WO2023103963A1 - Cellules modifiées et leurs utilisations pour l'administration d'agents - Google Patents

Cellules modifiées et leurs utilisations pour l'administration d'agents Download PDF

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WO2023103963A1
WO2023103963A1 PCT/CN2022/136600 CN2022136600W WO2023103963A1 WO 2023103963 A1 WO2023103963 A1 WO 2023103963A1 CN 2022136600 W CN2022136600 W CN 2022136600W WO 2023103963 A1 WO2023103963 A1 WO 2023103963A1
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cell
agent
sortase
phospholipid
cells
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Xiaofei GAO
Yanjie HUANG
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Westlake Therapeutics (Shanghai) Co., Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
    • A61K49/0047—Green fluorescent protein [GFP]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0097—Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
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    • 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
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    • 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
    • C12N5/0641—Erythrocytes
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G01N33/555—Red blood cell
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/445—Staphylococcus aureus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/2207—Sortase A (3.4.22.70)

Definitions

  • the present disclosure relates generally to modified cells, and more particularly to membrane modified cells via phospholipid and use of the same for delivering agents such as drugs and probes.
  • Red blood cells (RBCs) , the most common cell type in the human body, have been widely investigated as an ideal in vivo drug delivery system for over three decades due to their unique biological properties, including: (i) widespread circulation range throughout the body; (ii) good biocompatibility as a biological material with long in vivo survival time; (iii) large surface to volume ratio; and (iv) no nucleus, mitochondria, and other cellular organelles.
  • RBCs have been developed as drug delivery carriers by direct encapsulation, noncovalent attachment of foreign peptides, or through attachment of proteins by fusion to antibodies specific for RBC surface proteins. It has been demonstrated that such modified RBCs have limitations for applications in vivo. For instance, encapsulation will disrupt cell membranes, which subsequently affects in vivo survival rates of engineered cells. In addition, the non-covalent attachment of polymeric particles to RBCs dissociates readily, and the payloads will be rapidly degraded in vivo.
  • Bacterial sortases are transpeptidases capable of modifying proteins in a covalent and site-specific manner (see J. M. Antos, et al., “Site-Specific Protein Labeling via Sortase-Mediated transpeptidation, ” 2017) .
  • Wild type sortase A from Staphylococcus aureus (wt SrtA) recognizes an LPXTG motif and cleaves between the threonine and glycine to form a covalent acyl-enzyme intermediate between the enzyme and the substrate protein. This intermediate is resolved by a nucleophilic attack by a peptide or protein having three consecutive glycine residues (3 ⁇ glycines, G 3 ) at the N-terminus.
  • the disclosure provides a cell linker.
  • the cell linker comprises a structure of GlymXn-optional linker-phospholipid, wherein the phospholipid is incorporated into the cell membrane of the cell, in which Glym represents m glycines with m preferably being 1-5, Xn represents n spacing amino acids with n preferably being 0-10, and the optional linker is preferably a PEG linker.
  • the phospholipid is a PE series phospholipid, such as 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) , 1, 2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) , and 1, 2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) .
  • DSPE 2-distearoyl-sn-glycero-3-phosphoethanolamine
  • DEPE 2-dielaidoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DMPE 2-dimyristoyl-sn-glycero-3-phosphoethanolamine
  • the disclosure provides a cell modified by an agent, wherein the agent is conjugated to a phospholipid via a sortase recognition motif, and the phospholipid with the conjugated agent is incorporated into the cell membrane of the cell.
  • the phospholipid with the conjugated agent comprises a structure of A 1 -L 1 -Gly m X n -optional linker-phospholipid, in which A 1 represents the agent, L 1 represents the residual part of a sortase recognition motif after a sortase-mediated reaction, Gly m represents m glycines with m preferably being an integer from 1 to 5, X n represents n spacing amino acids with n preferably being an integer from 0 to 10, and the optional linker is preferably a PEG linker.
  • the sortase recognition motif comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X is any amino acid; preferably the sortase recognition motif is LPETG.
  • the sortase recognition motif comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein *represents the optionally substituted hydroxyl carboxylic acid described above, and X and Y independently represent any amino acid.
  • the sortase recognition motif comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, preferably the sortase recognition motif is LPET*G, with *being 2-hydroxyacetic acid.
  • L 1 is selected from the group consisting of LPXT, LPXA, LPXS, LPXL, LPXV, LGXT, LAXT, LSXT, NPXT, MPXT, IPXT, SPXT, VPXT, and YPXR, with X being any amino acid; , preferably L 1 is LPET.
  • the phospholipid is a PE series phospholipid, such as 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) , 1, 2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) , and 1, 2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) .
  • DSPE 2-distearoyl-sn-glycero-3-phosphoethanolamine
  • DEPE 2-dielaidoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DMPE 2-dimyristoyl-sn-glycero-3-phosphoethanolamine
  • the agent comprises a binding agent, a therapeutic agent, or a detection agent, including for example a protein, a peptide such as an extracellular domain of oligomeric ACE2, an antibody or its functional antibody fragment, an antigen or epitope such a tumor antigen, a MHC-peptide complex, a drug such as a small molecule drug (e.g., an antitumor agent such as a chemotherapeutic agent) , an enzyme (e.g., a functional metabolic or therapeutic enzyme) , a hormone, a cytokine, a growth factor, an antimicrobial agent, a probe, a ligand, a receptor, an immunotolerance-inducing peptide, a targeting moiety, a prodrug, or any combination thereof.
  • a small molecule drug e.g., an antitumor agent such as a chemotherapeutic agent
  • an enzyme e.g., a functional metabolic or therapeutic enzyme
  • the sortase is a Sortase A (SrtA) such as a Staphylococcus aureus transpeptidase A, e.g., Staphylococcus aureus transpeptidase A variant (mgSrtA) .
  • SertA Sortase A
  • mgSrtA Staphylococcus aureus transpeptidase A variant
  • the cell is selected from the group consisting of red blood cells, T cells, B cells, monocytes, NK cells, and megakaryocytes.
  • the disclosure provides a method for modifying a cell, comprising:
  • the phospholipid with the conjugated agent comprises a structure of A 1 -L 1 -Gly m X n -optional linker-phospholipid, in which A 1 represents the agent, L 1 represents the residual part of a sortase recognition motif after a sortase-mediated reaction, Gly m represents m glycines with m preferably being 1-5, X n represents n spacing amino acids with n preferably being 0-10, and the optional linker is preferably a PEG linker.
  • the phospholipid linked to Gly m comprises a structure of Gly m X n -optional linker-phospholipid, wherein the X n represents n spacing amino acids with n preferably being 0-10 and the optional linker is preferably a PEG linker, and/or the sortase substrate comprises a structure of A 1 -M, in which A 1 represents the agent, and M represents the sortase recognition motif.
  • the sortase recognition motif comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X is any amino acid.
  • the sortase recognition motif comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein *represents the optionally substituted hydroxyl carboxylic acid; and X and Y independently represent any amino acid.
  • the sortase recognition motif comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein M preferably is LPET*G, with *being 2-hydroxyacetic acid.
  • the phospholipid is a PE series phospholipid, such as 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) , 1, 2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) , and 1, 2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) .
  • DSPE 2-distearoyl-sn-glycero-3-phosphoethanolamine
  • DEPE 2-dielaidoyl-sn-glycero-3-phosphoethanolamine
  • DOPE 2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DMPE 2-dimyristoyl-sn-glycero-3-phosphoethanolamine
  • the agent comprises a binding agent, a therapeutic agent, or a detection agent, including for example a protein, a peptide such as an extracellular domain of oligomeric ACE2, an antibody or its functional antibody fragment, an antigen or epitope such a tumor antigen, a MHC-peptide complex, a drug such as a small molecule drug (e.g., an antitumor agent such as a chemotherapeutic agent) , an enzyme (e.g., a functional metabolic or therapeutic enzyme) , a hormone, a cytokine, a growth factor, an antimicrobial agent, a probe, a ligand, a receptor, an immunotolerance-inducing peptide, a targeting moiety, a prodrug, or any combination thereof.
  • a small molecule drug e.g., an antitumor agent such as a chemotherapeutic agent
  • an enzyme e.g., a functional metabolic or therapeutic enzyme
  • the sortase is a Sortase A (SrtA) such as a Staphylococcus aureus transpeptidase A, e.g., Staphylococcus aureus transpeptidase A variant (mgSrtA) .
  • SertA Sortase A
  • mgSrtA Staphylococcus aureus transpeptidase A variant
  • the cell is selected from the group consisting of red blood cells, T cells, B cells, monocytes, NK cells, and megakaryocytes.
  • the disclosure provides a cell obtained by the method as described herein.
  • the disclosure provides a composition comprising the cell as described herein and optionally a physiologically acceptable carrier.
  • the disclosure provides a method for diagnosing, treating, or preventing a disorder, condition, or disease in a subject in need thereof, comprising administering the cell or composition as described herein to the subject.
  • the disorder, condition, or disease is selected from the group consisting of tumors or cancers, metabolic diseases such as lysosomal storage disorders (LSDs) , bacterial infections, virus infections such as coronavirus infection for example SARS-COV or SARS-COV-2 infection, autoimmune diseases, and inflammatory diseases.
  • the disclosure provides a method of delivering an agent to a subject in need thereof, comprising administering the cell or composition as described herein to the subject.
  • the disclosure provides a method of increasing the circulation time or plasma half-life of an agent in a subject, comprising attaching the agent to a cell according to the method of as described herein.
  • the disclosure provides a use of the cell or composition as described herein in the manufacture of a medicament for diagnosing, treating, or preventing a disorder, condition. or disease, or a diagnostic agent for diagnosing a disorder, condition. or disease, or for delivering an agent.
  • the disorder, condition, or disease is selected from a group consisting of tumors or cancers, metabolic diseases such as lysosomal storage disorders (LSDs) , bacterial infections, virus infections such as coronavirus infection for example SARS-COV or SARS-COV-2 infection, autoimmune diseases, and inflammatory diseases.
  • the medicament is a vaccine.
  • the disclosure provides the cell or composition as described herein for use in diagnosing, treating, or preventing a disorder, condition, or disease in a subject in need thereof.
  • the disorder, condition, or disease is selected from a group consisting of tumors or cancers, metabolic diseases such as lysosomal storage disorders (LSDs) , bacterial infections, virus infections such as coronavirus infection for example SARS-COV or SARS-COV-2 infection, autoimmune diseases and inflammatory diseases.
  • metabolic diseases such as lysosomal storage disorders (LSDs)
  • bacterial infections such as coronavirus infection for example SARS-COV or SARS-COV-2 infection
  • autoimmune diseases inflammatory diseases.
  • Figs. 1A-1B illustrate two exemplary processes for labeling cells with peptide in vitro according to the present disclosure.
  • Fig. 2 shows the structural formula of an exemplary phospholipid linked to a glycine “GAAS-PEG-phospholipid” according to the present disclosure.
  • Figs. 3A-3B show efficient labeling of eGFP-LPETGAAS-PEG-DSPE on the surface of natural RBCs that was detected by flow cytometry.
  • Control groups Unlabeled RBCs (Red) ; and eGFP-LPETG (Blue) ; Treatment Group: RBCs labeled with eGFP-LPETGAAS-PEG-DSPE (Orange) .
  • Histograms show eGPF signals on the RBCs’ surface after their incubation with eGFP-LPETGAAS-PEG-DSPE, which are significantly higher than the unlabeled RBCs and the RBCs incubated with eGFP-LPETG.
  • Fig. 4 shows the percentage of RBCs with eGFP signals in vivo. 10 9 mouse RBCs were labeled with eGFP-LPETGAAS-PEG-DSPE. The labeled RBCs were stained with cell trace Far Red dye and injected intravenously into the mice. Mice were bled at 21 days post transfusion. Blood samples were analyzed by flow cytometry. Far Red positive cells were selected for analyzing the percentage of RBCs with eGFP signals.
  • Figs. 5A-5B show the percentage of eGFP positive cells in the circulation and the label stability of these RBCs in different days.
  • Fig. 6 shows the efficient labeling of eGFP-LPETGAAS-PEG-DSPE on the surface of various mammalian cells.
  • eGFP-LPETGAAS-PEG-DSPE After labeling eGFP-LPETGAAS-PEG-DSPE with T cells, Monocytes, NK cells, B cells, and Megakaryocytes, respectively, the labeling efficacy was detected by flow cytometry.
  • (a) Anti-CD3 positive indicates T cells;
  • Anti-CD14 positive indicates monocytes;
  • Anti-CD16 positive indicates NK cells;
  • Anti-CD19 positive indicates B cells; and
  • Anti-CD41 positive indicates megakaryocytes.
  • Fig. 7 shows that SARS-CoV-2 enters host cells through binding with ACE2 by its S protein.
  • Fig. 8 shows red blood cells (RBCs) with trimeric ACE2 engineered on the surface.
  • nucleic acids are written left to right in 5' to 3' orientation; and amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • the term “consisting essentially of” in the context of an amino acid sequence means the recited amino acid sequence together with additional one, two, three, four, or five amino acids at the N-or C-terminus.
  • the terms “patient, ” “individual, ” and “subject” are used in the context of any mammalian recipient of a treatment or composition disclosed herein. Accordingly, the methods and composition disclosed herein may have medical and/or veterinary applications. In a preferred form, the mammal is a human.
  • sequence identity is meant to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) 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 (i.e., the window size) , and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) .
  • Lipids have been used as an anchor to conjugate a protein on the cell membrane of RBCs (Civenni et al., 1998) . This approach resulted in the formation of functional constructs but they were released more easily than the inserted proteins in native RBCs.
  • the present disclosure is at least partially based on a surprising finding that by means of a phospholipid linked to a Gly (1-3) at its hydrophilic end as a membrane anchor to conjugate an agent via a sortase-mediated reaction, the labelled cells have the same lifespan as native RBCs and sustained signals in circulation for up to 28 days.
  • the inventors therefore developed a new strategy to modify cells, e.g., blood cells, especially natural RBCs, with an agent, e.g., peptides and/or small molecules, through a sortase-mediated reaction.
  • the technology allows for producing RBC products by directly modifying natural RBCs instead of HSPCs, which are limited by their resources.
  • the modified RBCs preserve their original biological properties well and remain as stable as their native state.
  • the technology also achieves a significantly higher labelling efficiency.
  • the present disclosure provides a cell modified by an agent, wherein the agent is conjugated to a phospholipid via a sortase recognition motif, and the phospholipid with the conjugated agent is incorporated into the cell membrane of the cell.
  • the animal cell is a mammalian cell, e.g., a human cell.
  • the cell is an immune system cell, e.g., lymphocytes (e.g., a T cell or NK cell) or dendritic cells.
  • the cell is a cytotoxic cell.
  • the cell is a non-immortalized cell.
  • the cell is a primary cell.
  • the cell is a natural cell.
  • the cell is not genetically engineered to express a polypeptide comprising a sortase recognition sequence, a sequence comprising one or more glycines or alanines at its terminus, or both.
  • the cell is a mature red blood cell (RBC) .
  • RBC is a human RBC, such as a human natural RBC.
  • the RBC is a red blood cell that has not been genetically engineered to express a protein comprising a sortase recognition motif or a nucleophilic acceptor sequence. In some embodiments the RBC has not been genetically engineered.
  • non-engineered, “non-genetically modified, ” and “non-recombinant” as used herein are interchangeable and refer to not being genetically engineered, absence of genetic modification, etc.
  • Non-engineered membrane proteins encompass endogenous proteins.
  • a non-genetically engineered red blood cell does not contain a non-endogenous nucleic acid, e.g., DNA or RNA that originates from a vector, from a different species, or that comprises an artificial sequence, e.g., DNA or RNA that was introduced artificially.
  • a non-engineered cell has not been intentionally contacted with a nucleic acid that is capable of causing a heritable genetic alteration under conditions suitable for uptake of the nucleic acid by the cells.
  • the cell has not been genetically engineered for sortagging.
  • a cell is considered “not genetically engineered for sortagging” if the cell has not been genetically engineered to express a protein comprising a sortase recognition motif or a nucleophilic acceptor sequence in a sortase-catalyzed reaction.
  • the present disclosure provides a cell modified by an agent, wherein the agent is conjugated to a phospholipid via a sortase recognition motif, and the phospholipid with the conjugated agent is incorporated into the cell membrane of the cell, wherein the phospholipid with the conjugated agent comprises a structure of A 1 -L 1 -Gly m X n -optional linker-phospholipid, in which A 1 represents the agent, L 1 represents the residual part of a sortase recognition motif after a sortase-mediated reaction, Gly m represents m glycines with m preferably being 1-5, X n represents n spacing amino acids with n preferably being 0-10, and the optional linker is preferably a PEG linker.
  • conjugation refers to an association of two molecules, for example, two proteins or a protein and a small molecule or other entity, with one another in a way that they are linked by a direct or indirect covalent or non-covalent interaction.
  • the term “incorporate” or “incorporating” or similar terminology refers to an insertion of a phospholipid into the lipid bilayer of a cell membrane in a way that the phospholipid functions as an anchor to conjugate an agent of interest.
  • the present disclosure provides a cell modified by an agent, wherein the agent is conjugated to a phospholipid via a sortase recognition motif, and the phospholipid with the conjugated agent is incorporated into the cell membrane of the cell.
  • a composition comprising a plurality of such cells is provided.
  • at least a selected percentage of the cells in the composition are modified, i.e., having an agent conjugated by sortase to a phospholipid incorporated into the cell membrane.
  • the cells have an agent conjugated thereto.
  • the conjugated agent may be one or more of the agents described herein.
  • the present disclosure provides a cell that comprises an agent conjugated via a sortase-mediated reaction to a phospholipid incorporated into the cell membrane.
  • a sortase-mediated reaction to a phospholipid incorporated into the cell membrane.
  • two, three, four, five, or more different phospholipids incorporated into the cell membrane have an agent conjugated thereto via a sortase-mediated reaction.
  • the agents attached to different phospholipids may be the same or the cell may be sortagged with a plurality of different agents.
  • the agent is linked via a sortase recognition motif to a phospholipid incorporated into the cell membrane.
  • the sortase recognition motif may be selected from the group consisting of LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X is any amino acid.
  • the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein * represents the optionally substituted hydroxyl carboxylic acid; and X and Y independently represent any amino acid.
  • the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein the sortase recognition motif preferably is LPET*G with *preferably being 2-hydroxyacetic acid.
  • the phospholipid with the conjugated agent comprises a structure of A 1 -L 1 -Gly m X n -optional linker-phospholipid, in which L 1 represents the residual part of a sortase recognition motif after a sortase-mediated reaction, and may be selected from the group consisting of LPXT, LPXA, LPXS, LPXL, LPXV, LGXT, LAXT, LSXT, NPXT, MPXT, IPXT, SPXT, VPXT, and YPXR.
  • genetically engineered cells are modified by using sortase to attach a sortase substrate to a phospholipid incorporated into the cell membrane of the cells.
  • the cells e.g., RBCs may, for example, have been genetically engineered to express any of a wide variety of products, e.g., polypeptides or noncoding RNAs, may be genetically engineered to have a deletion of at least a portion of one or more genes, and/or may be genetically engineered to have one or more precise alterations in the sequence of one or more endogenous genes.
  • a phospholipid incorporated into the cell membrane of such a genetically engineered cell is sortagged with any of the various agents described herein.
  • the present disclosure contemplates using autologous cells, e.g., red blood cells, that are isolated from an individual to whom such isolated cells, after modified in vitro, are to be administered.
  • autologous cells e.g., red blood cells
  • the present disclosure contemplates using immuno-compatible red blood cells that are of the same blood group as an individual to whom such cells are to be administered (e.g., at least with respect to the ABO blood type system and, in some embodiments, with respect to the D blood group system) or may be of a compatible blood group.
  • phospholipid also known as phosphatides, refers to a class of lipids whose molecule has a hydrophilic "head” containing a phosphate group, and two hydrophobic "tails” derived from fatty acids, joined by a glycerol molecule.
  • the phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine.
  • phospholipids may be PC series such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC; PG series such as DMPG, DPPG, DSPG, and POPG; PA series such as DMPA, DPPA, and DSPA; PE series such as 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) , 1, 2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , and 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) , and 1, 2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) ; and PS series such as DOPS.
  • DSPE 2-distearoyl-sn-glycero-3-phosphoethanolamine
  • the phospholipids may be PE series such as 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) , 1, 2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , and 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) , and 1, 2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) .
  • PE series such as 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) , 1, 2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , and 1, 2-dimyristoyl-sn-gly
  • a linker is used to link Gly m X n (n preferably being 0-10) and phospholipids, via, e.g., forming a peptide bond (-C (O) -NH-) .
  • linkers useful to contribute to the water solubility of phospholipids are contemplated.
  • the linker is a PEG linker, and accordingly PEG attached phospholipids (PEGylated phospholipids) are used.
  • PEG2000-PEG5000 such as PEG2500, PEG3000, PEG3500, PEG4000, or PEG4500 are contemplated.
  • the linker may have a free carboxyl group (e.g., a carboxyl end) and a free amino group (e.g., an amino end) .
  • the PEG linker may comprise a structure of NH 2 -PEG-COOH such as NH 2 - (CH 2 CH 2 O) n -COOH.
  • the phospholipid is linked to Gly m X n (n preferably being 0-10) at the amino group of the hydrophilic end (i.e., the hydrophilic "head" containing a phosphate group) of the phospholipid, in a way as illustrated in Fig. 2.
  • the phospholipid linked to Gly m comprises a structure of Gly m X n -optional linker-phospholipid, wherein Gly m represents m glycines with m preferably being 1-5 and X n represents n spacing amino acids with n preferably being 0-10, and the optional linker is preferably a PEG linker. It is to be understood that when the phospholipid is linked to a Gly m X n (n preferably being 0-10) , an agent to be conjugated may have a C-terminal sortase recognition motif.
  • the phospholipid may be linked to a sortase recognition motif as described herein at the amino group of the hydrophilic end (i.e., the hydrophilic "head" containing a phosphate group) of the phospholipid.
  • the phospholipid linked to a sortase recognition motif comprises a structure of M-X n -optional linker- phospholipid, wherein M represents the sortase recognition motif, X n represents n spacing amino acids with n preferably being 0-10, and the optional linker is preferably a PEG linker. It is to be understood that when the phospholipid is linked to a sortase recognition motif, an agent to be conjugated may have an N-terminal Gly m .
  • Gly m refers to m glycines with m preferably being 1-5, such as one, two, or three glycines.
  • X n represents n optional spacing amino acids which can be any amino acids. In some embodiments, n can be 0-10 or more, such as 0-5, 1-4, or 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the spacing amino acids may be any natural or unnatural amino acids, such as Gly, Ala, Ser, Lys, Asn, Thr, Glu, or Gln. In some embodiments, X n may be AAS or AASK.
  • Sortases Enzymes identified as “sortases” have been isolated from a variety of Gram-positive bacteria. Sortases, sortase-mediated transacylation reactions, and their use in protein engineering are well known to those of ordinary skills in the art (see, e.g., PCT/US2010/000274 (WO/2010/087994) , and PCT/US2011/033303 (WO/2011/133704) ) .
  • Sortases have been classified into 4 classes, designated A, B, C, and D, based on sequence alignment and phylogenetic analysis of 61 sortases from Gram-positive bacterial genomes (Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol. 156 (3) : 289-97, 2005) . Those skilled in the art can readily assign a sortase to the correct class based on its sequence and/or other characteristics such as those described in Drami, et al., supra.
  • sortase A refers to a class A sortase, usually named SrtA in any particular bacterial species, e.g., SrtA from S. aureus or S. pyogenes.
  • sortase also known as transamidases refers to an enzyme that has transamidase activity. Sortases recognize substrates comprising a sortase recognition motif, e.g., the amino acid sequence LPXTG. A molecule recognized by a sortase (i.e., comprising a sortase recognition motif) is sometimes termed a “sortase substrate” herein. Sortases tolerate a wide variety of moieties in proximity to the cleavage site, thus allowing for the versatile conjugation of diverse entities so long as the substrate contains a suitably exposed sortase recognition motif and a suitable nucleophile is available.
  • sortase-mediated transacylation reaction “sortase-catalyzed transacylation reaction, ” “sortase-mediated reaction, ” “sortase-catalyzed reaction, ” “sortase reaction, ” “sortase-mediated transpeptide reaction, ” and like terms, are used interchangeably herein to refer to such a reaction.
  • sortase recognition motif, ” “sortase recognition sequence, ” and “transamidase recognition sequence, ” with respect to sequences recognized by a transamidase or sortase are used interchangeably herein.
  • nucleophilic acceptor sequence refers to an amino acid sequence capable of serving as a nucleophile in a sortase-catalyzed reaction, e.g., a sequence comprising an N-terminal glycine (e.g., 1, 2, 3, 4, or 5 N-terminal glycines) .
  • sortase A is used, such as SrtA from S. aureus.
  • sortases may utilize different sortase recognition sequences and/or different nucleophilic acceptor sequences.
  • the sortase is a sortase A (SrtA) .
  • SrtA recognizes the motif LPXTG, with common recognition motifs being, e.g., LPKTG, LPATG, and LPNTG.
  • LPETG is used.
  • motifs falling outside this consensus may also be recognized.
  • the motif comprises an ‘A, ’ ‘S, ’ ‘L, ’ or ‘V’ rather than a ‘T’ at position 4, e.g., LPXAG, LPXSG, LPXLG, or LPXVG, e.g., LPNAG or LPESG, LPELG or LPEVG.
  • the motif comprises an ‘A’ rather than a ‘G’ at position 5, e.g., LPXTA, e.g., LPNTA.
  • the motif comprises a ‘G’ or ‘A’ rather than ‘P’ at position 2, e.g., LGXTG or LAXTG, e.g., LGATG or LAETG.
  • the motif comprises an ‘I’ or ‘M’ rather than ‘L’ at position 1, e.g., MPXTG or IPXTG, e.g., MPKTG, IPKTG, IPNTG, or IPETG.
  • Diverse recognition motifs of sortase A are described in Pishesha et al. 2018.
  • the sortase recognition sequence is LPXTG, wherein X is a standard or non-standard amino acid.
  • X is selected from D, E, A, N, Q, K, or R.
  • the recognition sequence is selected from LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, wherein X may be any amino acids, such as those selected from D, E, A, N, Q, K, or R in certain embodiments.
  • the sortase recognizes a motif comprising an unnatural amino acid, preferably located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif.
  • the unnatural amino acid is a substituted hydroxyl carboxylic acid and in some further embodiments, the hydroxyl carboxylic acid is substituted by one or more substituents selected from halo, C 1-6 alkyl, C 1-6 haloalkyl, hydroxyl, C 1-6 alkoxy, and C 1-6 haloalkoxy.
  • halo or “halogen” means fluoro, chloro, bromo, or iodo, and preferred are fluoro and chloro.
  • alkyl by itself or as part of another substituent refers to a hydrocarbyl radical of Formula C n H 2n+1 wherein n is a number greater than or equal to 1.
  • alkyl groups useful in the present disclosure comprise from 1 to 6 carbon atoms, preferably from 1 to 4 carbon atoms, more preferably from 1 to 3 carbon atoms, still more preferably 1 to 2 carbon atoms.
  • Alkyl groups may be linear or branched and may be further substituted as indicated herein.
  • C x-y alkyl refers to alkyl groups which comprise from x to y carbon atoms.
  • Suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and tert-butyl, pentyl and its isomers (e.g.
  • n-pentyl, iso-pentyl) n-pentyl
  • hexyl and its isomers e.g. n-hexyl, iso-hexyl
  • Preferred alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, and tert-butyl.
  • haloalkyl alone or in combination, refers to an alkyl radical having the meaning as defined above, wherein one or more hydrogens are replaced with a halogen as defined above.
  • Non-limiting examples of such haloalkyl radicals include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1, 1, 1-trifluoroethyl, and the like.
  • the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein *represents the optionally substituted hydroxyl carboxylic acid; and X and Y independently represent any amino acid.
  • the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein the sortase recognition motif is preferably LPET*G with *preferably being 2-hydroxyacetic acid.
  • the present disclosure contemplates using a variant of a naturally occurring sortase.
  • the variant is capable of mediating a glycine (n) conjugation, with n preferably being 1 or 2.
  • Such variants may be produced through processes such as directed evolution, site-specific modification, etc.
  • sortase enzymes e.g., sortase A enzymes
  • sortase A enzymes is available, including NMR or crystal structures of SrtA alone or bound to a sortase recognition sequence (see, e.g., Zong Y, et al. J. Biol Chem. 2004, 279, 31383-31389) .
  • the active site and substrate binding pocket of S. aureus SrtA have been identified.
  • S. aureus SrtA may be those described in CN106191015A and CN109797194A. In some embodiments, the S.
  • aureus SrtA variant can be a truncated variant with, e.g., 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids being removed from N-terminus (as compared to the wild type S. aureus SrtA) .
  • a functional variant of S. aureus SrtA useful in the present disclosure may be a S. aureus SrtA variant comprising one or more mutations on amino acid positions of D124, Y187, E189, and F200 of D124G, Y187L, E189R and F200L and optionally further comprising one or more mutations of P94S/R, D160N, D165A, K190E, and K196T.
  • the S. aureus SrtA variant comprising one or more mutations on amino acid positions of D124, Y187, E189, and F200 of D124G, Y187L, E189R and F200L and optionally further comprising one or more mutations of P94S/R, D160N, D165A, K190E, and K196T.
  • aureus SrtA variant may comprise D124G; D124G and F200L; P94S/R, D124G, D160N, D165A, K190E, and K196T; P94S/R, D160N, D165A, Y187L, E189R, K190E, and K196T; P94S/R, D124G, D160N, D165A, Y187L, E189R, K190E, and K196T; D124G, Y187L, E189R, and F200L; or P94S/R, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • the S is selected from D150N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • aureus SrtA variants have 59 or 60 (e.g., 25, 30, 35, 40, 45, 50, 55, 59, or 60) amino acids being removed from N-terminus.
  • the mutated amino acid positions above are numbered according to the numbering of a wild type S. aureus SrtA, e.g., as shown in SEQ ID NO: 1.
  • the full-length nucleotide sequence of the wild type S. aureus SrtA is shown as in, e.g., SEQ ID NO: 2.
  • SEQ ID NO: 1 full length, GenBank Accession No. : CAA3829591.1
  • SEQ ID NO: 2 full length, wild type
  • the S. aureus SrtA variant may comprise one or more mutations at one or more of the positions corresponding to 94, 105, 108, 124, 160, 165, 187, 189, 190, 196, and 200 of SEQ ID NO: 1.
  • the S. aureus SrtA variant may comprise one or more mutations corresponding to P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • the S. aureus SrtA variant may comprise one or more mutations corresponding to D124G, Y187L, E189R, and F200L and optionally further comprises one or more mutations corresponding to P94S/R, D160N, D165A, K190E, and K196T, and optionally further one or more mutations corresponding to E105K and E108A.
  • the S. aureus SrtA variant may comprise one or more mutations corresponding to D124G, Y187L, E189R, and F200L and optionally further comprises one or more mutations corresponding to P94S/R, D160N, D165A, K190E, and K196T, and optionally further one or more mutations corresponding to E105K and E108A.
  • the S. aureus SrtA variant may comprise one or more mutations corresponding to D124G, Y187L, E189R, and F200L and optionally further comprises one or more mutations corresponding to P94S/R, D160N, D
  • aureus SrtA variant may comprise mutations corresponding to D124G; D124G and F200L; P94S/R, D124G, D160N, D165A, K190E, and K196T; P94S/R, D160N, D165A, Y187L, E189R, K190E, and K196T; P94S/R, D124G, D160N, D165A, Y187L, E189R, K190E, and K196T; D124G, Y187L, E189R, and F200L; or P94S/R, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • the S is selected from the S.
  • aureus SrtA variant may comprise one or more mutations of P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1.
  • the S. aureus SrtA variant may comprise D124G, Y187L, E189R, and F200L and optionally further comprises one or more mutations of P94S/R, D160N, D165A, K190E, and K196T and optionally further comprises E105K and/or E108A relative to SEQ ID NO: 1.
  • the S. aureus SrtA variant may comprise one or more mutations of P94S/R, E160N, D165A, K190E, and K196T and optionally further comprises E105K and/or E108A relative to SEQ ID NO: 1.
  • aureus SrtA variant may, comprise, relative to SEQ ID NO: 1, D124G; D124G and F200L; P94S/R, D124G, D160N, D165A, K190E, and K196T; P94S/R, D160N, D165A, Y187L, E189R, K190E, and K196T; P94S/R, D124G, D160N, D165A, Y187L, E189R, K190E, and K196T; D124G, Y187L, E189R, and F200L; or P94S/R, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • mutations E105K and/or E108A/Q allows the sortase-mediated reaction to be Ca 2+ independent.
  • the S. aureus SrtA variants as described herein may have 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids removed from N-terminus.
  • the mutated amino acid positions above are numbered according to the numbering of a full length of a wild type S. aureus SrtA, e.g., as shown in SEQ ID NO: 1.
  • a functional variant of S. aureus SrtA useful in the present disclosure may be a S. aureus SrtA variant comprising one or more mutations of P94S/R, E105K, E108A/Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • the S. aureus SrtA variant comprising one or more mutations of P94S/R, E105K, E108A/Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • the S. aureus SrtA variant comprising one or more mutations of P94S/R, E105K, E108A/Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • the S. aureus SrtA variant comprising one or more mutations of
  • aureus SrtA variant may comprise P94S/R, E105K, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L; or P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L.
  • the S. aureus SrtA variant may comprise one or more mutations of P94S/R, E105K, E108A/Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1.
  • the S. aureus SrtA variant may comprise P94S/R, E105K, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1; or P94S/R, E105K, E108A, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L relative to SEQ ID NO: 1.
  • the S. aureus SrtA variants have 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids being removed from N-terminus.
  • the mutated amino acid positions above are numbered according to the numbering of a wild type S. aureus SrtA, e.g., as shown in SEQ ID NO: 1.
  • an S. aureus SrtA variant comprising, or consisting essentially of, or consisting of an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher) identity to an amino acid sequence as shown in SEQ ID NO: 3.
  • SEQ ID NO: 3 is a truncated SrtA and the mutations corresponding to wild type SrtA are shown in bold and underlined below.
  • the SrtA variant comprises or consists essentially of or consists of an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher) identity to an amino acid sequence as shown in SEQ ID NO: 3 and comprises the mutations of P94R/S, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L and optionally E105K and/or E108A/Q (numbered according to the numbering of SEQ ID NO: 1) .
  • the present disclosure provides a nucleic acid encoding the S. aureus SrtA variant, and in some embodiments the nucleic acid is set forth in SEQ ID NO: 4.
  • the S. aureus SrtA variant can be a truncated variant with, e.g., 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids being removed from N-terminus (as compared to the wild type S. aureus SrtA) .
  • the truncated variant comprises, or consists essentially of, or consists of an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher such as 100%) identity to an amino acid sequence as set forth in SEQ ID NO: 5 or 7.
  • the nucleic acids encoding SEQ ID NOs: 5 and 7 are set forth in SEQ ID NOs: 6 and 8 below.
  • a sortase A variant may comprise any one or more of the following: an S residue at position 94 (S94) or an R residue at position 94 (R94) , a K residue at position 105 (K105) , an A residue at position 108 (A108) or a Q residue at position 108 (Q 108) , a G residue at position 124 (G124) , an N residue at position 160 (N160) , an A residue at position 165 (A165) , a R residue at position 189 (R189) , an E residue at position 190 (E190) , a T residue at position 196 (T196) , and an L residue at position 200 (L200) (numbered according to the numbering of a wild type SrtA, e.g., SEQ ID NO: 1) , optionally with about 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids being removed from N-
  • a sortase A variant comprises two, three, four, or five of the afore-mentioned mutations relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) .
  • a sortase A variant comprises an S residue at position 94 (S94) or an R residue at position 94 (R94) , and also an N residue at position 160 (N160) , an A residue at position 165 (A165) , and a T residue at position 196 (T196) relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) .
  • a sortase A variant comprises P94S or P94R, and also D160N, D165A, and K196T relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) .
  • a sortase A variant comprises an S residue at position 94 (S94) or an R residue at position 94 (R94) and also an N residue at position 160 (N160) , an A residue at position 165 (A165) , an E residue at position 190, and a T residue at position 196 relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) .
  • a sortase A variant comprises P94S or P94R, and also D160N, D165A, K190E, and K196T relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) .
  • a sortase A variant comprises an R residue at position 94 (R94) , an N residue at position 160 (N160) , an A residue at position 165 (A165) , an E residue at position 190, and a T residue at position 196 relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) .
  • a sortase comprises P94R, D160N, D165A, K190E, and K196T relative to a wild type S. aureus SrtA (e.g., SEQ ID NO: 1) .
  • the S. aureus SrtA variants may have 25-60 (e.g., 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, or 60) amino acids being removed from N-terminus.
  • a sortase A variant having higher transamidase activity than a naturally occurring sortase A may be used.
  • the activity of the sortase A variant is at least about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200 times as high as that of wild type S. aureus sortase A.
  • such a sortase variant is used in a composition or method of the present disclosure.
  • a sortase variant comprises any one or more of the following substitutions relative to a wild type S.
  • aureus SrtA P94S/R, E105K, E108A, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L mutations.
  • the SrtA variant may have 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids being removed from N-terminus.
  • the amino acid mutation positions are determined by an alignment of a parent S. aureus SrtA (from which the S. aureus SrtA variant as described herein is derived) with the polypeptide of SEQ ID NO: 1, i.e., the polypeptide of SEQ ID NO: 1 is used to determine the corresponding amino acid sequence in the parent S. aureus SrtA.
  • Methods for determining an amino acid position corresponding to a mutation position as described herein is well known in the art. Identification of the corresponding amino acid residue in another polypeptide can be confirmed by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.
  • the sortase variant may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative amino acid mutations.
  • Conservative amino acid mutations that will not substantially affect the activity of a protein are well known in the art.
  • sortases preferably mg SrtA
  • sortases preferably mg SrtA
  • this may have certain constraints, since, for example, different types of cells have different types of membrane proteins, and the number of proteins containing N-terminal glycine (s) (e.g., G1 for mg SrtA) is also different.
  • the strategy of the present disclosure allows the possibility of using various sortases to modify various cells with agents.
  • the present disclosure contemplates using a sortase recognition motif comprising an unnatural amino acid, preferably located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif.
  • the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*Y, LPXA*Y, LPXS*Y, LPXL*Y, LPXV*Y, LGXT*Y, LAXT*Y, LSXT*Y, NPXT*Y, MPXT*Y, IPXT*Y, SPXT*Y, VPXT*Y, and YPXR*Y, wherein * represents the optionally substituted hydroxyl carboxylic acid; and X and Y independently represent any amino acid.
  • the sortase recognition motif comprising an unnatural amino acid may be selected from the group consisting of LPXT*G, LPXA*G, LPXS*G, LPXL*G, LPXV*G, LGXT*G, LAXT*G, LSXT*G, NPXT*G, MPXT*G, IPXT*G, SPXT*G, VPXT*G, YPXR*G, LPXT*S, and LPXT*A, wherein the sortase recognition motif is preferably LPET*G, with * preferably being 2-hydroxyacetic acid.
  • Leu-Pro-Glu-Thr-2-hydroxyacetic acid-Gly is used as a linker to ensure that the byproduct would make the reaction irreversible.
  • the sortase recognition motif comprising an unnatural amino acid as a linker is chemically synthesized and can be directly conjugated to an agent such as a protein or polypeptide.
  • a sortase substrate may comprise a sortase recognition motif and an agent.
  • an agent such as a polypeptide can be modified to include a sortase recognition motif at or near their C-terminus, thereby allowing them to serve as substrates for sortase.
  • the sortase recognition motif need not be positioned at the very C-terminus of a substrate but should typically be sufficiently accessible by the enzyme to participate in the sortase reaction.
  • a sortase recognition motif is considered to be “near” a C-terminus if there are no more than 5, 6, 7, 8, 9, 10 amino acids between the most N-terminal amino acid in the sortase recognition motif (e.g., L) and the C-terminal amino acid of the polypeptide.
  • a polypeptide comprising a sortase recognition motif may be modified by incorporating or attaching any of a wide variety of moieties (e.g., peptides, proteins, compounds, nucleic acids, lipids, small molecules, and sugars) thereto.
  • the present disclosure provides a sortase substrate comprising a structure of A 1 -M, in which A 1 represents an agent, and M represents the sortase recognition motif.
  • the sortase recognition motif comprises an unnatural amino acid located at position 5 from the direction of N-terminal to C-terminal of the sortase recognition motif as set forth herein.
  • an agent may comprise a protein, a peptide (e.g., an extracellular domain of oligomeric ACE2) , an antibody or its functional antibody fragment, an antigen or epitope, a MHC-peptide complex, a drug such as a small molecule drug (e.g., an antitumor agent such as a chemotherapeutic agent) , an enzyme (e.g., a functional metabolic or therapeutic enzyme) , a hormone, a cytokine, a growth factor, an antimicrobial agent, a probe, a ligand, a receptor, an immunotolerance-inducing peptide, a targeting moietyor any combination thereof.
  • a drug such as a small molecule drug (e.g., an antitumor agent such as a chemotherapeutic agent)
  • an enzyme e.g., a functional metabolic or therapeutic enzyme
  • the agent in addition to a therapeutically active domain such as an enzyme, a drug, a small molecule (such as a small molecule drug (e.g., an antitumor agent such as a chemotherapeutic agent) ) , a therapeutic protein and a therapeutic antibody as described herein, the agent may further comprise a targeting moiety for targeting the cells and/or agent to a site in the body where the therapeutic activity is desired.
  • the targeting moiety binds to a target present at such a site. Any targeting moiety may be used, e.g., an antibody.
  • the site may be any organ or tissue, e.g., respiratory tract (e.g., lung) , bone, kidney, liver, pancreas, skin, cardiovascular system (e.g., heart) , smooth or skeletal muscle, gastrointestinal tract, eye, blood vessel surfaces, etc.
  • respiratory tract e.g., lung
  • bone e.g., kidney
  • liver e.g., pancreas
  • cardiovascular system e.g., heart
  • smooth or skeletal muscle e.g., smooth or skeletal muscle
  • gastrointestinal tract e.g., eye
  • blood vessel surfaces e.g., etc.
  • a protein is an enzyme such as a functional metabolic or therapeutic enzyme, e.g., an enzyme that plays a role in metabolism or other physiological processes in a mammal.
  • a protein is an enzyme that plays a role in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, porphyrin metabolism, purine or pyrimidine metabolism, and/or lysosomal storage. Deficiencies of enzymes or other proteins can lead to a variety of diseases, e.g., diseases associated with defects in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, purine or pyrimidine metabolism, lysosomal storage disorders, and blood clotting, among others.
  • Metabolic diseases are characterized by the lack of functional enzymes or excessive intake of metabolites.
  • the metabolites deposition in the circulation and tissues causes tissue damage.
  • the present disclosure contemplates modifying membrane proteins of blood cells, such as RBCs, with functional metabolic enzymes.
  • the enzyme targeted blood cells, such as RBCs will uptake metabolites in plasma of patients.
  • Exemplary enzymes include urate oxidase for gout, phenylalanine ammonia-lyase for Phenylketonuria, acetaldehyde dehydrogenase for alcoholic hepatitis, butyrylcholinesterase for cocaine metabolite, and the like.
  • red blood cells having urate oxidase conjugated thereto may be administered to a subject in need of treatment of chronic hyperuricemia, e.g., a patient with gout, e.g., gout that is refractory to other treatments.
  • Enzyme replacement therapy has been a specific treatment for patients with e.g. lysosomal storage disorders (LSDs) over the past three decades.
  • LSDs lysosomal storage disorders
  • the therapeutic enzymes are rapidly cleared in human body for their extensive catabolism.
  • the present disclosure contemplates binding the therapeutic enzymes to RBC membrane proteins through the sortase reaction as described herein.
  • the use of blood cells such as RBCs as carriers will target the functional enzymes to macrophages in liver, where blood cells such as RBCs are cleared, and also reduce the dosage and frequency of drug interventions for the enhanced half-time of enzymes.
  • Exemplary enzymes include glucocerebrosidase for Gaucher disease, ⁇ -galactosidase for Fabry disease, alanine glycoxylate aminotransferase, and glyoxylate reductase /hydroxypyruvate reductase for primary hyperoxaluria.
  • the agent may comprise a peptide.
  • Various functional peptides can be contemplated in the present disclosure.
  • the peptide may comprise an oligomeric ACE2 extracellular domain.
  • SARS-CoV-2 which causes a respiratory disease named COVID-19, belongs to the same coronaviridea as SARS-CoV.
  • the genome of SARS-CoV-2 is very similar to SARS-CoV sharing ⁇ 80%nucleotide sequence identity and 94.6%amino acid sequence identity in the ORF encoding the spike protein.
  • SARS-CoV-2 and SARS-CoV spike proteins have very similar structures, both entering human cells through spike protein interaction with ACE2 as shown in Fig. 7.
  • ACE2 spike protein interaction with ACE2
  • Fig. 7 Unfortunately, seventeen years after the SARS pandemic, no effective detection (except RT-PCR) , prevention, or treatment approaches were developed from SARS-CoV that could be readily applied to SARS-CoV-2.
  • SARS-CoV-2 specific antibodies vaccines, protease inhibitors, and RNA-dependent RNA polymerase inhibitors to detect and combat the SARS-CoV-2 infection disease “COVID-19. ”
  • SARS-CoV-2 specific antibodies vaccines, protease inhibitors, and RNA-dependent RNA polymerase inhibitors to detect and combat the SARS-CoV-2 infection disease “COVID-19. ”
  • SARS-CoV-2 if developed quickly enough (probably within 2-3 months) .
  • the lack of cross-reactivity between several SARS-CoV specific antibodies and SARS-CoV-2 is a clear demonstration of this.
  • detection devices or therapeutic agents which are not only useful for SARS-CoV-2, but also could be readily applied to future coronavirus are highly desirable for development.
  • coronavirus infected patients show respiratory syndromes especially in the lung.
  • coronavirus infected patients show respiratory syndromes and some even develop ARDS suggests supplementing ACE2 could also alleviate respiratory syndromes for virus infection treatment.
  • the present disclosure contemplates using blood cells, such as red blood cells, as oligomeric ACE2 carriers for effective virus neutralization (Fig. 8) , by use of the new strategy to modify natural blood cells, such as RBCs, with peptides and/or small molecules through a sortase mediated reaction as described herein.
  • blood cells such as red blood cells
  • oligomeric ACE2 carriers for effective virus neutralization (Fig. 8)
  • the agent may comprise an antibody, including an antibody, an antibody chain, an antibody fragment, e.g., scFv, an antigen-binding antibody domain, a VHH domain, a single-domain antibody, a camelid antibody, a VNAR, a nanobody, an adnectin, or an anticalin.
  • the red blood cells having antibodies attached thereto may be used as a delivery vehicle for the antibodies and/or the antibodies may serve as a targeting moiety.
  • Exemplary antibodies include anti-tumor antibodies such as PD-1 antibodies, e.g., Nivolumab and Pembrolizumab, which both are monoclonal antibodies for human PD-1 protein and are now the forefront treatment for melanoma, non-small cell lung carcinoma, and renal-cell cancer.
  • the heavy chains of the antibodies modified with a sortase recognition motif, such as LPETG can be expressed and purified.
  • PD-L1 antibodies such as Atezolizum, Avelumab, and Durvalumab targeting PD-L1 for treating urothelial carcinoma and metastatic merkel cell carcinoma can be modified.
  • Adalimumab, Infliximab, Sarilumab, and Golimumab which are FDA approved therapeutic monoclonal antibodies for curing rheumatoid arthritis can be modified by using the method as described herein.
  • the agent may comprise an antigen or epitopes or a binding moiety that binds to an antigen or epitope.
  • an antigen is any molecule or complex comprising at least one epitope recognized by a B cell and/or by a T cell.
  • An antigen may comprise a polypeptide, a polysaccharide, a carbohydrate, a lipid, a nucleic acid, or combination thereof.
  • An antigen may be naturally occurring or synthetic, e.g., an antigen naturally produced by and/or genetically encoded by a pathogen, an infected cell, a neoplastic cell (e.g., a tumor or cancer cell) , a virus, bacteria, fungus, or parasite.
  • an antigen is an autoantigen or a graft-associated antigen.
  • an antigen is an envelope protein, capsid protein, secreted protein, structural protein, cell wall protein or polysaccharide, capsule protein or polysaccharide, or enzyme.
  • an antigen is a toxin, e.g., a bacterial toxin.
  • An antigen or epitope may be modified, e.g., by conjugation to another molecule or entity (e.g., an adjuvant) .
  • red blood cells having an epitope, antigen, or portion thereof conjugated thereto as described herein may be used as vaccine components.
  • an antigen conjugated to red blood cells as described herein may be any antigen used in a conventional vaccine known in the art.
  • an antigen is a surface protein or polysaccharide of, e.g., a viral capsid, envelope, or coat, or bacterial, fungal, protozoal, or parasite cell.
  • exemplary viruses may include, e.g., coronaviruses (e.g., SARS-CoV and SARS-CoV-2) , HIV, dengue viruses, encephalitis viruses, yellow fever viruses, hepatitis virus, Ebola viruses, influenza viruses, and herpes simplex virus (HSV) 1 and 2.
  • an antigen is a tumor antigen (TA) , which can be any antigenic substance produced by cells in a tumor, e.g., tumor cells or in some embodiments tumor stromal cells (e.g., tumor-associated cells such as cancer-associated fibroblasts or tumor-associated vasculature) .
  • TA tumor antigen
  • an antigen is a peptide.
  • Peptides may bind directly to MHC molecules expressed on cell surfaces, may be ingested and processed by APC and displayed on APC cell surfaces in association with MHC molecules, and/or may bind to purified MHC proteins (e.g., MHC oligomers) .
  • a peptide contains at least one epitope capable of binding to an appropriate MHC class I protein and/or at least one epitope capable of binding to an appropriate MHC class II protein.
  • a peptide comprises a CTL epitope (e.g., the peptide can be recognized by CTLs when bound to an appropriate MHC class I protein) .
  • the agent may comprise an MHC-peptide complex, which may comprise an MHC and a peptide such as an antigenic peptide or an antigen as described herein for activating immune cells.
  • the antigenic peptide is associated with a disorder and is able to activate CD8 + T cells when presented by an MHC class I molecule.
  • Class-I major histocompatibility complex presents antigen peptides to and activates immune cells, particularly CD8 + T cells, which are important for fighting against cancers, infectious diseases, etc.
  • MHC-peptide complexes with sortase recognition motifs, such as LPETG can be expressed and purified exogenously through eukaryotic or prokaryotic systems.
  • the purified MHC-peptide complexes will be bound to blood cells such as RBCs by sortase-mediated reactions as described herein.
  • MHC-I-OT1 complex was used as an example.
  • Mouse MHC-I-OT1 protein was expressed by E. coli and purified by histidine-tagged affinity chromatography.
  • the purified MHC-I-OT1 complexes were successfully conjugated to the phospholipid incorporated in the cell membrane of cells such as RBCs.
  • MHC-II presents antigen peptides to and activates immune cells, particularly CD4 + T cells, and thus a MHC complex comprising MHC-II and an antigen or an antigenic peptide can be bound to RBCs by sortase-mediated reactions as described herein.
  • This strategy of MHC complex can be used to treat or prevent diseases caused by viruses, such as HPV (e.g., targeting E6 /E7) , coronavirus (e.g., targeting SARS-CoV or SARS-CoV-2 Spike protein) , and influenza virus (e.g., targeting H antigen /N antigen) .
  • viruses such as HPV (e.g., targeting E6 /E7) , coronavirus (e.g., targeting SARS-CoV or SARS-CoV-2 Spike protein) , and influenza virus (e.g., targeting H antigen /N antigen) .
  • This strategy of MHC complex can also be used to target tumor mutations, for example Kras with mutations such as V8M and/or G12D, Alk with a mutation such as E1171D, Braf with a mutation such as W487C, Jak2 with a mutation such as E92K, Stat3 with a mutation such as M28I, Trp53 with mutations such as G242V and/or S258I, Pdgfra with a mutation such as V88I, and Brca2 with a mutation such as R2066K, for tumor treatment.
  • Kras with mutations such as V8M and/or G12D Alk with a mutation such as E1171D
  • Braf with a mutation such as W487C
  • Jak2 with a mutation such as E92K
  • Stat3 with a mutation such as M28I
  • Trp53 with mutations such as G242V and/or S258I
  • Pdgfra with a mutation such as V88I
  • the agent may comprise a growth factor.
  • the agent may comprise a growth factor for one or more cell types.
  • Growth factors include, e.g., members of the vascular endothelial growth factor (VEGF, e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D) , epidermal growth factor (EGF) , insulin-like growth factor (IGF; IGF-1, IGF-2) , fibroblast growth factor (FGF, e.g., FGF1-FGF22) , platelet derived growth factor (PDGF) , or nerve growth factor (NGF) families.
  • VEGF vascular endothelial growth factor
  • VEGF-A vascular endothelial growth factor
  • VEGF-B vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor
  • VEGF-D epidermal growth factor
  • EGF epidermal growth factor
  • IGF insulin-like growth
  • the agent may comprise a cytokine or the biologically active portion thereof.
  • a cytokine is an interleukin (IL) e.g., any of IL-1 to IL-38 (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12) , interferons (e.g., a type I interferon, e.g., IFN- ⁇ ) , and colony stimulating factors (e.g., G-CSF, GM-CSF, M-CSF) .
  • Cytokine (such as recombinant IL-2, recombinant IL-7, recombinant IL-12) loaded RBCs is a therapeutic delivery system for increasing tumor cytotoxicity and IFN- ⁇ production.
  • the agent may comprise a small molecule, e.g., those used as targeting moieties, immunomodulators, detection agents, therapeutic agents, or ligands (such as CD19, CD47, TRAIL, TGF, CD44) to activate or inhibit a corresponding receptor.
  • a small molecule e.g., those used as targeting moieties, immunomodulators, detection agents, therapeutic agents, or ligands (such as CD19, CD47, TRAIL, TGF, CD44) to activate or inhibit a corresponding receptor.
  • the agent may comprise a receptor or receptor fragment.
  • the receptor is a cytokine receptor, growth factor receptor, interleukin receptor, or chemokine receptor.
  • a growth factor receptor is a TNF ⁇ receptor (e.g., Type I TNF- ⁇ receptor) , VEGF receptor, EGF receptor, PDGF receptor, IGF receptor, NGF receptor, or FGF receptor.
  • a receptor is TNF receptor, LDL receptor, TGF receptor, or ACE2.
  • an agent to be conjugated may comprise an anti-cancer or anti-tumor agent, for example, a chemotherapy drug.
  • cells such as red blood cells are conjugated both with an anti-tumor agent and a targeting moiety, wherein the targeting moiety targets the cells such as red blood cell to a cancer.
  • Anti-cancer agents are conventionally classified in one of the following group: radioisotopes (e.g., Iodine-131, Lutetium-177, Rhenium-188, Yttrium-90) , toxins (e.g., diphtheria, pseudomonas, ricin, gelonin) , enzymes, enzymes to activate prodrugs, radio-sensitizing drugs, interfering RNAs, superantigens, anti-angiogenic agents, alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, anti-metabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones and anti-androgens.
  • radioisotopes e.g., Iodine-131, Lutetium-177, Rhenium-188, Yttrium-90
  • an anti-tumor agent is a protein such as a monoclonal antibody or a bispecific antibody such as anti-receptor tyrosine kinases (e.g., cetuximab, panitumumab, trastuzumab) , anti-CD20 (e.g., rituximab and tositumomab) and others for example alemtuzumab, aevacizumab, and gemtuzumab; an enzyme such as asparaginase; a chemotherapy drug including, e.g., alkylating and alkylating-like agents such as nitrogen mustards; platinum agents (e.g., alkylating-like agents such as carboplatin, cisplatin) , busulfan, dacarbazine, procarbazine, temozolomide, thioTEPA, treosulfan, and uramustine; purines such as cladribine, clofar
  • a tumor is a malignant tumor or a “cancer. ”
  • tumor includes malignant solid tumors (e.g., carcinomas, sarcomas) and malignant growths with no detectable solid tumor mass (e.g., certain hematologic malignancies) .
  • cancer is generally used interchangeably with “tumor” herein and/or to refer to a disease characterized by one or more tumors, e.g., one or more malignant or potentially malignant tumors.
  • Cancer includes, but is not limited to: breast cancer; biliary tract cancer; bladder cancer; brain cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma; adult T-cell leukemia/lymphoma; intraepithelial neoplasms; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastoma; melanoma, oral cancer including squamous cell carcinoma; ovarian cancer including ovarian cancer arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; neuroblastoma, pancreatic cancer; prostate cancer; rectal cancer; sarcomas including
  • an agent to be conjugated may comprise an anti-microbial agent.
  • An anti-microbial agent may include compounds that inhibit proliferation or activity of, destroy, or kill bacteria, viruses, fungi, or parasites.
  • the red blood cells are conjugated with an anti-microbial agent against a bacteria, virus, fungi, or parasite, and with a targeting moiety, wherein the targeting moiety targets the cell to the bacteria, virus, fungi, or parasite.
  • the anti-microbial agent may include ⁇ -lactamase inhibitory proteins or metallo-beta-lactamase for treating bacterial infections.
  • an agent to be conjugated may comprise probes, which can be used as for example diagnostic tools.
  • probes which can be used as for example diagnostic tools.
  • Molecular imaging has been demonstrated as an efficient way for tracking disease progression such as in cancer.
  • Small molecular probes such as fluorescein can be labeled on cells through the methods as described herein, instead of conventional chemical reaction which may cause damage to cells.
  • an agent to be conjugated may comprise a prodrug.
  • prodrug refers to a compound that, after in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically, or therapeutically active form of the compound.
  • a prodrug may be designed to alter the metabolic stability or the transport characteristics of a compound, to mask side effects or toxicity, to improve the flavor of a compound and/or to alter other characteristics or properties of a compound.
  • a prodrug is preferably a compound that, after in vivo administration, converts to its active form via enzymatic catalysis.
  • Sortase can recognize specific sortase recognition motifs like sequence LPXTG, and connect the LPXTG at the C-terminus of a protein with the G at the N-terminus of another protein through a transpeptidation reaction.
  • Sortase recognition motifs like sequence LPXTG, and connect the LPXTG at the C-terminus of a protein with the G at the N-terminus of another protein through a transpeptidation reaction.
  • the present disclosure provides a method for modifying a cell, comprising: providing a phospholipid having an agent conjugated thereto via a sortase recognition motif; and treating the cell with the phospholipid with the conjugated agent to incorporate the phospholipid into the cell membrane of the cell.
  • the phospholipid with the conjugated agent comprises a structure of A 1 -L 1 -Gly m X n -optional linker-phospholipid, in which A 1 represents the agent, L 1 represents the residual part of a sortase recognition motif after a sortase-mediated reaction, Gly m represents m glycines with m preferably being 1-5, X n represents n spacing amino acids with n preferably being 0-10, and the optional linker is preferably a PEG linker.
  • the present disclosure provides a method for modifying a cell, comprising: pretreating the cell with a phospholipid linked to Gly m to incorporate the phospholipid into the cell membrane of the cell, wherein Gly m represents m glycines with m preferably being 1-5; and contacting the pre-treated cell with a sortase substrate that comprises a sortase recognition motif and an agent, in the presence of a sortase under conditions suitable for the sortase to conjugate the sortase substrate to the Gly m by a sortase-mediated reaction.
  • the phospholipid linked to Gly m comprises a structure of Gly m X n -optional linker-phospholipid, wherein the X n represents n spacing amino acids with n preferably being 0-10 and the optional linker is preferably a PEG linker, and/or the sortase substrate comprises a structure of A 1 -M, in which A 1 represents the agent, and M represents the sortase recognition motif.
  • Modified cells described herein have a number of uses.
  • the modified cells may be used as a vaccine component, a delivery system, or a diagnostic tool.
  • the modified cells may be used to treat or prevent various disorders, conditions or diseases as described herein such as tumors or cancers, metabolic diseases such as lysosomal storage disorders (LSDs) , bacterial infections, virus infections such as coronavirus for example SARS-COV or SARS-COV-2 infection, autoimmune diseases or inflammatory diseases.
  • the modified cells may be used in cell therapy.
  • therapy is administered for treatment of cancer, infections such as bacterial or virus infections, autoimmune diseases, or enzyme deficiencies.
  • the cells modified with peptides for inducing immunotolerances may be used to modulate immune response such as inducing immunotolerance.
  • the administered cells may originate from the individual to whom they are administered (autologous) , may originate from different genetically identical individual (s) of the same species (isogeneic) , may originate from different non-genetically identical individual (s) of the same species (allogeneic) , or may originate from individual (s) of a different species.
  • allogeneic cells may originate from an individual who is immuno-compatible with the subject to whom the cells are administered.
  • the modified cells are used as a delivery vehicle or system for the agent.
  • the modified cells that have a protein conjugated to phospholipid incorporated into their cell membrane may serve as delivery vehicles for the protein.
  • Such cells may be administered to a subject suffering from a deficiency of the protein or who may benefit from increased levels of the protein.
  • the cells are administered to the circulatory system, e.g., by infusion. Examples of various diseases associated with deficiency of various proteins, e.g., enzymes, are provided above.
  • using the modified cells as a delivery system can achieve a retention release, for example, for delivering hormones like glucocorticoids, insulin, and/or growth hormones in a retention release profile.
  • the present disclosure provides a method for diagnosing, treating, or preventing a disorder, condition, or disease in a subject in need thereof, comprising administering the cell or composition as described herein to the subject.
  • the disorder, condition, or disease is selected from a group consisting of tumors or cancers, metabolic diseases such as lysosomal storage disorders (LSDs) , bacterial infections, virus infections such as coronavirus for example SARS-COV or SARS-COV-2 infection, autoimmune diseases, and inflammatory diseases.
  • LSDs lysosomal storage disorders
  • treating, ” “treat, ” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a pathogen-associated disease, disorder or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.
  • preventing, ” “prevent, ” or “prevention” refers to a course of action initiated prior to infection by, or exposure to, a pathogen or molecular components thereof and/or before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to prevent infection and/or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject.
  • a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder, or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder, or condition.
  • the method as described herein further comprises administering the modified cells to a subject, e.g., directly into the circulatory system, e.g., intravenously, by injection or infusion.
  • a method of delivering an agent to a subject in need thereof comprising administering the modified cell or the composition as described herein to the subject.
  • delivery or “delivering” refers to transportation of a molecule or agent to a desired cell or tissue site. Delivery can be to the cell surface, cell membrane, cell endosome, within the cell membrane, nucleus or within the nucleus, or any other desired area of the cell.
  • a method of increasing the circulation time or plasma half-life of an agent in a subject comprising attaching the agent to a cell according to the method as described herein.
  • the method further comprises administering the cell having the agent attached thereto to the subject, e.g., directly into the circulatory system, e.g., intravenously or by injection or infusion.
  • a subject receives a single dose of cells, or receives multiple doses of cells, e.g., between 2 and 5, 10, 20, or more doses, over a course of treatment.
  • a dose or total cell number may be expressed as cells/kg.
  • a dose may be about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cells/kg.
  • a course of treatment lasts for about 1 week to 12 months or more e.g., 1, 2, 3, or 4 weeks or 2, 3, 4, 5, or 6 months.
  • a subject may be treated about every 2-4 weeks.
  • the number of cells, doses, and/or dosing interval may be selected based on various factors such as the weight, and/or blood volume of the subject, the condition being treated, response of the subject, etc.
  • the exact number of cells required may vary from subject to subject, depending on factors such as the species, age, weight, sex, and general condition of the subject, the severity of the disease or disorder, the particular cell (s) , the identity and activity of agent (s) conjugated to the cells, mode of administration, concurrent therapies, and the like.
  • the present disclosure provides a composition comprising the modified cell as described herein and optionally a physiologically acceptable carrier, such as in the form of a pharmaceutical composition, a delivery composition or a diagnostic composition or a kit.
  • a physiologically acceptable carrier such as in the form of a pharmaceutical composition, a delivery composition or a diagnostic composition or a kit.
  • the composition may comprise a plurality of cells such as blood cells, e.g., RBCs.
  • at least a selected percentage of the cells in the composition are modified, i.e., having an agent conjugated thereto by the method as described herein. For example, in some embodiments at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the cells have an agent conjugated thereto.
  • two or more red blood cells or red blood cell populations conjugated with different agents are included.
  • a composition comprises the modified cells such as blood red cells, wherein the cells are modified with any agent of interest.
  • a composition comprises an effective amount of cells, e.g., up to about 10 14 cells, e.g., about 10, 10 2 , 10 3 , 10 4 , 10 5 , 5 ⁇ 10 5 , 10 6 , 5 ⁇ 10 6 , 10 7 , 5 ⁇ 10 7 , 10 8 , 5 ⁇ 10 8 , 10 9 , 5 ⁇ 10 9 , 10 10 , 5 ⁇ 10 10 , 10 11 , 5 ⁇ 10 11 , 10 12 , 5 ⁇ 10 12 , 10 13 , 5 ⁇ 10 13 , or 10 14 cells.
  • the number of cells may range between any two of the afore-mentioned numbers.
  • an effective amount refers to an amount sufficient to achieve a biological response or effect of interest, e.g., reducing one or more symptoms or manifestations of a disease or condition or modulating an immune response.
  • a composition administered to a subject comprises up to about 10 14 cells, e.g., about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 cells, or any intervening number or range.
  • physiologically acceptable carrier is meant a solid or liquid filler, diluent, or encapsulating substance that may be safely used in systemic administration.
  • a variety of carriers, diluents, and excipients well known in the art may be used.
  • These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.
  • sugars starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free
  • Mg SrtA (SEQ ID NO: 7/8) and eGFP-LPETG cDNA (SEQ ID NO: 9/10) were cloned in pET vectors and transformed in E. coli BL21 (DE3) cells for protein expression.
  • Transformed cells were cultured at 37 °C until the OD 600 reached 0.6, and then 500 ⁇ M IPTG (Isopropyl-beta-D-thiogalactopyranoside) was added. The cells were cultured with IPTG for 4 hrs at 37 °C until harvested by centrifugation and subjected to lysis by precooled lysis buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl) .
  • the lysates were sonicated on ice (5s on, 5s off, 60 cycles, 25%power, Branson Sonifier 550 Ultrasonic Cell Disrupter) . All supernatants were filtered by 0.45 ⁇ M filter after centrifugation at 14,000 g for 40 min at 4 °C. Filtered supernatants were loaded onto HisTrap FF 1 mL column (GE Healthcare) connected to the design chromatography systems. The proteins were eluted with the elution buffer containing 20 mM Tris-HCl, pH 7.8, 500 mM NaCl and 300 mM imidazole. All eluted fractions were analyzed on an SDS-PAGE gel.
  • amino acid sequence of eGFP-LEPTG is as shown in SEQ ID NO: 9 below:
  • eGFP-LEPTG The nucleotide sequence of eGFP-LEPTG is as shown in SEQ ID NO: 10 below:
  • Red blood cells were separated from peripheral blood of B6/C57 mice by density gradient centrifugation. The separated red blood cells were washed with PBS 3 times.
  • GAAS-PEG2000-DSPE linker (Synthesized by RUIXIBIO; see Fig. 2) was synthesized at a purity of more than 99%.
  • GAAS-PEG2000-DSPE was dissolved in 37°C phosphate buffer to a final concentration of 100 ⁇ M. Then 1 ⁇ 10 9 RBCs were pretreated with 50 ⁇ M GAAS-PEG2000-DSPE for 30 mins at 37°C. Then the pretreated RBCs were washed with PBS 3 times. The pretreated RBCs were used immediately or after storage for only a short time.
  • Reactions were performed in a total volume of 200 ⁇ L at room temperature for 2 hrs in PBS buffer while being rotated at a speed of 10 rpm.
  • the concentration of GAAS-PEG2000-DSPE-RBCs in the reaction was 1 ⁇ 10 9 /mL.
  • the concentration of mg SrtA was 10 ⁇ M and the concentrations of eGFP-LEPTG substrates were in the range of 10 ⁇ M.
  • the labeling efficiency of RBCs was analyzed by Beckman Coulter CytoFLEX LX.
  • Reactions were performed in a total volume of 1 mL at room temperature for 1 hr in PBS buffer while being rotated at a speed of 10 rpm.
  • concentration of mgSrtA was 10 ⁇ M
  • concentrations of GAAS-PEG2000-DSPE and eGFP-LPETG protein were 1 mM and 1 mM, respectively.
  • the eGFP-LPETGAAS-PEG2000-DSPE products were collected by removal of excess linker via dialysis and ultrafiltration.
  • Reactions were performed in a total volume of 200 ⁇ L at room temperature for 1 hr in PBS buffer while being rotated at a speed of 10 rpm.
  • the concentration of RBCs in the reaction was 1 ⁇ 10 9 /mL.
  • the labeling efficiency of RBCs was analyzed by Beckman Coulter CytoFLEX LX.
  • eGFP-LPETGAAS-PEG2000-DSPE-labeled in vitro results showed that 100%of natural RBCs were eGFP-LPETGAAS-PEG2000-DSPE-labeled in vitro, and the signal intensity was dose-dependent (see Figs. 3A-3B) .
  • Fig. 3A (Method 1) and Fig. 3B (Method 2) eGPF signals on the RBCs’ surface after their incubation with eGFP-LPETGAAS-PEG-DSPE were significantly higher than the unlabeled RBCs and the RBCs incubated with eGFP-LPETG.
  • eGFP-LPETGAAS-PEG-DSPE labelled mouse RBCs (dosage: 1 ⁇ 10 9 /mouse) , which were simultaneously labeled by a fluorescent dye cell trace Far Red, into recipient mice.
  • the percentage of Far Red and eGFP-LPETGAAS-PEG-DSPE positive RBCs in vivo was analyzed periodically.
  • eGFP-LPETGAAS-PEG-DSPE labeled RBCs not only showed the same lifespan as that of the control groups (Mice that transfused RBCs without eGFP-LPETGAAS-PEG-DSPE label) (see Fig. 5A) but also exhibited sustained signals in circulation for 28 days (see 5B) . No significant hemolysis was observed.
  • PBMCs peripheral blood mononuclear cells
  • the purpose of this step was to achieve a density gradient centrifugation of the cell components by the lymphocyte separation solution, and to separate the PBMCs from different cells such as red blood cells and platelets to ensure subsequent enrichment of the T cells.
  • T cell isolation the antigen on the cell surface was bound to the corresponding biotin-labeled antibody, and the biotin was bound to the streptavidin-labeled magnetic beads to express the lineage on the cell surface under the action of magnetic force. Cells with specific surface markers were isolated by magnetic beads.

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Abstract

L'invention concerne un lieur cellulaire et une cellule modifiée par un agent lié à celui-ci, l'agent étant conjugué à un phospholipide par l'intermédiaire d'un motif de reconnaissance de sortase, et le phospholipide avec l'agent conjugué étant incorporé dans la membrane cellulaire de la cellule. L'invention concerne également un procédé d'obtention de la cellule modifiée, ainsi que l'utilisation des cellules modifiées pour l'administration d'agents tels que des médicaments et des sondes.
PCT/CN2022/136600 2021-12-06 2022-12-05 Cellules modifiées et leurs utilisations pour l'administration d'agents WO2023103963A1 (fr)

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Citations (4)

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CN103204911A (zh) * 2005-12-09 2013-07-17 布拉科瑞士有限公司 靶向载体-磷脂缀合物
US20170183380A1 (en) * 2015-09-12 2017-06-29 Mahmoud Reza Jaafari Cancer targeting by anti-egfr peptides and applications thereof
WO2021185359A1 (fr) * 2020-03-20 2021-09-23 Westlake Therapeutics (Hangzhou) Co. Limited Globules rouges modifiés et leurs utilisations pour l'administration d'agents
WO2022089605A1 (fr) * 2020-10-30 2022-05-05 Westlake Therapeutics (Hangzhou) Co. Limited Globules rouges modifiés et utilisations correspondantes pour l'administration d'agents

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CN103204911A (zh) * 2005-12-09 2013-07-17 布拉科瑞士有限公司 靶向载体-磷脂缀合物
US20170183380A1 (en) * 2015-09-12 2017-06-29 Mahmoud Reza Jaafari Cancer targeting by anti-egfr peptides and applications thereof
WO2021185359A1 (fr) * 2020-03-20 2021-09-23 Westlake Therapeutics (Hangzhou) Co. Limited Globules rouges modifiés et leurs utilisations pour l'administration d'agents
WO2022089605A1 (fr) * 2020-10-30 2022-05-05 Westlake Therapeutics (Hangzhou) Co. Limited Globules rouges modifiés et utilisations correspondantes pour l'administration d'agents

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HUANG J; JIANG H; HE Z MA J ZHANG X HUANG J; WU Z; WU X WU Y WANG Y: "Preparation and biocompatibility evaluation of PEG-PLL/RGD-PEG-DSPE/phospholipid/CaP nanoparticles", JOURNAL OF BIOMEDICAL NANOTECHNOLOGY, AMERICAN SCIENTIFIC PUBLISHERS, US, vol. 14, no. 1, 1 January 2018 (2018-01-01), US , pages 98 - 113, XP009533759, ISSN: 1550-7033, DOI: 10.1166/jbn.2018.2460 *
J. SHI, L. KUNDRAT, N. PISHESHA, A. BILATE, C. THEILE, T. MARUYAMA, S. K. DOUGAN, H. L. PLOEGH, H. F. LODISH: "Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 111, no. 28, 15 July 2014 (2014-07-15), pages 10131 - 10136, XP055189994, ISSN: 00278424, DOI: 10.1073/pnas.1409861111 *

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