypeptide on the luminal surface of the EV, optionally linked via a Scaffold X.
  • the EV comprises a FLT3L polypeptide on the exterior surface of the EV, optionally linked via a Scaffold X, and a FLT3L polypeptide on the luminal surface of the EV, optionally linked via a Scaffold Y.
  • the EV comprises a FLT3L polypeptide on the exterior surface of the EV, optionally linked via a Scaffold Y, and a FLT3L polypeptide on the luminal surface of the EV, optionally linked via a Scaffold Y.
  • One or more scaffold moieties can be used to anchor a FLT3L polypeptide to the EV of the present disclosure.
  • the FLT3L polypeptide is linked to the scaffold moiety.
  • the EV comprises more than one scaffold moiety.
  • a first FLT3L polypeptide is linked to a first scaffold moiety and a second FLT3L polypeptide is linked to a second scaffold moiety.
  • the first scaffold moiety and the second scaffold moiety are the same type of scaffold moiety, e.g, the first and second scaffold moieties are both a Scaffold X protein.
  • the first scaffold moiety and the second scaffold moiety are different types of scaffold moiety, e.g. , the first scaffold moiety is a Scaffold Y protein and the second scaffold moiety is a Scaffold X protein.
  • the first scaffold moiety is a Scaffold Y, disclosed herein.
  • the first scaffold moiety is a Scaffold X, disclosed herein.
  • the second scaffold moiety is a Scaffold Y, disclosed herein.
  • the second scaffold moiety is a Scaffold X, disclosed herein.
  • the EV comprises one or more scaffold moieties, which are capable of anchoring a FLT3L polypeptide to the EV, e.g. , exosome, (e.g, either on the luminal surface or on the exterior surface).
  • the scaffold moiety is a polypeptide ("scaffold protein").
  • the scaffold protein comprises an exosome protein or a fragment thereof.
  • scaffold moieties are non-polypeptide moieties.
  • scaffold proteins include various membrane proteins, such as transmembrane proteins, integral proteins and peripheral proteins, enriched on the exosome membranes. They can include various CD proteins, transporters, integrins, lectins, and cadherins.
  • a scaffold moiety (e.g, scaffold protein) comprises Scaffold X.
  • a scaffold moiety (e.g, exosome protein) comprises Scaffold Y.
  • a scaffold moiety (e.g, exosome protein) comprises both a Scaffold X and a Scaffold Y.
  • EVs, e.g, exosomes, of the present disclosure comprise a membrane modified in its composition.
  • their membrane compositions can be modified by changing the protein, lipid, or glycan content of the membrane.
  • the surface-engineered EVs e.g, exosomes
  • the surface-engineered EVs, e.g, exosomes are generated by genetic engineering. EVs, e.g, exosomes, produced from a genetically-modified producer cell or a progeny of the genetically-modified cell can contain modified membrane compositions.
  • surface-engineered EVs e.g, exosomes
  • have scaffold moiety e.g, exosome protein, e.g, Scaffold X
  • higher or lower density e.g, higher number
  • surface (e.g, Scaffold X)-engineered EVs can be produced from a cell (e.g, HEK293 cells) transformed with an exogenous sequence encoding a scaffold moiety (e.g, exosome proteins, e.g, Scaffold X) or a variant or a fragment thereof.
  • EVs including scaffold moiety expressed from the exogenous sequence can include modified membrane compositions.
  • scaffold moiety modified to have enhanced affinity to a binding agent can be used for generating surface-engineered EV that can be purified using the binding agent.
  • Scaffold moieties modified to be more effectively targeted to EVs and/or membranes can be used.
  • Scaffold moieties modified to comprise a minimal fragment required for specific and effective targeting to exosome membranes can be also used.
  • Scaffold moieties can be engineered to be expressed as a fusion molecule, e.g, fusion molecule of Scaffold X to an FLT3L polypeptide.
  • the fusion molecule can comprise a scaffold moiety disclosed herein (e.g, Scaffold X, e.g, PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2, ATP transporter, or a fragment or a variant thereof) linked to a FLT3L polypeptide.
  • the surface (e.g, Scaffold X)-engineered EVs described herein demonstrate superior characteristics compared to EVs known in the art.
  • surface (e.g, Scaffold X)-engineered contain modified proteins more highly enriched on their surface than naturally occurring EVs or the EVs produced using conventional exosome proteins.
  • the surface (e.g, Scaffold X)-engineered EVs of the present disclosure can have greater, more specific, or more controlled biological activity compared to naturally occurring EVs or the EVs produced using conventional exosome proteins.
  • the Scaffold X comprises Prostaglandin F2 receptor negative regulator (the PTGFRN polypeptide).
  • the PTGFRN protein can be also referred to as CD9 partner 1 (CD9P-1), Glu-Trp-Ile EWI motif-containing protein F (EWI-F), Prostaglandin F2- alpha receptor regulatory protein, Prostaglandin F2-alpha receptor-associated protein, or CD315.
  • CD9P-1 CD9 partner 1
  • EWI-F Glu-Trp-Ile EWI motif-containing protein F
  • Prostaglandin F2- alpha receptor regulatory protein Prostaglandin F2-alpha receptor-associated protein
  • the full length amino acid sequence of the human PTGFRN protein (Uniprot Accession No. Q9P2B2) is shown at Table 2 as SEQ ID NO: 1.
  • the PTGFRN polypeptide contains a signal peptide (amino acids 1 to 25 of SEQ ID NO: 1), the extracellular domain (amino acids 26 to 832 of SEQ ID NO: 1), a transmembrane domain (amino acids 833 to 853 of SEQ ID NO: 1), and a cytoplasmic domain (amino acids 854 to 879 of SEQ ID NO: 1).
  • the mature PTGFRN polypeptide consists of SEQ ID NO: 1 without the signal peptide, i.e., amino acids 26 to 879 of SEQ ID NO: 1.
  • a PTGFRN polypeptide fragment useful for the present disclosure comprises a transmembrane domain of the PTGFRN polypeptide.
  • a PTGFRN polypeptide fragment useful for the present disclosure comprises the transmembrane domain of the PTGFRN polypeptide and (i) at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 amino acids at the N terminus of the transmembrane domain, (ii) at least five, at least 10, at least 15, at least 20, or at least 25 amino acids at the C terminus of the transmembrane domain, or both (i) and (ii).
  • the fragments of PTGFRN polypeptide lack one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 26 to 879 of SEQ ID NO: 1.
  • the Scaffold X comprises an amino acid sequence at least about 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 33.
  • the Scaffold X comprises the amino acid sequence of SEQ ID NO: 33, except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
  • the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
  • the Scaffold X comprises the amino acid sequence of SEQ ID NO: 33 and 1 amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids or longer at the N terminus and/or C terminus of SEQ ID NO: 33.
  • the Scaffold X comprises an amino acid sequence at least about 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 1, 9, 14, 21, 22, 23, or 33.
  • the Scaffold X comprises the amino acid sequence of SEQ ID NO: 1, 9, 14, 21, 22, 23, or 33, except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
  • the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
  • a Scaffold X comprises Basigin (the BSG protein), represented by SEQ ID NO: 9.
  • the BSG protein is also known as 5F7, Collagenase stimulatory factor, Extracellular matrix metalloproteinase inducer (EMMPRIN), Leukocyte activation antigen M6, OK blood group antigen, Tumor cell-derived collagenase stimulatory factor (TCSF), or CD 147.
  • the Uniprot number for the human BSG protein is P35613.
  • the signal peptide of the BSG protein is amino acid 1 to 21 of SEQ ID NO: 9. Amino acids 138-323 of SEQ ID NO: 9 is the extracellular domain, amino acids 324 to 344 is the transmembrane domain, and amino acids 345 to 385 of SEQ ID NO: 9 is the cytoplasmic domain.
  • the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 22 to 385 of SEQ ID NO: 9.
  • the fragments of BSG polypeptide lack one or more functional or structural domains, such as IgV, e.g ., amino acids 221 to 315 of SEQ ID NO: 9.
  • a Scaffold X comprises Immunoglobulin superfamily member 8 (IgSF8 or the IGSF8 protein), which is also known as CD81 partner 3, Glu-Trp-Ile EWI motif- containing protein 2 (EWI-2), Keratinocytes-associated transmembrane protein 4 (KCT-4), LIR- Dl, Prostaglandin regulatory-like protein (PGRL) or CD316.
  • IgSF8 or the IGSF8 protein Immunoglobulin superfamily member 8
  • EWI-2 Glu-Trp-Ile EWI motif- containing protein 2
  • KCT-4 Keratinocytes-associated transmembrane protein 4
  • LIR- Dl LIR- Dl
  • Prostaglandin regulatory-like protein PGRL
  • the human IGSF8 protein has a signal peptide (amino acids 1 to 27 of SEQ ID NO: 14), an extracellular domain (amino acids 28 to 579 of SEQ ID NO: 14), a transmembrane domain (amino acids 580 to 600 of SEQ ID NO: 14), and a cytoplasmic domain (amino acids 601 to 613 of SEQ ID NO: 14).
  • the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 28 to 613 of SEQ ID NO: 14.
  • the IGSF8 protein lack one or more functional or structural domains, such as IgV.
  • a Scaffold X for the present disclosure comprises Immunoglobulin superfamily member 3 (IgSF3 or the IGSF3 protein), which is also known as Glu-Trp-Ile EWI motif-containing protein 3 (EWI-3).
  • the human IGSF3 protein has a signal peptide (amino acids 1 to 19), an extracellular domain (amino acids 20 to 1124), a transmembrane domain (amino acids 1125 to 1145), and a cytoplasmic domain (amino acids 1146 to 1194).
  • the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 28 to 613 of IgSF3.
  • the IGSF3 protein lack one or more functional or structural domains, such as IgV.
  • a Scaffold X for the present disclosure comprises Integrin beta-1 (the ITGB1 protein), which is also known as Fibronectin receptor subunit beta, Glycoprotein Ila (GPIIA), VLA-4 subunit beta, or CD29, and is shown as the amino acid sequence of SEQ ID NO: 21.
  • the human ITGB1 protein has a signal peptide (amino acids 1 to 20 of SEQ ID NO: 21), an extracellular domain (amino acids 21 to 728 of SEQ ID NO: 21), a transmembrane domain (amino acids 729 to 751 of SEQ ID NO: 21), and a cytoplasmic domain (amino acids 752 to 798 of SEQ ID NO: 21).
  • the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 21 to 798 of SEQ ID NO: 21.
  • the ITGB1 protein lack one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ITGA4 protein, which comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 22 without the signal peptide (amino acids 1 to 33 of SEQ ID NO: 22).
  • the ITGA4 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the SLC3A2 protein, which comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 23 without the signal peptide.
  • the SLC3A2 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP1A1 protein without the signal peptide.
  • the ATP1A1 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP1A2 protein without the signal peptide.
  • the ATP1A2 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP1 A3 protein without the signal peptide.
  • the ATP 1 A3 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP1 A4 protein without the signal peptide.
  • the ATP1A4 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP1 A5 protein without the signal peptide. In some aspects, the ATP1A5 protein lacks one or more functional or structural domains, such as IgV. [0142] In other aspects, the Scaffold X comprises the ATP2B1 protein without the signal peptide. In some aspects, the ATP2B1 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP2B2 protein without the signal peptide.
  • the ATP2B2 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP2B3 protein without the signal peptide.
  • the ATP2B3 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the ATP2B4 protein without the signal peptide.
  • the ATP2B4 protein lacks one or more functional or structural domains, such as IgV.
  • the Scaffold X comprises the IGSF2 protein without the signal peptide.
  • the IGSF2 protein lacks one or more functional or structural domains, such as IgV.
  • Non-limiting examples of other Scaffold X proteins can be found at US Patent No. US10195290B1, issued Feb. 5, 2019, which is incorporated by reference in its entireties.
  • the sequence encodes a fragment of the scaffold moiety lacking at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the N-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety lacking at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from the C-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety lacking at least 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, or 800 amino acids from both the N- terminus and C-terminus of the native protein. In some aspects, the sequence encodes a fragment of the scaffold moiety lacking one or more functional or structural domains of the native protein.
  • the scaffold moieties e.g ., Scaffold X, e.g. , a PTGFRN protein
  • the one or more heterologous proteins can be linked to the N-terminus of the scaffold moieties.
  • the one or more heterologous proteins can be linked to the C-terminus of the scaffold moieties.
  • the one or more heterologous proteins are linked to both the N-terminus and the C-terminus of the scaffold moieties.
  • the heterologous protein is a mammalian protein.
  • the heterologous protein is a human protein.
  • Scaffold X can be used to link any moiety, e.g, a FLT3L polypeptide, to the luminal surface and on the exterior surface of the EV, e.g, exosome, at the same time.
  • the PTGFRN polypeptide can be used to link a FLT3L polypeptide inside the lumen (e.g, on the luminal surface) in addition to the exterior surface of the EV, e.g, exosome. Therefore, in certain aspects, Scaffold X can be used for dual purposes, e.g., a FLT3L polypeptide on the luminal surface and a FLT3L polypeptide on the exterior surface of the EV, e.g, exosome.
  • Scaffold X is a scaffold protein that is capable of anchoring the FLT3L polypeptide on the luminal surface of the EV and/or on the exterior surface of the EV.
  • EVs e.g, exosomes
  • EVs comprise an internal space (i.e., lumen) that is different from that of the naturally occurring EVs.
  • the EV can be changed such that the composition in the luminal surface of the EV, e.g, exosome has the protein, lipid, or glycan content different from that of the naturally-occurring exosomes.
  • engineered EVs e.g, exosomes
  • a cell transformed with an exogenous sequence encoding a scaffold moiety e.g, exosome proteins, e.g, Scaffold Y
  • a modification or a fragment of the scaffold moiety that changes the composition or content of the luminal surface of the EV, e.g, exosome.
  • Various modifications or fragments of the exosome protein that can be expressed on the luminal surface of the EV, e.g, exosome can be used for the aspects of the present disclosure.
  • the exosome proteins that can change the luminal surface of the EVs include, but are not limited to, the myristoylated alanine rich Protein Kinase C substrate (MARCKS) protein, the myristoylated alanine rich Protein Kinase C substrate like 1 (MARCKSL1) protein, the brain acid soluble protein 1 (BASP1) protein, or any combination thereof.
  • MARCKS myristoylated alanine rich Protein Kinase C substrate
  • MARCKSL1 myristoylated alanine rich Protein Kinase C substrate like 1
  • BASP1 brain acid soluble protein 1
  • Scaffold Y comprises the MARCKS protein (Uniprot accession no. P29966).
  • the MARCKS protein is also known as protein kinase C substrate, 80 kDa protein, light chain.
  • the full-length human MARCKS protein is 332 amino acids in length and comprises a calmodulin-binding domain at amino acid residues 152-176.
  • Scaffold Y comprises the MARCKSL1 protein (Uniprot accession no. P49006).
  • the MARCKSLl protein is also known as MARCKS-like protein 1, and macrophage myristoylated alanine-rich C kinase substrate.
  • the full-length human MARCKSLl protein is 195 amino acids in length.
  • the MARCKSLl protein has an effector domain involved in lipid-binding and calmodulin-binding at amino acid residues 87-110.
  • the Scaffold Y comprises the BASP1 protein (Uniprot accession number P80723).
  • the BASP1 protein is also known as 22 kDa neuronal tissue-enriched acidic protein or neuronal axonal membrane protein NAP-22.
  • the full-length human BASP1 protein sequence (isomer 1) is 227 amino acids in length. An isomer produced by an alternative splicing is missing amino acids 88 to 141 from SEQ ID NO: 49 (isomer 1).
  • Table 3 provides the full-length sequences for the exemplary Scaffold Y disclosed herein ( i.e ., the MARCKS, MARCKSLl, and BASP1 proteins).
  • the mature BASP1 protein sequence is missing the first Met from SEQ ID NO: 49 and thus contains amino acids 2 to 227 of SEQ ID NO: 49.
  • the mature MARCKS and MARCKSLl proteins also lack the first Met from SEQ ID NOs: 47 and 48, respectively. Accordingly, the mature MARCKS protein contains amino acids 2 to 332 of SEQ ID NO: 47.
  • the mature MARCKSLl protein contains amino acids 2 to 227 of SEQ ID NO: 48.
  • Scaffold Y useful for the present disclosure comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 2 to 227 of SEQ ID NO: 49.
  • a Scaffold Y useful for the present disclosure comprises the amino acid sequence of SEQ ID NO: 49, except one amino acid mutation, two amino acid mutations, three amino acid mutations, four amino acid mutations, five amino acid mutations, six amino acid mutations, or seven amino acid mutations.
  • the mutations can be a substitution, an insertion, a deletion, or any combination thereof.
  • the protein sequence of any of SEQ ID NOs: 47-49 without Met at amino acid residue 1 of the SEQ ID NOs: 47-49 is sufficient to be a Scaffold Y for the present disclosure (e.g ., scaffold moiety linked to a FLT3L polypeptide.
  • a Scaffold Y useful for the present disclosure comprises a peptide with the GXKLSKKK, where X is alanine or any other amino acid (SEQ ID NO: 204).
  • an EV e.g., exosome, comprises a peptide with sequence of (G)(u) ⁇ (®/u)(S/A/G/N)(+)(+), wherein each parenthetical position represents an amino acid, and wherein p is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), x is any amino acid selected from the group consisting of (Asn, Gin, Ser, Thr, Asp, Glu, Lys, His, Arg), F is any amino acid selected from the group consisting of (Val, lie, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position
  • an exosome described herein comprises a peptide with sequence of ( ⁇ )(p)(C)(F/p)(p)(+)(+), wherein each parenthetical position represents an amino acid, and wherein p is any amino acid selected from the group consisting of (Pro, Gly, Ala, Ser), X is any amino acid, F is any amino acid selected from the group consisting of (Val, lie, Leu, Phe, Trp, Tyr, Met), and (+) is any amino acid selected from the group consisting of (Lys, Arg, His); and wherein position five is not (+) and position six is neither (+) nor (Asp or Glu). See Aasland et al., FEBS Letters 513 (2002) 141-144 for amino acid nomenclature.
  • the Scaffold X comprises an amino acid sequence 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 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NO: 47-49 without Met at amino acid residue 1 of the SEQ ID NOs: 47-49.
  • Scaffold Y-engineered EVs e.g, exosomes described herein can be produced from a cell transformed with a sequence set forth in SEQ ID NOs: 47-49 without Met at amino acid residue 1 of the SEQ ID NOs: 47-49.
  • the Scaffold Y protein useful for the present disclosure comprises an "N-terminus domain” (ND) and an "effector domain”(ED), wherein the ND and/or the ED are associated with the luminal surface of the EV, e.g., an exosome.
  • the Scaffold Y protein useful for the present disclosure comprises an intracellular domain, a transmembrane domain, and an extracellular domain; wherein the intracellular domain comprises an "N-terminus domain” (ND) and an "effector domain” (ED), wherein the ND and/or the ED are associated with the luminal surface of the EV, e.g, an exosome.
  • ND N-terminus domain
  • ED effector domain
  • the term "associated with” refers to the interaction between a scaffold protein with the luminal surface of the EV, e.g, and exosome, that does not involve covalent linking to a membrane component.
  • the scaffolds useful for the present disclosure can be associated with the luminal surface of the EV, e.g, via a lipid anchor (e.g, myristic acid), and/or a polybasic domain that interacts electrostatically with the negatively charged head of membrane phospholipids.
  • a lipid anchor e.g, myristic acid
  • a polybasic domain that interacts electrostatically with the negatively charged head of membrane phospholipids.
  • the Scaffold Y protein comprises an N-terminus domain (ND) and an effector domain (ED), wherein the ND is associated with the luminal surface of the EV and the ED are associated with the luminal surface of the EV by an ionic interaction, wherein the ED comprises at least two, at least three, at least four, at least five, at least six, or at least seven contiguous basic amino acids, e.g, lysines (Lys), in sequence.
  • ND N-terminus domain
  • ED effector domain
  • the ED comprises at least two, at least three, at least four, at least five, at least six, or at least seven contiguous basic amino acids, e.g, lysines (Lys), in sequence.
  • the Scaffold Y protein comprises an N-terminus domain (ND) and an effector domain (ED), wherein the ND is associated with the luminal surface of the EV, e.g, exosome, and the ED is associated with the luminal surface of the EV by an ionic interaction, wherein the ED comprises at least two, at least three, at least four, at least five, at least six, or at least seven contiguous basic amino acids, e.g, lysines (Lys), in sequence.
  • ND N-terminus domain
  • ED effector domain
  • the ND is associated with the luminal surface of the EV, e.g, an exosome, via lipidation, e.g, via myristoylation.
  • the ND has Gly at the N terminus.
  • the N-terminal Gly is myristoylated.
  • the ED is associated with the luminal surface of the EV, e.g, an exosome, by an ionic interaction. In some aspects, the ED is associated with the luminal surface of the EV, e.g, an exosome, by an electrostatic interaction, in particular, an attractive electrostatic interaction.
  • the ED comprises (i) a basic amino acid (e.g, lysine), or (ii) two or more basic amino acids (e.g, lysine) next to each other in a polypeptide sequence.
  • the basic amino acid is lysine (Lys; K), arginine (Arg, R), or Histidine (His, H).
  • the basic amino acid is (Lys)n, wherein n is an integer between 1 and 10.
  • the ED comprises at least a lysine and the ND comprises a lysine at the C terminus if the N terminus of the ED is directly linked to lysine at the C terminus of the ND, i.e., the lysine is in the N terminus of the ED and is fused to the lysine in the C terminus of the ND.
  • the ED comprises at least two lysines, at least three lysines, at least four lysines, at least five lysines, at least six lysines, or at least seven lysines when the N terminus of the ED is linked to the C terminus of the ND by a linker, e.g., one or more amino acids.
  • a linker e.g., one or more amino acids.
  • the ED comprises K, KK, KKK, KKKK (SEQ ID NO: 205), KKKKK (SEQ ID NO: 206), R, RR, RRR, RRRR (SEQ ID NO: 207); RRRRR (SEQ ID NO: 208), KR, RK, KKR, KRK, RKK, KRR, RRK, (K/R)(K/R)(K/R) (SEQ ID NO: 209), (K/R)(K/R)(K/R)(K/R)(K/R) (SEQ ID NO: 210), or any combination thereof.
  • the ED comprises KK, KKK, KKKK (SEQ ID NO: 205), KKKKK (SEQ ID NO: 206), or any combination thereof.
  • the ND comprises the amino acid sequence as set forth in G:X2:X3:X4:X5:X6, wherein G represents Gly; wherein represents a peptide bond; wherein each of the X2 to the X6 independently represents an amino acid; and wherein the X6 represents a basic amino acid.
  • the X6 amino acid is selected is selected from the group consisting of Lys, Arg, and His.
  • the X5 amino acid is selected from the group consisting of Pro, Gly, Ala, and Ser.
  • the X2 amino acid is selected from the group consisting of Pro, Gly, Ala, and Ser.
  • the X4 is selected from the group consisting of Pro, Gly, Ala, Ser, Val, lie, Leu, Phe, Trp, Tyr, Gin, and Met.
  • the Scaffold Y protein comprises an N-terminus domain (ND) and an effector domain (ED), wherein the ND comprises the amino acid sequence as set forth in G:X2:X3:X4:X5:X6, wherein G represents Gly; wherein represents a peptide bond; wherein each of the X2 to the X6 is independently an amino acid; wherein the X6 comprises a basic amino acid, and wherein the ED is linked to X6 by a peptide bond and comprises at least one lysine at the N terminus of the ED.
  • ND N-terminus domain
  • ED effector domain
  • the ND of the Scaffold Y protein comprises the amino acid sequence of G:X2:X3:X4:X5:X6, wherein G represents Gly; represents a peptide bond; the X2 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser; the X3 represents any amino acid; the X4 represents an amino acid selected from the group consisting of Pro, Gly, Ala, Ser, Val, lie, Leu, Phe, Trp, Tyr, Gin, and Met; the X5 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser; and the X6 represents an amino acid selected from the group consisting of Lys, Arg, and His.
  • the X3 amino acid is selected from the group consisting of Asn, Gin, Ser, Thr, Asp, Glu, Lys, His, and Arg.
  • the ND and ED are joined by a linker.
  • the linker comprises one or more amino acids.
  • the term "linker" refers to a peptide or polypeptide sequence (e.g ., a synthetic peptide or polypeptide sequence) or to a non-polypeptide, e.g., an alkyl chain.
  • two or more linkers can be linked in tandem.
  • linkers provide flexibility or prevent/ameliorate steric hindrances. Linkers are not typically cleaved; however, in certain aspects, such cleavage can be desirable.
  • a linker can comprise one or more protease-cleavable sites, which can be located within the sequence of the linker or flanking the linker at either end of the linker sequence.
  • the ED comprise at least two lysines, at least three lysines, at least four lysines, at least five lysines, at least six lysines, or at least seven lysines.
  • the linker is a peptide linker.
  • the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, 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, or at least about 100 amino acids.
  • the linker is a glycine/serine linker.
  • the peptide linker is glycine/serine linker according to the formula [(Gly)n-Ser]m where n is any integer from 1 to 100 and m is any integer from 1 to 100.
  • the glycine/serine linker is according to the formula [(Gly)x-Seryjz wherein x in an integer from 1 to 4, y is 0 or 1, and z is an integer from 1 to 50.
  • the peptide linker comprises the sequence Gn, where n can be an integer from 1 to 100.
  • the peptide linker can comprise the sequence (GlyAla)n, wherein n is an integer between 1 and 100. In other aspects, the peptide linker can comprise the sequence (GlyGlySer)n, wherein n is an integer between 1 and 100.
  • the peptide linker is synthetic, i.e., non-naturally occurring.
  • a peptide linker includes peptides (or polypeptides) (e.g, natural or non-naturally occurring peptides) which comprise an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature.
  • the peptide linker can comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g comprising a mutation such as an addition, substitution or deletion).
  • the peptide linker can comprise non-naturally occurring amino acids.
  • the peptide linker can comprise naturally occurring amino acids occurring in a linear sequence that does not occur in nature.
  • the peptide linker can comprise a naturally occurring polypeptide sequence.
  • the present disclosure also provides an isolated extracellular vesicle (EV), e.g., an exosome, comprising a FLT3L polypeptide linked to a Scaffold Y protein, wherein the Scaffold Y protein comprises ND— ED, wherein: ND comprises G:X2:X3:X4:X5:X6; wherein: G represents Gly; represents a peptide bond; X2 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser; X3 represents any amino acid; X4 represents an amino acid selected from the group consisting of Pro, Gly, Ala, Ser,Val, lie, Leu, Phe, Trp, Tyr, Glu, and Met; X5 represents an amino acid selected from the group consisting of Pro, Gly, Ala, and Ser; X6 represents an amino acid selected from the group consisting of Lys, Arg, and His; "— " represents an optional linker; and ED is an effector domain comprising (EV),
  • the X2 amino acid is selected from the group consisting of Gly and Ala.
  • the X3 amino acid is Lys.
  • the X4 amino acid is Leu or Glu.
  • the X5 amino acid is selected from the group consisting of Ser and Ala.
  • the X6 amino acid is Lys.
  • the X2 amino acid is Gly, Ala, or Ser; the X3 amino acid is Lys or Glu; the X4 amino acid is Leu, Phe, Ser, or Glu; the X5 amino acid is Ser or Ala; and X6 amino acid is Lys.
  • the "— " linker comprises a peptide bond or one or more amino acids.
  • the ED in the scaffold protein comprises Lys (K), KK, KKK, KKKK (SEQ ID NO: 205), KKKKK (SEQ ID NO: 206), Arg (R), RR, RRR, RRRR (SEQ ID NO: 207); RRRRR (SEQ ID NO: 208), KR, RK, KKR, KRK, RKK, KRR, RRK, (K/R)(K/R)(K/R) (SEQ ID NO: 209), (K/R)(K/R)(K/R)(K/R)(K/R) (SEQ ID NO: 210), or any combination thereof.
  • the Scaffold Y protein comprises an amino acid sequence selected from the group consisting of (i) GGKLSKK (SEQ ID NO: 211), (ii) GAKLSKK (SEQ ID NO: 212), (iii) GGKQSKK (SEQ ID NO: 213), (iv) GGKLAKK (SEQ ID NO: 214), or (v) any combination thereof.
  • the ND in the Scaffold Y protein comprises an amino acid sequence selected from the group consisting of (i) GGKLSK (SEQ ID NO: 215), (ii) GAKLSK (SEQ ID NO: 216), (iii) GGKQSK (SEQ ID NO: 217), (iv) GGKLAK (SEQ ID NO: 218), or (v) any combination thereof and the ED in the scaffold protein comprises K, KK, KKK, KKKG (SEQ ID NO: 219), KKKGY (SEQ ID NO: 220), KKKGYN (SEQ ID NO: 221), KKKGYNV (SEQ ID NO: 222), KKKGYNVN (SEQ ID NO: 223), KKKGY S (SEQ ID NO: 224), KKKGYG (SEQ ID NO: 225), KKKGYGG (SEQ ID NO: 226), KKKGS (SEQ ID NO: 227), KKKGSG (SEQ ID NO: 215), (
  • the polypeptide sequence of a Scaffold Y protein useful for the present disclosure consists of an amino acid sequence selected from the group consisting of (i) GGKLSKK (SEQ ID NO: 211), (ii) GAKLSKK (SEQ ID NO: 212), (iii) GGKQSKK (SEQ ID NO: 213), (iv) GGKLAKK (SEQ ID NO: 214), or (v) any combination thereof.
  • the Scaffold Y protein comprises an amino acid sequence selected from the group consisting of (i) GGKLSKKK (SEQ ID NO: 238), (ii) GGKLSKK S (SEQ ID NO: 239), (iii) GAKLSKKK (SEQ ID NO: 240), (iv) GAKLSKK S (SEQ ID NO: 241), (v) GGKQSKKK (SEQ ID NO: 242), (vi) GGKQSKK S (SEQ ID NO: 243), (vii) GGKLAKKK (SEQ ID NO: 244), (viii) GGKLAKKS (SEQ ID NO: 245), and (ix) any combination thereof.
  • the polypeptide sequence of a Scaffold Y protein useful for the present disclosure consists of an amino acid sequence selected from the group consisting of (i) GGKLSKKK (SEQ ID NO: 238), (ii) GGKLSKK S (SEQ ID NO: 239), (iii) GAKLSKKK (SEQ ID NO: 240), (iv) GAKLSKK S (SEQ ID NO: 241), (v) GGKQSKKK (SEQ ID NO: 242), (vi) GGKQSKK S (SEQ ID NO: 243), (vii) GGKLAKKK (SEQ ID NO: 244), (viii) GGKLAKKS (SEQ ID NO: 245), and (ix) any combination thereof.
  • the Scaffold Y protein is at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 50, at least about 46, at least about 47, at least about 48, at least about 49, 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 85, at least about 90, at least about 95,
  • the Scaffold Y protein is between about 5 and about 10, between about 10 and about 20, between about 20 and about 30, between about 30 and about 40, between about 40 and about 50, between about 50 and about 60, between about 60 and about 70, between about 70 and about 80, between about 80 and about 90, between about 90 and about 100, between about 100 and about 110, between about 110 and about 120, between about 120 and about 130, between about 130 and about 140, between about 140 and about 150, between about 150 and about 160, between about 160 and about 170, between about 170 and about 180, between about 180 and about 190, between about 190 and about 200, between about 200 and about 210, between about 210 and about 220, between about 220 and about 230, between about 230 and about 240, between about 240 and about 250, between about 250 and about 260, between about 260 and about 270, between about 270 and about 280, between about 280 and about 290, between about 290 and about 300, between about 300 and about 310, between about 310, between about 310, between
  • the Scaffold Y protein comprises (i) GGKLSKKKKGYNVN (SEQ ID NO: 246), (ii) GAKLSKKKKGYNVN (SEQ ID NO: 247), (iii) GGKQ SKKKKGYNVN (SEQ ID NO: 248), (iv) GGKLAKKKKGYNVN (SEQ ID NO: 249), (v) GGKL SKKKKGY S GG (SEQ ID NO: 250), (vi) GGKL SKKKKGS GGS (SEQ ID NO: 251), (vii) GGKLSKKKKSGGSG (SEQ ID NO: 252), (viii) GGKL SKKKS GGS GG (SEQ ID NO: 253), (ix) GGKL SKK S GGS GGS (SEQ ID NO: 254), (x) GGKLSKSGGSGGSV (SEQ ID NO: 255), or (xi) GAKK SKKRF SFKK
  • the polypeptide sequence of a Scaffold Y protein useful for the present disclosure consists of (i) GGKLSKKKKGYNVN (SEQ ID NO: 246), (ii) GAKLSKKKKGYNVN (SEQ ID NO: 247), (iii) GGKQ SKKKKGYNVN (SEQ ID NO: 248), (iv) GGKLAKKKKGYNVN (SEQ ID NO: 249), (v) GGKL SKKKKGY S GG (SEQ ID NO: 250), (vi) GGKLSKKKKGSGGS (SEQ ID NO: 251), (vii) GGKLSKKKKSGGSG (SEQ ID NO: 252), (viii) GGKL SKKKS GGS GG (SEQ ID NO: 253), (ix) GGKL SKK S GGS GGS (SEQ ID NO: 254), (x) GGKLSKSGGSGGSV (SEQ ID NO: 255),
  • Non-limiting examples of the Scaffold Y protein useful for the present disclosure are listed below.
  • the Scaffold Y protein comprises an amino acid sequence set forth in Table 4.
  • the Scaffold Y protein consists of an amino acid sequence set forth in Table 4.
  • the Scaffold Y protein useful for the present disclosure does not contain an N-terminal Met.
  • the Scaffold Y protein comprises a lipidated amino acid, e.g ., a myristoylated amino acid, at the N-terminus of the scaffold protein, which functions as a lipid anchor.
  • the amino acid residue at the N-terminus of the scaffold protein is Gly.
  • the presence of an N-terminal Gly is an absolute requirement for N- myristoylation.
  • the amino acid residue at the N-terminus of the scaffold protein is synthetic.
  • the amino acid residue at the N-terminus of the scaffold protein is a glycine analog, e.g ., allylglycine, butylglycine, or propargylglycine.
  • the lipid anchor can be any lipid anchor known in the art, e.g. , palmitic acid or glycosylphosphatidylinositols.
  • lipid anchors known in the art, e.g. , palmitic acid or glycosylphosphatidylinositols.
  • some other fatty acids including shorter-chain and unsaturated, can be attached to the N-terminal glycine.
  • myristate has been reported to be attached posttranslationally to internal serine/threonine or tyrosine residues via a hydroxyester linkage.
  • Membrane anchors known in the art are presented in the following table:
  • extracellular vesicles (EVs) of the present disclosure can comprises one or more linkers that link a molecule of interest (e.g, a FLT3L polypeptide) to the EVs (e.g, to the exterior surface or on the luminal surface).
  • a FLT3L polypeptide is linked to the EVs directly or via a scaffold moiety (e.g, Scaffold X or Scaffold Y).
  • the FLT3L polypeptide is linked to the scaffold moiety by a linker.
  • the FLT3L polypeptide is linked to the second scaffold moiety by a linker.
  • a FLT3L polypeptide is linked to the exterior surface of an exosome via Scaffold X. In further aspects, a FLT3L polypeptide is linked to the luminal surface of an exosome via Scaffold X or Scaffold Y.
  • the linker can be any chemical moiety known in the art.
  • linker refers to a peptide or polypeptide sequence (e.g ., a synthetic peptide or polypeptide sequence) or to a non-polypeptide, e.g., an alkyl chain.
  • two or more linkers can be linked in tandem. When multiple linkers are present, each of the linkers can be the same or different.
  • linkers provide flexibility or prevent/ameliorate steric hindrances. Linkers are not typically cleaved; however, in certain aspects, such cleavage can be desirable.
  • a linker can comprise one or more protease-cleavable sites, which can be located within the sequence of the linker or flanking the linker at either end of the linker sequence.
  • the linker is a peptide linker.
  • the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, 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, or at least about 100 amino acids v
  • the peptide linker is synthetic, i.e., non-naturally occurring.
  • a peptide linker includes peptides (or polypeptides) (e.g, natural or non-naturally occurring peptides) which comprise an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature.
  • the peptide linker can comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g, comprising a mutation such as an addition, substitution or deletion).
  • Linkers can be susceptible to cleavage ("cleavable linker”) thereby facilitating release of the biologically active molecule (e.g, a FLT3L polypeptide).
  • the linker is a "reduction-sensitive linker.” In some aspects, the reduction-sensitive linker contains a disulfide bond. In some aspects, the linker is an "acid labile linker.” In some aspects, the acid labile linker contains hydrazone. Suitable acid labile linkers also include, for example, a cis-aconitic linker, a hydrazide linker, a thiocarbamoyl linker, or any combination thereof.
  • the linker comprises a non-cleavable linker.
  • EVs e.g ., exosomes
  • EVs can be produced from a cell grown in vitro or a body fluid of a subject.
  • various producer cells e.g, HEK293 cells, CHO cells, and MSCs, can be used.
  • a producer cell is not a dendritic cell, macrophage, B cell, mast cell, neutrophil, Kupffer-Browicz cell, cell derived from any of these cells, or any combination thereof.
  • the producer cell can be genetically modified to comprise exogenous sequences encoding a FLT3L polypeptide to produce EVs described herein.
  • the genetically-modified producer cell can contain the exogenous sequence by transient or stable transformation.
  • the exogenous sequence can be transformed as a plasmid.
  • the exogenous sequence is a vector.
  • the exogenous sequences can be stably integrated into a genomic sequence of the producer cell, at a targeted site or in a random site.
  • a stable cell line is generated for production of lumen-engineered exosomes.
  • the exogenous sequences can be inserted into a genomic sequence of the producer cell, located within, upstream (5’ -end) or downstream (3’ -end) of an endogenous sequence encoding an exosome protein.
  • Various methods known in the art can be used for the introduction of the exogenous sequences into the producer cell.
  • cells modified using various gene editing methods e.g, methods using a homologous recombination, transposon-mediated system, loxP-Cre system, CRISPR/Cas9 or TALEN are within the scope of the present disclosure.
  • the exogenous sequences can comprise a sequence encoding a scaffold moiety disclosed herein or a fragment or variant thereof.
  • An extra copy of the sequence encoding a scaffold moiety can be introduced to produce an exosome described herein (e.g, having a higher density of a scaffold moiety on the surface or on the luminal surface of the EV, e.g. , exosome).
  • An exogenous sequence encoding a modification or a fragment of a scaffold moiety can be introduced to produce a lumen-engineered and/or surface-engineered exosome containing the modification or the fragment of the scaffold moiety.
  • a producer cell can be modified, e.g, transfected, with one or more vectors encoding a scaffold moiety linked to a FLT3L polypeptide.
  • a producer cell disclosed herein is further modified to comprise an additional exogenous sequence.
  • an additional exogenous sequence can be introduced to modulate endogenous gene expression, or produce an exosome including a certain polypeptide as a payload (e.g, a FLT3L polypeptide).
  • the producer cell is modified to comprise two exogenous sequences, one encoding a scaffold moiety (e.g., Scaffold X and/or Scaffold Y), or a variant or a fragment thereof, and the other encoding a payload e.g, a FLT3L polypeptide).
  • the producer cell is modified to comprise two exogenous sequences, one encoding a scaffold moiety disclosed herein, or a variant or a fragment thereof, and the other encoding a protein conferring the additional functionalities to exosomes.
  • the producer cell is further modified to comprise one, two, three, four, five, six, seven, eight, nine, or ten or more additional exogenous sequences.
  • EVs e.g, exosomes, of the present disclosure (e.g, surface- engineered and/or lumen-engineered exosomes) can be produced from a cell transformed with a sequence encoding a full-length, mature scaffold moiety disclosed herein or a scaffold moiety linked to a FLT3L polypeptide.
  • Any of the scaffold moieties described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome or other exogenous nucleic acid, such as a synthetic messenger RNA (mRNA).
  • mRNA synthetic messenger RNA
  • compositions comprising an EV, e.g, exosome, of the present disclosure having the desired degree of purity, and a pharmaceutically acceptable carrier or excipient, in a form suitable for administration to a subject.
  • Pharmaceutically acceptable excipients or carriers can be determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising a plurality of extracellular vesicles. (See, e.g, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)).
  • a pharmaceutical composition comprises one or more therapeutic agents and an exosome described herein.
  • the EVs e.g ., exosomes
  • the FLT3L polypeptide and the one or more additional therapeutic agents for the present disclosure can be administered in the same EV.
  • the FLT3L polypeptide and the one or more additional therapeutic agents for the present disclosure are administered in different EVs.
  • the present disclosure includes a pharmaceutical composition comprising an EV comprising an FLT3L polypeptide and an EV comprising an additional therapeutic agent.
  • the pharmaceutical composition comprising the EV e.g. , exosome
  • the pharmaceutical composition comprising the EV is administered prior to administration of the additional therapeutic agent(s).
  • the pharmaceutical composition comprising the EV e.g. , exosome
  • the pharmaceutical composition comprising the EV, e.g. , exosome is administered concurrently with the additional therapeutic agent(s).
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g, animals or humans) at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutacetate,
  • Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the extracellular vesicles described herein, use thereof in the compositions is contemplated. Supplementary therapeutic agents can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • the EVs e.g.
  • exosomes can be administered by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular route or as inhalants.
  • the pharmaceutical composition comprising exosomes is administered intravenously, e.g. by injection.
  • the EVs, e.g. , exosomes can optionally be administered in combination with other therapeutic agents that are at least partly effective in treating the disease, disorder or condition for which the EVs, e.g. , exosomes, are intended.
  • Solutions or suspensions can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition is generally sterile and fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, e.g. , water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, e.g, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be added to the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the EVs, e.g, exosomes, in an effective amount and in an appropriate solvent with one or more ingredients enumerated herein or known in the art, as desired.
  • dispersions are prepared by incorporating the EVs, e.g. , exosomes, into a sterile vehicle that contains a basic dispersion medium and any desired other ingredients.
  • EVs e.g. , exosomes
  • methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the EVs e.g. , exosomes
  • compositions comprising exosomes can also be by transmucosal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, e.g. , for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of, e.g. , nasal sprays.
  • the pharmaceutical composition comprising EVs, e.g, exosomes is administered intravenously into a subject that would benefit from the pharmaceutical composition.
  • the composition is administered to the lymphatic system, e.g., by intralymphatic injection or by intranodal injection (see e.g., Senti el al, PNAS 105( 46): 17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus, or into the liver.
  • the pharmaceutical composition comprising exosomes is administered as a liquid suspension.
  • the pharmaceutical composition is administered as a formulation that is capable of forming a depot following administration.
  • the depot slowly releases the EVs, e.g, exosomes, into circulation, or remains in depot form.
  • compositions are highly purified to be free of contaminants, are biocompatible and not toxic, and are suited to administration to a subject. If water is a constituent of the carrier, the water is highly purified and processed to be free of contaminants, e.g, endotoxins.
  • the pharmaceutically-acceptable carrier can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro- crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto.
  • the pharmaceutical composition can further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and/or a preservative.
  • compositions described herein comprise the EVs, e.g ., exosomes, described herein and optionally a pharmaceutically active or therapeutic agent.
  • the therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent.
  • Dosage forms are provided that comprise a pharmaceutical composition comprising the EVs, e.g. , exosomes, described herein.
  • the dosage form is formulated as a liquid suspension for intravenous injection.
  • the dosage form is formulated as a liquid suspension for intratumoral injection.
  • the preparation of exosomes is subjected to radiation, e.g. , X rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays in order to damage residual replication-competent nucleic acids.
  • radiation e.g. , X rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays in order to damage residual replication-competent nucleic acids.
  • the preparation of exosomes is subjected to gamma irradiation using an irradiation dose of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100 kGy.
  • the preparation of exosomes is subjected to X-ray irradiation using an irradiation dose of more than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or greater than 10000 mSv.
  • kits comprising one or more exosomes described herein.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more exosomes provided herein, optional an instruction for use.
  • the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.
  • the kit further comprises instructions to administer the EV according to any method disclosed herein.
  • the kit is for use in the treatment of a disease or condition associated with hematopoiesis. VII. Methods of Producing EVs
  • the present disclosure is also directed to methods of producing EVs described herein.
  • the method comprises: obtaining the EV, e.g ., exosome from a producer cell, wherein the producer cell contains one or more components of the EV, e.g. , exosome (e.g, a FLT3L polypeptide); and optionally isolating the obtained EV, e.g, exosome.
  • the method comprises: modifying a producer cell by introducing one or more components of an EV disclosed herein (e.g, a FLT3L polypeptide); obtaining the EV, e.g, exosome, from the modified producer cell; and optionally isolating the obtained EV, e.g., exosome.
  • the method comprises: obtaining an EV from a producer cell; isolating the obtained EV; and modifying the isolated EV.
  • the method further comprises formulating the isolated EV into a pharmaceutical composition.
  • a method of producing an EV comprises modifying a producer cell with one or more moieties (e.g, a FLT3L polypeptide).
  • the one or more moieties comprise a FLT3L polypeptide.
  • the one or more moieties further comprise a scaffold moiety disclosed herein (e.g, Scaffold X or Scaffold
  • the producer cell can be a mammalian cell line, a plant cell line, an insect cell line, a fungi cell line, or a prokaryotic cell line.
  • the producer cell is a mammalian cell line.
  • mammalian cell lines include: a human embryonic kidney (HEK) cell line, a Chinese hamster ovary (CHO) cell line, an HT-1080 cell line, a HeLa cell line, a PERC-6 cell line, a CEVEC cell line, a fibroblast cell line, an amniocyte cell line, an epithelial cell line, a mesenchymal stem cell (MSC) cell line, and combinations thereof.
  • the mammalian cell line comprises HEK-293 cells, BJ human foreskin fibroblast cells, fHDF fibroblast cells, AGE.HN ® neuronal precursor cells, CAP ® amniocyte cells, adipose mesenchymal stem cells, RPTEC/TERT1 cells, or combinations thereof.
  • the producer cell is a primary cell.
  • the primary cell can be a primary mammalian cell, a primary plant cell, a primary insect cell, a primary fungi cell, or a primary prokaryotic cell.
  • the producer cell is not an immune cell, such as an antigen presenting cell, a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a T helper cell, or a regulatory T cell (Treg cell).
  • the producer cell is not an antigen presenting cell (e.g ., dendritic cells, macrophages, B cells, mast cells, neutrophils, Kupffer-Browicz cell, or a cell derived from any such cells).
  • the one or more moieties can be a transgene or mRNA, and introduced into the producer cell by transfection, viral transduction, electroporation, extrusion, sonication, cell fusion, or other methods that are known to the skilled in the art.
  • the one or more moieties is introduced to the producer cell by transfection.
  • the one or more moieties can be introduced into suitable producer cells using synthetic macromolecules, such as cationic lipids and polymers (Papapetrou et al ., Gene Therapy 12: S118-S130 (2005)).
  • the cationic lipids form complexes with the one or more moieties through charge interactions.
  • the positively charged complexes bind to the negatively charged cell surface and are taken up by the cell by endocytosis.
  • a cationic polymer can be used to transfect producer cells.
  • the cationic polymer is polyethylenimine (PEI).
  • chemicals such as calcium phosphate, cyclodextrin, or polybrene, can be used to introduce the one or more moieties to the producer cells.
  • the one or more moieties can also be introduced into a producer cell using a physical method such as particle-mediated transfection, "gene gun", biolistics, or particle bombardment technology (Papapetrou et al ., Gene Therapy 12: S118-S130 (2005)).
  • a reporter gene such as, for example, beta-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein can be used to assess the transfection efficiency of the producer cell.
  • the one or more moieties are introduced to the producer cell by viral transduction.
  • viruses can be used as gene transfer vehicles, including moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses, and spumaviruses.
  • the viral mediated gene transfer vehicles comprise vectors based on DNA viruses, such as adenovirus, adeno-associated virus and herpes virus, as well as retroviral based vectors.
  • the one or more moieties are introduced to the producer cell by electroporation. Electroporation creates transient pores in the cell membrane, allowing for the introduction of various molecules into the cell.
  • DNA and RNA as well as polypeptides and non-polypeptide therapeutic agents can be introduced into the producer cell by electroporation.
  • the one or more moieties introduced to the producer cell by microinjection. In some aspects, a glass micropipette can be used to inject the one or more moieties into the producer cell at the microscopic level.
  • the one or more moieties are introduced to the producer cell by extrusion.
  • the one or more moieties are introduced to the producer cell by sonication.
  • the producer cell is exposed to high intensity sound waves, causing transient disruption of the cell membrane allowing loading of the one or more moieties.
  • the one or more moieties are introduced to the producer cell by cell fusion. In some aspects, the one or more moieties are introduced by electrical cell fusion. In other aspects, polyethylene glycol (PEG) is used to fuse the producer cells. In further aspects, sendai virus is used to fuse the producer cells.
  • PEG polyethylene glycol
  • the one or more moieties are introduced to the producer cell by hypotonic lysis.
  • the producer cell can be exposed to low ionic strength buffer causing them to burst allowing loading of the one or more moieties.
  • controlled dialysis against a hypotonic solution can be used to swell the producer cell and to create pores in the producer cell membrane. The producer cell is subsequently exposed to conditions that allow resealing of the membrane.
  • the one or more moieties are introduced to the producer cell by detergent treatment.
  • producer cell is treated with a mild detergent which transiently compromises the producer cell membrane by creating pores allowing loading of the one or more moieties. After producer cells are loaded, the detergent is washed away thereby resealing the membrane.
  • the one or more moieties introduced to the producer cell by receptor mediated endocytosis are introduced to the producer cell by receptor mediated endocytosis.
  • producer cells have a surface receptor which upon binding of the one or more moieties induces internalization of the receptor and the associated moieties.
  • the one or more moieties are introduced to the producer cell by filtration.
  • the producer cells and the one or more moieties can be forced through a filter of pore size smaller than the producer cell causing transient disruption of the producer cell membrane and allowing the one or more moieties to enter the producer cell.
  • the producer cell is subjected to several freeze thaw cycles, resulting in cell membrane disruption allowing loading of the one or more moieties.
  • a method of producing an EV comprises modifying the isolated EV by directly introducing one or more moieties into the EVs.
  • the one or more moieties comprise a FLT3L polypeptide.
  • the one or more moieties comprise a scaffold moiety disclosed herein (e.g., Scaffold X or Scaffold Y).
  • the one or more moieties are introduced to the EV by transfection.
  • the one or more moieties can be introduced into the EV using synthetic macromolecules such as cationic lipids and polymers (Papapetrou et al, Gene Therapy 12: S118-S130 (2005)).
  • chemicals such as calcium phosphate, cyclodextrin, or polybrene, can be used to introduce the one or more moieties to the EV.
  • the one or more moieties are introduced to the EV by electroporation.
  • EVs are exposed to an electrical field which causes transient holes in the EV membrane, allowing loading of the one or more moieties.
  • the one or more moieties are introduced to the EV by microinjection.
  • a glass micropipette can be used to inject the one or more moieties directly into the EV at the microscopic level.
  • the one or more moieties are introduced to the EV by extrusion.
  • the one or more moieties are introduced to the EV by sonication.
  • EVs are exposed to high intensity sound waves, causing transient disruption of the EV membrane allowing loading of the one or more moieties.
  • one or more moieties can be conjugated to the surface of the EV. Conjugation can be achieved chemically or enzymatically, by methods known in the art.
  • the EV comprises one or more moieties that are chemically conjugated. Chemical conjugation can be accomplished by covalent bonding of the one or more moieties to another molecule, with or without use of a linker. The formation of such conjugates is within the skill of artisans and various techniques are known for accomplishing the conjugation, with the choice of the particular technique being guided by the materials to be conjugated.
  • polypeptides are conjugated to the EV.
  • non-polypeptides such as lipids, carbohydrates, nucleic acids, and small molecules, are conjugated to the EV.
  • the one or more moieties are introduced to the EV by hypotonic lysis.
  • the EVs can be exposed to low ionic strength buffer causing them to burst allowing loading of the one or more moieties.
  • controlled dialysis against a hypotonic solution can be used to swell the EV and to create pores in the EV membrane. The EV is subsequently exposed to conditions that allow resealing of the membrane.
  • the one or more moieties are introduced to the EV by detergent treatment.
  • extracellular vesicles are treated with a mild detergent which transiently compromises the EV membrane by creating pores allowing loading of the one or more moieties. After EVs are loaded, the detergent is washed away thereby resealing the membrane.
  • the one or more moieties are introduced to the EV by receptor mediated endocytosis.
  • EVs have a surface receptor which upon binding of the one or more moieties induces internalization of the receptor and the associated moieties.
  • the one or more moieties are introduced to the EV by mechanical firing.
  • extracellular vesicles can be bombarded with one or more moieties attached to a heavy or charged particle such as gold microcarriers.
  • the particle can be mechanically or electrically accelerated such that it traverses the EV membrane.
  • extracellular vesicles are subjected to several freeze thaw cycles, resulting in EV membrane disruption allowing loading of the one or more moieties.
  • VILC Methods of Isolating EV, e.g., Exosome
  • methods of producing EVs disclosed herein comprises isolating the EV from the producer cells.
  • the EVs released by the producer cell into the cell culture medium it is contemplated that all known manners of isolation of EVs are deemed suitable for use herein.
  • physical properties of EVs can be employed to separate them from a medium or other source material, including separation on the basis of electrical charge (e.g., electrophoretic separation), size (e.g, filtration, molecular sieving, etc.), density (e.g, regular or gradient centrifugation), Svedberg constant (e.g, sedimentation with or without external force, etc.).
  • isolation can be based on one or more biological properties, and include methods that can employ surface markers (e.g, for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non specific ligand binding, affinity purification etc.).
  • surface markers e.g, for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non specific ligand binding, affinity purification etc.
  • Isolation and enrichment can be done in a general and non-selective manner, typically including serial centrifugation.
  • isolation and enrichment can be done in a more specific and selective manner, such as using EV or producer cell-specific surface markers.
  • specific surface markers can be used in immunoprecipitation, FACS sorting, affinity purification, and magnetic separation with bead-bound ligands.
  • size exclusion chromatography can be utilized to isolate the EVs. Size exclusion chromatography techniques are known in the art. Exemplary, non-limiting techniques are provided herein.
  • a void volume fraction is isolated and comprises the EVs of interest.
  • the EVs can be further isolated after chromatographic separation by centrifugation techniques (of one or more chromatography fractions), as is generally known in the art.
  • density gradient centrifugation can be utilized to further isolate the extracellular vesicles.
  • the isolation of EVs can involve combinations of methods that include, but are not limited to, differential centrifugation, size-based membrane filtration, immunoprecipitation, FACS sorting, and magnetic separation.
  • Example 1 Flow Cytometry Analysis of a THP-1 Cell Population Containing pERK
  • FLT3L constructs were prepared comprising FLT3L fused to an exosome protein (FIGs. 1A-1B and 2A-2B).
  • FLT3L was fused as either a monomer (997, 998, 999, 1000, 1001, and 1251) or as a forced dimer (1081, 1082, and 1252) to a full length PrX (1000, 1001, and 1082), a fragment of PrX (997, 998, 999, and 1081), or to CD81 (1251 and 1252) .
  • THP-1 cells were harvested, diluted, and plated at a density of 100,000 cells per well.
  • a fixation buffer was warmed to 37C, and True-Phos Perm Buffer was cooled to -20C.
  • Cells were incubated with a stimulation cocktail comprising PMA and lonomycin, and following a 10 minute incubation, an equal volume of fixation buffer is added to each well, mixed, and incubated at 37C for 15 minutes. After incubation, the samples are centrifuged at 350 x g for 5 minutes, and the supernatant is decanted.
  • the FLT3L constructs 1001 and 1082 localized to exosomes, as shown in FIG. 3 and were found to have the highest activity of the tested samples (normalized to rhFLT3L as 100% activity), based on phosphorylation results as analyzed by an in-vitro activity assay (FIGs. 4A-4B).
  • FIGs. 6A and 6B The phosphorylation results as analyzed by cytometry can be seen in FIGs. 6A and 6B.
  • the results in FIGs. 6A and 6B were also normalized to rhFLT3L activity.
  • the pCB-1082 showed a signal higher than pCB-1001 as seen in FIG 6A.
  • a higher percentage of phosphorylated ERK was also seen at particle concentrations above approximately 10 10 as seen in FIG. 6B.
  • a 50% effective concentration analysis can be seen in FIG. 7A with respect the tested concentrations (particles/mL) for samples pCB-1001, pCB-1082, and rhFLT3L.
  • a signal to noise analysis of two assays used to evaluate the samples, the pERK/THP-1 assay and the CD1 lc + XCR1 + assay described in Example 2 can be seen in FIG. 7B with respect the tested concentrations for samples pCB-1001, pCB-1082, and rhFLT3L.
  • the higher values of the bone marrow assay indicated in FIG. 7B support a higher sensitivity of the assay as compared to the pERK/THP-1 assay.
  • Example 2 Flow Cytometry Analysis of a Cell Population Containing CD1 lc + XCR1 + Cells
  • pCB-1082 had a higher CD45 + IA/IE + count as compared to pCB-1001 or rhFLT3L as seen in FIGs. 8A-8D.

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Abstract

La présente invention concerne des vésicules extracellulaires, par exemple, des exosomes, comprenant un polypeptide FLT3L. L'invention concerne également des procédés d'induction d'une hématopoïèse chez un sujet en ayant besoin, comprenant l'administration d'une vésicule extracellulaire comprenant un polypeptide FLT3L.
PCT/US2020/040746 2019-07-03 2020-07-02 Procédés d'induction de l'hématopoïèse WO2021003425A1 (fr)

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