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WO2026035680A1 - Compositions et procédés d'administration d'agents - Google Patents

Compositions et procédés d'administration d'agents

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Publication number
WO2026035680A1
WO2026035680A1 PCT/US2025/040643 US2025040643W WO2026035680A1 WO 2026035680 A1 WO2026035680 A1 WO 2026035680A1 US 2025040643 W US2025040643 W US 2025040643W WO 2026035680 A1 WO2026035680 A1 WO 2026035680A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
nucleic acid
cell
lipid
functionalized
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/040643
Other languages
English (en)
Inventor
Jieni XU
Ryan Francis LANDIS
Cafer OZDEMIR
Burak YILMAZ
Yusuf ERKUL
Manfred Kraus
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kernal Biologics Inc
Original Assignee
Kernal Biologics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kernal Biologics Inc filed Critical Kernal Biologics Inc
Publication of WO2026035680A1 publication Critical patent/WO2026035680A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/13—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • lipid nanoparticles as delivery vehicles for biologically active substances. There remains a poor understanding of which compositions and methods of lipid nanoparticles provide specific tissue targeted delivery.
  • the present disclosure recognizes a need for compositions, preparations, and methods of use of lipid nanoparticles (LNPs) for the targeted delivery of nucleic acids.
  • LNPs lipid nanoparticles
  • the present disclosure recognizes that specific compositions, preparations, and methods of use of nanoparticles are useful for the targeted delivery of nucleic acids to particular tissues and cell types in vivo.
  • the present disclosure provides methods and compositions for delivering a nucleic acid to a tissue of a subject.
  • a tissue is extrahepatic.
  • a tissue is immune tissue.
  • the present disclosure provides compositions and methods of use of nanoparticles for the delivery of nucleic acids to immune cells.
  • the present disclosure documents surprisingly efficient delivery of payload(s), including expression of nucleic acid payload(s) to immune cells.
  • payload(s) including expression of nucleic acid payload(s) to immune cells.
  • such delivery is to T cells.
  • such delivery is to B cells.
  • delivery and expression of nucleic acid payloads by LNPs of the present disclosure is achieved by functionalization of the LNPs.
  • functionalization is achieved by direct conjugation of a targeting entity to the surface of an LNP.
  • the present disclosure provides a functionalized nucleic acid lipid particle composition
  • a functionalized nucleic acid lipid particle composition comprising: lipid components that encapsulate a Attorney Docket No.: 2013260-0047 payload to form a particle; and a targeting entity.
  • a payload is an RNA.
  • an RNA encodes a therapeutic agent.
  • an RNA encodes a chimeric antigen receptor.
  • lipid components comprise an ionizable lipid, a sterol, a helper lipid, and a PEG-lipid.
  • a PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, Maleimide-PEG-DSPE, Azide-PEG-DSPE and DBCO-PEG-DSPE.
  • a helper lipid is a phospholipid.
  • a helper lipid is DSPC.
  • a targeting entity comprises an antibody agent.
  • an antibody agent is covalently linked to the surface of the nucleic acid lipid particle by DTT reduction, SATA thio conjugation or DBCO conjugation.
  • an antibody agent binds CD19. In some embodiments, an antibody agent comprises an anti-CD19 antibody or antibody fragment. In some embodiments, an antibody agent binds CD3. In some embodiments, an antibody agent comprises an anti-CD3 antibody or antibody fragment. In some embodiments, and antibody agent binds CD79b. In some embodiments, an antibody agent comprises an anti-CD79b antibody or antibody fragment.
  • the present disclosure provides a method of delivering a nucleic acid to a tissue or a cell of a subject, the method comprising administering to the subject a functionalized nucleic acid lipid particle described herein.
  • a tissue is or comprises immune tissue.
  • a cell is or comprises an immune cell.
  • the immune tissue is spleen or thymus.
  • the immune cell is a B-cell or T-cell.
  • the present disclosure provides a method of treating a subject suffering from a disease, the method comprising administering to the subject a functionalized nucleic acid lipid particle as described herein.
  • a disease is a cancer.
  • a disease is a disease effecting a T-cell or B-cell.
  • the present disclosure provides a method of manufacturing a nucleic acid lipid particle comprising combining a nucleic acid with lipid components including an ionizable lipid, a helper lipid, a sterol, a PEG-lipid.
  • the present disclosure provides a method of manufacturing a functionalized nucleic acid lipid particle comprising combining a payload with lipid components and directly conjugating a targeting moiety to the lipid nanoparticle encapsulating the pay load.
  • Figures 1A-1C depict mAb reduction condition optimization and characterization of reduced mAb.
  • Figure 2 depicts mAb reduction condition optimization and characterization of reduced mAb.
  • Figure 3 depicts biodistribution of various LNP compositions in B6 Albino mice.
  • Figure 4 depicts biodistribution of T-mAb-LNPs with Lipid 5 versus LIP091 peripheral core compositions in B6 Albino mice.
  • Figures 5A-5C depict surface-functionalized T-MAB-LNPs with mAb 2- LNP demonstrates selective T cell targeting over off-target cells in human PBMCs.
  • Figures 6A and 6B depicts T-cell targeted CD19 CAR T-mAb 2-LNP uptake in cultured human PBMCs results in T-cell activation.
  • Figure 7 demonstrates B-cell depletion in mouse peripheral blood after CD19 CAR T-LNP in situ treatment.
  • Figures 8A-8F demonstrate Thy 1.1 RNA outperformed mCherry as the reporter RNA in vivo.
  • a and B Representative FACS histogram plots for mCherry vs Thy 1.1 signal in A20 cells.
  • A20 cells were transfected with aCD19 and ISO-LNP loaded with mCherry or Thy 1.1 RNA at 0.2 mg/mL. After 24 h, cells were washed, and protein expression was measured through flow cytometry.
  • C-F Representative FACS histogram Attorney Docket No.: 2013260-0047 plots and quantification of mCherry vs Thyl.l signal in spleen B-cells in vivo.
  • FIGS 9A-9C depict screening of anti-CD19 antibodies in vitro and in vivo.
  • 9A In vitro transfection efficiency (mCherry MFI) of anti-CD19 antibodies of different clones from different vendors.
  • A20 cells were transfected with aCD19-RG7-Peripheral and ISO-RG7-Peripheral LNPs loaded with mCherry at 0.2 mg/mL. After 24 h, cells were washed, and protein expression was measured through flow cytometry.
  • 9B and 9C In vivo cell binding (DiD+%) and protein expression (mCherry+%) of the top 3 candidates from in vitro screening versus the initial 1D3 antibody in spleen and bone marrow B-cells.
  • FIGS 10A-10L depict screening of targeting antibodies to improve B-cell delivery in vitro and in vivo.
  • 10A to 10F In vitro screening of B-cell targeting antibodies. Splenocytes were harvested from 8-week-old BALB/c mice and transfected with mAh functionalized LNP loaded with Thy 1. 1 RNA and DiD dye at 0.2 mg/mL. After 4h, cells were washed, stained with fluorescent labeled anti-Thyl. l mAb. The protein expression and cell uptake were measured by Thyl.l (A to C) and DiD (D to F) fluorescent signals in B- cells, myeloid cells and T-cells through flow cytometry. 10G-10L) In vivo validation of B cell targeting antibodies.
  • FIGS 11A-11J demonstrate dose titration of aCD79b-RG7-Peripheral LNP in spleen and bone marrow B-cell subpopulations and non-B-cells.
  • Eight-week-old BALB/c mice were injected with DiD dye and Thy 1.1 encoding RNA co-encapsulated LNPs through tail vein at 0.1 and 0.033 mg/kg.
  • spleens and bone marrows were harvested, processed into single cells, and subsequently stained with viability dye and surface markers. Dead cells and red blood cells were excluded by viability dye and TER- 119.
  • B-cells, developing B-cells, mature B-cells, T-cells and myeloid cells were identified as B220+CD3- , B220i»CD3-, B220h, g hCD3-, B220-CD3+, and B220-CD3-, respectively.
  • N 3-4/group.
  • Figures 12A-12D demonstrate Optimization of core LNP compositions for potent and selective B-cell targeting in vivo.
  • a and B Quantification of percentage of positive cells (A) and mean fluorescent intensity (B) for DiD and Thyl. 1 of standard LNP and aCD79b-standard LNP versus peripheral LNP and aCD79b-peripheral LNP in spleen B- cells in vivo.
  • C and D Quantification of cell uptake and protein expression by mean fluorescent intensity of DiD (C) and Thy 1. 1 (D) of LNP in B-cells, myeloid cells and T- cells. 10- week-old BALB/c mice were injected with Thy 1.1 RNA LNP through tail vein injection at 0.1 mg/kg.
  • B-cells, myeloid cells and T-cells were identified as B220+CD3-, B220-CD3-, and B220-CD3+, respectively.
  • N 2-3/group.
  • Figures 13A- 13F demonstrate direct conjugation of aCD79b onto the peripheral LNP compared to the previous two-layer method in vivo.
  • a and D Thyl.l RNA expression after LNPs treatments in spleen mature and developing B-cells. (One-way ANOVA with Dunnett’s multiple comparison test to the aCD79b-Peripheral LNP).
  • B and E B-cell size change indicated by FSC after LNPs treatments in spleen mature and developing B-cells.
  • C and F IgM expression after LNPs treatments in spleen mature and developing B- Attorney Docket No.: 2013260-0047 cells.
  • Figures 14A-14B demonstrate tissue level distribution of aCD79b-Peripheral LNP versus standard LNP core and peripheral LNP core.
  • A) Representative IVIS bioluminescence images of all organs. 10-week-old B6- Albino mice were injected with fLuc RNA LNP through tail vein injection at 0.3 mg/kg. After 18 hours, D-luciferin was injected intraperitoneally at 150 mg/kg. 10 min later. The thymus, lungs, livers, spleens, and lymph nodes were then harvested for ex vivo imaging. N 5/group.
  • FIGS 15 -15F demonstrate in vitro transfection of human PMBCs by anti-human B-cell targeting mAbs functionalized on peripheral LNPs.
  • Human PBMCs were plated into U-shape 96- well plate in RPMI-1640 containing 10% FBS at 105 cells per well.
  • Administration typically refers to the administration of a composition to a subject or system.
  • routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • administration may be ocular, oral, parenteral, topical, etc..
  • administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc.
  • bronchial e.g., by bronchial instillation
  • buccal which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc
  • enteral intra-arterial, intradermal, intragastric,
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • agent may refer to a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.
  • agent may refer to a compound, molecule, or entity that comprises a polymer.
  • the term may refer to a compound or entity that comprises one or more polymeric moieties.
  • agent may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety.
  • the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.
  • Amelioration refers to the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes, but does not Attorney Docket No.: 2013260-0047 require complete recovery or complete prevention of a disease, disorder or condition (e.g., radiation injury).
  • amino acid refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
  • an amino acid has the general structure H2N-C(H)(R)-COOH.
  • an amino acid is a naturally-occurring amino acid.
  • an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid.
  • Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide can contain a structural modification as compared with the general structure above.
  • an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure.
  • such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid.
  • such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
  • the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
  • an analog refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways.
  • an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance.
  • an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance.
  • an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.
  • Antagonist Those skilled in the art will appreciate that the term
  • an antagonist may be used to refer to an agent, condition, or event whose presence, level, degree, type, or form correlates with decreased level or activity of another agent (i.e., the inhibited agent, or target).
  • an antagonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity.
  • an antagonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an antagonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level or activity of the target is altered).
  • an antibody agent refers to an agent that specifically binds to a particular antigen.
  • the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody.
  • CDR complementarity determining region
  • an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference Attorney Docket No.: 2013260-0047
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR.
  • an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR.
  • an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.
  • an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.
  • an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilled Attorney Docket No.: 2013260-0047 in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc.
  • an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains).
  • an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®;
  • SMIPsTM Small Modular ImmunoPharmaceuticals
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc. ].
  • a covalent modification e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc]
  • other pendant group e.g., poly-ethylene glycol, etc.
  • Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
  • a particular disease, disorder, or condition e.g., across a relevant Attorney Docket No.: 2013260-0047 population.
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
  • Biologically active refers to an observable biological effect or result achieved by an agent or entity of interest.
  • a specific binding interaction is a biological activity.
  • modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity.
  • presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.
  • a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • precancerous e.g., benign
  • malignant pre-metastatic
  • metastatic metastatic
  • non-metastatic e.g., metastatic
  • present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant.
  • a relevant cancer may be characterized by a solid tumor.
  • a relevant cancer may be characterized by a hematologic tumor.
  • examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, Attorney Docket No.: 2013260-0047 breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.
  • hematopoietic cancers including le
  • chemotherapeutic agent has used herein has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example specifically including agents utilized and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation.
  • chemotherapeutic agents are useful in the treatment of cancer.
  • a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more an thracy clines, one or more cytoskeletal disruptors (e.g.
  • microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity).
  • HDACs histone deacetylase inhibitors
  • topoisomerase inhibitors e.g., inhibitors of topoisomerase I and/or topoisomerase II
  • kinase inhibitors e.g., inhibitors of topoisomerase
  • a chemotherapeutic agent may be or comprise one or more of Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and/or analogs thereof (e.g.
  • DM1 Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof.
  • a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate.
  • a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLLl -doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7-Pro-2-P-Dox, hMN-14- Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLLl-Pro-2- P-Dox, P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedot
  • Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
  • the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
  • “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
  • combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • composition may be used to refer to a discrete physical entity that comprises one or more specified components.
  • a composition may be of any form - e.g., gas, gel, liquid, solid, etc.
  • composition or method described herein as “comprising” one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method.
  • any composition or method described as “comprising” (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method “consisting essentially of” (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method.
  • composition or method described herein as “comprising” or “consisting essentially of” one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method “consisting of” (or “consists of”) the named elements or steps to the exclusion of any other unnamed element or step.
  • known or disclosed equivalents of any named essential element or step may be substituted for that element or step.
  • the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents.
  • Encapsulated The term “encapsulated” is used herein to refer to substances that are completely surrounded by another material.
  • Engineered In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be Attorney Docket No.: 2013260-0047
  • an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence.
  • a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell.
  • a particular agent of interest e.g., a protein, a nucleic acid, and/or a particular form thereof
  • progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • Excipient refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
  • suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • a gene product can be a transcript.
  • a gene product can be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and/or (4) post- translational modification of a polypeptide or protein.
  • Human In some embodiments, a human is an embryo, a fetus, an infant, a child, a teenager, an adult, or a senior citizen.
  • an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc).
  • comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • In vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Nanoparticle refers to a discrete entity of small size, e.g., typically having a longest dimension that is shorter than about 1000 nanometers (nm) and often is shorter than 500 nm, or even 100 nm or less. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 50 nm to 200nm, or between about 1 pm and about 500 nm, or between about 1 nm and 1000 nm.
  • a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is below about 1000 nm, about 750 nm, about 500 nm, about 200 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm.
  • a nanoparticle may be substantially spherical (e.g., so that its longest dimension may be its diameter).
  • a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health.
  • nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen).
  • a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and/or biodegradable polymer.
  • Nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
  • a "nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA.
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
  • a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.
  • a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
  • a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds.
  • a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine).
  • adenosine thymidine, guanosine, cytidine
  • uridine deoxyadenosine
  • deoxythymidine deoxy guanosine
  • deoxy cytidine deoxy cytidine
  • a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl- uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases,
  • a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.
  • a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
  • Payload In general, the term “payload”, as used herein, refers to an agent that may be delivered or transported by association with another entity. In some embodiments, such association may be or include a covalent linkage; in some embodiments such association may be or include non-covalent interaction(s). In some embodiments, association may be direct; in some embodiments, association may be indirect.
  • association may be direct; in some embodiments, association may be indirect.
  • a payload is not limited to a particular chemical identity or type; for example, in some embodiments, a payload may be or comprise, for example, an entity of any chemical class including, for example, a lipid, a metal, a nucleic acid, a polypeptide, a saccharide (e.g., a polysaccharide), small molecule, or a combination or complex thereof.
  • a payload may be or comprise a biological modifier, a detectable agent (e.g., a dye, a fluorophore, a radiolabel, etc.), a detecting agent, a nutrient, a therapeutic agent, etc., or a combination thereof.
  • a payload may be or comprise a cell or organism, or a fraction, extract, or component thereof.
  • a pay load may be or comprise a natural product in that it is found in and/or is obtained from nature; alternatively or additionally, in some embodiments, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature.
  • an pay load may be or comprise an agent in isolated or pure form; in some embodiments, such agent may be in crude form.
  • composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
  • active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or nonaqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or nonaqueous solutions or suspensions),
  • Reference As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
  • Subject refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms).
  • a subject is suffering from a relevant disease, disorder or condition.
  • a subject is susceptible to a disease, disorder, or condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • Therapeutic agent refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
  • the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • treat refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • treatment may be phrophylactic; for example may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
  • treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition and/or for delaying onset or decreasing rate of development or worsening of one or more features of a disease, disorder and/or condition.
  • the present disclosure provides lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • the present disclosure provides, at least in part, the discovery that surface functionalization of a lipid nanoparticle enhances association of a LNP with particular tissues.
  • the present disclosure provides functionalized lipid Attorney Docket No.: 2013260-0047 nanoparticles which exhibit increased delivery of a payload to specific tissues relative to other tissues.
  • the present disclosure provides functionalized lipid nanoparticles which exhibit tropism for specific tissues relative to other tissues.
  • the present disclosure provides functionalized lipid nanoparticles which exhibit increased delivery of a payload to specific cells relative to other cells.
  • the present disclosure provides functionalized lipid nanoparticles which exhibit tropism for specific tissues relative to other tissues.
  • the present disclosure provides functionalized LNPs comprising an ionizable lipid. In some embodiments, the present disclosure provides functionalized LNPs comprising at least one cationic lipid. In some embodiments, the present disclosure provides functionalized LNPs comprising at least one cationic ionizable lipid. In some embodiments, the term “cationic ionizable lipid” refers to lipid and lipid- like molecules with nitrogen atoms that can acquire charge (pKa). In some embodiments, a cationic ionizable lipid for use in accordance with the present disclosure has a pKa of 5, 6, 7, 8, 9, 10, 11 at physiological pH.
  • a cationic ionizable lipid comprises one or more groups which is protonated at physiological pH but deprotonates and has no charge at a pH above 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments a cationic ionizable lipid comprises one or more groups which are protonated and have a charge at a pH above 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the ionizable cationic group may contain one or more protonatable amines which are able to form a cationic group at physiological pH.
  • a cationic ionizable lipid has a high pKA.
  • a high pKa is a pKa greater than 7.
  • a high pKa is a pKa greater than 7.4.
  • a cationic ionizable lipid for use in accordance with the present disclosure has a pKa of between 7 and 8, 7.5 and 8.5, 8 and 9, 8.5 and 9.5, 9 and 10.
  • a cationic ionizable lipid for use in accordance with the present disclosure has a pKa of between 7.2 and 8.2, 7.4 and 8.4, 7.6 and 8.6, 7.8 and 8.8, 8.0 to 9.0, 8.2 to 9.2, 8.4 to 9.4, 8.6 to 9.6, 8.8 to 9.8, 9.0 to 10.0.
  • a useful cationic ionizable lipid has a pKa of 7. 1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
  • a cationic ionizable lipid with a high pKA (e.g., >7) is disclosed or described in WO2020219876; US20210162053; US20160317676; and WO2021113365 each of which is incorporated herein in their entirety.
  • a cationic ionizable lipid with a high pKA (e.g., >7 is one selected from those listed in Table 1.
  • functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to particular tissues or cells.
  • functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to tissues other than the liver (i.e., extrahepatic delivery).
  • functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to immune tissue.
  • functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to immune cells.
  • functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to B cells.
  • functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to T cells.
  • the present disclosure provides functionalized LNPs comprising a at least first cationic ionizable lipid and a second cationic ionizable lipid.
  • more than one (e.g., each) of such at least first and second cationic ionizable lipids has a high pKa (e.g., greater than , e.g., greater than 7.4) as described herein.
  • only one (i.e., a first) cationic ionizable lipid has such a high pKa (e.g., greater than 7, e.g., greater than 7.4) as described herein.
  • a second cationic ionizable lipid is a non-high-pKa lipid, e.g., in that it has a pKa below 7, e.g., about 6.8, 6.6, 6.4, 6.2, 6.0, 5.8, 5.6, 5.4, 5.2, 5.0 or lower.
  • a non-high-pKa lipid e.g., a second lipid
  • a non-high-pKa lipid e.g., a second lipid
  • a non-high-pKa lipid e.g., a second lipid
  • a cationic ionizable lipid with a non-high pKa (e.g., ⁇ 7) is disclosed or described in Finn et al., 2018 Cell Reports 22, 2227-2235; Jayaraman et al., Angew. Chem. Int. Ed. 2012, 51, 8529 -8533; Hassett et al., Molecular Therapy: Nucleic Acids Vol.
  • a cationic ionizable lipid with a non-high pKA is one selected from those listed in Table 4 Attorney Docket No.: 2013260-0047 Attorney Docket No.: 2013260-0047 Attorney Docket No.: 2013260-0047 Attorney Docket No.: 2013260-0047
  • a functionalized LNP comprises a lipid selected from
  • a functionalized LNP comprises about 0-80 mol % of high pKa cationic ionizable lipid as described herein; in some embodiments, a functionalized LNP comprises two or more high pKa cationic ionizable lipids that, together make up such 0-80 mol % of the functionalized LNP. In some embodiments, a functionalized LNP comprises about 20-80 mol % of high pKa cationic ionizable lipid. In some embodiments, a functionalized LNP comprises about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 7-, 75, 80 mol % of high pKa cationic ionizable lipid.
  • a functionalized LNP comprises about 0-60 mol % of cationic ionizable lipid that is not high pKa (e.g., that is characterized by a pKa below about Attorney Docket No.: 2013260-0047
  • a functionalized LNP comprises two or more non-high pKa cationic ionizable lipids that, together, make up such 0-60 mol % of the functionalized LNP.
  • a functionalized LNP comprises about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mol % of a non-high-pKa cationic ionizable lipid.
  • the present disclosure provides functionalized LNPs comprising at least one sterol.
  • a functionalized LNP comprises about 0-40 mol% of a sterol.
  • a functionalized LNP comprises about 0, 5, 10, 15, 20, 25, 30, 35, 40 mol% of a sterol.
  • a sterol is cholesterol, or a variant or derivative thereof.
  • a cholesterol is modified, for example oxidized. Unmodified cholesterol can be acted upon by enzymes to form variants that are side-chain or ring oxidized.
  • a cholesterol can be oxidized on the beta-ring structure or on the hydrocarbon tail structure.
  • Exemplary cholesterols that are considered for use in the disclosed functionalized LNPs include but are not limited to 25 -hydroxy cholesterol (25- OH), 20a-hydroxycholesterol (20a-OH), 27 -hydroxy cholesterol, 6-keto-5a- hydroxycholesterol, 7-ketocholesterol, 7
  • side-chain oxidized cholesterol can enhance cargo delivery relative to other cholesterol variants.
  • a cholesterol is an unmodified cholesterol.
  • the present disclosure provides functionalized LNPs comprising at least one helper lipid.
  • a helper lipid is a phospholipid.
  • a functionalized LNP comprises about 0-20 mol% of a helper lipid. In some embodiments, a functionalized LNP comprises about 0, 5, 10, 15, 20 mol% of a helper lipid.
  • Exemplary phospholipids include but are not limited to 1 ,2-distearoyL snglycero-3 -phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn- glycerophosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- Attorney Docket No.: 2013260-0047 dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC) , 1 ,2-diundecanoyl-sn- glycerophosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-diste
  • a phospholipid comprises l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(succinyl) (succinyl PE), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE),
  • DOPE 1.2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DMPC l,2-dimyristoyl-sn-glycero-3- phosphocholine
  • DPPC l,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • the present disclosure provides functionalized LNPs comprising at least one PEGylated lipid.
  • a functionalized LNP comprises about 0-25 mol% of a PEGylated lipid.
  • a functionalized LNP comprises about 0, 5, 10, 15, 20, 25 mol% of a PEGylated lipid.
  • inclusion of a PEGylated lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo.
  • PEGylation is reversible in that the PEG moiety is gradually released in blood circulation.
  • Exemplary PEGylated- lipids include but are not limited to PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20, PEG-modified-28- phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEGylated-lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEGDSPE lipid.
  • the present disclosure provides functionalized LNPs comprising at least one PEG-ligand.
  • a functionalized LNP comprises about 0-2 mol% of a PEG-ligand.
  • a functionalized LNP comprises about 0, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 1.5, 1.75, 2 mol% of a PEG-ligand.
  • a ligand e.g., as included in a PEG-ligand
  • a ligand is or comprises: antibodies targeting cell surface proteins, hyaluronic acid, small molecules, peptides, and/or peptides that target integrins.
  • the present disclosure provide functionalized LNPs directed to particular tissues.
  • functionalized LNPs of the present disclosure are directed to particular tissues by functionalization of the LNPs.
  • LNPs of the present disclosure are functionalized by conjugation of a targeting entity directly to the surface of an LNP described herein.
  • a targeting entity is an agent.
  • a targeting entity is an antibody agent.
  • an antibody agent is directly conjugated to an LNP.
  • an antibody agent is or comprises an antibody or antibody fragment that binds or targets a specific antigen expressed by and/or present on a desired cell type or tissue.
  • a targeting entity comprises a primary antibody agent and a secondary antibody agent.
  • a secondary antibody agent binds an Fc region.
  • a secondary antibody agent is or comprises an anti-human Fc, anti-mouse Fc, anti-rabbit Fc, or anti-hamster Fc antibody or antibody fragment.
  • a secondary antibody agent is or comprises, RG7/1.30 for Fc domain of Rat IgG2a, RG7/11.1 for Fc domain of Rat IgG2b.
  • a primary antibody or fragment is an antibody or fragment that binds or targets a specific antigen of the desired cell type or tissue.
  • a primary antibody or fragment binds or targets a B cell surface receptor.
  • a primary antibody or fragment binds or targets CD19, CD79b, CD20, CD23, CD38, or CD138.
  • antibody agents are modified by DTT, TCEP, SATA ((N-succinimidyl S -acetylthioacetate), SATP (N-succinimidyl-S-acetylthiopropionate), NHS-PEG4-DBCO, NHS-PEG4-Azide, or NHS-PEG4-ester to expose functional groups such as sulfhydryl, DBCO, and Azide groups.
  • an antibody agent contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 functional groups.
  • functionalized LNPs of the present disclosure target particular tissues or cells. In some embodiments, functionalized LNPs of the present disclosure target immune tissues and/or immune cells. In some embodiments, a functionalized LNP of the present disclosure targets a particular tissue or cell based on a targeting entity. In some embodiments a targeting entity binds a particular tissue or cell.
  • an immune tissue is spleen, bone marrow, thymus.
  • an immune cell is a B-cell or a T-cell.
  • a targeting entity binds a B-cell.
  • a targeting entity binds a T-cell.
  • a targeting entity binds or targets a B-cell surface receptor.
  • a targeting entity binds or targets a T-cell surface receptor.
  • a targeting entity binds or targets CD19, CD79b CD20, CD23, CD38, or CD138.
  • a targeting entity binds or targets CD3, CD62L, CD197, CD45.
  • functionalized LNPs of the present disclosure comprises a payload. In some embodiments, functionalized LNPs of the present disclosure encapuslate a payload. In some embodiments a payload is an agent
  • a payload is a nucleic acid.
  • exemplary nucleic acids may be or include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) RNA, analogs, and/or combinations thereof.
  • RNA ribonucleic acid
  • a nucleic acid may be or comprise single-stranded RNA, single-stranded DNA, double-stranded RNA, double stranded DNA, triple-stranded DNA, siRNA, shRNA, sgRNA, mRNA, miRNA, and/or antisense DNA.
  • a nucleic acid may include one or more non-natural residues as is known in the art.
  • a nucleic acid comprises a nucleic acid sequence that encodes a therapeutic.
  • a nucleic acid comprises a nucleic acid sequence that encodes a chimeric antigen receptor (CAR).
  • CARs are known in the art (see, e.g., Gill et al., Immunol. Rev. 263:68-89 (2015); Stauss et al., Curr. Opin. Pharmacol. 24: 113-118 (2015)).
  • a nucleic acid comprises a nucleic acid sequence that encodes a CAR that binds CD19 (e.g., a CAR19).
  • a nucleic acid comprises a nucleic acid sequence that encodes a CAR that binds BCMA.
  • a nucleic acid may be sequence engineered, for example to remove immunogenic sequence motifs.
  • a nucleic acid is sequence engineered to remove TLR7 or TLR8 stimulation motifs.
  • a nucleic acid is sequence engineered to remove motifs selected from the group consisting of KNUNDK motifs, UCW motifs, UNU motifs, UWN motifs, USU motifs, KWUNDK motifs, KNUWDK motifs, UNUNDK motifs, KNUNUK motifs, and combinations thereof.
  • a nucleic acid is sequence engineered as described in W02020/033720 the entirety of which is incorporated herein by reference.
  • a payload is a therapeutic or detection agent.
  • a detection agent is a visual marker (e.g., colormetric or fluorescent).
  • a detection agent is a surface expressed protein (e.g., Thy 1.1). Attorney Docket No.: 2013260-0047
  • functionalized LNPs of the present disclosure target or attach to specific tissues.
  • the present disclosure recognizes that inclusion of a targeting entity on the surface of an LNP results in more effective targeting of LNPs and thus delivery of a payload to specific tissues or cells.
  • functionalized LNPs as described herein preferentially target tissues other than liver tissues (i.e., extrahepatic).
  • functionalized LNPs as described herein preferentially target immune tissue or immune cells relative to other tissues.
  • functionalized LNPs as described herein preferentially target B cells or T cells, e.g., relative to liver tissue or cells.
  • provided functionalized LNPs achieve extrahepatic delivery at a level that is significantly higher than that observed with an appropriate reference LNP. In some embodiments, provided functionalized LNPs achieve immune tissue and/or immune cell delivery at a level that is significantly higher than that observed with an appropriate reference LNP.
  • provided functionalized LNPs achieve preferential extrahepatic, immune tissue and/or immune cell delivery relative to liver delivery to a degree greater than that observed with an appropriate reference LNP.
  • an RNA delivered with a provided functionalized LNP is preferentially expressed in immune tissue and/or immune cells relative to liver at ratios that may be 5, 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or more greater than those observed with a reference LNP.
  • whole body liver; tumorliver, spleemliver, B- celhliver, T-cell:liver, B-cell: T-cell, B-cell or T-cell/ myeloid cell expression ratio is 1, 2, 3, or 4 orders of magnitude greater for a provided functionalized LNP than for a relevant reference LNP.
  • provided functionalized LNPs achieve whole body, immune tissue, and/or immune cell delivery to a level that is as least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of its level of liver delivery.
  • provided functionalized LNPs achieve B- Attorney Docket No.: 2013260-0047 cell or T-cell delivery to a level that is as least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of its level of myeloid cell delivery.
  • provided functionalized LNPs achieve immune tissue, and/or immune cell delivery that is reasonably comparable to its liver delivery. In some embodiments, provided functionalized LNPs achieve preferential whole body, immune tissue, or immune cell delivery relative to liver delivery. In some embodiments, provided functionalized LNPs achieve preferential whole body, immune tissue, or immune cell delivery relative to myeloid cell delivery. In some embodiments, provided functionalized LNPs achieve whole body, immune tissue, or immune cell delivery at a level that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold or more that of its level of liver delivery.
  • provided functionalized LNPs achieve whole body, immune tissue, or immune cell delivery at a level that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold or more that of its level of myeloid cell delivery.
  • provided functionalized LNPs achieve both material levels of whole immune tissue and/or immune cell expression and significant preference for delivery to immune tissue and/or immune cell e.g., relative to liver (i.e., extrahepatic).
  • functionalized LNPs as described herein delivering nucleic acids to extrahepatic tissues can be selected from one of the following compositions: Attorney Docket No.: 2013260-0047
  • functionalized LNPs as described herein can be used to treat cancer. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents for the treatment of cancer. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids for the treatment of cancer. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents to cancers of immune tissue and/or immune cells. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids to cancers of immune tissue and/or immune cells.
  • functionalized LNPs as described herein can be used to treat diseases of T-cells. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents to T-cells cells. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids to T-cells cells.
  • functionalized LNPs as described herein can be used to treat diseases of B cells. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents to B cells. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids to B cells. Attorney Docket No.: 2013260-0047
  • disclosed functionalized LNPs can be used to treat a subject suffering from hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non- Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas.
  • leukemias lymphomas (Hodgkin’s and non- Hodgkin’s), myel
  • disclosed functionalized LNPs can be used to treat a subject suffering from autoimmune disorders, including but not limited to rheumatoid arthritis, multiple sclerosis, type I diabetes, Addison disease, celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, and systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis.
  • autoimmune disorders including but not limited to rheumatoid arthritis, multiple sclerosis, type I diabetes, Addison disease, celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, and systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic
  • functionalized LNPs used to treat a subject suffering from a disease can be administered as combination therapy.
  • a functionalized LNP used to treat a subject suffering from a disease can be administered to a subject in combination with other methods of treatment (e.g., standard of care treatment) for the disease.
  • functionalized LNPs of the present disclosure are evaluated for potency, selectivity, and/or tolerability.
  • potency is measured by level of RNA expression in a specific targeted tissue (e.g., T-cell, B-cell).
  • selectivity is measured by ratio of RNA expression in a specific targeted tissue relative to another tissue or a control tissue (e.g., immune tissue or cells/liver).
  • potency and selectivity are evaluated through in vivo and/or ex vivo measurements of RNA expression.
  • potency and selectivity are evaluated through in vivo and/or ex vivo measurements of expression of the protein encoded by an RNA.
  • tolerability is evaluated with respect to common markers of immunogenicity, complement activation, and liver toxicity.
  • markers may Attorney Docket No.: 2013260-0047 include, but are not limited to, IFNg, IFNa, IL- lb, IL-6, MCP-1, TNFa, IP- 10, sC5b-9, C3a, ALT, and AST.
  • lipid nanoparticles are manufactured using microfluidics.
  • microfluidics For exemplary methods of using microfluidics to form lipid nanoparticles, see Leung, A.K.K, et al., J Phys Chem, 116:18440-18450 (2012), Chen, Dminister et al., J Am Chem Soc, 134:6947- 6951 (2012), and Belliveau, N.M., et al., Molecular Therapy- Nucleic Acids, 1: e37 (2012). Briefly, the payload, such as a nucleic acid, is prepared in one buffer.
  • the other lipid nanoparticle components e.g., an ionizable lipid; a sterol; a helper lipid; and a PEGylated lipid
  • a syringe pump introduces the two solutions into a microfluidic device. The two solutions come into contact within the microfluidic device to form lipid nanoparticles encapsulating the cargo.
  • a functionalized LNP of the present disclosure is provided in a pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising a functionalized LNP as described herein (e.g., a functionalized LNP comprising a nucleic acid) and excipients or accessory ingredients.
  • a functionalized LNP as described herein e.g., a functionalized LNP comprising a nucleic acid
  • excipients or accessory ingredients e.g., a functionalized LNP comprising a nucleic acid
  • Pharmaceutical compositions, LNP formulations and method of administration of LNPs are known in the art. Some formulations and methods of administration are described in, for example, WO2012135805 and WO201711286 each of which are incorporated herein in their entirety. In addition, techniques for formulation and administration of LNPs may be found in “ Remington ' s Pharmaceutical Sciences” Mack Publishing Co, Easton , Pa .latest edition .
  • antibody agents are conjugated to an LNP (e.g., directly conjugated to an LNP toe make a functionalized LNP).
  • an antibody agent prior to interacting with an LNP, an antibody agent is modified to expose the functional groups in a buffer for 1, 2, 4, and 24 h hrs.
  • antibody agents are purified by desalting or dialysis.
  • LNPs are then incubated with antibody or Attorney Docket No.: 2013260-0047 antibody fragments in the buffer for 2, 4, or 24 h.
  • unconjugated antibody agents are removed by gel fdtration.
  • a method for manufacturing functionalized LNPs is optimized to achieve the best targeting efficiency.
  • adjusted parameters include but are not limited to the ratio between the lipids in the lipid composition of the LNP core, the number of the functional groups per antibody agent, , the time length of various steps, the purification process, or any combination thereof.
  • RNA CAR T cell therapy provides a major opportunity to reduce therapy cost as RNA manufacturing has significantly lower COGS than traditional CAR T cell therapies.
  • CAR T therapies are presently only approved for treatment of heme malignancies, this therapeutic modality has also demonstrated great value in the treatment of autoimmune disorders.
  • Autoreactive B cells are intricately involved in the pathogenesis of many autoimmune diseases, hence CAR T therapy directed against circulating B cells can also address autoimmune diseases through rapid depletion of autoreactive B cells.
  • Recent clinical evidence suggests that transient deep depletion of B-cells not only enables disease remission but can also allow for the recovery of B-cell numbers without reappearance of autoreactive B cells.
  • CAR T therapy for autoimmune diseases has shown signs of safety and efficacy in early-stage clinical trials for systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis, myasthenia gravis (NCT04146051), and MS, with more clinical trials ongoing (NCT06428188, NCT06420154, NCT06056921, NCT06373081, NCT06350110, NCT05085431, NCT05859997).
  • SLE systemic lupus erythematosus
  • NCT04146051 myasthenia gravis
  • MS myasthenia gravis
  • CAR T cells are targeted toward CD 19 or BCMA cell surface markers on healthy and self-reactive B cells/plasma cells.
  • the present example describes T cell-targeting lipid nanoparticles (T-mAb- LNPs).
  • T-mAb- LNPs T cell-targeting lipid nanoparticles
  • the present example demonstrates robust lipid nanoparticles with conjugated antibody agents that enable protein transfection (i.e., RNA encoding a protein delivery) to T cells in human PBMCs in vitro, avoiding both B and myeloid cells.
  • protein transfection i.e., RNA encoding a protein delivery
  • LIP003 Lipid 5 was purchased from DC Chemicals (Shanghai, China). LIP091 was synthesized in house. Cholesterol was purchased from Millipore Sigma Attorney Docket No.: 2013260-0047
  • Mouse lgG2A (aCD5, Clone 205919), Mouse IgG2b (aCD8 alpha, Clone 37006), and Mouse IgG2a (aCD8 alpha, Clone 1033123) were purchased from R&D Systems (Minneapolis, USA).
  • LNP Core Formulation Lipids consisting of ionizable lipid, DSPC, cholesterol, PEG-lipid were dissolved in ethanol. RNA was dissolved in 50mM Citrate buffer pH 5.0. The ratio of cationic ionizable amines to RNA phosphates in the LNP was 6. In general, the lipid composition is 50.0/27.5/22.0/0.5 of ionizable lipid/cholesterol/helper lipid/PEG lipid. The organic and aqueous phases were combined at a flow rate of 12 mL/min on a Precision Nanosystems NanoAssemblr at a ratio of 3:1 (aqueous: organic).
  • All LNP core for antibody functionalization also included a lipid dye DiD in the ethanol phase at 0.1 mol% to the total lipids. After mixing, the LNPs were immediately diluted to 16.5% ethanol using MilliQ water. The diluted product was dialyzed using Slide- A-Lyzer 10K MWCO cassettes (ThermoFisher) against a volume of IX PBS that was 300-fold in excess at 4 °C overnight. The dialyzed product was concentrated using Amicon Ultra lOOkDa centrifuge tubes and sterile filtered. Particle size and PDI were determined with Anton Paar Litesizer DLS 500 while RNA concentration and encapsulation were assessed by the Quant- IT Ribogreen assay (Invitrogen).
  • Mouse anti-human CD3 (clone OKT3, isotype mouse IgG2a) was reduced in IX PBS containing various concentrations (0.2 mM, 1 mM and 5 mM) of dithiothreitol (DTT) and 5 mM EDTA for different durations (0.5 h, 1 h and 2 h) at room temperature.
  • the reduced RG7 was then purified by buffer exchange to IX PBS with 5 mM EDTA using a 7K Zeba spin desalting column to remove excess DTT.
  • the reduced antibody was quantified by Nanodrop.
  • Rat anti-mouse CD3 (clone KT3, isotype rat IgG2a) was reduced in IX PBS containing various concentrations 1 mM DTT and 5 mM EDTA for Ih at room temperature.
  • the reduced antibodies were heated at 65 °C for 5 min and loaded on a 10-well Mini Protean TGX precast gels 4-15% under non-reducing conditions. The gel was stained with SimplyBlueTM SafeStain.
  • PBMCs ATCC PCS-800-01 1
  • RPMI-1640 Medium Gibco
  • Penicillin/Streptomycin Gibco
  • HI-FBS Gibco
  • Cells were maintained at 37°C and 5% CO2.
  • PBMCs were plated at a density of 100,000 cells per well in a 96-well U-bottom plate in RPMI-1640 medium.
  • LNPs were then added to the cells at a final concentration of 2, 0.2, and 0.02 ug/mL. Eighteen hours after transfection, cells were washed with lx DPBS three times.
  • Thy 1.1 RNA was used, cells were stained with PE-conjugated CD90.1 Antibody, anti-mouse/rat, REAfinityTM (Miltenyi Biotec) in 1:50 dilution in IX PEB (PBS/EDTA/BSA) buffer for 10 min in the dark at 4°C. Then, cells were washed with 1XPEB buffer three times and subjected to flow cytometry analysis.
  • Cells were then stained with 50 uL of predetermined diluted surface markers solution (2 uL per antibody in total 50 uL 1XPEB buffer) for an additional 8 minutes in the dark at room temperature. Cells were then washed three times with 300 uL 1XPEB buffer and resuspended in 200 uL 1XPEB buffer for flow cytometry analysis on Attune CytPix Cytometer (Thermo Fisher).
  • Figure 1 demonstrates mAb reduction condition optimization and characterization of reduced mAb.
  • Figure 1A Ellman’s assay demonstrates the current reaction condition (ImM DTT for 1 hr at r.t.) is optimal to expose ⁇ eight thiol groups on each antibody, likely attributed to the cleavage of four inter-chain disulfide bonds.
  • Figure 1A C Binding affinities of intact versus reduced antibodies under optimal conditions are comparable, indicating functionality of the antibody is preserved after chemical manipulation.
  • FIG. 2 demonstrates splenic T-cell targeting efficiency and selectivity of T mAb-LNPs in C57/BL6 mice.
  • Targeting T cells using T mAb-LNPs resulted in > 90% Attorney Docket No.: 2013260-0047 delivery and protein expression.
  • T mAb-LNP A shows significant (365 - fold, p ⁇ 0.0001) increase in mean fluorescence intensity (MFI) of protein marker expression in splenic T-cells while largely no difference in that of myeloid and B cells.
  • MFI mean fluorescence intensity
  • Selectivity to T cells was found to be as high as 25-fold and 64-fold (p ⁇ 0.0001) over myeloid and b cells respectively.
  • FIG. 3 demonstrates biodistribution of various LNP compositions in B6 Albino mice. Modification from a standard formulation to a peripheral composition resulted in a 14-fold decrease in liver expression. T-mAb-LNPs further reduced liver signal by 3-fold compared to the peripheral core. In stark contrast to the peripheral core, T-mAb-LNPs exhibit a 10-fold increase in spleen expression. An increase (2-fold) in thymus expression was also observed.
  • FIG. 4 demonstrates biodistribution of T-mAb-LNPs with Lipid 5 versus LIP091 peripheral core compositions in B6 Albino mice.
  • T-mAb-LNPs using lipid 5 or LIP091 as the ionizable lipid showed a comparable biodistribution pattern of shifting from livers to spleens compared to their untargeted counterparts.
  • FIG. 5 demonstrates surface-functionalized T-mAB-LNPs with mAb 2 demonstrate selective T cell targeting over off-target cells in human PBMCs.
  • Human PBMCs were transfected in vitro with 2, 0.2, or 0.02 ug/ml of Thy 1.1 RNA T-mAb-LNPs.
  • T, B, and myeloid cells were analyzed for Thy 1.1 expression by flow cytometry.
  • Geometric mean (Thy 1.1 MFI, A, B, C) for T, B, and myeloid cells are shown, respectively.
  • mAb 2 T- MAB-LNP performed the best with a selectivity of D) 50-fold and E) ⁇ 130-fold for T cells over both B cells and myeloid cells respectively.
  • RNA delivery via Lipofectamine MessengerMAX transfection reagent achieved very low expression in B and T cells.
  • FIG. 6 demonstrates T-cell targeted CD19 CAR T-mAb 2-LNP uptake in cultured human PBMCs results in T-cell activation.
  • Human PBMCs were treated with 0.02- 2 ug/ml CD 19 CAR T-mAb 2-LNP, control, or CD 19 CAR RNA T-LNP surface- functionalized with antibody isotype control mAb. 24h later cells were collected, stained and analyzed by FACS.
  • Figure 7 demonstrates B cell depletion in mouse peripheral blood after CD 19 CAR T-LNP in situ treatment.
  • CD19 CAR T-LNPaCD3-002 and T-LNPaCD3-155 achieved 83% and 81% depletion in B cell counts, respectively.
  • the murine CD 19 CAR RNA sequence was designed based on protein sequence from Kochenderfer et al.
  • B lymphocytes possess multifaceted roles that are fundamental to immune function in vivo. At the forefront of these functions is their capacity to produce antibodies and cytokines, a cornerstone in orchestrating immune responses. B-cells exhibit a remarkable ability to generate memory cells, thereby fostering immunological memory and enhancing the body's capacity to mount a rapid and specific defense upon re-exposure to a pathogen. Furthermore, B lymphocytes play a critical role in T cell activation by serving as adept antigen-presenting cells and instigate the cascade of immune responses. Modulating B cell function has recently emerged as a promising avenue for advancing targeted therapeutic strategies in diverse immune-related disorders, ranging from cancer and inflammation to autoimmune conditions.
  • non-myeloid phagocytic system (MPS) immune cells including B, T, and natural killer (NK) cells
  • MPS non-myeloid phagocytic system
  • NK natural killer
  • Anderson et al. introduced an ionizable lipidoid, OF-Deg-Lin, featuring ester linkages, achieving efficient transfection of B-cells in the spleen, with 7% Attorney Docket No.: 2013260-0047
  • Harashima et al. utilized a DODAP/DOPE combination with specific lipid ratios, resulting in nanoparticles of 200 nm size and a zeta potential of ⁇ -20 mV, demonstrating a 60% DiD-i- uptake in B-cells.
  • Siegwart and colleagues incorporated a negatively charged lipid (18PA) into their SORT lipid nanoparticles (LNPs), enabling spleen- specific delivery and detecting 12% tdTomato-i- splenic B-cells at 0.3 mg/kg of Cre RNA in Cre-loxP tdTomato reporter mice.
  • the present examples demonstrates surface functionalization of LNPs with the anti-CD79b antibody (mAb) to achieve targeted expression of B cell RNA and proteins in vivo.
  • CD79b also known as the B-cell antigen receptor complex-associated protein beta chain, is expressed on the surfaces of nearly all B-cells.
  • CD79b in conjunction with CD79a and surface immunoglobulin, forms a heterodimeric signal-transduction unit. CD79b can undergo internalization upon antibody binding, presenting a unique opportunity for selective delivery of molecules of interest to B- cells.
  • the anti-CD79b mAb has previously found application in antibody-drug conjugates designed for the prolonged depletion of proliferating B-cells, offering therapeutic benefits in the treatment of non-Hodgkin's lymphoma (NHL).
  • NDL non-Hodgkin's lymphoma
  • Polivy® an FDA-approved anti-CD79b-monomethyl auristatin E (MMAE) conjugate, sanctioned in 2019 for the treatment of diffuse large B-cell lymphoma.
  • the lead antibody was then directly conjugated to the LNP core using its optimum antibody surface densities to eliminate the unnecessary secondary mAb (RG7) component in the system.
  • the biodistribution of the B-cell targeted LNP was evaluated using IVIS imaging.
  • anti-human B-cell mAbs were conjugated on the LNP to validate the potential for translation.
  • RNA-encoded reporter protein were encapsulated to assess the functional RNA delivery to B-cells in a cell-specific and cost-effective manner.
  • Two common reporter RNAs for indication of functional RNA expression in vitro and in vivo were evaluated.
  • mCherry is a widely used fluorescent reporter protein. However, it is believed to be immunogenic, as its precursor dsRed has one D(b)-restricted peptide sequence (SSLQDGCFI) that acted as an epitope in C57BL/6 mice. Also, the brightness of mCherry is mild, which is 47 % of eGFP.
  • Thyl.l is a highly conserved glycoprotein that is anchored to the surface of cells. Most mouse strains don’t express Thyl.l except AKR/J and PL strains. Thyl.l protein is reported to not elicit an immune response when injected in mice. Moreover, it can be stained with antibodies with different fluorophores to maximize the signal.
  • Isotype control-RG7-Peripheral LNP or aCD19-RG7-Peripheral LNP were tested in the A20 cell line, and protein expression was assessed with flow cytometry 24 h post-transfection.
  • aCD19-RG7-Peripheral LNP showed very high levels of protein expression (>90%) in vitro in both mCherry and Thyl.l settings, while LNPs conjugated with isotype control were not effectively transfecting cells (Figure 8A and B).
  • Thyl. l RNA encapsulated aCD19-RG7-Peripheral LNP maintained >60% transfection efficiency Attorney Docket No.: 2013260-0047 and MFI levels at 10 A 3. Therefore, Thy 1.1 was selected as the reporter RNA for the following screening efforts.
  • aCD79b-LNPs outperformed aCD19-LNPs in transfecting splenic B-cells in vivo ( Figure 10 G and J), showing >80% Thy 1.1+ at 0.5 mg/kg.
  • aCD79b was selected as the lead targeting mAb for the future development. It’ s worth mentioning that, in Figure 10 H, despite a 6-fold lower of MFI than B-cells, all three mAb-conjugated LNPs expressed Thyl. 1 in around 70% splenic myeloid cells, suggesting the compromised selectivity at this dose. In the next study, we tested whether lowering the dose could improve selectivity.
  • aCD79b-RG7-Peripheral LNPs at 0.1 mg/kg still maintained the 70% transfection efficiency in spleen B-cells, which was significantly higher than the core and ISO counterparts at the same concentration (Figure 11A), while reduced the myeloid cell expression to around 50% ( Figure 11G). Further decreasing the dose to 0.033 mg/kg reduced the splenic B cell protein expression to ⁇ 40%, which might be too low for effective targeting. Thus, 0.1 mg/kg was selected for future formulation screening and development.
  • aCD79b mAb-Peripheral LNP exhibited a higher expression in mature B-cells than developing B-cells, as CD79b has a more prevalent Attorney Docket No.: 2013260-0047 expression on the mature B-cells ( Figure 11C and HE).
  • Figure 11C and HE the overall B cell targeting efficiency of aCD79b-RG7-Peripheral LNPs was lower in the bone marrow than in the spleens
  • LNP core composition optimization with targeting antibody screening.
  • B-cells originate in the bone marrow and activate in the secondary lymphoid organs such as the spleen and lymph nodes, which might not be reached by the standard LNP formulation and therefore require optimized LNP formulation that targets away from the liver.
  • peripheral LNPs functionalized with B cell-targeting mAbs would outperform the standard liver-targeting LNPs.
  • the aCD79b functionalized on the standard LNP reached 60% and functionally expressed in 50% spleen B-cells, while the untargeted standard LNP core had a low capability of transfecting B-cells ( ⁇ 5%).
  • the unconjugated peripheral LNP core itself showed about 25% Thy 1.1 expression, which was higher than its standard LNP core counterpart.
  • the level of B cell delivery was greatly enhanced by the incorporation of aCD79b, aCD79b-RG7-Peripheral LNP achieved delivery and protein expression in 70-80% of spleen B-cells in vivo at only 0.1 mg/kg, outperforming any other nanoparticle systems reported up to date.
  • aCD79b-RG7-Peripheral LNP exhibited around 100-fold and 15- fold protein expression over the unconjugated peripheral LNP core and aCD79b-RG7- Standard LNP, respectively.
  • This suggested our DOE-optimized LNP core formulation is a Attorney Docket No.: 2013260-0047 good foundation for targeting antibody surface functionalization, and achieving high levels of cell uptake and protein translation.
  • aCD79b-RG7 -Peripheral LNP only improved the transfection efficiency in B-cells, however not myeloid cells and T-cells, thus presented a high selectivity toward non-B-cells ( Figure 12C and D).
  • the protein expression was 6-fold over myeloid cells and 44-fold over T-cells.
  • aCD79b-RG7-Standard LNP could only bring the B-cells expression to the same level as the myeloid cells, an indication of suboptimal selectivity.
  • Rat IgG2a due to the binding affinity limitation of RG7, all targeting antibodies previously screened were Rat IgG2a.
  • B-targeted LNPs generated by the direct conjugation method showed a higher transfection efficiency than the previous two-layer method (aCD79b- RG7-Peripheral LNP) in spleen mature B-cells.
  • aCD79b-Peripheral LNP the previous two-layer method
  • aCD79b- RG7-Peripheral LNP the previous two-layer method
  • Multiple aCD79b’s on the LNP surface may crosslink several CD79b receptors on B-cells, causing the co-internalization with adjacent IgM and subsequent signal advance for the functional consequences, activating B-cells and triggering B cell proliferation.
  • Figure 13D to E the tendency in spleen developing B- cells was consistent with that of the mature B-cells, with a relatively lower protein expression level and a more moderate B cell size increase, which again was related to the less expression of CD79b on the developing B cell populations.
  • SN-8 or its Fab and scFv will be selected if we will test the anti-human aCD79b-peripheral LNPs in humanized mouse model.
  • viability of PMBC was maintained >80% across all tested groups, except MassengerMax at 2 ug/mL, which indicated our B-cell targeted LNP platform was more tolerable than the golden- standard of in vitro transfection.
  • SN-8 and CB3-1 were more prone to drive B -cells activation (CD69+) than 683023, which equipped us with the options of activating T-cells or not depending on the applicable indications.
  • LIP003 (Lipid 5) was purchased from DC Chemicals (Shanghai, China). Cholesterol was purchased from Millipore Sigma (Burlington, USA).
  • DSPC, PEG-DMG, Maleimide-PEG-DSPE were purchased from Avanti Polar lipids Inc. (Alabaster, USA).
  • Mouse anti-rat IgG2a (clone RG7/1.30), rat IgG2a isotype control (clone 2A3), Rat antimouse CD19 mAb (clone 1D3), and Rat anti-mouse CD38 (NIMR-5) were purchased from Bio X Cell (Lebanon, USA).
  • Rat anti-mouse CD 19 mAb (clone 1D3) was purchased from Novus Biologies (Centennial, USA). Rat anti-mouse CD19 mAb (clone 1D3), Rat antimouse CD19 mAb (clone 6D5), Rat anti-mouse CD19 mAb (clone 6OMP31), and Rat antimouse CD20 mAb (AISB12) were purchased from Invitrogen (Waltham, USA). Rat antimouse CD 19 mAb (clone 6D5), Rat anti-mouse CD38 (NIMR-5) and Rat anti-mouse CD38 mAb (clone 90) were purchased from SouthernBiotech (Birmingham, USA).
  • Rat anti-mouse CD 19 mAb (clone 6D5) and Rat anti-mouse CD38 mAb (clone 90) were purchased from Biolegend (San Diego, USA).
  • Rat anti-mouse CD79b mAb (clone 90) was purchased from R&D Systems (Minneapolis, USA).
  • Rat anti-mouse CD79b mAb (clone AT107-2) was Attorney Docket No.: 2013260-0047 purchased from Bio-Rad (Hercules, USA).
  • Hamster anti-mouse CD79b mAb (clone HM79- 11) was purchased from Thermo Scientific (Waltham, USA).
  • DTT and EDTA were purchased from Thermo Scientific (Waltham, USA). DiD was purchased from Invitrogen (Waltham, USA).
  • Formulation for LNP core-. Thyl.l, mCherry or Luciferase RNA was formulated in LNPs containing a cationic ionizable LIP003 (Lipid 5), cholesterol, DS PC, PEG2K-DMG and DSPE-PEG2K-Maleimimde at a ratio of 50/38.5/10/1/0.5 for the standard LNP core and 50/27.5/22/0.5 for the peripheral LNP core.
  • the ratio of cationic ionizable amines to RNA phosphates in the LNP was 6. Lipids were dissolved in ethanol and RNA was dissolved in 50mM Citrate buffer pH 5.0.
  • the organic and aqueous phases were combined at a flow rate of 12 mL/min on a Precision Nanosystems NanoAssemblr at a ratio of 3:1 (aqueous: organic).
  • the product was immediately diluted to 16.5% ethanol using MilliQ water.
  • the diluted product was dialyzed against a volume of IX PBS that was 300- fold in excess.
  • the dialyzed product was concentrated using Amicon Ultra lOOkDa centrifuge tubes and sterile filtered. RNA concentration and encapsulation were assessed by the Invitrogen Ribogreen assay.
  • Anti-IgG secondary antibody (RG7) or targeting antibodies was reduced in IX PBS containing 1 mM dithiothreitol (DTT) and 5 mM EDTA for 1 hour at room temperature.
  • the reduced antibodies were then purified by buffer exchange to IX PBS with 5 mM EDTA using a 7K Zeba spin desalting column (Thermo Scientific) to remove excess DTT.
  • the reduced antibody was quantified by Nanodrop. The reduced antibody was added to LNP at various rations of antibody/maleimide ratios and incubated for 2 hours at room temperature.
  • RG7 concentration was assessed by the Pierce BCA protein assay kit (Thermo Fisher Scientific). RNA concentration and encapsulation were assessed by the Ribogreen assay (Invitrogen).
  • Attaching targeting mAb (if the two-layer method was used): The targeting (primary) antibody was added to RG7-LNPs at various primary/secondary weight or molar ratios and incubated for 30 min at room temperature.
  • A20 cell culture and LNP transfection A20 cells (ATCC, TIB-208) were cultured in RPMI-1640 Medium (Gibco) supplemented with 10% HI-FBS (Gibco). Cells were maintained at 37°C and 5% CO .
  • RPMI-1640 Medium Gibco
  • HI-FBS Gibco
  • LNPs were then added to the cells at a final concentration of 0.2 ug/mL. Eighteen hours after transfection, cells were washed with lx DPBS three times.
  • Thy 1.1 RNA was used, cells were stained with PE-conjugated CD90.1 Antibody, anti-mouse/rat, REAfinityTM (Miltenyi Biotec) in 1:50 dilution in IX PEB (PBS/EDTA/BSA) buffer for 10 min in the dark at 4°C. Then, cells were washed with 1XPEB buffer three times and subjected to flow cytometry analysis.
  • PBMCs Human primary blood mononuclear cells
  • ATCC PCS -800 -01 1 Human primary blood mononuclear cells
  • RPMI-1640 Medium Gibco
  • Penicillin/Streptomycin Gibco
  • HI-FBS Gibco
  • PBMCs were plated at a density of 100,000 cells per well in a 96-well U-bottom plate in RPMI-1640 medium.
  • LNPs were then added to the cells at a final concentration of 2, 0.2, and 0.02 ug/mL.
  • mice Eight-week-old C57/BL6 mice were injected with DiD dye and mCherry or Thy 1.1 encoding RNA co-encapsulated LNPs through the tail vein. After 18 hours, spleens were harvested from mice and preserved in MACS® Tissue Storage Solution on ice. Spleens were mechanistically disrupted by pressing then through a 70 pm strainer. Cells were then spun down at 350g for 10 min and washed with 1XDPBS three times. Cells were then plated into a 96-well V-shape plate at 100,000 cells/well.
  • T-cells, B-cells and myeloid cells were identified as CD3+B220- , B220+CD3-, and CD3-B220-, respectively.
  • N 3-4/group.

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Abstract

La présente divulgation concerne des compositions de nanoparticules lipidiques et des procédés d'utilisation. Entre autres, la présente divulgation concerne des compositions de nanoparticules lipidiques qui ont une spécificité accrue pour des cellules ou des tissus spécifiques. La présente divulgation concerne des procédés d'utilisation des nanoparticules lipidiques divulguées.
PCT/US2025/040643 2024-08-06 2025-08-05 Compositions et procédés d'administration d'agents Pending WO2026035680A1 (fr)

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