In VIVO delivery of therapeutic agents to t cells

Abstract

The disclosure provides targeted LNPs comprising a targeting moiety a lipid nanoparticle (LNP) encapsulating a therapeutic agent (e.g., payload) for delivery to immune cells. The targeted LNPs 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

Attorney Docket No.: 25205-20027.40IN VIVO DELIVERY OF THERAPEUTIC AGENTS TO T CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63 / 729,083, filed on December 6, 2024; U.S. Provisional Application 63 / 752,513, filed on January 31, 2025; and U.S. Provisional Application 63 / 792,735, filed on April 22, 2025, 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 (252052002740seqlist.xml; Size: 499,740 bytes; and Date of Creation: December 3, 2025) 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 pay loads 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) must be able to disassociate from the endosome so it can be expressed in sufficient quantities in the targeted cell.1MF-364968774Attorney Docket No.: 25205-20027.40

[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] Furthermore, for in vivo delivery of appreciable amounts of nucleic acid payloads to T cells, activation of the T cells can be advantageous. In the context of LNPs, activation of T cells can be achieved by introducing an activating ligand on the surface of the T cell, such as a targeting moiety that binds to CD3 on the surface of the T cell. Activation of T cells, however, can potentially invoke an immunological response that can be toxic to the cells, thus limiting the use of LNPs in in vivo applications. Therefore, currently, ex vivo approaches remain the standard in developing T cell-based therapeutics, such as CAR-T.

[0008] Accordingly, there exists a need to develop LNPs for safe and effective delivery to immune cells, particularly using in vivo delivery methodsSUMMARY OF THE INVENTION

[0009] The disclosure provides a lipid nanoparticle (LNP) comprising a lipid component comprising an ionizable lipid and a helper lipid and a first targeting moiety and a second targeting moiety conjugated to the surface of the lipid component, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T cells selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8 and CD28, and wherein the second targeting moiety is present in a greater molar amount than the first targeting moiety. The LNPs of the disclosure will also be referred to herein as targeted LNPs or tLNPs,

[0010] As set forth herein, the inventors of the present application surprisingly discovered that significant amounts of payloads (e.g., therapeutic agents) can be delivered in vivo to T cells and subsequently expressed in the T cells using LNPs as delivery vehicles, wherein the quantity of a CD3 binder on the surface of the LNP is less than the quantity previously thought to be required for appreciable amounts of delivery and expression of the payload in the T cells, provided that a second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) is also present on the surface of the LNP. Without being bound by theory, the CD3 binder provides sufficient activation of the T cells that enhances delivery of a payload to cells, while the second targeting moiety, which is present in higher amounts than the CD3 binder on the surface of the LNP, further contributes to T cell binding, transduction and / or expression of the payload in the T cells. As shown in the examples, the second targeting moiety by itself is not capable of2MF-364968774Attorney Docket No.: 25205-20027.40 achieving high levels of transduction or expression of the pay load. Likewise, low levels of the CD3 binder, without the second targeting moiety present in the LNP, results in inadequate delivery and expression of the payload in the T cells. However, a synergy is observed when the CD3 binder and the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) are both present on the surface of the LNP. The synergy allows for sufficient expression of therapeutic pay loads, in some embodiments with less concurrent toxicity often associated with in vivo administration of LNPs. Accordingly, the present invention is applicable in different aspects of in vivo T cell-based therapeutics, including CAR-T.

[0011] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.2:2. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.4: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.6: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.8:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 2:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 3:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 4: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 5:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 6:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 7: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 8: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least3MF-364968774Attorney Docket No.: 25205-20027.4010: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 15:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 20: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 50: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 100: 1.

[0012] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of from about 1.2:1 to about 100:1, from about 1.2:1 to about 20:1, from about 1.2:1 to about 10: 1, from about 1.5: 1 to about 50:1, from about 1.5:1 to about 20:1, from about 1.5:1 to about 10: 1, from about 1.5:1 to about 5: 1, from about 2:1 to about 100: 1, from about 2: 1 to about 50: 1, from about 2: 1 to about 20:1, from about 2:1 to about 10:1, from about 2: 1 to about 5:1, from about 2: 1 to about 4:1, from about 5:1 to about 40:1, from about 5: 1 to about 20:1, from about 5:1 to about 10:1, or from about 10: 1 to about 20:1.

[0013] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.2:2. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.4:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.6: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.8: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 2:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 3:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 4: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at4MF-364968774Attorney Docket No.: 25205-20027.40 a mass ratio of at least 5: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 6: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 7: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 8: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 10: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 15:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 20: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 50: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 100: 1.

[0014] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of from about 1.2: 1 to about 100:1, from about 1.2: 1 to about 20:1, from about 1.2: 1 to about 10: 1, from about 1.5: 1 to about 50:1, from about 1.5:1 to about 20:1, from about 1.5:1 to about 10: 1, from about 1.5: 1 to about 5: 1, from about 2:1 to about 100: 1, from about 2: 1 to about 50:1, from about 2: 1 to about 20:1, from about 2:l to about 10:1, from about 2:l to about 5: 1, from about 2: 1 to about 4: 1, from about 5:1 to about 40:1, from about 5: 1 to about 20:1, from about 5:1 to about 10:1, or from about 10: 1 to about 20:1.

[0015] In other aspects, the disclosure, provides a lipid nanoparticle (LNP) comprising:(i) a lipid component comprising an ionizable lipid and a helper lipid;(ii) a first targeting moiety and a second target moiety conjugated to the surface of the lipid component of the LNP, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T5MF-364968774Attorney Docket No.: 25205-20027.40 cells selected from the group consisting of CD2, 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, wherein the LNP is prepared by chemically reacting the lipid component of the LNP with the first targeting moiety and the second targeting moiety, wherein the molar quantity of second targeting moiety reacting with the lipid component of the LNP is greater than the molar quantity of the first targeting moiety reacting with the lipid component of the LNP.

[0016] In some embodiments, the molar quantity of the second targeting moiety used to react with the lipid component of the LNP is at least 1.2, 1.4, 1.6, 1.8, 2, 3, 5, 5, 6, 7, 8, 9 10, or 20 times greater than the molar quantity of the first targeting moiety used to react with the lipid component of the LNP.

[0017] In some embodiments, the mass quantity of the second targeting moiety used to react with the lipid component of the LNP is at least 1.2, 1.4, 1.6, 1.8, 2, 3, 5, 5, 6, 7, 8, 9 10, or 20 times greater than the molar quantity of the first targeting moiety used to react with the lipid component of the LNP.

[0018] In some embodiments, 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.

[0019] In some embodiments, the targeted LNPs of the disclosure 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.

[0020] Alternatively, the targeted LNPs can be added to isolated immune cells (e.g., T cells) using an in vitro or ex vivo procedure. Cells that can be derived from the blood, bone marrow, lymphoid organs, or lymph are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. 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 the6MF-364968774Attorney Docket No.: 25205-20027.40 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 targeted LNPs of the disclosure are capable of activating the rested immune cells (e.g., T cells).

[0021] In some embodiments, the payload (e.g., therapeutic agent) 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.

[0022] In some embodiments, the pay load 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.

[0023] 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, a heavy-chain only IgG, 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 a receptor (e.g., a receptor on the surface of T cells).

[0024] 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 for7MF-364968774Attorney Docket No.: 25205-20027.40 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 a heterologous sequence into the genome, 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.

[0025] In some embodiments, the mass ratio of the first targeting moiety (CD3 binder) to the therapeutic agent is from about 0.001 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.5: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.25:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.1 : 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.025:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.01 : 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 :1.0 to about 0.5: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.25: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.05:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.025: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.005: 1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.01 : 1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.025:1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.05: 1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.1 : 1.8MF-364968774Attorney Docket No.: 25205-20027.40

[0026] In some embodiments, the mass ratio of the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8, or CD28 binder) to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.5:1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 0.5:1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 0.4: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4:1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 1.0: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 1.0: 1.0 to about 1.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.3 : 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.4:1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.5 : 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 1.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 1.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 2.0: 1.0.

[0027] In some embodiments, the mass ratio of the first targeting moiety (CD3 binder) to the therapeutic agent is from about 0.001 : 1.0 to about 1.0:1.0 and the mass ratio of the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8, or CD28 binder) to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.5: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.5: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.5: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic9MF-364968774Attorney Docket No.: 25205-20027.40 agent is from about 0.01 : 1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.05: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.05:1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4:1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 :1.0 to about 0.025:1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4:1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.025: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 1.0: 1.0.

[0028] It will be understood that in the foregoing embodiments, the mass of the therapeutic agent refers to the total mass of the payload (e.g., nucleic acids) encapsulated in the LNP. In embodiments where the therapeutic agent comprises more than one nucleic acid, the mass ratio of the targeting moiety to the therapeutic agent refers to the ratio between the mass of the targeting moiety and the total mass of the nucleic acid molecules. For instance, in embodiments where the LNP encapsulates two RNA molecules (e.g., an RNA encoding a gene modifying polypeptide and a guide or template RNA), a 2.0: 1.0 mass ratio of targeting moiety to therapeutic agent refers to 2 parts targeting moiety by weight to 1 part total RNA (RNA encoding gene modifying peptide and guide or template RNA) by weight.

[0029] In one aspect, the disclosure provides a targeted LNP encapsulating one or more nucleic acids encoding components of a system for modifying or altering DNA (e.g., genomic DNA). 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 acids10MF-364968774Attorney Docket No.: 25205-20027.40 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).

[0030] 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 aa targeted LNP described herein 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 a targeted LNP described herein.

[0031] 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 a targeted 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 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) from the isolated immune cell (e.g., T cell) population comprise a genome that has been modified or altered.

[0032] 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 chimeric antigen receptor (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).

[0033] 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 be11MF-364968774Attorney Docket No.: 25205-20027.40 generated using the tLNPs described herein to target B cell markers such as CD 19, CD20, and BCMA. In some embodiments, the autoimmune disease is systemic lupus erythematosus, refractory antisynthetase syndrome, myasthenia gravis, systemic scleroderma, and multiple sclerosis.

[0034] In some embodiments, the targeted LNPs 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, naive T cells, stem cell memory T cells, central memory T cells, or a mixture of subpopulations. In some embodiments, the targeted LNPs may be used to deliver or modify a sequence encoding a T-cell receptor (TCR) in a T cell. In some embodiments, the targeted LNPs may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T-cells. For instance, in specific embodiments, the targeted LNPs can be used to deliver an RNA encoding a CAR to T-cells. In some cases, the targeted LNPs are formulated with an mRNA encoding a CAR. Such targeted LNPs 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 targeted LNPs 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 targeted LNPs can be contacted with T cells to deliver the DNA molecule encoding the CAR, resulting in cellular expression of the CAR. In some embodiments, the targeted LNPs may be used to deliver at least one sequence encoding a CAR to natural killer (NK) cells. In some embodiments, the targeted LNPs may be used to deliver at least one sequence encoding a CAR to natural killer T (NKT) cells. In some embodiments, the targeted LNPs 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.

[0035] In some embodiments, the targeted LNPs 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 targeted LNPs 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 targeted LNPs 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 targeted LNPs 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.12MF-364968774Attorney Docket No.: 25205-20027.40

[0036] In some embodiments, the targeted LNPs 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 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 can be used to deliver the components one or more gene modifying systems. For instance, one targeted LNP 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 targeted LNP 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 targeted LNP, 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 targeted LNP, as described herein, can be used to deliver a heterologous gene modifying system to an immune cell, e.g., a T cell.

[0037] In some embodiments, tLNPs of the disclosure comprising dual binders can be administered in vivo, in vitro, or ex vivo to deliver more than one CAR into the genome of T cells. For instance, a single LNP can deliver two RNA molecules that each encodes a CAR. In other embodiments, separate LNPs can deliver each of two RNA molecules that each encodes a CAR. Alternatively, a single LNP can deliver a gene modifying system to T cells, wherein the gene modifying system comprises two template RNAs that each encodes a different CAR. The gene modifying system are capable of generating dual-specific CAR-T cells by multiplex insertion of the two different CARs into the genome. As a result, the T cells, following administration of the LNP, express two different CARs on their surface. The two different13MF-364968774Attorney Docket No.: 25205-20027.40CARs preferentially target two different target antigens. In some embodiments, the target antigens are cancer antigens.

[0038] In some embodiments, the immune cells comprise two CARs 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), LI CAM (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), and HPV (human papilloma virus).

[0039] In some embodiments, the two CARs delivered by the tLNPs of the disclosure are CD19-targeted CARs (CD19 CARs) and CD20-targeted CARs (CD20 CARs). In some embodiments, the two CARs delivered by the tLNPs of the disclosure are CD19-targeted CARs (CD 19 CARs) and BCMA-targeted CARs (BCMA CARs). In some embodiments, the two CARs delivered by the tLNPs of the disclosure are CD20-targeted CARs (CD20 CARs) and BCMA-targeted CARs (BCMA CARs). In some embodiments, the two CARs delivered by the tLNPs of the disclosure are CD19-targeted CARs (CD 19 CARs) and GPRC5D-targeted CARs (GPRC5D CARs). In some embodiments, the two CARs delivered by the tLNPs of the disclosure are CD20-targeted CARs (CD20 CARs) and GPRC5D-targeted CARs (GPRC5D CARs). In some embodiments, the two CARs delivered by the tLNPs of the disclosure are BCMA-targeted CARs (BCMA CARs) and GPRC5D-targeted CARs (GPRC5D CARs).14MF-364968774Attorney Docket No.: 25205-20027.40

[0040] In some embodiments, the two CARs delivered by the tLNPs of the disclosure are any of the two CAR molecules listed in Table 17.

[0041] In some embodiments, the targeted LNPs of the disclosure may be used to simultaneously deliver gene modifying components to T cells that are capable of inserting a sequence encoding at two CARs 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 such embodiments, the two CARs delivered by the tLNPs of the disclosure are any of the two CAR molecules listed in Table 17.

[0042] In some embodiments, a targeted LNP of the present disclosure comprises the ionizable lipid V003, depicted below.Lipid V003

[0043] In some embodiments, a targeted LNP, of the disclosure comprises an ionizable lipid in Table 1. In some embodiments, a targeted LNP, of the disclosure comprises an ionizable lipid in Table 2. In some embodiments, a targeted LNP, of the disclosure comprises an ionizable lipid in Table 3. 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 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 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, a targeted 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).

[0044] In some embodiments, the ionizable lipid has one of the structures depicted below:15MF-364968774Attorney Docket No.: 25205-20027.40Lipid A316MF-364968774Attorney Docket No.: 25205-20027.40Lipid A1417MF-364968774Attorney Docket No.: 25205-20027.40Lipid A8

[0045] In some embodiments, the ionizable lipid has one of the structures depicted below:18MF-364968774Attorney Docket No.: 25205-20027.40Lipid A2019MF-364968774Attorney Docket No.: 25205-20027.40

[0046] In some embodiments, the 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.

[0047] 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., Cl 8 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 Cl 6 PEG lipid anchors or Cl 8 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).

[0048] In some embodiments, the pegylated lipid has at least one Cl 6 (palmitoyl) PEG lipid anchor. In some embodiments, the pegylated lipid has two Cl 6 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 -methylpoly oxy ethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is Cl 6 PEG ceramide. In some embodiments, the targeted LNPs comprising the Cl 6 pegylated lipids show significantly reduced liver uptake than otherwise identical LNPs comprising Cl 4 pegylated lipids.20MF-364968774Attorney Docket No.: 25205-20027.40

[0049] In some embodiments, the pegylated lipid has at least one Cl 8 PEG lipid anchor. In some embodiments, the pegylated lipid has two Cl 8 PEG lipid anchors (i.e., dialkyl chains of 18 carbons long). In some embodiments, the Cl 8 pegylated lipid is 1 ,2-distearoyl-sn-glycero-3- phosphoetlianolamine-N-[methoxy(poly ethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the Cl 8 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG-PEG2000).

[0050] 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.

[0051] In some embodiments, a targeted LNP of the present disclosure 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.

[0052] In some embodiments, 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 helper lipid (e.g., DSPC or sphingomyelin) in the 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 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 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 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 targeted LNPis 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 from about 10% to about 20%. 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%, about21MF-364968774Attorney Docket No.: 25205-20027.4032%, about 33%, about 34% or about 35%. In other embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. As set forth in the examples below, in vivo delivery of certain pay loads 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.

[0053] 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.

[0054] 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%).22MF-364968774Attorney Docket No.: 25205-20027.40

[0055] 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 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%).

[0056] 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%).

[0057] 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 23MF-364968774Attorney Docket No.: 25205-20027.40 is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 50% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 30% to about 40%. In some of the foregoing embodiments, the mol% of cholesterol in the targeted LNP is from about 25%-40%,

[0058] In some embodiments, the targeted LNP comprises Lipid A2. In some embodiments, the targeted LNP comprises Lipid A2 and DSPC. In some embodiments, the targeted LNP, comprises Lipid A2 and a sphingomyelin. In some embodiments, the mol% 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 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 targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). In some such embodiments, the LNP is DPPE- PEG2000 or DPG-PEG2000.

[0059] In some embodiments, the targeted LNP comprises Lipid Al . In some embodiments, the targeted LNP, comprises Lipid Al and DSPC. In some embodiments, the targeted LNP, comprises Lipid Al and a sphingomyelin. In some embodiments, the mol% 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 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 Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted 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.

[0060] In some embodiments, the targeted LNP comprises Lipid A10. In some embodiments, the targeted LNP comprises Lipid A10 and DSPC. In some embodiments, the targeted LNP comprises Lipid A10 and a sphingomyelin. In some embodiments, the mol% 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 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 targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 424MF-364968774Attorney Docket No.: 25205-20027.40 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE- PEG2000 or DPG-PEG2000.

[0061] In some embodiments, the targeted LNP, comprises Lipid A3. In some embodiments, the targeted LNP, comprises Lipid A3 and DSPC. In some embodiments, the targeted LNP, comprises Lipid A3 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE-PEG2000 or DPG-PEG2000.

[0062] In some embodiments, the targeted LNP comprises Lipid Al l . In some embodiments, the targeted LNP comprises Lipid Al 1 and DSPC. In some embodiments, the targeted LNP comprises Lipid Al 1 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 4 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the LNP is DPPE- PEG2000 or DPG-PEG2000.

[0063] In some embodiments, the targeted LNP comprises Lipid Al 2. In some embodiments, the targeted LNP, comprises Lipid Al 2 and DSPC. In some embodiments, the targeted LNP, comprises Lipid Al 2 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 425MF-364968774Attorney Docket No.: 25205-20027.40 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE- PEG2000 or DPG-PEG2000.

[0064] In some embodiments, the targeted LNP comprises Lipid Al 3. In some embodiments, the targeted LNP comprises Lipid Al 3 and DSPC. In some embodiments, the targeted LNP comprises Lipid Al 3 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 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the targeted LNP is DPPE-PEG2000 or DPG-PEG2000.

[0065] In some embodiments, the targeted LNP comprises Lipid Al 4. In some embodiments, the targeted LNP comprises Lipid Al 4 and DSPC. In some embodiments, the targeted LNP comprises Lipid Al 4 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one Cl 4 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE-PEG2000 or DPG- PEG2000.

[0066] In some embodiments, the targeted LNP comprises Lipid Al 5. In some embodiments, the targeted LNP comprises Lipid Al 5 and DSPC. In some embodiments, the targeted LNP comprises Lipid Al 5 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 426MF-364968774Attorney Docket No.: 25205-20027.40 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE- PEG2000 or DPG-PEG2000.

[0067] In some embodiments, the targeted LNP comprises Lipid Al 6. In some embodiments, the targeted LNP comprises Lipid Al 6 and DSPC. In some embodiments, the targeted LNP comprises Lipid Al 6 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 4 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE- PEG2000 or DPG-PEG2000.

[0068] In some embodiments, the targeted LNP comprises Lipid Al 7 In some embodiments, the targeted LNP comprises Lipid Al 7 and DSPC. In some embodiments, the targeted LNP comprises Lipid Al 7 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the targeted LNP is DPPE-PEG2000 or DPG-PEG2000.

[0069] In some embodiments, the targeted LNP, comprises Lipid A8. In some embodiments, the targeted LNP comprises Lipid A8 and DSPC. In some embodiments, the targeted LNP, comprises Lipid A8 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g.,27MF-364968774Attorney Docket No.: 25205-20027.40 two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE-PEG2000 or DPG-PEG2000.

[0070] In some embodiments, the targeted LNP, comprises Lipid A9. In some embodiments, the targeted LNP comprises Lipid A9 and DSPC. In some embodiments, the targeted LNP, comprises Lipid A9 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE-PEG2000 or DPG-PEG2000.

[0071] In some embodiments, the targeted LNP comprises Lipid V003. In some embodiments, the targeted LNP comprises Lipid V003 and DSPC. In some embodiments, the targeted LNP comprises Lipid V003 and a sphingomyelin. In some embodiments, the mol% 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%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 6 alkyl chain (e.g., two Cl 6 alkyl chains). PEG2000. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one Cl 4 alkyl chain (e.g., two Cl 4 alkyl chains). In some such embodiments, the targeted LNP is DPPE- PEG2000 or DPG-PEG2000.

[0072] In any of the foregoing embodiments, the targeted LNP 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 ratio28MF-364968774Attorney Docket No.: 25205-20027.40 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.

[0073] In some embodiments, the targeted LNPs are formed via a Click reaction between a first Click handle on the targeting moiety (e.g., antibody, Fab fragment, or scFv) and a second Click handle on the LNP, thereby generating a Click product. The second Click handle can be on one or more of the lipids comprising the LNP. In some embodiments, the second Click handle is covalently bonded to a pegylated lipid comprising the LNP.

[0074] 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. 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.

[0075] 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 to29MF-364968774Attorney Docket No.: 25205-20027.40 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 ID: 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.

[0076] In some embodiments, the targeting moiety that targets CD3 on the surface of the T cell is selected from SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab, visilizumabm, glofitamab, plamatomab, HPN-536, pasotuxizumab, flotetuzumab, cevostamab, elranatamab, ERY974 (Ishiguro et al., Sci. Trani. Med. 9, eaal4291 (2017)), SAR440234, GBR 1302, or an antigen-binding portion thereof. In some embodiments, the targeting moiety that binds to CD3 is an antibody, Fab fragment, ScFv, or VHH domain. In certain embodiments, the targeting moiety is SP34 or an antigen-binding portion thereof (e.g., a CD3 -binding portion thereof). In some embodiments, the targeting moiety is a modified SP34 or an antigen-binding portion thereof, that comprises the six complementarity-determining regions (CDRs) of SP34 (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In other embodiments, the targeting moiety is teclistamab or an antigen-binding portion thereof (e.g., a CD3 -binding portion thereof). In other embodiments, the targeting moiety is mosunetuzumab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is odronextamab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is tebentafusp or an antigenbinding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is muromonab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is visilizumab or an antigenbinding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is tepilizumab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is glofitamab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is plamatomab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is HPN-536 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is pasotuxizumab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is flotetuzumab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is cevostamab30MF-364968774Attorney Docket No.: 25205-20027.40 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is elranatamab or an antigen-binding portion thereof (e.g., a CD3 -binding portion thereof). In other embodiments, the targeting moiety is ERY974 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is SAR440234 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the targeting moiety is GBR 1302 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof).

[0077] In some embodiments, the 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.

[0078] In some embodiments, the targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD5. In some embodiments, the targeted LNP 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 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.

[0079] In further embodiments, the targeted LNP comprises a third targeting moiety. In some such embodiments, the first targeting moiety binds to CD3, the second targeting moiety binds toto CD7 and the third targeting moiety binds to CD28. 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 embodiments, the targeting moiety is a modified SP34 or an antigen-binding portion thereof, that comprises the six complementarity-determining regions (CDRs) of SP34 (i.e. CDR-H1, CDR- H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some of the foregoing embodiments, the targeting moiety that binds to CD3 is teclistamab or an antigen-binding portion.

[0080] In some aspects, the disclosure provides a lipid nanoparticle (LNP) comprising:31MF-364968774Attorney Docket No.: 25205-20027.40(i) a lipid component comprising an ionizable lipid and a helper lipid;(ii) a first targeting moiety and a second target moiety conjugated to the surface of the lipid component of the LNP, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T cells selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8 and CD28, and wherein the second targeting moiety is present in a greater molar amount than the first targeting moiety.(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.

[0081] 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.

[0082] 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. a first targeting moiety and a second target moiety conjugated to the surface of the lipid component of the LNP, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T cells selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8 and CD28, and wherein the second targeting moiety is present in a greater molar amount than the first targeting moiety; 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.32MF-364968774Attorney Docket No.: 25205-20027.40

[0083] 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.

[0084] 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.

[0085] 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 system for 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.33MF-364968774Attorney Docket No.: 25205-20027.40

[0086] 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 of modifying at least 10% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 15% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 20% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 25% of the genomes of the T cells. In some embodiments, the LNPs comprising the targeting moiety are capable of modifying at least 30% of the genomes of the T cells.

[0087] 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 comprising34MF-364968774Attorney Docket No.: 25205-20027.40 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.

[0088] 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 some embodiments, the T cells are not activated prior to mixing with the pharmaceutical composition comprising targeted LNPs.

[0089] 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

[0090] 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.

[0091] In some embodiments, the disclosure provides a lipid nanoparticle (LNP) comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid; (ii) a first targeting moiety and a second target moiety conjugated to the surface of the lipid component of the LNP, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T cells selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8 and CD28, and wherein the second targeting moiety is present in a greater molar amount than the35MF-364968774Attorney Docket No.: 25205-20027.40 first targeting moiety; (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 CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 or Table 22 or Table 24 or Table 34 (or CDR sequences (bolded) selected from Table 20 or Table 22 or Table 24 or Table 34) In some embodiments, the second targeting moiety of the targeted 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 21 (or CDR sequences (bolded) selected from Table 21). In some embodiments, the second targeting moiety of the targeted 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 21 (or CDR sequences (bolded) selected from Table 21). In some embodiments, the second targeting moiety of the targeted 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 21 (or CDR sequences (bolded) selected from Table 21). In some embodiments, the second targeting moiety of the targeted LNP comprises a CD28 targeting moiety. In some embodiments, the second targeting moiety of the targeted LNP comprises a CD4 targeting moiety. In some embodiments, the second targeting moiety of the targeted LNP comprises a CD8 targeting moiety. 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% 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 40 mol% of the lipid component, and the helper lipid is present at about 10 mol% to about 20 mol% of the lipid component. In some embodiments, the template RNA comprises a sequence present in Table 6B or a sequence selected from SEQ ID NOs: 395-404.36MF-364968774Attorney Docket No.: 25205-20027.40

[0092] 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) ) a first targeting moiety and a second target moiety conjugated to the surface of the lipid component of the LNP, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T cells selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8 and CD28, and wherein the second targeting moiety is present in a greater molar amount than the first targeting moiety; 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 CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences selected from Table 20 or Table 22 or Table 24 or Table 34 (or CDR sequences (bolded) selected from Table 20 or Table 22 or Table 24 or Table 34) In some embodiments, the second targeting moiety of the targeted 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 21 (or CDR sequences (bolded) selected from Table 21). In some embodiments, the second targeting moiety of the targeted 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 21 (or CDR sequences (bolded) selected from Table 21). In some embodiments, the second targeting moiety of the targeted 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 21 (or CDR sequences (bolded) selected from Table 21). In some embodiments, the second targeting moiety of the targeted LNP comprises a CD28 targeting moiety. In some embodiments, the LNP comprises a CD4 targeting moiety. In some embodiments, the second targeting moiety of the targeted LNP comprises a CD8 targeting moiety. 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, the37MF-364968774Attorney Docket No.: 25205-20027.40 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 6B or a sequence selected from SEQ ID NOs: 395-404.

[0093] 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 to CD3; (iii) means for binding a cell surface receptor to of CD2, CD4, CD5, CD6, CD7, CD8 or CD28; and (iv) 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.

[0094] 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 6B or a sequence selected from SEQ ID NOs: 395-404. In some embodiments, the gene modifying polypeptide comprises an amino acid sequence of Table 5 or Table 6A 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 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. In some embodiments, the template RNA comprises both of a 5’UTRretro and a 3’ UTRretro. In some embodiments, the 5’UTRretro and the 3’ UTRretro comprise 5’ or 3’ sequences of Table 5 (e.g., SEQ ID NO: 44 and / or SEQ ID NO: 55) or any of Examples 4-11 or 15-17.38MF-364968774Attorney Docket No.: 25205-20027.40BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1 provides a bar graph showing that higher levels of transfection of targetedLNPs in activated T cells are achieved in the absence of serum.

[0096] 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).

[0097] 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 nontargeted base LNPs, both in the presence and absence of serum.

[0098] 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.

[0099] 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.

[0100] FIGs. 6A-6D provides bar graphs showing the percentage of rested T cells that express GFP (FIGs, 6A and 6B) and the GFP expression levels (FIGs. 6C and 6D) when the tested tLNPs are transfected into rested T cells in culture in the presence or FIGs. 6A and 6D) or absence (FIGs. 6B and 6C) of serum (FBS). The targeted LNPs conjugated to anti-CD7, anti- CD5, and anti-CD3 targeting moieties outperformed non-targeted base LNPs, both with and without serum.

[0101] 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 the39MF-364968774Attorney Docket No.: 25205-20027.40 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.

[0102] 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 Lipid A2 or Lipid A3 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 Lipid A3 LNPs resulted in the highest GFP expression levels in the cells, followed by LNPs comprising Lipid A2. 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. The Lipid A2 and Lipid A3 LNPs transfected the largest numbers of cells at the lOOng 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 Lipid A3.

[0103] FIG. 9 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. 9A 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 A2 tLNPs comprising an anti-CD3 targeting moiety and an exemplary gene modifying system. FIG. 9B 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.

[0104] FIGs. 10A -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. 10A, respectively) and the GFP expression levels (MFI) at 4 days or 7 days post-administration in these cells (FIGs. 10B and 10C, respectively). Delivery of the gene modifying system payload to rested T cells using targeted LNPs formulated with Lipid A3 and a higher percentage of helper lipid (22% DSPC) generated40MF-364968774Attorney Docket No.: 25205-20027.40 many more cells that expressed GFP (%GFP+) and at higher levels (MFI) relative to the baseline control tLNP (“Std” in FIG. 10) that was the identical except that it was formulated with 8% DSPC.

[0105] FIG. 11A 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 pay load to rested T cells using targeted LNPs formulated with higher helper lipid (22% DSPC) generated many more CAR-T cells (%CAR+) at all dose levels relative to the baseline control tLNP that was the identical except that it was formulated with 8% DSPC. FIGs. 11B and 11C 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. 11 A. 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.

[0106] FIG. 12 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.

[0107] FIG. 13 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.

[0108] FIG. 14 is a schematic showing an enzymatic approach of site-specifically introducing a Tz ring onto a sugar moiety of an antibody.

[0109] FIG. 15 is a schematic showing an example of a light-induced crosslinking approach of site-specifically introducing a Tz ring onto an antibody.

[0110] FIG. 16 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.

[0111] FIG. 17 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).41MF-364968774Attorney Docket No.: 25205-20027.40

[0112] FIG. 18 is a schematic showing a Fab fragment modified with a free cysteine designed to react with a maleimide group.

[0113] FIG. 19 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.

[0114] FIG. 20 provides a bar graph showing the percentage of human T cells that express a CAR after human PBMCs were either transfected with an anti-CD3 tLNP comprising an exemplary retrotransposon gene modifying system comprising a template RNA encoding a CAR sequence for 4 hours with TransAct (right side) or mock treated as a control (left side).

[0115] FIG. 21 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. 20 compared to the percentage of tumor cells killed by the mock treated T cells.

[0116] FIG. 22 provides a bar graph showing the quantification of the IFN-y cytokine in the 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).

[0117] FIG. 23 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) /

[0118] FIG. 24 provides a bar graph showing the quantification of CAR expression levels (MFI) in the CAR-T cells described in FIG. 23.

[0119] FIG. 25 provides a bar graph showing the quantification of the percentage of BCMA tumor cells killed by the CAR-T cells described in FIG. 23 in a tumor killing assay.

[0120] FIG. 26 is a schematic showing an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).42MF-364968774Attorney Docket No.: 25205-20027.40

[0121] FIG. 27A 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. 27B is a schematic showing the assembly of an LNP encapsulating a therapeutic payload using a postinsertion technique.

[0122] FIG. 28 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 thiolreactive group conjugated to the surface of the LNP.

[0123] FIG. 29 is an exemplary schematic showing conjugate formation. In FIG. 29, 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.

[0124] FIG. 30 is an exemplary schematic showing conjugate formation. In FIG. 30, 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.

[0125] FIG. 31 is an exemplary schematic showing conjugate formation using DBM as a bridging agent.

[0126] FIG. 32 is an exemplary schematic showing conjugate formation. In FIG. 32, a first Fab fragment (Fabl) 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 cysteine43MF-364968774Attorney Docket No.: 25205-20027.40 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.

[0127] FIG. 33 is a schematic showing some examples of a pegylated lipid bonded to a maleimide moiety.

[0128] FIG. 34 is a schematic showing some examples of non-pegylated lipid bonded to a maleimide moiety.

[0129] FIG. 35 is a schematic showing some examples of ionizable lipid bonded to a maleimide moiety.

[0130] FIG. 36 is a schematic showing some examples of sterols bonded to a maleimide moiety.

[0131] FIG. 37 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.

[0132] FIG. 38 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.

[0133] FIG. 39 shows that the tLNPs comprising higher amounts of anti-CD3 targeting moiety (in the absence of an anti-CD7 targeting moiety) on the surface generated more CAR-T cells after administration to resting T cells at the 1 pg dose. However, in the presence of an anti- CD7 targeting moiety, much lower amounts of the anti-CD3 targeting moiety can be used to achieve CAR-T generation.

[0134] FIG. 40 shows that fewer Jurkat NFAT reporter cells expressed GFP, indicating less activation, when the cells were transfected with the dual tLNPs comprising fewer anti-CD3 targeting moieties on the surface. As the density of anti-CD3 targeting moieties was increased from 0.01 :1 to 0.025:1 to 0.05:1 (mg of targeting moiety to mg of total RNA in the LNP) on the dual targeted LNPs, the Jurkat cells progressively became more activated, as shown via more cells expressing GFP as a readout for NFAT transactivation.44MF-364968774Attorney Docket No.: 25205-20027.40

[0135] FIG. 41A shows that almost 70% of the population of T cells comprised the less differentiated naive and TCM subpopulations when the anti-CD3 (lower density) / anti-CD7 dual tLNPs had been administered to the resting T cells. By contrast, the majority of the population of T cells comprised the more differentiated effector T cells subpopulations (TEM and TCEM) when the tLNPs comprising a higher density of anti-CD3 targeting moieties had been administered to the resting T cells. FIG. 41B shows that the population of T cells that had received the anti-CD3 (lower density) / anti-CD7 dual tLNPs were more effective at killing cancer cells in an in vitro tumor cell killing assay (as described above) than the population of T cells that had received the tLNPs comprising the higher density of anti-CD3 targeting moieties (0.4 mg of targeting moiety to mg of total RNA).

[0136] FIG. 42 shows that administration of each of the tLNPs carrying an exemplary all- RNA gene modifying system to the tumor-bearing mice led to the generation of human CD45+ CAR-T cells in peripheral blood in vivo, starting at around Day 6 post-administration and peaking at around 20% of the CD45+ T cells comprising a CAR at around Day 13, as determined by flow cytometry. The CAR-T cells persisted through about Day 27 post-administration.

[0137] FIG. 43 also shows that administration of the dual tLNPs generated human CAR-T cells in the mouse model, with over 60 CD3+ / CD5+ CAR-T cells present per ul of peripheral blood at 13 days after administering the anti-CD3 / anti-CD7 dual tLNPs and over 80 CD3+ / CD5+ CAR-T cells present per ul of peripheral blood at 13 days after administering the anti-CD3 / anti-CD2 dual tLNPs and over 100 cells per ul when treated with the anti-CD3 / anti- CD5 dual tLNPs.

[0138] FIG. 44 shows that tumor growth was substantially reduced in the mice following administration of the dual tLNPs and sustained over the course of 27 days post-administration. By contrast, the tumor cells continued to expand in mice that were administered the vehicle control only resulting in termination at day 15.

[0139] FIG. 45 shows that the mice that were treated with the dual tLNPs had a greater number of total T cells over the course of the first 18 days of the study than the mice that were treated with the vehicle control.

[0140] FIG. 46 shows that approximately 20% and 29% of the T cells comprised a CAR following administration of the tLNPs comprising 0.025:1 and 0.075: 1 of anti-CD3 targeting moiety (mg of total RNA) (“tLNPl” and “tLNP2” in the figure), respectively.

[0141] FIG. 47 also shows that administration of the dual targeted LNPs generated human CAR-T cells in the mouse model, as about 4 CD3+ / CD5+ CAR-T cells were present per ul of 45MF-364968774Attorney Docket No.: 25205-20027.40 peripheral blood at 11 days after administering tLNPl and over 10 CD3+ / CD5+ CAR-T cells were present per ul of peripheral blood on the same day after administering tLNP2. Administration of the vehicle control did not generate any CAR-T cells in these mice.

[0142] FIG. 48 shows that administration of either of the dual targeted LNPs to the naive humanized mice resulted in depletion of human B cells in the mice over the course of 40 days. Starting at around Day 4 post-administration, the number of B cells dropped substantially (over 50%) and were largely undetectable by Day 11 post-administration. By contrast, administration of the vehicle control did not lead to a reduction in the number of human B cells.

[0143] FIG. 49 shows that administration of dual tLNPs resulted in the generation of CAR-T cells at 7 days following LNP transfection of the rested T cells isolated from two donors.

[0144] FIG. 50 shows that administration of the dual tLNPs carrying the exemplary all-RNA gene modifying system resulted in the generation of human CAR-T cells in the peripheral blood of the mice engrafted with human CD34+ HSCs at all doses tested.

[0145] FIG. 51 shows that administration of CD3 / CD7 dual tLNPs formulated with the exemplary gene modifying system to the naive humanized mice resulted in depletion of human B cells over the course of over 30 days for all doses tested.

[0146] FIG. 52 shows that 11 days after administration of the dual tLNPs carrying the exemplary all-RNA gene modifying system, greater proportion of the T cells population was comprised of the less differentiated naive and TCM subpopulations relative to higher doses at the same time point.

[0147] FIG. 53 shows that 32 days after administration of the dual tLNPs carrying the exemplary all-RNA gene modifying system, the subtypes of T cells are similar to non-treated animals.

[0148] FIG. 54 shows that 32 days after administration of the dual tLNPs carrying the exemplary all-RNA gene modifying system, there was an increase in the number of CD3+ T cells present in the spleen at all tLNPs doses tested as well as a near complete clearance of B cells as determined by CD20 staining.

[0149] FIG. 55 shows that administration of the anti-CD3 / antiCD7 dual tLNPs formulated with the exemplary gene modifying system comprising two template RNAs encoding the CD 19 and CD20 CARs to resting T cells resulted in the production of dual-specific CAR-T cells expressing both CARs (CD19+ / CD20+ cells).46MF-364968774Attorney Docket No.: 25205-20027.40

[0150] FIG. 56 shows that the dual-specific CAR-T cells that expressed both the CD19 and the CD20 CARs killed both the CD20- and CD 19- Raji cells in the mixed cancer cell population to near complete clearance.

[0151] FIG. 57 shows that targeted anti-CD3 / antiCD7 dual tLNPs formulated with various ionizable lipids and an exemplary all-RNA gene modifying system administered to resting T effectively delivered the gene modifying system into the cells to enable TPRT-mediated insertion of a CAR sequence into the genomic DNADETAILED DESCRIPTIONI. DEFINITIONS

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.47MF-364968774Attorney Docket No.: 25205-20027.40

[0156] 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, 3 A, 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.

[0157] 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.48MF-364968774Attorney Docket No.: 25205-20027.40

[0158] 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.

[0159] 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.

[0160] 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 but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector). In some49MF-364968774Attorney Docket No.: 25205-20027.40 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).

[0161] 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 PCT7US2021 / 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 a sequence 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.50MF-364968774Attorney Docket No.: 25205-20027.40

[0162] 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.

[0163] 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, Table 16), 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., peptide51MF-364968774Attorney Docket No.: 25205-20027.40 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).

[0164] 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.

[0165] It is understood that aspects and embodiments described herein as “comprising” include “consisting of’ and “consisting essentially of’ embodiments.II. TARGETED LIPID NANOPARTICLES

[0166] In one aspect, the disclosure provides a targeted LNP comprising an ionizable lipid), 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). The targeted LNP comprises two targeting moieties (a first targeting moiety and a second targeting moiety) that each independently binds to a protein (e.g., a protein receptor) on an immune cell (e.g., a T cell). The52MF-364968774Attorney Docket No.: 25205-20027.40 first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of immune cells (e.g., T cells) selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8 and CD28. The second targeting moiety is present in a greater molar amount than the first targeting moiety.

[0167] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.2:2. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.4: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.6: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 1.8: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 2:1.. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 3:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 4: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 5: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 6:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 7: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 8: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 10:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 15: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6,53MF-364968774Attorney Docket No.: 25205-20027.40CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 20: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 50: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of at least 100: 1.

[0168] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a molar ratio of from about 1.2:1 to about 100:1, from about 1.2:1 to 20:1, from about 1.2: 1 to about 10: 1. from about 1.5:1 to about 50:1, from about 1.5:1 to about 20:1, from about 1.5: 1 to about 10: 1, from about 1.5:1 to about 5: 1, from about 2:1 to about 100: 1, from about 2: 1 to about 50: 1, from about 2: 1 to about 20:1, from about 2:1 to about 10: 1, from about 2:l to about 5: 1, from about 2: 1 to about 4:1, from about 5:1 to about 40:1, from about 5: 1 to about 20:1, from about 5:1 to about 10:1, or from about 10: 1 to about 20:1

[0169] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.2:2. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.4:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.6: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 1.8:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 2:1.. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 3:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 4: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 5:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 6:1. In some embodiments, the second targeting moiety54MF-364968774Attorney Docket No.: 25205-20027.40(CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 7: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 8: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 10: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 15:1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 20: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 50: 1. In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of at least 100: 1.

[0170] In some embodiments, the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8 or CD28 binder) and the first targeting moiety (CD3 binder) are present in the LNP at a mass ratio of from about 1.2: 1 to about 100:1, from about 1.2: 1 to 20:1, from about 1.2:1 to about 10: 1. from about 1.5: 1 to about 50:1, from about 1.5:1 to about 20:1, from about 1.5:1 to about 10: 1, from about 1.5: 1 to about 5: 1, from about 2:1 to about 100: 1, from about 2: 1 to about 50:1, from about 2: 1 to about 20:1, from about 2:l to about 10:1, from about 2: l to about 5:1, from about 2: 1 to about 4: 1, from about 5:1 to about 40:1, from about 5: 1 to about 20:1, from about 5:1 to about 10:1, or from about 10: 1 to about 20:1

[0171] In some embodiments, the mass ratio of the first targeting moiety (CD3 binder) to the therapeutic agent is from about 0.001 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.5: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.25:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.1 : 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.025:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.001 : 1.0 to about 0.01 : 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 :1.0 to about 0.5:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from 55MF-364968774Attorney Docket No.: 25205-20027.40 about 0.01 : 1.0 to about 0.25:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.05: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.025:1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.005: 1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.01 : 1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.025: 1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.05: 1. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is about 0.1 :1.

[0172] In some embodiments, the mass ratio of the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8, or CD28 binder) to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 0.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 0.4: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4:1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 1.0: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is from about 1.0: 1.0 to about 1.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.3 : 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.4:1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.5 : 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 0.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 1.0: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 1.5: 1.0. In some embodiments, the mass ratio of the second targeting moiety to the therapeutic agent is about 2.0: 1.0.56MF-364968774Attorney Docket No.: 25205-20027.40

[0173] In some embodiments, the mass ratio of the first targeting moiety (CD3 binder) to the therapeutic agent is from about 0.001 : 1.0 to about 1.0: 1.0 and the mass ratio of the second targeting moiety (CD2, CD4, CD5, CD6, CD7, CD8, or CD28 binder) to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.5: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.5: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.5: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 :1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.1 : 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.1 : 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4:1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.05:1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.05:1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 1.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.025: 1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 2.0: 1.0. In some embodiments, the mass ratio of the first targeting moiety to the therapeutic agent is from about 0.01 : 1.0 to about 0.025:1.0 and the mass ratio of the second targeting moiety to the therapeutic agent is from about 0.4: 1.0 to about 1.0: 1.0.

[0174] It will be understood that in the foregoing embodiments, the mass of the therapeutic agent refers to the total mass of the payload (e.g., nucleic acids) encapsulated in the LNP. In57MF-364968774Attorney Docket No.: 25205-20027.40 embodiments where the therapeutic agent comprises more than one nucleic acid, the mass ratio of the targeting moiety to the therapeutic agent refers to the ratio between the mass of the targeting moiety and the total mass of the nucleic acid molecules. For instance, in embodiments where the LNP encapsulates two RNA molecules (e.g., an RNA encoding a gene modifying polypeptide and a guide or template RNA), a 2.0:1.0 mass ratio of targeting moiety to therapeutic agent refers to 2 parts targeting moiety by weight to 1 part total RNA (RNA encoding gene modifying peptide and guide or template RNA) by weight.

[0175] In some embodiments, each of the targeting moieties is an antibody or antigen binding fragment thereof. In some instances, each of the targeting moieties 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, each of the targeting moieties 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).

[0176] The targeted LNPs described herein may be used to target and modify immune cells. In some embodiments, the targeted LNPs 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, naive T cells, stem cell memory T cells, central memory T cells, effector T cells, cytotoxic T cells, helper T cells (e.g., Thl cells, Th2 cells, Thl7 cells, etc.), regulatory T cells, tumor-infiltrating T cells, tissue-resident T cells, or a mixture of subpopulations. In some embodiments, the targeted LNPs may be used to deliver or modify a sequence encoding a T-cell receptor (TCR) in a T cell. In some embodiments, the targeted LNPs may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T cells. For instance, in specific embodiments, the targeted LNPs can be used to deliver an RNA encoding a CAR to T cells. In some embodiments, the targeted LNPs may be used to deliver at least one sequence encoding a CAR to natural killer (NK) cells. In some embodiments, the targeted LNPs be used to deliver at least one sequence encoding a CAR to natural killer T (NKT) cells. In some embodiments, the targeted LNPs 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 a combination 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 targetable58MF-364968774Attorney Docket No.: 25205-20027.40 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), LI CAM (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.

[0177] 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.

[0178] In some embodiments, the targeted LNP comprises at least two targeting moieties that each independently targets a receptor on the surface of the T cell, wherein the first targeting moiety binds CD3 and the second targeting moiety binds a receptor selected from CD2, CD4, CD5, CD6, CD7, CD8, and CD28. In some embodiments, the second targeting moiety targets a CD7 molecule on the surface of the T cell. In some embodiments, the second targeting moiety targets a CD5 molecule on the surface of the T cell. In some embodiments, second targeting moiety targets a CD2 molecule on the surface of the T cell. In some embodiments, the second targeting moiety targets a CD8 molecule on the surface of the T cell. In some embodiments, the 59MF-364968774Attorney Docket No.: 25205-20027.40 second targeting moiety targets a CD28 molecule on the surface of the T cell. In some embodiments, the targeted LNP comprises about 2 to about 10 targeting moieties, such as about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting moieties, wherein one of the targeting moieties targets a CD3 receptor on the surface of the T cell.

[0179] In some embodiments, the targeted LNP comprises at least two targeting moieties, wherein one targeting moiety targets a CD3 molecule on the surface of the T cell, and wherein the CD3 -targeting moiety is selected from SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab, visilizumabm, glofitamab, plamatomab, HPN-536, pasotuxizumab, flotetuzumab, cevostamab, elranatamab, ERY974 (Ishiguro et al., Sci. Trani. Med. 9, eaal4291 (2017)), SAR440234, GBR 1302, or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In some embodiments, the targeting moiety that binds to CD3 is an antibody, Fab fragment,, ScFv, or VHH domain. In certain embodiments, the CD3 -targeting moiety is SP34 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In some embodiments, the targeting moiety is a modified SP34 or an antigen-binding portion thereof, that comprises the six complementarity-determining regions (CDRs) of SP34 (i.e. CDR- Hl, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In other embodiments, the CD3- targeting moiety is teclistamab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3 -targeting moiety is mosunetuzumab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3-targeting moiety is odronextamab or an antigen-binding portion thereof (e.g., a CD3- binding portion thereof). In other embodiments, the CD3 -targeting moiety is tebentafusp or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3-targeting moiety is muromonab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3 -targeting moiety is visilizumab or an antigenbinding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3- targeting moiety is tepilizumab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3 -targeting moiety is glofitamab or an antigenbinding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3- targeting moiety is plamotamab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3-targeting moiety is HPN-536 or an antigenbinding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3- targeting moiety is pasotuxizumab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3 -targeting moiety is flotetuzumab or an antigenbinding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3-60MF-364968774Attorney Docket No.: 25205-20027.40 targeting moiety is cevostamab or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3 -targeting moiety is elranatamab or an antigenbinding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3- targeting moiety is ERY974 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3-targeting moiety is SAR440234 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof). In other embodiments, the CD3-targeting moiety is GBR 1302 or an antigen-binding portion thereof (e.g., a CD3-binding portion thereof).

[0180] In some embodiments, a targeted LNP for delivery of a therapeutic agent to immune cells (e.g., T cells) as described herein, comprises: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any or the lipids in Table 1, Table 2 and Table 3; a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties bind to at least two targeting moieties on a T cell, wherein at least one targeting moiety targets the CD3 molecule on the surface of a T cell; and a therapeutic agent encapsulated within the LNP.

[0181] In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD2. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD4. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD5. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD6. 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 CD8. 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.

[0182] In some embodiments, the targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD5. In some embodiments, the targeted LNP 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 comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other 61MF-364968774Attorney Docket No.: 25205-20027.40 targeting moiety binds to CD28. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD3, a second targeting moiety binds to CD5, and a third targeting moiety binds to CD28. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD3, a second targeting moiety binds to CD7, and a third targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD).

[0183] 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

[0184] 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.

[0185] In some embodiments, the CD2 targeting moiety includes an antibody or antigenbinding 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-62MF-364968774Attorney Docket No.: 25205-20027.40 specific antibody or fragment thereof. In some embodiments, the CD2 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0186] Exemplary anti-CD2 binders, antibodies, or antigen-binding fragments thereof include Siphzumab (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- Ec, 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 antigenbinding 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; W02024079046; W02024079046; etc., each hereby incorporated by reference in its entirety.

[0187] 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 21, with complementary determining regions (CDRs) marked in bold.

[0188] 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-63MF-364968774Attorney Docket No.: 25205-20027.40H3 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.

[0189] 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 ammo 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 least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:277 or 278, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:279.

[0190] 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 64MF-364968774Attorney Docket No.: 25205-20027.4094%, 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.

[0191] 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-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: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:290.65MF-364968774Attorney Docket No.: 25205-20027.40CD 3 Targeting Moieties

[0192] 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 (s), CD3-gamma (y), CD3-delta (8), and CD3-zeta (Q) to form a CD3y£-CD38s-CD3^ 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.

[0193] In some embodiments, the CD3 targeting moiety includes an antibody or antigenbinding 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 multispecific antibody or fragment thereof. In some embodiments, the CD3 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0194] Exemplary anti-CD3 targeting moieties can include an 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. etal., 3. 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 etal., J. Immunol. 137: 1097-1100: 1986; US 8,846,042; US 11,013,800; and US 10,870,701), Cns-7 monoclonal antibody (Reinherz, E. L. etal. (eds.), Leukocyte typing II, Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti etal. (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 etal. (2003) J. Clin. Invest. 11 :409), visilizumab (Carpenter et al. (2002) Blood 99:2712), glofitamab, mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, muromonab, plamotamab, HPN-536, pasotuxizumab, flotetuzumab, cevostamab, elranatamab, ERY974 (Ishiguro et al., (2017) Sci. Trani. Med. 9, eaal4291), SAR440234, GBR 1302, 145-2C11 monoclonal antibody (Hirsch et a / . (1988) J. Immunol. 140: 3766), and anti-CD3 -binding fragments thereof. Further CD 3 binding molecules66MF-364968774Attorney Docket No.: 25205-20027.40 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 W02004 / 106380; W02010 / 037838; W02008 / 119567; W02007 / 042261; W02010 / 0150918; the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the exemplary anti- CD3 targeting moiety is a \ monocolonal antibody or CD-3 -binding fragment thereof. In some embodiments, the exemplary anti-CD3 targeting moiety is a CD3 -binding portion of a bispecific or multi-specific antibody or fragment thereof. In some embodiments, the exemplary anti-CD3 targeting moiety is a CD3 -binding portion of an antibody drug composition (ADC).

[0195] In some embodiments, the targeting moiety is a modified SP34 or an antigen-binding portion thereof, that comprises the six complementarity-determining regions (CDRs) of SP34 (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3).

[0196] 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 CD367MF-364968774Attorney Docket No.: 25205-20027.40 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:238 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:239. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:248 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:249. 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 20, 22, 24, and 34, with complementary determining regions (CDRs) marked in bold.

[0197] 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 ammo 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.

[0198] 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%) sequence68MF-364968774Attorney Docket No.: 25205-20027.40 identity to the amino acid sequence of SEQ ID NO: 152, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 153. 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.

[0199] 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 ammo 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 or69MF-364968774Attorney Docket No.: 25205-20027.40161, 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.

[0200] 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 ammo 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.

[0201] 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), a70MF-364968774Attorney Docket No.: 25205-20027.40CDR-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 antigenbinding fragment thereof.

[0202] 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 ammo acid sequence YINPRSGYTHYNQKLKD (SEQ ID NO: 199), a CDR- H3 comprising an amino acid sequence SAYYDYDGFAY (SEQ ID N0:200), 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.

[0203] 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%) sequence71MF-364968774Attorney Docket No.: 25205-20027.40 identity to the amino acid sequence of SEQ ID NO:207, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:208. 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 ammo 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.

[0204] 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 ammo acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR-H3 comprising an amino acid sequence HGNFGNSYI SYWAY (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 acid72MF-364968774Attorney Docket No.: 25205-20027.40 sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:217. In some embodiments, the CD3 targeting moiety is SP34 or an antigen-binding fragment thereof.

[0205] 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 ammo acid sequence RIRSKYNNYATYYAD S VKG (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.

[0206] 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 ammo acid sequence RIRSKYNNYATYYAD QVKD (SEQ ID NO:241), a73MF-364968774Attorney Docket No.: 25205-20027.40CDR-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 HPN-536 or an antigen-binding fragment thereof.

[0207] 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 ammo acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR-H3 comprising an amino acid sequence HGNFGNSYI SYWAY (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 antigenbinding fragment thereof.74MF-364968774Attorney Docket No.: 25205-20027.40

[0208] 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 ammo acid sequence RIRSKYNNYATYYAD S VKD (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

[0209] 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, gpl 50) 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.75MF-364968774Attorney Docket No.: 25205-20027.40

[0210] In some embodiments, the CD5 targeting moiety includes an antibody or antigenbinding 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 multispecific antibody or fragment thereof. In some embodiments, the CD5 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0211] 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.

[0212] 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 21, with complementary determining regions (CDRs) marked in bold.

[0213] 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 ammo 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 CD576MF-364968774Attorney Docket No.: 25205-20027.40 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:354 or 355, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:356.CD7 Targeting Moieties

[0214] 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.

[0215] In some embodiments, the CD7 targeting moiety includes an antibody or antigenbinding 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 multispecific antibody or fragment thereof. In some embodiments, the CD7 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0216] 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; W02003051926 WO2023185256; Wang et al. (2024) Biomolecules 14(1): 106; etc., each hereby incorporated by reference in its entirety.

[0217] 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 region77MF-364968774Attorney Docket No.: 25205-20027.40 comprising the amino acid sequence of SEQ ID NO:314. In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:324 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:325. In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:335 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:336. 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 21, with complementary determining regions (CDRs) marked in bold.

[0218] 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 ammo acid sequence WINTYTGEPTYADDFKG (SEQ ID NO:316), a CDR- H3 comprising an ammo 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.

[0219] 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%) sequence78MF-364968774Attorney Docket No.: 25205-20027.40 identity to the amino acid sequence of SEQ ID NO: 324, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:325. In some embodiments, the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NY AMS (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.

[0220] 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., at79MF-364968774Attorney Docket No.: 25205-20027.40 least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:334.

[0221] 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 RY AMS (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

[0222] 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.

[0223] In some embodiments, the CD28 targeting moiety includes an antibody or antigenbinding 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,80MF-364968774Attorney Docket No.: 25205-20027.40DARPIN, 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.

[0224] 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; W02002051871 ; Tan et al. (2002) J Immunol 169:1119-1125; Elsyed et al. (2023) Mabs 15(l):2220839; etc., each hereby incorporated by reference in its entirety.

[0225] 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

[0226] In some embodiments, the targeted LNP comprises two or more targeting moieties, wherein at least a first targeting moiety targets CD3, and wherein at least a second targeting moiety targets a receptor on the surface of the T cell is selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8, 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-81MF-364968774Attorney Docket No.: 25205-20027.40H3 of any one of SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab, visilizumabm, glofitamab, plamatomab, HPN-536, pasotuxizumab, flotetuzumab, cevostamab, elranatamab, ERY974, SAR440234, and GBR 1302, and a VL comprising a CDR- Ll, a CDR-L2, and a CDR-L3 of any one SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab, visilizumabm, glofitamab, plamatomab, HPN-536, pasotuxizumab, flotetuzumab, cevostamab, elranatamab, ERY974, SAR440234, and GBR 1302. In some embodiments, the first targeting moiety comprises a CD3-binding domain comprising the VH and the VL of any one of SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab, visilizumabm, glofitamab, plamatomab, HPN-536, pasotuxizumab, flotetuzumab, cevostamab, elranatamab, ERY974, SAR440234, and GBR 1302. Exemplary sequences for the first targeting moiety and / or the second targeting moiety can be found, e.g., in Tables 20, 22, 24, and 34.

[0227] In some embodiments, the 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 targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD4. In some embodiments, the targeted LNP 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 comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD6. In some embodiments, the targeted LNP 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 comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD8. In some embodiments, the 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).

[0228] In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD2, a second targeting moiety binds to CD28, and a third targeting moiety binds to CD3. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD3, a second targeting moiety binds to CD28, and a third targeting moiety binds to CD3. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD4, a second targeting moiety binds to CD28, and a third targeting moiety binds to CD3. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting82MF-364968774Attorney Docket No.: 25205-20027.40 moiety binds to CD5, a second targeting moiety binds to CD28, and a third targeting moiety binds to CD3. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD6, a second targeting moiety binds to CD28, and a third targeting moiety binds to CD3. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD7, a second targeting moiety binds to CD28, and a third targeting moiety binds to CD3. In some embodiments, the targeted LNP comprises three targeting moieties, wherein a first targeting moiety binds to CD8, a second targeting moiety binds to CD28, and a third targeting moiety binds to CD3. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD).

[0229] 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 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 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.

[0230] 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 the83MF-364968774Attorney Docket No.: 25205-20027.40 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.

[0231] 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 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 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.

[0232] 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 SEQ84MF-364968774Attorney Docket No.: 25205-20027.40ID NO:219, and the second targeting moiety that binds to CD28 comprises the amino acid sequence of SEQ ID NO:365.Methods of Making Targeted LNPs

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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,85MF-364968774Attorney Docket No.: 25205-20027.40 the tetrazine rung is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl substituted tetrazine.

[0238] 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.

[0239] 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 some embodiments 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 86MF-364968774Attorney Docket No.: 25205-20027.40 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%.

[0240] 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.

[0241] In some embodiments, the first or second Click handle is a tetrazine derivative having one of the following structures:87MF-364968774Attorney Docket No.: 25205-20027.40wherein <4WV represents the point of attachment to the linker of the conjugate or to the LNP.

[0242] In some embodiments, the first or second Click handle is a TCO derivative having one of the following structures:88MF-364968774Attorney Docket No.: 25205-20027.40

[0243] 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.

[0244] 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 some89MF-364968774Attorney Docket No.: 25205-20027.40 embodiments 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%.

[0245] 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.

[0246] 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.

[0247] 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. 12). The glycine and the histidine residues are removed during the transpeptidation reaction.90MF-364968774Attorney Docket No.: 25205-20027.40

[0248] 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.

[0249] 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.

[0250] FIG. 12 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.

[0251] In one embodiment, the targeted LNP 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 the91MF-364968774Attorney Docket No.: 25205-20027.40LNP. 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.

[0252] In some embodiments, the targeted LNPs 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).

[0253] 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 targeted LNPs 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).

[0254] In some embodiments, the targeted LNPs 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 -92MF-364968774Attorney Docket No.: 25205-20027.40LNP, wherein variables n and Z are defined as above. In particular embodiments, the dihydropyridazine moiety is a 1 ,4- dihydropyridazine.

[0255] 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.

[0256] In some embodiments of step (i) the LplA acceptor peptide is covalently bonded to one or more C-terminus of the antibody or Fab fragment. In other embodiments of step i) the LplA acceptor peptide is covalently bonded to the C-terminus of the scFv.

[0257] In some embodiments, step (i) involves covalently bonding the peptide with the LplA 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.

[0258] 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.

[0259] 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 in93MF-364968774Attorney Docket No.: 25205-20027.40 step (ii) is a fatty acid, for instance a C7-C19 fatty acid. In some such embodiments, the fatty acid is selected from decanoic acid, palmitic acid, lauric acid or octanoic acid.

[0260] 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 — 120.94MF-364968774Attorney Docket No.: 25205-20027.4095MF-364968774Attorney Docket No.: 25205-20027.40

[0261] FIG. 13 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.96MF-364968774Attorney Docket No.: 25205-20027.40

[0262] 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 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 targeted LNP 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 targeted LNP comprises a scFv, wherein the C-terminus of the scFv is bonded to the linker.

[0263] 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 targeted LNPs 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).

[0264] 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 targeted LNPs, 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 surface modified with a Fab fragment is depicted below, where R is a lipid group (e.g., C2-C3097MF-364968774Attorney Docket No.: 25205-20027.40 alkyl group) and the LplA accepter peptide comprises the sequence of GFEIDKVWYDLDA (SEQ ID NO: 24).Fab-Z-GFEIDKVWYDLDA

[0265] 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.

[0266] 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.

[0267] 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. 14 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.98MF-364968774Attorney Docket No.: 25205-20027.40

[0268] 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. 15 shows one particular embodiment using oYo-Link Tz. In FIG. 15, 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.

[0269] 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. 16 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.

[0270] 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. 17 shows one particular embodiment of introducing a first Click handle (e.g., Tz) onto a Fab fragment. In FIG. 17, 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. 17). 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,

[0271] In some embodiments, the Click product can be formed using any suitable photoinduced 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).

[0272] In some embodiments, the Click product can be formed using oxime and hydrazone ligations. In some embodiments, a ketone or aldehyde can react with a effect amine, such as99MF-364968774Attorney Docket No.: 25205-20027.40 hydroxylamine, hydrazine and hydrazide (see, e.g., Agten et al., ChemBioChem 2013, 14 (18), 2431-2434 and Dirksen et al., J. Am. Chem. Soc. 2006, 128 (49), 15602-15603).

[0273] Exemplary lipids and cholesterol molecules bonded to Click handles that can be used to make targeted LNPs are depicted below.100MF-364968774Attorney Docket No.: 25205-20027.40101MF-364968774Attorney Docket No.: 25205-20027.40102MF-364968774Attorney Docket No.: 25205-20027.40Interchain CL-CH1 disulfide reduction followed by conjugation

[0274] 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 (CHI , CH2, and CH3). The Fab region of the antibody includes the VL, CL, VH, and CHI domains. The Fc region includes the constant heavy chain domains CH2, and CH3. A hinge region of the IgG antibody covalently links the CHI 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 IgGl 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 Cysl27 (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 Cysl35 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.103MF-364968774Attorney Docket No.: 25205-20027.40

[0275] Proteolytic cleavage of an IgG antibody results in the formation of a Fab fragment known as a F(ab’)2 fragment. The F(ab’)2 fragment does not include the CH2 domain or the CH3 domain. However, the hinge region of the antibody is retained in a F(ab’)2 fragment. The F(ab’)2 fragment includes disulfide bonds that covalently link two Fab fragments. Reduction of the disulfide bond in the F(ab’)2 generates two F(ab’) fragments. The sulfhydryl (thiol) groups of the F(ab’) could potentially react with a thiol-reactive group on the surface of an LNP, hence generating a conjugate. 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’)2 to theF(ab’) fragments could also disrupt the natural interchain disulfide bonds between the CL and CHI regions of the Fab fragments, hence further compromising sitespecific conjugation.

[0276] 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.

[0277] 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 CL and CHI 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 targeted LNPs 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. 26.

[0278] 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 forth104MF-364968774Attorney Docket No.: 25205-20027.40 herein, 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.

[0279] 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.

[0280] 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 (CHI ), 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.

[0281] 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 (CHI ), 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.

[0282] 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 LNP105MF-364968774Attorney Docket No.: 25205-20027.40 with the modified lipid. For instance, as shown in FIG. 27A, 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. 27A). 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. 27B, 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.

[0283] 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.

[0284] 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.

[0285] 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.106MF-364968774Attorney Docket No.: 25205-20027.40

[0286] 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.

[0287] 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.

[0288] A schematic showing the reduction of an interchain disulfide bond in a Fab fragment is depicted in FIG. 28. In some embodiments, the Fab fragment depicted in FIG. 28 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. 28, 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.

[0289] FIG. 29 shows an exemplary schematic of conjugate formation, as described herein. In FIG. 29, 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 conjugated107MF-364968774Attorney Docket No.: 25205-20027.40 to the surface of the LNP via a linkage through at least one of the free cysteine residues of the Fab fragment.

[0290] 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. 30. In some embodiments, the targeted LNPs formed by methods disclosed herein include at least one, at least two or all three orientations shown in FIG. 30. 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.

[0291] In some embodiments, the thiol-reactive group is maleimide, as shown in FIG. 30. 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.

[0292] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM) as shown in FIG. 31. 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. 31). 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.108MF-364968774Attorney Docket No.: 25205-20027.40

[0293] 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 thiolreactive 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. 32 shows some examples of a pegylated lipid bonded to a thiol-reactive group, in this case a maleimide moiety.

[0294] In some embodiments, the thiol-reactive group is bonded to at least one of the non- pegylated phospholipids (helper lipids) comprising the LNP. FIG. 33 shows some examples of a non-pegylated lipid bonded to a maleimide moiety.

[0295] In some embodiments, the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP. FIG. 34 shows some examples of an ionizable lipid bonded to a maleimide moiety.

[0296] In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. FIG. 35 shows some examples of sterols bonded to a maleimide moiety.

[0297] The disclosed methods provide stable conjugates that display excellent ability to transduce specific targeted cells. In some aspects, the disclosure provides a conjugate109MF-364968774Attorney Docket No.: 25205-20027.40 comprising an LNP and a Fab fragment, wherein the LNP is covalently bonded to either or both a first cysteine residue in the constant region of the heavy chain of the Fab fragment and a second cysteine residue in the constant region of light chain of the Fab fragment. 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.

[0298] 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.

[0299] 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.110MF-364968774Attorney Docket No.: 25205-20027.40

[0300] 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.

[0301] 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. 32. In FIG. 32, two Fab fragments (Fabl 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 Fabl and Fab2 are conjugated to the surface of the LNP. Despite reduction of the disulfide bonds in Fabl and Fab2, the heavy chain and light chain in Fabl 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 Fabl associate with the heavy or light chain of Fab2 on the surface of the LNP.

[0302] 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 some111MF-364968774Attorney Docket No.: 25205-20027.40 embodiments, a total of four different Fab fragments can be conjugated to the surface of the LNP.

[0303] 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.

[0304] In some embodiments, targeted LNPs 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 targeted LNP can comprise a plurality of Fab fragments conjugated to the LNP surface. In some embodiments, the targeted LNP can comprise more than 10 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise more than 20 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise more than 30 Fab fragments. In some embodiments, the targeted LNP can comprise more than 50 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise more than 75 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise more than 100 Fab fragments. In some embodiments, the targeted LNP can comprise from about 50 to about 200 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise from about 100 to about 200 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise from about 100 to about 230 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise from about 10 to about 150 Fab fragments per LNP. In some embodiments, the targeted LNP can comprise from about 10 to about 30 Fab fragments per LNP.112MF-364968774Attorney Docket No.: 25205-20027.40

[0305] Targeted LNPs prepared by site-specific methods (e.g., FIGs. 12-18 and 26-32) have a high density of the targeting moiety on the surface of the LNP. For instance, the targeted LNP can comprise more than one targeting moiety (e.g., antibody, Fab fragment or scFv) per LNP. In some embodiments, the targeted LNP can comprise more than 10 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise more than 20 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise more than 30 targeting moieties (e.g., antibodies, Fab fragments or scFvs). In some embodiments, the targeted LNP can comprise more than 50 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise more than 75 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise more than 100 targeting moieties (e.g., antibodies, Fab fragments or scFvs). In some embodiments, the targeted LNP can comprise from about 50 to about 200 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise from about 100 to about 200 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise from about 100 to about 230 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise from about 10 to about 150 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the targeted LNP can comprise from about 10 to about 30 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP.

[0306] 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.

[0307] In some embodiments, the targeted LNPs prepared by site-specific methods (e.g., FIGs. 12-18 and 26-32) can have two or more different targeting moieties on the surface of the LNP. For example, the targeted LNP can have two different targeting moieties. In some embodiments, the targeted LNP can have three different targeting moieties. In some embodiments, the targeted LNP 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, 20:1.113MF-364968774Attorney Docket No.: 25205-20027.40III. LIPID NANOPARTICLES

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] 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 about114MF-364968774Attorney Docket No.: 25205-20027.4035 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.

[0313] 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 1 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.

[0314] 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 poly dispersity index of a LNP may be from about 0.10 to about 0.20.115MF-364968774Attorney Docket No.: 25205-20027.40

[0315] 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...

Claims

Attorney Docket No.: 25205-20027.40CLAIMS1. A lipid nanoparticle (LNP) comprising:(i) a lipid component comprising an ionizable lipid and a helper lipid;(ii) a first targeting moiety and a second target moiety conjugated to the surface of the lipid component of the LNP, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T cells selected from the group consisting of CD2, CD4, CD5, CD6, CD7, CD8 and CD28, and wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 1.5: 1; and(iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises one or more nucleic acid molecules.

2. The LNP of claim 1, wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 2: 1.

3. The LNP of claim 1, wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 3: 1.

4. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 4: 1.

5. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 8: 1.

6. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 10:1.

7. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 15:1.

8. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 20: 1.

9. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 50: 1.430MF-364968774Attorney Docket No.: 25205-20027.4010. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of at least 100: 1.

11. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of from about 2: 1 to about 100: 1.

12. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of from about 2: 1 to about 50:1.

13. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of from about 5 : 1 to about 40: 1.

14. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of from about 5 : 1 to about 20: 1.

15. The LNP of claim 1 , wherein the second targeting moiety and first target moiety are present at a molar ratio of from about 5 : 1 to about 10:1.

16. The LNP of any one of claims 1-14, wherein the second targeting moiety binds to CD717. The LNP of any one of claims 1-14, wherein the second targeting moiety binds to CD 5 or CD2.

18. The LNP of any one of claims 1-17, wherein the helper lipid is present at about 22 mol% to about 36 mol% of the lipid component.

19. The LNP of any one of claims 1-17, wherein the helper lipid is present at about 20 mol% to about 30 mol% of the lipid component.

20. The LNP of any one of claims 1-19, wherein the helper lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidy Icholine (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),431MF-364968774Attorney Docket No.: 25205-20027.40 dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1- 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.

21. The LNP of claim 20, wherein the helper lipid is DSPC.

22. The LNP of claim 20, wherein the helper lipid is sphingomeylin.

23. The LNP of any one of claims 1-22, further comprising a cholesterol molecule.

24. The LNP of claim 23, wherein the cholesterol molecule is selected from the group consisting of beta-sitoesterol, hydroxy cholesterol, stigmastanol, and any combination thereof.

25. The LNP of any one of claims 1-24, wherein the CD3 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 20 or Table 22 or Table 24 or Table 34, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

26. The LNP of any one of claims 1-25, wherein the second targeting moiety is a CD7 targeting moiety, and wherein the CD7 targeting moiety is an antibody or fragment thereof comprising variable heavy and variable light chain sequences of Table 21, or variable heavy and light chain sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

27. The LNP of any one of claims 1-26, wherein the ionizable lipid is a compound of formula (I):432MF-364968774Attorney Docket No.: 25205-20027.40(I), or a pharmaceutically acceptable salt thereof, wherein:X is -O- or -CH2-; m is 0, 1, 2, or 3;R1is Ci-4alkyl;R2is Ci-4alkyl; n is 1, 2, 3, or 4;R3is C4-ioalkyl;R4is C4-ioalkyl; p is 2, 3, 4, 5, or 6;R5is C4-ioalkyl; andR6is C4-ioalkyl.

28. The LNP of any one of claims 1-26, wherein the ionizable lipid is a compound of one of the structures depicted below:Lipid A2433MF-364968774Attorney Docket No.: 25205-20027.40Lipid All434MF-364968774Attorney Docket No.: 25205-20027.40Lipid A15435MF-364968774Attorney Docket No.: 25205-20027.40Lipid A829. The LNP of any one of claims 1 -26, wherein the ionizable lipid is a compound of one of the structures depicted below:Lipid A9436MF-364968774Attorney Docket No.: 25205-20027.40Lipid A2030. The LNP of any one of claims 1-29, wherein the therapeutic agent comprises one nucleic acid molecule, wherein the one nucleic acid molecule is an RNA molecule.

31. The LNP of any one of claims 1 -29, wherein the therapeutic agent comprises two nucleic acid molecules, wherein the two nucleic acid molecules are RNA molecules.

32. The LNP of claim 31, wherein one RNA molecule is an mRNA and the other RNA molecule is a template RNA.

33. The LNP of claim 32, wherein the mRNA encodes a gene modifying polypeptide or a heterologous gene modifying polypeptide.437MF-364968774Attorney Docket No.: 25205-20027.4034. The LNP of claim 32 or 33, wherein the template RNA is configured to insert a heterologous sequence encoding a protein into the genome of an immune cell.

35. The LNP of any one of claims 32-34, wherein the template RNA encodes a chimeric antigen receptor (CAR).

36. The LNP of any one of claims 1 -29, wherein the therapeutic agent comprises three nucleic acid molecules.

37. The LNP of any one of claims 1 -29, wherein the therapeutic agent comprises an mRNA encoding a CAR.

38. The LNP of any one of claims 1-29, wherein the therapeutic agent comprises a first mRNA encoding a first CAR and a second mRNA encoding a second CAR.

39. The LNP of claim 38, wherein the first CAR and the second CAR are 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), kappalight chain, KDR (kinase insert domain receptor), LeY (Lewis Y), LI CAM (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 (prostatespecific 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), and HPV (human papilloma virus).438MF-364968774Attorney Docket No.: 25205-20027.4040. The LNP of claim 38, wherein the first CAR is a CD19-targeted CAR and the second CAR is a BCMA-targeted CAR.

41. The LNP of claim 38, wherein the first CAR is a CD20-targeted CAR and the second CAR is a BCMA-targeted CAR.

42. The LNP of claim 38, wherein the first CAR is a CD 19-targeted CAR and the second CAR is a CD20-targered CAR.

43. The LNP of claim 38, wherein the first CAR is a CD 19-targeted CAR and the second CAR is a GPRC5D-targeted CAR.

44. The LNP of claim 38, wherein the first CAR is a CD20-targeted CAR and the second CAR is a GPRC5D-targeted CAR.

45. The LNP of claim 38, wherein the first CAR is a BCMA-targeted CAR and the second CAR is a GPRC5D-targeted CAR.

46. The LNP of claim 38, wherein the first CAR and the second CAR are any two CAR molecules listed in Table 17.

47. The LNP of any one of claims 1 -29, wherein the therapeutic agent comprises a DNA molecule (e.g., a DNA molecule encoding a CAR).

48. The LNP of any one of claims 1 -29, wherein the therapeutic agent comprises three nucleic acid molecules, wherein the three nucleic acid molecules are a first RNA molecule, a second RNA molecule, and a third RNA molecule.

49. The LNP of claim 48, wherein the first RNA molecule is an mRNA, the second RNA molecule is a first template RNA and the third RNA molecule is a second template RNA.

50. The LNP of claim 49, wherein the mRNA encodes a gene modifying polypeptide or a heterologous gene modifying polypeptide.

51. The LNP of claim 49 or 50, wherein the first template RNA and the second template RNA are each configured to insert a heterologous sequence encoding a protein into the genome of an immune cell.

52. The LNP of any one of claims 49-51 , wherein the first template RNA encodes a first chimeric antigen receptor (CAR) and the second template RNA encodes a second CAR.439MF-364968774Attorney Docket No.: 25205-20027.4053. The LNP of claim 52, wherein the first CAR and the second CAR are 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), kappalight chain, KDR (kinase insert domain receptor), LeY (Lewis Y), LI CAM (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 (prostatespecific 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), and HPV (human papilloma virus).

54. The LNP of claim 52, wherein the first CAR is a CD19-targeted CAR and the second CAR is a BCMA-targeted CAR.

55. The LNP of claim 52, wherein the first CAR is a CD20-targeted CAR and the second CAR is a BCMA-targeted CAR.

56. The LNP of claim 52, wherein the first CAR is a CD19-targeted CAR and the second CAR is a CD20-targered CAR.

57. The LNP of claim 52, wherein the first CAR is a CD19-targeted CAR and the second CAR is a GPRC5D-targeted CAR.

58. The LNP of claim 52, wherein the first CAR is a CD20-targeted CAR and the second CAR is a GPRC5D-targeted CAR.440MF-364968774Attorney Docket No.: 25205-20027.4059. The LNP of claim 52, wherein the first CAR is a BCMA-targeted CAR and the second CAR is a GPRC5D-targeted CAR.

60. The LNP of claim 52, wherein the first CAR and the second CAR are any two CAR molecules listed in Table 17.

61. A lipid nanoparticle (LNP) comprising:(i) a lipid component comprising an ionizable lipid and a helper lipid;(ii) a first targeting moiety and a second target moiety conjugated to the surface of the lipid component of the LNP, wherein the first targeting moiety is an antibody or fragment thereof configured to bind CD3 and the second targeting moiety is an antibody or fragment thereof configured to bind to a receptor on the surface of T cells selected from the group consisting of CD2, 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, wherein the LNP is prepared by chemically reacting the lipid component of the LNP with the first targeting moiety and the second targeting moiety, wherein the molar quantity of second targeting moiety reacting with the lipid component of the LNP is greater than the molar quantity of the first targeting moiety reacting with the lipid component of the LNP.

62. The LNP of claim 61 , wherein the molar quantity of the second targeting moiety used to react with the lipid component of the LNP is at least 1.2, 1.4, 1.6, 1.8, 2, 3, 5, 5, 6, 7, 8, 9 10, or 20 times greater than the molar quantity of the first targeting moiety used to react with the lipid component of the LNP.441MF-364968774

Images