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WO2025090525A1 - Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des lymphocytes t - Google Patents

Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des lymphocytes t Download PDF

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Publication number
WO2025090525A1
WO2025090525A1 PCT/US2024/052438 US2024052438W WO2025090525A1 WO 2025090525 A1 WO2025090525 A1 WO 2025090525A1 US 2024052438 W US2024052438 W US 2024052438W WO 2025090525 A1 WO2025090525 A1 WO 2025090525A1
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WIPO (PCT)
Prior art keywords
lnp
lipid
targeting moiety
mol
amino acid
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/US2024/052438
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English (en)
Inventor
Athanasios Dimitry DOUSIS
Chunxi ZENG
Zhan Wang
Rui Zhang
Han GU
Apiwat WANGWEERAWONG
Lorenzo TOZZI
Lei Liu
Rahul Palchaudhuri
Mohit Gupta
Michael Monte
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.)
Tessera Therapeutics Inc
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Tessera Therapeutics Inc
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Publication of WO2025090525A1 publication Critical patent/WO2025090525A1/fr
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
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    • C07K16/2806—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 against CD2
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C—CHEMISTRY; METALLURGY
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Definitions

  • LNPs lipid nanoparticles
  • payloads such as nucleic acids (e.g., mRNA or siRNA).
  • LNPs are generally comprised of multiple components including an ionizable lipid, a PEGylated lipid, a helper lipid and cholesterol, all of which play important roles in effectively delivering the payload to diseased tissue.
  • T cells immune cells
  • therapeutic agents e.g., a genetic medicine payload, such as a gene therapy or gene editing agent
  • T cells be difficult as any therapeutic agent delivered systemically must evade or overwhelm myriad mechanisms that will prevent its delivery to the relevant microenvironment (for example, liver uptake, phagocytic cell uptake, complement activation, opsonization, phagocytic cell uptake, etc.).
  • the payload(s) after delivery to the target cell, the payload(s) must be able to disassociate from the endosome so it can be expressed in sufficient quantities in the targeted cell.
  • LNPs lipid nanoparticles
  • conjugates comprising a lipid nanoparticle (LNP) encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or reporter construct, for delivery to immune cells such as T cells.
  • a therapeutic agent e.g., a gene modifying system
  • reporter construct for delivery to immune cells such as T cells.
  • the LNPs and conjugates described herein can deliver the payload to immune cell more effectively than a baseline conjugate or baseline LNP.
  • the disclosure also provides conjugates comprising an LNP encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or reporter construct, for delivery to immune cells (e.g., T cells) wherein the LNP has at least one targeting moiety on its surface.
  • a therapeutic agent e.g., a gene modifying system
  • reporter construct for delivery to immune cells (e.g., T cells) wherein the LNP has at least one targeting moiety on its surface.
  • the conjugates having at least one targeting moiety described herein will also be referred to as targeted LNPs or tLNPs.
  • the disclosure provides a lipid nanoparticle (LNP) for delivery of a therapeutic agent to immune cells (e.g., T cells) in vivo, in vitro, or ex vivo, wherein the LNP comprises an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or from the lipids in Table 1, Table 2 and Table 3, as set forth in the disclosure below, and a therapeutic agent encapsulated within the LNP.
  • the disclosure provides conjugates comprising an LNP and a targeting moiety.
  • the targeted LNPs can be formulated in a pharmaceutical composition and can be directly administered to a subject (e.g., patient) in need thereof (e.g., by in vivo administration). Once administered, the compositions can deliver significant quantities of a therapeutic agent to the immune cells (e.g., T cells) of the subject.
  • the conjugates can be added to isolated immune cells (e.g., T cells) using an in vitro or ex vivo procedure.
  • Cells that can be derived from the blood, bone marrow, lymphoid organs, or lymph are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
  • blood is first collected from a subject (e.g., a human patient).
  • the immune cells can be activated and expanded. Delivery of the LNPs of the disclosure to the activated T cells results in transduction of the desired payload into the immune cells (e.g., T cells).
  • the activated immune cells e.g., T cells
  • the activated immune cells can then be administered to the patient.
  • the LNPs of the disclosure can be delivered to immune cells (e.g., T cells) without activating the immune cells/T cells.
  • T cells that have not been activated prior to mixing with LNPs are referred to herein as rested T cells.
  • delivery of conjugates of the disclosure are capable of activating the rested immune cells (e.g., T cells).
  • the payload can be one of more nucleic acid molecules.
  • the one or more nucleic acid molecules can be RNA molecules.
  • the one or more nucleic acid molecules can be DNA molecules.
  • both nucleic acid molecules can be RNA molecules.
  • both nucleic acid molecules can be DNA molecules.
  • the payload comprises one nucleic acid molecule can be an RNA molecule and one nucleic acid molecule can be a DNA molecule.
  • the payload can be a nucleic acid encoding a reporter gene, such as green fluorescent protein (GFP).
  • the payload can be a therapeutic agent, for example, a therapeutic peptide or protein, a nucleic acid comprising a therapeutic agent, or a nucleic acid encoding a therapeutic agent.
  • the therapeutic agent can be a genetic medicine, wherein the therapeutic agent is capable of modifying, altering or effecting a change in the genomic DNA of a cell (e.g., a cell in the subject).
  • the therapeutic agent is a gene therapy agent or gene editing agent.
  • the therapeutic agent is a gene modifying polypeptide.
  • the therapeutic agent is a gene modifying system.
  • the targeting moiety is an antibody, Fab fragment or single chain variable fragment (scFv), a DARPIN, a VHH domain antibody, a FN3 domain, a nanobody, a single domain antibody or a Centyrin.
  • the targeting moiety is a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin or a N-Acetylgalactosamine (GalNac).
  • the targeting moiety is a peptide or protein, such as a ligand or part of a ligand, that binds to a receptor (e.g., a receptor on the surface of a T cell).
  • the targeting moiety is a peptide or protein that binds to a receptor or ligand on the surface of an immune cells (e.g., T cells), wherein the affinity of the peptide or protein targeting moiety for the receptor or ligand on the surface of the immune cell (e.g., T cell) is modulated via phage display.
  • the payload (e.g., therapeutic agent) delivered by the LNP or targeted LNP can be a small molecule, peptide or protein, non-coding RNA (ncRNA), gRNA, siRNA or miRNA, a nucleic acid (e.g., mRNA) encoding a peptide or protein (e.g., a protein for replacement gene therapy, or a protein for modifying or altering the genome or epigenome, e.g., a protein for gene editing), a nucleic acid encoding or comprising one or more components of a system for altering a genome (e.g., one or more components of a ribonucleoprotein (RNP) complex for editing or altering the genome or epigenome (e.g., for introducing insertion-deletion mutations (indels), base editing, epigenetic editing, or target-primed reverse transcription (TPRT), e.g., by a mechanism that requires a recombin
  • ncRNA non-
  • the therapeutic agent (i.e., payload) delivered by the LNP or targeted LNP can be a gene modifying protein, a nucleic acid encoding a gene modifying protein, or a gene modifying system, as described herein.
  • the disclosure provides a targeted LNP (conjugate) encapsulating one or more nucleic acids encoding components of a system for modifying or altering DNA (e.g., genomic DNA), wherein the targeted LNP comprises one or more targeting moieties, such as antibodies, Fab fragments, or scFvs, capable of binding to one or more proteins on the cell surface of an immune cell (e.g., T cell).
  • the targeted LNP is delivered in vivo to a subject (e.g., a human patient) in need thereof.
  • the targeted LNP is delivered using an ex vivo or in vitro procedure.
  • the nucleic acids encoding components of a system for modifying or altering a genome can be expressed, resulting in altering of the genome (e.g., gene editing) of the immune cell (e.g., T cell).
  • LNPs comprising particular targeting moieties (e.g., antibodies, Fab fragments or scFvs) or particular combinations of targeting moieties (e.g., antibodies, Fab fragments or scFvs), ionizable lipids, and/or helper lipids are capable of enhancing delivery of a therapeutic payload, (e.g., a system capable of altering the genome, such as a gene modifying system) to immune cells (e.g., T cells) following in vivo administration to a subject or ex vivo delivery, result in enhanced levels of therapeutic activity in the immune cells (e.g., T cells.)
  • a therapeutic payload e.g., a system capable of altering the genome, such as a gene modifying system
  • immune cells e.g., T cells
  • an LNP or targeted LNP (conjugate) comprising an ionizable lipid as described herein is capable of delivering a payload (e.g., a therapeutic payload
  • an LNP comprising an ionizable lipid as described herein is capable of delivering a payload (e.g., a therapeutic payload) to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% more immune cells (e.g., T cells) than an LNP comprising a baseline lipid (such as V003) in an animal model, as described herein.
  • the LNP is a targeted LNP.
  • a payload (e.g., a therapeutic payload) delivered to immune cells (e.g., T cells) by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as V003).
  • a baseline lipid such as V003
  • a payload (e.g., a therapeutic payload) delivered to immune cells (e.g., T cells) by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as V003).
  • the LNP is a targeted LNP.
  • At least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% immune cells (e.g., T cells) in a subject comprise a genome that have been modified or altered following in vivo administration of an LNP described herein, such as a targeted LNP encapsulating a therapeutic agent (e.g., a system for modifying, altering or editing a genome).
  • a targeted LNP encapsulating a therapeutic agent e.g., a system for modifying, altering or editing a genome.
  • from about 5% to about 50%, from about 5% to about 20%, from about 10% to about 30%, from about 20% to about 50%, from about 20% to about 40%, or from about 20% to about 30% of immune cells (e.g., T cells) in a subject comprise a genome that has been modified or altered following in vivo administration of an LNP described herein, such as a targeted LNP.
  • immune cells e.g., T cells
  • immune cells comprise a genome that have been modified or altered following ex vivo incubation of isolated activated or inactivated immune cells (e.g., T cells) with an LNP described herein, such as a targeted LNP encapsulating a therapeutic agent (e.g., a system for modifying, altering or editing a genome).
  • an LNP described herein such as a targeted LNP encapsulating a therapeutic agent (e.g., a system for modifying, altering or editing a genome).
  • the isolated immune cell (e.g., T cell) population comprise a genome that has been modified or altered.
  • immune cells e.g., T cells
  • the high level of genome modification or gene editing following in vivo administration or ex vivo delivery of the targeted LNPs of the disclosure e.g., tLNPs comprising a gene modifying system configured to insert a CAR into the genome of a T cell
  • tLNPs comprising a gene modifying system configured to insert a CAR into the genome of a T cell
  • the cancer is an advanced leukemia or lymphoma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), or Diffuse Large B-Cell Lymphoma (DLBCL).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • NHL non-Hodgkin lymphoma
  • CAR-T cells can be generated using the tLNPs described herein to target B cell markers such as CD19, CD20, and BCMA.
  • the autoimmune disease is systemic lupus erythematosus, refractory antisynthetase syndrome, myasthenia gravis, systemic scleroderma, and multiple sclerosis.
  • the targeted LNPs (conjugates) of the disclosure may be used to modify T cells.
  • T-cells may include any subpopulation of T-cells, e.g., CD4+, CD8+, gamma-delta, na ⁇ ve T cells, stem cell memory T cells, central memory T cells, or a mixture of subpopulations.
  • the conjugates may be used to deliver or modify a sequence encoding a T-cell receptor (TCR) in a T cell.
  • the conjugates may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T-cells.
  • CAR chimeric antigen receptor
  • the conjugates can be used to deliver an RNA encoding a CAR to T-cells.
  • the conjugates are formulated with an mRNA encoding a CAR. Such conjugates can be contacted with T cells to deliver the mRNA molecule encoding the CAR, resulting in cellular expression of the CAR.
  • the conjugates are formulated with a DNA molecule encoding a CAR, e.g., a plasmid or linear (e.g., a closed-ended linear DNA) encoding the CAR.
  • a DNA molecule encoding a CAR e.g., a plasmid or linear (e.g., a closed-ended linear DNA) encoding the CAR.
  • the conjugates may be used to deliver at least one sequence encoding a CAR to natural killer (NK) cells.
  • the conjugates may be used to deliver at least one sequence encoding a CAR to natural killer T (NKT) cells.
  • the conjugates may be used to deliver at least one sequence encoding a CAR to a progenitor cell, e.g., a progenitor cell of T, NK, or NKT cells.
  • the conjugates can be used to deliver at least one gene modifying system (e.g., a retrotransposon gene modifying system) comprising a heterologous sequence encoding a CAR to an immune cell, e.g., a T cell.
  • the conjugates can be used to deliver at least one gene modifying system (e.g., a retrotransposon gene modifying system) to insert a heterologous sequence encoding a CAR into the genome of an immune cell, e.g., a T cell.
  • the conjugates can further be used to deliver at least one gene modifying system (e.g., a heterologous gene modifying system) to the immune cell, e.g., T cell.
  • the conjugates can further be used to deliver at least one gene modifying system (e.g., a heterologous gene modifying system) to modify or alter the TRAC locus and/or B2M locus in the genome of the immune cell, e.g., T cell.
  • modification or alteration of the TRAC locus and/or B2M locus results in a reduction or loss of expression of the TRAC and/or B2M locus.
  • the targeted LNPs (conjugates) of the disclosure may be used to simultaneously deliver gene modifying components to T cells that are capable of inserting a sequence encoding a CAR into the genome of the T cells and altering the T cell receptor alpha constant (TRAC) locus and/or the B2M locus in T cells, hence disrupting the TRAC gene and/or B2M gene.
  • simultaneous insertion of a CAR sequence and knock out of a TRAC and/or B2M gene can be accomplished via LNP delivery of two or more gene modifying systems, e.g., a retrotransposon gene modifying system and a heterologous gene modifying system.
  • a single targeted LNP can deliver all of the components of a gene modifying system including (i) a gene modifying polypeptide; (ii) a template RNA (or DNA encoding the template RNA) that binds to the gene modifying polypeptide and a heterologous CAR sequence; and (iii) an additional gene modifying system for modifying DNA to install a mutation (e.g., resulting in knock down or knock out of the TRAC gene and/or B2M gene).
  • a mutation e.g., resulting in knock down or knock out of the TRAC gene and/or B2M gene.
  • multiple targeted LNPs can be used to deliver the components one or more gene modifying systems.
  • one conjugate can be used to deliver including (i) a gene modifying polypeptide; and (ii) a template RNA (or DNA encoding the template RNA) that binds to the gene modifying polypeptide and a heterologous CAR sequence and a second conjugate can be used to deliver an additional gene modifying system for modifying DNA to install a mutation (e.g., resulting in knock down or knock out of the TRAC gene and/or B2M gene).
  • a first conjugate, as described herein can be used to deliver a retrotransposon gene modifying system to an immune cell, e.g., a T cell
  • a second conjugate, as described herein can be used to deliver a heterologous gene modifying system to an immune cell, e.g., a T cell.
  • an LNP of the present disclosure such as a targeted LNP, comprises the ionizable lipid V003, depicted below.
  • an LNP of the present disclosure, such as a targeted LNP comprises an ionizable lipid in Table 1.
  • the LNP (e.g., a targeted LNP) comprises an ionizable lipid in Table 2. In some embodiments, the LNP (e.g., a targeted LNP) comprises an ionizable lipid in Table 3.
  • the LNPs and targeted LNPs detailed herein in some embodiments comprise any one or more ionizable lipid as detailed in Tables 1, 2, and 3. In some embodiments, the LNP and targeted LNPs detailed herein comprises 2 or more ionizable lipids, such as any two or more of V003 and the ionizable lipids in Tables 1, 2 and 3.
  • the LNP and targeted LNPs as detailed herein comprise only one ionizable lipid selected from the group consisting of ionizable lipids in Tables 1, 2 and 3.
  • an LNP containing s an ionizable lipid described herein exhibits higher levels of transduction in immune cells (e.g., T cells) and/or higher expression of a payload protein in immune cells (e.g., T cells) relative to LNPs that contain V003 as the ionizable lipid.
  • the ionizable lipids described herein provide higher levels of transduction and/or protein expression relative to V003 when used as a component of targeted LNP for in vivo or ex vivo delivery to immune cells (e.g., T cells).
  • the ionizable lipid has one of the structures depicted below:
  • the LNPs of the present disclosure comprise one or more pegylated lipid molecules.
  • the targeting moiety e.g., antibody, Fab fragment or scFv
  • the targeted LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the targeting moiety.
  • the PEG spacer between the lipid and the targeting moiety comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units.
  • the PEG spacer comprises about 10-120 ethylene glycol units.
  • the molecular weight of the pegylated lipid bonded to the targeting moiety is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the targeting moiety is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300).
  • the lipid component of the pegylated lipid bonded to the targeting moiety is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.
  • Pegylated lipids in the targeted LNPs of the disclosure can imbue different properties to the LNPS, including pharmacokinetics, distribution and endosomal release.
  • LNPs comprising different pegylated lipids not covalently bound to a targeting moiety could show different toxicological properties when all other components of the LNPs (e.g., ionizable lipid) and relative proportions are the same.
  • LNPs comprising pegylated lipids with PEG lipid anchors having 16 carbon atoms (i.e., C16 PEG lipid anchors) or 18 carbon atoms (i.e., C18 PEG lipid anchors) could potentially reduce or eliminate the toxicity associated with identical LNPs other than the pegylated lipid component.
  • administration of the LNPs comprising C16 PEG lipid anchors or C18 PEG lipid anchors does not result in substantial liver toxicity.
  • administration of the targeted LNPs results in no or minimal increases in serum levels of the secreted liver enzyme alanine aminotransferase (ALT).
  • ALT alanine aminotransferase
  • the pegylated lipid has at least one C16 (palmitoyl) PEG lipid anchor.
  • the pegylated lipid has two C16 PEG lipid anchors (i.e., dialkyl chains of 16 carbons long).
  • the pegylated lipid is 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000.). In some embodiments, the pegylated lipid is 1,2-Dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is C16 PEG ceramide. In some embodiments, the targeted LNPs comprising the C16 pegylated lipids show significantly reduced liver uptake than otherwise identical LNPS comprising C14 pegylated lipids.
  • the pegylated lipid has at least one C18 PEG lipid anchor. In some embodiments, the pegylated lipid has two C18 PEG lipid anchors (i.e., dialkyl chains of 18 carbons long). In some embodiments, the C18 pegylated lipid is 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the C18 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG-PEG2000).
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG-PEG2000.
  • an LNP of the present disclosure e.g., a targeted LNP, comprises one or more non-pegylated lipids (e.g. non-pegylated phospholipids). Such lipids are often referred to as helper lipids.
  • the targeting moiety (e.g., antibody, Fab fragment or scFv) is associated with or chemically bonded to at least one of the non-pegylated lipids.
  • the non-pegylated lipid (helper lipid) is selected from POPC, DOPC, DOPE, and DSPC.
  • the non-pegylated lipid (helper lipid) is a sphingolipid.
  • the non-pegylated lipid is a sphingomyelin.
  • the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide.
  • the sphingomyelin is egg sphingomyelin.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 36%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 22% to about 28%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 20% to about 25%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 25% to about 30%. some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 30% to about 35%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34% or about 35%.
  • helper lipid e.g., DSPC or sphingomyelin
  • mol% of the helper lipid refers to the mol% of the total lipid component of the LNP, which does not include the therapeutic agent (i.e., payload) or the targeting moiety.
  • the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 1:1 to about 7:1. In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 4:1.
  • the molar ratio between the ionizable lipid and the non-pegylated helper lipid is from about 1:1 to about 3:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 1:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 2:1.
  • the molar ratio between the ionizable lipid and the non-pegylated helper lipid is from about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 2.5:1.
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 40%.
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%.
  • the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%).
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%m about 34% or about 35%).
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%).
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%).
  • the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).
  • the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 60% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 60% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 40%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 50% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 50% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 40%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of cholesterol in the targeted LNP is from about 25%-40%, [0039]
  • the LNP, e.g., targeted LNP comprises Lipid 092.
  • the LNP, e.g., targeted LNP comprises Lipid 092 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 092 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP e.g., targeted LNP, comprises Lipid 093.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 093 and DSPC.
  • the LNP e.g., targeted LNP
  • the LNP comprises Lipid 093 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the LNP is DPPE-PEG2000 or DPG-PEG2000. [0041] In some embodiments, the targeted LNP comprises Lipid 153. In some embodiments, the targeted LNP comprises Lipid 153 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 153 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 154.
  • the LNP, e.g., targeted LNP comprises Lipid 154 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 154 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 155.
  • the LNP, e.g., targeted LNP comprises Lipid 155 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 155 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 162.
  • the LNP, e.g., targeted LNP comprises Lipid 162 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 162 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 163.
  • the LNP, e.g., targeted LNP comprises Lipid 163 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 163 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 169.
  • the LNP, e.g., targeted LNP comprises Lipid 169 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 169 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 176.
  • the LNP, e.g., targeted LNP comprises Lipid 176 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 176 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 178.
  • the LNP, e.g., targeted LNP comprises Lipid 178 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 178 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 183
  • the LNP, e.g., targeted LNP comprises Lipid 183 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 183 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid 232.
  • the LNP, e.g., targeted LNP comprises Lipid 232 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 232 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP, e.g., targeted LNP comprises Lipid V003.
  • the LNP, e.g., targeted LNP comprises Lipid V003 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid V003 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C16 alkyl chain (e.g., two C16 alkyl chains). PEG2000.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the LNP is DPPE-PEG2000 or DPG-PEG2000.
  • the LNP or conjugate can comprise one or more cholesterol molecules.
  • the molar ratio between the cholesterol molecule and the non-pegylated helper lipid e.g., DSPC or sphingomyelin
  • the non-pegylated helper lipid is from about 6:1 to about 0.5:1.
  • the molar ratio between the cholesterol molecule and the non-pegylated helper lipid is from about 3:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1.5:1 to about 0.5:1.
  • the molar ratio between the cholesterol molecule and the non-pegylated helper lipid is from about 1:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:2 to about 0.8:1.
  • the targeted LNPs are formed via a Click reaction between a first Click handle on the targeting moiety (e.g., antibody, Fab fragment, or scFv) and a second Click handle on the LNP, thereby generating a Click product.
  • the second Click handle can be on one or more of the lipids comprising the LNP.
  • the second Click handle is covalently bonded to a pegylated lipid comprising the LNP.
  • the Click product can be formed using a copper-catalyzed Click reaction.
  • the first or second Click handle comprises a cyclic derivative of the alkynyl group.
  • the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne. Cyclooctyne, and difluorinated cyclooctyne,
  • the click chemistry involves strain promoted cycloaddition of azides.
  • the click chemistry is based upon reaction of strained alkenes.
  • the Click product can be formed using copper-free Click chemistry.
  • the Click product can be formed between an azide and dibenzocyclooctene (DBCO).
  • DBCO dibenzocyclooctene
  • the Click product can be formed using a Staudinger reaction between an azide and a phosphine, hence producing an aza-ylide.
  • the Click product can be formed from an inverse electron demand Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the first or second Click handle.
  • TCO trans-cyclooctene
  • the first Click handle comprises a tetrazine (Tz) ring
  • the second Click handle comprises a TCO moiety.
  • the tetrazine ring is unsubstituted.
  • the tetrazine rung is methyltetrazine.
  • the tetrazine ring is a 6-methyl substituted tetrazine.
  • the Click product is formed by conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence.
  • an antibody, Fab fragment or single chain variable fragment (scFv) can be covalently linked to a first Click handle through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle on the LNP, thereby generating a Click product.
  • the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide.
  • the antibody Fab fragment or scFv is directly bonded to the enzyme recognition sequence.
  • the antibody Fab fragment or scFv is bonded to the enzyme recognition sequence via one or more amino acid residues.
  • Particular amino acid residues added that can be covalently attached to the C-terminus of the antibody, Fab fragment or scFv include, but are not limited to (GGGGS) v , (G) v , (EAAAK) v , (PAPAP) v , (AP) v and A(EAAAK) u ALEA(EAAAK) v A, wherein u is 1-10 and v is 1-10.
  • the targeting moiety is a T-cell targeting moiety, for example, an antibody, Fab fragment or scFv that binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and r IL-15 receptor.
  • the CD80 targeting moiety is a CD80 extracellular domain (ECD).
  • the targeted LNP comprises a targeting moiety that targets (binds) a receptor on the surface of the T cell selected from CD2, CD3, CD4, CD5, CD6, CD7, and CD8.
  • the targeting moiety targets a CD3 receptor on the surface of the T cell.
  • the targeting moiety targets a CD7 receptor on the surface of the T cell.
  • the targeting moiety targets a CD5 receptor on the surface of the T cell.
  • the targeting moiety targets a CD2 receptor on the surface of the T cell.
  • the targeting moiety targets a CD8 receptor on the surface of the T cell.
  • the targeted LNP comprises a targeting moiety that targets CD3 on the surface of the T cell, wherein the targeting moiety is an antibody, Fab fragment or scFv selected from SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab and visilizumabm, or an antigen-binding portion thereof.
  • the targeting moiety is SP34 or an antigen-binding portion thereof.
  • the targeting moiety is teclistamab or an antigen-binding portion thereof.
  • the targeting moiety is mosunetuzumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is odronextamab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is tebentafusp or an antigen-binding portion thereof. In other embodiments, the targeting moiety is muromonab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is visilizumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is tepilizumab or an antigen-binding portion thereof.
  • a targeted LNP for delivery of a therapeutic agent to immune cells (e.g., T cells) as described herein, comprises: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any or the lipids in Table 1, Table 2 and Table 3; a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties bind to at least one targeting moiety on a T cell; and a therapeutic agent encapsulated within the LNP.
  • the plurality of targeting moieties bind to one or more T-cell antigens selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR) ⁇ ,TCR - ⁇ , TCR - ⁇ / ⁇ , TCR - ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor.
  • the targeted LNP comprises a targeting moiety that targets a receptor on the surface of the T cell selected from CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • a targeted LNP in some embodiments comprises two or more different targeting moieties, each of which may bind to the same or a different target.
  • a targeted LNP having two different targeting moieties which bind to the same or different targets may be referred to an a “dual binder” or “dual binding” LNP.
  • a targeted LNP comprises a plurality of targeting moieties that bind to two or more T-cell antigens selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor [0062]
  • the plurality of targeting moieties can be added to the surface of the LNP by methods described in Section II of the Detailed Description.
  • each targeting moiety of the dual binding LNP is conjugated to the lipid nanoparticle through a linker, wherein each linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the targeting moieties are antibodies or antigen binding fragments thereof.
  • the targeting moieties are scFvs.
  • the targeting moieties are Fab fragments.
  • the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD5.
  • the targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD7. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD8. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD5 and the other targeting moiety binds to CD28.
  • the targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD7 and the other targeting moiety binds to CD28.
  • the CD28 targeting moiety is a CD80 extracellular domain (ECD).
  • the targeted LNP (conjugate) comprises three targeting moieties, wherein one targeting moiety binds to CD3.
  • one of the targeting moieties binds to CD7 and the other targeting moiety binds to CD28.
  • one of the targeting moieties binds to CD5 and the other targeting moiety binds to CD28.
  • the CD28 targeting moiety is a CD80 extracellular domain (ECD).
  • the targeting moiety that binds to CD3 is SP34 or an antigen-binding portion. In some of the foregoing embodiments, the targeting moiety that binds to CD3 is teclistamab or an antigen-binding portion.
  • the targeted LNP comprises two targeting moi eties, wherein each targeting moiety binds to the same target (e.g., receptor) on the T cell.
  • both targeting moi eties of the conjugate bind to CD3.
  • one of the targets is SP34 or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof.
  • one of the targets is SP34 or an antigen-binding portion thereof and the other is visilizumab or an antigenbinding portion thereof.
  • one of the targets is SP34 or an antigenbinding portion thereof and the other is tepilizumab or an antigen-binding portion thereof.
  • one of the targets is visilizumab or an antigen-binding portion thereof and the other is tepilizumab or an antigen-binding portion thereof.
  • one of the targets is visilizumab or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof.
  • both targeting moieties of the conjugate bind to CD3. In some embodiments, both targeting moieties of the conjugate bind to CD7.
  • lipid nanoparticle comprising:
  • lipid component comprising an ionizable lipid and a helper lipid
  • one or two targeting moieties conjugated to the LNP wherein the one or two targeting moieties are configured to target one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; and
  • a first template RNA that binds to the first gene modifying polypeptide and comprises a heterologous object sequence.
  • heterologous object sequence encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a first intracellular signaling domain, and a second intracellular signaling domain.
  • the heterologous sequence comprises SEQ ID NO: 7.
  • the disclosure provides a targeted lipid nanoparticle (LNP) for ex vivo or in vivo delivery of a therapeutic agent to immune cells (e.g., T cells), wherein the targeted LNP comprises: i. an ionizable lipid; ii. a helper lipid; iii.
  • each of the two different targeting moieties binds to a target on the surface of the immune cells (e.g., T cells); and iv. one or more nucleic acids (e.g., two or more nucleic acids) encoding components of a system for modifying or altering genomic DNA (e.g., a gene modifying system), wherein the one or more nucleic acids (e.g., two or more nucleic acids) are RNA molecules.
  • the two different targeting moieties can be added to the surface of the LNP by methods described in Section II of the Detailed Description.
  • each of the targeting moieties is conjugated to the lipid nanoparticle through a linker, wherein each linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the targeting moieties are antibodies or antigen binding fragments thereof.
  • the targeting moieties are scFvs.
  • the targeting moieties are Fab fragments.
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is an mRNA encoding a gene modifying polypeptide, as described herein.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a retrotransposon.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9- RT fusion protein.
  • one of the RNA molecules comprising a component of a system for modifying or altering genomic DNA is a guide RNA (gRNA).
  • one of the RNA molecules comprising a component of a system for modifying or altering genomic DNA is a template RNA encoding a heterologous nucleic acid for use with a gene modifying polypeptide to insert the heterologous nucleic acid sequence into a DNA sequence, e.g., the genomic DNA of a cell.
  • the components of a system for modifying or altering genomic DNA have nuclease activity, e.g., nickase activity.
  • the components of a system for modifying or altering genomic DNA do not have nuclease activity.
  • the components of a system for modifying or altering genomic DNA do not elicit a double-stranded break in the genomic DNA. In such embodiments, the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA.
  • system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous sequence into the genomic DNA.
  • TPRT target-primed reverse transcription
  • the ionizable lipid is V003 or a lipid from Table 1, Table 2 or Table 3.
  • the ionizable lipid is Lipid 092.
  • the ionizable lipid is Lipid 154.
  • the helper lipid of the targeted LNP is DSPC or sphingomyelin. .
  • the mo1% of the DSPC or sphingomyelin in the targeted LNP is from about 20% to about 40%.
  • the mo1% of the DSPC or sphingomyelin in the targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the targeted LNP further comprises a pegylated lipid. In some embodiments, the pegylated lipid comprises at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.
  • the pegylated lipid comprises at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated is DPPE-PEG2000 or DPG-PEG2000.
  • the targeted delivery (ex vivo or in vivo) of a system for modifying or altering genomic DNA (e.g., a gene modifying system) with an LNP comprising a T cell specific targeting moiety (e.g., an anti-CD3 targeting moiety) conjugated to the LNP results in gene editing or modification of the target T cells.
  • the LNPs comprising the targeting moiety are capable of modifying at least 5% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 10% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 15% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 20% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 25% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 30% of the genomes of the T cells.
  • the targeted delivery (ex vivo or in vivo) of a system for modifying or altering genomic DNA (e.g., a gene modifying system) with an LNP comprising two or more T cells specific targeting moieties conjugated to the LNP results in synergistic enhancement of gene editing or modification of the target T cells.
  • the LNPs comprising two or more T cells specific targeting moieties are capable of modifying at least 5% of the genomes of the T cells.
  • the LNPs comprising two or more T cells specific targeting moieties are capable of modifying at least 10% of the genomes of the T cells.
  • the LNPs comprising two or more T cells specific targeting moieties are capable of modifying at least 15% of the genomes of the T cells. In some embodiments, the LNPs comprising two or more T cells specific targeting moieties are capable of modifying at least 20% of the genomes of the T cells. In some embodiments, the LNPs comprising two or more T cells specific targeting moieties are capable of modifying at least 25% of the genomes of the T cells. In some embodiments, the LNPs comprising two or more T cells specific targeting moieties are capable of modifying at least 30% of the genomes of the T cells. In some embodiments, the LNPs comprising two or more T cells specific targeting moieties are capable of modifying at least 30% of the genomes of the T cells.
  • the LNPs comprising two or more T cells specific targeting moieties are capable of modifying from about 10% to about 30% of the genomes of the T cells. In some embodiments, the LNPs comprising two or more T cells specific targeting moieties are capable of modifying from about 20% to about 30% of the genomes of the T cells. In some embodiments, the LNPs comprising two or more T cells specific targeting moieties are capable of modifying from about 10% to about 20% of the genomes of the T cells.
  • the disclosure provides a method of delivering a therapeutic agent to the T cells of a subject, said method comprising, isolating T cells from the subject; mixing a pharmaceutical composition comprising targeted LNPs of the disclosure with the T cells for a sufficient time to allow for encapsulation of the therapeutic agent in the T cells; and administering the T cells encapsulating the therapeutic agent to the subject.
  • the T cells are not activated prior to mixing with the pharmaceutical composition comprising targeted LNPs.
  • the targeted LNPs include at least one CD3 binder (e.g., antibody or fragment thereof) on their surface.
  • the targeted LNPs include at least one CD3 binder (e.g., antibody or fragment thereof) on their surface and a CD2, CD3, CD4, CD5, CD7 or CD28 binder (e.g., antibody or fragment thereof) on their surface.
  • the targeted LNPs include a CD3 binder (e.g., antibody or fragment thereof) and a CD28 binder (e.g., antibody or fragment thereof).
  • the disclosure provides a method for administering a therapeutic composition to a patient, comprising, collecting a blood fraction comprising lymphocytes from the patient; isolating T cells from the blood fraction; contacting the isolated T cells with targeted lipid nanoparticles (LNPs) of the disclosure, thereby producing T cells encapsulating a therapeutic payload; and reinfusing the T cells of step into the patient.
  • the T cells are not activated prior to mixing with the pharmaceutical composition comprising targeted LNPs.
  • the targeted LNPs include at least one CD3 binder (e.g., antibody or fragment thereof) on their surface.
  • the targeted LNPs include at least one CD3 binder (e.g., antibody or fragment thereof) on their surface and a CD2, CD3, CD4, CD5, CD7 or CD28 binder (e.g., antibody or fragment thereof) on their surface.
  • the targeted LNPs include a CD3 binder (e.g., antibody or fragment thereof) and a CD28 binder (e.g., antibody or fragment thereof).
  • the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA.
  • a system for modifying or altering genomic DNA includes a gene modifying polypeptide and a template RNA for a nucleic acid sequence to be inserted at a specific location of the genomic DNA, hence resulting in modification of the genomic DNA.
  • the gene modifying system comprises a retrotransposon.
  • the gene modifying system comprises a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • a system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous nucleic acid sequence into a DNA sequence, e.g., genomic DNA.
  • TPRT target-primed reverse transcription
  • the disclosure provides a lipid nanoparticle (LNP) comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid; (ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting moieties are antibodies or fragments thereof configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; (iii) an mRNA encoding a gene modifying polypeptide; and (iv) a template RNA comprising (a) a sequence that binds to the polypeptide and (b) a heterologous object sequence.
  • LNP lipid nanoparticle
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 20 mol% to about 40 mol% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component. In some embodiments, the ionizable lipid is present at about 20 mol% to about 30 mol% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component. In some embodiments, the ionizable lipid is present at about 20 mol% to about 40 mol% of the lipid component, and the helper lipid is present at about 25 mol% to about 40 mol% of the lipid component.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, or about 20 mol% to about 40 mol% of the lipid component, or about 20 mol% to about 30 mol% of the lipid component or about 25 mol% to about 40 mol% of the lipid component and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.
  • the template RNA comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component; (ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting moieties are antibodies or fragments thereof configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.
  • the therapeutic agent comprises a DNA molecule.
  • the therapeutic agent comprises an mRNA molecule.
  • the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA.
  • the therapeutic agent comprises a gene modifying system. In some embodiments, the therapeutic agent comprises a heterologous gene modifying system. In some embodiments, the therapeutic agent comprises a gene modifying system, and the template RNA of the system comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component; (ii) means for binding a cell surface receptor selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or more nucleic acid molecules (e.g., one or more RNA molecules).
  • the therapeutic agent comprises one or more nucleic acid molecules (e.g., one or more RNA molecules
  • the means for binding the cell surface receptor is present on the surface of the LNP.
  • the means for binding a cell surface receptor comprises a targeting moiety, such as an antibody or fragment thereof.
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.
  • the therapeutic agent comprises a DNA molecule.
  • the therapeutic agent comprises an mRNA molecule.
  • the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA.
  • the therapeutic agent comprises a gene modifying system. In some embodiments, the therapeutic agent comprises a heterologous gene modifying system. In some embodiments, the therapeutic agent comprises a gene modifying system, and the template RNA of the system comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404. [0083] In some embodiments, the disclosure provides a gene modifying system comprising a gene modifying polypeptide (or a nucleic acid, e.g., a DNA or mRNA molecule, encoding the gene modifying polypeptide) and a template RNA, wherein the template RNA comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.
  • the gene modifying polypeptide comprises an amino acid sequence of Table 4 or Table 5A or a sequence having no more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid differences thereto, or a nucleic acid (e.g., DNA or mRNA) encoding the gene modifying polypeptide.
  • the gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 32, 33, 64 or 65, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the template RNA comprises both of a 5’UTR retro and a 3’ UTR retro .
  • the 5’UTR retro and the 3’ UTR retro comprise 5’ or 3’ sequences of Table 4 (e.g., SEQ ID NO: 44 and/or SEQ ID NO: 55) or any of Examples 4-11 or 15-17.
  • FIG.1 provides a bar graph showing that higher levels of transfection of targeted LNPs in activated T cells are achieved in the absence of serum.
  • FIGs.2A and 2D provide bar graphs showing the percent of activated T cells that express GFP and the GFP expression levels (MFI), respectively, when the anti-CD3 tLNPs are transfected into activated T cells in culture in the presence of serum (FBS).
  • FIGs.2B and 2C provide bar graphs showing the percent of T cells that express GFP and the GFP expression levels (MFI), respectively, when the anti-CD3 tLNPs are transfected into activated T cells in culture in the absence of serum (FBS).
  • FIGs.3A-3D provide bar graphs showing the percent of rested T cells that express GFP (FIGs.3A and 3B) and the GFP expression levels (MFI) (FIGs.3C and 3D) when the anti- CD3 tLNPs are transfected into rested T cells in culture in the presence (FIGs.3A and 3D) or absence (FIGs.3B and 3C) of serum (FBS).
  • FIG.4 provides a bar graph showing that tLNPs comprising the anti-CD3 targeting moieties induced variable expression (MFI) of the T cell activation marker CD25.
  • MFI induced variable expression
  • FIGs.5A-5D provide bar graphs showing the percent of activated T cells that express GFP (FIGs.5A and 5B) and the GFP expression levels (FIGs.5C and 5D) when the tested tLNPs are transfected into activated T cells in culture in the presence (FIGs.3A and 3D) or absence (FIGs.3B and 3C) of serum (FBS). All of the anti-CD2, anti-CD7, anti-CD3, and anti- CD5 targeted tLNPs outperform non-targeted base LNPs in activated T cells, either when the cells were cultured with serum or without serum.
  • FIGs.6A-6D provides bar graphs showing the percentage of rested T cells that express GFP (FIGs, 6A and 6B) and the GFP expression levels (FIGs.6C and 6D) when the tested tLNPs are transfected into rested T cells in culture in the presence or FIGs.6A and 6D) or absence (FIGs.6B and 6C) of serum (FBS).
  • the targeted LNPs conjugated to anti-CD7, anti- CD5, and anti-CD3 targeting moieties outperformed non-targeted base LNPs, both with and without serum.
  • FIGs.7A and 7B provide bar graphs showing the surface expression levels of the CD25 (FIG.7A) and CD69 (FIG.7B) cell activation markers following administration of the tLNPs comprising an anti-CD3 targeting moiety or dual targeted LNPs conjugated either to the anti-CD3 targeting moiety and the CD80 ECD or to two different anti-CD3 targeting moieties to rested T cells.
  • the dual tLNPs drove increased expression of surface activation markers CD25 and CD69 relative to non-targeted base LNPs, and increased expression of the activation markers by at least two-fold relative to tLNPs with the anti-CD3 targeting moiety alone.
  • FIG.8A provides a graph showing the percentage of activated T cells that expressed GFP following transfection with the various tested LNPs comprising different ionizable lipids at different doses. Close to 100% of living activated T cells transfected with LNPs comprising Lipid092 or Lipid154 expressed GFP, starting at the lowest dose. Fewer T cells were transfected with the other LNPs tested across all dose levels.
  • FIG.8B provides a graph showing the GFP expression levels (MFI) in the activated T cells transfected with the various tested LNPs comprising different ionizable lipids at different doses. Transfection with the Lipid154 LNPs resulted in the highest GFP expression levels in the cells, followed by LNPs comprising Lipid092.
  • MFI GFP expression levels
  • FIG.8C provides a graph showing the percentage of rested T cells that expressed GFP following transfection with the various tested LNPs comprising different ionizable lipids at different doses.
  • TheLipid092 and Lipid154 LNPs transfected the largest numbers of cells at the 100ng to 400 ng doses.
  • FIG.8D provides a graph showing the GFP expression levels (MFI) in the rested T cells transfect with the various tested LNPs comprising different ionizable lipids at different doses. Transfection with the Lipid092 LNPs resulted in the highest GFP expression levels, followed by LNPs with Lipid154.
  • MFI GFP expression levels
  • FIG.9A provides a bar graph showing the percentage of activated T cells that expressed GFP at 4 days following transfection with the Lipid 154, Lipid 092 or V003 LNPs at different doses. Substantially more activated T cells expressed GFP at all doses when Lipid092 LNPs or Lipid154 LNPs delivered the gene modifying system compared to activated T cells that were contacted with the V003 LNPs.
  • FIG.9B provides a bar graph showing the GFP expression levels (MFI) in activated T cells at 4 days following transfection with the Lipid 154, Lipid0092 or V003 LNPs at different doses.
  • MFI GFP expression levels
  • FIG.10A provides a bar graph showing the percentage of activated T cells that expressed GFP at 4 days following transfection with the Lipid 154, Lipid 092 or V003 anti-CD3 tLNPs or the Lipid 092 anti-CD3/CD80 ECD dual tLNPs at different doses.
  • the tLNPs formulated with Lipid092 and Lipid154 both delivered the gene modifying system more effectively to activated T cells than the baseline tLNP formulated with the V003 lipid.
  • FIG. 10B provides a bar graph showing the GFP expression levels (MFI) in activated T cells at 4 days following transfection with the Lipid 154, Lipid 092 or V003 anti-CD3 tLNPs or the Lipid 092 anti-CD3/CD80 ECD dual tLNPs at different doses.
  • the activated T cells transduced with the Lipid092 or Lipid154 tLNPs expressed GFP at higher levels (higher MFI) relative to activated T cells transduced with tLNPs comprising the V003 ionizable lipid.
  • FIG.11 shows that the tLNPs carrying an all-RNA gene modifying system generated human T cells expressing GFP in the engrafted mice, as determined by flow cytometry.
  • FIG. 11A provides a bar graph showing the percentage of engrafted human T cells (total T cells, CD4+ T cells or CD8+ T cells) that expressed GFP in mice following administration of Lipid 092 tLNPs comprising an anti-CD3 targeting moiety and an exemplary gene modifying system.
  • FIG.11B shows that engrafted human cells, including CD4+ and CD8+ T cells, expressed GFP at higher levels when tLNPs comprising the gene modifying system were administered to the humanized mice compared to engrafted cells in mice that were administered the negative control tLNP.
  • FIGs.12A-D provide bar graphs showing the percentage of activated T cells expressing GFP at 4 days or 7 days following administration of the tested LNPs comprising the exemplary gene modifying system (FIGs.12A and 12C, respectively) and the GFP expression levels (MFI) in these cells (FIGs.12B and 12D). Delivery of the gene modifying system payload to activated cells using targeted LNPs formulated with Lipid154 and 22% DSPC generated more cells that expressed GFP (%GFP+) and at higher levels (MFI) relative to the baseline control tLNP that was the identical except that it was formulated with 8% DSPC.
  • FIGs.13A -C provide bar graphs showing the percentage of rested T cells expressing GFP at 4 days following administration of the tested LNPs comprising the exemplary gene modifying system (with or without TransAct) (FIGs.13A, respectively) and the GFP expression levels (MFI) at 4 days or 7 days post-administration in these cells (FIGs.13B and 13, respectively).
  • FIG.14A provides a bar graph showing the percentage of resting T cells that express a CAR at 3 days and 7 days following administration of anti-CD3 tLNPs formulated with either 8% helper lipid or 22% helper lipid and comprising an exemplary gene modifying system (with or without coadministration of TransAct).
  • FIGs.14B and 14C provide bar graphs showing the CAR expression levels (MFI) in the rested T cells at 3 days and 7 days following administration of the anti-CD3 tLNPs of FIG.14A.
  • FIG.15A provides a bar graph showing the percentages of cells comprising an edit after bulk T cells were co-transfected with tLNPs formulated with a heterologous gene modifying system and a retrotransposon gene modifying system designed to integrate a GFP sequence into the genome of cells.
  • FIG.15B provides a bar graph showing the percentages of cells comprising an edit after bulk T cells were co-transfected with tLNPs formulated with a heterologous gene modifying system and a retrotransposon gene modifying system designed to integrate a CAR sequence into the genome of cells.
  • FIG.16 is a schematic showing the formation of an LNP conjugated to the C- terminus of a Fab fragment through a sortase mediated ligation followed by a Click reaction.
  • FIG.17 is a schematic showing the formation of an LNP conjugated to the C- terminus of a Fab fragment through a lipoic acid ligase mediated ligation followed by a Click reaction.
  • FIG.18 is a schematic showing an enzymatic approach of site-specifically introducing a Tz ring onto a sugar moiety of an antibody.
  • FIG.19 is a schematic showing an example of a light-induced crosslinking approach of site-specifically introducing a Tz ring onto an antibody.
  • FIG.20 is a schematic showing an example of using a non-natural amino acid to site- specifically introduce a Tz ring onto an antibody or Fab fragment.
  • FIG.21 is a schematic showing an example of using a cysteine-maleimide reaction to site-specifically introduce a first Click handle onto a Fab fragment, which subsequently reacts with an LNP bonded to a second Click handle, thereby generating a targeted LNP (conjugate).
  • FIG.22 is a schematic showing a Fab fragment modified with a free cysteine designed to react with a maleimide group.
  • FIG.23A shows a schematic example for producing a site-specific conjugate through the reaction of an anti-CD117 Fab fragment with site-specific sortase recognition motif and an LNP. In the schematic, the squiggly lines represent PEG bonded to a lipid of the LNP.
  • FIG.23B shows a schematic of producing a conjugate through the reaction of a Fab fragment that has been modified using a random approach (NHS modification) and an LNP.
  • FIGs.24A-24F provide schematics showing the synthesis of Lipid 092.
  • FIGs.25A-25C provide schematics showing the synthesis of Lipid 093.
  • FIGs.26A-26C provide schematics showing the synthesis of Lipid 162.
  • FIGs.27A-27C provide schematics showing the synthesis of Lipid 153.
  • FIGs.28A-28F provide schematics showing the synthesis of Lipid 154.
  • FIGs.29A-29E provide schematics showing the synthesis of Lipid 155.
  • FIGs.30A-30F provide schematics showing the synthesis of Lipid 176.
  • FIG.31 provides a bar graph showing that a site-specific conjugation process, as described herein, can efficiently conjugate two targeting moieties (anti-CD117 Fab and anti- CD45 Fab, in this case) to the surface of LNPs at specific ratios.
  • FIG.32 provides a bar graph showing the percentage of human CD3+ T cells comprising indels or a designed edit (via target primed reverse transcription mediated rewriting) at the B2M locus following administration of anti-CD5 tLNPs comprising either a gene editing system or a gene modifying system, respectively, to mice engrafted with human T cells.
  • FIG.33 provides a bar graph showing the percentage of human T cells that express a CAR after human PBMCs were either transfected with an anti-CD3 tLNP comprising an exemplary retrotransposon gene modifying system comprising a template RNA encoding a CAR sequence for 4 hours with TransAct (right side) or mock treated as a control (left side).
  • FIG.34 provides a bar graph showing the percentage of BCMA tumor cells killed in a tumor cell killing assay using the CAR-T cells generated by the exemplary retrotransposon gene modifying system of FIG.33 compared to the percentage of tumor cells killed by the mock treated T cells.
  • FIG.35 provides a bar graph showing the quantification of the IFN- human T cells that express a CAR after human PBMCs were either transfected with an anti-CD3 tLNP comprising an exemplary retrotransposon gene modifying system comprising a template RNA encoding a CAR sequence for 4 hours with TransAct (right side) or mock treated as a control (left side).
  • FIG.36 provides a bar graph showing the quantification of the percentage of edited cells expressing a CAR at 8 days following a 4-hour incubation with tLNPs conjugated to one of four different anti-CD3 targeting moieties (fab fragments) tested.
  • FIG.37 provides a bar graph showing the quantification of CAR expression levels (MFI) in the CAR-T cells described in FIG.36.
  • FIG.38 provides a bar graph showing the quantification of the percentage of BCMA tumor cells killed by the CAR-T cells described in FIG.36 in a tumor killing assay.
  • FIG.39 is a schematic showing an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).
  • FIG.40A is a schematic showing the assembly of an LNP encapsulating a therapeutic payload with a functional group (e.g., maleimide, 2,3-dibromomaleimide, sortase tag, etc.) to be reacted with an antibody or antigen-binding fragment thereof.
  • FIG.40B is a schematic showing the assembly of an LNP encapsulating a therapeutic payload using a post- insertion technique.
  • FIG.41 is an exemplary schematic showing the reduction of an interchain disulfide bond in a Fab fragment. After reduction, each cysteine residue is available to react with a thiol- reactive group conjugated to the surface of the LNP.
  • FIG.42 is an exemplary schematic showing conjugate formation.
  • a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)).
  • the Fab fragment is contacted with an LNP comprising a plurality of thiol-reactive groups (e.g., a plurality of maleimide or DBM groups) conjugated to the surface of the LNP, whereby a thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the Fab fragment.
  • a thiol-reactive groups e.g., a plurality of maleimide or DBM groups
  • FIG.43 is an exemplary schematic showing conjugate formation.
  • a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)).
  • the Fab fragment is contacted with an LNP comprising a plurality of maleimide groups conjugated to the surface of the LNP, whereby a maleimide group of the plurality reacts with one of the two free cysteine residues of the Fab fragment.
  • FIG.44 is an exemplary schematic showing conjugate formation using DBM as a bridging agent.
  • FIG.45 is an exemplary schematic showing conjugate formation.
  • a first Fab fragment (Fab1) and a second Fab fragment (Fab2) are each contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide between the heavy and light chain of each Fab.
  • each Fab comprises two free cysteine residues with one free cysteine residue on the heavy chain and one free cysteine residue on the light chain.
  • step (ii) the first Fab fragment and the second Fab fragment are contacted with an LNP comprising a plurality of thiol-reactive groups (e.g., maleimide or DBM) conjugated to the surface of the LNP, whereby a thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the first Fab Fragment and another thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the second Fab Fragment.
  • FIG.46 is a schematic showing some examples of a pegylated lipid bonded to a maleimide moiety.
  • FIG.47 is a schematic showing some examples of non-pegylated lipid bonded to a maleimide moiety.
  • FIG.48 is a schematic showing some examples of ionizable lipid bonded to a maleimide moiety.
  • FIG.49 is a schematic showing some examples of sterols bonded to a maleimide moiety.
  • FIG.50 provides a bar graph showing the percentage of human T cells that express CAR, as assayed by flow cytometry, at 16 hours following the administration of tLNPs formulated with 8% helper lipid and an exemplary all-RNA gene modifying system to mice that were engrafted with human T cells.
  • FIG.51 provides a bar graph showing the percentage of human T cells that express CAR, as assayed by flow cytometry, at 16 hours following the administration of tLNPs formulated with 22% helper lipid and an exemplary all-RNA gene modifying system to mice that were engrafted with human T cells.
  • the tLNPs were conjugated with an anti-CD2, anti-CD5 or anti-CD7 targeting moiety. DETAILED DESCRIPTION I.
  • Antigen binding domain refers to that portion of antibody or a chimeric antigen receptor which binds an antigen.
  • an antigen binding domain binds to a cell surface antigen of a cell.
  • an antigen binding domain binds an antigen characteristic of a cancer, e.g., a tumor associated antigen in a neoplastic cell.
  • an antigen binding domain binds an antigen characteristic of an infectious disease, e.g. a virus associated antigen in a virus infected cell.
  • an antigen binding domain binds an antigen characteristic of a cell targeted by a subject’s immune system in an autoimmune disease, e.g., a 0-antigen.
  • an antigen binding domain is or comprises an antibody or antigen-binding portion thereof.
  • an antigen binding domain is or comprises an scFv or Fab.
  • gRNA spacer refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid.
  • gRNA scaffold refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid.
  • the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.
  • Gene modifying polypeptide A “gene modifying polypeptide,” and “retrotransposon gene modifying polypeptide” as used herein interchangeably to refer to a polypeptide comprising a retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to said domains, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell).
  • a nucleic acid sequence e.g., a sequence provided on a template nucleic acid
  • target DNA molecule e.g., in a mammalian host cell, such as a genomic DNA molecule in
  • the endonuclease domain is a catalytically inactive endonuclease domain.
  • the retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain are derived from the same retrotransposase.
  • the gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery.
  • the gene modifying polypeptide integrates a sequence into a random position in a genome, and in some embodiments, the gene modifying polypeptide integrates a sequence into a specific target site.
  • a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the template nucleic acid into the target DNA.
  • Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence.
  • Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain.
  • Exemplary gene modifying polypeptides, and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in WO/2021/178717, which is incorporated herein by reference, including Tables 10, 11, X, 3A, 3B, and Z1 therein.
  • a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene.
  • a “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid. [0140] Gene modifying system: A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide, or a nucleic acid (e.g., an mRNA) encoding the gene modifying polypeptide, and a template nucleic acid.
  • Domain refers to a structure of a biomolecule that contributes to a specified function of the biomolecule.
  • a domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolecule.
  • protein domains include, but are not limited to, an endonuclease domain, a DNA binding domain, a reverse transcriptase domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain.
  • Exogenous when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell, or organism by the hand of man.
  • a nucleic acid that is as added into an existing genome, cell, tissue, or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.
  • Heterologous The term “heterologous”, when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described.
  • a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions.
  • a heterologous regulatory sequence e.g., promoter, enhancer
  • a heterologous domain of a polypeptide or nucleic acid sequence e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide
  • a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both.
  • heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).
  • a domain is heterologous relative to another domain, if the first domain is not naturally comprised in the same polypeptide as the other domain (e.g., a fusion between two domains of different proteins from the same organism).
  • heterologous gene modifying polypeptide refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell).
  • a nucleic acid sequence e.g., a sequence provided on a template nucleic acid
  • target DNA molecule e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell.
  • the heterologous gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery.
  • the heterologous gene modifying polypeptide integrates a sequence into a random position in a genome, and in some embodiments, the heterologous gene modifying polypeptide integrates a sequence into a specific target site.
  • the sequence that is integrated comprises a deletion, substitution, or insertion relative to the target DNA molecule.
  • a heterologous gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the template nucleic acid into the target DNA.
  • Heterologous gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence.
  • Heterologous gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain.
  • heterologous gene modifying polypeptides and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in PCT/US2021/020948, which is incorporated herein by reference with respect to heterologous gene modifying polypeptides that comprise a retroviral reverse transcriptase domain.
  • a heterologous gene modifying polypeptide integrates a sequence into a gene.
  • a heterologous gene modifying polypeptide integrates a sequence into a sequence outside of a gene.
  • a “heterologous gene modifying system,” as used herein, refers to a system comprising a heterologous gene modifying polypeptide and a template nucleic acid.
  • Mutation or Mutated when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence.
  • a nucleic acid sequence may be mutated by any method known in the art. In some embodiments a mutation occurs naturally. In some embodiments a desired mutation can be produced by a system described herein.
  • Nucleic acid molecule refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein.
  • the nucleic acid molecule can be double-stranded or single-stranded, circular, or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand.
  • nucleic acid comprising SEQ ID NO:1 refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complimentary to SEQ ID NO:1.
  • the choice between the two is dictated by the context in which SEQ ID NO:1 is used. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.
  • Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.).
  • uncharged linkages for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • RNA molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs).
  • the nucleic acids are in operative association with additional genetic elements, such as tissue-specific expression-control sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats/direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (5 ⁇ , 3 ⁇ , or both 5 ⁇ and 3 ⁇ UTRs), and various combinations of the foregoing.
  • tissue-specific expression-control sequence(s) e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences
  • additional elements such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats
  • the nucleic acid elements of the systems provided by the invention can be provided in a variety of topologies, including single-stranded, double-stranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), closed-ended DNA (ceDNA).
  • dbDNA doggybone DNA
  • ceDNA closed-ended DNA
  • PBS sequence refers to a portion of a template RNA capable of binding to a region comprised in a target nucleic acid sequence.
  • a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to the region comprised in the target nucleic acid sequence.
  • the primer region comprises at least 5, 6, 7, 8 bases with 100% identity to the region comprised in the target nucleic acid sequence.
  • the PBS sequence binds to a region comprised in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.
  • the disclosure provides an LNP (conjugate) comprising an ionizable lipid as described herein (e.g., in Table 1 or Table 2), wherein the LNP can deliver a payload, such as a therapeutic agent (e.g., a gene modifying system, such as a retrotransposon gene modifying system and/or a heterologous gene modifying system, as described herein) to an immune cell (e.g., a T cell).
  • a therapeutic agent e.g., a gene modifying system, such as a retrotransposon gene modifying system and/or a heterologous gene modifying system, as described herein
  • an immune cell e.g., a T cell
  • the LNP (conjugate) comprises a targeting moiety that binds to a protein (e.g., a protein receptor) on an immune cell (e.g., a T cell), as described herein.
  • an LNP comprises both an ionizable lipid and a targeting moiety, as described herein.
  • the disclosure provides targeted LNPs (conjugates) comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system), wherein the targeting moiety binds to a protein (e.g., protein receptor) on an immune cell (e.g., T cell).
  • a payload e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system
  • the targeting moiety binds to a protein (e.g., protein receptor) on an immune cell (e.g., T cell).
  • the targeting moiety is an antibody or antigen binding fragment thereof.
  • the targeting moiety is an antibody, a Fab fragment, a scFv, a DARPIN, a VHH domain antibody, a FN3 domain, a nanobody, a single domain antibody or a Centyrin.
  • the targeting moiety is a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin or a N- Acetylgalactosamine (GalNac).
  • the payload e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system
  • the payload is capable of modifying one or more genes of the target immune cell (e.g., T cell).
  • the conjugates described herein may be used to target and modify immune cells. In some embodiments, the conjugates may be used to modify T cells.
  • T cells may include any subpopulation of T cells, e.g., CD4+ T cells, CD8+ T cells, gamma-delta T cells, alpha-beta T cells, na ⁇ ve T cells, stem cell memory T cells, central memory T cells, effector T cells, cytotoxic T cells, helper T cells (e.g., Th1 cells, Th2 cells, Th17 cells, etc.), regulatory T cells, tumor-infiltrating T cells, tissue-resident T cells, or a mixture of subpopulations.
  • the conjugates may be used to deliver or modify a sequence encoding a T-cell receptor (TCR) in a T cell.
  • TCR T-cell receptor
  • the conjugates may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T cells.
  • CAR chimeric antigen receptor
  • the conjugates can be used to deliver an RNA encoding a CAR to T cells.
  • the conjugates may be used to deliver at least one sequence encoding a CAR to natural killer (NK) cells.
  • NK natural killer
  • the conjugates be used to deliver at least one sequence encoding a CAR to natural killer T (NKT) cells.
  • the conjugates may be used to deliver at least one sequence encoding a CAR to a progenitor cell, e.g., a progenitor cell of T, NK, or NKT cells.
  • cells modified with at least one CAR are used to treat a condition as identified in the targetable landscape of CAR therapies in MacKay, et al. Nat Biotechnol 38, 233-244 (2020), incorporated by reference herein in its entirety.
  • the immune cells comprise a CAR specific to a tumor or a pathogen antigen selected from a group consisting of AChR (fetal acetylcholine receptor), ADGRE2, AFP (alpha fetoprotein), BAFF-R, BCMA, CAIX (carbonic anhydrase IX), CCR1, CCR4, CEA (carcinoembryonic antigen), CD3, CD5, CD8, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD30, CD33, CLLI, CD34, CD38, CD41, CD44, CD49f, CD56, CD61, CD64, CD68, CD70, CD74, CD99, CD117, CD123, CD133, CD138, CD44v6, CD267, CD269, CDS, CLEC12A, CS1, EGP-2 (epithelial glycoprotein-2), EGP-40 (epithelial glycoprotein-40), EGFR (HER1), EGFR-VIII, EpCAM (
  • immune cells e.g., T cells, NK cells, NKT cells, or progenitor cells are modified ex vivo and then delivered to a patient.
  • a nucleic acid e.g., RNA, such as mRNA
  • immune cells e.g., T cells, NK cells, NKT cells, or progenitor cells are modified in vivo in the patient.
  • the targeting moiety is a T-cell targeting moiety, for example, an antibody, Fab fragment, or scFv that binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor.
  • a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, or IL
  • the targeting moiety is a T-cell targeting moiety, for example, an antibody, Fab fragment, or scFv that binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor.
  • the CD28 targeting moiety is a CD80 extracellular domain (ECD).
  • the targeted LNP comprises a targeting moiety that targets CD3 on the surface of the T cell, wherein the targeting moiety is an antibody, Fab fragment, or scFv selected from SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab, visilizumab, Plamotamab, HPN536, Pasotuxizumab, and Flotetuzumab, or an antigen-binding portion thereof.
  • the targeting moiety is SP34 or an antigen-binding portion thereof.
  • the targeting moiety is teclistamab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is mosunetuzumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is odronextamab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is tebentafusp or an antigen-binding portion thereof. In other embodiments, the targeting moiety is muromonab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is visilizumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is tepilizumab or an antigen-binding portion thereof.
  • the targeting moiety is Plamotamab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is HPN536 or an antigen-binding portion thereof. In other embodiments, the targeting moiety is Pasotuxizumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is Flotetuzumab or an antigen-binding portion thereof.
  • a targeted LNP for delivery of a therapeutic agent to immune cells (e.g., T cells) as described herein, comprises: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any or the lipids in Table 1, Table 2 and Table 3; a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties bind to at least one targeting moiety on a T cell; and a therapeutic agent encapsulated within the LNP.
  • the plurality of targeting moieties bind to two or more T-cell antigens selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD80, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor.
  • the targeted LNP comprises a targeting moiety that targets a receptor on the surface of the T cell selected from CD2, CD3, CD4, CD5, CD6, CD7, and CD28.
  • a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD7. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD28. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD3. In some embodiments, the one targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the other targeting moiety that binds to CD3.
  • the targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD5. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD7. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD5 and the other targeting moiety binds to CD28.
  • the targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD7 and the other targeting moiety binds to CD28.
  • the CD28 targeting moiety is a CD80 extracellular domain (ECD).
  • a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD3.
  • the one targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the other targeting moiety that binds to CD3.
  • the targeted LNP comprises two targeting moieties, wherein each targeting moiety binds to the same target (e.g., receptor) on the T cell.
  • both targeting moieties of the conjugate bind to CD3.
  • one of the targets is SP34 or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof.
  • one of the targets is SP34 or an antigen-binding portion thereof and the other is visilizumab or an antigen- binding portion thereof.
  • one of the targets is SP34 or an antigen- binding portion thereof and the other is tepilizumab or an antigen-binding portion thereof.
  • one of the targets is visilizumab or an antigen-binding portion thereof and the other is tepilizumab or an antigen-binding portion thereof.
  • one of the targets is visilizumab or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof.
  • both targeting moieties of the conjugate bind to CD3. In some embodiments, both targeting moieties of the conjugate bind to CD7.
  • the targeting moiety binds to a CD4+ and/or CD8+ T cell. In other embodiments, the targeting moiety binds to a natural killer (NK) cell. In other embodiments, the targeting moiety binds to a hematopoietic stem cell. In other embodiments, the targeting moiety binds to a lymphoid progenitor cell. In other embodiments, the targeting moiety binds to a myeloid cell. In other embodiments, the targeting moiety binds to a macrophage.
  • CD2 Targeting Moieties [0162] In some embodiments, the target molecule is CD2. In some embodiments, the target cell is CD2+.
  • the glycoprotein CD2 is a costimulatory receptor expressed mainly on T cells, NK cells, thymocytes, and dendritic cells that binds to lymphocyte-associated antigen 3 (LFA3; also known as CD58) which is expressed on the surface of B cells, T cells, monocytes, granulocytes, thymic epithelial cells.
  • LFA3 lymphocyte-associated antigen 3
  • CD2 also binds to CD48, albeit with a relatively lower affinity.
  • CD2 has an important role in the formation and organization of the immunological synapse that is formed between T cells and antigen-presenting cells upon cell-cell conjugation and associated intracellular signaling. CD2 expression is upregulated on memory T cells as well as activated T cells and plays an important role in activation of memory T cells.
  • the CD2 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD2.
  • the CD2 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’) 2 , Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin).
  • the CD2 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.
  • the CD2 targeting moiety is an anti-CD2 antibody or antigen binding fragment thereof.
  • the CD2 targeting moiety is an IgA, IgG, IgE, or IgM antibody.
  • the CD2 targeting moiety is a bispecific or multi- specific antibody or fragment thereof.
  • the CD2 targeting moiety is a humanized antibody or antigen-binding fragment thereof.
  • Exemplary anti-CD2 binders, antibodies, or antigen-binding fragments thereof include Siplizumab (i.e., MEDI-507 or TCD601, ITB-Med LLC), BTI-322 (Lo-CD2a), Alefacept (i.e., a chimeric fusion protein consisting of the CD2-binding portion of human LFA3- Fc, Biogen) CB.219 (e.g., BioXCell), UMCD2 (e.g., Santa Cruz Biotechnology), TS1/8, RPA- 2.10, TS1/18, TS1/18.1.1, TS2/18, AB75, and ZR100, as well as anti-CD2 antibodies or antigen- binding fragments thereof disclosed in any of: US 5,730,979; US 5,928,643; US 5,951,983; US 6,764,681; US 7,858,095; US 6,162,432; US 11,732,042; US 12,037,378; US2021003230
  • the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:269 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:270. In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:280 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:281. In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:291 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:292.
  • the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:302 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:292.
  • SEQ ID NOs:269, 270, 280, 281, 291, 292, and 302 are shown in Table 20, with complementary determining regions (CDRs) marked in bold.
  • the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:269, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:270.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:270.
  • the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence SYWVN (SEQ ID NO:271), a CDR- H2 comprising an amino acid sequence RIDPYDSETHYNQKFTD (SEQ ID NO:272), a CDR- H3 comprising an amino acid sequence SPRDSSTNLAD (SEQ ID NO:273), a CDR-L1 comprising an amino acid sequence RASQSISDYLH (SEQ ID NO:274), a CDR-L2 comprising an amino acid sequence YASQSIS (SEQ ID NO:275), and a CDR-L3 comprising an amino acid sequence QNGHSFPLT (SEQ ID NO:276).
  • the CD2 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:266 or 267, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:268.
  • the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:280, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:281.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:281.
  • the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RYWIH (SEQ ID NO:282), a CDR- H2 comprising an amino acid sequence NIDPSDSETHYNQKFKD (SEQ ID NO:283), a CDR- H3 comprising an amino acid sequence EDLYYAMEY (SEQ ID NO:284), a CDR-L1 comprising an amino acid sequence KSSQSVLYSSNQKNYLA (SEQ ID NO:285), a CDR-L2 comprising an amino acid sequence WASTRES (SEQ ID NO:147), and a CDR-L3 comprising an amino acid sequence HQYLSSHT (SEQ ID NO:287).
  • the CD2 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:277 or 278, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:279.
  • the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:291, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:292.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:292.
  • the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence EYYMY (SEQ ID NO:293), a CDR- H2 comprising an amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO:294), a CDR- H3 comprising an amino acid sequence GKFNYRFAY (SEQ ID NO:295), a CDR-L1 comprising an amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO:296), a CDR-L2 comprising an amino acid sequence LVSKLES (SEQ ID NO:297), and a CDR-L3 comprising an amino acid sequence MQFTHYPYT (SEQ ID NO:298).
  • the CD2 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:288 or 289, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:290.
  • the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:302, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:292.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:292.
  • the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence EYYMY (SEQ ID NO:293), a CDR- H2 comprising an amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO:294), a CDR- H3 comprising an amino acid sequence GKFNYRFAY (SEQ ID NO:295), a CDR-L1 comprising an amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO:296), a CDR-L2 comprising an amino acid sequence LVSKLES (SEQ ID NO:297), and a CDR-L3 comprising an amino acid sequence MQFTHYPYT (SEQ ID NO:298).
  • the CD2 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:299 or 300, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:290.
  • CD3 Targeting Moieties [0170]
  • the target molecule is CD3.
  • the target cell is CD3+.
  • CD3 is a multimeric protein complex made up of four polypeptide chains (CD3- epsilon ( ⁇ ), CD3 -gamma ( ⁇ ), CD3 -delta ( ⁇ ), and CD3 -zeta ( ⁇ )) to form a CD3 ⁇ –CD3 ⁇ –CD3 ⁇ signaling hexamer that associates with the T cell receptor (TCR).
  • TCR T cell receptor
  • the CD3/TCR complex is critical for T cells to recognize foreign antigens and activate T-cell adaptive immunity.
  • CD3 is expressed by all T cells and is a defining marker of the T lymphocyte lineage.
  • the CD3 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD3.
  • the CD3 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’) 2 , Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin).
  • the CD3 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.
  • the CD3 targeting moiety is an anti-CD3 antibody or antigen binding fragment thereof.
  • the CD3 targeting moiety is an IgA, IgG, IgE, or IgM antibody.
  • the CD3 targeting moiety is a bispecific or multi- specific antibody or fragment thereof.
  • the CD3 targeting moiety is a humanized antibody or antigen-binding fragment thereof.
  • Exemplary anti-CD3 binders, antibodies, or antigen-binding fragments thereof include SP34 mouse monoclonal antibody (see, for example, Pressano, S. The EMBO J.4:337- 344, 1985; Alarcon, B. EMBO J.10:903-912, 1991; Salmeron A. et al., J. Immunol.147:3047- 52, 1991; Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad M L. et al., Cytometry 71A:925-33, 2007; Yang et al., J.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:141 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:142.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:152 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:153.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:163 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:164. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:174 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:175. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:185 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:186.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:196 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:197. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:207 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:208. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:218 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:219.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:228 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:229. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:238 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:239. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:248 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:249.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:258 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:259.
  • SEQ ID NOs:141-142, 152-153, 163-163, 174-175, 185-186, 196-197, 207-208, 218-219, 228-229, 238-239, 248-249, and 258-259 are shown in Tables 19, 30, and 32, with complementary determining regions (CDRs) marked in bold.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:141, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:142.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:142.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NYYIH (SEQ ID NO:143), a CDR- H2 comprising an amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO:144), a CDR- H3 comprising an amino acid sequence DSYSNYYFDY (SEQ ID NO:145), a CDR-L1 comprising an amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO:146), a CDR-L2 comprising an amino acid sequence WASTRES (SEQ ID NO:147), and a CDR-L3 comprising an amino acid sequence TQSFILRT (SEQ ID NO:148).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:138 or 139, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:140.
  • the CD3 targeting moiety is mosunetuzumab or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:152, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:153.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence DYTMH (SEQ ID NO:154), a CDR- H2 comprising an amino acid sequence GISWNSGSIGYADSVKG (SEQ ID NO:155), a CDR- H3 comprising an amino acid sequence DNSGYGHYYYGMDV (SEQ ID NO:156), a CDR-L1 comprising an amino acid sequence RASQSVSSNLA (SEQ ID NO:157), a CDR-L2 comprising an amino acid sequence GASTRAT (SEQ ID NO:158), and a CDR-L3 comprising an amino acid sequence QHYINWPLT (SEQ ID NO:159).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:149 or 150, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:151.
  • the CD3 targeting moiety is odronextamab or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:163, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:164.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence GYTMN (SEQ ID NO:165), a CDR- H2 comprising an amino acid sequence LINPYKGVSTYNQKFKD (SEQ ID NO:166), a CDR- H3 comprising an amino acid sequence SGYYGDSDWYFDV (SEQ ID NO:167), a CDR-L1 comprising an amino acid sequence RASQDIRNYLN (SEQ ID NO:168), a CDR-L2 comprising an amino acid sequence YTSRLES (SEQ ID NO:169), and a CDR-L3 comprising an amino acid sequence QQGNTLPWT (SEQ ID NO:170).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:160 or 161, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:162.
  • the CD3 targeting moiety is tebentafusp or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:174, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:175.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RYTMH (SEQ ID NO:176), a CDR- H2 comprising an amino acid sequence YINPSRGYTNYNQKVKD (SEQ ID NO:177), a CDR- H3 comprising an amino acid sequence YYDDHYCLDY (SEQ ID NO:178), a CDR-L1 comprising an amino acid sequence SASSSVSYMN (SEQ ID NO:179), a CDR-L2 comprising an amino acid sequence DTSKLAS (SEQ ID NO:180), and a CDR-L3 comprising an amino acid sequence QQWSSNPFT (SEQ ID NO:181).
  • the CD3 targeting moiety is teplizumab or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:185, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:186.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NTYAMN (SEQ ID NO:187), a CDR- H2 comprising an amino acid sequence RIRSKYNNYATYYAASVKG (SEQ ID NO:188), a CDR-H3 comprising an amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:189), a CDR-L1 comprising an amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:190), a CDR- L2 comprising an amino acid sequence GTNKRAP (SEQ ID NO:191), and a CDR-L3 comprising an amino acid sequence ALWYSNLWV (SEQ ID NO:192).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:182 or 183, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:184.
  • the CD3 targeting moiety is teclistamab or an antigen- binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:196, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:197.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence SYTMH (SEQ ID NO:198), a CDR- H2 comprising an amino acid sequence YINPRSGYTHYNQKLKD (SEQ ID NO:199), a CDR- H3 comprising an amino acid sequence SAYYDYDGFAY (SEQ ID NO:200), a CDR-L1 comprising an amino acid sequence SASSSVSYMN (SEQ ID NO:179), a CDR-L2 comprising an amino acid sequence DTSKLAS (SEQ ID NO:180), and a CDR-L3 comprising an amino acid sequence QQWSSNPPT (SEQ ID NO:203).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:193 or 194, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:195.
  • the CD3 targeting moiety is visilizumab or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:207, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:208.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RYTMH (SEQ ID NO:176), a CDR- H2 comprising an amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID NO:210), a CDR- H3 comprising an amino acid sequence YYDDHYCLDY (SEQ ID NO:178), a CDR-L1 comprising an amino acid sequence SASSSVSYMN (SEQ ID NO:179), a CDR-L2 comprising an amino acid sequence DTSKLAS (SEQ ID NO:180), and a CDR-L3 comprising an amino acid sequence QQWSSNPFT (SEQ ID NO:181).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:204 or 205, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:206.
  • the CD3 targeting moiety is muromonab or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:218, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:219.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence KYAMN (SEQ ID NO:220), a CDR- H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR-H3 comprising an amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:222), a CDR-L1 comprising an amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:223), a CDR- L2 comprising an amino acid sequence GTKFLAP (SEQ ID NO:224), and a CDR-L3 comprising an amino acid sequence VLWYSNRWV (SEQ ID NO:225).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:215 or 216, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:217.
  • the CD3 targeting moiety is SP34 or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:228, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:229.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence TYAMN (SEQ ID NO:230), a CDR- H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKG (SEQ ID NO:231), a CDR-H3 comprising an amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO:232), a CDR-L1 comprising an amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO:233), a CDR- L2 comprising an amino acid sequence GTNKRAP (SEQ ID NO:191), and a CDR-L3 comprising an amino acid sequence ALWYSNHWV (SEQ ID NO:235).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:226, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:227.
  • the CD3 targeting moiety is plamotamab or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:238, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:239.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence KYAIN (SEQ ID NO:240), a CDR- H2 comprising an amino acid sequence RIRSKYNNYATYYADQVKD (SEQ ID NO:241), a CDR-H3 comprising an amino acid sequence HANFGNSYISYWAY (SEQ ID NO:242), a CDR-L1 comprising an amino acid sequence ASSTGAVTSGNYPN (SEQ ID NO:243), a CDR- L2 comprising an amino acid sequence GTKFLVP (SEQ ID NO:244), and a CDR-L3 comprising an amino acid sequence TLWYSNRWV (SEQ ID NO:245).
  • a CDR-H1 comprising an amino acid sequence KYAIN (SEQ ID NO:240)
  • a CDR- H2 comprising an amino acid sequence RIRSKYNNYATYYADQVKD
  • SEQ ID NO:241 comprising an amino acid sequence HANFGNSYIS
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:236, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:237.
  • the CD3 targeting moiety is HPN536 or an antigen-binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:218, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:249.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence KYAMN (SEQ ID NO:220), a CDR- H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR-H3 comprising an amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:222), a CDR-L1 comprising an amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:223), a CDR- L2 comprising an amino acid sequence GTKFLAP (SEQ ID NO:224), and a CDR-L3 comprising an amino acid sequence VLWYSNRWV (SEQ ID NO:225).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:246, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:247.
  • the CD3 targeting moiety is pasotuxizumab or an antigen- binding fragment thereof.
  • the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:258, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:259.
  • the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence TYAMN (SEQ ID NO:230), a CDR- H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR-H3 comprising an amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:189), a CDR-L1 comprising an amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:190), a CDR- L2 comprising an amino acid sequence GTNKRAP (SEQ ID NO:191), and a CDR-L3 comprising an amino acid sequence ALWYSNLWV (SEQ ID NO:192).
  • the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:256, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:257.
  • the CD3 targeting moiety is flotetuzumab or an antigen-binding fragment thereof.
  • CD5 Targeting Moieties [0186]
  • the target molecule is CD5.
  • the target cell is CD5+.
  • CD5 is a type-I transmembrane glycoprotein with an extracellular region composed of three scavenger receptor cysteine-rich (SRCR) domains.
  • SRCR scavenger receptor cysteine-rich
  • CD5 ligands have been reported such as CD72, the IgV(H) frame-work region and several polypeptides (gp40-80, gp150) whose identity remains undetermined. CD5 regulates T cell functions and development, including negative regulation of TCR signaling.
  • CD5 is an activation marker of T cells, wherein the expression of CD5 increases according to the magnitude of the signal delivered by the TCR. Consequently, CD5 expression reflects the heterogeneity of the signal strength associated with each individual TCR within a polyclonal T cell population. See, e.g., Voisinne et al. (2016) Front. Immunol.9:2900, hereby incorporated by reference in its entirety.
  • the CD5 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD5.
  • the CD5 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’) 2 , Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin).
  • the CD5 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.
  • the CD5 targeting moiety is an anti-CD5 antibody or antigen binding fragment thereof.
  • the CD5 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD5 targeting moiety is a bispecific or multi- specific antibody or fragment thereof. In some embodiments, the CD5 targeting moiety is a humanized antibody or antigen-binding fragment thereof.
  • Exemplary anti-CD5 binders, antibodies, or antigen-binding fragments thereof include AFM 16 (e.g., Affimed Therapeutics); AFM 17 (e.g., Affimed Therapeutics), RM354, L17F12, CRIS-1, UCHT2, RM314, SP19, and CD5-5D7, as well as anti-CD5 antibodies or antigen-binding fragments thereof disclosed in any of: US 10,786,549; US20110250203; Dai et al. (2021) Mol Ther.29(9)2707-2722; etc., each hereby incorporated by reference in its entirety.
  • the CD5 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:357 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:358.
  • SEQ ID NOs:357 and 358 are shown in Table 20, with complementary determining regions (CDRs) marked in bold.
  • the CD5 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:357, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:358.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:358.
  • the CD5 targeting moiety comprises a CDR-H1 comprising an amino acid sequence TSGMGVG (SEQ ID NO:359), a CDR-H2 comprising an amino acid sequence HIWWDDDVYYNPSLKS (SEQ ID NO:360), a CDR-H3 comprising an amino acid sequence RRATGTGFDY (SEQ ID NO:361), a CDR-L1 comprising an amino acid sequence QASQDVGTAVA (SEQ ID NO:362), a CDR-L2 comprising an amino acid sequence WTSTRHT (SEQ ID NO:363), and a CDR-L3 comprising an amino acid sequence HQYNSYNT (SEQ ID NO:364).
  • the CD5 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:354 or 355, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:356.
  • the target molecule is CD7.
  • the target cell is CD7+.
  • CD7 also known as GP40, LEU-9, Tp40, and TP41
  • CD7 is a transmembrane glycoprotein expressed by T cells, NK cells, and their precursors. It is present in >95% of lymphoblastic T-cell leukemias and lymphomas and a subset of PTCLs.
  • CD7 has a costimulatory role in T-cell activation and cytokine production (e.g., IL-2) upon binding to its ligand, K12/SECTM1.
  • the CD7 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD7.
  • the CD7 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’) 2 , Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin).
  • the CD7 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.
  • the CD7 targeting moiety is an anti-CD7 antibody or antigen binding fragment thereof.
  • the CD7 targeting moiety is an IgA, IgG, IgE, or IgM antibody.
  • the CD7 targeting moiety is a bispecific or multi- specific antibody or fragment thereof.
  • the CD7 targeting moiety is a humanized antibody or antigen-binding fragment thereof.
  • Exemplary anti-CD7 binders, antibodies, or antigen-binding fragments thereof include SP94 (e.g., Roche Diagnostics), A20153E (e.g., BioLegend), 4H9/CD7 (e.g., BioLegend), 124-1D1, CD7-6B7, B-F12, 4H9, 3A1E, LT7, MEM-186, and MG34, as well as anti-CD7 antibodies or antigen-binding fragments thereof disclosed in any of: US 11,440,958; US 11,390,658; US20240075143; US20230128800; US20230399398; US20230159636; WO2003051926 WO2023185256; Wang et al.
  • SP94 e.g., Roche Diagnostics
  • A20153E e.g., BioLegend
  • 4H9/CD7 e.g., BioLegend
  • the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:313 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:314. In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:324 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:325. In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:335 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:336.
  • the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:346 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:347.
  • SEQ ID NOs:313-314, 324-325, 335-336, and 346-347 are shown in Table 20, with complementary determining regions (CDRs) marked in bold.
  • the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:313, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:314.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:314.
  • the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NYGMN (SEQ ID NO:315), a CDR- H2 comprising an amino acid sequence WINTYTGEPTYADDFKG (SEQ ID NO:316), a CDR- H3 comprising an amino acid sequence WAYFYGSSPYFFDY (SEQ ID NO:317), a CDR-L1 comprising an amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:190), a CDR-L2 comprising an amino acid sequence GTNNRAP (SEQ ID NO:319), and a CDR-L3 comprising an amino acid sequence ALWCSNHLV (SEQ ID NO:320).
  • the CD7 targeting moiety i5s a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:310 or 311, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:312.
  • the CD7 targeting moiety is grisnilimab or an antigen-binding fragment thereof.
  • the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:324, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:325.
  • the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NYAMS (SEQ ID NO:326), a CDR- H2 comprising an amino acid sequence TISGSGGSTYYADSAK (SEQ ID NO:327), a CDR-H3 comprising an amino acid sequence GGLLYFGEFHFDY (SEQ ID NO:328), a CDR-L1 comprising an amino acid sequence RASQGISNYLA (SEQ ID NO:329), a CDR-L2 comprising an amino acid sequence AASSLQS (SEQ ID NO:330), and a CDR-L3 comprising an amino acid sequence QHYNSYPLT (SEQ ID NO:331).
  • the CD7 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:321 or 322, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:323.
  • the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:335, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:336.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:336.
  • the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NAWMS (SEQ ID NO:337), a CDR- H2 comprising an amino acid sequence RIKSKTDGGTTDYAAPVKG (SEQ ID NO:338), a CDR-H3 comprising an amino acid sequence TIEAVAGHFDY (SEQ ID NO:339), a CDR-L1 comprising an amino acid sequence RASQSISSWLA (SEQ ID NO:340), a CDR-L2 comprising an amino acid sequence KASSLES (SEQ ID NO:341), and a CDR-L3 comprising an amino acid sequence QQYNNYSPT (SEQ ID NO:342).
  • the CD7 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:332 or 333, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:334.
  • the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:346, and/or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:347.
  • a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:347.
  • the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RYAMS (SEQ ID NO:348), a CDR- H2 comprising an amino acid sequence SISASGATTFYADPVKG (SEQ ID NO:349), a CDR- H3 comprising an amino acid sequence DQDFDILTGYLNWFDP (SEQ ID NO:350), a CDR-L1 comprising an amino acid sequence RVSQSVSSYLA (SEQ ID NO:351), a CDR-L2 comprising an amino acid sequence DTSNRAT (SEQ ID NO:352), and a CDR-L3 comprising an amino acid sequence QQRRNWPLT (SEQ ID NO:353).
  • the CD7 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:343 or 344, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:345.
  • the target molecule is CD28.
  • the target cell is CD28+.
  • CD28 is a T-cell costimulatory molecule. It is a homodimeric glycoprotein member of the Ig gene superfamily and has a single IgV domain. It is expressed on T cells where it is activated upon binding to its ligands B7-1 or B7-2 (CD80 or CD86), which are expressed on professional antigen-presenting cells. CD28 does not affect T cell activation unless the T-cell receptor is first engaged by cognate antigen.
  • the CD28 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.
  • the CD28 targeting moiety is an anti-CD28 antibody or antigen binding fragment thereof.
  • the CD28 targeting moiety is an IgA, IgG, IgE, or IgM antibody.
  • the CD28 targeting moiety is a bispecific or multi-specific antibody or fragment thereof.
  • the CD28 targeting moiety is a humanized antibody or antigen-binding fragment thereof.
  • anti-CD28 binders, antibodies, or antigen-binding fragments thereof include Theralizumab (i.e., TGN1412, TAB08, or CD28-SuperMAB, e.g., TeGenero), davoceticept (i.e., ALPN-202, e.g., Alpine Immune Sciences, Inc.), FPT155 (Five Prime Therapeutics, Inc.), 10F3, RM404, 15E8, CD28.3, Leu-2, 9.3, EX5.3D10, YTH913.12, S20013F, S20013B, and QA17A12, as well as anti-CD28 antibodies or antigen-binding fragments thereof disclosed in any of: US 7,175,843; US 8,168,759; US 8,785,138; US 8,785,604; US 10,273,281; US 11,117,949; US20180112000; US20230227530; US20230348600; US20230382972; WO1994029436; WO
  • the CD28 targeting moiety is a CD28 receptor ligand.
  • the CD28 receptor ligand is CD80.
  • the CD28 targeting moiety is CD80.
  • CD80 is a costimulatory molecule known for its role in T-cell activation and also in regulating the activity of normal and malignant B cells. Surface CD80 is expressed transiently on activated B cells, macrophages, and DCs.
  • the CD28 targeting moiety is a CD80 extracellular domain (ECD), for example a CD80 ECD comprising an amino acid sequence of SEQ ID NO:365 or comprising at least about 80% (such as about any of 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:365.
  • ECD extracellular domain
  • the targeted LNP comprises two or more targeting moieties, wherein each targeting moiety independently binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD80, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor.
  • T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD80, CD137, CD45, T-cell receptor (TCR)- ⁇ ,TCR- ⁇ , TCR- ⁇ / ⁇ , TCR- ⁇ / ⁇ , PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor.
  • the targeted LNP comprises two or more targeting moieties, wherein each targeting moiety independently targets a receptor on the surface of the T cell selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, and CD28.
  • the targeted LNP comprises two or more targeting moieties, wherein at least a first targeting moiety targets CD3, and wherein at least a second targeting moiety targets a receptor on the surface of the T cell is selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, and CD28.
  • the first targeting moiety comprises a CD3-binding domain comprising a VH comprising a CDR-H1, a CDR-H2, a CDR-H3 of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab.
  • the first targeting moiety comprises a CD3-binding domain comprising the VH and the VL of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab.
  • Exemplary sequences for the first targeting moiety and/or the second targeting moiety can be found, e.g., in Tables 19, 20, 30, and 32.
  • the targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD5.
  • the targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD7.
  • the targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD28.
  • the targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD5 and a second targeting moiety binds to CD28.
  • the targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD7, and a second targeting moiety binds to CD28.
  • the CD28 targeting moiety is a CD80 extracellular domain (ECD).
  • a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD3.
  • the first targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the second targeting moiety that binds to CD3.
  • a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD5.
  • the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259
  • the second targeting moiety that binds to CD5 comprises a VH comprising the amino acid of SEQ ID NO:357, and a VL comprising the amino acid sequence of SEQ ID NO:358.
  • a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD2.
  • the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259
  • the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO:269, and a VL comprising the amino acid sequence of SEQ ID NO:270.
  • the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259
  • the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO:280, and a VL comprising the amino acid sequence of SEQ ID NO:281.
  • the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259
  • the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO:291, and a VL comprising the amino acid sequence of SEQ ID NO:292.
  • the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259
  • the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO:301, and a VL comprising the amino acid sequence of SEQ ID NO:292.
  • a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD7.
  • the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259
  • the second targeting moiety that binds to CD7 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 313, 324, 335, and 346, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 314, 325,
  • the first targeting moiety comprises a CD3-binding domain comprising the VH and the VL of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab
  • the second targeting moiety comprises a CD7-binding domain comprising the VH and the VL of grisnilimab.
  • a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD28.
  • the CD28 targeting moiety is a CD80 extracellular domain (ECD).
  • the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259
  • the second targeting moiety that binds to CD28 comprises the amino acid sequence of SEQ ID NO:365.
  • the first targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO:218 and a VL comprising the amino acid sequence of SEQ ID NO:219
  • the second targeting moiety that binds to CD28 comprises the amino acid sequence of SEQ ID NO:365.
  • a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD3.
  • the first targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the second targeting moiety that binds to CD3.
  • the first targeting moiety and the second targeting moiety each independently comprises a CD3-binding domain comprising the VH and the VL of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab, wherein the first targeting moiety comprises a different a CD3-binding domain than the second targeting moiety.
  • the first targeting moiety and the second targeting moiety each independently comprises a CD3-binding domain comprising a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, wherein the first targeting moiety comprises a different a CD3-binding domain than the second targeting moiety.
  • the first targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO:196 and a VL comprising the amino acid sequence of SEQ ID NO:197
  • the second targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO:218 and a VL comprising the amino acid sequence of SEQ ID NO:219.
  • the LNP generally includes a lipid that has polyethylene glycol (PEG) spacer functionalized with a reactive moiety such as a thiol, amine, maleimide or carboxylic acid group.
  • PEG polyethylene glycol
  • the functionalized lipid of the LNP reacts with a complementary group that is covalently bonded to a targeting moiety, hence generating a conjugate of the LNP and the targeting moiety.
  • the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the targeting moiety is an antibody or antigen binding fragment thereof.
  • the targeting moiety is a scFv. In some embodiments, the targeting moiety is a Fab fragment.
  • the Click product can be formed using a copper-catalyzed Click reaction.
  • One such copper-catalyzed Click reaction is a Huisgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne.
  • the first or second Click handle comprises a cyclic derivative of the alkynyl group.
  • the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne.
  • the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes.
  • the Click product can be formed using copper-free Click chemistry. For example, the Click product can be formed between an azide and dibenzocyclooctene (DBCO). Alternatively, the Click product can be formed using a Staudinger reaction between an azide and a phosphine, hence producing an aza-ylide.
  • the Click product can be formed from an inverse electron demand Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the first or second Click handle.
  • TCO trans-cyclooctene
  • the first Click handle comprises a tetrazine (Tz) ring and the second Click handle comprises a TCO moiety.
  • the tetrazine ring is unsubstituted.
  • the tetrazine rung is methyltetrazine.
  • the tetrazine ring is a 6-methyl substituted tetrazine.
  • the targeting moiety e.g., antibody, Fab fragment or scFv
  • an enzyme recognition sequence can site-specifically introduce the first Click handle onto the targeting moiety through covalent attachment.
  • the first Click handle can next react with the second Click handle on the LNP to produce the targeted LNP.
  • an antibody, Fab fragment or single chain variable fragment (scFv) that is covalently linked to a first Click handle through a linker comprising an enzyme recognition sequence is reacted with an LNP comprising a second Click handle, thereby forming a Click reaction product that conjugates the antibody, Fab fragment or scFv to the LNP.
  • the antibody Fab fragment or scFv is directly bonded to the enzyme recognition sequence.
  • the targeting moiety e.g., antibody Fab fragment or scFv
  • the enzyme recognition sequence via one or more amino acid residues.
  • Particular amino acid residues added that can be covalently attached to the C-terminus of the targeting moiety include, but are not limited to (GGGGS) v (SEQ ID NO:1), (G) v , (EAAAK) v (SEQ ID NO:3), (PAPAP) v (SEQ ID NO:4), (AP) v and A(EAAAK) u ALEA(EAAAK) v A (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.
  • the enzyme recognition sequence is a sortase recognition motif or an LplA acceptor peptide.
  • the C-terminus of one or more of the heavy or light chains of the antibody is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide) either directly or through a linker comprising one or more amino acid residues, a set forth herein.
  • the enzyme recognition sequence e.g., sortase recognition motif or LplA acceptor peptide
  • the C- terminus of the heavy or light chain of the Fab fragment is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide).
  • the C-terminus of the scFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide).
  • the first Click handle comprises a tetrazine ring or TCO moiety and the second Click handle comprises a tetrazine ring or TCO moiety.
  • the first Click handle comprises a tetrazine ring and the second Click handle comprises a TCO moiety.
  • the tetrazine ring is a methyltetrazine.
  • the conjugation efficiency achieved by the disclosed method is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the conjugation efficiency achieved by the disclosed method is from about 60% to about 95%. In some embodiments, the conjugation efficiency achieved by the disclosed methods are from about 70% to about 85%.
  • the linker further comprises a spacer between the targeting moiety and the Click product.
  • the spacer can include additional functional groups that indirectly link the targeting moiety to the Tz group.
  • the spacer may also include additional amino acid residues that indirectly links the targeting moiety to the Tz group.
  • the first or second Click handle is a tetrazine derivative having one of the following structures:
  • the first or second Click handle is a TCO derivative having one of the following structures:
  • the spacer that links the targeting moiety to the Tz ring is an enzyme recognition sequence. Accordingly, the disclosure provides methods of conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence, wherein said conjugating is accomplished via a Click reaction between a Tz ring covalently bound to the targeting moiety and a TCO moiety bound to the LNP.
  • the disclosure provides methods of conjugating an LNP to an antibody, Fab fragment or single chain variable fragment (scFv), wherein the antibody, Fab fragment or scFv is covalently linked to a first Click handle (Tz ring) through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle (TCO moiety), said method comprising contacting the LNP with an antibody, Fab fragment or scFv such that first Click handle reacts with the second Click handle to form a Click reaction product (dihydropyridazine) that conjugates the antibody, Fab fragment or scFv to the LNP.
  • a Click reaction product dihydropyridazine
  • the antibody, Fab fragment or scFv is directly bonded to the enzyme recognition sequence.
  • the antibody Fab fragment or scFv is bonded to the enzyme recognition sequence via one or more amino acid residues.
  • Particular amino acid residues added that can be covalently attached to the C-terminus of the antibody, Fab fragment or scFv include, but are not limited to (GGGGS) v (SEQ ID NO:1), (G) v , (EAAAK) v (SEQ ID NO:3), (PAPAP) v (SEQ ID NO:4), (AP) v and A(EAAAK) u ALEA(EAAAK) v A (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.
  • the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide.
  • the C-terminus of one or more of the heavy or light chains of the antibody is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide) either directly or through a linker comprising one or more amino acid residues, a set forth herein.
  • the C- terminus of the heavy or light chain of the Fab fragment is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide).
  • the C-terminus of the scFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide).
  • the conjugation efficiency achieved by the disclosed method is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.
  • lipid nanoparticles conjugated to an antibody, Fab fragment or scFv in a site-specific manner are produced according to the following steps: (i) covalently bonding a peptide with a sortase recognition site to one or more C- terminus of the antibody or Fab fragment, or to the C-terminus of the scFv; (ii) contacting the product of step (i) with a first Click handle comprising a Tz ring covalently bound to three or more glycine residues in the presence of a sortase enzyme under conditions suitable for the sortase enzyme to ligate the antibody, Fab fragment or scFv to the first Click handle; (iii) contacting the product of step (ii) with a lipid nanoparticle (LNP), where
  • step (i) involves covalently bonding the peptide with the sortase recognition site to a linker covalently bonded to one or more C-terminus of the antibody or Fab fragment, or to the C-terminus of the scFv.
  • the linker comprises one or more amino acid residues.
  • Particular amino acid residues added that can be covalently attached to the C-terminus of the antibody, Fab fragment or scFv include, but are not limited to (GGGGS) v (SEQ ID NO:1), (G) v , (EAAAK) v (SEQ ID NO:3), (PAPAP) v (SEQ ID NO:4), (AP) v and A(EAAAK) u ALEA(EAAAK) v A (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.
  • the sortase recognition motif bonded to the C-terminus of the antibody, Fab fragment or scFv is bonded to one or more amino acid residues.
  • the glycine (G) of the LPXTG motif (SEQ ID NO:8) can be bonded to one or more (e.g., between 1-10) histidine (H) residues (see FIG.16). The glycine and the histidine residues are removed during the transpeptidation reaction.
  • step (ii) i.e., the sortase-catalyzed reaction
  • Sortase A5 is an engineered pentamutant variant of the wild-type sortase from Staphylococcus aureus that is significantly more active than the wild-type sortase. See U.S.
  • the sortase recognition site bond to the antibody, Fab fragment or scFv in step (i) has the sequence LPXTG (SEQ ID NO:8), where X is any amino acid residue.
  • sortase A catalyzes the transpeptidation reaction between the LPXTG recognition motif (SEQ ID NO:8) and a glycine residue bound to the first Click handle by cleaving the sortase recognition site between the threonine and the glycine residues.
  • step (ii) is carried out in the presence of the sortase enzyme Streptococcus pyogenes sortase A (SpSrtA WT).
  • the sortase recognition site bond to the antibody, Fab fragment or scFv in step (i) has the sequence LPXTA (SEQ ID NO:9), where X is any amino acid residue.
  • SpSrtA catalyzes the transpeptidation reaction between the LPXTA recognition motif (SEQ ID NO:9) and a glycine residue bound to the first Click handle by cleaving the sortase recognition site between the threonine and the alanine residues.
  • FIG.16 shows the formation of an LNP conjugated to the C-terminus of a Fab fragment through a sortase mediated ligation followed by a Click reaction.
  • a C-terminus of the Fab fragment is first covalently modified with a sortase recognition motif covalently bonded to six histidine (H) residues.
  • the sortase modified Fab fragment is then reacted with a first Click handle (Click handle 1) covalently bonded to glycine (G) residues.
  • the resultant Fab fragment modified with the first Click handle is then reacted with an LNP that comprises at least one lipid that is covalently bonded to a second Click handle (Click handle 2), thus affording an LNP site-specifically conjugated to the Fab fragment.
  • the conjugate comprises a protein targeting moiety as set forth above (e.g., antibody, Fab fragment or scFv) and a lipid nanoparticle (LNP), wherein a C- terminus of the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a sortase recognition motif and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • a protein targeting moiety as set forth above (e.g., antibody, Fab fragment or scFv) and a lipid nanoparticle (LNP), wherein a C- terminus of the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a sortase recognition motif and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the C-terminus of one or more of the heavy or light chains of the antibody, the C-terminus of the heavy or light chain of the Fab fragment, or C-terminus of the scFv is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a sortase recognition motif and a Click product formed from a Click reaction between a first Click handle on the antibody, Fab fragment, or scFv and a second Click handle on the LNP.
  • the sortase recognition motif comprises an LPXT motif, wherein X is any amino acid residue.
  • the sortase recognition motif comprises an LPET motif (SEQ ID NO:10).
  • the linker comprises 3 or more glycine residues between the sortase recognition motif and the Click product. In some embodiments, the linker comprises between 3 and 50 glycine residues. In some embodiments, the linker comprises between 3 and 10 glycine residues. In some such embodiments, the linker comprises 3, 4, 5, or 6 glycine residues.
  • the conjugates have the structure Antibody-LPXT(G)n-Click product-LNP, Fab fragment- LPXT(G) n -Click product-LNP, or scFv- LPXT(G) n -Click product- LNP, wherein X is any amino acid residue and n is between 1 and 20 (e.g., between 3 and 10 or between 3 and 6).
  • the leucine (L) residue of the sortase recognition motif is bonded to at least one C–terminus of the antibody or Fab fragment or to the C-terminus of the scFv.
  • X is E.
  • the linker between the antibody (or Fab fragment or scFv) and the Click includes the following amino acid residues: LPETGGG (SEQ ID NO:12), LPETGGGG (SEQ ID NO:13), LPETGGGGG (SEQ ID NO:14), LPETAGGG (SEQ ID NO:15), or LPETGGGGGG (SEQ ID NO:16).
  • the linker comprises additional amino acid residues between the antibody, Fab fragment or scFv and the sortase recognition motif.
  • a C- terminus of the antibody, Fab Fragment or scFv can be covalently modified with one or more amino acid residues prior to covalently linking the sortase recognition motif.
  • the conjugates have the structure Antibody-Z-LPXT(G)n-Click product- LNP, Fab fragment-Z-LPXT(G) n -Click product-LNP, or scFv-Z-LPXT(G) n -Click product-LNP, wherein Z is a linker between the antibody (or Fab fragment or scFv) and the leucine of the sortase recognition motif.
  • Z comprises one or more amino acid residues.
  • Z is (GGGGS) v (SEQ ID NO:1), (G) v , (EAAAK) v (SEQ ID NO:3), (PAPAP) v (SEQ ID NO:4), (AP) v and A(EAAAK) u ALEA(EAAAK) v A (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.
  • Z is GG, GGG, GGGG (SEQ ID NO:18), GGGGG (SEQ ID NO:19), GGGGGG (SEQ ID NO:20), and GGGGGGG (SEQ ID NO:21) or GGGGGS (SEQ ID NO:22).
  • the conjugates have the structure Antibody-LPXT(G) n - dihydropyridazine-LNP, Fab fragment- LPXT(G)n- dihydropyridazine -LNP, or scFv- LPXT(G) n - dihydropyridazine -LNP, Antibody-Z-LPXT(G) n - dihydropyridazine-LNP, Fab fragment-Z-LPXT(G) n - dihydropyridazine -LNP, or scFv-Z-LPXT(G) n - dihydropyridazine - LNP, wherein variables n and Z are defined as above.
  • the dihydropyridazine moiety is a 1,4- dihydropyridazine.
  • the LNP lipid is conjugated to an antibody, Fab fragment or scFv in a site-specific manner, comprising (i) covalently bonding a LplA acceptor peptide site to the antibody, Fab fragment or scFv; (ii) contacting the product of step (i) with a carboxylic acid compound that includes a first Click handle comprising a Tz ring in the presence of a lipoic acid ligase under conditions suitable for lipoic acid ligase to ligate the antibody, Fab fragment or scFv to the first Click handle; and (iii) contacting the product of step (ii) with a lipid nanoparticle (LNP), wherein one or more lipids comprising the LNP includes a second Click handle capable of reacting with the first Click handle, wherein the second Click handle comprises
  • step (i) the Lp1A acceptor peptide is covalently bonded to one or more C-terminus of the antibody or Fab fragment. In other embodiments of step i) the Lp1A acceptor peptide is covalently bonded to the C-terminus of the scFv. [0234] In some embodiments, step (i) involves covalently bonding the peptide with the Lp1A acceptor peptide to a linker covalently bonded to one or more C-terminus of the antibody or Fab fragment, or to the C-terminus of the scFv. In some embodiments, the linker comprises one or more amino acid residues.
  • step (ii) i.e., the lipoic acid ligase catalyzed reaction
  • step (ii) is carried out in the presence of a mutated lipoic acid ligase, wherein the tryptophan reside (W) at position of the lipoic acid ligase is mutated.
  • W37 mutants are described in Cohen et al., 2012, ChemBioChem, 13, 888-894.
  • the mutant lipoic acid ligase is selected from W37V, W37I, W37T, W37L, W37C, W37A, and W37G.
  • the carboxylic acid in step (ii) is a C3-C20 carboxylic acid.
  • the carboxylic acid in step (ii) is a fatty acid, for instance a C 7 -C 19 fatty acid.
  • the fatty acid is selected from decanoic acid, palmitic acid, lauric acid or octanoic acid.
  • FIG. 17 depicts the formation of an LNP conjugated to the C-terminus of a Fab fragment through a LplA ligation followed by a Click reaction.
  • a C-terminus of the Fab fragment is first covalently modified with a LplA acceptor peptide.
  • a first Click handle (Click handle 1) comprising a Tz ring is first introduced by chemically reacting a carboxylic acid to the lysine residue of the LplA acceptor peptide.
  • the resultant Fab fragment modified with the first Click handle is then reacted with an LNP that comprises at least one lipid that is covalently bonded to a second Click handle comprising a TCO moiety (Click handle 2), thus affording an LNP site-specifically conjugated to the Fab fragment.
  • the conjugate produced by methods disclosed herein comprises a protein targeting moiety as set forth above (e.g., antibody, Fab fragment or scFv), conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a lipoic acid ligase (LplA) acceptor peptide and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the linker further comprises one or more additional amino acid residues between the protein targeting moiety (e.g., antibody, Fab fragment or scFv) and the LplA acceptor peptide.
  • the LplA acceptor peptide has the sequence GFEDKVWYDLDA (SEQ ID NO:23).
  • the conjugate comprises an antibody, wherein the C-terminus of one or more of the heavy or light chains of the antibody is bonded to the linker.
  • the conjugate comprises a Fab fragment, the C- terminus of the heavy or light chain of the Fab fragment is bonded to the linker.
  • the conjugate comprises a scFv, wherein the C-terminus of the scFv is bonded to the linker.
  • the linker comprises additional amino acid residues between the targeting moiety (e.g., antibody, Fab fragment or scFv) and the LplA acceptor peptide.
  • a C-terminus of the targeting moiety e.g., antibody, Fab Fragment or scFv
  • the conjugates have the structure targeting moiety-Z-LplA acceptor peptide-Click product-LNP (e.g., Antibody-Z- LplA acceptor peptide -Click product-LNP, Fab fragment-Z- LplA acceptor peptide -Click product- LNP, or scFv-Z- LplA acceptor peptide -Click product-LNP), wherein Z is a linker between the antibody (or Fab fragment or scFv) and the glycine residue of the LplA acceptor peptide.
  • Z comprises one or more amino acid residues.
  • Z is (GGGGS) v (SEQ ID NO:1), (G) v , (EAAAK) v (SEQ ID NO:3), (PAPAP) v (SEQ ID NO:4), (AP) v and A(EAAAK) u ALEA(EAAAK) v A (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.
  • Z is GG, GGG, GGGG (SEQ ID NO:18), GGGGG (SEQ ID NO:19), GGGGGG (SEQ ID NO:20), and GGGGGGG (SEQ ID NO:21) or GGGGGS (SEQ ID NO:22).
  • the lysine (K) residue of the LplA acceptor peptide is covalently linked to Click product, which is covalently linked to the LNP.
  • the side chain lysyl group reacts with the carboxylic acid compound that includes the first Click handle.
  • the resultant modified targeting moieties (antibodies or Fab fragments or scFvs) are reacted with an LNP that has been modified with a second Click handle, as disclosed herein, thereby generating a Click product.
  • LNP surface modified with a Fab fragment is depicted below, where R is a lipid group (e.g., C 2 -C 30 alkyl group) and the LplA accepter peptide comprises the sequence of GFEIDKVWYDLDA (SEQ ID NO:24).
  • the enzyme recognition sequence is a transglutaminase enzyme recognition sequence (LLQG, SEQ ID NO:25).
  • the transglutaminase enzyme recognition sequence (LLQG, SEQ ID NO:25) is also referred to as Q-tag.
  • the Q-tag may be present on or can be inserted at one or more locations of targeting moiety, (e.g., antibody, Fab fragment or scFv), for instance at a C-terminus.
  • the transglutamine enzyme catalyzes the reaction between a side-chain amide group on the Q-tag and an alkyl-primary amine on a component of the LNP (e.g., a lipid), thus linking the antibody to the LNP through an amide bond.
  • the enzyme recognition sequence is a sequence recognized by formylglycine generating enzyme, specifically CXPXR, wherein each X is any amino acid.
  • the CXPXR sequence can be inserted at one or more locations of the targeting moiety (e.g., antibody, Fab fragment or scFv), for instance at a C-terminus.
  • the formylglycine generating enzyme converts the cysteine thiol of CXPXR into an aldehyde group, which can be reacted with an aminooxy or hydrazine group covalently bonded to a component of the LNP.
  • the lipid nanoparticle is conjugated to the targeting moiety, e.g., antibody, Fab fragment or scFv, in a site-specific manner through a sugar moiety on a glycosylated antibody.
  • FIG.18 shows one particular embodiment of introducing a Tz Click handle through a sugar moiety on an antibody.
  • an azide moiety is site-specifically introduced into a sugar moiety of the targeting antibody catalyzed by the enzymes galactosidase and transferase.
  • the azide functionality then reacts with a dibenzocyclooctene (DBCO)-MeTz via a Click reaction.
  • DBCO dibenzocyclooctene
  • the Click reaction is used to introduce the Tz ring, in this case MeTz onto the antibody, which can then be reacted with a TCO moiety on an LNP (not shown) in a second Click reaction, hence affording a conjugate.
  • the LNP is conjugated to a targeting moiety (e.g., antibody, Fab fragment or scFv) in a site-specific manner through a light-induced crosslinking reaction.
  • a targeting moiety e.g., antibody, Fab fragment or scFv
  • FIG.19 shows one particular embodiment using oYo-Link Tz.
  • a Tz ring e.g., MeTz
  • oYo link site specific antibody label
  • the oYo-Link tetrazine covalently bound to the antibody reacts with TCO (not shown) bound to an LNP, hence generating the surface- modified LNP.
  • the lipid nanoparticle is conjugated to the targeting moiety (e.g., antibody, Fab fragment or scFv) in a site-specific manner by mutating an amino acid residue on the targeting moiety and subsequently reacting the mutated targeting moiety with a compound that includes a Tz ring.
  • FIG.20 shows particular embodiments where an azide functionality is introduced during antibody or Fab fragment production. The azide group then undergoes a Click reaction with a DBCO group that is linked to a tetrazine ring. The tetrazine ring bound to the antibody reacts with TCO (not shown) bound to an LNP, hence generating the surface modified LNP.
  • the lipid nanoparticle is conjugated to the targeting moiety (e.g., antibody, Fab fragment or scFv) in a site-specific manner by using a cysteine-maleimide reaction to introduce the first Click handle onto the targeting moiety (antibody, Fab fragment or scFv).
  • FIG.21 shows one particular embodiment of introducing a first Click handle (e.g., Tz) onto a Fab fragment.
  • a hinge with a free cysteine is first introduced at a C-terminus of the Fab fragment.
  • an engineered inter-chain disulfide bound away from the C-terminus and buried in the CL-CH1 interface is introduced for improved stability (FIG.22).
  • the resultant modified Fab fragment is reacted with a maleimide moiety with a first Click handle (Click handle 1) covalently bonded to the maleimide nitrogen.
  • the first Click handle than reacts with a second Click handle (Click handle 2) on the LNP, hence generating the surface modified targeted LNP.
  • Click handle 1 is a TZ and Click handle 2 is a TCO moiety
  • the Click product can be formed using any suitable photo- induced Click chemistry reaction.
  • the Click product can be formed using photoinducible 1,3-dipolar cycloaddition reaction between a tetrazole and an alkene (see, e.g., Song et al., Angew.
  • the Click product can be formed using oxime and hydrazone hydroxylamine, hydrazine and hydrazide (see, e.g., Agten et al., ChemBioChem 2013, 14 (18), 2431-2434 and Dirksen et al., J. Am. Chem. Soc.2006, 128 (49), 15602-15603).
  • Exemplary lipids and cholesterol molecules bonded to Click handles that can be used to make targeted LNPs are depicted below.
  • IgG antibodies consist of four polypeptide chains linked by disulfide bonds.
  • the two polypeptide chains of low molecular weight are call light chains (L).
  • the light chains consist of a variable light chain domain (VL) and a constant light chain domain (C L ).
  • the heavy chains consist of a variable heavy light domain (VH) and three constant heavy chain domains (C H 1, C H 2, and C H 3).
  • the Fab region of the antibody includes the V L , C L , V H , and C H 1 domains.
  • the Fc region includes the constant heavy chain domains C H 2, and C H 3.
  • a hinge region of the IgG antibody covalently links the C H 1 domain to the C H 2 domain.
  • the two heavy chains of IgG antibodies are connected in the hinge region by a variable number of disulfide bonds depending on the IgG subclass. Different subclasses of IgG antibodies have varying numbers of interchain disulfide bonds. Additionally, the light chain is covalently linked to the heavy chain via a disulfide bond between the light chain and the heavy chain. Using standard IgG nomenclature, this natural interchain disulfide bond is also referred to as the C L -C H 1 disulfide bond to distinguish it from disulfide bonds present in the hinge region.
  • Therapeutic antibodies of type IgG1 possess an intermolecular disulfide bond between Cys233 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.
  • Therapeutic antibodies of type IgG4 possess an intermolecular disulfide bond between Cys127 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.
  • Therapeutic antibodies of type IgG2 possess an intermolecular disulfide bond between Cys135 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.
  • F(ab’) 2 fragment Fab fragment known as a F(ab’) 2 fragment.
  • the F(ab’) 2 fragment does not include the C H 2 domain or the C H 3 domain. However, the hinge region of the antibody is retained in a F(ab’) 2 fragment.
  • the F(ab’)2 fragment includes disulfide bonds that covalently link two Fab fragments. Reduction of the disulfide bond in the F(ab’)2 generates two F(ab’) fragments.
  • the sulfhydryl (thiol) groups of the F(ab’) could potentially react with a thiol-reactive group on the surface of an LNP, hence generating a conjugate.
  • site-specific conjugation is challenging.
  • the reduction of the F(ab’) 2 to the F(ab’) fragments could also disrupt the natural interchain disulfide bonds between the C L and C H 1 regions of the Fab fragments, hence further compromising site- specific conjugation.
  • Fab fragment e.g., a Fab fragment that binds to CD164
  • LNP precursor lipid nanoparticle
  • the term “precursor LNP” or “base LNP” refers to an LNP that has been functionalized with a reactive moiety (e.g., thiol-reactive group or polyglycine) prior to reacting with the Fab fragment.
  • the process for conjugating a targeting moiety involves reducing the natural interchain disulfide bond between the C L and C H 1 domains of a Fab fragment, and reacting the reduced Fab fragment with a thiol-reactive group (e.g., a maleimide or DBM group) covalently bonded to the surface of a precursor LNP, thus forming a conjugate.
  • a thiol-reactive group e.g., a maleimide or DBM group
  • the reduced Fab fragment can be reacted with a lipid that has been chemically modified (functionalized) with a thiol-reactive group (e.g., maleimide or DBM group).
  • the resultant lipid can then be inserted into a preexisting LNP, thus generating a conjugate.
  • Fab fragments used for conjugation may be used by recombinant methods.
  • the Fab fragments generated recombinant are designed not to include a hinge region at the C-terminus.
  • the recombinantly generated Fab fragments include only one disulfide bond between the C L -C H 1 and domains.
  • the C L -C H 1 can then be reduced and the resultant free thiol groups can be used as anchors to conjugate the Fab fragment to the surface of an LNP.
  • the Fab fragment is of the IgG class, the IgM class, or the IgA class.
  • the Fab fragment is of the IgG class and has an IgGl, IgG2, IgG3, or IgG4 isotype.
  • the Fab fragment is a native protein.
  • the Fab fragment is an engineered protein.
  • the disclosure provides methods of making a targeted LNP, said method comprising: (i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (C L ) and the constant heavy chain domain 1 (C H 1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.
  • the disclosure provides a conjugate produced by a method comprising: (i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (C L ) and the constant heavy chain domain 1 (C H 1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.
  • the thiol-reactive group e.g., maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl
  • a lipid molecule to create a modified lipid wherein the thiol-reactive group is covalently attached to the lipid where it is capable of reacting with at least one free cysteine residue of the reduced Fab fragment (either on the heavy or light chain of the Fab fragment).
  • the reaction between the thiol-reactive group and the at least one free cysteine residue can be completed prior to or after formation of the LNP with the modified lipid.
  • the various components comprising the LNP and a therapeutic payload can be mixed with lipid molecules, including one or more lipids that comprise a thiol-reactive group, thus generating an LNP that comprises a plurality of thiol-reactive groups (each thiol-reactive group shown schematically as a “functional group” in FIG.40A).
  • the thiol-reactive group can then be reacted with at least one free cysteine residue of the Fab fragment, hence generating a conjugate.
  • a lipid that has been modified with the thiol-reactive group can be directly reacted with at least one free cysteine residue of a Fab fragment.
  • the resultant modified lipid attached to the Fab fragment can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.
  • Any suitable reducing reagent can be used to reduce the interchain disulfide bond of the Fab fragment. Examples of reducing reagents include, but are not limited to, 2- mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP), and combinations thereof.
  • the reducing reagent is a mild reducing reagent.
  • mild reducing reagents include, e.g., DTT, TCEP, and DTE.
  • the reducing reagent is TCEP.
  • Any suitable reaction conditions can be used for the reduction of the interchain disulfide bond in step (i).
  • the reduction reaction can occur in water, aqueous buffer, or cell culture media.
  • the reduction reaction is performed at physiological pH (e.g., about 7.4).
  • the reduction reaction is performed at physiological temperature (e.g., about 37° C).
  • the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reduction reaction is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the reduction reaction is performed at about 0° C to about 4° C. [0261] In some embodiments, excess reducing agent is removed following step (i), prior to conjugation to the precursor LNP. In some embodiments, excess reducing agent is not removed following step (i), prior to conjugation to the precursor LNP.
  • the thiol-reactive groups on the LNP comprises any suitable reactive group, including but not limited to, maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl.
  • the thiol-reactive group is maleimide.
  • maleimide reacts with one of the two free cysteine residues of the Fab fragment (either on the heavy or light chain) to form a thiosuccinimide moiety.
  • maleimide reacts with a free cysteine residue on the heavy chain of the Fab fragment.
  • maleimide reacts with a free cysteine residue on the light chain of the Fab fragment.
  • two maleimide groups each react with the Fab fragment, wherein one maleimide reacts with a free cysteine residue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.
  • Any suitable conditions can be used for the reaction between the thiol-reactive group and at least one of the two free cysteine residues of the Fab fragment in step (ii).
  • the reaction can occur in water, aqueous buffer, or cell culture media.
  • the reaction is performed at physiological pH (e.g., about 7.4).
  • the reaction is performed at physiological temperature (e.g., about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reaction is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the reaction is performed at about 0° C to about 4° C. [0265] A schematic showing the reduction of an interchain disulfide bond in a Fab fragment is depicted in FIG.41.
  • the Fab fragment depicted in FIG.41 can be produced recombinantly without a hinge region at the C-terminus. Accordingly, in such embodiments, the Fab fragment only comprises a single interchain disulfide bond, which is located in the C L -C H 1 interface of the Fab fragment. As shown in FIG.41, the interchain disulfide bond is located between the heavy chain and the light chain of the Fab fragment. The reduction reaction breaks the covalent linkage forming the disulfide bond, thereby generating two free cysteine residues that can react in a subsequent step with a thiol-reactive group. [0266] FIG.42 shows an exemplary schematic of conjugate formation, as described herein.
  • a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)).
  • the reduced Fab fragment is reacted with an LNP comprising a plurality of thiol-reactive groups (e.g., maleimide or DBM) conjugated to the surface of the LNP, whereby the thiol-reactive groups react with the free cysteine residues of the reduced Fab fragment.
  • the Fab fragment is site-specifically conjugated to the surface of the LNP via a linkage through at least one of the free cysteine residues of the Fab fragment.
  • the heavy chain, light chain or both the heavy chain and light chain of the Fab fragment are conjugated to the surface of the LNP.
  • the various orientations are depicted in FIG. 43.
  • the conjugates formed by methods disclosed herein include at least one, at least two or all three orientations shown in FIG. 43.
  • the concentration of the thiolreactive group e.g., maleimide
  • increasing the number of thiol -reactive groups on the LNP increases the number of Fab fragments conjugated to two thiol -reactive groups.
  • decreasing the number of thiol -reactive groups on the LNP decreases the number of Fab fragments conjugated to two thiol -reactive groups. Regardless of the orientation, the heavy chain and the light chain remain intact on the surface of the LNP, thus forming a functional Fab fragment that is capable of engaging with a receptor on a targeted cell.
  • the thiol -reactive group is maleimide, as shown in FIG. 43.
  • maleimide reacts with one of the two free cysteine residues of the antibody or antigen-binding fragment thereof.
  • maleimide reacts with one of the two free cysteine residues of the Fab fragment to form a thiosuccinimide moiety.
  • maleimide reacts with a free cysteine residue on the heavy chain of the Fab fragment.
  • maleimide reacts with a free cysteine residue on the light chain of the Fab fragment.
  • two maleimide groups on the LNP each react with the Fab fragment, wherein one maleimide reacts with a free cysteine residue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.
  • the thiol -reactive group is 2,3-dibromomaleimide (DBM) as shown in FIG. 44.
  • DBM 2,3-dibromomaleimide
  • the reduced Fab fragment is added to DBM covalently bonded to a lipid (represented by squiggly line in FIG. 44).
  • the lipid may be part of an LNP or may be post-inserted into an LNP following reaction with the Fab fragment. Both of the free cysteine residues displace the two bromine groups of DBM, hence generating a dithiomalemide.
  • the dithiolmalemide can be converted to the corresponding maleamic acid via hydrolysis.
  • the thiol-reactive group can be introduced onto any of the lipids comprising the LNP.
  • the conjugate can comprise one or more pegylated lipid molecules.
  • the thiol-reactive group is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid- PEGx-thiol-reactive group, wherein x is 2-120 ethylene glycol units.
  • at least one free cysteine residue of a Fab fragment reacts with the thiol-reactive group bonded to the one or more of the pegylated lipids comprising the LNP.
  • the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the thiol- reactive group.
  • the PEG spacer between the lipid and the thiol-reactive group comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000).
  • the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300).
  • the thiol-reactive group is bonded to at least one of the non-pegylated phospholipids comprising the LNP.
  • the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP.
  • the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP.
  • the lipid portion of the pegylated lipid bonded to the thiol-reactive group is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.
  • FIG.45 shows some examples of a pegylated lipid bonded to a thiol-reactive group, in this case a maleimide moiety.
  • the thiol-reactive group is bonded to at least one of the non- pegylated phospholipids (helper lipids) comprising the LNP.
  • FIG.46 shows some examples of a non-pegylated lipid bonded to a maleimide moiety.
  • the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP.
  • FIG.47 shows some examples of an ionizable lipid bonded to a maleimide moiety.
  • the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP.
  • FIG.48 shows some examples of sterols bonded to a maleimide moiety.
  • the disclosed methods provide stable conjugates that display excellent ability to transduce specific targeted cells.
  • the disclosure provides a conjugate comprising an LNP and a Fab fragment, wherein the LNP is covalently bonded to either or both a first cysteine residue in the constant region of the heavy chain of the Fab fragment and a second cysteine residue in the constant region of light chain of the Fab fragment.
  • the Fab fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Fab fragment and the constant region of the light chain of the Fab fragment.
  • both the constant region of the heavy chain constant region of the heavy chain and the constant region of the light chain of the Fab fragment are covalently bonded to the LNP.
  • the Fab fragment is linked to the LNP through a thiosuccinimide moiety. In other of the foregoing embodiments, the Fab fragment is linked to the LNP through a dithiomalemide moiety In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through a maleamic acid moiety.
  • the Fab fragment conjugated to the LNP is an IgGl Fab fragment.
  • the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab fragment is covalently bonded to the LNP.
  • cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment are covalently bonded to the LNP.
  • the Fab fragment conjugated to the LNP is an IgG2 Fab fragment.
  • the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently bonded to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment are covalently bonded to the LNP.
  • the Fab fragment conjugated to the LNP is an IgG4 Fab fragment.
  • the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently to the LNP.
  • the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently to the LNP.
  • the processes described herein also enable the ability to conjugate two or more different Fab fragments to the surface of an LNP.
  • An embodiment showing the conjugation of two different Fab fragments (which can bind to the same or different antigen) to the surface of an LNP is shown in FIG.45.
  • two Fab fragments (Fab1 and Fab2) are reduced (step (i)) and reacted (step (ii)) with a precursor LNP comprising a thiol-reactive group (e.g., maleimide or DBM).
  • a precursor LNP comprising a thiol-reactive group (e.g., maleimide or DBM).
  • both Fab1 and Fab2 are conjugated to the surface of the LNP.
  • the heavy chain and light chain in Fab1 and the heavy and light chain in Fab2 remain together on the surface of the LNP. In other words, neither the heavy chain or light chain of Fab1 associate with the heavy or light chain of Fab2 on the surface of the LNP.
  • the first Fab fragment and the second Fab fragment can be reduced in the same reaction (e.g., the first and second Fab fragments are mixed in a reaction vessel and contacted with the same reducing reagent).
  • the first Fab fragment and the second Fab fragment are reduced separately (e.g., the first and second Fab fragments thereof are each contacted with a reducing reagent in separate reaction vessels).
  • the first Fab fragment is contacted with the reducing reagent prior to step (ii) (wherein the reduced first Fab fragment is conjugated to the LNP surface).
  • the second Fab fragment is contacted with the reducing reagent after step (ii) (wherein the reduced second Fab fragment is conjugated to the LNP surface).
  • the reduced first Fab fragment and the reduced second Fab fragment are contacted with the LNP simultaneously.
  • the reduced first Fab fragment thereof and the reduced second Fab fragment are contacted with the LNP sequentially (in either order). It can be contemplated that any number of Fab fragments thereof can be implemented in the method or process (e.g., a third, fourth, fifth, etc. Fab fragment). In some embodiments, a total of three different Fab fragments can be conjugated to the surface of the LNP.
  • a total of four different Fab fragments can be conjugated to the surface of the LNP.
  • the reaction between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP forms at least one covalent bond.
  • the formation of at least one covalent bond between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is reversible.
  • the formation of at least one covalent bond between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is irreversible.
  • the reaction efficiency between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is greater than 5%, greater than 10%, greater than 25%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the reaction efficiency between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is from about 5% to about 30%, about 10% to about 20%, about 25% to about 50%, about 30% to about 40%, about 50% to about 80%, about 60% to about 70%, about 70% to about 95%, or about 80% to about 90%.
  • the conjugate product of the disclosed method can be purified from remaining intermediate product using any suitable technique such as, but not limited to, ultrafiltration and diafiltration.
  • conjugates prepared by the method or process disclosed herein have a high density of the Fab fragment on the surface of the LNP.
  • the conjugate can comprise a plurality of Fab fragments conjugated to the LNP surface.
  • the conjugate can comprise more than 10 Fab fragments per LNP.
  • the conjugate can comprise more than 20 Fab fragments per LNP.
  • the conjugate can comprise more than 30 Fab fragments.
  • the conjugate can comprise more than 50 Fab fragments per LNP.
  • the conjugate can comprise more than 75 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 100 Fab fragments. In some embodiments, the conjugate can comprise from about 50 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 Fab fragments per LNP.
  • Conjugates prepared by site-specific methods have a high density of the targeting moiety on the surface of the LNP.
  • the conjugate can comprise more than one targeting moiety (e.g., antibody, Fab fragment or scFv) per LNP.
  • the conjugate can comprise more than 10 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP.
  • the conjugate can comprise more than 20 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP.
  • the conjugate can comprise more than 30 targeting moieties (e.g., antibodies, Fab fragments or scFvs). In some embodiments, the conjugate can comprise more than 50 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 75 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 100 targeting moieties (e.g., antibodies, Fab fragments or scFvs).
  • targeting moieties e.g., antibodies, Fab fragments or scFvs.
  • the conjugate can comprise from about 50 to about 200 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP.
  • targeting moieties e.g., antibodies, Fab fragments or scFvs
  • the conjugate can comprise from about 10 to about 30 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP.
  • the weight ratio between a targeting moiety on the surface of the LNP and the payload (e.g., RNA) encapsulated in the LNP can be 1:20, 1:15, 1:10, 1:9, 1:8, 1:7.1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1.
  • the conjugates prepared by site-specific methods e.g., FIGs.
  • the conjugate 16-23 and 39-49 can have two or more different targeting moieties on the surface of the LNP.
  • the conjugate can have two different targeting moieties.
  • the conjugate can have three different targeting moieties.
  • the conjugate can have two different targeting moieties, wherein the mass ratio of the two targeting moieties on the surface of the LNP is 20:1, 15:1, 10:1, 9:1, 8:1, 7:1.6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, or 1:20.
  • the weight ratio between the one or more targeting moieties on the surface of the LNP and the payload (e.g., RNA) encapsulated in the LNP can be 1:20, 1:15, 1:10, 1:9, 1:8, 1:7.1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1.
  • Lipids that can be used in nanoparticle formations include, for example, those described in Table 4 of WO2019217941, which is incorporated by reference—e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in table 4 of WO2019217941.
  • Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in table 5 of WO2019217941, incorporated by reference.
  • conjugated lipids when present, can include one or more of PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distea
  • DAG PEG-di
  • sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009/127060 or US2010/0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference. [0289]
  • the lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties.
  • the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 35 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids.
  • the ratio of total lipid to nucleic acid can be varied as desired.
  • the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1.
  • the average LNP diameter of the targeted LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS).
  • the average LNP diameter of the targeted LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.
  • the average LNP diameter of the targeted LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm.
  • the average LNP diameter of the targeted LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the targeted LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation ranges from about l mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.
  • An LNP described herein e.g., a targeted LNP, may, in some instances, be relatively homogenous.
  • a polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles.
  • a small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • a LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.
  • the polydispersity index of a LNP may be from about 0.10 to about 0.20.
  • the zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of a LNP.
  • the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV
  • the efficiency of encapsulation of a protein and/or nucleic acid describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with an LNP after preparation, relative to the initial amount provided.
  • the encapsulation efficiency is desirably high (e.g., close to 100%).
  • the encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents.
  • An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution.
  • Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution.
  • the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the encapsulation efficiency may be at least 80%.
  • the encapsulation efficiency may be at least 90%.
  • the encapsulation efficiency may be at least 95%.
  • An LNP of the disclosure may optionally comprise one or more coatings.
  • an LNP may be formulated in a capsule, film, or tablet having a coating.
  • a capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density.
  • Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.
  • in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio).
  • LNPs are formulated using the GenVoy ILM ionizable lipid mix (Precision NanoSystems).
  • targeted LNPs of the disclosure are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.
  • the LNPs of the disclosure comprise one or more ionizable lipids.
  • an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated.
  • the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.
  • Exemplary cationic lipids include one or more amine group(s), which bear the positive charge.
  • the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol and polymer conjugated lipids.
  • the cationic lipid may be an ionizable cationic lipid.
  • An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0.
  • a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid.
  • a lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid, and a therapeutic agent as described herein (e.g., one or more nucleic acids (e.g., RNA) comprising a gene modifying system) encapsulated within or associated with the lipid nanoparticle.
  • the therapeutic agent e.g., one or more nucleic acids
  • the therapeutic agent is co-formulated with the cationic lipid.
  • the therapeutic agent may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid.
  • the therapeutic agent e.g., one or more nucleic acids
  • the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent.
  • the LNP formulation is biodegradable.
  • a lipid nanoparticle comprising one or more lipid described herein encapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule (e.g., an mRNA molecule).
  • an RNA molecule e.g., an mRNA molecule
  • the lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1 : 1 to about 25: 1, from about 10: 1 to about 14: 1, from about 3 :1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.
  • the amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher.
  • the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg/ml to about 30 mg/mL.
  • the LNP (e.g., targeted LNP) comprises the ionizable lipid V003, depicted below. V003 is described in U.S. Patent No.10,059,655.
  • the LNP (e.g., targeted LNP) comprises the ionizable lipid shown in Table 1.
  • an LNP containing an ionizable lipid of Table 1 exhibits higher levels of transduction in immune cells (e.g., T cells) and/or higher expression of a payload protein in immune cells (e.g., T cells) relative to an LNP that contains V003 as the ionizable lipid.
  • Lipid 092 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 093 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 153 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid154 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 155 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 162 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 163 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 169 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 176 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 178 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • Lipid 183 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).
  • the LNP e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (I): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; R 3 and R 4 are independently C 1-4 alkyl; and Y is a lipophilic tail, branched or unbranched. [0303] In some embodiments of the formula (I), R 1 is C 6-8 alkyl. In some embodiments, R 1 is C6alkyl.
  • R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y is In some embodiments, Y is In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n- propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl.
  • R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • compounds of formula (I) are compounds of formula (I-A): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle; and Y is a lipophilic tail, branched or unbranched. [0305] In some embodiments of the formula (I-A), R 1 is C 6-8 alkyl. In some embodiments, R 1 is C 6 alkyl. In some embodiments, R 2 is C 3 alkyl.
  • R 2 is C 4 alkyl.
  • Y is In some embodiments, Y is In some embodiments, Y is In some embodiments, Y is In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl.
  • R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • compounds of formula (I) are compounds of formula (I-B): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ;FTab m is 1, 2, or 3; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C 6-8 alkyl.
  • R 1 is C 6 alkyl.
  • R 2 is C 3 alkyl.
  • R 2 is C 4 alkyl.
  • X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine.
  • R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • compounds of formula (I) are compounds of formula (I-C): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C 6-8 alkyl. In some embodiments, R 1 is C6alkyl.
  • R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine.
  • compounds of formula (I) are compounds of formula (I-D): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C 6-8 alkyl. In some embodiments, R 1 is C 6 alkyl. In some embodiments, R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl.
  • R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine. [0312] In some embodiments, compounds of formula (I) are compounds of formula (I-E):
  • m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • compounds of formula (I) are compounds of formula (I-F): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C 6-8 alkyl.
  • R 1 is C 6 alkyl.
  • R 2 is C 3 alkyl.
  • R 2 is C 4 alkyl.
  • X is O.
  • X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine.
  • R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • compounds of formula (I) are compounds of formula (I-G): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C 6-8 alkyl. In some embodiments, R 1 is C 6 alkyl.
  • R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine.
  • compounds of formula (I) are compounds of formula (I-H): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; R 1 is C 4-10 alkyl; R 2 is C 1-4 alkyl; and R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle.
  • R 1 is C 6-8 alkyl. In some embodiments, R 1 is C6alkyl. In some embodiments, R 2 is C 3 alkyl. In some embodiments, R 2 is C 4 alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl.
  • R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (II): or a pharmaceutically acceptable salt thereof, wherein: X is O or CH 2 ; m is 1, 2, or 3; and Y is a lipophilic tail, branched or unbranched.
  • Y is In some embodiments, Y is . In some embodiments, Y is In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.
  • compounds of formula (II) are compounds of formula (II-A): or a pharmaceutically acceptable salt thereof, wherein: Y is a lipophilic tail, branched or unbranched.
  • Y is .
  • the LNP e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (III): or a pharmaceutically acceptable salt thereof, wherein: R 3 and R 4 are independently C 1-4 alkyl, or R 3 and R 4 are taken together with the nitrogen to which they are attached to form a heterocycle; and Y is a lipophilic tail, branched or unbranched.
  • Y is . In some embodiments, Y is . In some embodiments, Y is In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R 3 and R 4 are taken to together with the N to which they are attached to form a piperidine.
  • R 3 and R 4 are taken to together with the N to which they are attached to form a piperazine.
  • compounds of formula (III) are compounds of formula (III-A): or a pharmaceutically acceptable salt thereof, wherein: Y is a lipophilic tail, branched or unbranched.
  • Y is a lipophilic tail, branched or unbranched.
  • the compound of formula (I) is a compound selected from the exemplary compounds of Table 2. Table 2: Exemplary Ionizable Lipids
  • the LNP e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (IV): or a pharmaceutically acceptable salt thereof, wherein: X is -O- or -CH 2 -; m is 0, 1, 2, or 3; R 1 is C 1-4 alkyl; R 2 is C 1-4 alkyl; n is 1, 2, 3, or 4; R 3 is C 4-10 alkyl; R 4 is C 4-10 alkyl; p is 2, 3, 4, 5, or 6; R 5 is C 4-10 alkyl; and R 6 is C 4-10 alkyl.
  • formula (IV) or a pharmaceutically acceptable salt thereof, wherein: X is -O- or -CH 2 -; m is 0, 1, 2, or 3; R 1 is C 1-4 alkyl; R 2 is C 1-4 alkyl; n is 1, 2, 3, or 4; R 3 is C 4-10 alkyl; R 4 is C 4-10 alkyl; p is 2, 3, 4, 5, or 6; R 5 is C 4
  • compounds of formula (IV) are compounds of formula (IV- A): or a pharmaceutically acceptable salt thereof, wherein: p is 2, 3, 4, 5, or 6; R 5 is C 4-10 alkyl; and R 6 is C 4-10 alkyl.
  • compounds of formula (IV) are compounds of formula (IV- B):
  • exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013/0178541; I of US2013/0303587 or US2013/0123338; I of US2015/0141678; II, III, IV, or V of US2015/0239926; I of US2017/0119904; I or II of WO2017/117528; A of US2012/0149894; A of US2015/0057373; A of WO2013/116126; A of US2013/0090372; A of US2013/
  • the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28,3 l-tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3- nonyldocosa-l3, l6-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety).
  • the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US9,867,888(incorporated by reference herein in its entirety).
  • the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of WO2015/095340(incorporated by reference herein in its entirety).
  • the ionizable lipid is Di((Z)-non-2-en-1-yl) 9-((4- dimethylamino)butanoyl)oxy)heptadecanedioate (L319), , e.g. as synthesized in Example 7, 8, or 9 of US2012/0027803(incorporated by reference herein in its entirety).
  • the ionizable lipid is 1,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010/053572(incorporated by reference herein in its entirety).
  • the ionizable lipid is; Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946 (incorporated by reference herein in its entirety).
  • ICE Imidazole cholesterol ester
  • the mol% of the ionizable lipid in the LNP is from about 25% to about 65%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 35% to about 60%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 40% to about 50%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 45% to about 50%.
  • the mol% of the ionizable lipid in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 25% to about 40%. [0337] %. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 15% to about 30%.
  • the compounds disclosed herein may include an asymmetric center and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms.
  • the present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another and “diastereomers,” which refers to stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.
  • the present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms.
  • Optically active (+) and (-), or (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization.
  • Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate using, for example, chiral high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • the LNPs e.g., targeted LNPs, of the disclosure comprise one or more ionizable lipids.
  • exemplary helper lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10- C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl.
  • Additional exemplary lipids include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.
  • non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl
  • non-cationic lipids are described in WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety.
  • the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018/0028664, incorporated herein by reference in its entirety.
  • the helper lipid is a sphingolipid.
  • the non-pegylated lipid is a sphingomyelin.
  • the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide.
  • the sphingomyelin is egg sphingomyelin.
  • the helper lipid comprises 5-40% (mol), 8%-30%, 10%-28%, 20%-40%, 20%-36%, 24%-38%, 22%-32%, or 10-15% (mol) of the total lipid present in the lipid nanoparticle.
  • the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 18% to about 32%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 24% to about 38%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 20% to about 25%.
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 25% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 28%.
  • the helper lipid (e.g., DSPC or sphingomyelin) in the LNP is from about 25% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted L
  • the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 21% to about 23%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%.
  • in vivo delivery of certain payloads following administration of the disclosed LNPs (e.g., targeted LNPs) with these percentages of helper lipids provides enhanced transduction and expression of the payloads relative to targeted LNPs with smaller or larger quantities of helper lipid.
  • the disclosure provides a lipid nanoparticle (LNP) comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid; (ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting moieties are antibodies or fragments thereof configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; (iii) an mRNA encoding a gene modifying polypeptide; and (iv) a template RNA comprising (a) a sequence that binds to the polypeptide and (b) a heterologous object sequence.
  • LNP lipid nanoparticle
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.
  • the template RNA comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395- 404.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component; (ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting moieties are antibodies or fragments thereof configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.
  • the therapeutic agent comprises a DNA molecule.
  • the therapeutic agent comprises an mRNA molecule.
  • the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA.
  • the therapeutic agent comprises a gene modifying system. In some embodiments, the therapeutic agent comprises a heterologous gene modifying system. In some embodiments, the therapeutic agent comprises a gene modifying system, and the template RNA of the system comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component; (ii) means for binding a cell surface receptor selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or more nucleic acid molecules (e.g., one or more RNA molecules).
  • the therapeutic agent comprises one or more nucleic acid molecules (e.g., one or more RNA molecules
  • the means for binding the cell surface receptor is present on the surface of the LNP.
  • the means for binding a cell surface receptor comprises a targeting moiety, such as an antibody or fragment thereof.
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, about 20 mol% to about 40 mol% of the lipid component, about 20 mol% to about 30 mol% of the lipid component or about 25% to about 40% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.
  • the therapeutic agent comprises a DNA molecule.
  • the therapeutic agent comprises an mRNA molecule.
  • the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA.
  • the therapeutic agent comprises a gene modifying system. In some embodiments, the therapeutic agent comprises a heterologous gene modifying system. In some embodiments, the therapeutic agent comprises a gene modifying system, and the template RNA of the system comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 65 mol% of the lipid component, and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component; (ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting moieties are antibodies or fragments thereof configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or more nucleic acid molecules (e.g., one or more RNA molecules).
  • the therapeutic agent comprises one or more nucleic acid molecules (e.g., one or more RNA molecules).
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component or is present at about 40 mol% to about 50 mol% of the lipid component or is present at about 20 mol% to about 40 mol% of the lipid component, or is present at about 20 mol% to about 30 mol% of the lipid component or is present at about 40 mol% to about 50 mol% of the lipid component and the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.
  • the therapeutic agent comprises a DNA molecule. In some embodiments, the therapeutic agent comprises an mRNA molecule.
  • the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA.
  • the therapeutic agent comprises a gene modifying system.
  • the therapeutic agent comprises a heterologous gene modifying system.
  • the therapeutic agent comprises a gene modifying system
  • the template RNA of the system comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.
  • the ionizable lipid is present at about 47% of the lipid component and the helper lipid is present at about 8% of the lipid component.
  • the ionizable lipid is present at about 47% of the lipid component, the helper lipid is present at about 8% of the lipid component, and the cholesterol is present at between about 42% and 44% of the lipid component.
  • the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 65 mol% of the lipid component, and the helper lipid is present at about 5 mol% to about 15 mol% of the lipid component; (ii) means for binding a cell surface receptor selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or more nucleic acid molecules (e.g., one or more RNA molecules).
  • a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 65 mol% of the lipid component, and the helper lipid is present at about 5 mol% to about
  • the means for binding the cell surface receptor is present on the surface of the LNP.
  • the means for binding a cell surface receptor comprises a targeting moiety, such as an antibody or fragment thereof.
  • the LNP comprises a CD3 targeting moiety, optionally wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32).
  • the LNP comprises a CD2 targeting moiety, optionally wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD5 targeting moiety, optionally wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD7 targeting moiety, optionally wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20).
  • the LNP comprises a CD28 targeting moiety.
  • the LNP comprises a CD4 targeting moiety.
  • the LNP comprises a CD8 targeting moiety.
  • the LNP comprises two targeting moieties configured to bind one or more receptors on the surface of T cells selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.
  • the two targeting moieties are configured to bind to CD3 and CD2, CD3 and CD4, CD3 and CD5, CD3 and CD7, CD3 and CD8 or CD3 and CD28.
  • the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32
  • the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20.
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component or is present at about 40 mol% to about 50 mol% of the lipid component, and the helper lipid is present at about 5 mol% to about 10 mol% of the lipid component (e.g., 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol% or 10 mol% of the lipid component).
  • the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, and the helper lipid is present at about 10 mol% to about 15 mol% of the lipid component (e.g., 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol% or 15 mol% of the lipid component).
  • the therapeutic agent comprises a DNA molecule.
  • the therapeutic agent comprises an mRNA molecule.
  • the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA.
  • the therapeutic agent comprises a gene modifying system.
  • the therapeutic agent comprises a heterologous gene modifying system.
  • the therapeutic agent comprises a gene modifying system
  • the template RNA of the system comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.
  • the ionizable lipid is present at about 47% of the lipid component and the helper lipid is present at about 8% of the lipid component.
  • the ionizable lipid is present at about 47% of the lipid component
  • the helper lipid is present at about 8% of the lipid component
  • the cholesterol is present at between about 42% and 44% of the lipid component.
  • the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP is from about 1:1 to about 7:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 4:1.
  • the molar ratio between the ionizable lipid and the non-pegylated helper lipid is from about 1:1 to about 3:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 1:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 2:1.
  • the molar ratio between the ionizable lipid and the non-pegylated helper lipid is from about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 2.5:1.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid in Table 1, Table 2 or Table 3 and DSPC.
  • the LNP e.g., targeted LNP
  • the LNP comprises an ionizable lipid in Table 1, Table 2 or Table 3 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 40%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 25%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP e.g., targeted LNP, comprises an ionizable lipid of Formula I and DSPC.
  • the LNP e.g., targeted LNP
  • the LNP comprises an ionizable lipid of Formula I and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 25%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%.
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 25% to about 35%.
  • the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%).
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%m about 34% or about 35%).
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%).
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%).
  • the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).
  • the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 60% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 60% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 40%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 50% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 50% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 40%.
  • the helper lipid e.g., DSPC or sphingomyelin
  • the mol% of cholesterol in the targeted LNP is from about 25%-40%, [0356]
  • the LNP, e.g., targeted LNP comprises Lipid 093 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 093 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 40%.
  • the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 25%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 20%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 092 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 092 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 153 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 153 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 154 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 154 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 155 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 155 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 162 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 163 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 169 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 169 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 176 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 176 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 178 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 178 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 183 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 183 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid V003 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid V003 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • the LNP, e.g., targeted LNP comprises Lipid 232 and DSPC.
  • the LNP, e.g., targeted LNP comprises Lipid 232 and a sphingomyelin.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 40%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%.
  • the mol% of the DSPC or sphingomyelin in the LNP is from about 22% to about 28%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 36%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 24% to about 38%.
  • the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 27%, about 38%, about 39%, or about 40%.
  • Sterols [0368]
  • the LNPs, e.g., targeted LNPs, of the disclosure can comprise a component, such as a sterol, to provide membrane integrity.
  • a sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof.
  • Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53-coprostanol, choiesteryl-(2 , -hydroxy)-ethyl ether, choiesteryl-(4'- hydroxy)-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p- cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue, e.g., choiesteryl-(4 '-hydroxy)-buty1 ether.
  • the component providing membrane integrity such as a sterol
  • the component providing membrane integrity can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle.
  • such a component is 20-50% (mol) 30- 40% (mol) of the total lipid content of the lipid nanoparticle.
  • the molar ratio between the cholesterol molecule and the non- pegylated helper lipid is from about 6:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 3:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 0.5:1.
  • the ratio between the cholesterol molecule and the non-pegylated helper lipid is from about 1.5:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:2 to about 0.8:1.
  • the LNPs e.g., targeted LNPs
  • the LNPs can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule.
  • PEG polyethylene glycol
  • conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof.
  • the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
  • PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), 1,2-dimyristoyl-sn-glycerol, methoxypoly ethylene glycol (DMG-PEG-2K), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'- di(tetradecanoyloxy)propyl-l-0-
  • exemplary PEG- lipid conjugates are described, for example, in US5,885,6l3, US6,287,59l, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety.
  • a PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety.
  • a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety.
  • the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG- dipalmityloxypropyl, or PEG-distearyloxypropyl.
  • the PEG-lipid can be one or more of PEG- DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG- dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (l-[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'- dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG- DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1,2- dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-
  • the PEG-lipid comprises PEG-DMG, 1,2- dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].
  • the PEG-lipid comprises a structure selected from: .
  • lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid.
  • polyoxazoline (POZ)-lipid conjugates, polyamide- lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.
  • the pegylated lipid has at least one C16 (palmitoyl) PEG lipid anchor. In some embodiments, the pegylated lipid has two C16 PEG lipid anchors (i.e., dialkyl chains of 16 carbons long).
  • the pegylated lipid is 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000.). In some embodiments, the pegylated lipid is 1,2-Dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is C16 PEG ceramide. In some embodiments, the targeted LNPs comprising the C16 pegylated lipids show reduced liver uptake than otherwise identical LNPs comprising C14 pegylated lipids.
  • the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains).
  • the pegylated lipid is DMG-PEG2000.
  • the pegylated lipid has at least one C18 PEG lipid anchor.
  • the pegylated lipid has two C18 PEG lipid anchors (i.e., dialkyl chains of 18 carbons long).
  • the C18 pegylated lipid is 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the C18 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG-PEG2000). [0378] In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle.
  • one or more additional compounds can also be included in the LNPs, e.g., targeted LNPs, of the disclosure. Those compounds can be administered separately, or the additional compounds can be included in the lipid nanoparticles of the invention.
  • the lipid nanoparticles can contain other compounds, such as a payload, e.g., a therapeutic agent, as described herein.
  • the lipid nanoparticles can contain one or more nucleic acids.
  • the lipid nanoparticles can contain at least a first nucleic acid and a second nucleic acid, wherein the second nucleic acid is different than the first nucleic acid.
  • lipid nanoparticle or a formulation comprising lipid nanoparticles
  • reactive impurities e.g., aldehydes or ketones
  • comprises less than a preselected level of reactive impurities e.g., aldehydes or ketones
  • a lipid reagent is used to make a lipid nanoparticle formulation, and the lipid reagent may comprise a contaminating reactive impurity (e.g., an aldehyde or ketone).
  • a lipid regent may be selected for manufacturing based on having less than a preselected level of reactive impurities (e.g., aldehydes or ketones).
  • aldehydes can cause modification and damage of RNA, e.g., cross-linking between bases and/or covalently conjugating lipid to RNA (e.g., forming lipid-RNA adducts).
  • a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.
  • a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • a lipid nanoparticle formulation is produced using a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • the lipid nanoparticle formulation is produced using a plurality of lipid reagents, and each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph. [0382] In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.
  • each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph.
  • the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or
  • the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • the lipid nanoparticle formulation comprises: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.
  • one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
  • any single reactive impurity e.g., aldehyde
  • total aldehyde content and/or quantity of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS/MS), e.g., according to the method described in Example 34.
  • LC liquid chromatography
  • MS/MS tandem mass spectrometry
  • reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleic acid molecule (e.g., an RNA molecule, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents.
  • a nucleic acid molecule e.g., an RNA molecule, e.g., as described herein
  • reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleotide or nucleoside (e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents.
  • a nucleotide or nucleoside e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein
  • reactive impurities e.g., aldehydes
  • LNPs e.g., targeted LNPs
  • the LNPs can be used to deliver payloads to cells, e.g., immune cells (e.g., T cells).
  • the LNPs can be used to deliver a payload into T cells.
  • a targeted LNP as described herein can be used to deliver a payload to cells expressing cell-surface receptors targeted by the targeting moiety, e.g., antibody, Fab fragment, scFv or nanobody component, of the targeted LNP (conjugate).
  • the conjugates disclosed herein can be used to deliver a therapeutic of interest to a cell.
  • the conjugates disclosed herein can be used to deliver a nucleic acid (e.g., mRNA) encoding a vaccine.
  • the conjugates disclosed herein can be used to deliver a nucleic acid (e.g., mRNA) encoding an enzyme.
  • the conjugates disclosed herein can be used to deliver a nucleic acid, e.g., a DNA or RNA molecule, encoding a chimeric antigen receptor (CAR) to T cells.
  • the payload is one or more nucleic acids.
  • the payload is one or more RNA molecules.
  • the payload is an mRNA (e.g., an mRNA encoding an enzyme).
  • the RNA molecule is a non-coding RNA (ncRNA).
  • the payload is an RNA template (for example, an RNA template for reverse transcription, e.g., for Target Primed Reverse Transcription (TPRT)).
  • TPRT Target Primed Reverse Transcription
  • the payload is a siRNA or a microRNA (miRNA).
  • the payload is a guide RNA.
  • the payload is a tRNA.
  • the payload is an antisense oligonucleotide (ASO).
  • the payload is a DNA molecule, for example, a DNA plasmid, closed-ended DNA (ceDNA), or a small circular DNA (e.g., a minicircle or nanoplasmid).
  • Nucleic acid payloads can be linear, circular, covalently closed, single-stranded, double-stranded, or hybrid RNA/DNA molecules.
  • the payload is a small molecule.
  • the payload is a peptide or protein.
  • the conjugates disclosed herein can include two or more payloads, for example, selected from mRNA, ncRNA, guide RNA, siRNA, miRNA, ASO, DNA vector, small molecule, peptide, or protein.
  • the LNPs described herein, e.g., targeted LNPs can be used to deliver a therapeutic of interest to a cell, e.g., an immune cell (e.g., T cell).
  • the LNP e.g., targeted LNP, contains a payload that is a therapeutic agent.
  • the therapeutic agent can be a therapeutic peptide or protein, a nucleic acid comprising a therapeutic agent, or a nucleic acid encoding a therapeutic agent.
  • the therapeutic agent can be a genetic medicine (e.g., for gene therapy or gene editing), wherein the therapeutic agent is capable of modifying, altering or effecting a change in the genomic DNA of a cell (e.g., an immune cell (e.g., T cell)).
  • the therapeutic agent is a gene therapy agent or gene editing agent.
  • the therapeutic agent is a gene modifying polypeptide, as described herein.
  • the therapeutic agent is a gene modifying system, as described herein.
  • the therapeutic agent can be a peptide or protein, such as an enzyme, or a nucleic acid (e.g., mRNA or DNA) encoding the peptide or protein (e.g., an enzyme).
  • the enzyme can be a nuclease, recombinase, integrase, transposase, retrotransposase, helicase, transcriptase, polymerase, reverse transcriptase, deaminase, methylase, demethylase, or ligase, or can have a combination of enzymatic activities thereof.
  • the therapeutic agent can be a peptide or protein, or a nucleic acid encoding the peptide or protein, for use as a replacement gene therapy.
  • the therapeutic agent can be a peptide or protein, or a nucleic acid encoding the peptide or protein, for use in modifying or altering the genome or epigenome of a cell, e.g., an immune cell of a subject.
  • the therapeutic agent can comprise one or more components of a system for modifying or altering the genome or epigenome of a cell, e.g., an immune cell (e.g., T cell) of a subject.
  • the system for modifying or altering the genome or epigenome of a cell of a subject comprises one or more proteins, one or more nucleic acids (e.g., RNA and/or DNA), or combinations thereof.
  • the therapeutic agent can be one or more components of a ribonucleoprotein (RNP) complex for modifying or altering the genome or epigenome of a cell, e.g., immune cell such as a T cell.
  • RNP ribonucleoprotein
  • the therapeutic agent can be a protein, or a nucleic acid (e.g., mRNA) encoding the protein, and/or an RNA molecule (e.g., a gRNA) for guiding the protein to a particular location in the genome or epigenome, wherein the protein is capable of modifying or altering the genome or epigenome as a nuclease, recombinase, integrase, transposase, helicase, reverse transcriptase, deaminase, methylase, demethylase, or ligase, or combinations thereof.
  • the therapeutic agent is a nuclease.
  • the nuclease cleaves DNA (e.g., both strands of the DNA), thereby introducing insertion and/or deletion (indel) mutations in DNA, e.g., genomic DNA.
  • the nuclease is a nickase (i.e., it cleaves a single stand of DNA).
  • the nuclease is mutated such that it is inactive or comprises reduced nuclease activity.
  • the therapeutic agent can be used to introduce a substitution in DNA, e.g., genomic DNA, via base editing.
  • the therapeutic agent can be used for epigenome editing.
  • the therapeutic agent can be used to introduce an indel or a substitution in DNA, e.g., genomic DNA, by inducing target-primed reverse transcription (TPRT).
  • the therapeutic agent (i.e., payload) delivered by the LNP e.g., targeted LNP
  • the therapeutic agent can be a gene modifying protein, a nucleic acid encoding a gene modifying protein, or a gene modifying system, as described herein.
  • the therapeutic agent can be a small molecule.
  • the therapeutic agent can be an siRNA or miRNA
  • the LNPs, e.g., targeted LNPs, of the disclosure can be used to deliver gene editing components into immune cells (e.g., T cells).
  • the LNPs, e.g., targeted LNPs can be used to deliver a CRISPR-Cas system into immune cells (e.g., T cells).
  • the LNPs, e.g., targeted LNPs can be used to deliver a Class 1 (type I, type III, or type IV) CRISPR system into immune cells (e.g., T cells).
  • the LNPs can be used to deliver a Class 2 (type II, type V, or type VI) CRISPR system into immune cells (e.g., T cells).
  • the LNPs e.g., targeted LNPs
  • the LNPs can be used to deliver a CRISPR-Cas12 system (e.g., a Cas12a system), or a nucleic acid encoding one or more components of the CRISPR-Cas12 system, into immune cells (e.g., T cells).
  • the LNPs e.g., targeted LNPs
  • the Cas is Cas9 or Cas12a.
  • the Cas is Cas9.
  • the Cas is Cas12a.
  • the gRNA is a single guide RNA (sgRNA).
  • the LNPs e.g., targeted LNPs, comprise a payload consisting of or comprising a Cas9 or an mRNA encoding a Cas9.
  • the LNPs, e.g., targeted LNPs comprise a payload consisting of or comprising a Cas12 (e.g., Cas12a) or an mRNA encoding a Cas12 (e.g., Cas12a).
  • the payload further consists of or comprises a gRNA.
  • the mRNA component of a gene modifying system comprises a recombinant nuclease, or a nucleic acid encoding the nuclease, for example a restriction endonuclease, meganuclease, homing endonuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).
  • a restriction endonuclease for example a restriction endonuclease, meganuclease, homing endonuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • the therapeutic agent (i.e., payload) delivered by the LNP can be a gene modifying system, or one or more nucleic acids (e.g., RNA molecules) comprising the gene modifying system (e.g., an mRNA encoding a gene modifying polypeptide and a template RNA), as described herein.
  • the gene modifying system is a retrotransposon gene modifying system, as described herein.
  • the gene modifying system is a heterologous gene modifying system, as described herein.
  • the system comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises: (i) an endonuclease and/or DNA binding domain; and (ii) a reverse transcriptase (RT) domain, where (i) and (ii) are both derived from a retrotransposon (e.g., from the same retrotransposon or different retrotransposons); and (B) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence.
  • a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide comprises: (i) an endonuclease and/or DNA binding domain; and (ii) a reverse transcriptase (RT) domain, where (i) and (ii) are both derived from a retrotransposon (e.
  • a gene modifying polypeptide acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery.
  • the heterologous object sequence may include, e.g., a coding sequence, a regulatory sequence, or a gene expression unit.
  • Gene modifying polypeptides [0394]
  • Non-long terminal repeat (LTR) retrotransposons are a type of mobile genetic elements that are widespread in eukaryotic genomes.
  • APE apurinic/apyrimidinic endonuclease
  • RLE restriction enzyme-like endonuclease
  • PLE Penelope-like element
  • APE class retrotransposons are comprised of two functional domains: an endonuclease/DNA binding domain, and a reverse transcriptase domain. Examples of APE-class retrotransposons can be found, for example, in Table 1 of PCT Application No. PCT/US2019/048607, incorporated herein by reference in its entirety, including the sequence listing and sequences referred to in Table 1 therein.
  • the RLE class are comprised of three functional domains: a DNA binding domain, a reverse transcription domain, and an endonuclease domain.
  • RLE-class retrotransposons can be found, for example, in Table 2 of PCT Application No. PCT/US2019/048607, incorporated herein by reference in its entirety, including the sequence listing and sequences referred to in Table 2 therein.
  • Penelope-like elements are distinct from both LTR and non-LTR retrotransposons. PLEs generally comprise a reverse transcriptase domain distinct from that of APE and RLE elements, but similar to that of telomerases and Group II introns, and an optional GIY-YIG endonuclease domain.
  • RTE e.g., RTE-1_MD, RTE-3_BF, and RTE-25_LMi
  • CR1 e.g., CR1-1_PH
  • Crack e.g., Crack-28_RF
  • L2 e.g., L2-2_Dre and L2-5_GA
  • Vingi e.g., Vingi-1_Acar
  • an amino acid sequence encoded by an element of Table 4 is an amino acid sequence encoded by the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the full-length sequence of an element listed in Table 4 may comprise one or more (e.g., all of) of a 5’ UTR, polypeptide-encoding sequence, or 3’ UTR of a retrotransposon as described herein.
  • an amino acid sequence of Table 4 is an amino acid sequence encoded by the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • a 5’ UTR of an element of Table 4 comprises a 5’ UTR of the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • a 3’ UTR of an element of Table 4 comprises a 3’ UTR of the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • Table 4 Also indicated in Table 4 are the host organisms from which the nucleic acid sequences were obtained and a listing of domains present within the polypeptide encoded by the open reading frame of the nucleic acid sequence.
  • a template RNA described herein comprises one or both of a first homology domain comprising a sequence of a 5' Human Homology Arm of Table 4 (or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto) and a second homology domain comprising a sequence of a 3' Human Homology Arm of Table 4 (or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto).
  • a gene modifying system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 1, 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 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides).
  • a gene modifying system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases).
  • Template RNA component of retrotransposon gene modifying systems [0403]
  • the gene modifying polypeptides described herein can be used together with a template RNA, to install a mutation into a target RNA.
  • the template RNA comprises a 5’ UTR, a heterologous object sequence, and a 3’ UTR.
  • the template RNA has a 3’ untranslated region derived from a retrotransposon, e.g., a retrotransposon described herein.
  • the template RNA has a 3’ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the 3’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g. a retrotransposon in Table 4.
  • the template RNA has a 5’ untranslated region derived from a retrotransposon, e.g., a retrotransposon described herein.
  • the template RNA has a 5’ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, or 200 or more bases of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater homology to the 5’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g., a retrotransposon described in Table 4.
  • the template RNA may have some homology to the target DNA.
  • the template RNA has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of exact homology to the target DNA at the 3’ end of the RNA.
  • the template RNA has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, 180, or 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the target DNA, e.g., at the 5’ end of the template RNA.
  • the template RNA has a poly-A tail at the 3’ end. In some embodiments, the template RNA does not have a poly-A tail at the 3’ end.
  • a system or method described herein comprises a single template RNA. In some embodiments, a system or method described herein comprises a plurality of template RNAs.
  • the template nucleic acid may comprise one or more UTRs (e.g., a 5’ UTR or a 3’ UTR, e.g., from an R2-type retrotransposon). In some embodiments, the UTR facilitates interaction of the template with the reverse transcriptase domain of the polypeptide.
  • the template possesses one or more sequences aiding in association of the template with the gene modifying polypeptide. In some embodiments, these sequences may be derived from retrotransposon UTRs.
  • the UTRs may be located flanking the desired insertion sequence.
  • a sequence with target site homology may be located outside of one or both UTRs.
  • the sequence with target site homology can anneal to the target sequence to prime reverse transcription.
  • the 5’ and/or 3’ UTR may be located terminal to the target site homology sequence.
  • the gene modifying system may result in the insertion of a desired payload without any additional sequence (e.g., a gene expression unit without UTRs used to bind the gene modifying protein).
  • the template RNA comprises one or more chemically modified nucleotides.
  • the template RNA comprises a backbone modification, e.g., a modification to a sugar or phosphate group in the backbone. In some embodiments, the template RNA comprises a nucleobase modification.
  • the heterologous object sequence encodes a membrane protein, e.g., a CAR or a membrane protein other than a CAR, and/or an endogenous human membrane protein. In some embodiments, the heterologous object sequence encodes an extracellular protein. In some embodiments, the heterologous object sequence encodes an enzyme, a structural protein, a signaling protein, a regulatory protein, a transport protein, a sensory protein, a motor protein, a defense protein, or a storage protein.
  • an LNP described herein comprises a retrotransposon gene modifying system, as described herein.
  • the template RNA of the retrotransposon gene modifying system comprises a template RNA sequence present in Table 5B or a template RNA sequence comprising 80% to 99% sequence identity (e.g., 80%, 85%, 90%, 95%, or 99% sequence identity) to a template RNA sequence present in Table 5B.
  • the template RNA of the retrotransposon gene modifying system comprises a heterologous object sequence comprising a CAR coding sequence present in Table 5B or a heterologous object sequence comprising 80% to 99% sequence identity (e.g., 80%, 85%, 90%, 95%, or 99% sequence identity) to a CAR coding sequence present in Table 5B.
  • the heterologous object sequence encodes a chimeric antigen receptor (CAR) comprising an antigen binding domain.
  • the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a single intracellular signaling domain.
  • the CAR is or comprises a second generation CAR comprising an antigen binding domain, a transmembrane domain, and two intracellular signaling domains (e.g., a first intracellular signaling domain and a second intracellular signaling domain).
  • the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three intracellular signaling domains.
  • a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four intracellular signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene.
  • the antigen binding domain is or comprises an scFv, Fab, a diabody, a D domain binder, centyrins (e.g., antibody-like scaffolds, e.g., a CARTyrin), one or more single domain antibodies such as VHH domains (e.g., comprises two VHH binding domains).
  • a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, a cell surface antigen is characteristic of one type of cell. In some embodiments, a cell surface antigen is characteristic of more than one type of cell. In some embodiments, the CAR antigen binding domain binds to two epitopes of the target antigen (e.g., is a biepitopic binding domain). In some embodiments, the CAR comprises two antigen binding domains, such that each antigen binding domain binds to a different target antigen on a cell, e.g., a neoplastic cell.
  • the antigen binding domain targets an antigen characteristic of a neoplastic cell.
  • the antigen characteristic of a neoplastic cell is selected from a receptor listed in Table 6, or an antigenic fragment or antigenic portion thereof.
  • the antigen binding domain binds one or more antigens of a blood cancer (e.g., a leukemia or a lymphoma, or a multiple myeloma).
  • the blood cancer antigen is a B cell antigen.
  • the antigen binding domain binds an antigen of a solid tumor.
  • the antigen binding domain targets an antigen characteristic of a T-cell.
  • the antigen characteristic of a T-cell is selected from an exemplary T cell antigen listed in Table 7, or an antigenic fragment thereof. Table 7: Exemplary T-cell Antigens
  • the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder.
  • the autoimmune or inflammatory disorder is selected from chronic graft-vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purrpura, neuromyelits optica, Evan's syndrome, IgM mediated neuropathy, cyroglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticarial
  • GVHD chronic graft-v
  • the antigen characteristic of an autoimmune or inflammatory disorder is selected from an exemplary antigen listed in Table 8, or an antigenic fragment thereof.
  • a CAR antigen binding domain binds to a ligand expressed on B cells, plasma cells, plasmablasts.
  • the antigen expressed on B cells, plasma cells, or plasmablasts is selected from an exemplary antigen listed in Table 9, or an antigenic fragment thereof.
  • Table 9 Exemplary B cell, Plasma Cell, and Plasmablast Antigens
  • the antigen binding domain targets an antigen characteristic of an infectious disease.
  • the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma-associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Eptstein-Barr virus, CMV, human papillomavirus.
  • the antigen characteristic of an infectious disease is selected from an exemplary antigen listed in Table 10, or an antigenic fragment thereof.
  • the CAR transmembrane domain comprises at least a transmembrane region of an exemplary transmembrane domain listed in Table 11, or a functional fragment thereof.
  • Table 11 Exemplary Transmembrane Domains
  • the transmembrane domain is a CD8 transmembrane domain.
  • the CD8 transmembrane domain has an amino acid sequence of a CD8 transmembrane domain listed in Table C6A, or sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the CD8 transmembrane domain is encoded by a nucleic acid sequence of a CD8 transmembrane domain listed in Table 12, or sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • Table 12 Sequences of Exemplary Transmembrane Domains Intracellular Signaling Domains
  • the CAR comprises at least one signaling domain selected from one or more intracellular signaling domains listed in Table 13, or a functional fragment thereof.
  • the CAR comprises a first intracellular signaling domain and a second intracellular signaling domain.
  • the first intracellular signaling domain mediates downstream signaling during T-cell activation.
  • the second intracellular signaling domain is a costimulatory domain.
  • Table 13 Exemplary Intracellular Signaling Domains
  • the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.
  • the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof, and/or (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.
  • the CAR comprises a CD28z co-stimulatory domain.
  • the CAR comprises a CD3z signaling domain.
  • intracellular signaling domain comprises an intracellular signaling domain listed in Table 14, or sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • Table 14 Sequences of Exemplary Intracellular Signaling Domains
  • the CAR further comprises one or more spacers, e.g., wherein the spacer is a first spacer between the antigen binding domain and the transmembrane domain.
  • the first spacer includes at least a portion of an immunoglobulin constant region or variant or modified version thereof.
  • the spacer is a second spacer between the transmembrane domain and a signaling domain.
  • the second spacer is an oligopeptide, e.g., wherein the oligopeptide comprises glycine-serine doublets.
  • the CAR further comprises a hinge domain.
  • the hinge domain is a CD8 hinge domain.
  • the CD8 hinge domain has an amino acid sequence of a CD8 hinge domain in Table 15, or sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • the CAR comprises a sequence of a CAR listed in Table 16, or sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
  • Table 16 Sequences of Exemplary CAR Molecules
  • a system described herein may comprise:
  • RNA or DNA encoding the template RNA
  • a template RNA or DNA encoding the template RNA
  • a heterologous object sequence encoding a chimeric antigen receptor (CAR)
  • the CAR comprises an antigen-binding domain, a transmembrane domain, a first intracellular signaling domain, and a second intracellular signaling domain, as disclosed herein.
  • the CAR comprises an antigen binding domain that binds one or more antigens of a blood cancer (e.g., a leukemia or lymphoma), wherein optionally the antigen is a B cell antigen;
  • a blood cancer e.g., a leukemia or lymphoma
  • the antigen is a B cell antigen
  • the CAR comprises an antigen binding domain that binds one or more antigens of a solid tumor
  • the CAR comprise an antigen binding domain of any one of Tables 5-10 or 16;
  • the CAR comprises a transmembrane domain of Table 11 or 12;
  • the CAR comprises a hinge domain (e.g., a hinge domain of Table 15);
  • the CAR comprises an intracellular signaling domain of Table 13 or 14;
  • the CAR comprises a costimulatory domain of Table 13 or 14;
  • the CAR comprises an antigen binding domain which comprises an scFv;
  • the CAR comprises an amino acid sequence according to of Table 16; (x) wherein the CAR comprises a first intracellular signaling domain and a second intracellular signaling domain.
  • the method involves contacting the cell (e.g., an immune effector cell) with a composition disclosed herein.
  • the immune effector cell is a cell that expresses one or more Fc receptors and mediates one or more effector functions.
  • the immune effector cell may include, but may not be limited to, one or more of a monocyte, macrophage, neutrophil, dendritic cell, eosinophil, mast cell, platelet, large granular lymphocyte, Langerhans' cell, natural killer (NK) cell, T-lymphocyte (e.g., T-cell), a Gamma delta T cell, B-lymphocyte (e.g., B-cell) and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • the mammalian cell is a T cell, e.g., a primary T cell.
  • the method is performed ex vivo or in vitro.
  • a retrotransposon gene modifying system described herein may be used in a multiplex format.
  • the retrotransposon gene modifying system may be used to install a large sequence, such as a CAR sequence, and at the same time a heterologous gene modifying system may be used to make a smaller edit.
  • a heterologous gene modifying system may comprise a heterologous gene modifying polypeptide and a template RNA.
  • the heterologous gene modifying polypeptide may comprise a Cas domain, a linker, and a reverse transcriptase domain derived from a retrovirus.
  • the template RNA compatible with the heterologous gene modifying polypeptide may comprise (e.g., from 5' to 3') (i) a gRNA spacer that binds a target site, (ii) a gRNA scaffold that binds the heterologous gene modifying polypeptide, e.g., the Cas domain of the heterologous gene modifying polypeptide, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence.
  • a gRNA spacer that binds a target site
  • a gRNA scaffold that binds the heterologous gene modifying polypeptide, e.g., the Cas domain of the heterologous gene modifying polypeptide
  • PBS primer binding site
  • a heterologous gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide that comprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain, wherein the RT domain is C-terminal of the Cas domain; and a linker disposed between the RT domain and the Cas domain.
  • a Cas domain e.g., a Cas nickase domain, e.g., a Cas9 nickase domain
  • RT reverse transcriptase
  • the heterologous gene modifying polypeptide comprises a sequence of SEQ ID NO:126 which comprises the first NLS and the Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • the heterologous gene modifying polypeptide comprises a sequence of SEQ ID NO:127 which comprises the second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
  • N-terminal NLS-Cas9 domain MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
  • a heterologous gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 17, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 17, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
  • a heterologous gene modifying polypeptide comprises an amino acid sequence according to Table 18, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. Table 17.
  • a heterologous gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 10 of International Application WO/2023/039440 (which Table is incorporated herein by reference in its entirety).
  • a template RNA molecule for use in the system comprises, from 5 to 3 (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence.
  • Is a gRNA spacer of ⁇ 18-22 nt e.g., is 20 nt
  • Is a gRNA scaffold comprising one or more hairpin loops, e.g., 1, 2, of 3 loops for associating the template with a Cas domain, e.g., a nickase Cas9 domain.
  • a Cas domain e.g., a nickase Cas9 domain.
  • the heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nt or, 80-90 nt in length.
  • the PBS sequence that binds the target priming sequence after nicking occurs is e.g., 3-20 nt, e.g., 7-15 nt, e.g., 12-14 nt.
  • the PBS sequence has 40-60% GC content.
  • the template RNA is configured to alter a genomic sequence at nucleotide TRAC and/or B2M genetic locus to disrupt the TRAC gene and/or B2M gene.
  • the gRNA spacer of the template RNA is complementary to a portion of the human TRAC gene
  • the heterologous object sequence installs a mutation in the TRAC gene
  • the PBS is complementary to another portion of the human TRAC gene.
  • the template RNA directs a mutation that leads to a loss of TRAC mRNA.
  • the mutation leads to an inactive TRAC protein.
  • the gRNA spacer of the template RNA is complementary to a portion of the human B2M gene
  • the heterologous object sequence installs a mutation in the B2M gene
  • the PBS is complementary to another portion of the human B2M gene.
  • the template RNA directs a mutation that leads to a loss of B2M mRNA.
  • the mutation leads to an inactive B2M protein.
  • a retrotransposase gene modifying system used to insert a heterologous object sequence (e.g., a CAR) into the genome of a cell, may be combined with an additional system for modifying DNA to install a mutation (e.g., resulting in knock down or knock out of a target gene, or a correction or deletion of an aberrant gene) into a human gene.
  • the additional system may be a heterologous gene modifying system.
  • two systems may be utilized to introduce multiple edits to the same cell.
  • the two systems may be introduced into cells simultaneously or separately.
  • editing efficiency when inserting whole genes using the retrotransposase gene modifying system is not affected by the co-delivery of another exemplary system containing a heterologous gene modifying polypeptide (e.g., directing a short edit, e.g., an indel or substitution in the DNA).
  • editing efficiency when making short edits e.g., editing of 5 nucleotides or fewer
  • a retrotransposase gene modifying system e.g., directing the insertion of a gene.
  • treating T cells with both the first and second systems results in high level of cooccurrence of the two editing events in the same cell.
  • no skewing in the distribution of the subpopulations or phenotypes of T cells is observed when comparing mock treated T cells, T cells edited by just the first gene modifying system, and T cells edited by both the first and second gene modifying systems.
  • Targeted LNPs for delivering gene modifying systems to immune cells
  • multiple components of a gene modifying system may be prepared as a single LNP formulation, e.g., an LNP formulation comprises mRNA encoding for the gene modifying polypeptide and an RNA template. Ratios of nucleic acid components may be varied in order to maximize the properties of a therapeutic. In some embodiments, the ratio of RNA template to mRNA encoding a gene modifying polypeptide is about 1 : 1 to 100: 1, e.g., about 1 : 1 to 20: 1, about 20: 1 to 40: 1, about 40: 1 to 60: 1, about 60: 1 to 80: 1, or about 80: 1 to 100: 1, by molar ratio.
  • a system of multiple nucleic acids may be prepared by separate formulations, e.g., one LNP formulation comprising a template RNA and a second LNP formulation comprising an mRNA encoding a gene modifying polypeptide.
  • the system may comprise more than two nucleic acid components formulated into LNPs.
  • the system may comprise a protein, e.g., a gene modifying polypeptide, and a template RNA formulated into at least one LNP formulation.
  • the targeted LNPs (conjugates) of the disclosure may be used to simultaneously deliver gene modifying components to T cells that are capable of inserting CAR into the genome of the T cells and altering the T cell receptor alpha constant (TRAC) locus and/or the B2M genetic locus in T cells, hence disrupting the TRAC gene and/or B2M gene.
  • simultaneous insertion of a CAR sequence and knock out of a TRAC and/or B2M gene can be accomplished via LNP delivery of a retrotransposase gene modifying system.
  • a single targeted LNP can deliver all of the components of the gene modifying system including (i) a gene modifying polypeptide; (ii) a template RNA (or DNA encoding the template RNA) that binds to the gene modifying polypeptide and a heterologous CAR sequence; and (iii) an additional system for modifying DNA to install a mutation (e.g., resulting in knock down or knock out of the TRAC gene and/or B2M gene.
  • multiple targeted LNPs can be used to deliver the components of the gene modifying system.
  • one conjugate can be used to deliver including (i) a gene modifying polypeptide; and (ii) a template RNA (or DNA encoding the template RNA) that binds to the gene modifying polypeptide and a heterologous CAR sequence and a second conjugate can be used to deliver an additional system for modifying DNA to install a mutation (e.g., resulting in knock down or knock out of the TRAC gene and/or B2M gene.
  • a mutation e.g., resulting in knock down or knock out of the TRAC gene and/or B2M gene.
  • the disclosure provides a targeted lipid nanoparticle (LNP) for ex vivo or in vivo delivery of a therapeutic agent to immune cells (e.g., T cells), wherein the targeted LNP comprises: i. an ionizable lipid; ii. a helper lipid; iii. two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target on the surface of the immune cells (e.g., T cells); and iv.
  • a targeted lipid nanoparticle for ex vivo or in vivo delivery of a therapeutic agent to immune cells (e.g., T cells)
  • the targeted LNP comprises: i. an ionizable lipid; ii. a helper lipid; iii. two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target on the surface of the immune cells (e.g., T cells); and
  • each of the targeting moieties is conjugated to the lipid nanoparticle through a linker, wherein each linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP.
  • the targeting moieties are antibodies or antigen binding fragments thereof.
  • the targeting moieties are scFvs.
  • the targeting moieties are Fab fragments.
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is an mRNA encoding a gene modifying polypeptide, as described herein.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9-RT fusion protein.
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is a guide RNA (gRNA).
  • one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA is a template RNA encoding a heterologous nucleic acid for use with a gene modifying polypeptide to insert the heterologous nucleic acid sequence into a DNA sequence, e.g., the genomic DNA of a cell.
  • the components of a system for modifying or altering genomic DNA have nuclease activity, e.g., nickase activity.
  • the components of a system for modifying or altering genomic DNA do not have nuclease activity.
  • the components of a system for modifying or altering genomic DNA do not elicit a double-stranded break in the genomic DNA.
  • the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA.
  • a system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous sequence into the genomic DNA.
  • TPRT target-primed reverse transcription
  • the ionizable lipid is V003 or a lipid from Table 1, Table 2, or Table 3. In some embodiments, the ionizable lipid is Lipid 093. In some embodiments, the ionizable lipid is Lipid 092. In some embodiments, the ionizable lipid is Lipid 154.
  • the helper lipid of the targeted LNP is DSPC or sphingomyelin. In some embodiments, the mo1% of the DSPC or sphingomyelin in the targeted LNP is from about 20% to about 30%.
  • the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.
  • the targeted LNP further comprises a pegylated lipid.
  • the pegylated lipid comprises at least one C16 alkyl chain (e.g., two C16 alkyl chains).
  • the pegylated is DPPE-PEG2000 or DPG-PEG2000.
  • the targeted delivery (ex vivo or in vivo) of a system for modifying or altering genomic DNA (e.g., a gene modifying system) with an LNP comprising two or more T cells specific binders conjugated to the LNP results in synergistic enhancement of gene editing or modification of the target T cells.
  • the LNPs comprising dual binders are capable of modifying at least 5% of the genomes of the T cells.
  • the LNPs comprising dual binders are capable of modifying at least 10% of the genomes of the T cells.
  • the LNPs comprising dual binders are capable of modifying at least 15% of the genomes of the T cells.
  • the LNPs comprising dual binders are capable of modifying at least 20% of the genomes of the T cells. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 25% of the genomes of the T cells. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the T cells. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the T cells. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 30% of the genomes of the T cells.
  • the LNPs comprising dual binders are capable of modifying from about 20% to about 30% of the genomes of the T cells. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 20% of the genomes of the T cells.
  • the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA. In some such embodiments, a system for modifying or altering genomic DNA includes a gene modifying polypeptide and a template RNA for a nucleic acid sequence to be inserted at a specific location of the genomic DNA, hence resulting in modification of the genomic DNA.
  • the gene modifying system comprises a Cas9 nickase fused to a reverse transcriptase (RT) domain.
  • a system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous nucleic acid sequence into a DNA sequence, e.g., genomic DNA.
  • TPRT target-primed reverse transcription
  • an LNP e.g., targeted LNP, disclosed herein is delivered ex vivo to isolated immune cells (e.g., T cells) of a subject (e.g., human patient) in need thereof.
  • the immune cells are collected from the blood or bone marrow of a subject. Following isolation of the immune cells (e.g., T cells) the LNPs of the disclosure can be mixed with the immune cells (e.g., T cells), thereby resulting in effective transduction of the payload (e.g., gene modifying system) into the immune cells (e.g., T cells).
  • the modified immune cells e.g., T cells
  • the immune cells are activated, e.g., with an activating agent (such as TransAct (Miltenyi Biotec)), prior to incubation with the LNPs.
  • the immune cells are not activated prior to incubation with the LNPs.
  • an LNP e.g., targeted LNP, as disclosed herein can be introduced into cells, tissues and/or multicellular organisms.
  • the LNPs e.g., targeted LNPs, are delivered to the cells via mechanical means or physical means.
  • Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).
  • the LNP described herein e.g., targeted LNPs
  • the high level of transduction and protein expression following in vivo administration allows for sufficient levels of transduction and expression of proteins (e.g., gene modifying proteins), and hence can be used to treat various diseases.
  • an LNP, e.g., targeted LNP, described herein is delivered to a tissue or cell from or in the blood or bone marrow.
  • the LNP e.g., targeted LNP
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous, intradermal, epidural, intracerebral, intracerebroventricular, epicutaneous, nasal, intra-arterial, intra-articular, intracavernous, intraocular, intraosseous infusion, intraperitoneal, intrathecal, intrauterine, intravaginal, intravesical, perivascular, or transmucosal administration.
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous, intradermal, epidural, intracerebral, intracerebroventricular, epicutaneous, nasal, intra-arterial, intra-articular, intracavernous, intraocular, intraosseous infusion, intraperitoneal, intrathecal, intrauterine, intravaginal, intravesical, perivascular, or transmucosal administration.
  • an LNP, e.g., targeted LNP of the disclosure delivers a therapeutic
  • an LNP e.g., targeted LNP
  • a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide
  • immune cells e.g., T cells
  • an LNP, e.g., targeted LNP of the disclosure delivers a gene modifying system to immune cells (e.g., T cells) of a patient following in vivo administration.
  • an LNP, e.g., targeted LNP, of the disclosure delivers gene editing components to immune cells (e.g., T cells) of a patient following in vivo administration. Exemplary gene editing systems are described above.
  • the nucleic acids encoding components of a system for modifying or altering a genome can be expressed, resulting in in vivo gene editing of the immune cells (e.g., T cells)
  • the immune cells e.g., T cells
  • particular combinations of targeting moieties e.g., antibodies, Fab fragments or scFvs
  • ionizable lipids in particular LNP formulations e.g., comprising particular amounts of helper lipid
  • LNP formulations e.g., comprising particular amounts of helper lipid
  • the LNPs described herein result in enhanced levels of modification or alteration of the genomic DNA of immune cells (e.g., T cells), e.g., enhanced levels of gene editing in the immune cells (e.g., T cells).
  • the percentage of immune cells (e.g., T cells) exhibiting modified or altered (e.g., edited) genomic DNA in the subject following ex vivo incubation or after administration of the LNPs, e.g., targeted LNPs is at least 5%.
  • the percentage of immune cells (e.g., T cells) exhibiting modified or altered (e.g., edited) genomic DNA in the subject following ex vivo incubation or after administration of the LNPs, e.g., targeted LNPs is at least 10%. In some embodiments, the percentage of immune cells (e.g., T cells) exhibiting modified or altered (e.g., edited) genomic DNA in the subject following ex vivo incubation or after administration of the LNPs, e.g., targeted LNPs, is at least 15%.
  • the percentage of immune cells (e.g., T cells) exhibiting modified or altered (e.g., edited) genomic DNA in the subject following ex vivo incubation or after administration of the LNPs, e.g., targeted LNPs is at least 20%.
  • the LNPs described herein result in enhanced levels of modification or alteration of the genomic DNA of immune cells (e.g., T cells) e.g., enhanced levels of gene editing in the immune cells (e.g., T cells).
  • the percentage of immune cells (e.g., T cells) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs is at least 30%.
  • the percentage of immune cells (e.g., T cells) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs is at least 40%. In some embodiments, the percentage of immune cells (e.g., T cells) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 50%. In some embodiments, the percentage of immune cells (e.g., T cells) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 60%.
  • the percentage of immune cells (e.g., T cells) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs is at least 70%. In some embodiments, the percentage of immune cells (e.g., T cells) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is from about 20% to about 50%, from about 20% to about 40%, or from about 20% to about 30%.
  • 10% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 15% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 20% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 25% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 30% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 35% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 40% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 45% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • a therapeutic agent e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • 50% more immune cells have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent.
  • the number of immune cells (e.g., T cells) comprising modified or altered (e.g., edited) genomic DNA can be determined by DNA sequencing.
  • the LNPs described herein can deliver greater amounts of a payload, e.g., a therapeutic agent or a reporter, to a immune cells (e.g., T cells) when administered ex vivo or in vivo to a subject compared to a baseline LNP (e.g., V003).
  • a baseline LNP e.g., V003
  • the targeted LNPs described herein can deliver greater amounts of a payload, e.g., a therapeutic agent or a reporter, to an immune cell (e.g., T cell) when administered in vivo to a subject compared to a baseline LNP (e.g., an LNP comprising V003) comprising the same targeting moieties as the targeted LNP.
  • the LNPs described herein can deliver greater amounts of a nucleic acid, e.g., a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide or one or more nucleic acids comprising a gene modifying system), to an immune cell (e.g., T cell) when administered in vivo to a subject compared to a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid e.g., mRNA
  • an immune cell e.g., T cell
  • a baseline LNP e.g., an LNP comprising V003
  • a nucleic acid payload e.g., a therapeutic agent such as a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system
  • a nucleic acid payload that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) is expressed at higher levels relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 10% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 15% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 20% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 25% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 30% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 35% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 40% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload (e.g., mRNA) that is delivered to immune cells (e.g., T cells) in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 45% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003).
  • a nucleic acid payload e.g., mRNA
  • immune cells e.g., T cells
  • an LNP as described herein e.g., a targeted LNP
  • exhibits 50% higher expression relative to the same payload delivered using a baseline LNP e.g., an LNP comprising V003
  • a nucleic acid payload e.g., mRNA
  • immune cells e.g., T cells
  • an LNP as described herein e.g., a targeted LNP
  • the level of expression of the payload can be determined by measuring protein levels, e.g., by flow cytometry.
  • the expression level of the payload can be measured by determining the level of modification or alteration of the genomic DNA of the immune cells (e.g., T cells) e.g., by DNA sequencing.
  • a lipid nanoparticle (LNP) for delivery of a therapeutic agent to immune cells ex vivo or in vivo comprising: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from the lipids in Table 1, Table 2 and Table 3; and a therapeutic agent encapsulated within the LNP.
  • LNP of embodiment 1, wherein the ionizable lipid has the structure: .
  • LNP of embodiment 1, wherein the ionizable lipid has the structure: .
  • the LNP of embodiment 5, wherein the nucleic acid molecule is an mRNA molecule or a DNA molecule.
  • the LNP of embodiment 5, wherein the nucleic acid molecule is an mRNA molecule.
  • the LNP of embodiment 5, wherein the nucleic acid molecule is a siRNA or miRNA molecule.
  • the LNP of embodiment 5, wherein the therapeutic agent comprises two nucleic acid molecules.
  • the LNP of embodiment 9, wherein the two nucleic acid molecules are RNA molecules.
  • the LNP of embodiment 11, wherein one nucleic acid molecule comprises an mRNA molecule and one nucleic acid molecule comprises a guide RNA.
  • the Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).
  • the mRNA encodes a gene modifying polypeptide.
  • the mRNA encodes a gene modifying polypeptide and the RNA encodes a template RNA, e.g., a template RNA encoding a CAR.
  • the LNP of embodiment 20, wherein the mol% of ionizable lipids in the LNP ranges from about 40% to about 50%.
  • the LNP of embodiment 20, wherein the mol% of ionizable lipids in the LNP ranges from about 45% to about 50%.
  • helper lipid is selected from the group consisting of distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohex
  • the LNP of embodiment 23, wherein the helper lipid is DSPC. [0486] The LNP of embodiment 23, wherein the helper lipid is sphingomeylin. [0487] The LNP of embodiment 25, wherein the sphingomeylin has a head group selected from phosphocholine, phosphoethanolamine and ceramide. [0488] The LNP of embodiment 26, wherein the sphingomeylin is egg sphingomeylin. [0489] The LNP of any one of embodiments 23-27, wherein the mol% of helper lipid is from about 18% to about 32%. [0490] The LNP of embodiment 28, wherein the mol% of helper lipid is from about 20% to about 25%. [0491] The LNP of embodiment 28, wherein the mol% of helper lipid is from about 22% to about 28%.
  • a conjugate for targeted delivery of a therapeutic agent to T cells ex vivo or in vivo comprising: a lipid nanoparticle (LNP) comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from the lipids in Table 1, Table 2 and Table 3; a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties is configured to target T cells.; and a therapeutic agent encapsulated within the LNP.
  • LNP lipid nanoparticle
  • conjugate of any one of embodiments 31-47, wherein the plurality of targeting moieties comprises antibodies or fragments thereof.
  • nucleic acid molecule is an mRNA molecule or a DNA molecule.
  • nucleic acid molecule is a siRNA or miRNA molecule.
  • nucleic acid molecule comprises an mRNA molecule and one nucleic acid molecule comprises an RNA molecule.
  • nucleic acid molecule comprises an mRNA molecule and one nucleic acid comprises a guide RNA.
  • nucleic acid molecule is an mRNA molecule and one nucleic acid molecule is a DNA molecule.
  • nucleic acid molecules encodes at least one component for altering a genome.
  • the at least one component comprises an mRNA encoding a CRISPR-associated nuclease (Cas) and a guide RNA.
  • Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).
  • the therapeutic agent comprises a gene modifying system, e.g., a retrotransposon gene modifying system or a heterologous gene modifying system.
  • the mRNA encodes a gene modifying polypeptide.
  • the mRNA encodes a gene modifying polypeptide and the RNA encodes a template RNA, e.g., a template RNA encoding a CAR.
  • the conjugate of embodiment 58, wherein the guide RNA is a single guide RNA.
  • the helper lipid is selected from the group consisting of distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohe
  • the conjugate of embodiment 79 or embodiment 80, wherein the PEGylated lipids each comprise a lipid component, wherein the lipid component of the PEGylated lipids is selected from the group consisting of DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, stearic acid, and any combination thereof.
  • the lipid component of the PEGylated lipids is DSPE.
  • Example 1 In vitro screen for anti-CD3 targeting moieties
  • a screen was conducted to identify anti-CD3 targeting moieties that can be conjugated to the surface of an LNP to create targeted LNPs (tLNPs) capable of enhanced delivery of a payload (e.g., a therapeutic agent) to T cells.
  • a payload e.g., a therapeutic agent
  • a base LNP was conjugated to select anti-CD3 targeting moieties to generate tLNPs.
  • T Cell Activation and Culture [0581] Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume.
  • the complete T cell media was comprised of CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-2 at 20ng/mL (Miltenyi Biotec #130-097-746), human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362), and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764).
  • TransAct Research Grade T cell activation reagent (Miltenyi Biotec #130-111-160) was added to cell suspension at a volume of 10uL per 1x106 cells.
  • Activated cells were transferred to flasks and incubated at 37°C, 5% CO 2 for 72 hours (“Activated” condition).
  • additional T cells from the same donor were thawed and resuspended in complete T cell media as above, without TransAct stimulation. These cells were transferred to flasks and incubated at 37°C, 5% CO 2 for 24 hours (“Rested” condition).
  • Preparation of T Cells for Transfection [0583] Following the incubation periods above, the activated and rested T cells were collected separately and counted. Two aliquots of 1.5x10 7 cells each were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated.
  • the cell pellets were resuspended in CTS AIM V SFM at a concentration of 1.33x10 6 cells/mL, supplemented with human IL-2, human IL-7, and human IL-15, at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.
  • One aliquot per condition (activated and rested) was resuspended in basal media without serum, while the second aliquot was resuspended in basal media with 5% fetal bovine serum (FBS, Gibco #A31604-01).
  • Targeted LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (V003), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO.
  • aqueous phase was composed of mRNA (encoding eGFP) dissolved in 25 mM acetate buffer.
  • the anti-CD3 targeting moiety was added to the LNP solution at 1:1 ratio of mRNA:targeting moiety.
  • the mixture was incubated at 25 C for 2 hours, followed by overnight incubation at 4 C.
  • Each anti-CD3 targeting moiety was produced with an additional sortase tag (LPETG, SEQ ID NO:11) on the C terminus of the heavy chain (i.e., GGGGSLPETGHHHHHH, SEQ ID NO:17).10uM anti-CD3 Fab- LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction.
  • sortase buffer 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4
  • the resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding eGFP) of 1:1, except where otherwise noted (Table 1).
  • the solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane.
  • the resulting antibody-LNP product was further concentrated using an Amicon column. Table 19.
  • Targeted LNPs were screened with a dose titration ranging from 0.125ug/1x10 6 cells up to 4ug/1x10 6 cells.
  • tLNPs were diluted to a top concentration of 16ug/mL in basal CTS AIM V SFM media (with no serum or cytokines), then serially diluted 1:1 in additional basal CTS AIM V SFM media to the lowest concentration of 0.5ug/mL.
  • tLNPs were further diluted 1 in 4 upon addition to the plated cells, for the final concentration range of 0.125ug/mL to 4ug/mL.
  • LNP Transfection 50uL of diluted tLNPs were added to plated activated and rested T cells, in singlicate, and mixed gently by pipetting. After addition of the tLNPs, all plated cells were at a final density of 1x10 6 cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. Transfected cells were returned to incubation at 37°C, 5% CO 2 overnight.
  • FIGs.2B and 2C show that in the absence of serum, all anti-CD3 targeting moieties screened improved transfection of activated T cells when conjugated to an LNP relative to the base LNP (non-conjugated to an anti-CD3 targeting moiety).
  • the tLNPs with the anti-CD3-8 targeting moiety boosted GFP expression by 75-fold relative to non-targeted LNPs (FIG.2C) and increased the proportion of transfected cells from 53.6% to 99.4% of cells at the highest doses tested (FIG.2B).
  • the tLNPs conjugated to the other tested anti-CD3 targeting moieties also increased both GFP expression and the proportion of GFP+ cells (to GFP- cells) relative to the base LNP, with the tLNPs having the anti-CD3-5 targeting moiety exhibiting the second highest GFP expression levels after the anti-CD3-8 tLNPs (FIGs.2B and 2C).
  • FIGs.2A and 2D when FBS was present, the transfection efficiency in activated T cells appeared more normalized across the anti-CD3 targeting moieties. The increase in MFI between anti-CD3-8 tLNPs and non-targeted base LNPs was less than 4-fold higher.
  • FIG.2D shows that the overall levels of expression were enhanced in the presence of serum for most anti-CD3 targeting moieties screened, except for the anti-CD3-8 and anti- CD3-5 targeting moieties.
  • FIGs.3A-3D show that in rested T cells, all tLNPs conjugated to the anti-CD3 targeting moieties that were screened enhanced transfection efficiency above that of non-targeted base LNPs, both in the presence and absence of serum. As in activated cells, rested cells transfected with tLNPs conjugated to the anti-CD3-8 or the anti-CD3-5 targeting moieties had higher GFP expression in serum-free conditions compared to cells transfected in the presence of FBS.
  • Targeted LNPs comprising the anti-CD3 targeting moieties induced variable expression of the T cell activation marker CD25 (FIG.4A).
  • Anti-CD3-2 was minimally activating, even at the highest dose tested, while anti-CD3-4 and anti-CD3-6 induced high levels of CD25 expression.
  • Targeted LNPs comprising the anti-CD3-1, anti-CD3-5, anti-CD3-7, or anti-CD3-8 targeting moieties induced moderately high levels of CD25 expression.
  • Example 2 In vitro screen for anti-CD2, anti-CD5 and anti-CD7 targeting moieties [0597] A screen was conducted to identify anti-CD2, anti-CD5, and anti-CD7 targeting moieties that can be conjugated to the surface of an LNP to create targeted LNPs (tLNPs) capable of enhanced delivery of a payload (e.g., a therapeutic agent) to T cells. A base LNP was conjugated to select anti-CD2, anti-CD5, or anti-CD7 Fab fragments to generate tLNPs.
  • a payload e.g., a therapeutic agent
  • T cells were cultured and activated as described in Example 1.
  • Targeted LNPs were prepared as described in Example 1 except that the base LNP was conjugated to one of the targeting moieties present in Table 20 (an anti-CD2, anti-CD7, anti-CD5, or anti-CD3 targeting moiety or a CD28 targeting protein derived from the CD80 extracellular domain (“CD80 ECD”) at a 1:1 antibody weight: mRNA weight ratio.
  • both the anti-CD3 targeting moiety and CD80 ECD were both conjugated to the surface of the base LNP at a 1:1 molar ratio (anti-CD3 targeting moiety:CD80 ECD:mRNA) to create dual targeted LNPs.
  • Table 20 provides the targeting moieties that were tested.
  • Anti-CD2, anti-CD5, anti-CD7, and-CD3 targeting moieties and CD80 ECD tested with targeted LNPs were tested.
  • T cells were transfected with LNPs as described in Example 1. Following overnight incubation, cells in plates were pelleted by centrifugation at 800xg for 2 minutes, washed once with 100uL of 1X PBS (Gibco #), then resuspended in 50uL PBS containing LIVE/DEADTM Fixable Near-IR Dead Cell Stain (Invitrogen # L34976) at 1:1,000 dilution, plus the following antibodies: mouse anti-human CD3 PE (clone OKT3, Biolegend # 317308), mouse anti-human CD25 Alexa Fluor 700 (clone BC96, Biolegend # 302622), mouse anti-human CD69 APC (clone FN50, Biolegend # 310910)and mouse anti-human LDLR PE (clone C7, BD Biosciences # 565653).
  • FIGs.5A-5D show that all of the anti-CD2, anti-CD7, anti-CD3, and anti-CD5 targeted tLNPs outperform non-targeted base LNPs in activated T cells, either when the cells were cultured with serum (FBS) or without serum. All of the tLNPs conjugated to anti-CD7 targeting moieties drove higher GFP expression when transfected in activated T cells in the presence or absence of serum compared to tLNPs conjugated with anti-CD2 targeting moieties, especially at higher LNP concentrations (FIGs.5C-5D).
  • FIGs.6A-6D show that targeted LNPs conjugated to anti-CD7, anti-CD5, and anti-CD3 targeting moieties outperformed non-targeted base LNPs, both with and without serum.
  • FIGs.6C and 6D show that the tLNPs with the anti-CD3 targeting moiety resulted in an average four-fold higher GFP expression than the tLNPs conjugated to the anti-CD7 targeting moieties.
  • Dual targeted LNPs comprising both the anti-CD3 targeting moiety and the CD80 extracellular domain (ECD) on their surface boosted transfection capacity in both activated and resting T cells relative to the other tLNPs tested (FIGs.5A-5D and FIGs.6A-6D).
  • the tLNPs with the CD80 ECD alone provided a minimal increase to transfection efficiency compared to non-targeted LNPs
  • the tLNPs with both the anti-CD3 targeting moiety and the CD80 ECD increased GFP expression at least two-fold compared to tLNPs with the anti-CD3 targeting moiety alone in all conditions tested.
  • the dual targeted anti-CD3/CD80 ECD tLNPs generated higher GFP expression levels in activated T cells without serum and in the rested T cells with or without serum (FIGs.5C, 6C, and 6D).
  • FIGs.7A and 7B show that dual targeted LNPs conjugated to the anti-CD3 targeting moiety and the CD80 ECD were also capable of activating rested T cells. These dual tLNPs drove increased expression of surface activation markers CD25 and CD69 relative to non-targeted base LNPs, and increased expression of the activation markers by at least two-fold relative to tLNPs with the anti-CD3 targeting moiety alone.
  • a dual targeted LNP comprising two different anti-CD3 targeting moieties similarly increased expression of the CD25 and CD69 activation markers relative to the base LNP and tLNPs with a single anti-CD3 targeting moiety (FIGs.7A-7B). None of the tLNPs conjugated to the anti-CD2, anti-CD7, or anti-CD5 targeting moieties increased the activation markers.
  • Example 3 Screening for LNPs comprising novel ionizable lipids that enhance delivery to activated and resting T cells [0606] A screen was conducted to identify novel ionizable lipids that can be formulated in LNPs to enhance delivery of a payload to human T cells.
  • Base LNPs (lacking targeting moieties) were formulated with Lipid092, Lipid110, Lipid133, Lipid134, or Lipid154, and an mRNA encoding GFP, and then the transduction efficiency of each LNP was tested in activated and resting T cells. LNPs containing the V003, SM102, or Lipid177 ionizable lipids were also generated to establish baseline transduction controls for the purpose of comparing transduction efficiency.
  • T Cell Activation and Culture [0607] Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume.
  • the complete T cell media was comprised of CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-2 at 20ng/mL (Miltenyi Biotec #130-097-746), human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362), and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764).
  • TransAct Research Grade T cell activation reagent (Miltenyi Biotec #130-111-160) was added to cell suspension at a volume of 10uL per 1x106 cells.
  • Activated cells were transferred to flasks and incubated at 37°C, 5% CO 2 for 72 hours (“Activated” condition).
  • additional T cells from the same donor were thawed and resuspended in complete T cell media as above, without TransAct stimulation. These cells were transferred to flasks and incubated at 37°C, 5% CO 2 for 24 hours (“Rested” condition).
  • Preparation of T Cells for Transfection [0609] Following the incubation periods above, the activated and rested T cells were collected separately and counted.2.0x10 7 cells were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated.
  • the cell pellets were resuspended in CTS AIM V SFM at a concentration of 1.33x10 6 cells/mL, supplemented with 6.67% fetal bovine serum (FBS, Gibco #A31604-01), and human IL-2, human IL7, and human IL-15, at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.
  • FBS fetal bovine serum
  • human IL-2, human IL7, and human IL-15 at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.
  • the resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates (Falcon # 08-772-54) at a density of 2x10 5 cells (150uL) per well, and returned to incubation at 37°C, 5% CO 2 while the LNPs were prepared.
  • LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid092, Lipid110, Lipid133, Lipid134, Lipid154, Lipid177, V003, or SM102), 8% DSPC, 42.5% cholesterol, 2% DMG-PEG2K and 0.5% DSPE-PEG2K- TCO. An aqueous phase was composed of mRNA (encoding eGFP) dissolved in 25 mM acetate buffer.
  • ipid092, Lipid110, Lipid133, Lipid134, Lipid154, Lipid177, V003, or SM102 8% DSPC, 42.5% cholesterol, 2% DMG-PEG2K and 0.5% DSPE-PEG2K- TCO.
  • An aqueous phase was composed of mRNA (encoding eGFP) dissolved in 25 mM acetate buffer.
  • the LNPs were then subjected to overnight dialysis in Tris/sucrose buffer at 4 C followed by centrifugation (100k MWCO) and/or via Tangential Flow Filtration (TFF) to the desired concentration.
  • LNP Transfection 50uL of diluted LNPs were added to plated activated and rested T cells at 100ng, 200ng, 400ng, 600ng and 800ng of LNP per 2 x10 5 cells, and mixed gently by pipetting. After addition of the LNPs, all plated cells were at a final density of 1x10 6 cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. Transfected cells were returned to incubation at 37°C, 5% CO 2 overnight.
  • FIG.8A shows that close to 100% of activated T cells transfected with LNPs comprising Lipid092 or Lipid154 expressed GFP, starting at the lowest dose. Fewer T cells were transfected with the other LNPs tested, including the baseline control LNPs, across all dose levels.
  • FIG.8B shows that transfection with Lipid154 LNPs resulted in the highest GFP expression levels (MFI) in the cells, followed by LNPs comprising Lipid092.
  • MFI GFP expression levels
  • the MFI levels increased in a dose-dependent fashion for these LNPs.
  • Activated T cells transfected with the other LNPs exhibited much lower GFP expression levels at all doses tested.
  • the base LNPs formulated with Lipid092 and Lipid154 also exhibited the highest transfection levels in rested T cells out of all the LNPs tested.
  • FIG.8C shows that Lipid092 and Lipid154 LNPs transfected the largest numbers of cells at the 100ng to 400 ng doses (per 2x10 5 cells), but then the percentage of GFP+ cells fell at higher doses of the Lipid154 LNP.
  • FIG.8D shows that transfection of LNPs with Lipid092 GFP expression resulted in the highest levels of GFP expression at most doses, followed by LNPs with Lipid154.
  • LNPs formulated with V003 induced the lowest GFP expression levels out of all LNPs tested.
  • Example 4 Testing of ionizable lipids for LNP delivery of an exemplary gene modifying system to activated T cells in vitro
  • the Lipid092 and Lipid154 ionizable lipids that exhibited enhanced delivery to T cells in the lipid screen described in Example 3 were next formulated in base LNPs with a gene modifying system comprised of mRNA encoding an exemplary gene modifying polypeptide and a template RNA encoding GFP for use with the gene modifying polypeptide.
  • Activated T cells were then transfected to determine whether these base LNPs could deliver the gene modifying system to activated T cells to enable insertion of the heterologous GFP gene into the genomic DNA of the cells.
  • T Cell Activation and Culture Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume. Cells were counted on a Cellometer K2 Cell Counter (Nexcelom), then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10 6 cells/mL.
  • the complete T cell media was comprised of CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-2 at 20ng/mL (Miltenyi Biotec #130-097-746), human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362), and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764).
  • TransAct Research Grade T cell activation reagent (Miltenyi Biotec #130-111-160) was added to cell suspension at a volume of 10uL per 1x106 cells.
  • Activated cells were transferred to flasks and incubated at 37°C, 5% CO 2 for 72 hours (“Activated” condition).
  • Preparation of T Cells for Transfection [0618] Following the incubation periods above, the activated T cells were collected separately and counted.2.0x10 7 cells were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated.
  • the cell pellets were resuspended in CTS AIM V SFM at a concentration of 1.33x10 6 cells/mL, supplemented with 6.67% fetal bovine serum (FBS, Gibco #A31604-01), and human IL-2, human IL-7, and human IL-15, at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.
  • FBS fetal bovine serum
  • human IL-2, human IL-7, and human IL-15 at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.
  • the resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates (Falcon # 08-772-54) at a density of 2x10 5 cells (150uL) per well, and returned to incubation at 37 C, 5% CO 2 while the LNPs were prepared.
  • LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid092, Lipid154, or V003), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA encoding the exemplary gene modifying polypeptide and a template RNA encoding GFP dissolved in 25 mM acetate buffer.
  • the LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).
  • TFF Tangential Flow Filtration
  • centrifugation 100k MWCO
  • the gene modifying polypeptide comprised the amino acid sequence of: MDSTAHPNQGRGLEKVSQTLPALQTPGQHTAAGGSSPLSGRNQRKNTKKLLLGAWNIR TLLDRENTPRPERRTALIGKELARYNIDIAALSETRLPEEGSLSEPTTGYTFFWKGRASNE DRIHGVGLAIKTSLLKQLPDLPVGISERLMKIRLPLSKDRYATIISAYAPTLTSTEETIEQFY SDLSAVLHSVPTNDKLILLGDFNARVGQDHERWKGVLGKHGVGKMNNNGLLLLSKCS EFELTITNTVFRMANKYKTTWMHPRSKQWHLIDYIIVRRRDIQDVKITRAMRGAECWT DHRLVRATLQMRIAPRHPKRAQTVRAFYNVSRLRDPSYLQTFQSCLDDKLSAKGPLTGS STEKWNQFRDAVKETSKAVLGPKQRNHQDWFDENNTAIEDLLSKKNKAFMEWQNNPN SAPKKDRFKSL
  • the gene modifying system further comprised an exemplary template RNA comprising the following sequences from 5’ to 3’: 5’UTR retro , GFP reporter, MND promoter, 3’UTR retro .
  • the template RNA comprised a 5’UTR having the sequence of: GGGUGUAUGGGGUGCUCAGGGAGGGGUAGUAUCUCUGGUAUGGAGGGCUUGUCG UGCCCUCCUAGGGCAGCUCUCUCCAGCCUCUGACCCCCACCUGACACCCAGCUCUCAC UUGUGGCUCCCAGUAGCUGCUAGCAUGUGGCAGCGGCCACACCCCGGGCAACGGC UUCGACAGGCCGGCUAAACCUUGUGAGGGUAGCCAUCGGGUCAUCGACCCCUGGU GAACCAGGGCUUUGCUCACCCAGCAUGUGAAGACUGCUUCGGCUGAACAGACGGA AGAAACCAAUAAGAAGGUUCAACGGCUGAGAGGGCGACGCAGCAAAGCACUGUG GAGUGCUUAGGGCGUGUUGGAGCACAAAGGACAACACGGCCAUCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCACUGG
  • FIG.9A shows that at 4 days following transfection, substantially more activated T cells expressed GFP at all doses when Lipid092 LNPs or Lipid154 LNPs delivered the gene modifying system compared to activated T cells that were contacted with the V003 LNPs, with the Lipid154 LNPs showing the highest levels of delivery.
  • the peak dose for the LNPs was at 2.5 ⁇ g per 1 x10 6 cells.
  • FIG.9B shows that activated T cells transduced with the Lipid092 or Lipid154 expressed GFP at higher levels (higher MFI) relative to activated T cells transduced with LNPs comprising the V003 ionizable lipid.
  • LNPs formulated with the Lipid092, Lipid154 or V003 ionizable lipids were all capable of delivering an exemplary all-RNA gene modifying system to activated T cells to insert a heterologous gene encoded by template RNA.
  • LNPs formulated with Lipid092 or Lipid154 can deliver a gene modifying system more effectively to activated T cells to enable higher insertion rates of a heterologous gene into genomic DNA.
  • Example 5 Testing of ionizable lipids for targeted LNP delivery of an exemplary gene modifying system to activated T cells in vitro [0636] Base LNPs formulated with the Lipid092 and Lipid154 ionizable lipids were conjugated to the anti-CD3-8 targeting moiety, as described in Example 1, to generate targeted LNPs.
  • the tLNPs were then tested to determine whether they could more effectively deliver a gene modifying system (mRNA encoding an exemplary gene modifying polypeptide and a template RNA encoding GFP) to activated T cells relative to the base LNPs.
  • the efficiency of LNP delivery and insertion of the heterologous GFP sequence encoded by the template RNA using these tLNPs was compared to the efficiency of delivery and GFP insertion using a tLNP formulated with the V003 ionizable lipid.
  • Dual targeted LNPs were also generated that comprised the Lipid092 ionizable lipid conjugated to both the anti-CD3-8 targeting moiety and a CD80 ECD and compared to the tLNPs conjugated to a single targeting moiety.
  • T Cell Activation and Culture Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume. Cells were counted on a Cellometer K2 Cell Counter (Nexcelom), then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10 6 cells/mL.
  • the complete T cell media was comprised of CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-2 at 20ng/mL (Miltenyi Biotec #130-097-746), human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362), and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764).
  • TransAct Research Grade T cell activation reagent (Miltenyi Biotec #130-111-160) was added to cell suspension at a volume of 10uL per 1x106 cells.
  • Activated cells were transferred to flasks and incubated at 37°C, 5% CO 2 for 72 hours (“Activated” condition).
  • Preparation of T Cells for Transfection [0638] Following the incubation periods above, the activated T cells were collected separately and counted.2.0x10 7 cells were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated.
  • the cell pellets were resuspended in CTS AIM V SFM at a concentration of 1.33x10 6 cells/mL, supplemented with 6.67% fetal bovine serum (FBS, Gibco #A31604-01), and human IL-2, human IL-7, and human IL-15, at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.
  • FBS fetal bovine serum
  • human IL-2, human IL-7, and human IL-15 at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.
  • the resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates (Falcon # 08-772-54) at a density of 2x10 5 cells (150uL) per well, and returned to incubation at 37°C, 5% CO 2 while the LNPs were prepared.
  • LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid092, Lipid154, or V003), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA encoding the exemplary gene modifying polypeptide and a template RNA encoding GFP dissolved in 25 mM acetate buffer.
  • the LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).
  • the anti-CD3-8 targeting moiety was produced with an additional sortase tag (LPETG, SEQ ID NO:11) on the C terminus of the heavy chain.
  • 10uM anti-CD3 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm.
  • the resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding eGFP) of 1:1, except where otherwise noted (Table 19).
  • the solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane.
  • the resulting antibody-LNP product was further concentrated using an Amicon column.
  • the anti-CD3-8 Fab and the CD80 ECD were produced with sortase tags and conjugated to the surface of the base LNP as described above, but at a 1:1:1 molar ratio (anti-CD3:CD80 ECD:mRNA) to create the dual targeted LNPs.
  • the gene modifying system formulated in the LNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprised the following components.
  • the gene modifying polypeptide comprised the amino acid sequence of: MDSTAHPNQGRGLEKVSQTLPALQTPGQHTAAGGSSPLSGRNQRKNTKKLLLGAWNIR TLLDRENTPRPERRTALIGKELARYNIDIAALSETRLPEEGSLSEPTTGYTFFWKGRASNE DRIHGVGLAIKTSLLKQLPDLPVGISERLMKIRLPLSKDRYATIISAYAPTLTSTEETIEQFY SDLSAVLHSVPTNDKLILLGDFNARVGQDHERWKGVLGKHGVGKMNNNGLLLLSKCS EFELTITNTVFRMANKYKTTWMHPRSKQWHLIDYIIVRRRDIQDVKITRAMRGAECWT DHRLVRATLQMRIAPRHPKRAQTVRAFYNVSRLRDPSYLQTFQSCLDDKLSAKGPLTGS STEKWNQFRDAVKETSKAVLGPKQRNHQDWFDENNTAIEDLLSKKNKAFMEWQNNPN SAPKKDRFKSL
  • the full- length sequences are provided in Table 21 (Example 4).
  • the gene modifying system further comprised a template RNA comprising the following sequences from 5’ to 3’: 5’UTR retro , GFP reporter, MND promoter, 3’UTR retro .
  • the template RNA comprised a 5’UTR having the sequence of: GGGUGUAUGGGGUGCUCAGGGAGGGGUAGUAUCUCUGGUAUGGAGGGCUUGUCG UGCCCUCCUAGGGCAGCUCUCUCCAGCCUCUGACCCCCACCUGACACCCAGCUCUCAC UUGUGGCUCCCAGUAGCUGCUAGCAUGUGGCAGCGGCCACACCCCGGGCAACGGC UUCGACAGGCCGGCUAAACCUUGUGAGGGUAGCCAUCGGGUCAUCGACCCCUGGU GAACCAGGGCUUUGCUCACCCAGCAUGUGAAGACUGCUUCGGCUGAACAGACGGA AGAAACCAAUAAGAAGGUUCAACGGCUGAGAGGGCGACGCAGCAAAGCACUGUG GAGUGCUUAGGGCGUGUUGGAGCACAAAGGACAACACGGCCAUCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCACUGG
  • tLNPs After addition of the tLNPs, all plated cells were at a final density of 1x10 6 cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL- 2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL.
  • Transfected cells were returned to incubation at 37°C, 5% CO 2 for 4 hours. After the 4 hour tLNP incubation, cells were centrifuged at 350xg for 7min, supernatant aspirated, and cells resuspended in complete T cell media as described above at 1e6 cells/mL in flat bottom plates.
  • FIG.10A shows that targeted LNPs formulated with Lipid092 and Lipid154 both delivered the exemplary gene modifying system more effectively to activated T cells than the baseline tLNP formulated with the V003 lipid.
  • FIG.10A shows that at 4 days following transfection, substantially more activated T cells expressed GFP at all doses when Lipid092 tLNPs or Lipid154 tLNPs delivered the gene modifying system relative to activated T cells that were contacted with the V003 tLNPs.
  • FIG.10B shows that activated T cells transduced with the Lipid092 or Lipid154 tLNPs expressed GFP at higher levels (higher MFI) relative to activated T cells transduced with tLNPs comprising the V003 ionizable lipid.
  • All tLNPs tested (comprising the Lipid092, Lipid154, or V003 ionizable lipids) delivered the gene modifying system to substantially more activated T cells, resulting in increases in GFP insertion into the genomic DNA of T cells and GFP expression, relative to the base LNPs formulated without the CD3 targeting moiety (compare FIGs.9A-9B to FIGs.10A- 10B).
  • the addition of the anti-CD3 targeting moiety to the surface of the LNPs increases delivery of a gene modifying system payload to activated T cells.
  • Example 6 Targeted LNP delivery of an exemplary gene modifying system to human T cells in a humanized mouse model
  • Targeted LNPs formulated with the Lipid092 ionizable lipid were tested to determine whether they could deliver a gene modifying system comprising mRNA encoding an exemplary gene modifying polypeptide and a template RNA encoding GFP to human T cells in a humanized mouse model (na ⁇ ve human T cells engrafted 2-3 weeks prior to study) effectively enough to insert the exogenous GFP gene into the genomic DNA of the T cells.
  • the tLNPs were conjugated to the anti-CD3-8 targeting moiety, as described in Example 1.
  • LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid092), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA encoding the gene modifying polypeptide and a template RNA encoding GFP dissolved in 25 mM acetate buffer.
  • the LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).
  • the anti-CD3-8 targeting moiety was produced with an additional sortase tag (LPETG, SEQ ID NO:11) on the C terminus of the heavy chain.
  • 10uM anti-CD3 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm.
  • the resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for targeting moiety-LNP conjugation, with a mass ratio between targeting moiety and mRNA (encoding eGFP) of 1:1, except where otherwise noted (Table 1).
  • the solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane.
  • the resulting targeting moiety-LNP product was further concentrated using an Amicon column.
  • the gene modifying system formulated in the LNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprised the following components.
  • the gene modifying polypeptide comprised the amino acid sequence of: MDSTAHPNQGRGLEKVSQTLPALQTPGQHTAAGGSSPLSGRNQRKNTKKLLLGAWNIR TLLDRENTPRPERRTALIGKELARYNIDIAALSETRLPEEGSLSEPTTGYTFFWKGRASNE DRIHGVGLAIKTSLLKQLPDLPVGISERLMKIRLPLSKDRYATIISAYAPTLTSTEETIEQFY SDLSAVLHSVPTNDKLILLGDFNARVGQDHERWKGVLGKHGVGKMNNNGLLLLSKCS EFELTITNTVFRMANKYKTTWMHPRSKQWHLIDYIIVRRRDIQDVKITRAMRGAECWT DHRLVRATLQMRIAPRHPKRAQTVRAFYNVSRLRDPSYLQTFQSCLDDKLSAKGPLTGS STEKWNQFRDAVKETSKAVLGPKQRNHQDWFDENNTAIEDLLSKKNKAFMEWQNNPN SAPKKDRFKSL
  • the full- length sequences are provided in Table 21 (Example 4).
  • the gene modifying system further comprised a template RNA comprising the following sequences from 5’ to 3’: 5’UTR retro , GFP reporter, MND promoter, 3’UTR retro .
  • the template RNA comprised a 5’UTR having the sequence of: GGGUGUAUGGGGUGCUCAGGGAGGGGUAGUAUCUCUGGUAUGGAGGGCUUGUCG UGCCCUCCUAGGGCAGCUCUCUCCAGCCUCUGACCCCCACCUGACACCCAGCUCUCAC UUGUGGCUCCCAGUAGCUGCUAGCAUGUGGCAGCGGCCACACCCCGGGCAACGGC UUCGACAGGCCGGCUAAACCUUGUGAGGGUAGCCAUCGGGUCAUCGACCCCUGGU GAACCAGGGCUUUGCUCACCCAGCAUGUGAAGACUGCUUCGGCUGAACAGACGGA AGAAACCAAUAAGAAGGUUCAACGGCUGAGAGGGCGACGCAGCAAAGCACUGUG GAGUGCUUAGGGCGUGUUGGAGCACAAAGGACAACACGGCCAUCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCACUGG
  • mice Following acclimation to the vivarium for 3-5 days, animals received a tail vein injection of 5 X 10 6 human CD3+ T cells (Source) and waited for 2-3 weeks for T cell engraftment.
  • “humanized” NSG mice were randomized into groups of 3-4 animals and baseline weights were obtained. Animals were then treated via intravenous tail vein injection with 100 ul of candidate tLNPs formulated with gene modifying system at a dose of 4 mg/kg. Following a 16-hour incubation, animals were euthanized via CO2 asphyxiation, weighed and their spleens were collected and the splenic cells were isolated.
  • FIG.11 shows that the tLNPs carrying an all-RNA gene modifying system generated human T cells expressing GFP in the engrafted mice, as determined by flow cytometry.
  • 11A shows that an average of at least 5 percent of human cells expressed GFP, including about 5 percent of human CD4+ T cells and about 5 percent of CD8+ T cells, when the gene modifying polypeptide mRNA and the GFP template RNA were formulated within the same tLNP.
  • the percentages of GFP positive cells were slightly lower, on average, when the gene modifying polypeptide mRNA and the GFP template RNA were formulated in separate tLNPs.
  • FIG.11B shows that engrafted human cells, including CD4+ and CD8+ T cells, expressed GFP at higher levels when tLNPs comprising the gene modifying system were administered to the humanized mice compared to engrafted cells in mice that were administered the negative control tLNP.
  • Example 7 Improved LNP formulation for delivery of an exemplary gene modifying system to activated T cells
  • Alternative formulations of targeted LNPs were tested to determine whether they could improve delivery of a gene modifying system payload to activated T cells, leading to increased insertion of an exogenous gene in the cells.
  • LNPs were formulated with the Lipid154 ionizable lipid and a higher amount of helper lipid (22% DSCP or sphingomyelin) relative to the LNPs in prior examples (formulated with 8% helper lipid) and then conjugated to the anti-CD3-8 targeting moiety, as described in Example 1.
  • the LNPs were formulated with an all-RNA gene modifying system composed of an mRNA encoding an exemplary gene modifying polypeptide and a template RNA encoding GFP.
  • a baseline control anti-CD3 tLNP comprising Lipid154 and 8% helper lipid (8% DSPC) was also prepared as described in prior examples.
  • Negative control tLNPs were formulated to carry either the gene modifying polypeptide mRNA payload only or a GFP template RNA payload only.
  • T Cell Activation and Culture [0675] Cryopreserved primary T cells from two human donors (HemaCare, donor D328798 and donor DXXX405) were thawed and transferred into PBS with 2% BSA by volume.
  • the complete T cell media was comprised of CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-2 at 20ng/mL (Miltenyi Biotec #130-097-746), human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362), and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764).
  • TransAct Research Grade T cell activation reagent (Miltenyi Biotec #130-111-160) was added to cell suspension at a volume of 10uL per 1x10 6 cells.
  • Activated cells were transferred to flasks and incubated at 37°C, 5% CO 2 for 72 hours.
  • Preparation of T Cells for Transfection [0676] Following the incubation periods above, the activated T cells were collected separately and counted.2.0x10 7 cells were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated.
  • the cell pellets were resuspended in CTS AIM V SFM at a concentration of 2x10 6 cells/mL, supplemented with 10% fetal bovine serum (FBS, Gibco #A31604-01), and human IL-2, human IL-7, and human IL-15, at concentrations of 40ng/mL, 20ng/mL and 10ng/mL, respectively.
  • FBS fetal bovine serum
  • human IL-2, human IL-7, and human IL-15 at concentrations of 40ng/mL, 20ng/mL and 10ng/mL, respectively.
  • the resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates (Falcon # 08-772-54) at a density of 2x10 5 cells (100uL) per well, and returned to incubation at 37°C, 5% CO 2 while the LNPs were prepared.
  • LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid154), 22% helper lipid (DSPC or egg sphingomyelin), 28.5% cholesterol, 2% DMG-PEG-2K and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA encoding the gene modifying polypeptide and a template RNA encoding GFP dissolved in 25 mM acetate buffer.
  • lipids containing 47% ionizable lipid (Lipid154), 22% helper lipid (DSPC or egg sphingomyelin), 28.5% cholesterol, 2% DMG-PEG-2K and 0.5% DSPE-PEG2K-TCO.
  • An aqueous phase was composed of mRNA encoding the gene modifying polypeptide and a template RNA encoding GFP dissolved in 25 mM acetate buffer.
  • the LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).
  • the control tLNP was prepared using the same approach, except the ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid154), 8% DSPC, 42.5% cholesterol, 2% DMG-PEG2K and 0.5% DSPE-PEG2K-TCO.
  • the negative control tLNPs an aqueous phase containing either the gene modifying polypeptide mRNA only or the GFP template RNA only was mixed with the ethanol phase followed by the process described earlier.
  • the anti-CD3-8 targeting moiety was produced with an additional sortase tag (LPETG, SEQ ID NO:11) on the C terminus of the heavy chain.
  • 10uM anti-CD3 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm.
  • the resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding eGFP) of 1:1, except where otherwise noted (Table 19).
  • the solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane.
  • the resulting antibody-LNP product was further concentrated using an Amicon column.
  • the gene modifying system formulated in the LNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprised the following components.
  • the gene modifying polypeptide comprised the amino acid sequence of: MDSTAHPNQGRGLEKVSQTLPALQTPGQHTAAGGSSPLSGRNQRKNTKKLLLGAWNIR TLLDRENTPRPERRTALIGKELARYNIDIAALSETRLPEEGSLSEPTTGYTFFWKGRASNE DRIHGVGLAIKTSLLKQLPDLPVGISERLMKIRLPLSKDRYATIISAYAPTLTSTEETIEQFY SDLSAVLHSVPTNDKLILLGDFNARVGQDHERWKGVLGKHGVGKMNNNGLLLLSKCS EFELTITNTVFRMANKYKTTWMHPRSKQWHLIDYIIVRRRDIQDVKITRAMRGAECWT DHRLVRATLQMRIAPRHPKRAQTVRAFYNVSRLRDPSYLQTFQSCLDDKLSAKGPLTGS STEKWNQFRDAVKETSKAVLGPKQRNHQDWFDENNTAIEDLLSKKNKAFMEWQNNPN SAPKKDRFKSL
  • the full- length sequences are provided in Table 21 (Example 4).
  • the gene modifying system further comprised a template RNA comprising the following sequences from 5’ to 3’: 5’UTR retro , GFP reporter, MND promoter, 3’UTR retro .
  • the template RNA comprised a 5’UTR having the sequence of: GGGUGUAUGGGGUGCUCAGGGAGGGGUAGUAUCUCUGGUAUGGAGGGCUUGUCG UGCCCUCCUAGGGCAGCUCUCUCCAGCCUCUGACCCCCACCUGACACCCAGCUCUCAC UUGUGGCUCCCAGUAGCUGCUAGCAUGUGGCAGCGGCCACACCCCGGGCAACGGC UUCGACAGGCCGGCUAAACCUUGUGAGGGUAGCCAUCGGGUCAUCGACCCCUGGU GAACCAGGGCUUUGCUCACCCAGCAUGUGAAGACUGCUUCGGCUGAACAGACGGA AGAAACCAAUAAGAAGGUUCAACGGCUGAGAGGGCGACGCAGCAAAGCACUGUG GAGUGCUUAGGGCGUGUUGGAGCACAAAGGACAACACGGCCAUCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCACUGG
  • Negative control tLNPs were dosed at 2.5 ⁇ g of LNP per 1 x10 6 cells. After addition of the tLNPs, all plated cells were at a final density of 1x10 6 cells/mL in 200uL, with a final concentration of 5% FBS (for serum- containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. Transfected cells were returned to incubation at 37°C, 5% CO 2 for 4 hours.
  • FIG.12A-D show that delivery of a gene modifying system payload to activated cells using targeted LNPs formulated with Lipid154 and 22% DSPC generated more cells that expressed GFP (%GFP+) and at higher levels (MFI) relative to the baseline control tLNP that was the identical except that it was formulated with 8% DSPC.
  • FIGs.12A and B At four days (FIGs.12A and B) and at 7 days (FIGs.12C and D) following transfection of tLNPs comprising 22% DSPC at all doses tested, more activated T cells expressed GFP at higher levels relative to the cells transfected with tLNPs comprising 8% DSPC.
  • the differences in percent GFP+ cells and MFI were especially large at lower doses.
  • the tLNPs formulated with 22% egg sphingomyelin also generated more cells expressing GFP relative to the tLNPs formulated with 8% DSPC at 4 days and 7 days post-transfection, but the expression differences were not as large when cells were transfected with tLNPs comprising 22% DSPC.
  • the negative control tLNPs comprising the gene modifying polypeptide mRNA alone or the GFP template RNA alone did not produce any GFP-expressing cells.
  • delivery of a gene modifying system to activated T cells using tLNPs comprising higher helper lipid levels can increase the number of cells that are engineered by the gene modifying system to comprise genomic integration of an exogenous nucleic acid sequence.
  • Example 8 Improved LNP formulation for delivery of an exemplary gene writer system to resting T cells [0693]
  • the tLNP formulations comprising higher helper lipid of Example 7 were tested to determine whether they could improve delivery of a gene modifying system payload to rested T cells, leading to increased levels of exogenous gene insertion in the cells.
  • LNPs were formulated with the Lipid154 ionizable lipid and a higher amount of helper lipid (22% DSPC or sphingomyelin) relative to the LNPs in prior examples (formulated with 8% helper lipid) and then conjugated to the anti-CD3-8 targeting moiety, as described in Example 1.
  • the LNPs were formulated with an all-RNA gene modifying system composed of an mRNA encoding an exemplary gene modifying polypeptide and a template RNA encoding GFP.
  • Negative control tLNPs were also formulated with either the gene modifying polypeptide mRNA payload alone or a GFP template RNA payload alone, as described in Example 7.
  • T Cell Culture [0694] Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume.
  • T cells were counted on a Cellometer K2 Cell Counter (Nexcelom), then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10 6 cells/mL.
  • the T cells were resuspended and cultured overnight in CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362) and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764).
  • T Cells for Transfection All cells were pelleted by centrifugation and the supernatant aspirated. The cell pellets were resuspended in CTS AIM V SFM at a concentration of 2x10 6 cells/mL. The resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates (Falcon # 08-772-54) at a density of 2x105 cells (100uL) per well, and returned to incubation at 37°C, 5% CO 2 while the LNPs were prepared. Preparation of Targeted LNPs for Transfection [0696] LNPs were formulated as follows.
  • An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid154), 22% helper lipid (DSPC or egg sphingomyelin), 28.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO.
  • An aqueous phase was composed of mRNA encoding the gene modifying polypeptide and a template RNA encoding GFP dissolved in 25 mM acetate buffer.
  • the LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).
  • the base and baseline control tLNPs were prepared using the same approach, except the ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid154), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO.
  • the negative control tLNPs an aqueous phase containing either the gene modifying polypeptide mRNA only or the GFP template RNA only was mixed with the ethanol phase.
  • the anti-CD3-8 targeting moiety was produced with an additional sortase tag (LPETG, SEQ ID NO:11) on the C terminus of the heavy chain.
  • 10uM anti-CD3 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm.
  • the resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding eGFP) of 1:1, except where otherwise noted (Table 1).
  • the solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane.
  • the resulting antibody-LNP product was further concentrated using an Amicon column.
  • the gene modifying system formulated in the LNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprised the following components.
  • the gene modifying polypeptide comprised the amino acid sequence of: MDSTAHPNQGRGLEKVSQTLPALQTPGQHTAAGGSSPLSGRNQRKNTKKLLLGAWNIR TLLDRENTPRPERRTALIGKELARYNIDIAALSETRLPEEGSLSEPTTGYTFFWKGRASNE DRIHGVGLAIKTSLLKQLPDLPVGISERLMKIRLPLSKDRYATIISAYAPTLTSTEETIEQFY SDLSAVLHSVPTNDKLILLGDFNARVGQDHERWKGVLGKHGVGKMNNNGLLLLSKCS EFELTITNTVFRMANKYKTTWMHPRSKQWHLIDYIIVRRRDIQDVKITRAMRGAECWT DHRLVRATLQMRIAPRHPKRAQTVRAFYNVSRLRDPSYLQTFQSCLDDKLSAKGPLTGS STEKWNQFRDAVKETSKAVLGPKQRNHQDWFDENNTAIEDLLSKKNKAFMEWQNNPN SAPKKDRFKSL
  • the full- length sequences are provided in Table 21 (Example 4).
  • the gene modifying system further comprised a template RNA comprising the following sequences from 5’ to 3’: 5’UTR retro , GFP reporter, MND promoter, 3’UTR retro .
  • the template RNA comprised a 5’UTR having the sequence of: GGGUGUAUGGGGUGCUCAGGGAGGGGUAGUAUCUCUGGUAUGGAGGGCUUGUCG UGCCCUCCUAGGGCAGCUCUCUCCAGCCUCUGACCCCCACCUGACACCCAGCUCUCAC UUGUGGCUCCCAGUAGCUGCUAGCAUGUGGCAGCGGCCACACCCCGGGCAACGGC UUCGACAGGCCGGCUAAACCUUGUGAGGGUAGCCAUCGGGUCAUCGACCCCUGGU GAACCAGGGCUUUGCUCACCCAGCAUGUGAAGACUGCUUCGGCUGAACAGACGGA AGAAACCAAUAAGAAGGUUCAACGGCUGAGAGGGCGACGCAGCAAAGCACUGUG GAGUGCUUAGGGCGUGUUGGAGCACAAAGGACAACACGGCCAUCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCACUGG
  • Negative control tLNPs were dosed at 2.5 ⁇ g of LNP per 1 x10 6 cells.
  • TransAct Research Grade T cell activation reagent (Miltenyi Biotec #130-111-160) was also added to the rested T cell suspension with the tLNPs at a volume of 10uL per 1x10 6 cells.
  • all plated cells were at a final density of 1x10 6 cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL.
  • Transfected cells were returned to incubation at 37°C, 5% CO 2 for four hours.
  • cells were centrifuged at 350xg for 7min and resuspended in CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362) and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764) at 1e6 cells/mL and returned to 37°C, 5% CO 2 incubation.
  • FIG.13A -C show that delivery of a gene modifying system payload to rested T cells using targeted LNPs formulated with Lipid154 and a higher percentage of helper lipid (22% DSPC) generated many more cells that expressed GFP (%GFP+) and at higher levels (MFI) relative to the baseline control tLNP that was the identical except that it was formulated with 8% DSPC.
  • FIG.13A At four days (FIG.13A) and at 7 days (FIG.13B) following transfection of tLNPs comprising higher helper lipid (22% DSPC) at all doses tested, more rested T cells expressed GFP at higher levels relative to the cells transfected with Lipid154 tLNPs comprising 8% DSPC.
  • the tLNPs formulated with 22% egg sphingomyelin also generated more cells expressing GFP relative to the tLNPs formulated with 8% DSPC at 4 days and 7 days post-transfection, but the effects were not as pronounced compared to the effects generated using tLNPs formulated with 22% DSPC.
  • FIG.13C shows that the GFP+ cells generated using tLNPs comprising 22% DSPC expressed the GFP at higher levels than cells transfected with the baseline tLNPs.
  • the negative control tLNPs comprising a gene modifying polypeptide mRNA alone or the GFP template RNA alone did not produce any GFP-expressing cells.
  • tLNPs comprising a combination of Lipid154 as the ionizable lipid and a higher percentage of helper lipid (such as DSPC or sphingomyelin), and an anti-CD3 targeting moiety with a gene modifying system enables effective insertion of an exogenous nucleic acid sequence into the genomes of rested T cells.
  • T cells At Day 4 post-transfection, up to approximately 30% of the T cells expressed the exogenous GFP nucleic acid sequence, while up to over 40% of the T cells expressed GFP at Day 7 post-transfection.
  • the addition of TransAct can further increase the percentage of cells expressing the exogenous nucleic acid.
  • Example 9 Improved LNP formulation for delivery of an exemplary gene writer system to resting T cells to produce CAR-T cells
  • the targeted LNPs of Example 8 comprising Lipid154 and a higher helper lipid percentage (22% DSPC) and conjugated to the anti-CD3-8 targeting moiety were tested to determine whether they could deliver a gene modifying system payload into rested T cells to integrate an exogenous chimeric antigen receptor (CAR) into the genomic DNA of the rested T cells.
  • the tLNPs were formulated with an all-RNA gene modifying system composed of an mRNA encoding an exemplary gene modifying polypeptide and a template RNA encoding a CAR.
  • a control tLNP comprising Lipid154 and 8% DSPC and conjugated to the anti-CD3-8 targeting moieties was also generated to establish a baseline CAR insertion level for purposes of comparison.
  • T Cell Culture Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume. Cells were counted on a Cellometer K2 Cell Counter (Nexcelom), then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10 6 cells/mL.
  • the complete T cell media was comprised of CTS AIM V SFM basal media (Gibco #0870112DK) with 5% human serum AB (Gemini Bio #100-512) and the following cytokines: human IL-7 at 10ng/mL (Miltenyi Biotec #130-095-362), and human IL-15 at 5ng/mL (Miltenyi Biotec #130-095-764).
  • CTS AIM V SFM basal media Gibco #0870112DK
  • human serum AB Gibco #100-512
  • cytokines human IL-7 at 10ng/mL
  • human IL-15 at 5ng/mL
  • Preparation of T Cells for Transfection [0714] 2.0x10 7 cells were pelleted by centrifugation and the supernatant aspirated. The cell pellets were resuspended in CTS AIM V SFM at a concentration of 2x10 6 cells/mL.
  • LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid154), 22% DSPC, 28.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO.
  • aqueous phase was composed of mRNA encoding the gene modifying polypeptide and a template RNA encoding the CAR dissolved in 25 mM acetate buffer.
  • the LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).
  • the baseline control tLNP was prepared using the same approach, except the ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid154), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO.
  • the negative control tLNPs an aqueous phase containing either the gene modifying polypeptide mRNA only or the GFP template RNA only was mixed with the ethanol phase.
  • the anti-CD3-8 targeting moiety was produced with an additional sortase tag (LPETG, SEQ ID NO:11) on the C terminus of the heavy chain.
  • 10uM anti-CD3 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm.
  • the resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding eGFP) of 1:1, except where otherwise noted (Table 1).
  • the solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane.
  • the resulting targeting moiety-LNP product was further concentrated using an Amicon column.
  • the gene modifying system formulated in the tLNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprised the following components.
  • the gene modifying polypeptide comprising the amino acid sequence of: MDSTAHPNQGRGLEKVSQTLPALQTPGQHTAAGGSSPLSGRNQRKNTKKLLLGAWNIR TLLDRENTPRPERRTALIGKELARYNIDIAALSETRLPEEGSLSEPTTGYTFFWKGRASNE DRIHGVGLAIKTSLLKQLPDLPVGISERLMKIRLPLSKDRYATIISAYAPTLTSTEETIEQFY SDLSAVLHSVPTNDKLILLGDFNARVGQDHERWKGVLGKHGVGKMNNNGLLLLSKCS EFELTITNTVFRMANKYKTTWMHPRSKQWHLIDYIIVRRRDIQDVKITRAMRGAECWT DHRLVRATLQMRIAPRHPKRAQTVRAFYNVSRLRDPSYLQTFQSCLDDKLSAKGPLTGS STEKWNQFRDAVKETSKAVLGPKQRNHQDWFDENNTAIEDLLSKKNKAFMEWQNNPN SAPKKDRFKSL
  • the full- length sequences are provided in Table N (Example 21).
  • the gene modifying system further comprised a template RNA with the following sequences from 5’ to 3’: 5’UTR retro , BCMA CAR, MND promoter, 3’UTR retro .
  • the template RNA comprised a 5’UTR having the sequence of: GGGUGUAUGGGGUGCUCAGGGAGGGGUAGUAUCUCUGGUAUGGAGGGCUUGUCG UGCCCUCCUAGGGCAGCUCUCUCCAGCCUCUGACCCCCACCUGACACCCAGCUCUCAC UUGUGGCUCCCAGUAGCUGCUAGCAUGUGGCAGCGGCCACACCCCGGGCAACGGC UUCGACAGGCCGGCUAAACCUUGUGAGGGUAGCCAUCGGGUCAUCGACCCCUGGU GAACCAGGGCUUUGCUCACCCAGCAUGUGAAGACUGCUUCGGCUGAACAGACGGA AGAAACCAAUAAGAAGGUUCAACGGCUGAGAGGGCGACGCAGCAAAGCACUGUG GAGUGCUUAGGGCGUGUUGGAGCACAAAGGACAACACGGCCAUCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCAAUGCAGCU GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCACUGG
  • Negative conrtol tLNPs were dosed at 2.5 ⁇ g of LNP per 1 x10 6 cells.
  • TransAct Research Grade T cell activation reagent (Miltenyi Biotec #130-111-160) was also added to the rested T cell suspension at the same time as the tLNPs at a volume of 10uL per 1x10 6 cells.
  • all plated cells were at a final density of 1x10 6 cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL.
  • Transfected cells were returned to incubation at 37°C, 5% CO 2 for four hours. After the 4-hour tLNP incubation, cells were centrifuged at 350xg for 7min, supernatant aspirated, and cells resuspended in complete T cell media as previously described at 1e6 cells/mL in flat bottom plates. Flow cytometry was conducted on the cells, as described in Example 1, at 4 days, 7 days, and 10 days following tLNP transfection.
  • FIG.14A shows that delivery of an exemplary gene modifying system payload to rested T cells using targeted LNPs formulated with Lipid154 and higher helper lipid (22% DSPC) generated many more CAR-T cells (%CAR+) at all dose levels at 3 and 7 days post transfection relative to the baseline control tLNP that was the identical except that it was formulated with 8% DSPC.
  • FIGs.14B and 14C show that the rested T cells transfected with the tLNPs comprising higher helper lipid exhibited higher CAR expression levels (MFI) relative to the cells transfected with the baseline control tLNPs at most doses at 3 and 7 days, respectively, following LNP transfection.
  • MFI CAR expression levels
  • tLNPs comprising a combination of Lipid154 as the ionizable lipid and a higher percentage of helper lipid (such as DSPC), and an anti-CD3 targeting moiety with a gene modifying system enables effective insertion of an exogenous nucleic acid sequence encoding a CAR into the genomes of rested T cells.
  • the T cells expressed the exogenous CAR nucleic acid sequence at the 1 ug dose (or higher) without TransAct and over 10% expressed the CAR at doses of 2.5 ug or higher.
  • the addition of TransAct further increased CAR expression levels in the cells.
  • the CAR-T cells generated using these tLNPs were functional, as they were capable of killing target tumor cells in an in vitro tumor cell killing assay (data not shown).
  • Example 10 Testing a dual targeted LNP conjugated to anti-CD3 and anti-CD7 targeting moieties for delivery of an exemplary gene writer system to resting T cells to produce CAR- T cells
  • Dual targeted LNPs conjugated to anti-CD3 and anti-CD7 targeting moieties were administered to resting T cells to determine whether they could effectively deliver an exemplary gene modifying system into the cells to enable TPRT-mediated insertion of a CAR sequence into the genomic DNA of the cells.
  • the tLNPs were formulated with an all-RNA gene modifying system composed of an mRNA encoding an exemplary gene modifying polypeptide and a template RNA encoding a CAR.
  • Targeted LNPs were also generated that were identical except that they were conjugated to either an anti-CD3 targeting moiety or an anti-CD7 targeting moiety alone, as described herein, for the purposes of comparing delivery efficiency of the gene modifying system in the rested T cells.
  • T Cell Culture Cryopreserved primary peripheral blood monocyte cells (PBMCs) from two human donors (HemaCare, donor D354 and D448) were thawed and transferred into PBS with 2% BSA by volume.
  • PBMCs peripheral blood monocyte cells
  • T cell media was comprised of Tex MACS (Miltenyi Biotec # 130-097-196 ) with 2% Human serum albumin (AkronBio: AK8228-0100).
  • the resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates (Falcon # 08-772-54) and returned to incubation at 37°C, 5% CO 2 while the LNPs were prepared.

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Abstract

L'invention concerne des conjugués comprenant une fraction de ciblage, et une nanoparticule lipidique (LNP) encapsulant un agent thérapeutique (par exemple, une charge utile) pour l'administration à des cellules immunitaires. Les conjugués peuvent être administrés à des cellules immunitaires ex vivo ou formulés dans une composition pharmaceutique et peuvent être directement administrés à un sujet ayant besoin (c'est-à-dire, par administration in vivo ).
PCT/US2024/052438 2023-10-22 2024-10-22 Nanoparticules lipidiques pour l'administration de charges utiles thérapeutiques à des lymphocytes t Pending WO2025090525A1 (fr)

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US20220378700A1 (en) * 2019-10-18 2022-12-01 The Trustees Of The University Of Pennsylvania Lipid Nanoparticles and Formulations Thereof for CAR mRNA Delivery
WO2023039424A2 (fr) * 2021-09-08 2023-03-16 Flagship Pioneering Innovations Vi, Llc Procédés et compositions pour moduler un génome
WO2023056282A1 (fr) * 2021-09-28 2023-04-06 The Trustees Of The University Of Pennsylvania Compositions et méthodes pour l'administration d'agents thérapeutiques ciblée à des lymphocytes t

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220378700A1 (en) * 2019-10-18 2022-12-01 The Trustees Of The University Of Pennsylvania Lipid Nanoparticles and Formulations Thereof for CAR mRNA Delivery
WO2023039424A2 (fr) * 2021-09-08 2023-03-16 Flagship Pioneering Innovations Vi, Llc Procédés et compositions pour moduler un génome
WO2023056282A1 (fr) * 2021-09-28 2023-04-06 The Trustees Of The University Of Pennsylvania Compositions et méthodes pour l'administration d'agents thérapeutiques ciblée à des lymphocytes t

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