Lipid nanoparticles for delivery of therapeutic payloads to t cells

Abstract

The disclosure provides conjugates comprising a targeting moiety a lipid nanoparticle (LNP) encapsulating a therapeutic agent (e.g., payload) for delivery to immune cells. The conjugates can be delivered to immune cells ex vivo or formulated in a pharmaceutical composition and can be directly administered to a subject in need of (i.e., via in vivo administration).

Description

LIPID NANOPARTICLES FOR DELIVERY OF THERAPEUTIC PAYLOADS TO T CELLSCROSS REFERNCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63 / 592,197, filed on October 22, 2023; U.S. Provisional Application 63 / 598,347, filed on November 13, 2023; U.S. Provisional Application 63 / 641,887, filed on May 2, 2024; and U.S. Provisional Application 63 / 677,895, filed on July 31, 2024; the contents of each of which are hereby incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (252052001640seqlist.xml; Size: 441,952 bytes; and Date of Creation: October 21, 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0003] The development of lipid nanoparticles (LNPs) has recently made significant advances towards intracellular delivery of 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.

[0004] Despite advances in the development of LNPs, substantial challenges still remain. Specifically, development of LNPs that provide a sufficient quantity of a payload to target cells has been challenging. In particular, development of LNPs that can deliver larger payloads and / or multiple payloads, such as those used in gene editing, is an important goal.

[0005] Additionally, delivery of therapeutic payloads to immune cells such as T cells is currently being investigated but can be challenging. For example, specific delivery of therapeutic agents (e.g., a genetic medicine payload, such as a gene therapy or gene editing agent), to 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.). Furthermore, after delivery to the target cell, the payload(s) mustbe able to disassociate from the endosome so it can be expressed in sufficient quantities in the targeted cell.

[0006] Moreover, LNPs often exhibit unfavorable toxicological profiles, that precludes their use as therapeutic delivery vehicles. LNPs may accumulate and deliver payloads to cells other than the intended target, which results in potential toxicity.

[0007] Accordingly, there exists a need to develop LNPs for safe and effective delivery to immune cells. SUMMARY OF THE INVENTION

[0008] The disclosure provides lipid nanoparticles (LNPs) or 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. In some cases, the LNPs and conjugates described herein can deliver the payload to immune cell more effectively than a baseline conjugate or baseline LNP. In some embodiments, 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. The conjugates having at least one targeting moiety described herein will also be referred to as targeted LNPs or tLNPs.

[0009] In one aspect, 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.

[0010] In another aspect, the disclosure provides conjugates comprising an LNP and a targeting moiety. In some cases, inclusion of a targeting moiety of a targeted LNP enhances the ability of the conjugate or LNP to deliver the payload to immune cells (e.g., T cells). In some embodiments, 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.

[0011] Alternatively, 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 oradaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. In some embodiments, blood is first collected from a subject (e.g., a human patient). In some embodiments, following isolation of the immune cells (e.g., T cells), 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) can then be administered to the patient. In other embodiments, 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. In some embodiments, delivery of conjugates of the disclosure are capable of activating the rested immune cells (e.g., T cells).

[0012] In some embodiments, the payload can be one of more nucleic acid molecules. In some embodiments, the one or more nucleic acid molecules can be RNA molecules. In some embodiments, the one or more nucleic acid molecules can be DNA molecules. In some embodiments where the payload comprises two nucleic acid molecules, both nucleic acid molecules can be RNA molecules. In some embodiments where the payload comprises two nucleic acid molecules, both nucleic acid molecules can be DNA molecules. In some embodiments where the payload comprises one nucleic acid molecule can be an RNA molecule and one nucleic acid molecule can be a DNA molecule.

[0013] In some embodiments, the payload can be a nucleic acid encoding a reporter gene, such as green fluorescent protein (GFP). In some embodiments, 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. In some embodiments, 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). In some embodiments, the therapeutic agent is a gene therapy agent or gene editing agent. In some embodiments, the therapeutic agent is a gene modifying polypeptide. In some embodiments, the therapeutic agent is a gene modifying system.

[0014] In some embodiments, 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. In other embodiments, 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). In some embodiments, the targeting moiety is a peptide or protein, such as a ligand or part of a ligand, that binds to areceptor (e.g., a receptor on the surface of a T cell). In some embodiments, 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.

[0015] In certain embodiments, 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 recombinase, transposase, retrotransposase, helicase, reverse transcriptase, polymerase, deaminase, methylase, demethylase, or ligase function), or combinations thereof. In certain embodiments, 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.

[0016] In one aspect, 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). In some embodiments, the targeted LNP is delivered in vivo to a subject (e.g., a human patient) in need thereof. In some embodiments, the targeted LNP is delivered using an ex vivo or in vitro procedure. Following in vivo, ex vivo, or in vitro administration, 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). It has surprisingly been discovered that 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.)

[0017] In some embodiments, an LNP or targeted LNP (conjugate) 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 some embodiments, 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. In some embodiments, the LNP is a targeted LNP.

[0018] In some embodiments, 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). In some embodiments, 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). In some embodiments, the LNP is a targeted LNP.

[0019] In some embodiments, 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). In some embodiments, 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.

[0020] In some embodiments, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or at least 50% of immune cells (e.g., T 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). In some embodiments, following ex vivo incubation, from about 5% to about50%, 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) from the isolated immune cell (e.g., T cell) population comprise a genome that has been modified or altered.

[0021] 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) enables effective treatment of particular diseases such as cancer. In some embodiments, 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).

[0022] 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) also enables effective treatment of autoimmune diseases. In some embodiments, CAR-T cells can be generated using the tLNPs described herein to target B cell markers such as CD19, CD20, and BCMA. In some embodiments, the autoimmune disease is systemic lupus erythematosus, refractory antisynthetase syndrome, myasthenia gravis, systemic scleroderma, and multiple sclerosis.

[0023] In some embodiments, the targeted LNPs (conjugates) of the disclosure may be used to modify T cells. In some embodiments, T-cells may include any subpopulation of T-cells, e.g., CD4+, CD8+, gamma-delta, naïve T cells, stem cell memory T cells, central memory T cells, or a mixture of subpopulations. In some embodiments, the conjugates may be used to deliver or modify a sequence encoding a T-cell receptor (TCR) in a T cell. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T-cells. For instance, in specific embodiments, the conjugates can be used to deliver an RNA encoding a CAR to T-cells. In some cases, 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. In some cases, 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. Such conjugates can be contacted with T cells to deliver the DNA molecule encoding the CAR, resulting in cellular expression of the CAR. Insome embodiments, the conjugates may be used to deliver at least one sequence encoding a CAR to natural killer (NK) cells. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a CAR to natural killer T (NKT) cells. In some embodiments, 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.

[0024] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0025] In some embodiments, 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. In some embodiments, 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. In some such embodiments, a single targeted LNP (conjugate) 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). Systems 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 are discussed in Section IV. In alternative embodiments, multiple targeted LNPs (conjugates) can be used to deliver the components one or more gene modifying systems. For instance, 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). In some embodiments, 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, and 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. In some embodiments, an LNP of the present disclosure, such as a targeted LNP, comprises the ionizable lipid V003, depicted below.

[0026] In some embodiments, an LNP of the present disclosure, such as a targeted LNP,comprises an ionizable lipid in Table 1. In some embodiments, 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. In some embodiments, 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. In some cases, 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. In some cases, some of 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). In some embodiments, the ionizable lipid has one of the structures depicted below:

[0027] In some embodiments, the LNPs of the present disclosure, e.g., targeted LNPs, comprise one or more pegylated lipid molecules. In some embodiments, the targeting moiety(e.g., antibody, Fab fragment or scFv) of a targeted LNP is associated with or chemically bonded to at least one of the pegylated lipid molecules. In some embodiments, the targeted LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the targeting moiety. In some embodiments, 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. 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 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). In some embodiments, 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.

[0028] Pegylated lipids in the targeted LNPs of the disclosure can imbue different properties to the LNPS, including pharmacokinetics, distribution and endosomal release. Surprisingly, it has been discovered that 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. In particular, it has been discovered that 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. In some embodiments, administration of the LNPs comprising C16 PEG lipid anchors or C18 PEG lipid anchors does not result in substantial liver toxicity. In some embodiments, administration of the targeted LNPs results in no or minimal increases in serum levels of the secreted liver enzyme alanine aminotransferase (ALT).

[0029] In some embodiments, 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). In some embodiments, 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.

[0030] In some embodiments, 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).

[0031] 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 pegylated lipid is DMG-PEG2000.

[0032] In some embodiments, 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. In some embodiments, 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. In some embodiments, the non-pegylated lipid (helper lipid) is selected from POPC, DOPC, DOPE, and DSPC. In some embodiments, the non-pegylated lipid (helper lipid) is a sphingolipid. In some such embodiments, the non-pegylated lipid is a sphingomyelin. In some embodiments, the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0033] 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 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%. 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%. 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%. 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%. 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%, about 32%, about 33%, about 34% or about 35%. As set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs with these percentages of helper lipids provides enhanced transduction and / or expression of the payloads relative to LNPs with smaller or larger quantities of helper lipid. It will be understood that mol% of the helper lipid as used herein 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.

[0034] In some embodiments, 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. 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 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. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) 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.

[0035] In some embodiments, 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%. 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%. 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%. In some of the foregoing embodiments, 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%).

[0036] In some embodiments, 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 thetargeted 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%. 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%. 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%. In some of the foregoing embodiments, 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%).

[0037] In some embodiments, 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%). 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 30% to about 35% (e.g., about 31%, about 32%, about 33%m about 34% or 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 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or 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 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%). In some of the foregoing embodiments, 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%).

[0038] 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 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%. 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 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 orsphingomyelin) in the targeted LNP, is from about 30% to about 40%. In some of the foregoing embodiments, the mol% of cholesterol in the targeted LNP is from about 25%-40%,

[0039] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and a sphingomyelin. In some embodiments, 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). In some such embodiments, the LNP is DPPE-PEG2000 or DPG-PEG2000.

[0040] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and a sphingomyelin. In some embodiments, 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. 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. In some embodiments, 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. 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.

[0042] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 154. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 154 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 154 and a sphingomyelin. In some embodiments, 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. 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.

[0043] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 155. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 155 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 155 and a sphingomyelin. In some embodiments, 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. 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.

[0044] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 162. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 162 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 162 and a sphingomyelin. In some embodiments, 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. 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.

[0045] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 163. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 163 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 163 and a sphingomyelin. In some embodiments, 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. 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.

[0046] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 169. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 169 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 169 and a sphingomyelin. In some embodiments, 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. 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.

[0047] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 176. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 176 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 176 and a sphingomyelin. In some embodiments, 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. 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.

[0048] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 178. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 178 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 178 and a sphingomyelin. In some embodiments, 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. 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.

[0049] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 183 In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 183 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 183 and a sphingomyelin. In some embodiments, 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. 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.

[0050] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 232. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 232 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 232 and a sphingomyelin. In some embodiments, 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. 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.

[0051] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid V003. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid V003 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid V003 and a sphingomyelin. In some embodiments, 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. 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.

[0052] In any of the foregoing embodiments, the LNP or conjugate can comprise one or more cholesterol molecules. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 6:1 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 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. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 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.

[0053] In some embodiments, the targeted LNPs (i.e., conjugates) 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. In some embodiments, the second Click handle is covalently bonded to a pegylated lipid comprising the LNP.

[0054] In one embodiment, 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. In some embodiments, the first or second Click handle comprises a cyclic derivative of the alkynyl group. In some embodiments, the cyclicderivative of the alkynyl group is selected from dibenzocyclooctyne. Cyclooctyne, and difluorinated cyclooctyne, In some embodiments, the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes. In another embodiment, 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. In some embodiments, 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. In some embodiments, the first Click handle comprises a tetrazine (Tz) ring and the second Click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is unsubstituted. In some such embodiments, the tetrazine rung is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl substituted tetrazine.

[0055] In some embodiments, the Click product is formed by conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence. For instance, 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. In some embodiments, the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide. In some embodiments, the antibody Fab fragment or scFv is directly bonded to the enzyme recognition sequence. In some embodiments, 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)vand A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0056] In certain embodiments, 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)-β,TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and r IL-15 receptor. In some embodiments the CD80 targeting moiety is a CD80 extracellular domain (ECD).

[0057] In some embodiments, the targeted LNP (conjugate) 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. In some embodiments, the targeting moiety targets a CD3 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD7 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD5 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD2 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD8 receptor on the surface of the T cell.

[0058] In some embodiments, the targeted LNP (conjugate) 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. In certain embodiments, the targeting moiety is SP34 or an antigen-binding portion thereof. In other embodiments, 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.

[0059] In some embodiments, a targeted LNP (conjugate) 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.

[0060] In some embodiments, 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)β,TCR -α, TCR -α / β, TCR -γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor. In some embodiments, the targeted LNP (conjugate) 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.

[0061] 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. In some embodiments, 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)-β,TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor

[0062] In some embodiments, the plurality of targeting moieties can be added to the surface of the LNP by methods described in Section II of the Detailed Description. For instance, in some embodiments, 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. In some such embodiments, the targeting moieties are antibodies or antigen binding fragments thereof. In other such embodiments, the targeting moieties are scFvs. In some embodiments, the targeting moieties are Fab fragments.

[0063] 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 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 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. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD7 and the other targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD).

[0064] In some embodiments, the targeted LNP (conjugate) comprises three targeting moieties, wherein one targeting moiety binds to CD3. In some such embodiments, one of the targeting moieties binds to CD7 and the other targeting moiety binds to CD28. In some such embodiments, one of the targeting moieties binds to CD5 and the other targeting moiety binds toCD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD). In some of the foregoing embodiments, 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.

[0065] In some embodiments, the targeted LNP (conjugate) comprises two targeting moi eties, wherein each targeting moiety binds to the same target (e.g., receptor) on the T cell. For instance, in some embodiments, both targeting moi eties of the conjugate bind to CD3. In some such embodiments, one of the targets is SP34 or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof. In other such embodiments, one of the targets is SP34 or an antigen-binding portion thereof and the other is visilizumab or an antigenbinding portion thereof. In other such embodiments, one of the targets is SP34 or an antigenbinding portion thereof and the other is tepilizumab or an antigen-binding portion thereof. In other such embodiments, one of the targets is visilizumab or an antigen-binding portion thereof and the other is tepilizumab or an antigen-binding portion thereof. In other such embodiments, one of the targets is visilizumab or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof. In some embodiments, both targeting moieties of the conjugate bind to CD3. In some embodiments, both targeting moieties of the conjugate bind to CD7.

[0066] In some aspects, 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 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(iii) a first mRNA encoding a first gene modifying polypeptide; and(iv) a first template RNA that binds to the first gene modifying polypeptide and comprises a heterologous object sequence.

[0067] In some embodiments, 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. In some embodiments, the heterologous sequence comprises SEQ ID NO: 7.

[0068] In other aspects, 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. 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.

[0069] In some embodiments, the two different targeting moieties can be added to the surface of the LNP by methods described in Section II of the Detailed Description. For instance, in some embodiments, 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. In some such embodiments, the targeting moieties are antibodies or antigen binding fragments thereof. In other such embodiments, the targeting moieties are scFvs. In some embodiments, the targeting moieties are Fab fragments.

[0070] In some embodiments, one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is an mRNA encoding a gene modifying polypeptide, as described herein. In some embodiments, one of the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is an mRNA encoding a retrotransposon. In some embodiments, one of the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is an mRNA encoding a Cas9 nickase fused to a reverse transcriptase (RT) domain. In some embodiments, one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9- RT fusion protein.

[0071] In some embodiments, one of the RNA molecules comprising a component of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is a guide RNA (gRNA). In some embodiments, one of the RNA molecules comprising a component of a systemfor modifying or altering genomic DNA (e.g., a gene modifying system) 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. In some embodiments, the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) have nuclease activity, e.g., nickase activity. In some embodiments, the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) do not have nuclease activity. In some embodiments, the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) 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. In some embodiments, system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous sequence into the genomic DNA.

[0072] In some embodiments, the ionizable lipid is V003 or a lipid from Table 1, Table 2 or Table 3. In some embodiments, the ionizable lipid is Lipid 092. In some embodiments, the ionizable lipid is Lipid 154.

[0073] In some embodiments, 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 40%. In some embodiments, 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%.

[0074] In some embodiments, 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. In some embodiments, 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.

[0075] In some embodiments, 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. In some embodiments, 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 ofmodifying 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.

[0076] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0077] In some embodiments, 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. In someembodiments, the T cells are not activated prior to mixing with the pharmaceutical composition comprising targeted LNPs. In some embodiments, the targeted LNPs include at least one CD3 binder (e.g., antibody or fragment thereof) on their surface. In some embodiments, 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. In some embodiments, the targeted LNPs include a CD3 binder (e.g., antibody or fragment thereof) and a CD28 binder (e.g., antibody or fragment thereof).

[0078] In some embodiments, 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. In some embodiments, the T cells are not activated prior to mixing with the pharmaceutical composition comprising targeted LNPs. In some embodiments, the targeted LNPs include at least one CD3 binder (e.g., antibody or fragment thereof) on their surface. In some embodiments, 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. In some embodiments, the targeted LNPs include a CD3 binder (e.g., antibody or fragment thereof) and a CD28 binder (e.g., antibody or fragment thereof).

[0079] In any of the foregoing embodiments, 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. In some embodiments, the gene modifying system comprises a retrotransposon. In some embodiments, the gene modifying system comprises a Cas9 nickase fused to a reverse transcriptase (RT) domain. In some embodiments, 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.

[0080] In some embodiments, 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 selectedfrom 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. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, the LNP comprises a CD28 targeting moiety. In some embodiments, the LNP comprises a CD4 targeting moiety. In some embodiments, the LNP comprises a CD8 targeting moiety. In some embodiments, 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. In some embodiments, 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. In some embodiments, the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32, and the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20. 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 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. In some embodiments, 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% toabout 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 template RNA comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395-404.

[0081] In some embodiments, 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 more nucleic acid molecules (e.g., one or more RNA molecules). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, the LNP comprises a CD28 targeting moiety. In some embodiments, the LNP comprises a CD4 targeting moiety. In some embodiments, the LNP comprises a CD8 targeting moiety. In some embodiments, 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. In some embodiments, 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. In some embodiments, the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table32, and the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20. In some embodiments, 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. In some embodiments, the therapeutic agent comprises a DNA molecule. In some embodiments, the therapeutic agent comprises an mRNA molecule. In some embodiments, the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA. In some embodiments, 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.

[0082] In some embodiments, 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). In some embodiments, the means for binding the cell surface receptor is present on the surface of the LNP. In some embodiments, the means for binding a cell surface receptor comprises a targeting moiety, such as an antibody or fragment thereof. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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 chainsequences selected from Table 20 (or CDR sequences (bolded) selected from Table 20). In some embodiments, 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). In some embodiments, the LNP comprises a CD28 targeting moiety. In some embodiments, the LNP comprises a CD4 targeting moiety. In some embodiments, the LNP comprises a CD8 targeting moiety. In some embodiments, 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. In some embodiments, 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. In some embodiments, the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32, and the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20. In some embodiments, 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. In some embodiments, the therapeutic agent comprises a DNA molecule. In some embodiments, the therapeutic agent comprises an mRNA molecule. In some embodiments, the therapeutic agent comprises two nucleic acid molecules, wherein one nucleic acid is an mRNA and the other nucleic acid is a template RNA. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gene modifying polypeptide comprises an amino acidsequence 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. In some embodiments, the template RNA comprises both of a 5’UTRretroand a 3’ UTRretro. In some embodiments, the 5’UTRretroand the 3’ UTRretrocomprise 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] 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.

[0085] 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).

[0086] 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). 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.

[0087] 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.

[0088] 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.

[0089] 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 thetested 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.

[0090] 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.

[0091] 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. 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.

[0092] 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. Those activated T cells that were transduced with the Lipid092or Lipid154 LNPs expressed GFP at higher levels (higher MFI) relative to activated T cells transduced with LNPs comprising the V003 ionizable lipid.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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). Delivery of the gene modifying system payload to rested T cells using targetedLNPs 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 (“Std” in FIG.13) that was the identical except that it was formulated with 8% DSPC.

[0097] 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). Delivery of the gene modifying system payload to rested T cells using targeted LNPs formulated with higher helper lipid (22% DSPC) generated many more CAR-T cells (%CAR+) at all dose levelsrelative to the baseline control tLNP that was the identical except that it was formulated with 8% DSPC. 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. 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. The coadministration of TransAct (“TA”) increased the number of CAR expressing cells and CAR expression levels for both tLNPs formulated with different helper liver percentages.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] FIG.18 is a schematic showing an enzymatic approach of site-specifically introducing a Tz ring onto a sugar moiety of an antibody.

[0102] FIG.19 is a schematic showing an example of a light-induced crosslinking approach of site-specifically introducing a Tz ring onto an antibody.

[0103] 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.

[0104] 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).

[0105] FIG.22 is a schematic showing a Fab fragment modified with a free cysteine designed to react with a maleimide group.

[0106] 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.

[0107] FIGs.24A-24F provide schematics showing the synthesis of Lipid 092.

[0108] FIGs.25A-25C provide schematics showing the synthesis of Lipid 093.

[0109] FIGs.26A-26C provide schematics showing the synthesis of Lipid 162.

[0110] FIGs.27A-27C provide schematics showing the synthesis of Lipid 153.

[0111] FIGs.28A-28F provide schematics showing the synthesis of Lipid 154.

[0112] FIGs.29A-29E provide schematics showing the synthesis of Lipid 155.

[0113] FIGs.30A-30F provide schematics showing the synthesis of Lipid 176.

[0114] 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.

[0115] 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.

[0116] 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 anexemplary 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).

[0117] 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.

[0118] 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).

[0119] 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. The tLNPs were formulated with an exemplary retrotransposon gene modifying system comprising a template RNA encoding a CAR sequence, and administered at 2 different doses (either 5ug or 2.5ug of LNPs per million cells) .

[0120] FIG.37 provides a bar graph showing the quantification of CAR expression levels (MFI) in the CAR-T cells described in FIG.36.

[0121] 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.

[0122] FIG.39 is a schematic showing an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).

[0123] 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.

[0124] 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.

[0125] FIG.42 is an exemplary schematic showing conjugate formation. In FIG.42, 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)). In step (ii), 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.

[0126] FIG.43 is an exemplary schematic showing conjugate formation. In FIG.43, 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)). In step (ii), 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.

[0127] FIG.44 is an exemplary schematic showing conjugate formation using DBM as a bridging agent.

[0128] FIG.45 is an exemplary schematic showing conjugate formation. In FIG.45, 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. After step (i), 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. In 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.

[0129] FIG.46 is a schematic showing some examples of a pegylated lipid bonded to a maleimide moiety.

[0130] FIG.47 is a schematic showing some examples of non-pegylated lipid bonded to a maleimide moiety.

[0131] FIG.48 is a schematic showing some examples of ionizable lipid bonded to a maleimide moiety.

[0132] FIG.49 is a schematic showing some examples of sterols bonded to a maleimide moiety.

[0133] 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. The tLNPs were conjugated with an anti-CD2, anti-CD3, anti-CD5, or anti-CD7 targeting moiety.

[0134] 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. DEFINITIONS

[0135] Antigen binding domain: The term “antigen binding domain” as used herein refers to that portion of antibody or a chimeric antigen receptor which binds an antigen. In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments an antigen binding domain binds an antigen characteristic of a cancer, e.g., a tumor associated antigen in a neoplastic cell. In some embodiments, an antigen binding domain binds an antigen characteristic of an infectious disease, e.g. a virus associated antigen in a virus infected cell. In some embodiments, 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. In some embodiments, an antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, an antigen binding domain is or comprises an scFv or Fab.

[0136] Expression cassette: The term “expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention.

[0137] gRNA spacer: A “gRNA spacer”, as used herein, 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.

[0138] gRNA scaffold: A “gRNA scaffold”, as used herein, 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. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.

[0139] 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). In some embodiments, the endonuclease domain is a catalytically inactive endonuclease domain. In some embodiments, the retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain are derived from the same retrotransposase. In some embodiments, the gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0141] Domain: The term “domain” as used herein 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. Examples of 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.

[0142] Exogenous: As used herein, the term “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. For example, 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.

[0143] 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. For example, 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. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, 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) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, 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. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome butinstead 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). In some embodiments, 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).

[0144] Heterologous gene modifying polypeptide: As used herein, the term “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). In some embodiments, the heterologous gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, 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. In some embodiments, the sequence that is integrated comprises a deletion, substitution, or insertion relative to the target DNA molecule. In some embodiments, 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. Exemplary 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. In some embodiments, a heterologous gene modifying polypeptide integrates asequence into a gene. In some embodiments, 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.

[0145] Mutation or Mutated: The term “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.

[0146] Nucleic acid molecule: “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. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “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.). Also included are chemically modified bases (see, for example, Table 13), backbones (see, for example 14, Table16), and modified caps. Also included are synthetic 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). 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). In various embodiments, 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. 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).

[0147] Primer Binding Sequence: The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region comprised in a target nucleic acid sequence. In some instances, 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. In some embodiments the primer region comprises at least 5, 6, 7, 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments when a template RNA comprises a PBS sequence and a heterologous object 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.

[0148] It is understood that aspects and embodiments described herein as “comprising” include “consisting of” and “consisting essentially of” embodiments. II. CONJUGATES

[0149] In one aspect, 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). In another aspect, 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. In some embodiments, an LNP (conjugate) comprises both an ionizable lipid and a targeting moiety, as described herein.

[0150] In one aspect, 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). In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In some instances, 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. In other embodiments, 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). In some embodiments, the payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system) is capable of modifying one or more genes of the target immune cell (e.g., T cell).

[0151] 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. In some embodiments, 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, naïve 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. In some embodiments, the conjugates may be used to deliver or modify a sequence encoding a T-cell receptor (TCR) in a T cell. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T cells. For instance, in specific embodiments, the conjugates can be used to deliver an RNA encoding a CAR to T cells. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a CAR to natural killer (NK) cells. In some embodiments, the conjugates be used to deliver at least one sequence encoding a CAR to natural killer T (NKT) cells. In some embodiments, 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. In some embodiments, cells modified with at least one CAR (e.g., CAR-T cells, CAR-NK cells, CAR-NKT cells), or acombination of cells modified with at least one CAR (e.g., a mixture of CAR-T / CAR-NK / CAR- NKT cells) 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. In some embodiments, 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 (epithelial cell adhesion molecule), EphA2, ERBB2 (HER2, human epidermal growth factor receptor 2), ERBB3, ERBB4, FBP (folate-binding protein), Flt3 receptor, folate receptor-a, GD2 (ganglioside G2), GD3 (ganglioside G3), GPC3 (glypican-3), GPI00, hTERT (human telomerase reverse transcriptase), ICAM-1, integrin B7, interleukin 6 receptor, IL13Ra2 (interleukin-13 receptor 30 subunit alpha-2), kappa-light chain, KDR (kinase insert domain receptor), LeY (Lewis Y), L1CAM (LI cell adhesion molecule), LILRB2 (leukocyte immunoglobulin like receptor B2), MARTI, MAGE-A1 (melanoma associated antigen Al), MAGE- A3, MSLN (mesothelin), MUC16 (mucin 16), MUCI (mucin I), KG2D ligands, NY-ESO-1 (cancer-testis antigen), PRI (proteinase 3), TRBCI, TRBC2, TFM-3, TACI, tyrosinase, survivin, hTERT, oncofetal antigen (h5T4), p53, PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), hRORl, TAG-72 (tumor- associated glycoprotein 72), VEGF-R2 (vascular endothelial growth factor R2), WT-1 (Wilms tumor protein), and antigens of HIV (human immunodeficiency virus), hepatitis B, hepatitis C, CMV (cytomegalovirus), EBV (Epstein-Barr virus), HPV (human papilloma virus), etc.

[0152] In some embodiments, immune cells, e.g., T cells, NK cells, NKT cells, or progenitor cells are modified ex vivo and then delivered to a patient. In some embodiments, a nucleic acid (e.g., RNA, such as mRNA) is delivered by one of the methods mentioned herein, and immune cells, e.g., T cells, NK cells, NKT cells, or progenitor cells are modified in vivo in the patient.

[0153] In certain embodiments, 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)-β,TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor.

[0154] In certain embodiments, 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)-β,TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD).

[0155] In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that targets a receptor on the surface of the T cell selected from CD2, CD3, CD4, CD5, CD6, CD7, and CD8. In some embodiments, the targeting moiety targets a CD3 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD7 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD5 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD2 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD8 receptor on the surface of the T cell.

[0156] In some embodiments, the targeted LNP (conjugate) 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. In certain embodiments, the targeting moiety is SP34 or an antigen-binding portion thereof. In other embodiments, 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. In other embodiments, 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.

[0157] In some embodiments, a targeted LNP (conjugate) 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.

[0158] In some embodiments, 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)-β,TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor. In some embodiments, the targeted LNP (conjugate) 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. In some embodiments, 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.

[0159] 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 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. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD7 and the other targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD). 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 onetargeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the other targeting moiety that binds to CD3.

[0160] In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein each targeting moiety binds to the same target (e.g., receptor) on the T cell. For instance, in some embodiments, both targeting moieties of the conjugate bind to CD3. In some such embodiments, one of the targets is SP34 or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof. In other such embodiments, one of the targets is SP34 or an antigen-binding portion thereof and the other is visilizumab or an antigen- binding portion thereof. In other such embodiments, one of the targets is SP34 or an antigen- binding portion thereof and the other is tepilizumab or an antigen-binding portion thereof. In other such embodiments, one of the targets is visilizumab or an antigen-binding portion thereof and the other is tepilizumab or an antigen-binding portion thereof. In other such embodiments, one of the targets is visilizumab or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof. In some embodiments, both targeting moieties of the conjugate bind to CD3. In some embodiments, both targeting moieties of the conjugate bind to CD7.

[0161] In certain embodiments, 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. 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. See, e.g., Binder et al. (2020) Front. Immunol.11:1090, hereby incorporated by reference in its entirety.

[0163] In some embodiments, the CD2 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD2. In some embodiments, 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). In other embodiments, 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. In some embodiments, the CD2 targeting moiety is an anti-CD2 antibody or antigen binding fragment thereof. In some embodiments, the CD2 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD2 targeting moiety is a bispecific or multi- specific antibody or fragment thereof. In some embodiments, the CD2 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0164] 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; US20210032308; US20030068320; US20230365687; WO1999058147; WO2014025198; WO2024180185; WO2023126445; WO2024079046; WO2024079046; etc., each hereby incorporated by reference in its entirety.

[0165] In some embodiments, 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. In some embodiments, 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.

[0166] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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.

[0167] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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 least96%, 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.

[0168] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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.

[0169] In some embodiments, 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. In some embodiments, 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-L2comprising an amino acid sequence LVSKLES (SEQ ID NO:297), and a CDR-L3 comprising an amino acid sequence MQFTHYPYT (SEQ ID NO:298). In some embodiments, 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] In some embodiments, the target molecule is CD3. In some embodiments, the target cell is CD3+. CD3 is a multimeric protein complex made up of four polypeptide chains (CD3- epsilon (ε), CD3 -gamma (γ), CD3 -delta (δ), and CD3 -zeta (ζ)) to form a CD3γε –CD3δε –CD3ζζ signaling hexamer that associates with the T cell receptor (TCR). 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. See, e.g., Dong et al. (2019) Nature 573:546-552, hereby incorporated by reference in its entirety.

[0171] In some embodiments, the CD3 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD3. In some embodiments, 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). In other embodiments, 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. In some embodiments, the CD3 targeting moiety is an anti-CD3 antibody or antigen binding fragment thereof. In some embodiments, the CD3 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD3 targeting moiety is a bispecific or multi- specific antibody or fragment thereof. In some embodiments, the CD3 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0172] 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. Immunol.137:1097-1100: 1986; US 8,846,042; US 11,013,800; and US 10,870,701), Cris-7 monoclonal antibody (Reinherz, E. L. et al. (eds.),Leukocyte typing II, Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med.172:1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med.313:337) and derivatives thereof such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest.11:409), visilizumab (Carpenter et al. (2002) Blood 99:2712), mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, muromonab, plamotamab, HPN536, pasotuxizumab, flotetuzumab, and 145-2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol.140: 3766). Further CD3 binding molecules contemplated herein include UCHT-1 (Beverley, P C and Callard, R. E. (1981) Eur. J. Immunol.11: 329-334) and CD3 binding molecules described in WO2004 / 106380; WO2010 / 037838; WO2008 / 119567; WO2007 / 042261; WO2010 / 0150918; the contents of each of which are incorporated herein by reference in their entirety.

[0173] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 CD3targeting 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. In some embodiments, 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.

[0174] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is mosunetuzumab or an antigen-binding fragment thereof.

[0175] In some embodiments, 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%) sequenceidentity 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is odronextamab or an antigen-binding fragment thereof.

[0176] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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 or161, 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. In some embodiments, the CD3 targeting moiety is tebentafusp or an antigen-binding fragment thereof.

[0177] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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:171 or 172, 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:173. In some embodiments, the CD3 targeting moiety is teplizumab or an antigen-binding fragment thereof.

[0178] In some embodiments, 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. In some embodiments, 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), aCDR-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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is teclistamab or an antigen- binding fragment thereof.

[0179] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is visilizumab or an antigen-binding fragment thereof.

[0180] In some embodiments, 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%) sequenceidentity 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is muromonab or an antigen-binding fragment thereof.

[0181] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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 acidsequence 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. In some embodiments, the CD3 targeting moiety is SP34 or an antigen-binding fragment thereof.

[0182] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is plamotamab or an antigen-binding fragment thereof.

[0183] In some embodiments, 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. In some embodiments, 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), aCDR-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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is HPN536 or an antigen-binding fragment thereof.

[0184] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is pasotuxizumab or an antigen- binding fragment thereof.

[0185] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD3 targeting moiety is flotetuzumab or an antigen-binding fragment thereof. CD5 Targeting Moieties

[0186] In some embodiments, the target molecule is CD5. In some embodiments, 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. Several 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. (2018) Front. Immunol.9:2900, hereby incorporated by reference in its entirety.

[0187] In some embodiments, the CD5 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD5. In some embodiments, 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). In other embodiments, 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. In some embodiments, the CD5 targeting moiety is an anti-CD5 antibody or antigen binding fragment thereof. In some embodiments, 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.

[0188] 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.

[0189] In some embodiments, 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.

[0190] In some embodiments, 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. In some embodiments, 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). In some embodiments, the CD5targeting 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. CD7 Targeting Moieties

[0191] In some embodiments, the target molecule is CD7. In some embodiments, the target cell is CD7+. CD7 (also known as GP40, LEU-9, Tp40, and TP41) 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.

[0192] In some embodiments, the CD7 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD7. In some embodiments, 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). In other embodiments, 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. In some embodiments, the CD7 targeting moiety is an anti-CD7 antibody or antigen binding fragment thereof. In some embodiments, the CD7 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD7 targeting moiety is a bispecific or multi- specific antibody or fragment thereof. In some embodiments, the CD7 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0193] 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. (2024) Biomolecules 14(1):106; etc., each hereby incorporated by reference in its entirety.

[0194] In some embodiments, 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 regioncomprising 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. In some embodiments, 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.

[0195] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the CD7 targeting moiety is grisnilimab or an antigen-binding fragment thereof.

[0196] In some embodiments, 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%) sequenceidentity 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. In some embodiments, 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). In some embodiments, 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.

[0197] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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., atleast 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.

[0198] In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. CD28 Targeting Moieties

[0199] In some embodiments, the target molecule is CD28. In some embodiments, 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. Upon antigen recognition, CD28 signaling strongly amplifies T-cell receptor signaling to activate T cells, and CD28 co-stimulation of T cells increases glucose uptake and glycolysis during an immune response.

[0200] In some embodiments, the CD28 targeting moiety includes an antibody or antigen- binding fragment thereof that binds to CD28. In some embodiments, the CD28 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). In other embodiments, 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. In some embodiments, the CD28 targeting moiety is an anti-CD28 antibody or antigen binding fragment thereof. In some embodiments, the CD28 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD28 targeting moiety is a bispecific or multi-specific antibody or fragment thereof. In some embodiments, the CD28 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0201] Exemplary 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; WO2002051871; Tan et al. (2002) J Immunol 169:1119-1125; Elsyed et al. (2023) Mabs 15(1):2220839; etc., each hereby incorporated by reference in its entirety.

[0202] In some embodiments, the CD28 targeting moiety is a CD28 receptor ligand. In some embodiments, the CD28 receptor ligand is CD80. Accordingly, in some embodiments, 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. In certain embodiments, 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. Exemplary Dual Targeting Moieties

[0203] In some embodiments, the targeted LNP (conjugate) 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)-β,TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor. In some embodiments, thetargeted LNP (conjugate) 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. In some embodiments, the targeted LNP (conjugate) 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. In some embodiments, 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. In some embodiments, 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.

[0204] In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD5. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD7. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD5 and a second targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD7, and a second targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD). In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD3. In some embodiments, 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.

[0205] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD5. In someembodiments, 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, and 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.

[0206] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD2. In some embodiments, 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, and 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. In some embodiments, 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, and 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. In some embodiments, 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, and 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. In some embodiments, 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, and 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.

[0207] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD7. In someembodiments, 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, and 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, 336, and 347. In some embodiments, 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, and the second targeting moiety comprises a CD7-binding domain comprising the VH and the VL of grisnilimab.

[0208] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD). In some embodiments, 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, and the second targeting moiety that binds to CD28 comprises the amino acid sequence of SEQ ID NO:365. In some embodiments, 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, and the second targeting moiety that binds to CD28 comprises the amino acid sequence of SEQ ID NO:365.

[0209] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD3. In some embodiments, 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. In some embodiments, 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. In some embodiments, the first targeting moiety and the second targeting moiety each independentlycomprises 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. In some embodiments, 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, and 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. Methods of Making Targeted LNPs (Conjugates)

[0210] Different approaches can be used to introduce a targeting moiety onto the surface of an LNP. For example, one approach relies on functionalizing a preformed LNP with a targeting moiety. 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. 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.

[0211] In some embodiments, 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. In some such embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In other such embodiments, the targeting moiety is a scFv. In some embodiments, the targeting moiety is a Fab fragment.

[0212] In one embodiment, 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. In some embodiments, the first or second Click handle comprises a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne. cyclooctyne, and difluorinated cyclooctyne. In some embodiments, the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes.

[0213] In another embodiment, the Click product can be formed using copper-free Click chemistry. For example, the Click product can be formed between an azide anddibenzocyclooctene (DBCO). Alternatively, the Click product can be formed using a Staudinger reaction between an azide and a phosphine, hence producing an aza-ylide.

[0214] In some embodiments, 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. In some embodiments, the first Click handle comprises a tetrazine (Tz) ring and the second Click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is unsubstituted. In some such embodiments, the tetrazine rung is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl substituted tetrazine.

[0215] In another embodiment, the targeting moiety (e.g., antibody, Fab fragment or scFv) is first selectively modified with an enzyme recognition sequence. An enzyme recognizing the 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. Hence, in one embodiment, 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. In some embodiments, the antibody Fab fragment or scFv is directly bonded to the enzyme recognition sequence. In some embodiments, the targeting moiety (e.g., 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 targeting moiety (e.g., 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)vand A(EAAAK)uALEA(EAAAK)vA (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.

[0216] In some embodiments, the enzyme recognition sequence is a sortase recognition motif or an LplA acceptor peptide. In some embodiments where the LNP is conjugated to an antibody, 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. In some embodiments where the LNP is conjugated to a Fab fragment, 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). In someembodiments where the LNP is conjugated to s scFv, the C-terminus of the scFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some of the foregoing embodiments, the first Click handle comprises a tetrazine ring or TCO moiety and the second Click handle comprises a tetrazine ring or TCO moiety. In some such embodiments, the first Click handle comprises a tetrazine ring and the second Click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is a methyltetrazine. In some embodiments, 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%.

[0217] In some embodiments, 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.

[0218] In some embodiments, the first or second Click handle is a tetrazine derivative having one of the following structures:wherein represents the point of attachment to the linker of the conjugate or to the LNP.

[0219] In some embodiments, the first or second Click handle is a TCO derivative having one of the following structures:

[0220] In some embodiments, 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. For instance, 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. In some embodiments, the antibody, Fab fragment or scFv is directly bonded to the enzyme recognition sequence. In some embodiments, 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)vand A(EAAAK)uALEA(EAAAK)vA (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.

[0221] In some embodiments, the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide. In some embodiments where the LNP is conjugated to an antibody, 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. In some embodiments where the LNP is conjugated to a Fab fragment, 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). In someembodiments where the LNP is conjugated to s scFv, the C-terminus of the scFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some embodiments, 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%.

[0222] In one embodiment, 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), wherein one or more lipids comprising the LNP include a second Click handle capable of reacting with the first Click handle, wherein the second Click handle comprises a TCO moiety, thereby generating a Click product.

[0223] In some embodiments, 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. In some embodiments, 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)vand A(EAAAK)uALEA(EAAAK)vA (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.

[0224] In some embodiments, 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. For instance, in some embodiments, 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.

[0225] In some embodiments, step (ii) (i.e., the sortase-catalyzed reaction) is carried out in the presence of sortase A5. 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. patent No.9,267,227. In such embodiments, 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. In step (ii), 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.

[0226] In other embodiments, step (ii) is carried out in the presence of the sortase enzyme Streptococcus pyogenes sortase A (SpSrtA WT). In such embodiments, 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. In step (ii), 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.

[0227] 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. In the schematic, 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.

[0228] In one embodiment, 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. In some embodiments, 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 theLNP. In particular embodiments, the sortase recognition motif comprises an LPXT motif, wherein X is any amino acid residue. In some such embodiments, the sortase recognition motif comprises an LPET motif (SEQ ID NO:10). In some embodiments, 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.

[0229] In some embodiments, 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). In these embodiments, 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. In some such embodiments, X is E. In some embodiments, 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).

[0230] In some embodiments, the linker comprises additional amino acid residues between the antibody, Fab fragment or scFv and the sortase recognition motif. In such embodiments, 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. For instance, in particular embodiments, 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. In some embodiments, Z comprises one or more amino acid residues. In some embodiments Z is (GGGGS)v(SEQ ID NO:1), (G)v, (EAAAK)v(SEQ ID NO:3), (PAPAP)v(SEQ ID NO:4), (AP)vand A(EAAAK)uALEA(EAAAK)vA (SEQ ID NO:2), wherein u is 1-10 and v is 1-10. In some embodiments, 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).

[0231] In some embodiments, 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. In particular embodiments, the dihydropyridazine moiety is a 1,4- dihydropyridazine.

[0232] In another embodiment, 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 a TCO moiety, thereby generating a Click product.

[0233] In some embodiments of 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. 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)vand A(EAAAK)uALEA(EAAAK)vA (SEQ ID NO:2), wherein u is 1-10 and v is 1-10.

[0235] In some embodiments, step (ii) (i.e., the lipoic acid ligase catalyzed reaction) 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. Specific W37 mutants are described in Cohen et al., 2012, ChemBioChem, 13, 888-894. In some embodiments, the mutant lipoic acid ligase is selected from W37V, W37I, W37T, W37L, W37C, W37A, and W37G.

[0236] In some embodiments, the carboxylic acid in step (ii) (i.e., the lipoic acid ligase catalyzed reaction) is a C3-C20 carboxylic acid. In some embodiments, the carboxylic acid instep (ii) is a fatty acid, for instance a C7-C19fatty acid. In some such embodiments, the fatty acid is selected from decanoic acid, palmitic acid, lauric acid or octanoic acid.

[0237] In other embodiments, the carboxylic acid in step (ii) has the formula COOH-R-Click handle, where R includes two lipid tales. In some such embodiments, the carboxylic acid as shown below, wherein n = 1—120.

[0238] 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. In the schematic, a C-terminus of the Fab fragment is first covalently modified with a LplA acceptor peptide. Specifically, 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.

[0239] In some embodiments, 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. In some embodiments, 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. In some embodiments, the LplA acceptor peptide has the sequence GFEDKVWYDLDA (SEQ ID NO:23). In some embodiments, 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. In some embodiments, the conjugate comprises a Fab fragment, the C- terminus of the heavy or light chain of the Fab fragment is bonded to the linker. In some embodiments, the conjugate comprises a scFv, wherein the C-terminus of the scFv is bonded to the linker.

[0240] In some embodiments, the linker comprises additional amino acid residues between the targeting moiety (e.g., antibody, Fab fragment or scFv) and the LplA acceptor peptide. In such embodiments, a C-terminus of the targeting moiety (e.g., antibody, Fab Fragment or scFv) can be covalently modified with one or more amino acid residues prior to covalently linking the LplA acceptor peptide. For instance, in particular embodiments, 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. In some embodiments, Z comprises one or more amino acid residues. In some embodiments Z is (GGGGS)v(SEQ ID NO:1), (G)v, (EAAAK)v(SEQ ID NO:3), (PAPAP)v(SEQ ID NO:4), (AP)vand A(EAAAK)uALEA(EAAAK)vA (SEQ ID NO:2), wherein u is 1-10 and v is 1-10. In some embodiments, 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).

[0241] It will be understood that in the forgoing embodiments, the lysine (K) residue of the LplA acceptor peptide is covalently linked to Click product, which is covalently linked to the LNP. Specifically, to generate conjugates, 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. An LNP surfacemodified with a Fab fragment is depicted below, where R is a lipid group (e.g., C2-C30alkyl group) and the LplA accepter peptide comprises the sequence of GFEIDKVWYDLDA (SEQ ID NO:24).

[0242] In other embodiments, 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.

[0243] In other embodiments, the enzyme recognition sequence is a sequence recognized by formylglycine generating enzyme, specifically CXPXR, wherein each X is any amino acid. In such embodiments, 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.

[0244] In another embodiment, 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. In step 1, 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. 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.

[0245] In another embodiment, 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. FIG.19 shows one particular embodiment using oYo-Link Tz. In FIG.19, a Tz ring (e.g., MeTz) is covalently bound to a site specific antibody label (oYo link) that specifically reacts with residues on the heavy chain of the antibody. The oYo-Link tetrazine covalently bound to the antibody reacts with TCO (not shown) bound to an LNP, hence generating the surface- modified LNP.

[0246] In another embodiment, 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.

[0247] In another embodiment, 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. In FIG.21, a hinge with a free cysteine is first introduced at a C-terminus of the Fab fragment. In some such embodiments, 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. In some embodiments, Click handle 1 is a TZ and Click handle 2 is a TCO moiety,

[0248] In some embodiments, the Click product can be formed using any suitable photo- induced Click chemistry reaction. In some embodiments, 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. Chem., Int. Ed.2008, 47 (15), 2832-2835).

[0249] In some embodiments, the Click product can be formed using oxime and hydrazonehydroxylamine, 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).

[0250] Exemplary lipids and cholesterol molecules bonded to Click handles that can be used to make targeted LNPs are depicted below.Interchain CL-CH1 disulfide reduction followed by conjugation

[0251] 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 (CL). The heavy chains consist of a variable heavy light domain (VH) and three constant heavy chain domains (CH1, CH2, and CH3). The Fab region of the antibody includes the VL, CL, VH, and CH1 domains. The Fc region includes the constant heavy chain domains CH2, and CH3. A hinge region of the IgG antibody covalently links the CH1 domain to the CH2 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 CL-CH1 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.

[0252] Proteolytic cleavage of an IgG antibody results in the formation of a Fab fragment known as a F(ab’)2fragment. The F(ab’)2fragment does not include the CH2 domain or the CH3 domain. However, the hinge region of the antibody is retained in a F(ab’)2fragment. 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. However, owing to the presence of multiple sulfhydryl groups in the hinge region of the F(ab’) fragment, site-specific conjugation is challenging. Moreover, the reduction of the F(ab’)2to the F(ab’) fragments could also disrupt the natural interchain disulfide bonds between the CLand CH1 regions of the Fab fragments, hence further compromising site- specific conjugation.

[0253] As set forth herein, Fab fragment (e.g., a Fab fragment that binds to CD164) can be site-selectively conjugated to the surface of a precursor lipid nanoparticle (LNP) through the natural interchain disulfide bond between the heavy chain and the light chain (i.e., the CL-CH1 disulfide bond) of the Fab fragment to make a targeted LNP (conjugate).

[0254] 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, as disclosed herein, involves reducing the natural interchain disulfide bond between the CLand CH1 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. Alternatively, 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. As described herein, despite the removal of the natural interchain disulfide bond linking the heavy and light chains of the Fab fragment, the resulting conjugates are able to effectively target specific cell types depending on the nature of the Fab targeting moiety. For instance, specific Fab fragments for targeting immune cells (e.g. T cells) as disclosed herein. A schematic of an LNP site-specifically conjugated to a Fab fragment is shown in FIG.39.

[0255] In one embodiment, Fab fragments used for conjugation may be used by recombinant methods. In particular embodiments, the Fab fragments generated recombinantly are designed not to include a hinge region at the C-terminus. Therefore, the recombinantly generated Fab fragments include only one disulfide bond between the CL-CH1 and domains. As set forthherein, the CL-CH1 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.

[0256] In some embodiments, the Fab fragment is of the IgG class, the IgM class, or the IgA class. In some embodiments, the Fab fragment is of the IgG class and has an IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fab fragment is a native protein. In some embodiments, the Fab fragment is an engineered protein.

[0257] In one aspect, 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 (CL) and the constant heavy chain domain 1 (CH1), 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.

[0258] In some aspects, 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 (CL) and the constant heavy chain domain 1 (CH1), 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.

[0259] In some embodiments, the thiol-reactive group (e.g., maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl) is chemically reacted with 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 LNPwith the modified lipid. For instance, as shown in FIG.40A, the various components (e.g., lipids) 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. Alternatively, 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. As depicted in FIG.40B, 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.

[0260] 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. In some embodiments, the reducing reagent is a mild reducing reagent. Examples of mild reducing reagents include, e.g., DTT, TCEP, and DTE. In some embodiments, the reducing reagent is TCEP. Any suitable reaction conditions can be used for the reduction of the interchain disulfide bond in step (i). In some embodiments, the reduction reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reduction reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reduction 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 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.

[0262] In some embodiments of the method or process, the thiol-reactive groups on the LNP (or lipid to be post-inserted into an LNP) comprises any suitable reactive group, including but not limited to, maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl.

[0263] In some embodiments, the thiol-reactive group is maleimide. In some embodiments, 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. In some embodiments, maleimide reacts with a free cysteine residue on the heavy chain of the Fab fragment. In some embodiments, maleimide reacts with a free cysteine residue on the light chain of the Fab fragment. In some embodiments, 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.

[0264] 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). In some embodiments, the reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, 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. In some embodiments, 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 CL-CH1 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. In FIG.42, 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)). In step (ii), 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 conjugatedto the surface of the LNP via a linkage through at least one of the free cysteine residues of the Fab fragment.

[0267] Following reaction of the reduced Fab fragment with the thiol -reactive group of the LNP, either 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. In some embodiments, 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) will likely determine which orientation is dominant. In some embodiments, increasing the number of thiol -reactive groups on the LNP increases the number of Fab fragments conjugated to two thiol -reactive groups. In some embodiments, 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.

[0268] In some embodiments, the thiol -reactive group is maleimide, as shown in FIG. 43. In some embodiments, maleimide reacts with one of the two free cysteine residues of the antibody or antigen-binding fragment thereof. In some embodiments, maleimide reacts with one of the two free cysteine residues of the Fab fragment to form a thiosuccinimide moiety. In some embodiments, maleimide reacts with a free cysteine residue on the heavy chain of the Fab fragment. In some embodiments, maleimide reacts with a free cysteine residue on the light chain of the Fab fragment. In some embodiments, 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.

[0269] In some embodiments, the thiol -reactive group is 2,3-dibromomaleimide (DBM) as shown in FIG. 44. Following reduction of the disulfide bond, the reduced Fab fragment is added to DBM covalently bonded to a lipid (represented by squiggly line in FIG. 44). As set forth above, 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. DBM reacts with a free cysteine residue on the heavy chain and a free cysteine residue on the light chain of the Fab fragment to form a bridge between the cysteine residues. Accordingly, the heavy and light chain of the Fab fragment are effectively bridged together following reaction with DBM.

[0270] In all embodiments discussed above, the thiol-reactive group can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, 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. In such embodiments, 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. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the thiol- reactive group. In some embodiments, 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). In some embodiments, 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). In some embodiments, the thiol-reactive group is bonded to at least one of the non-pegylated phospholipids comprising the LNP. In some embodiments, the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, 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.

[0271] In some embodiments, 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.

[0272] In some embodiments, 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.

[0273] In some embodiments, 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.

[0274] The disclosed methods provide stable conjugates that display excellent ability to transduce specific targeted cells. In some aspects, the disclosure provides a conjugatecomprising 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, only the constant region of the heavy chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the light chain remains associated with the covalently bond heavy chain on the surface of the LNP. In some embodiments, only the constant region of the light chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the heavy chain remains associated with the covalently bond light chain on the surface of the LNP. In some of the foregoing embodiments, 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.

[0275] In some embodiments, the Fab fragment conjugated to the LNP is an IgGl Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab fragment is covalently bonded to the LNP. In other such embodiments, the 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.

[0276] In some embodiments, the Fab fragment conjugated to the LNP is an IgG2 Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, 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.

[0277] In some embodiments, the Fab fragment conjugated to the LNP is an IgG4 Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently to the LNP. In other such embodiments, 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.

[0278] 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. 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). Following the reaction in step (ii), both Fab1 and Fab2 are conjugated to the surface of the LNP. Despite reduction of the disulfide bonds in Fab1 and Fab2, 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.

[0279] In some embodiments involving conjugating two Fab fragments (i.e., a first Fab fragment and a second Fab fragment) to the surface of an 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, the reduced first Fab fragment and the reduced second Fab fragment are contacted with the LNP simultaneously. In some embodiments, 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. In someembodiments, a total of four different Fab fragments can be conjugated to the surface of the LNP.

[0280] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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 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%. In some embodiments, 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.

[0281] In some embodiments, conjugates prepared by the method or process disclosed herein have a high density of the Fab fragment on the surface of the LNP. For instance, the conjugate can comprise a plurality of Fab fragments conjugated to the LNP surface. In some embodiments, the conjugate can comprise more than 10 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 20 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 30 Fab fragments. In some embodiments, the conjugate can comprise more than 50 Fab fragments per LNP. In some embodiments, 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.

[0282] Conjugates prepared by site-specific methods (e.g., FIGs.16-23 and 39-49) have a high density of the targeting moiety on the surface of the LNP. For instance, the conjugate can comprise more than one targeting moiety (e.g., antibody, Fab fragment or scFv) per LNP. In some embodiments, the conjugate can comprise more than 10 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 20 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, 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). In some embodiments, 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. In some embodiments, the conjugate can comprise from about 10 to about 30 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP.

[0283] In some embodiments, 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.

[0284] In some embodiments, the conjugates prepared by site-specific methods (e.g., FIGs. 16-23 and 39-49) can have two or more different targeting moieties on the surface of the LNP. For example, the conjugate can have two different targeting moieties. In some embodiments, the conjugate can have three different targeting moieties. In some embodiments, 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. In some embodiments, 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.III. LIPID NANOPARTICLES

[0285] Lipid nanoparticles, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids), also referred to herein as helper lipids; one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.

[0286] Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) 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.

[0287] In some embodiments, 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-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.

[0288] In some embodiments, 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] In some embodiments, 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. For example, in some embodiments, 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 about35 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 (e.g., comprising the therapeutic agent and / or encoding the gene modifying polypeptide, template nucleic acid, or gene modifying system) can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1.

[0290] In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0291] 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. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20.

[0292] 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. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, 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, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0293] The efficiency of encapsulation of a protein and / or nucleic acid (e.g., an mRNA encoding a polypeptide), 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. For the lipid nanoparticles described herein, 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%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0294] An LNP of the disclosure may optionally comprise one or more coatings. In some embodiments, 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.

[0295] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.

[0296] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, 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.

[0297] Additional specific LNP formulations useful for delivery of nucleic acids are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.Ionizable Lipids

[0298] The LNPs of the disclosure (e.g., targeted LNPs) comprise one or more ionizable lipids. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, 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. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) is co-formulated with the cationic lipid. The therapeutic agent (e.g., one or more nucleic acids) may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, thelipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, 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).

[0299] In some embodiments, 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. Generally, the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg / ml to about 30 mg / mL.

[0300] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid V003, depicted below. V003 is described in U.S. Patent No.10,059,655.

[0301] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid shown in Table 1. In some cases, 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. In other embodiments, Lipid 092 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 093 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 153 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid154 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 155 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 162 isused as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 163 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 169 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 176 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 178 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 183 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).Table 1: Exemplary Ionizable Lipids

[0302] In some embodiments, 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 CH2;m is 1, 2, or 3; n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1is C4-10alkyl; R2is C1-4alkyl; R3and R4are independently C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0303] In some embodiments of the formula (I), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y isIn some embodiments, Y isIn 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, R3is n- propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0304] In some embodiments, compounds of formula (I) are compounds of formula (I-A):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; R3and R4are independently C1-4alkyl, or R3and R4are 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), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, Y isIn some embodiments, Y isIn some embodiments, Y isIn 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0306] In some embodiments, compounds of formula (I) are compounds of formula (I-B):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2;FTab m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0307] In some embodiments of the formula (I-B), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl.In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0308] In some embodiments, compounds of formula (I) are compounds of formula (I-C):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0309] In some embodiments of the formula (I-C), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they areattached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0310] In some embodiments, compounds of formula (I) are compounds of formula (I-D):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0311] In some embodiments of the formula (I-D), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are 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):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0313] In some embodiments of the formula (I-E), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0314] In some embodiments, compounds of formula (I) are compounds of formula (I-F):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0315] In some embodiments of the formula (I-F), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0316] In some embodiments, compounds of formula (I) are compounds of formula (I-G):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0317] In some embodiments of the formula (I-G), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0318] In some embodiments, compounds of formula (I) are compounds of formula (I-H):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0319] In some embodiments of the formula (I-H), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0320] In some embodiments, 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 CH2; m is 1, 2, or 3; and Y is a lipophilic tail, branched or unbranched.

[0321] In some embodiments of the formula (II), Y isIn some embodiments, Y is. In some embodiments, Y isIn 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.

[0322] In some embodiments, 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.

[0323] In some embodiments of the formula (II-A), Y is. In

[0324] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (III):or a pharmaceutically acceptable salt thereof, wherein: R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle; andY is a lipophilic tail, branched or unbranched.

[0325] In some embodiments of the formula (III), Y is. In some embodiments, Y is. In some embodiments, Y isIn some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0326] In some embodiments, 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.

[0327] In some embodiments of the formula (III-A), Y is.In some embodiments, Y is. In some embodiments, Y is. ,

[0328] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 2. Table 2: Exemplary Ionizable Lipids

[0329] In some embodiments, 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 -CH2-; m is 0, 1, 2, or 3; R1is C1-4alkyl; R2is C1-4alkyl;n is 1, 2, 3, or 4; R3is C4-10alkyl; R4is C4-10alkyl; p is 2, 3, 4, 5, or 6; R5is C4-10alkyl; and R6is C4-10alkyl.

[0330] In some embodiments, 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; R5is C4-10alkyl; and R6is C4-10alkyl.

[0331] In some embodiments, compounds of formula (IV) are compounds of formula (IV- B):or a pharmaceutically acceptable salt thereof, wherein: R5is C4-6alkyl; and R6is C4-6alkyl.

[0332] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 3. Table 3: Exemplary Ionizable Lipids

[0333] In some embodiments, the ionizable lipid has one of the structures depicted below:

[0334] Other 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 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of W02013 / 016058; A of W02012 / 162210; I of US2008 / 042973; I, II, III, or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131; A of US2012 / 01011478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of US2013 / 0323269; I of US2011 / 0117125; I, II, or III of US2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242; XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 0116307; I, II, or III of US2013 / 0116307; I or II of US2010 / 0062967; I-X of US2013 / 0189351; I of US2014 / 0039032; V of US2018 / 0028664; I ofUS2016 / 0317458; I of US2013 / 0195920; 5, 6, or 10 of US10,221,127; III-3 of WO2018 / 081480; I-5 or I-8 of WO2020 / 081938; 18 or 25 of US9,867,888; A of US2019 / 0136231; II of WO2020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013; cKK-E12 / A6 of Miao et al (2020); C12-200 of WO2010 / 053572; 7C1 of Dahlman et al (2017); 304-O13 or 503-O13 of Whitehead et al; TS-P4C2 of US9,708,628; I of WO2020 / 106946; I of WO2020 / 106946.

[0335] In some embodiments, 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). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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).

[0336] In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted 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% ofthe 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%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted 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%.

[0338] The compounds disclosed herein (e.g., lipids in Table 1, Table 2, and Table 3), or their pharmaceutically acceptable salts, 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). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Helper Lipids

[0339] 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-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), l8-l-trans PE, l-stearoyl-2- oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The 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, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0340] Other examples of 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. Other 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. In some embodiments, 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.

[0341] In some embodiments, the helper lipid is a sphingolipid. In some such embodiments, the non-pegylated lipid is a sphingomyelin. In some embodiments, the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0342] In some embodiments, 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 thelipid nanoparticle. In embodiments, 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).

[0343] 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 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%. In some embodiments, 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%. 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%. 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%. In some embodiments, 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%. As set forth in the examples below, 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.

[0344] In some embodiments, 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. In some embodiments, 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 fromTable 19 or Table 32 (or CDR sequences (bolded) selected from Table 19 or Table 32). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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). In some embodiments, the LNP comprises a CD28 targeting moiety. In some embodiments, the LNP comprises a CD4 targeting moiety. In some embodiments, the LNP comprises a CD8 targeting moiety. In some embodiments, 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. In some embodiments, 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. In some embodiments, the CD3 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 19 or Table 32, and the CD2, CD5, or CD7 targeting moiety comprises variable heavy and light chain sequences (or CDR sequences (bolded)) selected from Table 20. In some embodiments, 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. In some embodiments, the template RNA comprises a sequence present in Table 5B or a sequence selected from SEQ ID NOs: 395- 404.

[0345] In some embodiments, 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 thegroup 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). In some embodiments, 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 sequ...

Claims

CLAIMS1. 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 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;(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.

2. 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 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;(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.

3. The LNP of claim 2, wherein the means for binding the cell surface receptor is present on the surface of the LNP.

4. The LNP of claim 2 or claim 3, wherein the LNP comprises means for binding a first cell surface receptor and means for binding a second cell surface receptor, wherein the first cell surface receptor and the second cell surface receptor are different and wherein thefirst cell surface receptor and the second cell surface receptor are each independently selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, CD8 and CD28.

5. The LNP of any one of claims 1-4, wherein the helper lipid is present at about 22 mol% to about 36 mol% of the lipid component.

6. The LNP of any one of claims 1-4, wherein the helper lipid is present at about 20 mol% to about 30 mol% of the lipid component.

7. The LNP of any one of claims 1-6, wherein the helper lipid is present at about 25 mol% to about 30 mol% of the lipid component.

8. The LNP of any one of claims 1-6, wherein the helper lipid is present at about 20 mol% to about 25 mol% of the lipid component.

9. The LNP of any one of claims 1-8, wherein ionizable lipid is present at about 40 mol% to about 50 mol% of the lipid component.

10. The LNP of any one of claims 1-9, wherein ionizable lipid is present at about 20 mol% to about 30 mol% of the lipid component.

11. The LNP of any one of claims 1-10, wherein 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)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O- dimethyl PE), l8-l-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

12. The LNP of claim 11, wherein the helper lipid is DSPC.

13. The LNP of claim 11, wherein the helper lipid is sphingomeylin.

14. The LNP of any one of claims 1-13, further comprising a cholesterol molecule.

15. The LNP of claim 14, wherein the cholesterol molecule is selected from the group consisting of beta-sitoesterol, hydroxycholesterol, stigmastanol, and any combination thereof.

16. The LNP of claim 14 or claim 15, wherein the cholesterol molecule is present at about 20 mol% to about 55 mol% of the lipid component.

17. The LNP of claim 14 or claim 15 wherein the cholesterol molecule is present at about 20 mol% to about 35 mol% of the lipid component.

18. The LNP of any one of claims 1 and 5-17, wherein the lipid component further comprises a PEGylated lipid.

19. The LNP of claim 18, wherein the one or two targeting moieties are conjugated to the LNP through the PEGylated lipid.

20. The LNP of claim 3 or claim 4, wherein the lipid component further comprises a PEGylated lipid.

21. The LNP of claim 20, wherein the means for binding the cell surface receptor is conjugated to the surface of the LNP through a PEGylated lipid.

22. The LNP of any one of claims 18-21, wherein the PEGylated lipid comprises a lipid portion selected from the group consisting of DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, stearic acid, and any combination thereof.

23. The LNP of claim 22, wherein the lipid portion of the PEGylated lipid is DSPE.

24. The LNP of any one of claims 18-23, wherein the PEGylated lipid is present at about 0.05 mol% to about 2.5 mol% of the lipid component.

25. The LNP of any one of claims 1, 5-19 and 22-24, wherein the LNP comprises one targeting moiety conjugated to the surface of the LNP.

26. The LNP of claim 25, wherein the one targeting moiety binds CD2.

27. The LNP of claim 25, wherein the one targeting moiety binds CD3.

28. The LNP of claim 25, wherein the one targeting moiety binds CD5.

29. The LNP of claim 25, wherein the one targeting moiety binds CD6.

30. The LNP of claim 25, wherein the one targeting moiety binds CD7.

31. The LNP of claim 25, wherein the one targeting moiety binds CD8.

32. The LNP of claim 25, wherein the one targeting moiety binds CD28.

33. The LNP of claim 25, wherein the one targeting moiety binds CD4.

34. The LNP of any one of claims 1, 5-19 and 22-24, wherein the LNP comprises two targeting moieties conjugated to the surface of the LNP.

35. The LNP of claim 34, wherein the two targeting moieties bind CD3 and CD2.

36. The LNP of claim 34, wherein the two targeting moieties bind CD3 and CD4.

37. The LNP of claim 34, wherein the two targeting moieties bind CD3 and CD5.

38. The LNP of claim 34, wherein the two targeting moieties bind CD3 and CD6.

39. The LNP of claim 34, wherein the two targeting moieties bind CD3 and CD7.

40. The LNP of claim 34, wherein the two targeting moieties bind CD3 and CD8.

41. The LNP of claim 34, wherein the two targeting moieties bind CD3 and CD28.

42. The LNP of any one of claims 35-40, further comprising a third targeting moiety, wherein the third targeting moiety binds CD28.

43. The LNP of any one of claims 1, 5-19 and 22-24, wherein the LNP comprises two different targeting moieties conjugated to the surface of the LNP, wherein the two different targeting moieties bind the same cell surface receptor.

44. The LNP of claim 43, wherein the cell surface receptor is CD3.

45. The LNP of claim 27 or any one of claims 35-42 or 44, wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 19 or Table 32, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

46. The LNP of claim 30 or claim 39, wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table20, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

47. The LNP of claim 26 or claim 35, wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

48. The LNP of claim 33 or claim 36, wherein the CD4 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20.

49. The LNP of claim 28 or claim 37, wherein the CD5 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

50. The LNP of claim 31 or claim 40, wherein the CD8 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20.

51. The LNP of any one of claims 1-50, wherein the ionizable lipid is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: X is -O- or -CH2-; m is 0, 1, 2, or 3; R1is C1-4alkyl; R2is C1-4alkyl;n is 1, 2, 3, or 4; R3is C4-10alkyl; R4is C4-10alkyl; p is 2, 3, 4, 5, or 6; R5is C4-10alkyl; and R6is C4-10alkyl.

52. The LNP of any one of claims 1-50, wherein the ionizable lipid is a compound of one of the structures depicted below:

53. The LNP of any one of claims 1-52, wherein the therapeutic agent comprises one nucleic acid molecules, wherein the one nucleic acid molecule is an RNA molecule.

54. The LNP of claim 53, wherein the therapeutic agent comprises two nucleic acid molecules, wherein the two nucleic acid molecules are RNA molecules.

55. The LNP of claim 54, wherein one RNA molecule is an mRNA and the other RNA molecule is a template RNA.

56. The LNP of claim 55, wherein the mRNA encodes a gene modifying polypeptide polypeptide or a heterologous gene modifying polypeptide.

57. The LNP of claim 56, wherein the mRNA encodes a gene modifying polypeptide.

58. The LNP of any one of claims 55-57, wherein the template RNA is configured to insert a heterologous sequence encoding a protein into the genome of an immune cell.

59. The LNP of any one of claims 55-58, wherein the template RNA encodes a chimeric antigen receptor (CAR).

60. The LNP of any one of claims 1-59, wherein the therapeutic agent comprises three nucleic acid molecules.

61. The LNP of any one of claims 1-52, wherein the therapeutic agent comprises an mRNA encoding a CAR.

62. The LNP of any one of claims 1-52, wherein the therapeutic agent comprises a DNA molecule encoding a CAR63. The LNP of claim 61 or claim 62, wherein the CAR comprises a sequence provided in Table 16.

64. 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 to 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) an mRNA encoding a gene modifying polypeptide; and(iv) a template RNA comprising (a) sequence that binds to the polypeptide and (b) a heterologous object sequence.

65. The LNP of claim 64, wherein the heterologous object sequence encodes a chimeric antigen receptor (CAR).

66. The LNP of claim 65, wherein(i) 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;(ii) the CAR comprises an antigen binding domain that binds one or more antigens of a solid tumor;(iii) the CAR comprises an antigen binding domain of any one of Tables 6-10 or 16;(iv) the CAR comprises a transmembrane domain of Table 11 or 12;(v) the CAR comprises a hinge domain (e.g., a hinge domain of Table 15);(vi) the CAR comprises an intracellular signaling domain of Table 13 or 14;(vii) the CAR comprises a costimulatory domain of Table 13 or 14;(viii) the CAR comprises an antigen binding domain which comprises an scFv, a Fab, a diabody, a D domain binder, a centryin, or one or more single domain antibodies (e.g., VHH domains); or(ix) the CAR comprises an amino acid sequence of Table 16 or an amino acid sequence according to any one of SEQ ID NOs: 74, 76-78, 80-87 or 102-125; and / or(x) wherein the CAR comprises a first intracellular signaling domain and a second intracellular signaling domain.

67. The LNP of claim 65 or claim 66, wherein the template RNA sequence comprises an RNA sequence of Table 5B or an RNA sequence of any one of SEQ ID NOs: 395-404.

68. The LNP of any one of claims 64-67, wherein 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.

69. The LNP of any one of claims 64-68, wherein the gene modifying polypeptide comprises an amino acid sequence listed in any of Examples 4-11 or 15-17 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.

70. The LNP of any one of claims 69, wherein 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.

71. The LNP of claim 64, wherein the sequence that binds the polypeptide comprises a 5’UTRretroor a 3’ UTRretro.

72. The LNP of claim 64, wherein the template RNA comprises both of a 5’UTRretroand a 3’ UTRretro.

73. The LNP of claim 71 or 72, wherein the 5’UTRretroand the 3’ UTRretrocomprise 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.

74. The LNP of any one of claims 64-73, wherein the helper lipid is present at about 20 mol% to about 40 mol% of the lipid component.

75. The LNP of any one of claims 64-73, wherein the helper lipid is present at about 22 mol% to about 36 mol% of the lipid component.

76. The LNP of any one of claims 64-73, wherein the helper lipid is present at about 20 mol% to about 30 mol% of the lipid component.

77. The LNP of any one of claims 64-73, wherein the ionizable lipid is present at about 40 mol% to about 50 mol% of the lipid component or about 20 mol% to about 30 mol% of the lipid component.

78. The LNP of any one of claims 64-77, wherein the LNP comprises one targeting moiety conjugated to the surface of the LNP.

79. The LNP of claim 78, wherein the one targeting moiety binds CD2.

80. The LNP of claim 78, wherein the one targeting moiety binds CD3.

81. The LNP of claim 78, wherein the one targeting moiety binds CD4.

82. The LNP of claim 78, wherein the one targeting moiety binds CD5.

83. The LNP of claim 78, wherein the one targeting moiety binds CD6.

84. The LNP of claim 78, wherein the one targeting moiety binds CD7.

85. The LNP of claim 78, wherein the one targeting moiety binds CD8.

86. The LNP of claim 78, wherein the one targeting moiety binds CD28.

87. The LNP of any one of claims 64-77. wherein the LNP comprises two targeting moieties conjugated to the surface of the LNP.

88. The LNP of claim 87, wherein the two targeting moieties bind CD3 and CD2.

89. The LNP of claim 87, wherein the two targeting moieties bind CD3 and CD4.

90. The LNP of claim 87, wherein the two targeting moieties bind CD3 and CD5.

91. The LNP of claim 87, wherein the two targeting moieties bind CD3 and CD6.

92. The LNP of claim 87, wherein the two targeting moieties bind CD3 and CD7.

93. The LNP of claim 87, wherein the two targeting moieties bind CD3 and CD8.

94. The LNP of claim 87, wherein the two targeting moieties bind CD3 and CD28.

95. The LNP of any one of claims 78-93, further comprising a third targeting moiety, wherein the third targeting moiety binds CD28.

96. The LNP of any one of claims 64-73, wherein the LNP comprises two different targeting moieties conjugated to the surface of the LNP, wherein the two different targeting moieties bind the same cell surface receptor.

97. The LNP of claim 96, wherein the cell surface receptor is CD3.

98. The LNP of claim 80 or any one of claims 88-94, wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 19 or Table 32, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

99. The LNP of claim 79 or claim 88, wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

100. The LNP of claim 82 or claim 90, wherein the CD2 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20.

101. The LNP of claim 84 or claim 92, wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

102. The LNP of any one of claims 64-101, wherein the ionizable lipid is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: X is -O- or -CH2-; m is 0, 1, 2, or 3; R1is C1-4alkyl; R2is C1-4alkyl; n is 1, 2, 3, or 4; R3is C4-10alkyl; R4is C4-10alkyl; p is 2, 3, 4, 5, or 6; R5is C4-10alkyl; and R6is C4-10alkyl.

103. The LNP of any one of claims 64-101, wherein the ionizable lipid is a compound of one of the structures depicted below:

104. The LNP of any one of claims 1-103, wherein the one or two targeting moieties are Fab fragments.

105. The LNP of claim 104, wherein the conjugation is performed by a method comprising: (i) contacting 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 (CL) and the constant heavy chain domain 1 (CH1), 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 conjugate.

106. The LNP of claim 105, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP).

107. The LNP of claim 105 or claim 106, wherein the thiol-reactive group is maleimide.

108. The LNP of claim 105 or claim 106, wherein the thiol-reactive group is 2,3- dibromomaleimide (DBM).

109. The LNP of any one of claims 105-108, wherein the thiol-reactive group is covalently bonded to one or more of the lipids comprising the precursor LNP.

110. A pharmaceutical composition comprising an LNP of any one of claims 1-109.

111. A method of delivering a therapeutic agent to the T cells of a subject, said method comprising in vivo administration to the subject of a pharmaceutical composition of claim 110.

112. The method of claim 111, wherein the subject is a human patient.

113. The method of claim 111 or claim 112, wherein the therapeutic agent is an anti-cancer agent or an immunosuppressant targeting an antigen associated with an autoimmune disease.

114. A method of treating cancer in a human patient in need thereof, said method comprising in vivo administration of a pharmaceutical composition of claim 110 to the human patient.

115. The method of claim 114, wherein the cancer is an advanced leukemia or lymphoma.

116. The method of claim 114, wherein cancer is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), or Diffuse Large B-Cell Lymphoma (DLBCL).

117. The method of any one of claims 111-116, wherein the pharmaceutical composition is administered parenterally.

118. The method of claim 117, wherein the pharmaceutical composition is administered intravenously, intramuscularly, subcutaneously.

119. A method of delivering a therapeutic agent to the T cells of a subject, said method comprising: a. isolating T cells from the subject, b. mixing a pharmaceutical composition of claim 110 with the T cells for a sufficient time to allow for encapsulation of the therapeutic agent in the T cells; and c. administering the T cells encapsulating the therapeutic agent to the subject.

120. The method of claim 119, wherein the subject is a human patient.

121. The method of claim 119 or 120, wherein the therapeutic agent is an anti-cancer agent or an immunosuppressant targeting an antigen associated with an autoimmune disease.

122. A method of treating cancer in a human patient in need thereof, said method comprising a. isolating T cells from the subject, b. mixing a pharmaceutical composition of claim 110 with the T cells for a sufficient time to allow for encapsulation of the therapeutic agent in the T cells; and c. administering the T cells encapsulating the therapeutic agent to the subject.

123. The method of claim 122, wherein the cancer is an advanced leukemia or lymphoma.

124. The method of claim 122 wherein cancer is acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), or Diffuse Large B-Cell Lymphoma (DLBCL).

125. The method of any one of claims 119-124, wherein the T cells are not activated prior to mixing with the pharmaceutical composition of claim 104.

126. The method of claim 125, wherein the T cells become activated after mixing with the pharmaceutical composition of claim 104.

127. The method of claim 125 or claim 126, wherein at least one or two targeting moieties conjugated to the LNP binds CD3.

128. The method of claim 125 or claim 126, wherein the LNP comprises two targeting moieties, wherein one targeting moiety binds CD3 and the other targeting moiety binds CD28.

129. The method of claim 125 or claim 126, wherein the LNP comprises two targeting moieties, wherein one targeting moiety binds CD3 and the other targeting moiety binds CD2, one targeting moiety binds CD3 and the other targeting moiety binds CD4, one targeting moiety binds CD3 and the other targeting moiety binds CD5, one targeting moiety binds CD3 and the other targeting moiety binds CD6, one targeting moiety binds CD3 and the other targeting moiety binds CD7, or one targeting moiety binds CD3 and the other targeting moiety binds CD8.

130. The method of claim 125 or claim 126, wherein the LNP comprises a first targeting moiety and a second targeting moiety, wherein the first targeting moiety and the second targeting moiety bind CD3.

131. A method for administering a therapeutic composition to a patient, comprising: a. collecting a blood fraction comprising lymphocytes from the patient; b. isolating T cells from the blood fraction; c. contacting the isolated T cells with lipid nanoparticles (LNPs) according to any one of claims 1-109, thereby producing T cells encapsulating a therapeutic payload; and d. reinfusing the T cells of step (c) into the patient.

132. A method for treating cancer in a patient, comprising: a. collecting a blood fraction comprising lymphocytes from the patient; b. isolating T cells from the blood fraction; c. contacting the isolated T cells with lipid nanoparticles (LNPs) according to any one of claims 1-109, thereby producing T cells encapsulating a therapeutic payload; and d. reinfusing the T cells of step (c) into the patient.

133. The method of claim 131 or claim 132, wherein the T cells are not activated prior to step (c).

134. The method of any one of claims 131-133, wherein the two targeting moieties bind CD3 and CD28.

135. The method of any one of claims 131-133, wherein the two targeting moieties bind CD3 and CD2.

136. The method of any one of claims 131-133, wherein the two targeting moieties bind CD3 and CD4.

137. The method of any one of claims 131-133, wherein the two targeting moieties bind CD3 and CD5.

138. The method of any one of claims 131-133, wherein the two targeting moieties bind CD3 and CD6.

139. The method of any one of claims 131-133, wherein the two targeting moieties bind CD3 and CD7.

140. The method of any one of claims 131-133, wherein the two targeting moieties bind CD3 and CD8.

141. A lipid nanoparticle (LNP) comprising: a. a lipid component comprising an ionizable lipid and a helper lipid,; b. one or two targeting moieties conjugated to the LNP, wherein the one or two targeting moieties are 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 c. an mRNA encoding a heterologous gene modifying polypeptide; and a template RNA (or DNA encoding the template RNA) comprising (1) a gRNA spacer, (2) a gRNA scaffold, (3) a heterologous object sequence, and (4) a primer binding site (PBS) sequence.

142. The LNP of claim 141, wherein the LNP further comprises a second template RNA (or DNA encoding the template RNA) comprising (1) a gRNA spacer, (2) a gRNA scaffold, (3) a heterologous object sequence, and (4) a primer binding site (PBS) sequence.

143. The LNP of any of claim 141 or claim 142, wherein the heterologous gene modifying polypeptide comprises:a reverse transcriptase (RT) domain (e.g., an RT domain from a retrovirus, or a polypeptide domain having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acids sequence identity thereto); and a Cas domain that binds to the bind DNA molecule and is heterologous to the RT domain (e.g., a Cas9 domain); and optionally, a linker disposed between the RT domain and the Cas domain.

144. The LNP of claim 143, wherein the RT domain comprises an amino acid sequence of Table 17, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

145. The LNP of claim 143 or claim 144, wherein the Cas domain comprises a Cas9.

146. The LNP of any one of claims 143-145, wherein the Cas domain comprises a Cas domain of SEQ ID NO: 126 and / or SEQ ID NO: 127, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

147. The LNP of any of any of claims 143-146, wherein the heterologous gene modifying polypeptide comprises a linker, optionally wherein the linker comprises an amino acid sequence of Table 17, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

148. The LNP of claim 143, wherein the template RNA is configured to alter a nucleotide sequence at the human TRAC or B2M locus to disrupt the TRAC or B2M gene.

149. The LNP of claim 144, wherein the second template is configured to alter a nucleotide sequence at the human TRAC or B2M locus to disrupt the TRAC or B2M gene.

150. The LNP of claim 148, wherein the template RNA comprises a sequence of Table 23 or Table 24.

151. The LNP of claim 149, wherein the second template RNA comprises a sequence of Table 23 or 24.

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