Constrained, ionizable cationic lipids and lipid nanoparticles

Abstract

Ionizable cationic lipids, methods of their synthesis, intermediates useful in the synthesis of ionizable cationic lipids, and methods of synthesizing intermediates are disclosed. Ionizable cationic lipids can be used as components of lipid nanoparticles (LNPs), which in turn can be used to deliver nucleic acids into cells, either in vivo or ex vivo. Also disclosed are LNP compositions, including LNPs comprising functionalized lipids to enable conjugation of binding moieties, and targeted LNPs (tLNPs), i.e., LNPs in which binding moieties have been conjugated to the functionalized lipids and can act as targeting moieties to direct the tLNPs to a desired tissue or cell type.

Description

Cross-reference to related applications

[0001] This application claims priority to the following U.S. provisional applications: U.S. Provisional Application No. 63 / 588,282, filed October 5, 2023; U.S. Provisional Application No. 63 / 632,931, filed April 11, 2024; and U.S. Provisional Application No. 63 / 654,744, filed May 31, 2024; the disclosure of each of these U.S. provisional applications is expressly incorporated herein by reference. Reference to electronic sequence listing

[0002] This application contains a sequence list, which has been electronically submitted and incorporated herein by reference in its entirety. The sequence list was created on September 27, 2024, named "24-0221-WO.xml", and has a size of 11,729 bytes. Background Technology

[0003] Lipid formulations have been used in the laboratory to deliver nucleic acids into cells. Early formulations based on the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and the ionizable, fused lipid dioleoylphosphatidylethanolamine (DOPE) had large particle sizes and presented problems when used in vivo, exhibiting excessively rapid clearance, pulmonary tropism, and toxicity. Lipid nanoparticles (LNPs) incorporating ionizable cationic lipids have been developed to address these issues, achieving RNA-based products such as siRNA and ONPATTRO. ® The extent to which two mRNA-based SARS-CoV-2 vaccines have been approved by regulatory authorities and entered the market.

[0004] However, the ability to control which tissues or cells take up LNPs after administration is limited. Intravenously administered LNPs are primarily absorbed in the liver, lungs, or spleen, largely depending on net charge and particle size. For example, a combination of formulation and intravenous administration can direct >90% of LNPs to the liver. Intramuscular administration can provide clinically useful levels of local delivery and expression. By conjugating to LNPs, a binding moiety specific to the target tissue or cell type, such as a polypeptide conjugated to an antigen-binding domain derived from an antibody, can redirect LNPs to other tissues or cell types. However, avoiding hepatic uptake remains a challenge. Furthermore, using current systems, only a small fraction of the encapsulated nucleic acids are successfully delivered to the cells of interest and into the cytoplasm. Current formulations release only 2% to 5% of the administered RNA into the cytoplasm (see, for example, Gilleron et al., 2013, Nat. Biotechnol. 31:638-646 and Munson et al., 2021, Commun. Biol. 4:211-224). Problems remain regarding off-target delivery, inefficient release of nucleic acids into the cytoplasm, and toxicity associated with the accumulation of component lipids.

[0005] Therefore, there is a need to address issues such as inefficient off-target delivery, low efficiency of nucleic acid release into the cytoplasm, and toxicity associated with the accumulation of component lipids. Summary of the Invention

[0006] This disclosure addresses the need to resolve issues of off-target delivery and low therapeutic agent release efficiency, and provides further related advantages.

[0007] In some respects, this disclosure provides constrained ionizable cationic lipids having the structure of formula M2 as set forth herein.

[0008] In some embodiments, this disclosure provides the ionizable cationic lipids CICL-207, CICL-215, CICL-216, CICL-217, CICL-218, CICL-219, CICL-220, CICL-221, CICL-222, CICL-223, CICL-224, CICL-225, CICL-238, CICL-239, CICL-242, CICL-243, CICL-244, CICL-245, CICL-246, CICL-247, CICL-248, and CICL-249.

[0009] In some respects, this disclosure provides methods for synthesizing ionizable cationic lipids as described herein (e.g., CICL-207, CICL-215, CICL-216, CICL-217, CICL-218, CICL-219, CICL-220, CICL-221, CICL-222, CICL-223, CICL-224, CICL-225, CICL-238, CICL-239, CICL-242, CICL-243, CICL-244, CICL-245, CICL-246, CICL-247, CICL-248 and CICL-249).

[0010] In some respects, this disclosure provides lipid nanoparticles (LNPs) and targeted lipid nanoparticles (tLNPs) incorporating the ionizable cationic lipids disclosed herein.

[0011] In some respects, this disclosure provides methods for preparing LNPs and tLNPs as described herein.

[0012] In some embodiments, this disclosure provides a method for delivering a bioactive payload (e.g., a nucleic acid molecule encoding a therapeutic agent) into a cell, the method comprising contacting the cell with an LNP or tLNP of this disclosure.

[0013] These and other features, objects, and advantages of this disclosure will become more readily apparent from the following description. In the description, reference is made to the accompanying drawings, which form a part of this document, in which embodiments of the disclosure are shown by way of illustration and not limitation. The description of preferred embodiments is not intended to limit the scope of this disclosure to all modifications, equivalents, and substitutions. Therefore, the scope of this disclosure should be interpreted with reference to the claims enumerated herein. Attached Figure Description

[0014] This disclosure will be better understood when taken into consideration the description of the following figures, and features, aspects and advantages other than those set forth above will become apparent.

[0015] Figures 1A to 1C The transfection rate (percentage of cells expressing mRNA) versus expression level (as equivalent soluble fluorescent dye molecule (MESF)) of four tLNP compositions varying in ionizable cationic lipid and / or PEG-lipid fractions was depicted, as noted. The full lipid compositions are described in Table 7. Splenic T cells from C57BL / 6 mice administered tLNP are shown. Figure 1A CD45 - hepatocytes ( Figure 1B ) and hepatic Kupffer cells (CD45) + / CD11 +Hepatocytes; Figure 1C The results in () show that the binding portion of tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry.

[0016] Figures 2A to 2C The transfection rate (percentage of cells expressing mRNA) versus expression level (as an equivalent soluble fluorescent dye molecule (MESF)) of two tLNP compositions, varying only in the ionizable cationic lipid fraction, was depicted as indicated. The full lipid compositions are described in Table 7. Splenic T cells from C57BL / 6 mice administered tLNP are shown. Figure 2A CD45 - hepatocytes ( Figure 2B ) and hepatic Kupffer cells (CD45) + / CD11 + Hepatocytes; Figure 2C The results in () show that the binding portion of tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry.

[0017] Figures 3A to 3B The summary transfection rate data (percentage of cells expressing mRNA) of tLNPs incorporating various ionizable cationic lipids or different amounts of ionizable cationic lipids were depicted. Figure 3A ) and expression level (MESF; Figure 3B The results were normalized to those obtained in each experiment for tLNPs containing 58% CICL-207 as ionizable cationic lipids. The binding moiety of the tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry.

[0018] Figures 4A to 4C The relationship between the transfection rate of tLNPs incorporating various ionizable cationic lipids and the measured pKa of tLNPs was depicted. The results were obtained from spleen T cells of C57BL / 6 mice administered tLNPs. Figure 4A CD45 - hepatocytes ( Figure 4B ) and hepatic Kupffer cells (CD45) + / CD11 + Hepatocytes; Figure 4C Table 7 describes the full lipid composition. The binding moiety of tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry.

[0019] Figures 5A to 5CThe transfection rate (percentage of cells expressing mRNA) versus expression level (as MESF or mean fluorescence intensity (MFI)) of the tLNP composition, varying only in the ionizable cationic lipid fraction, was depicted as indicated. The full lipid composition was CICL:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL {58:10:30.5:1.4:0.1}. Spinal T cells from C57BL / 6 mice administered tLNP are shown. Figure 5A CD45 - hepatocytes ( Figure 5B ) and hepatic Kupffer cells (CD45) + / CD11 + Hepatocytes; Figure 5C The results in () show that the binding portion of tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry.

[0020] Figures 6A to 6C Compensatory variations in transfection rate (percentage of cells expressing mRNA) and cholesterol content were depicted for two ionizable cationic lipids containing different amounts of ionizable cationic lipids. The full lipid compositions are described in Table 7. Splenic T cells from C57BL / 6 mice administered tLNP are shown. Figure 6A CD45 - hepatocytes ( Figure 6B ) and hepatic Kupffer cells (CD45) + / CD11 + Hepatocytes; Figure 6C The results in ).

[0021] Figures 7A to 7H The results were described in NCG mice implanted with human peripheral blood mononuclear cells (PBMCs) and administered tLNP compositions 58% CICL-1 (F9) and 58% CICL-207 (F50) that target CD8 and encapsulate mRNA encoding mCherry or anti-CD19 CAR. Figures 7A to 7B The bar graph depicts the effects of applying targeted CD8. + Following tLNP in cells, CD8 levels in the spleen and blood of NCG mice implanted with human PBMCs were observed. + The engineering rate of T cells with mCherry or anti-CD19 CAR. The results of engineering rates in spleen T cells and blood cells are shown in [the figures]. Figure 7A and Figure 7B middle. Figures 7C to 7F The bar graph depicts the effects of applying targeted CD8. + Following tLNP in cells, CD8 levels in the spleen and blood of NCG mice implanted with human PBMCs were observed.+ Anti-CD19 CAR expression in T cells (as MFI) and CAR molecules per cell (based on MESH). Results of CAR expression MFI in spleen T cells and blood cells were shown in [data missing]. Figure 7C and Figure 7D The results of CAR molecules in each cell of spleen T cells and blood cells were shown in the following figures. Figure 7E and Figure 7F middle. Figures 7G to 7H The depletion of B cells in the spleen of mice 6 hours after administration of tLNP is depicted using a bar graph. The number and percentage of B cells per μL of spleen cell suspension are shown in the figure. Figure 7G and Figure 7H middle.

[0022] Figures 8A to 8E The images present whole-animal images of C57BL / 6 mice administered BF1 LNP, CD5-targeting BF1 tLNP, F50 LNP, or CD5-targeting F50tLNP at 6 and 24 hours post-administration. Figures 8A to 8B Images of bioluminescence in various organs collected at 6 hours and 24 hours post-injection (in prone and supine positions, respectively) and in other positions (in supine and prone positions, respectively). Figures 8C to 8D ) and quantitative bioluminescence of various organs ( Figure 8E ).

[0023] Figure 9 The study described the effects of applying / transfecting mCherry mRNA with tLNP modified with anti-CD117 monoclonal antibody on humanized CD34. + CD34 in bone marrow of NCG mice + Cells (left image) and CD34 + CD117 + In vivo transfection efficiency in cells (right panel), as mCherry-positive cells %. tLNP contains a CICL-1 (F9) or CICL-207 (F50) lipid composition and targets CD117. + Hematopoietic stem cells and progenitor cells (HSPCs). The bar graph represents the average of results from three mice (F9 tLNP mCherry and F50 tLNP mCherry groups) or one mouse (PBS group).

[0024] Figure 10 Depicted on CD34 + In NCG mice, the transfection efficiency of tLNP targeting CD117 was expressed as the percentage of mCherry-positive cells. tLNP contains a CICL-1 (F9) or CICL-207 (F50) lipid composition, encapsulates mCherry mRNA, and targets CD117. +Hematopoietic stem cells and progenitor cells. Mapping bone marrow CD34. + CD38 低 Cells (top left image), CD117 + CD34 + CD38 低 Cells (top right image) and CD117 - CD34 + CD38 低 Data for cells (see image below).

[0025] Figure 11 The study depicted the effects of applying / transfecting humanized CD34 with a CD117-targeting tLNP (administered at 30 μg or 60 μg tLNP) containing a gene-editing payload (SpCas9 mRNA + sgRNA targeting the B2M locus). + CD34 in bone marrow of NCG mice + (Left image) or CD34 + CD38 低 The percentage of B2M (β-2-microglobulin) knockout in tLNP. tLNP contains either CICL-1 (F9) or CICL-207 (F50) lipid compositions. The bar graph represents the average from one to three mice, where the symbols indicate the number of mice implanted with human CD34 from three different donors (triangle, square, and circle). + Results of HSPC in mice.

[0026] Figure 12A Figure 12L depicts the levels of various liver enzymes, acute-phase proteins, and cytokines in rats administered F50 LNP. Figures 12A to 12B The levels of aspartate aminotransferase (AST) were plotted 24 hours after administration of the F50 LNP composition. Figure 12A ) and alanine aminotransferase (ALT) Figure 12B The average liver enzyme levels in rat serum. Figures 12C to 12E The levels of three acute-phase proteins in rat plasma were depicted at 6 and 24 hours after administration of the indicated dose of F50 LNP, specifically α1-acid glycoprotein ( Figure 12C ), α2-macroglobulin ( Figure 12D ) and haptoglobin ( Figure 12E ). Figures 12F to 12H The levels of three pro-inflammatory cytokines in rat plasma following administration of the indicated dose of F50 LNP at 6 and 24 hours post-administration are shown, specifically interleukin-6 (IL-6). Figure 12F ), interleukin-1β ( Figure 12G ), tumor necrosis factor-α ( ) Figure 12H ). Detailed Implementation

[0027] This disclosure provides constrained ionizable cationic lipids (hereinafter referred to as ionizable cationic lipids), methods for synthesizing them, and intermediates and methods for synthesizing these lipids. This disclosure provides ionizable cationic lipids as components of lipid nanoparticles (LNPs) that can be used to deliver bioactive payloads (e.g., nucleic acid molecules encoding therapeutic agents) into cells in vivo or in vitro. This disclosure also discloses LNP compositions comprising functionalized PEG-lipids to enable conjugation of binding moieties to generate targeting LNPs (tLNPs), i.e., LNPs containing binding moieties that direct tLNPs to desired tissue or cell types (e.g., immune cells such as T cells, or stem cells such as hematopoietic stem cells (HSCs)). This disclosure also discloses methods for delivering nucleic acids into cells, comprising contacting cells with the LNPs or tLNPs of this disclosure. The LNPs and tLNPs of this disclosure can be used for in vivo, in vitro, or extracellular transfection. This disclosure also discloses methods for preparing LNPs and tLNPs comprising the ionizable cationic lipids as described herein.

[0028] Before elaborating on this disclosure in more detail, it may be helpful to provide abbreviations and definitions for certain terms used herein. Additional abbreviations are set forth in this disclosure.

[0029] abbreviation

[0030] The abbreviations used in this article include:

[0031] BOC2O-Di-tert-butyl dicarbonate

[0032] CDCl3-deuterated chloroform

[0033] CDI-carbonyldiimidazole

[0034] CH3CN-acetonitrile

[0035] CHOL - Cholesterol

[0036] DMAP-4-Dimethylaminopyridine

[0037] DMG-Myristoyl Glycerin

[0038] DSG-distearate

[0039] DSPC-distearatephosphatidylcholine

[0040] DSPE-distearatephosphatidylethanolamine

[0041] EDC-HCl-1-Ethyl-3-(3'-Dimethylaminopropyl)carbodiimide·HCl

[0042] EtOAc - Ethyl acetate

[0043] EtOH - Ethanol

[0044] Et2O-Diethyl ether

[0045] Et3N-Triethylamine

[0046] MAL-maleimide

[0047] Me3N-Trimethylamine

[0048] MeOH-methanol

[0049] MeOTf-methyl trifluoromethanesulfonate

[0050] Pd / C-Palladium on Carbon

[0051] PEG-polyethylene glycol

[0052] PhCH3-Toluene

[0053] TFA-trifluoroacetic acid

[0054] THF-Tetrahydrofuran

[0055] While this disclosure can be implemented in various forms, the following description of several embodiments is made with the understanding that this disclosure is regarded as an example of the innovation disclosed herein and is not intended to limit this disclosure to the particular embodiments illustrated.

[0056] The headings are provided for convenience only and should not be construed as limiting the implementation in any way. The implementations illustrated under any heading may be combined with those illustrated under any other heading.

[0057] In the event of any conflict between any material incorporated herein by reference and this disclosure, this disclosure shall prevail.

[0058] definition

[0059] Before elaborating on this disclosure in more detail, definitions of certain terms used herein are provided. Further definitions are set forth in this disclosure.

[0060] As used in this specification and claims, unless the context clearly specifies otherwise, the singular forms “an” and “the” include plural references. It should be understood that, as used herein, the terms “an” and “a” mean “one or more” of the listed components.

[0061] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives.

[0062] As used in the context of numbers, the term "approximately" refers to a range centered on a number and spanning between 10% smaller and 10% larger than that number. In the context of ranges, the term "approximately" refers to an extended range spanning between 10% smaller than the lowest number listed in the range and 10% larger than the highest number listed in the range.

[0063] Throughout this disclosure, unless otherwise stated, any concentration range, percentage range, ratio range, or integer range shall be construed as including any integer value within the range, and, where appropriate, fractions of that integer (such as tenths and hundredths of an integer). Furthermore, unless otherwise stated, any numerical range of this disclosure relating to any physical characteristic (such as polymer subunits, size, or thickness) shall be construed as including any integer within the range. Throughout this disclosure, unless otherwise specifically stated, numerical ranges include their enumerated endpoints.

[0064] Unless the context otherwise requires, throughout this specification and claims, the word “comprise” and its variations (such as “comprises” and “comprising”) shall be construed as having an open, inclusive meaning, that is, as meaning “including but not limited to”. As used herein, the terms “comprise” and “comprising” are used synonymously.

[0065] The phrase “at least one of…” when followed by a list of items or elements refers to an open set of one or more elements in the list, which may, but does not necessarily, include more than one element.

[0066] As used herein, a “derivative” refers to a chemically or biologically modified form of a compound that is structurally similar to the parent compound and (in practice or theoretically) derived from it. Generally, a “derivative” differs from an “analogy” in that the parent compound can be the starting material for producing the “derivative,” while the parent compound is not necessarily used as the starting material for producing the “analogy.” A derivative can have different chemical or physical properties than the parent compound. For example, a derivative can be more hydrophilic or hydrophobic than the parent compound, or it can have altered reactivity. Derivatives can be obtained through physical (e.g., biological or chemical) modification of the parent compound, but derivatives can also be conceptually derived, for example, when a protein sequence is designed based on one or more known sequences, a nucleic acid encoding it is constructed, and a derived protein is obtained through the expression of the nucleic acid.

[0067] As used herein, “expansion” refers to cell proliferation, increasing their number. Activators can be used to stimulate proliferation (and other metabolic changes), but can also cause activation-induced death upon initial exposure, resulting in no immediate expansion. For T cells treated in vitro with activators such as IL-2 or CD3 / CD28 activators, the doubling time can be approximately 24 hours (which is fairly typical in mammalian cells in vitro); in vivo doubling times can be significantly shorter, depending on the presence and type of stimulus. Therefore, such protocols will be effectively expansion-free during limited in vitro manipulation, even when activators are used.

[0068] As used herein, “exogenous protein” means a synthetic, recombinant, or other peptide or protein that is not produced by wild-type cells of that type, or is expressed at a lower level in wild-type cells than in cells containing the exogenous peptide. In some embodiments, the exogenous peptide is a peptide or protein encoded by nucleic acid introduced into the cell, which optionally is not retained by the cell. As used herein, “peptide” means an amino acid chain of less than 50 amino acids, while “protein” and “polypeptide” mean an amino acid chain of at least 50 amino acids.

[0069] As used herein, “in vitro” refers to cells harvested or extracted from the body, such as peripheral blood or bone marrow cells, and the manipulation or modification of these cells prior to their intended return (reinfusion). Cell manipulation and modification typically involve cell separation and washing procedures, as well as exposure to activators (e.g., biological response modifiers (BRMs)) and transfection agents (e.g., LNP, tLNP) over time intervals of several hours, such as less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour; and spatially, this is performed within a single facility. Compared to ex vivo, the use of “in vitro” as used herein encompasses a wider range of manipulations, including extended cell culture and expansion cycles over days or longer, and / or cryopreservation or cryogenic storage or transportation.

[0070] As used herein, “transfection” or “transfecting” refers to the introduction of nucleic acids into cells via non-viral methods. Transfection can be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation, and lipid-based reagents. In the preferred embodiments disclosed herein, transfection is mediated by solid lipid nanoparticles (LNPs) comprising targeting LNPs (tLNPs) (which can also be used to deliver non-nucleic acid payloads into cells). The term transfection is used to distinguish it from transduction (the transfer of genetic material from cells to cells or from viruses to cells) and transformation (the uptake of extracellular genetic material by the cell’s natural processes). As used herein, phrases such as “delivering nucleic acids into cells” are synonymous with transfection.

[0071] As used in this article regarding immune cells, “reprogramming” refers to altering the antigen-specific function of immune cells by inducing the expression of exogenous T-cell receptors (TCRs), chimeric antigen receptors (CARs), or immune cell connectors (“reprogramming agents”). Typically, T lymphocytes and natural killer (NK) cells can be reprogrammed with TCRs, CARs, or immune cell connectors, while only CARs or immune cell connectors will be used to reprogram monocytes. As used in this article, regarding stem cells, such as hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs), “reprogramming” refers to correcting or improving genetic defects (e.g., hemoglobinopathies) so that the modified or corrected genes and gene products are reprogramming agents. Reprogramming can be transient or persistent, depending on the nature of the engineered agent.

[0072] As used herein, “engineering agent” refers to a reagent that enables immune cells (particularly non-B lymphocytes or monocytes) to express a reprogramming agent. Engineering agents may include nucleic acids encoding the reprogramming agent, including mRNA. Engineering agents may also include nucleic acids that are components of or encode gene editing systems, such as RNA-directed nucleases, guide RNA, and nucleic acid templates for knocking in reprogramming agents or knocking out endogenous antigen receptors. Gene editing systems include base editors, leader editors, or gene writers. RNA-directed nucleases include CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, and CasX. For transient expression of reprogramming agents (such as CARs), mRNA encoding the reprogramming agent can be used as an engineering agent. For persistent expression of reprogramming agents, such as exogenous, modified, or corrected genes (and their gene products), engineering agents may contain mRNA-encoded RNA-directed nucleases, guide RNA, nucleic acid templates, and other components of the gene / genome editing system.

[0073] Examples of gene editing components encoded by nucleic acid molecules include mRNA encoding RNA-directed nucleases, gene or base editing proteins, leader editing proteins, gene writing proteins (e.g., modified or modularized non-long terminal repeat (LTR) retrotransposons), retrotransposases, RNA writers, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), large-scale nucleases, transposases, retrotransposons, reverse transcriptases (e.g., M-MLV reverse transcriptase), nicking enzymes or inactive nucleases (e.g., Cas9, nCas9, dCas9), DNA recombinases, CRISPR nucleases (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), DNA nicking enzymes, Cas9 nicking enzymes (e.g., D10A or H840A), or any fusion or combination thereof. Other components include guide RNA (gRNA), single guide RNA (sgRNA), leader editing guide RNA (pegRNA), clustered regularly spaced short palindromic repeat (CRISPR) RNA (crRNA), trans-activating clustered regularly spaced short palindromic repeat (CRISPR) RNA (tracrRNA), or DNA molecules to be inserted or used as templates for double-strand break (DSB) repair at specific genomic loci. Genome, gene, and base editing technologies have been reviewed in Anzalone et al., Nature Biotechnology 38:824-844, 2020, Sakuma, Gene and Genome Editing 3-4:100017, 2022, and Zhou et al., MedComm 3(3):e155, 2022. All content taught in each of these publications regarding the components and uses of this technology is incorporated herein by reference to the extent that it does not conflict with this disclosure.

[0074] As used herein, "opsonizer" refers to a biological response modifier (BRM) that enhances the efficiency of engineered immune cells, expands the number of engineered immune cells available for use or the number of engineered cells in target tissues (e.g., tumors, fibrotic tissues, or tissues subjected to autoimmune attack), promotes the activity of engineered cells in target tissues, or broadens the scope of operational mechanisms that contribute to therapeutic immune responses. Opsonizers can be provided by delivering a nucleic acid encoding a tLNP. Exemplary BRMs include cytokines such as IL-7, IL-15, or IL-18.

[0075] As used herein, “immune cell” can refer to any cell of the immune system. However, certain aspects may exclude polymorphonuclear leukocytes and / or B cells, or limit to non-B lymphocytes such as T cells and / or NK cells, or limit to monocytes such as various forms of dendritic cells and / or macrophages.

[0076] As used herein, “lipid nanoparticles” (LNPs) refer to solid particles, which are distinct from liposomes having aqueous chambers. The core of an LNP, like the chamber of a liposome, is surrounded by a lipid layer, which may be, but is not necessarily, a continuous lipid monolayer, bilayer, or multilayer with three or more lipid layers.

[0077] As used herein, a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof capable of specifically binding to one or more targets. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinant binding conjugate of a biomolecule or another target of interest. Exemplary binding moieties of this disclosure include antibodies, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single-domain antibodies, VHH, VNAR, sdAb, nanobodies, receptor extracellular domains or their ligand-binding moieties, or ligands (e.g., cytokines, chemokines). A “Fab” (antigen-binding fragment) is a portion of an antibody that binds an antigen and includes a variable region and CH1 of a heavy chain linked to a light chain via interchain disulfide bonds. In other embodiments, the binding moiety comprises a receptor or a ligand-binding domain of a receptor ligand. In some embodiments, the binding moiety may have more than one specificity, including, for example, bispecific or multispecific binding agents. Various assays are known for identifying the binding moiety of this disclosure that specifically binds to a particular target, including Western blotting, ELISA, and Biacore. ® Analysis. Binding portions (such as binding portions containing variable domains of immunoglobulin light and heavy chains (e.g., scFv)) can be incorporated into a variety of protein scaffolds or structures as described herein (such as antibodies or their antigen-binding fragments, scFv-Fc fusion proteins, or fusion proteins containing two or more such immunoglobulin binding domains).

[0078] As used herein, “antibody” refers to a protein containing an immunoglobulin domain with a hypervariable region that determines the specificity of antibody binding to an antigen; the so-called complementarity-determining region (CDR). Therefore, the term antibody can refer to a complete antibody or an entire antibody, as well as antibody fragments and constructs containing the antigen-binding portion of the entire antibody. While typical natural antibodies have a pair of heavy and light chains, camelids (camels, alpacas, llamas, etc.) produce antibodies with typical structures and antibodies containing only the heavy chain. The variable region of camelid-only heavy-chain antibodies has a unique structure with an elongated CDR3, called VHH, or, when produced as a fragment, a nanobody. Antigen-binding fragments and constructs of antibodies include F(ab)2, F(ab), microantibodies, Fv, single-chain Fv (scFv), biantibodies, and VH. Such elements can be combined to produce bispecific and multispecific agents, such as bispecific T-cell adaptors (BiTEs). The term “monoclonal antibody” originated from hybridoma technology but is now used to refer to any single molecular species of antibody, regardless of its origin or production. Antibodies can be obtained through immunization, selection from natural or immune libraries (e.g., by phage display), alteration of the coding sequence of isolated antibodies, or any combination thereof. Many antibodies that can be used as binding moieties are known in the art. An excellent source of information (including sequence information) on antibodies for which International Nonproprietary Pharmaceutical Names (INNs) have been proposed or recommended is Wilkinson & Hale, 2022, MAbs 14(1):2123299, including its supplementary tables, all of which teaches about individual antibodies and the various antibody forms that can be constructed is incorporated herein by reference. U.S. Patent No. 11,326,182 (especially its Table 9, entitled “Cancer, Inflammation and Immune System Antibodies”) is a source of sequences and other information on a wide range of antibodies, including many that do not have INNs, and all of which teaches about individual antibodies is incorporated herein by reference.

[0079] If the antibody or other binding moiety (or its fusion protein) is equal to or greater than 10 5 M −1 If an antibody or other binding domain (or its fusion protein) binds to a target with a specific affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction in units of 1 / M), without significantly binding to other components present in the test sample, then the antibody or other binding moiety (or its fusion protein) "specifically binds" to the target. Binding domains (or their fusion proteins) can be classified as "high-affinity" binding domains (or their fusion proteins) and "low-affinity" binding domains (or their fusion proteins). A "high-affinity" binding domain is defined as one with a Ka of at least 10. 8 M −1 At least 109 M −1 At least 10 10 M −1 At least 10 11 M −1 At least 10 12 M −1 Or at least 10 13 M −1 Preferably at least 10 8 M −1 Or at least 10 9 M −1 Those binding domains. "Low affinity" binding domains refer to those with a Ka value as high as 10. 8 M -1 Up to 10 7 M -1 Up to 10 6 M -1 Up to 10 5 M -1 Those binding structural domains. Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction, in units of M (e.g., 10⁻⁶). -5 M to 10 -13 M). The affinity of the binding domain peptide and the fusion protein according to this disclosure can be readily determined using conventional techniques (see, for example, Scatchard et al., 1949, Ann. NYAcad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614 or equivalents thereof).

[0080] As used herein, "payload" refers to a negatively charged bioactive agent that can interact with cationic lipids (such as the ionizable cationic lipids of this disclosure) and is thereby encapsulated within lipid nanoparticles containing the cationic lipids. The negatively charged bioactive agent can be a small organic molecule or a large molecule, such as a nucleic acid, carbohydrate, or peptide or polypeptide. In many embodiments, the payload can be one or more nucleic acid molecules, RNA, or DNA, including mRNA and guide RNA (gRNA) molecules.

[0081] As used herein, “bioactive agent” means any substance or combination of substances that affects the metabolic or physiological responses of a living organism or its cultured cells.

[0082] As used in this article, a "therapeutic agent" is a substance whose biological activity has the potential to cure, improve, stabilize, prevent, or otherwise benefit a disease, pathological condition, or other ailment.

[0083] For simplicity, the chemical part is primarily defined and referred to throughout as the monovalent chemical part (e.g., alkyl, aryl, etc.). However, such terms may also be used to convey the corresponding polyvalent part where appropriate structures are clear to those skilled in the art. For example, while the "alkyl" part generally refers to a monovalent group (e.g., CH3-CH2-), in some cases the divalent linking part can be "alkyl," in which case those skilled in the art will understand that alkyl is a divalent group (e.g., -CH2-CH2-), which is equivalent to the term "alkylene". (Similarly, where a divalent part is required and is stated as "aryl," those skilled in the art will understand that the term "aryl" refers to the corresponding divalent part, arylene.) All atoms are understood to have their normal valence for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of S).

[0084] As used herein, the term "alkyl" refers to a saturated straight-chain and branched aliphatic group having 1 to 12 carbon atoms. Therefore, "alkyl" includes C1, C2, C3, C4, C5, C6, C7, C8, C9, C1 ...1, C1, C1, C1, C1, C1, C1 10 C 11 and C 12 Group.

[0085] As used herein, the term "alkenyl" refers to an unsaturated straight-chain or branched aliphatic group having one or more carbon-carbon double bonds and containing 2 to 12 carbon atoms. Therefore, "alkenyl" includes C2, C3, C4, C5, C6, C7, C8, C9, C16, C17, C18, C19, C19, C10, C11, C12, C13, C14, C15, C16, C17, C18 ... 10 C 11 and C 12 Group.

[0086] In some embodiments, the hydrocarbon chain is unsubstituted. In other embodiments, one or more hydrogen atoms of the alkyl or alkenyl group may be substituted with the same or different substituents.

[0087] An alkynyl acid is a carboxylic acid moiety containing one or more carbon-carbon triple bonds. In some embodiments, the hydrogen atoms are unsubstituted. In other embodiments, one or more hydrogen atoms of the alkynyl acid group may be substituted with the same or different substituents.

[0088] Amides are carboxylic acid derivatives that contain a carbonyl group of a carboxylic acid that is partially bonded to an amine.

[0089] An ester is a carboxylic acid derivative that contains a carbonyl group that bonds to an alkoxy group to form an ester bond -C(=O)-O-.

[0090] The head group refers to the hydrophilic or polar portion of a lipid.

[0091] Sterols are steroidal subgroups containing at least one hydroxyl (OH) group. Examples of sterols include, but are not limited to, cholesterol, ergosterol, β-sitosterol, stigmasterol, stigmasterol, 20-hydroxycholesterol, and 22-hydroxycholesterol.

[0092] As a standard, in describing absolute stereochemistry, bonds are represented as solid wedges extending above the plane, and bonds are represented as dashed wedges extending below the plane, throughout the text.

[0093] In contrast to similar molecules where the two branched tails are not connected to each other, in the constrained ionizable lipids described herein, a ring containing a nitrogen atom at the central branch point connects the two branched tail groups to each other, thereby reducing their degrees of freedom of movement. As used herein, "constrained" refers to this limited degree of freedom of movement of the two tail groups extending from the ring containing the nitrogen atom.

[0094] Ionizable cationic lipids

[0095] When designing compound families, a constant or nearly constant core is typically used, with variations in the side groups attached to it. A different approach was employed for the ionizable cationic lipids disclosed herein. A family of ionizable cationic lipids within the general structure of Formula 1b is provided, where R represents the hydrophobic tail of the lipid, n represents an integer from 0 to 4, and X constitutes a polar head group containing an ionizable amine.

[0096]

[0097] Such lipids can be used as components of lipid nanoparticles (LNPs) for delivering nucleic acids into cells. Drawing the lipid structure as shown here is both convenient and compact because it illustrates a cone shape believed to facilitate endosome escape from the contents of LNPs containing such lipids. However, this is only one possible conformation for lipids. Entropy will facilitate the use of multiple conformations.

[0098] The orientation of the three groups extending from the central branch point nitrogen atom in Formula 1b can be constrained by forming a ring containing the nitrogen atom and to which the two branch tail groups are attached, for example, Formula 1c:

[0099]

[0100] By introducing this constraint, the number of conformations that lipids can adopt is reduced. This can increase the physical stability of LNPs or tLNPs incorporating similar lipids that lack a constraint ring.

[0101] The ring size can be varied, as can the location of the tail groups, whether the tail groups are directly attached to the ring or inserted with additional carbon, whether the tail groups are cis or trans, whether the substituents are axial or elongated, or whether the tail groups are symmetrically or asymmetrically attached to the ring. However, these variations should preserve the high biodegradability believed to be conferred by these branched tail groups, and the head groups can be modified to obtain favorable c-pKa and cLogD.

[0102] In some respects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M2:

[0103] ,

[0104] Where X is , , , , , , , , , , , , , , , , , , , , or ;

[0105] Y is O, S, NH, or NCH3;

[0106] Z is O, NH, or NCH3;

[0107] Each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11 alkenyl;

[0108] Each A 1 A 2 A 3 and A 4 Independently selected from (CH2)0 and (CH2)1,

[0109] A 5 Selected from (CH2) 0-4 CH=CH and CH2-CH=CH-CH2; and

[0110] The wavy bond indicates that any relative or absolute stereoconfiguration or mixture of stereoconfigurations of the corresponding ring atom can be assumed.

[0111] In various implementation schemes, option A is selected. 1 To A 4 This results in only two main chain atoms between each nearest ester oxygen in the cyclic nitrogen and the nearest tail group.

[0112] In some implementations, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 (CH2) 1-4 Or CH2-CH=CH-CH2.

[0113] In some implementations, A 1 For (CH2)0, A 2 For (CH2)1, A 3 For (CH2)1, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0114] In some implementations, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)0.

[0115] In some implementations, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0116] In some implementations, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)2 or CH=CH.

[0117] The ionizable cationic lipids disclosed herein have a branched structure to impart a conical rather than cylindrical three-dimensional shape to the lipids, and this structure facilitates endosome cleavage activity. Greater endosome cleavage activity results in higher efficiency of nucleotide cargo release.

[0118] In some embodiments, the ionizable cationic lipid is substantially enantiomerically pure (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% pure). In some embodiments, the ionizable cationic lipid is a racemic mixture. In some embodiments, the ionizable cationic lipid is a mixture of two or more stereoisomers. In some embodiments, at least two of the two or more stereoisomers are diastereomers. In some embodiments, at least two of the two or more stereoisomers are enantiomers.

[0119] The ionizable cationic lipids described herein can be used as components of lipid nanoparticles for the delivery of nucleic acids, including DNA, mRNA, or siRNA, into cells. The ionizable cationic lipids may have a c-pKa (calculated pKa) in the range of about 6, 7, or 8 to about 9, 10, or 11. For example, in various embodiments as described herein, the ionizable cationic lipids have a c-pKa in the range of about 6 to about 10, about 7 to about 10, about 8 to about 10, about 8 to about 9, 6 to 10, 7 to 10, 8 to 10, or 8 to 9. In some embodiments, the ionizable cationic lipids have a c-pKa in the range of about 8.2 to about 9.0 or 8.2 to 9.0. In some embodiments, the ionizable cationic lipids have a c-pKa in the range of about 8.4 to about 8.7 or 8.4 to 8.7. The ionizable cationic lipids described herein may have a cLogD ranging from about 9 to about 18, for example, from about 10 to about 18, or from about 10 to about 16, from about 10 to about 14, or from about 11 to about 18, or from about 11 to about 15, or from about 11 to about 14. In some embodiments, the ionizable cationic lipids have a cLogD ranging from about 13.6 to about 14.4 or 13.6 to 14.4. In some embodiments, the ionizable cationic lipids described herein may have a c-pKa ranging from about 8 to about 11 or 8 to 11 and a cLogD ranging from about 9 to about 18 or 9 to 18. For example, in some embodiments, the ionizable cationic lipid has a c-pKa ranging from about 8.4 to about 8.7 or 8.4 to 8.7 and a cLogD ranging from about 13.6 to about 14.4 or 13.6 to 14.4. These ranges can result in a pKa range of about 6 to about 7 or 6 to 7 as measured in the LNP, which facilitates ionization in endosomes after delivery to cells.

[0120] In some embodiments, a slightly higher basicity may be desired and can be obtained from ionizable cationic lipids having c-pKa and cLogD within the said range. In some embodiments, cLogD is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or within a range defined by any pair of these values. Lipid design also considers the potential biodegradable pathways of the target lipids, utilizing esterases in plasma, liver, and other tissues. Another consideration in lipid design is the fate of the ionizable lipid fragments resulting from esterase cleavage. Preferably, the resulting fragments are rapidly cleared from the body without intervention by hepatic oxidative metabolism.

[0121] As used herein, cLogD is a calculated measure of lipophilicity that takes into account the ionization state of a molecule at a specific pH, predicting the partitioning of lipids between water and octanol as a function of pH. More specifically, cLogD at a given pH is calculated based on cLogP and c-pKa. (LogP is the partition coefficient of a molecule between an aqueous phase (e.g., water) and a lipophilic phase (e.g., octanol). Many software packages are available to provide cLogD values. When a higher basicity of ionizable lipids is required, it should be balanced by greater lipophilicity, represented by a higher cLogD value. A balance between basicity and lipophilicity is needed for LNPs to function optimally in terms of particle stability and the release of bioactive payloads (e.g., one or more nucleic acid molecules encoding therapeutic agents) after cellular uptake. Therefore, with R... 1 Increasing the C6 to C10 increases the overall lipophilicity of the ionizable cationic lipids, as indicated by cLogD. This can be balanced by changing X, which results in a higher c-pKa based on the basicity of the head group. Each ionizable cationic lipid described herein has cLogD and c-pKa values ​​within the desired range as described herein. Specific cLogD and c-pKa values ​​for the ionizable cationic lipids of this disclosure have been calculated using ACD Labs Structure Designer v 12.0. cLogP was calculated using ACD Labs Version B; cLogD was calculated at pH 7.4. Table 1 shows the cLogD and c-pKa for CICL-207, CICL-215, CICL-216, CICL-217, CICL-218, CICL-219, CICL-220, CICL-221, CICL-222, CICL-223, CICL-224, and CICL-225.

[0122] Table 1. CICL-207, CICL-215, CICL-216, CICL-217, CICL-218, CICL-219, CICL-220, CICL-221, CICL-222, CICL-223, CICL-224, CICL-2 25. cLogD and c-pKa of CICL-238, CICL-239, CICL-242, CICL-243, CICL-244, CICL-245, CICL-246, CICL-247, CICL-248 and CICL-249.

[0123]

[0124] X, Y, and R 1Different compositions allow for tuning of cLogD and c-pKa to achieve the desired pKa value measured within LNP or tLNP. For example, to prepare a head group with For lipids with lower basicity, the following head groups can be used instead: , , , , or .

[0125] Conversely, in order to prepare the head group as For lipids with higher alkalinity, the following head groups can be used instead: or .

[0126] Adding a CH2 group to X will tend to increase the basicity of the lipid, which in turn will tend to increase the measured pKa. In R 1 Adding a CH2 group will tend to increase the lipophilicity of lipids (cLogD), which in turn will tend to decrease the pKa of LNP or tLNP measurements.

[0127] The use of additional head groups to modify the calculated pKa and target the desired measured pKa range is illustrated in Example 65.

[0128] In some implementations as described herein, X is... For example, in some implementations, X is... .

[0129] In some implementations as described herein, X is... In some implementations, X is... .

[0130] In some implementations as described herein, X is... .

[0131] In some implementations as described herein, X is... .

[0132] In some implementations as described herein, X is... .

[0133] In some implementations as described herein, X is... .

[0134] In some implementations as described herein, X is... .

[0135] In some implementations as described herein, X is... .

[0136] In some implementations as described herein, X is... .

[0137] In some implementations as described herein, X is... .

[0138] In some implementations as described herein, X is... .

[0139] In some implementations as described herein, X is... .

[0140] In some implementations as described herein, X is... .

[0141] In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0142] In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0143] In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0144] In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0145] In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0146] In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0147] In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0148] In some implementations as described herein, X is... In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0149] In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0150] In some implementations as described herein, X is... In some implementations as described herein, X is... .

[0151] As described above, Y can be selected from O, S, NH, or NCH3. In some embodiments, Y is O. In other embodiments, Y is S.

[0152] In some implementation schemes, X is And Y is 0. In some implementations, X is... And Y is S.

[0153] As mentioned above, Z can be selected from O, NH, or NCH3. In some embodiments, Z is O.

[0154] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0155] In some implementation schemes, X is And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0156] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0157] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0158] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0159] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0160] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0161] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Y is 0. In some embodiments as described herein, X is... And Z is O.

[0162] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0163] In some implementations as described herein, X is... And Z is 0. In some embodiments as described herein, X is... And Z is O.

[0164] As mentioned above, each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11Alkenyl. In some embodiments, each R 1 Independently selected from C7-C 11 Alkyl groups, such as C7-C 10 Alkyl or C7-C9 alkyl. In some embodiments, each R 1 Independently selected from linear C7-C 11 Alkyl groups, such as straight-chain C7-C 10 Alkyl or straight-chain C7-C9 alkyl. In some embodiments as described herein, each R 1 Independently selected from (CH2) 6-8 CH3. In some of these and other embodiments, R1 is (CH2)7CH3. In some embodiments, each R 1 Independently selected from linear C7-C 11 Alkenyl groups, such as straight-chain C7-C 10 Alkenyl or straight-chain C7-C9 alkenyl. For example, in some embodiments, each R 1 It is a straight-chain C8 alkenyl group. In some other embodiments, each R 1 Independently selected from branch C7-C 11 Alkyl groups, such as C7-C 10 Alkyl or C7-C9 alkyl. For example, in some embodiments, each R 1 It is a branched C8 alkyl group. In some embodiments, each R 1 Independently selected from branch C7-C 11 Alkenyl groups, such as C7-C 10 Alkenyl or C7-C9 alkenyl. For example, in some embodiments, each R 1 It is a branched C8 alkenyl group. In some embodiments, R... 1 The ester carbonyl group is branched alkyl or alkenyl, and the position of the branch point is such that the carbonyl group is not in the α position relative to the branch point, but rather in the β position relative to the branch point.

[0165] In some implementations as described herein, each R 1 They are the same. In some implementations, each R closest to the common branch point... 1 They are the same, but those closest to the first common branch point are different from those closest to the second common branch point. In some implementations, each R closest to the common branch point... 1 They are different, but the pair of R closest to the first common branch point 1 It is the same as the pair closest to the second common branch point.

[0166] As described above, in some embodiments as illustrated herein, the ionizable cationic lipid has a structure of formula M2, wherein A 1 For (CH2)0, A2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 (CH2) 1-4 Or CH2-CH=CH-CH2. For example, in some embodiments as described herein, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 The structure is (CH2)1. In some embodiments as described herein, the ionizable cationic lipid has the following structure:

[0167]

[0168] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0169] In some embodiments of formula M2 as described herein, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 It is (CH2)2. For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0170]

[0171] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0172] In some embodiments of formula M2 as described herein, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 It is (CH2)3. For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0173]

[0174] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0175] In some embodiments of formula M2 as described herein, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 It is (CH2)4. For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0176]

[0177] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0178] In some embodiments of formula M2 as described herein, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 The structure is CH2-CH=CH-CH2. For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0179]

[0180] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0181] As described above, in some embodiments as illustrated herein, the ionizable cationic lipid has a structure of formula M2, wherein A 1 For (CH2)0, A 2 For (CH2)1, A 3 For (CH2)1, A 4 It is (CH2)0, and A 5 For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0182]

[0183] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0184] As described above, in some embodiments as illustrated herein, the ionizable cationic lipid has a structure of formula M2, wherein A 1 For (CH2)1, A 2 For (CH2)1, A 3For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)0. For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0185]

[0186] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0187] As described above, in some embodiments as illustrated herein, the ionizable cationic lipid has a structure of formula M2, wherein A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0188]

[0189] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0190] As described above, in some embodiments as illustrated herein, the ionizable cationic lipid has a structure of formula M2, wherein A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 For example, in some embodiments as described herein, A is (CH2)2 or CH=CH. 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 The form is (CH2)2. In some embodiments as described herein, the ionizable cationic lipid has the following structure:

[0191]

[0192] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0193] In some embodiments of formula M2 as described herein, A1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 For example, in some embodiments, the ionizable cationic lipid has the following structure:

[0194]

[0195] X is as described in this paper, and the wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

[0196] In some implementations, the ionizable cationic lipid has the structure CICL-207:

[0197] .

[0198] CICL-207 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207 (e.g., racemic mixtures, or other mixtures in various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-223 and CICL-224) and mixtures comprising two or more different stereoisomers.

[0199] In some implementations, the ionizable cationic lipid has the structure CICL-215:

[0200] .

[0201] CICL-215 is an example of a lipid as disclosed herein, wherein A 3 A 4 and A 5 Does not exist, A 1 and A 2 The form is CH2, and the tail group is symmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include its enantiomers and other mixtures thereof. Still other embodiments include diastereomers of these compounds (e.g., CICL-216) and mixtures comprising two or more different stereoisomers.

[0202] In some implementations, the ionizable cationic lipid has the structure CICL-216:

[0203] .

[0204] CICL-216 is an example of a lipid as disclosed herein, wherein A 3 A 4 and A 5 Does not exist, A 1 and A 2 The form is CH2, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-216 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-215) and mixtures comprising two or more different stereoisomers.

[0205] In some embodiments, the ionizable cationic lipid has the structure CICL-217:

[0206] .

[0207] CICL-217 is an example of a lipid as disclosed herein, wherein A 1 and A 4 Does not exist, A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. This lipid differs from CICL-207 in that Y is S instead of O. The structure drawn above shows the absolute stereochemistry of this lipid. Other embodiments include enantiomers and diastereomers of CICL-217, as well as mixtures of stereoisomers (e.g., racemic mixtures, or other mixtures of various stereoisomers in various ratios). Other embodiments include diastereomers of these compounds and mixtures comprising two or more different stereoisomers.

[0208] In some embodiments, the ionizable cationic lipid has the structure CICL-218:

[0209] .

[0210] CICL-218 is an example of a lipid as disclosed herein, wherein A 3 and A 4 Does not exist, A 1 and A 2 For CH2, A 5The structure is CH=CH, and the tail group is symmetrically positioned in a trans configuration relative to the cyclic nitrogen. CICL-218 was synthesized as a racemic mixture, therefore the structure drawn above indicates the relative stereochemistry of the lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomers, and mixtures thereof. Still other embodiments are diastereomers of these compounds (e.g., CICL-219) and mixtures comprising two or more different stereoisomers.

[0211] In some implementations, the ionizable cationic lipid has the structure CICL-219:

[0212] .

[0213] CICL-219 is an example of a lipid as disclosed herein, wherein A 3 and A 4 Does not exist, A 1 and A 2 For CH2, A 5 The tail group is CH=CH, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-219 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-218) and mixtures comprising two or more different stereoisomers.

[0214] In some implementations, the ionizable cationic lipid has the structure CICL-220:

[0215] .

[0216] CICL-220 is an example of a lipid as disclosed herein, wherein A 3 and A 4 Does not exist, A 1 A 2 and A 5 The form is CH2, and the tail group is symmetrically positioned in a trans configuration relative to the cyclic nitrogen. CICL-220 and the structure drawn above demonstrate the absolute stereochemistry of this lipid. Other embodiments include its enantiomers and other mixtures thereof. Still other embodiments include diastereomers of these compounds and mixtures comprising two or more different stereoisomers.

[0217] In some embodiments, the ionizable cationic lipid has the structure CICL-221:

[0218] .

[0219] CICL-221 is an example of a lipid as disclosed herein, wherein A 1 and A2 Does not exist, A 3 A 4 and A 5 The form is CH2, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-221 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-222) and mixtures comprising two or more different stereoisomers.

[0220] In some implementations, the ionizable cationic lipid has the structure CICL-222:

[0221] .

[0222] CICL-221 is an example of a lipid as disclosed herein, wherein A 1 and A 2 Does not exist, A 3 A 4 and A 5 The form is CH2, and the tail group is symmetrically positioned in an trans configuration relative to the cyclic nitrogen. CICL-222 was synthesized as a racemic mixture, therefore the structure drawn above indicates the relative stereochemistry of the lipid. Other embodiments include the absolute stereochemistry drawn above, its enantiomers, and mixtures thereof. Still other embodiments include diastereomers of these compounds (e.g., CICL-221) and mixtures comprising two or more different stereoisomers.

[0223] In some implementations, the ionizable cationic lipid has the structure CICL-223:

[0224] .

[0225] CICL-223 is an example of a lipid as disclosed herein, wherein A 1 and A 4 Does not exist, A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a cis configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers of CICL-223 (CICL-224) and mixtures of enantiomers (e.g., racemic mixtures, or other mixtures in various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-207 and CICL-225) and mixtures comprising two or more different stereoisomers.

[0226] In some implementations, the ionizable cationic lipid has the structure CICL-224:

[0227] .

[0228] CICL-224 is an example of a lipid as disclosed herein, wherein A 1 and A 4 Does not exist, A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a cis configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers of CICL-224 (CICL-223) and mixtures of enantiomers (e.g., racemic mixtures, or other mixtures in various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-207 and CICL-225) and mixtures comprising two or more different stereoisomers.

[0229] In some implementations, the ionizable cationic lipid has the structure CICL-225:

[0230] .

[0231] CICL-225 is an example of a lipid as disclosed herein, wherein A 1 and A 4 Does not exist, A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers of CICL-225 (CICL-207) and mixtures of enantiomers (e.g., racemic mixtures, or other mixtures in various ratios). Other embodiments include diastereomers of these compounds (e.g., CICL-223 and CICL-224) and mixtures comprising two or more different stereoisomers.

[0232] In some implementations, the ionizable cationic lipid has the structure CICL-238:

[0233]

[0234] CICL-238 is an example of a lipid as disclosed herein, wherein A 1 A 2 and A 5 For CH2, A 3 and A 4It does not exist, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-238 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-220) and mixtures comprising two or more different stereoisomers.

[0235] In some implementations, the ionizable cationic lipid has the structure CICL-239:

[0236]

[0237] CICL-239 is an example of a lipid as disclosed herein, wherein A 1 and A 4 Does not exist, A 2 A 3 and A 5 The group is CH2, and the tail group is asymmetrically positioned in a cis configuration relative to the cyclic nitrogen. Unlike many exemplary lipids, the group Y in CICL-239 is N-methyl-piperazine. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-239 (e.g., racemic mixtures, or other mixtures in various ratios). Other embodiments include diastereomers of these compounds and mixtures comprising two or more different stereoisomers.

[0238] In some implementations, the ionizable cationic lipid has the structure CICL-242:

[0239]

[0240] CICL-242 is an example of a lipid as disclosed herein, wherein A 1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5 The form is CH2CH2, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-242 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-243) and mixtures comprising two or more different stereoisomers.

[0241] In some implementations, the ionizable cationic lipid has the structure CICL-243:

[0242]

[0243] CICL-243 is an example of a lipid as disclosed herein, wherein A1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5 The form is CH2CH2, and the tail group is symmetrically positioned in a trans configuration relative to the cyclic nitrogen. CICL-243 is an optical enantiomer, therefore the structure drawn above indicates the absolute stereochemistry of the lipid. Other embodiments include: its enantiomers and mixtures of enantiomers (e.g., racemates, or other mixtures in various ratios). Still other embodiments include diastereomers of these compounds (e.g., CICL-242) and mixtures comprising two or more different stereoisomers.

[0244] In some implementations, the ionizable cationic lipid has the structure CICL-244:

[0245]

[0246] CICL-244 is an example of a lipid as disclosed herein, in which A 1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5 The form is CH2CH2CH2, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-244 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-245) and mixtures comprising two or more different stereoisomers.

[0247] In some implementations, the ionizable cationic lipid has the structure CICL-245:

[0248]

[0249] CICL-245 is an example of a lipid as disclosed herein, wherein A 1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5 The form is CH2CH2CH2, and the tail group is symmetrically positioned in a trans configuration relative to the cyclic nitrogen. CICL-245 is a racemic mixture, therefore the structure drawn above shows the relative stereochemistry of the lipid. Other embodiments include: its enantiomers and mixtures thereof. Still other embodiments include diastereomers of these compounds (e.g., CICL-244) and mixtures comprising two or more different stereoisomers.

[0250] In some implementations, the ionizable cationic lipid has the structure CICL-246:

[0251]

[0252] CICL-246 is an example of a lipid as disclosed herein, wherein A 1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5 The tail group is CH2CH=CHCH2, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-246 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-247) and mixtures comprising two or more different stereoisomers.

[0253] In some implementations, the ionizable cationic lipid has the structure CICL-247:

[0254]

[0255] CICL-247 is an example of a lipid as disclosed herein, wherein A 1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5 The structure is CH2CH=CHCH2, and the tail group is symmetrically positioned in a trans configuration relative to the cyclic nitrogen. CICL-247 is a racemic mixture, therefore the structure drawn above shows the relative stereochemistry of the lipid. Other embodiments include: its enantiomers and mixtures thereof. Still other embodiments include diastereomers of these compounds (e.g., CICL-246) and mixtures comprising two or more different stereoisomers.

[0256] In some implementations, the ionizable cationic lipid has the structure CICL-248:

[0257]

[0258] CICL-248 is an example of a lipid as disclosed herein, wherein A 1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5The form is CH2CH2CH2CH2, and the tail group is symmetrically positioned in a cis configuration relative to the cyclic nitrogen. CICL-248 is symmetrical, and the two nominal cis configurations are overlapping due to the identical tail group. Other embodiments include diastereomers of the compound (e.g., CICL-249) and mixtures comprising two or more different stereoisomers.

[0259] In some implementations, the ionizable cationic lipid has the structure CICL-249:

[0260]

[0261] CICL-249 is an example of a lipid as disclosed herein, wherein A 1 and A 2 Does not exist, A 3 and A 4 It is CH2, and A 5 The form is CH2CH2CH2CH2, and the tail group is symmetrically positioned in a trans configuration relative to the cyclic nitrogen. CICL-249 is a racemic mixture, therefore the structure drawn above shows the relative stereochemistry of the lipid. Other embodiments include: its enantiomers and mixtures thereof. Still other embodiments include diastereomers of these compounds (e.g., CICL-248) and mixtures comprising two or more different stereoisomers.

[0262] In some embodiments, the ionizable cationic lipid has the structure CICL-207-91:

[0263]

[0264] CICL-207-91 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-91 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-91 or mixtures thereof.

[0265] In some embodiments, the ionizable cationic lipid has the structure CICL-207-92:

[0266]

[0267] CICL-207-92 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-92 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-92 or mixtures thereof.

[0268] In some embodiments, the ionizable cationic lipid has the structure CICL-207-93:

[0269]

[0270] CICL-207-93 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-93 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-93 or mixtures thereof.

[0271] In some embodiments, the ionizable cationic lipid has the structure CICL-207-94:

[0272]

[0273] CICL-207-94 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-94 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-94 or mixtures thereof.

[0274] In some embodiments, the ionizable cationic lipid has the structure CICL-207-95:

[0275]

[0276] CICL-207-95 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-95 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-95 or mixtures thereof.

[0277] In some implementations, the ionizable cationic lipid has the structure CICL-207-96:

[0278]

[0279] CICL-207-96 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-96 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-96 or mixtures thereof.

[0280] In some embodiments, the ionizable cationic lipid has the structure CICL-207-97:

[0281]

[0282] CICL-207-97 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-97 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-97 or mixtures thereof.

[0283] In some implementations, the ionizable cationic lipid has the structure CICL-207-98:

[0284]

[0285] CICL-207-98 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-98 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-98 or mixtures thereof.

[0286] In some embodiments, the ionizable cationic lipid has the structure CICL-207-99:

[0287]

[0288] CICL-207-99 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-99 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-99 or mixtures thereof.

[0289] In some embodiments, the ionizable cationic lipid has the structure CICL-207-100:

[0290]

[0291] CICL-207-100 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-100 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-100 or mixtures thereof.

[0292] In some embodiments, the ionizable cationic lipid has the structure CICL-207-101:

[0293]

[0294] CICL-207-101 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-101 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-101 or mixtures thereof.

[0295] In some embodiments, the ionizable cationic lipid has the structure CICL-207-102:

[0296]

[0297] CICL-207-102 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-102 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-102 or mixtures thereof.

[0298] In some embodiments, the ionizable cationic lipid has the structure CICL-207-103:

[0299]

[0300] CICL-207-103 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-103 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-103 or mixtures thereof.

[0301] In some embodiments, the ionizable cationic lipid has the structure CICL-207-104:

[0302]

[0303] CICL-207-104 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-104 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-104 or mixtures thereof.

[0304] In some embodiments, the ionizable cationic lipid has the structure CICL-207-105:

[0305]

[0306] CICL-207-105 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-105 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-105 or mixtures thereof.

[0307] In some embodiments, the ionizable cationic lipid has the structure CICL-207-106:

[0308]

[0309] CICL-207-106 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-106 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-106 or mixtures thereof.

[0310] In some embodiments, the ionizable cationic lipid has the structure CICL-207-107:

[0311]

[0312] CICL-207-107 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-107 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-107 or mixtures thereof.

[0313] In some embodiments, the ionizable cationic lipid has a structure selected from the following:

[0314]

[0315]

[0316]

[0317]

[0318]

[0319] In some embodiments, the ionizable cationic lipid has the structure CICL-207-108:

[0320]

[0321] CICL-207-108 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-108 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-108 or mixtures thereof.

[0322] In some embodiments, the ionizable cationic lipid has the structure CICL-207-109:

[0323]

[0324] CICL-207-109 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-109 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-109 or mixtures thereof.

[0325] In some implementations, the ionizable cationic lipid has the structure CICL-207-110.

[0326]

[0327] CICL-207-110 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-110 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-110 or mixtures thereof.

[0328] In some embodiments, the ionizable cationic lipid has the structure CICL-207-111:

[0329]

[0330] CICL-207-111 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-111 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-111 or mixtures thereof.

[0331] In some embodiments, the ionizable cationic lipid has the structure CICL-297-112:

[0332]

[0333] CICL-207-112 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-112 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-112 or mixtures thereof.

[0334] In some embodiments, the ionizable cationic lipid has the structure CICL-207-113:

[0335]

[0336] CICL-207-113 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-113 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-113 or mixtures thereof.

[0337] In some embodiments, the ionizable cationic lipid has the structure CICL-207-114:

[0338]

[0339] CICL-207-114 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-114 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-114 or mixtures thereof.

[0340] In some embodiments, the ionizable cationic lipid has the structure CICL-207-115:

[0341]

[0342] CICL-207-115 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-115 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-115 or mixtures thereof.

[0343] In some embodiments, the ionizable cationic lipid has the structure CICL-207-116:

[0344]

[0345] CICL-207-116 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-116 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-116 or mixtures thereof.

[0346] In some embodiments, the ionizable cationic lipid has the structure CICL-207-117:

[0347]

[0348] CICL-207-117 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-117 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-117 or mixtures thereof.

[0349] In some embodiments, the ionizable cationic lipid has the structure CICL-207-118:

[0350]

[0351] CICL-207-118 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-118 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-118 or mixtures thereof.

[0352] In some embodiments, the ionizable cationic lipid has the structure CICL-207-119:

[0353]

[0354] CICL-207-119 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-119 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-119 or mixtures thereof.

[0355] In some embodiments, the ionizable cationic lipid has the structure CICL-207-120:

[0356]

[0357] CICL-207-120 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-120 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-120 or mixtures thereof.

[0358] In some embodiments, the ionizable cationic lipid has the structure CICL-207-121:

[0359]

[0360] CICL-207-121 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-121 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-121 or mixtures thereof.

[0361] In some embodiments, the ionizable cationic lipid has the structure CICL-207-122:

[0362]

[0363] CICL-207-122 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-122 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-122 or mixtures thereof.

[0364] In some embodiments, the ionizable cationic lipid has the structure CICL-207-123:

[0365]

[0366] CICL-207-123 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-123 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-123 or mixtures thereof.

[0367] In some embodiments, the ionizable cationic lipid has the structure CICL-207-124:

[0368]

[0369] CICL-207-124 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-124 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-124 or mixtures thereof.

[0370] In some embodiments, the ionizable cationic lipid has the structure CICL-207-125:

[0371]

[0372] CICL-207-125 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-125 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-125 or mixtures thereof.

[0373] In some embodiments, the ionizable cationic lipid has the structure CICL-207-126:

[0374]

[0375] CICL-207-126 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-126 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-126 or mixtures thereof.

[0376] In some embodiments, the ionizable cationic lipid has the structure CICL-207-127:

[0377]

[0378] CICL-207-127 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-127 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-127 or mixtures thereof.

[0379] In some embodiments, the ionizable cationic lipid has the structure CICL-207-128:

[0380]

[0381] CICL-207-128 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-128 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-128 or mixtures thereof.

[0382] In some implementations, the ionizable cationic lipid has the structure CICL-207-129:

[0383]

[0384] CICL-207-129 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-129 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-129 or mixtures thereof.

[0385] In some embodiments, the ionizable cationic lipid has the structure CICL-207-130:

[0386]

[0387] CICL-207-130 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-130 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-130 or mixtures thereof.

[0388] In some embodiments, the ionizable cationic lipid has the structure CICL-207-131:

[0389]

[0390] CICL-207-131 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-131 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-131 or mixtures thereof.

[0391] In some embodiments, the ionizable cationic lipid has the structure CICL-207-132:

[0392]

[0393] CICL-207-132 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-132 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-132 or mixtures thereof.

[0394] In some embodiments, the ionizable cationic lipid has the structure CICL-207-133:

[0395]

[0396] CICL-207-133 is an example of a lipid as disclosed herein, wherein A 1 and A 4 It does not exist, and A 2 A 3 and A 5 The form is CH2, and the tail group is asymmetrically positioned in a trans configuration relative to the cyclic nitrogen. The structure drawn above illustrates the absolute stereochemistry of this lipid. Other embodiments include enantiomers and mixtures of enantiomers of CICL-207-133 (e.g., racemic mixtures, or other mixtures in various ratios). Further embodiments include either diastereomers of CICL-207-133 or mixtures thereof.

[0397] In some embodiments, the ionizable cationic lipid has a structure selected from the following:

[0398]

[0399]

[0400]

[0401]

[0402] In other aspects of this disclosure, intermediate lipids of the ionizable cationic lipids disclosed herein are provided. In some aspects, the lipids of this disclosure (e.g., intermediate lipids) have a structure of formula M2-1:

[0403]

[0404] in

[0405] Each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11 alkenyl;

[0406] R 2 It is H or a protecting group;

[0407] Each A 1 A 2 A 3 and A 4 Independently selected from (CH2)0 and (CH2)1,

[0408] A 5 Selected from (CH2) 0-4 CH=CH and CH2-CH=CH-CH2; and

[0409] The wavy bond indicates that any relative or absolute stereoconfiguration or mixture of stereoconfigurations of the corresponding ring atom can be assumed.

[0410] In the various implementation schemes described herein, R 2 For H.

[0411] In the various implementation schemes described herein, R 2 The protecting group is (i.e., PG1). PG1 can be selected from protecting groups known in the art that are unstable in bases or acids. For example, in some embodiments, R 2 The protecting group is acid-labile, such as tert-butoxycarbonyl (BOC) or benzyloxycarbonyl (Cbz). In some other embodiments, R 2 The protecting group is unstable in bases, such as the trimethylsilylethoxycarbonyl moiety. In various embodiments of formula M2-1, R 1 A 1 A 2 A 3 A 4 and A 5As described elsewhere in this article.

[0412] For example, in various implementations of formula M2-1, A is selected. 1 To A 4 This results in only two main chain atoms between each nearest ester oxygen in the cyclic nitrogen and the nearest tail group.

[0413] In some embodiments of formula M2-1, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 (CH2) 1-4 Or CH2-CH=CH-CH2.

[0414] In some embodiments of formula M2-1, A 1 For (CH2)0, A 2 For (CH2)1, A 3 For (CH2)1, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0415] In some embodiments of formula M2-1, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)0.

[0416] In some embodiments of formula M2-1, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0417] In some embodiments of formula M2-1, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)2 or CH=CH.

[0418] In some respects, the lipids (e.g., intermediate lipids) of this disclosure have a structure of formula M2-2:

[0419]

[0420] Each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11 alkenyl;

[0421] Each A 1 A 2 A 3 and A 4 Independently selected from (CH2)0 and (CH2)1,

[0422] A 5 Selected from (CH2) 0-4 CH=CH and CH2-CH=CH-CH2; and

[0423] The wavy bond indicates that any relative or absolute stereoconfiguration or mixture of stereoconfigurations of the corresponding ring atom can be assumed.

[0424] In the various implementation schemes described herein, R 1 A 1 A 2 A 3 A 4 and A 5 As described elsewhere in this article.

[0425] For example, in various implementations of formula M2-2, A is selected. 1 To A 4 This results in only two main chain atoms between each nearest ester oxygen in the cyclic nitrogen and the nearest tail group.

[0426] In some implementations of formula M2-2, A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 (CH2) 1-4 Or CH2-CH=CH-CH2.

[0427] In some implementations of formula M2-2, A 1 For (CH2)0, A 2 For (CH2)1, A 3 For (CH2)1, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0428] In some implementations of formula M2-2, A 1 For (CH2)1, A2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)0.

[0429] In some implementations of formula M2-2, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)1.

[0430] In some implementations of formula M2-2, A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)2 or CH=CH.

[0431] To promote biodegradability and minimize the accumulation of the ionizable cationic lipids of this disclosure, the fatty acid tails are designed to contain the ester at positions that minimize steric hindrance for ester cleavage. For example, while a single fatty acid tail would tend to extend away from the ester carbonyl group to provide an energy-favorable position, the presence of two tails results in the tails extending in opposite directions to provide an energy-favorable conformation. In some other embodiments, the fatty acid tails may be located in energy-unfavorable positions. For example, in some embodiments, one of these tails extends toward the carbonyl group and sterically hinders ester cleavage. Therefore, large branches adjacent to the ester carbonyl group are avoided in the design of the cationic lipids disclosed herein. Thus, large branches adjacent to the ester carbonyl group are avoided. Therefore, in some embodiments, the ester carbonyl group is not at the α-position relative to the branch point, for example, they are at the β-position relative to the branch point. When positioning the ester within the lipid, potential degradation products must also be considered to avoid the formation of toxic compounds such as formaldehyde.

[0432] The advantage of relying at least in part on the ionizable cationic lipids of this disclosure is that they avoid the toxicity associated with quaternary ammonium cationic lipids. Therefore, in the various embodiments described herein, the LNP or tLNP does not contain quaternary ammonium (e.g., a quaternary nitrogen group). Some LNPs containing such lipids (which are essentially permanent cations) have shown lethal hyperacute toxicity in laboratory animals. The use of the ionizable cationic lipids of this disclosure in LNPs eliminates the need for quaternary ammonium cationic lipids and thereby mitigates or avoids potential LNP toxicity. In some embodiments, the use of the LNP or tLNP of this disclosure does not cause detectable toxicity to cells or subjects. In some embodiments, the toxicity to cells or subjects caused by the use of the LNP or tLNP of this disclosure does not exceed mild toxicity, which is asymptomatic or causes only mild symptoms that do not require intervention. In some embodiments, the toxicity to cells or subjects caused by the use of the LNP or tLNP of this disclosure does not exceed moderate toxicity, which may impair daily living activities but requires only minimal, local, or non-invasive intervention.

[0433] The relationship between drug efficacy and toxicity is typically expressed as the therapeutic window and the therapeutic index. The therapeutic window is the range of doses from the lowest dose that demonstrates a detectable therapeutic effect to the maximum tolerated dose (MTD); the highest dose that achieves the desired therapeutic effect without producing unacceptable toxicity. Most typically, the therapeutic index is calculated based on the LD50:ED50 ratio in animal studies and the TD50:ED50 ratio in human studies (although this calculation can also be derived from animal studies and is sometimes called the protection index), where LD50, TD50, and ED50 represent the doses that cause lethality, toxicity, and efficacy in 50% of the test population, respectively. These concepts apply regardless of whether toxicity is based on the active agent itself or some other component of the drug product (e.g., LNP or its components). For any inherent toxicity level of the disclosed lipid or LNP itself, increased efficiency in delivering nucleic acids into the cytoplasm improves the therapeutic window or index, as a smaller dose of LNP (and its component lipids) can deliver an effective amount of the bioactive payload (e.g., one or more nucleic acid molecules).

[0434] Sometimes, toxicity and adverse events are graded according to a 5-point scale. Grade 1 or mild toxicity is asymptomatic or causes only mild symptoms; it can be characterized by clinical or diagnostic observations alone; and does not indicate intervention. Grade 2 or moderate toxicity may impair activities of daily living (such as cooking, shopping, managing finances, using the telephone, etc.), but only indicates minimal, local, or non-invasive intervention. Grade 3 toxicity is medically significant but not immediately life-threatening; it indicates hospitalization or prolonged hospitalization; activities of daily living related to self-care (such as bathing, dressing and undressing, self-feeding, toileting, taking medications, and not being bedridden) may be impaired. Grade 4 toxicity is life-threatening and indicates urgent intervention. Grade 5 toxicity results in adverse event-related death. Therefore, in various implementations, toxicity is limited to Grade 2 or lower, Grade 1 or lower, or no observable toxicity by using the disclosed LNP and tLNP.

[0435] Tolerance

[0436] Conventional LNPs are primarily delivered to the liver. Hepatotoxicity is a major dose-limiting parameter observed with drugs containing LNPs. For example, ONPATTRO contains the ionizable lipid MC3. ® The NOAEL (no adverse reaction dose) observed after multiple administrations in rats was only 0.3 mg / kg. (SARS-CoV-2 vaccine COMIRNATY) ® The baseline LNPs used, comprising the ionizable cationic lipid ALC-0315, induced elevations in liver enzymes and acute-phase protein levels in rats at a single dose of ≥1 mg / kg. This elevation was partially reversed only by attaching the antibody to the baseline LNP, and the reversal was greater if the antibody directed the LNP to some other tissue (i.e., tLNP). However, the use of the highly biodegradable, ionizable cationic lipid CICL-1 (whose catabolism should be similar to those disclosed herein) resulted in a greater reduction in liver delivery and decreased associated liver enzyme and acute-phase protein levels for LNPs, antibody-conjugated LNPs, and tLNPs. tLNPs comprising CICL-207 disclosed herein are generally well-tolerated in rats at single doses up to at least 8 mg / kg.

[0437] Methods for preparing ionizable cationic lipids

[0438] Structural symmetry and convergent nonlinear synthetic pathways can be used to simplify the synthesis of ionizable lipids.

[0439] In some respects, this disclosure provides methods for synthesizing ionizable cationic lipids of the formula M2 (e.g., but not limited to, CICL-207, CICL-215, CICL-216, CICL-217, CICL-218, CICL-219, CICL-220, CICL-221, CICL-222, CICL-223, CICL-224 and CICL-225).

[0440] Table 2 provides a summary of the substituents in formula M2 of the ring structures CICL-207, CICL-215, CICL-216, CICL-217, CICL-218, CICL-219, CICL-220, CICL-221, CICL-222, CICL-223, CICL-224, CICL-225, CICL-238, CICL-239, CICL-242, CICL-243, CICL-244, CICL-245, CICL-246, CICL-247, CICL-248, and CICL-249.

[0441] Table 2. CICL-207, CICL-215, CICL-216, CICL-217, CICL-218, CICL-219, CICL-220, CICL-221, CICL-222, CICL-223, CICL-224, CICL-225, CICL-238, CICL-239, CICL-242, CICL- The head and ring structure of CICL-243, CICL-244, CICL-245, CICL-246, CICL-247, CICL-248 and CICL-249 Substituents in M2

[0442]

[0443]

[0444]

[0445]

[0446] In some embodiments, this disclosure provides a method for synthesizing an ionizable cationic lipid of formula M2, the method comprising a synthetic step as shown in scheme M2, wherein A - It is the anion of acid AH, and the remaining substituents are defined as in formula M2. In some embodiments, the method further includes the synthetic steps shown in scheme 4-A. Specific stereoisomers of the ionizable cationic lipid of formula M2 can be prepared using starting materials and / or intermediates having the same stereochemistry according to the synthetic methods disclosed herein.

[0447]

[0448] The synthetic steps shown in scheme M2 include reacting diester amine 6-A with 1,1'-carbonyldiimidazole (CDI) to provide imidazole carboxamide 7-A; and after activation of imidazole carboxamide 7-A, coupling it with the desired alcohol / thiol / amine (HX) to provide the corresponding carbamate / thiocarbamate / urea having the structure of formula M2, with all substituents defined as those in formula M2.

[0449] In some embodiments, the synthesis of imidazole carboxamide 7-A is carried out in an organic solvent (e.g., but not limited to CH2Cl2) in the presence of a basic catalyst (e.g., but not limited to trimethylamine). In some embodiments, the synthesis of carbamate / thiocarbamate / urea may include: first reacting imidazole carboxamide 7-A with methyl trifluoromethanesulfonate (MeOTf), and then reacting it with the desired alcohol / thiol / amine (HX) in the presence of a base (e.g., trimethylamine). The reaction may be carried out in an organic solvent (e.g., acetonitrile). In some embodiments as described herein, HX is an alcohol, wherein X is as described herein. In some embodiments as described herein, HX is a thiol, wherein X is as described herein. In some embodiments as described herein, HX is an amine, wherein X is as described herein. Thus, in some embodiments, HX is an alcohol / thiol / amine that provides the desired X group to the lipid of formula M2. For example, in various embodiments as described herein, HX is selected from... , , , , , , , , , , , , , , , , , , , , , or Where Y is O, S, or NH of NCH3, and Z is O, NH, or NCH3. The various HX compounds disclosed herein are commercially available, known in the scientific literature, or can be prepared using procedures familiar to those skilled in the art, provided from commercial sources, or prepared using the general procedures described in the following examples.

[0450] For example, in various implementation schemes, HX is selected from any of the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0451] In some embodiments, the method may further include the synthesis of diester amine 6-A, which involves deprotecting the protected diester amine 5-A to provide unprotected diester amine 6-A. Deprotection can be carried out under acidic or basic conditions, depending on the protecting group (PG1). For example, the deprotection step can be carried out in an organic solution (e.g., CH2Cl2) under acidic conditions (e.g., in the presence of an acid AH such as trifluoroacetic acid (TFA), when the protecting group is an acid-labile protecting group such as tert-butoxycarbonyl (BOC)). Other PG1 and deprotection steps as known to those skilled in the art can be used. For example, PG1 can be benzyloxycarbonyl (Cbz), which can be removed in the presence of hydrogen and a Pd / C catalyst. Base-labile PG1 (such as a trimethylsilylethoxycarbonyl moiety) can also be used and removed with nBuNH-4 or HF-pyridine.

[0452] In some embodiments, the method further includes synthesizing the protected diester amine 5-A, which involves coupling unprotected diester acid 2-A with a desired diol amine (diol-protected amine 4-A) protected by a first protecting group (PG1) to form the protected diester amine 5-A. In some embodiments, the coupling reaction is carried out in an organic solvent (e.g., acetonitrile) in the presence of a nucleophilic catalyst (e.g., DMAP) and an acidic catalyst (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl)). In some embodiments, PG1 may be -C(=O)-OC(CH3)3.

[0453] In some embodiments, the method further includes synthesizing a diol-protected amine 4-A as shown in scheme 4-A, which involves reacting benzyl diolamine 3-A with a dicarbonate (e.g., (PG1)2O, such as ditert-butyl dicarbonate (BOC2O)) in the presence of a catalyst (e.g., Pd(OH)2) and hydrogen to provide a diol-protected amine 4-A.

[0454]

[0455] Option 4-A

[0456] The synthesis is described using specific solvents, but in all cases, alternative solvents are known to those skilled in the art. THF can be substituted, for example, but not limited to, DMF, diethyl ether, methyl tert-butyl ether, dioxane, or 2-methylTHF. Ethyl acetate can be substituted, for example, but not limited to, isopropyl acetate, THF, 2-methylTHF, dioxane, or methyl tert-butyl ether. Dichloromethane can be substituted, for example, but not limited to, ethyl acetate, isopropyl acetate, THF, methyl tert-butyl ether, 2-methylTHF, dioxane, or heptane. Methanol can be substituted, for example, but not limited to, ethanol or an aqueous solution of THF. Acetonitrile can be substituted, for example, THF, 2-methylTHF, dichloromethane, ethyl acetate, isopropyl acetate, methyl tert-butyl ether, or toluene.

[0457] Numerous general references are available that provide commonly known chemical synthesis schemes and conditions for the synthesis of materials / intermediates used in the synthesis of the disclosed compounds (see, for example, Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Eighth Edition, Wiley-Interscience, 2019; or Furniss, Hannaford, Smith, Tatchelll, Vogel's Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fifth Edition, New York: Longman, 1989).

[0458] The compounds described herein can be purified by any method known in the art, including chromatographic techniques such as HPLC, preparative thin-layer chromatography, flash column chromatography, and ion-exchange chromatography. Any suitable stationary phase can be used, including normal and reversed-phase, as well as ion exchange resins. Most typically, the disclosed compounds are purified by silica gel and / or alumina chromatography. See, for example, Still, Kahn, Mitra, J. Org. Chem. 1978, 43, 2923-292, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. LR Snyder and JJ Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed. E. Stahl, Springer-Verlag, New York, 1969.

[0459] In any method of preparing the subject compound, it may be necessary and / or desirable to protect sensitive or reactive groups on any molecules involved. This can be achieved using conventional protecting groups as described in standard publications, such as JFW McOmie, “Protective Groups in Organic Chemistry,” Plenum Press, London and New York 1973; T PGM Wuts, “Greene's Protective Groups in Organic Synthesis,” Firth edition, Wiley, New York 2014; “The Peptides,” Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981; “Methoden der organischen Chemie,” Houben-Weyl, 4th edition, Vol. 15 / l, Georg Thieme Verlag, Stuttgart 1974; H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Proteine,” Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982; and / or Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate," Georg Thieme Verlag, Stuttgart 1974. Protecting groups can be removed in a convenient subsequent stage using methods known in the art.

[0460] The ionizable cationic lipid intermediates disclosed herein can be prepared using procedures familiar to those skilled in the art and as described herein. For example, compounds of structural formula M2 can be prepared according to scheme M2, scheme 4A, a general procedure (see examples below), and / or similar synthetic procedures. Those skilled in the art can adjust the reaction sequence of schemes M2 and 4A, the general procedure, and the examples to suit desired target molecules. Of course, in some cases, those skilled in the art will use different reagents to influence one or more individual steps or use the protected form of certain substituents. Furthermore, those skilled in the art will recognize that the compounds disclosed herein can be synthesized using entirely different pathways.

[0461] Lipid nanoparticles (LNPs) and targeted LNPs (tLNPs)

[0462] In some aspects, this disclosure provides LNPs comprising ionizable cationic lipids of formula M2. In some embodiments, the LNP comprises an ionizable cationic lipid of formula M2 and phospholipids, sterols, cofactor lipids, PEGylated lipids, or combinations thereof. In some embodiments, the PEG-lipid is not a functionalized PEG-lipid. In other embodiments, the PEG-lipid is a functionalized PEG-lipid. In some embodiments, the LNP comprises at least one functionalized PEG-lipid and at least one non-functionalized PEG-lipid.

[0463] In other respects, this disclosure provides targeted lipid nanoparticles (tLNPs) comprising an ionizable cationic lipid of formula M2. In some embodiments, the tLNP may further comprise one or more of phospholipids, sterols, accessory lipids, and PEG-lipids, or combinations thereof, as well as functionalized PEG-lipids. As used herein, "functionalized PEG-lipid" refers to a PEG-lipid in which the PEG portion has been derivatized with a chemically reactive group that can be used to conjugate the targeting portion to the PEG-lipid. The functionalized PEG-lipid can react with the binding portion after LNP formation, such that the binding portion conjugates to the PEG portion of the lipid. Thus, the conjugated binding portion can act as the targeting portion of the tLNP.

[0464] In various embodiments, the binding moiety of the LNP (or tLNP) includes an antigen-binding domain of an antibody, an antigen, a ligand-binding domain of a receptor, or a receptor ligand. In some embodiments, the binding moiety containing the antigen-binding domain of an antibody includes intact antibodies, F(ab)2, Fab, microantibodies, single-chain Fv (scFv), biantibodies, VH domains, or nanobodies, such as VHH or single-domain antibodies. In some embodiments, the receptor ligand is a carbohydrate, such as a carbohydrate containing terminal galactose or N-acetylgalactosamine units, which can be bound by desialylate glycoprotein receptors. These binding moieties constitute a means of LNP targeting. Some embodiments specifically include one or more of these binding moieties. Other embodiments explicitly exclude one or more of these binding moieties.

[0465] LNP and tLNP composition

[0466] LNP compositions contribute to the formation of stable LNPs and tLNPs, effectively encapsulate payloads, protect payloads from degradation until they are delivered into cells, and facilitate payload escape from endosomes into the cytoplasm. These functions are largely independent of the specificity of the binding moiety (or moieties) used to direct or deflect tLNPs to specific cell types. Additional LNP and tLNP compositions are substantially disclosed in PCT / US2024 / 032141 (titled LipidNanoparticle Formulations and Compositions), filed May 31, 2024, all of which teaches the design, formation, characterization, properties, and uses of LNPs and tLNPs and is incorporated herein by reference.

[0467] LNPs and / or tLNPs may contain sufficient amounts of various components to provide nanoparticles with the desired shape, flowability, and bioacceptability as described herein. Regarding the LNPs or tLNPs of this disclosure, in some embodiments, the LNP (or tLNP) comprises at least one ionizable cationic lipid (e.g., as described herein) in an amount ranging from about 35 mol% to about 65 mol%, or any integer bounded sub-range thereof, for example, about 40 mol% to about 65 mol%, about 40 mol% to about 60 mol%, or about 40 mol% to about 62 mol%. In some embodiments, the LNP or tLNP comprises about 58 mol%, about 60 mol%, or 62 mol% of an ionizable cationic lipid. In some embodiments, the LNP (or tLNP) comprises phospholipids in an amount ranging from about 7 mol% to about 30 mol%, or any integer bounded sub-range thereof, for example, about 13 mol% to about 30 mol%. In some embodiments, the LNP or tLNP comprises about 10 mol% phospholipids. In some embodiments, the LNP (or tLNP) comprises sterols in an amount ranging from about 20 mol% to about 50 mol%, or any integer bounded thereto, such as from about 20 mol% to about 45 mol%, or from about 30 mol% to about 50 mol%, or from about 30 mol% to about 45 mol%. In some embodiments, the LNP or tLNP comprises about 30.5 mol%, 26.5 mol%, or 23.5 mol% sterols. In some embodiments, the LNP (or tLNP) comprises at least one accessory lipid in an amount ranging from about 1 mol% to about 30 mol%. In some embodiments, the LNP or tLNP comprises total PEG-lipids in an amount ranging from about 1 mol% to about 5 mol%, or any integer × 10⁻⁶. -1Within the bounded subscale, for example, in amounts ranging from about 1 mol% to about 2 mol% of total PEG-lipids. In some embodiments, LNP (or tLNP) comprises at least one nonfunctionalized PEG-lipid in an amount of 0 mol% to about 5 mol%, or any integer thereof × 10⁻⁶. -1 Within the bounded range, for example, in amounts ranging from 0 mol% to about 3 mol%, or from about 0.1 mol% to about 5 mol%, or from about 0.5 mol% to about 5 mol%, or from about 0.5 mol% to about 3 mol%. In some embodiments, the LNP or tLNP comprises about 1.4 mol% of nonfunctionalized PEG-lipids. In some embodiments, the LNP or tLNP comprises at least one functionalized PEG-lipid in an amount ranging from about 0.1 mol% to about 5 mol%, or any integer × 10⁻⁶ of the same. -1 Within the bounded subatomic range, for example, in the range of about 0.1 mol% to 0.3 mol%. In some embodiments, the LNP or tLNP contains about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% of functionalized PEG-lipids. In some embodiments, the LNP or tLNP contains about 0.1 mol% of functionalized PEG-lipids. In some embodiments, the functionalized PEG-lipids are conjugated to the binding moiety. In some cases, the tLNP is an LNP that also contains an antibody (e.g., intact IgG) as the binding moiety, which is present at an antibody:mRNA ratio (w / w) of about 0.3 to about 1.0.

[0468] In some aspects, this disclosure provides an LNP or tLNP comprising about 35 mol% to about 65 mol% of an ionizable cationic lipid, about 0.5 mol% to about 3 mol% of a PEG-lipid (including unfunctionalized PEG-lipids and optionally functionalized PEG-lipids), about 7 mol% to about 13 mol% of a phospholipid, and about 30 mol% to about 50 mol% of a sterol. In some embodiments, the LNP or tLNP comprises a payload having a net negative charge, such as a peptide, polypeptide, protein, small molecule, or nucleic acid molecule, or a combination thereof. The payload is typically surrounded by or located within the LNP or tLNP. As disclosed herein, a dose always refers to the amount of payload provided. In some embodiments, the payload comprises one or more nucleic acid molecules. For tLNP-encapsulated mRNA, the dose is typically in the range of 0.05 mg / kg to 5 mg / kg, regardless of the recipient species. In some embodiments, the dose is in the range of 0.1 mg / kg to 1 mg / kg.

[0469] Regarding the LNP or tLNP of this disclosure, in some embodiments, the total lipid to nucleic acid ratio is from about 10:1 to about 50:1 by weight. In some embodiments, the total lipid to nucleic acid ratio is about 10:1, about 20:1, about 30:1, or about 40:1 to about 50:1, or 10:1 to 20:1, 30:1, 40:1, or 50:1, or any range defined by a pair of these ratios. The lipid to nucleic acid ratio can also be reported as an N / P ratio, which is the ratio of positively charged lipid amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. In some embodiments, the N / P ratio is from about 3 to about 9, from about 3 to about 7, from about 3 to about 6, from about 4 to about 6, from about 5 to about 6, or about 6. In some embodiments, the N / P ratio is 3 to 9, 3 to 7, 3 to 6, 4 to 6, 5 to 6, or 6. In some embodiments as described herein, the LNP (or tLNP) includes a binding portion comprising an antigen-binding domain of an antibody, and wherein the antibody is a whole antibody, and the lipid-to-nucleic acid ratio is in the range of about 0.3 w / w to about 1.0 w / w.

[0470] Due to physiological and manufacturing limitations, a hydrodynamic diameter of LNP or tLNP particles for in vivo use is ideally between about 50 nm and 150 nm. Therefore, in some embodiments, LNPs or tLNPs have a hydrodynamic diameter of 50 nm to 150 nm, and in some embodiments, the hydrodynamic diameter is ≤120 nm, ≤110 nm, ≤100 nm, or ≤90 nm. Particle size uniformity is also desirable, where a polydispersity index (PDI) of ≤0.2 (on a scale of 0 to 1) is acceptable. Both the hydrodynamic diameter and the polydispersity index are determined by dynamic light scattering (DLS). Particle size assessed by cryo-transmission electron microscopy (Cryo-TEM) can be smaller than the value determined by DLS.

[0471] Phospholipids

[0472] As described above, in various embodiments, LNPs and tLNPs comprise phospholipids. Phospholipids are amphiphilic molecules, as understood by those skilled in the art or of ordinary skill. Due to their amphiphilic nature, these molecules are known to form bilayers, and by including them in LNPs and tLNPs, as described herein, they can provide film formation, stability, and rigidity. As used herein, phospholipids comprise a hydrophilic head group and two hydrophobic tail groups derived from fatty acids, the hydrophilic head group comprising a functionalized phosphate group. For example, in various embodiments as described herein, phospholipids comprise phosphate groups functionalized with ethanolamine, choline, glycerol, serine, or inositol. As described above, phospholipids comprise two hydrophobic tail groups derived from fatty acids. These hydrophobic tail groups can be derived from unsaturated or saturated fatty acids. For example, the hydrophobic tail groups can be derived from C... 12 -C 20 Fatty acids. Regarding the LNP or tLNP of this disclosure, in various embodiments, the phospholipid includes dioleoylphosphatidylethanolamine (DOPE), myristoylphosphatidylcholine (DMPC), distearylphosphatidylcholine (DSPC), myristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), or 1,2-dicarachidonico-sn-glycerol-3-phosphate choline (DAPC), or combinations thereof. In various embodiments, the phospholipid is dioleoylphosphatidylethanolamine (DOPE), myristoylphosphatidylcholine (DMPC), distearylphosphatidylcholine (DSPC), myristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), or 1,2-dicarachidonico-sn-glycerol-3-phosphate choline (DAPC). In some embodiments, the phospholipid is distearylphosphatidylcholine (DSPC). Phospholipids can facilitate the formation of membranes surrounding a core of an LNP or tLNP, whether monolayer, bilayer, or multilayer. Furthermore, phospholipids (such as DSPC, DMPC, DPPC, and DAPC) impart structural stability and rigidity to the membrane. Phospholipids (such as DOPE) impart fusion. Other phospholipids (such as DMPG, which acquires a negative charge at physiological pH) promote charge regulation. Therefore, phospholipids constitute a means for promoting membrane formation, imparting membrane stability and rigidity, imparting fusion, and regulating charge.

[0473] In some embodiments, the LNP or tLNP has about 7 mol% to about 13 mol% phospholipids, about 7 mol% to about 10 mol% phospholipids, or about 10 mol% to about 13 mol% phospholipids. In some embodiments, the LNP has about 7 mol%, about 10 mol%, or about 13 mol% phospholipids. In some cases, the phospholipid is DSPC. In some cases, the phospholipid is DAPC.

[0474] Sterols

[0475] The disclosed LNPs and tLNPs contain sterols. A sterol is a steroidal subgroup containing at least one hydroxyl (OH) group. More specifically, it is an adenosine derivative in which the H at position 3 is replaced by an OH group, or in other words, but equivalently, a steroid in which the H at position 3 is replaced by an OH group. Examples of sterols include, but are not limited to, cholesterol, ergosterol, β-sitosterol, stigmasterol, stigmasterol, 20-hydroxycholesterol, 22-hydroxycholesterol, etc. Regarding the LNPs or tLNPs of this disclosure, in various embodiments, the sterol is cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, or phytosterol. In further embodiments, the phytosterol includes campesterol, sitosterol, or stigmasterol, or combinations thereof. In a preferred embodiment, the cholesterol is not of animal origin but is obtained through synthesis using phytosterols as a starting point. LNPs containing C-24 alkyl (such as methyl or ethyl) phytosterols have been reported to provide enhanced gene transfection. The length of the alkyl tail, the flexibility of the sterol ring, and the polarity associated with the retained C-3-OH group are important for achieving high transfection efficiency. While β-sitosterol and stigmasterol perform well, vitamin D2, D3, and calcipotriol (analogs lacking complete cholesterol bodies) and betulin, lupeol, ursolic acid, and oleanolic acid (containing the 5th ring) should be avoided. Sterols fill the spaces between other lipids in LNPs or tLNPs and influence the shape of LNPs or tLNPs. Sterols also control the flowability of lipid compositions and reduce temperature dependence. Therefore, sterols (such as cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, β-sitosterol, and stigmasterol) constitute a means of controlling LNP shape and flowability or a sterol means of increasing transfection efficiency. In designing lipid compositions for LNPs or tLNPs, in some embodiments, the sterol content can be selected to compensate for different amounts of other types of lipids, such as ionizable cationic lipids or phospholipids.

[0476] In some embodiments, the LNP or tLNP has about 27 mol% or about 30 mol% to about 50 mol% of sterols, or about 30 mol% to about 38 mol% of sterols. In some embodiments, the LNP or tLNP has about 30.5 mol%, about 33.5 mol%, or about 37.5 mol% of sterols. In some embodiments, the LNP or tLNP has 27 mol% or 30 mol% to 50 mol% of sterols, or 30 mol% to 38 mol% of sterols. In further embodiments, the LNP or tLNP has 30.5 mol%, 33.5 mol%, or 37.5 mol% of sterols. In some cases, the sterol is cholesterol. In some embodiments, the sterol is a mixture of sterols, such as cholesterol and β-sitosterol or cholesterol and 20-hydroxycholesterol. In some cases, the sterol component is about 25 mol% of 20-hydroxycholesterol and about 75 mol% of cholesterol. In some cases, the sterol component is approximately 25 mol% β-sitosterol and approximately 75 mol% cholesterol. In some cases, the sterol component is approximately 50 mol% β-sitosterol and approximately 50 mol% cholesterol. In some cases, the sterol component is 25 mol% 20-hydroxycholesterol and 75 mol% cholesterol. In a further case, the sterol component is 25 mol% β-sitosterol and 75 mol% cholesterol. In an even further case, the sterol component is 50 mol% β-sitosterol and 50 mol% cholesterol.

[0477] assist lipids

[0478] Regarding the LNP or tLNP of this disclosure, in some embodiments, the auxiliary lipid is absent, or includes ionizable lipids, anionic lipids, or cationic lipids. The auxiliary lipid can be used to modulate various properties of the LNP or tLNP, such as surface charge, fluidity, rigidity, size, stability, etc. In some embodiments, the auxiliary lipid is an ionizable lipid, such as cholesterol hemisuccinate (CHEMS) or the ionizable lipid of this disclosure. In some embodiments, the auxiliary lipid is a charged lipid, such as a lipid containing a quaternary ammonium head group. In some embodiments, lipids containing a quaternary ammonium head group include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3β-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof. In some embodiments, these compounds are chlorides, bromides, methanesulfonates, or toluenesulfonates. As mentioned above, lethal hyperacute toxicity has been observed in experimental animals when lipids containing quaternary ammonium head groups are included in LNPs or tLNPs. Therefore, when the auxiliary lipid is a lipid containing a quaternary ammonium head group, the amount of lipid containing the quaternary ammonium head group present does not exceed 50 mol% of the total cationic lipids, for example, 5% to 50% of the total cationic lipids. For illustration, if an LNP or tLNP has a cationic lipid content of 70 mol%, and wherein quaternary ammonium lipids account for 5 mol% to 50 mol% of the total cationic lipids, then the LNP or tLNP will have 3.5 mol% quaternary ammonium lipids and 66.5 mol% ionizable cationic lipids, up to 35 mol% each of the quaternary ammonium lipids and the ionizable cationic lipids.

[0479] When the ionizable lipids of the disclosed formula M2 have a measured pKa of 6 to 7, they are able to contribute substantial endosome release activity to LNPs or tLNPs containing the ionizable lipids. Ionizable lipids of formula M2 that are more acidic or more basic can contribute surface charge, thereby acting as accessory lipids as described above. In such cases, incorporating another lipid with fusion activity into the LNP or tLNP of this disclosure may be advantageous. Surface charge is known to affect the tissue tropism of LNPs or tLNPs; for example, positively charged LNPs or tLNPs have shown tropism towards the spleen and lungs.

[0480] PEG-lipids

[0481] Regarding the LNP or tLNP of this disclosure, the PEG-lipid is a lipid conjugated with polyethylene glycol (PEG). In some embodiments as described herein, the PEG-lipid is a C-lipid conjugated with PEG. 14 -C 20Lipids. For example, in the various embodiments described herein, PEG-lipids are C-lipids conjugated with PEG. 14 -C 20 Lipids, or C-type compounds conjugated with PEG 14 -C 18 Lipids, or C-type compounds conjugated with PEG 14 -C 16 Lipids. In some embodiments as described herein, PEG-lipids are fatty acids conjugated with PEG. The fatty acids in PEG-lipids can have various chain lengths. For each, in some embodiments, PEG-lipids are fatty acids conjugated with PEG, wherein the fatty acid chain length is in the range of C... 14 -C 20 Within the range (e.g., in C) 14 -C 18 Or C 14 -C 16 (within the range). If the fatty acid chain length of PEG-lipids is less than C... 14 If the fatty acid chain length is greater than C, it will be lost too quickly from the LNP of tLNP; 20 If not, difficulties may easily arise during the preparation process.

[0482] PEG can be produced in various sizes. In some embodiments, the PEG of the disclosed LNP and tLNP is PEG-1000 to PEG-5000. It should be understood that these sizes of polyethylene formulations are polydisperse, and the nominal size indicates the approximate average molecular weight of the distribution. Assuming the molecular weight of a single repeating unit of (OCH2CH2)n is 44, then a PEG molecule with n=22 will have a molecular weight of 986, a PEG molecule with n=45 will have a molecular weight of 1998, and a PEG molecule with n=113 will have a molecular weight of 4990. n≈22 to 113 is used to represent PEG-lipids containing the PEG portion in the range of PEG-1000 to PEG-5000, such as PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000, although some molecules from formulations at the average molecular weight boundary will have n outside that range. For a single formulation, n≈22 is used to represent PEG-lipids containing the PEG portion from PEG-1000, n≈45 is used to represent PEG-lipids containing the PEG portion from PEG-2000, n≈67 is used to represent PEG-lipids containing the PEG portion from PEG-3000, n≈90 is used to represent PEG-lipids containing the PEG portion from PEG-4000, and n≈113 is used to represent PEG-lipids containing the PEG portion from PEG-5000. Some embodiments include a PEG portion within a range defined by any pair of the aforementioned n or average molecular weight values. In some embodiments of the PEG-lipid, the PEG has a molecular weight (MW) of 500 Da to 5000 Da or 1000 Da to 5000 Da. For example, in some embodiments, the PEG-lipid has a PEG with a molecular weight in the range of 1500 Da to 5000 Da or 2000 Da to 5000 Da. In some embodiments as described herein, the PEG-lipid has a molecular weight in the range of 500 Da to 4000 Da, or 500 Da to 3000 Da, or 1000 Da to 4000 Da, or 1000 Da to 3000 Da, or 1000 Da to 2500 Da, or 1500 Da to 4000 Da, or 1500 Da to 3000 Da, or 1500 Da to 2500 Da. In some embodiments, the PEG portion is PEG-500, PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000. In some embodiments, the PEG unit has a MW of 2000 Da (sometimes abbreviated as PEG(2k)).Some embodiments include the PEG portion of PEG-1000, PEG-2000, or PEG-5000. In some cases, the PEG portion is PEG-2000. Some embodiments contain DSG-PEG, such as DSG-PEG-2000. Some embodiments contain DSPE-PEG, such as DSPE-PEG-2000. Some embodiments contain DSG-PEG-2000 and / or DSPE-PEG2000.

[0483] Common PEG-lipids are divided into two categories: diacylglycerols and diacylphospholipids. Examples of diacylglycerol PEG-lipids include DMG-PEG (1,2-dimyristoyl-glycerol-3-methoxy polyethylene glycol), DPG-PEG (1,2-dipalmitoyl-glycerol-3-methoxy polyethylene glycol), DSG-PEG (1,2-distearate-glycerol-3-methoxy polyethylene glycol), and DOG-PEG (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol). Examples of diacylphospholipids include DMPE-PEG (1,2-dimyristoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol), DPPE-PEG (1,2-dipalmitoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol), DSPE-PEG (1,2-distearate-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol), and DOPE-PEG (1,2-dioleoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol).

[0484] In some embodiments, the MW2000 PEG-lipid (e.g., a PEG-lipid containing PEG with a molecular weight of 2000 Da) comprises DMG-PEG2000 (1,2-dimyristoyl-glycerol-3-methoxy polyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycerol-3-methoxy polyethylene glycol-2000), DSG-PEG2000 (1,2-distearyl-glycerol-3-methoxy polyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol-2000), DM... PE-PEG200 (1,2-dimyristoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DPPE-PEG2000 (1,2-dispalmitoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DSPE-PEG2000 (1,2-distearate-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DOPE-PEG2000 (1,2-dioleoyl-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), or combinations thereof. In some embodiments, the PEG unit has a MW of 2000 Da. In some embodiments, MW 2000 PEG-lipids include DMRG-PEG2000 (1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000), DPrG-PEG2000 (1,2-dipalmitoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000), DSRG-PEG2000 (1,2-distearate-racemic-glycerol-3-methoxy polyethylene glycol-2000), DorG-PEG2000 (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol-racemic-2000), and DMPER-PEG200 (1,2-dioleoyl-glycerol-3-methoxy polyethylene glycol-racemic-2000). PEG-2000 (1,2-dipalmitoyl-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DPPEr-PEG2000 (1,2-distearatel-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DSPEr-PEG2000 (1,2-distearatel-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), DOPEr-PEG2000 (1,2-dioleoyl-racemic-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000), or combinations thereof. The glycerol in these lipids is chiral. Therefore, in some embodiments, the PEG-lipids are racemic. Alternatively, optically pure enantiomers of the glycerol moiety can be used, i.e., the glycerol moiety is purely chiral. As used herein with respect to the glycerol moiety, optically pure means ≥95% of a single enantiomer (D or L). In some implementations, the enantiomeric excess is ≥98%.In some embodiments, the enantiomeric excess is ≥99%. Other PEG-lipids (including achiral PEG-lipids constructed on symmetrical dihydroxyacetone scaffolds, symmetrical 2-(hydroxymethyl)butane-1,4-diol, or symmetrical glycerol scaffolds) are disclosed in U.S. Provisional Application No. 63 / 362,502, filed April 5, 2022, and PCT / US2023 / 017648 (WO 2023 / 196445A1), filed April 5, 2023, both entitled PEG-Lipids and Lipid Nanoparticles, the full text of which is incorporated herein by reference.

[0485] The above-described PEG-lipid examples are presented as methoxy polyethylene glycol, but the terminus is not necessarily methoxy. For any unfunctionalized PEG-lipid, in alternative embodiments, the PEG moiety of the PEG-lipid can be capped with methoxy, benzyloxy, 4-methoxybenzyloxy, or hydroxy (i.e., alcohol). The terminal hydroxyl group promotes functionalization. Methoxy, benzyloxy, and 4-methoxybenzyloxy groups are advantageously provided to PEG-lipids that will be used as components of an LNP without functionalization. However, all four of these alternatives can be used as (unfunctionalized) PEG-lipid components of an LNP. The 4-methoxybenzyloxy group, which is often used as a protecting group during the synthesis of PEG-lipids, is readily removed to generate the corresponding hydroxyl group. Therefore, the 4-methoxybenzyloxy group provides a convenient route to synthesize alcohols when they are not synthesized directly. Before incorporating the PEG-lipid into the LNP, the alcohol can be functionalized so that the binding moiety can be conjugated to it as the targeting moiety of the LNP (making it a tLNP). As used herein, the terminus of the PEG moiety and similar structures refer to the terminus of the PEG moiety that is not linked to the lipid.

[0486] The PEG moiety provides a hydrophilic surface on LNPs, inhibiting LNP aggregation or coalescence, thereby contributing to their stability and reducing polydispersity, i.e., reducing the heterogeneity of LNP dispersions. Additionally, the PEG moiety can hinder LNP binding, including binding to plasma proteins. These plasma proteins include apoE, which is understood to mediate hepatic uptake of LNPs, so inhibiting this binding can lead to an increased proportion of LNPs reaching other tissues. These plasma proteins also include opsonins, so inhibition of binding reduces recognition by the reticuloendothelial system. The PEG moiety can also be functionalized to act as an attachment site for the targeting moiety. Conjugating cell- or tissue-specific binding moieties to PEG moieties allows tLNPs to bypass the liver and bind to their target tissues or cell types, thereby significantly increasing the proportion of LNPs reaching the target tissue or cell type. Therefore, PEG-lipids can serve as a means of inhibiting LNP binding, and PEG-lipids conjugated to binding moieties can serve as a means of LNP targeting.

[0487] As used herein, the term "functionalized PEG-lipid" and similar constructs generally refer to both unreacted and reacted entities. Even after conjugation (formation of tLNP) has occurred, the lipid composition of the LNP can be described by reference to the reactive substance. For example, a lipid composition may be described as containing DSPE-PEG-maleimide and, moreover, a binding moiety, without explicitly stating that the maleimide has been converted to succinimide (or hydrolyzed succinimide) during the reaction to form the conjugate. Similarly, if the reactive group is bromomaleimide, it will be maleimide after conjugation. These differences in the chemical nomenclature of unreacted and reacted substances should be understood implicitly, even if not explicitly stated. Some embodiments contain DSG-PEG, such as DSG-PEG-2000. Some embodiments contain functionalized DSPE-PEG, such as functionalized DSPE-PEG-2000. Some embodiments contain both DSG-PEG-2000 and functionalized DSPE-PEG-2000. In some cases, functionalized PEG-lipids are partially functionalized with maleimide, such as DSPE-PEG-2000-MAL.

[0488] In some aspects, the LNP comprises one or more PEG-lipids and / or functionalized PEG-lipids; when both functionalized and non-functionalized PEG-lipids are present, they may be the same or different; and one or more ionizable cationic lipids; the LNP may also comprise phospholipids, sterols, accessory lipids, or any combination thereof. The term "functionalized PEG-lipid" refers to a PEG-lipid in which the PEG portion has been derivatized with a chemically reactive group that can be used to conjugate a targeting portion to the PEG-lipid. The functionalized PEG-lipid can react with the binding portion such that the binding portion conjugates to the PEG portion of the lipid. Thus, the conjugated binding portion can act as the targeting portion of the LNP to constitute the tLNP. In some embodiments, the binding portion is conjugated to the functionalized PEG-lipid after the formation of the LNP comprising the functionalized PEG-lipid. In other embodiments, the binding portion is conjugated to the PEG-lipid, and then the conjugate is inserted into the previously formed LNP.

[0489] In some embodiments, the LNP is a tLNP comprising one or more functionalized PEG-lipids conjugated to the binding moiety. In some embodiments, the tLNP also comprises unfunctionalized or unconjugated PEG-lipids. In some embodiments, the functionalization is a maleimide. In some embodiments, the functionalization is a bromomaleimide or bromomaleimide amide, alkynyl amide, or alkynylimide moiety at the terminal hydroxyl end of the PEG moiety. In some embodiments, the binding moiety comprises an antibody or its antigen-binding moiety thereof. In some embodiments, the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group. In some embodiments, the conjugation bond comprises a reaction product of the thiol in the binding moiety and the functionalized PEG-lipid. In some embodiments, the functionalization is a maleimide, azide, alkyne, dibenzocyclooctyne (DBCO), bromomaleimide or bromomaleimide amide, alkynyl amide, or alkynylimide. In some embodiments, the binding moiety comprises an antibody or its antigen-binding moiety thereof. In some embodiments, the binding portion is a polypeptide comprising a binding domain and an N- or C-terminal extension containing an accessible thiol group.

[0490] In some embodiments, the PEG-lipid and / or functionalized PEG-lipid comprises a scaffold selected from formula S1, S2, S3, or S4:

[0491]

[0492] in Indicates the ester junction with the fatty acid, and This indicates the ester (S1) or ether (S2, S3, and S4) formation site with the PEG moiety. In some embodiments, the fatty acid ester is C 14 -C 20 Linear alkyl fatty acids. In some embodiments, the PEG moiety is functionalized, and the fatty acid ester is C... 16 -C 20 Straight-chain alkyl fatty acids. For example, straight-chain alkyl fatty acids are C64-C ... 14 C 15 C 16 C 17 C 18 C 19 Or C 20 In some implementations, the fatty acid ester is C 14 -C 20 Symmetrically branched alkyl fatty acids. For example, branched alkyl fatty acids are C16-26-3 ... 14 C 15 C 16 C 17 C 18 C 19 Or C20 Symmetry refers to the fact that each alkyl branch has the same number of carbons. In some embodiments, the branches are located at positions 3, 4, 5, 6, or 7 of the fatty acid ester. The synthesis and uses of PEG-lipids constructed on scaffolds S1-S4 are disclosed in WO2023 / 196445A1, all of which teaches about PEG-lipids and their uses and is incorporated herein by reference.

[0493] Some embodiments of the disclosed ionizable cationic lipids have head groups with small (<250 Da) PEG moieties. These lipids are not the subject of the term PEG-lipid as used herein. Although these small PEG moieties may affect the lipophilicity of the ionizable cationic lipids, they are generally too small to impede binding to a similar degree as the larger PEG moieties in the PEG-lipids disclosed above. Furthermore, it is understood that PEG-lipids are primarily located in the outer surface thin layer, while the majority of the ionizable cationic lipids reside within the LNP.

[0494] In some embodiments, the functionalized PEG-lipid of LNP (or tLNP) includes one or more fatty acid tails, and for straight-chain fatty acids, each fatty acid tail is not shorter than C0. 16 And not longer than C 20 For branched-chain fatty acids, the length must be at least C. 14 Fatty acids and not longer than C 20 The tail is acceptable. In some implementations, the fatty acid tail is C... 16 In some implementations, the fatty acid tail is C. 18 In some embodiments, the functionalized PEG-lipids include dipalmitoyl lipids. In some embodiments, the functionalized PEG-lipids include distearate lipids. The fatty acid tail is used as a means to anchor the PEG-lipids in the tLNP to reduce or eliminate PEG-lipid detachment from the tLNP. This is a useful property regardless of whether the PEG-lipids are functionalized, but it is more significant for functionalized PEG-lipids because the targeting moiety is attached thereto, and the targeting function may be impaired if the PEG-lipid (with the conjugated binding moiety) detaches from the tLNP.

[0495] In some embodiments, the LNP or tLNP comprises about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol% of PEG-lipids, which include functionalized and non-functionalized PEG-lipids. In some embodiments, the LNP or tLNP comprises DSG-PEG. In other embodiments, the LNP or tLNP comprises DMG-PEG or DPG-PEG. In some embodiments, the LNP or tLNP comprises DSPE-PEG. In some embodiments, the functionalized and non-functionalized PEG-lipids are not the same PEG-lipid; for example, the non-functionalized PEG-lipid may be diacylglycerol, while the functionalized PEG-lipid may be diacylphospholipid. tLNPs with such a mixture show reduced expression in the liver, possibly due to reduced uptake. In some embodiments, the functionalized PEG-lipid is DSPE-PEG, and the non-functionalized PEG-lipid is DSG-PEG. In some embodiments, the LNP or tLNP comprises about 0.4 mol% to about 2.9 mol%, or about 0.9 mol% to about 1.4 mol% of non-functionalized PEG-lipids. In some embodiments, the LNP or tLNP comprises about 1.4 mol% or 1.4 mol% of nonfunctionalized PEG lipids. In some embodiments, the LNP or tLNP comprises about 0.1 mol% to about 0.3 mol% or 0.1 mol% to 0.3 mol% of functionalized lipids. In some cases, the functionalized lipid is DSPE-PEG. In some cases, the LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% of DSPE-PEG. In some cases, the LNP or tLNP comprises 0.1 mol%, 0.2 mol%, or 0.3 mol% of DSPE-PEG. In some cases, the functionalized PEG-lipid is conjugated with a binding moiety. As used herein, unless the context otherwise specifies, the phrase “conjugated with” and similar constructs are intended to convey a state of being, i.e., a structure, rather than a process.

[0496] Adhesion

[0497] Any suitable chemical method can be used to conjugate the PEG-lipid to the PEG, including maleimide chemistry (see Parhiz et al., 2018, Journal of Controlled Release 291:106-115) and click chemistry (see Kolb et al., 2001, Angewandte Chemie International Edition 40(11):2004–2021; and Evans, 2007, Australian Journal of Chemistry 60(6):384–395). Reagents used in such reactions include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid-PEG-dibenzocyclooctylene (DBCO), and lipid-PEG-azide. Further conjugation reactions utilize lipid-PEG-bromomaleimide, lipid-PEG-alkanoamide, PEG-alkynylimide, and lipid-PEG-alkynylene reactions, as disclosed in U.S. Provisional Application No. 63 / 362,502, filed April 5, 2022, and PCT / US2023 / 017648 (WO2023 / 196445A1), filed April 5, 2023, both entitled PEG-Lipids and Lipid Nanoparticles, the full text of which is incorporated herein by reference. On the conjugation side of the reaction, existing cysteine ​​thiol groups can be used, or the protein can be derived by adding a sulfur-containing carboxylic acid, for example, to the ε-amino group of lysine, to react with maleimide, bromomaleimide, alkanoamide, or alkynylimide. In some embodiments, to modify the ε-amino group of the lysine in the binding moiety for reaction with maleimide-functionalized PEG-lipids, the binding moiety (e.g., an antibody) can be reacted with N-succinimide-S-acetylthioacetate (SATA). The SATA is then deprotected, for example, using 0.5 M hydroxylamine, followed by removal of unreacted components via a G-25 Sephadex QuickSpin Protein column (Roche Applied Science, Indianapolis, IN). The reactive thiol group on the binding moiety is then conjugated to the maleimide moiety on the LNP of this disclosure using thioether conjugation chemistry. Alternatively, an alkyne can be added to the thiol group or ε-amino group of lysine to participate in click chemistry reactions.

[0498] Purification can be performed using a Sepharose CL-4B gel filtration column (Sigma-Aldrich). tLNPs (LNPs conjugated with the targeting antibody) can be stored frozen, for example, at -70°C or -80°C until needed. Others conjugate antibodies with free functionalized PEG-lipids and then incorporate the conjugated lipids into pre-formed LNPs. However, the procedure of the present invention has been found to be more controllable and produces more consistent results.

[0499] Several site-specific conjugation methods also exist. Particularly, but not exclusively, for truncated forms of antibodies, C-terminal extensions typically employ a natural or artificial sequence containing particularly accessible cysteine ​​residues. For example, partial reduction of cysteine ​​bonds in the antibody with tris(2-carboxy)phosphine (TCEP) can also generate thiol groups for conjugation, which can be site-specific when using a suitable antibody fragment under specific conditions. Alternatively, the C-terminal extension may contain the sorting enzyme A substrate sequence LPXTG (SEQ ID NO: 6), which can then be functionalized and conjugated with PEG-lipids in a reaction catalyzed by sorting enzyme A, including via click chemistry (see, e.g., Moliner-Morro et al., Biomolecules 10(12):1661, 2020, all of which teaches about antibody conjugation mediated by sorting enzyme A reactions and / or click chemistry is incorporated herein by reference). The use of click chemistry for conjugating target moieties (such as antibodies of various forms) is disclosed, for example, in WO2024 / 102,770, all of which teaches in that document and which does not contradict the present disclosure regarding the conjugation of target moieties with LNPs is incorporated herein by reference in its entirety.

[0500] For antibodies containing the Fc region and other forms, using AJICAP ®One type of reagent can achieve site-specific conjugation to either (or both) of two specific lysine residues (Lys248 and Lys288) without any alteration or extension of the natural antibody sequence (see, for example, Matsuda et al., Mol. Pharmaceutics 18:4058, 2021 and Fujii et al., Bioconjugate Chem. 34:728, 2023; all the teachings of these documents regarding antibody conjugation with the AJICAP reagent are incorporated herein by reference). The AJICAP reagent is a modified affinity peptide that binds to a specific locus on the Fc and reacts with an adjacent lysine residue to form an affinity peptide conjugate of the antibody. The peptide is then cleaved with a base to leave a thiol-functionalized lysine residue, which can then be conjugated via, for example, a maleimide or haloamide reaction. Functionalization with azides or dibenzocyclooctylene (DBCO) for conjugation via click chemistry is also possible. This technology and similar technologies are further described in US 2020 / 0190165 (corresponding to WO 2018 / 199337), US 2021 / 0139549 (corresponding to WO 2019 / 240287) and US 2023 / 0248842 (corresponding to WO 2020 / 184944), all of which teach about such modified affinity peptides and their uses and are incorporated herein by reference.

[0501] Therefore, in some embodiments, the binding moiety is conjugated to the PEG portion of the PEG-lipid via a thiol-modified lysine residue. In some embodiments, conjugation is achieved via cysteine ​​residues in a native or added antibody sequence. In some embodiments, specific cysteine ​​residues react preferentially or exclusively, such as cysteine ​​residues in the antibody hinge region. In a further embodiment, the binding moiety having conjugable cysteine ​​residues in the antibody hinge region is a Fab' or similar fragment. In other embodiments, conjugation is achieved via a sorting enzyme A substrate sequence. In other embodiments, conjugation is achieved via a specific lysine residue (Lys248 or Lys288) in the Fc region.

[0502] Combined part

[0503] Various disclosed aspects of tLNPs include binding moieties, such as antibodies or their antigen-binding domains or cell surface receptor ligands. As used herein, a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide or peptide, carbohydrate, nucleic acid, or combination thereof capable of specifically binding to one or more targets. Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinant binding conjugate of a biomolecule or another target of interest. Exemplary binding moieties of this disclosure include antibodies, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single-domain antibodies, VHH, VNAR, sdAb, nanobodies, receptor extracellular domains or their ligand-binding moieties, or ligands (e.g., cytokines, chemokines). A “Fab” (antigen-binding fragment) is a portion of an antibody that binds an antigen and includes a variable region and CH1 of a heavy chain linked to a light chain via interchain disulfide bonds. In other embodiments, the binding moieties include a receptor or a ligand-binding domain of a receptor ligand. In some embodiments, the binding moiety may have more than one specificity, including, for example, bispecific or multispecific binding agents. Various assays are known for identifying the binding moiety of this disclosure that specifically binds to a particular target, including Western blotting, ELISA, biolayer interferometry, and surface plasmon resonance. Binding moieties (such as those containing variable domains of immunoglobulin light and heavy chains (e.g., scFv)) may be incorporated into various protein scaffolds or structures as described herein, such as antibodies or their antigen-binding fragments, scFv-Fc fusion proteins, or fusion proteins containing two or more such immunoglobulin binding domains.

[0504] The fundamental ability of tLNPs to deliver payloads into the cytoplasm of cells is agnostic to and independent of specific binding specificity. Of course, the binding moiety is the determining factor in which cells the payload is delivered. Many known antibodies are specific for one or more cell surface markers associated with a particular cell type, which can be used as targets for the binding moiety on the disclosed tLNP, and several sources have compiled such information. An excellent source of information on antibodies for which International Nonproprietary Medicine Names (INNs) have been proposed or recommended is Wilkinson & Hale, MAbs 14(1):2123299, 2022, including its supplementary tables, all of which teaches about individual antibodies and the various antibody forms that can be constructed is incorporated herein by reference. US11,326,182 (especially its Table 9, Antibodies for Cancer, Inflammation, and the Immune System) is a source of sequences and other information on a broad range of antibodies, including many that do not have INNs, and all of which teaches about individual antibodies is incorporated herein by reference. For antibodies mentioned in this art, sequence information is not always readily available, even when commercially available. This is not necessarily an obstacle to their use. When an antibody or cell line is commercially available or available from its original developer, it can be used as a binding moiety for tLNPs without any sequence information. Even where sequence information is required, those skilled in the art are fully capable of sequencing antibody proteins (or having them sequenced by a contract laboratory), allowing the variable region of the antibody to be incorporated into scFv, biantibodies, microantibodies, or some other antibody form, or to be humanized. When selecting from available antibodies in this art for the development of reagents for humans, human antibodies are preferred over humanized antibodies, and more preferably over non-human antibodies, all other things being equal. Other factors may include antibody stability and ease of production, antibody affinity, lack of binding to non-target extracellular antigens and cell surface antigens, and cross-reactivity with homologous antigens in the model species to be used for product development.

[0505] In some embodiments, the binding portion may be an antibody or its antigen-binding portion; an antigen; a receptor ligand-binding domain; or a receptor ligand. In some embodiments, the binding portion may have more than one specificity, including, for example, bispecific or multispecific binding agents.

[0506] In some implementations, the binding portion comprises an antibody or its antigen-binding portion. As used herein, “antibody” refers to a protein containing an immunoglobulin domain having a hypervariable region that determines the specificity of antibody binding to an antigen, called a complementarity-determining region (CDR). Therefore, the term antibody can refer to complete (i.e., whole) antibodies and antibody fragments, as well as constructs containing the antigen-binding portion of the whole antibody. While typical natural antibodies have a pair of heavy and light chains, camelids (camels, alpacas, llamas, etc.) produce antibodies with typical structures and antibodies containing only the heavy chain. The variable region of camelid-only heavy-chain antibodies has a unique structure with an elongated CDR3, called a VHH, or, when produced as a fragment, a nanobody. Antigen-binding fragments and constructs of antibodies include F(ab')2, F(ab'), F(ab), microantibodies, Fv, single-chain Fv (scFv), biantibodies, and VH. Such elements can be combined to produce bispecific and multispecific agents, including various immune cell connectors such as BiTE (bispecific T-cell connector). The term “monoclonal antibody” originated from hybridoma technology but is now used to refer to any single molecular class of antibody, regardless of its origin or production. Antibodies can be obtained through immunization, selection from natural or immune libraries (e.g., by phage display), alteration of the coding sequence of isolated antibodies, or any combination thereof. Many antibodies that can be used as binding parts are known in the art. An excellent source of information (including sequence information) on antibodies for which International Nonproprietary Pharmaceutical Names (INNs) have been proposed or recommended is Wilkinson & Hale, 2022, MAbs 14(1):2123299, including its supplementary tables, all of which teaches about single antibodies and the various antibody forms that can be constructed is incorporated herein by reference. U.S. Patent No. 11,326,182 (especially its Table 9, entitled “Cancer, Inflammation and Immune System Antibodies”) is a source of sequence and other information on a wide range of antibodies, including many that do not have INNs, and all of which teaches about single antibodies and the antigens they bind is incorporated herein by reference. WO2024040195A1 is also a source of sequences and other information on a wide range of antibodies that are specific to various cell surface antigens of immune system cells and cancer cells, and all that this literature teaches about individual antibodies and the antigens they bind is incorporated herein by reference.

[0507] If the antibody or other binding moiety (or its fusion protein) is equal to or greater than 10 5 M −1When an antibody or other binding domain (or its fusion protein) binds to a target with an affinity or Ka (i.e., the equilibrium association constant of a specific binding interaction in units of 1 / molar or 1 / M), without significantly binding to other components present in the test sample, then the antibody or other binding moiety (or its fusion protein) "specifically binds" to the target. Binding domains (or their fusion proteins) can be classified as "high-affinity" binding domains (or their fusion proteins) and "low-affinity" binding domains (or their fusion proteins). A "high-affinity" binding domain is defined as one with a Ka of at least 10. 8 M −1 At least 10 9 M −1 At least 10 10 M −1 At least 10 11 M −1 At least 10 12 M −1 Or at least 10 13 M −1 Preferably at least 10 8 M −1 Or at least 10 9 M −1 Those binding domains. "Low affinity" binding domains refer to those with a Ka value as high as 10. 8 M −1 Up to 10 7 M −1 Up to 10 6 M −1 Up to 10 5 M −1 Those binding structural domains. Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction, in units of M (e.g., 10⁻⁶). -5 M to 10 -13 M). The affinity of the binding domain peptide and the fusion protein according to this disclosure can be readily determined using conventional techniques (see, for example, Scatchard et al., 1949, Ann. NY Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614 or equivalents thereof).

[0508] A biantibody is an scFv dimer in which each chain consists of V molecules linked by small peptide linkers. H and V L The region composition is such that the small peptide linker is too short to allow two domains on the same chain to pair. This arrangement forces the V of one chain to... H With the V of the second chain LThe antibodies pair to form a divalent, and typically bispecific, dimer. BiTE is a fusion protein of two scFvs with different antibodies (typically an antibody against a tumor-associated antigen and an antibody against CD3) on a single peptide chain, thereby forming a cytolytic synapse between a T cell and a cell carrying the target antigen. The term "antigen-binding moiety" can refer to a portion of an antibody as described, which has the ability to specifically recognize, associate with, bind to, or combine with a target molecule. Antigen-binding moieties include any naturally occurring, synthetic, semi-synthetic, or recombinant binding coupler against a specific antigen. Thus, antibodies and their antigen-binding moieties constitute a means of binding to cell surface molecules. In various embodiments, depending on the specificity of the antibody, the cell can be an immune cell, leukocyte, lymphocyte, monocyte, stem cell, HSC, or MSC.

[0509] In some implementations, the antibody or its antigen-binding portion may be derived from a mammalian species, such as a mouse, rat, or human. The variable regions of the antibody may be those derived from a single species, or they may be chimeric, containing segments from multiple species that can be further modified to optimize characteristics such as binding affinity or low immunogenicity. For human applications, it is desirable for the antibody to have a human sequence. In cases where the antibody or its antigen-binding portion is derived from a non-human species, the antibody or its antigen-binding portion may be humanized to reduce immunogenicity in human subjects. For example, if a human antibody with the desired specificity is not available, but such an antibody from a non-human species is available, the non-human antibody may be humanized, for example, through CDR transplantation, where a CDR from the non-human antibody is placed in a corresponding position within a compatible human antibody frame. Less preferred are antibodies in which only the constant regions of the non-human antibody are replaced by human sequences. Such antibodies are generally referred to as chimeric antibodies, as opposed to humanized antibodies.

[0510] In some embodiments, the antibody or its antigen-binding portion is non-immunogenic. In some embodiments, the antibody may be modified in its Fc region to reduce or eliminate secondary functions such as FcR binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC); this is commonly referred to as an Fc-silencing antibody.

[0511] The binding density on the LNP (or tLNP) can be defined based on the amount of antibody input based on the conjugation reaction or, as measured in the LNP (or tLNP) after conjugation, the ratio (w / w) of antibody (binding agent) to mRNA. For intact antibodies (e.g., whole IgG), in some embodiments, the preferred ratios for the input or final measured binding agent ratio are about 0.3 to about 1.0, about 0.3 to about 0.7, about 0.3 to about 0.5, about 0.5 to about 1.0, and about 0.5 to about 0.7. In some embodiments, the LNP (or tLNP) has antibody ratios of 0.3 to 1.0, 0.3 to 0.7, 0.3 to 0.5, 0.5 to 1.0, and 0.5 to 0.7 for the input or final measured binding agent ratio. In some embodiments, if the binding agent differs in size from the intact antibody (e.g., scFv, biantibody, or microantibody, etc.), the w / w ratio is adjusted for the different sizes of the binding agent.

[0512] In some implementations, the LNP (or tLNP) comprises a binding moiety derived from the following antibody: anti-CD40* ‡ Antibody, anti-LRRC15 †‡ Antibody, anti-CTSK antibody, anti-ADAM12 ‡ Antibody, anti-ITGA11 antibody, anti-FAP* †‡ Antibodies, anti-NOX4 antibody, anti-SGCD antibody, anti-SYNDIG1 antibody, anti-CDH11 ‡ Antibodies, anti-PLPP4 antibody, anti-SLC24A2 antibody, anti-PDGFRB* ‡ Antibody, anti-THY1 ‡ Antibodies, anti-ANTXR1 ‡ Antibodies, anti-GAS1 antibody, anti-CALHM5 antibody, anti-SDC1* ‡ Antibodies, anti-HER2* †‡ Antibody, anti-TROP2* †‡ Antibodies, anti-MSLN* ‡ Antibody, anti-Nectin4 †‡ Antibody or anti-MUC16* †‡ Antibody. In a further embodiment, the LNP (or tLNP) comprises a binding moiety specifically selected from immune cell antigens: CD1, CD2* †‡ CD3* †‡ CD4* †‡ CD5 †‡ CD7 †‡ CD8 † CD11b ‡ CD14 †‡ CD16, CD25 †‡ CD26*‡ 、CD27* †‡ 、CD28* †‡ 、CD30* †‡ 、CD32*、CD38* †‡ 、CD39 ‡ 、CD40* †‡ 、CD40L(CD154)* †‡ 、CD44* ‡ 、CD45 †‡ 、CD64* ‡ 、CD62 †‡ 、CD68、CD69 ‡ 、CD73 †‡ 、CD80* ‡ 、CD83 ‡ 、CD86* ‡ 、CD95 ‡ 、CD103 ‡ 、CD119 ‡ 、CD126 ‡ 、CD137(4-1BB) †‡ 、CD150 ‡ 、CD153 ‡ 、CD161 ‡ 、CD166 ‡ 、CD183(CXCR3) ‡ 、CD183(CXCR5) ‡ 、CD223(LAG-3)* †‡ 、CD254 ‡ 、CD275 ‡ 、CD45RA、CTLA-4* † * † 、DEC205、OX40 † 、PD-1* †‡ 、GITR † 、TIM-3* †‡ 、FasL* ‡ 、IL18R1、ICOS(CD278) ‡ 、leu-12、TCR † 、TLR1、TLR2 †‡ 、TLR3* ‡ 、TLR4 †‡ 、TLR6、TREM2 ‡ 、NKG2D ‡ 、CCR、CCR1(CD191) ‡ 、CCR2(CD192)* †‡ 、CCR4(CD194)*†‡ CCR6 (CD196) ‡ CCR7 ‡ Low affinity IL-2 receptor †‡ IL-7 receptor ‡ IL-12 receptor ‡ IL-15 receptor ‡ IL-18 receptor ‡ and IL-21 receptor ‡ In a further embodiment, tLNP includes a binding moiety specifically selected from HSC surface molecules: CD117 † CD34* ‡ CD44* ‡ CD90 (Thy1) ‡ CD105 ‡ CD133 ‡ BMPR2 ‡ and Sca-1; or specific binding sites to MSC surface molecules selected from the following: CD70* ‡ CD105 ‡ CD73 ‡ Stro-1 ‡ SSEA-3 ‡ SSEA-4 ‡ CD271 ‡ CD146 ‡ GD2* †‡ ,SUSD2,Stro-4,MSCA-1,CD56 ‡ CD200* †‡ PODXL ‡ CD13 ‡ CD29* ‡ CD44* ‡ and CD10 ‡ In various embodiments, the binding portion is an antibody or its antigen-binding portion. (* indicates an exemplary antibody having the indicated specificity, from which the binding portion can be derived, and can be found in Table 9 or Table 10 of U.S. Patent No. 11,326,182B2.) † Exemplary antibodies with the indicated specificity are shown, from which the binding moiety can be derived; these can be found in Wilkinson & Hale, 2022. Both references cited above are incorporated herein by reference. ‡Exemplary antibodies with the specificity shown are indicated, from which the binding moiety can be derived (which can be found in the Therapeutic Antibody Database (TABS) at tabs.craic.com). Other suitable antibodies can be found in Appendix A or WO2024040195A1, all of which teach in each of these articles about a single antibody and the antigen it binds to, and are incorporated herein by reference.

[0513] The following paragraphs provide a non-exhaustive list of known antibodies that bind to cell surface markers / antigens on immune cells (lymphocytes and monocytes) and stem cells (HSCs and MSCs). These antibodies, or their antigen-binding domains, can be used as binding portions targeting the disclosed LNPs. Similarly, these antibodies can contribute their antigen-binding domains to immune cell reprogramming agents, such as CARs and ICEs. While immune cell reprogramming agents are typically expressed in immune cells, biological response modulators (opsonizers) or immune cell reprogramming agents, such as ICEs, can also be expressed in tumor cells. When cells expressing a certain antigen play a role in the pathology of certain diseases or conditions, those immune cell and stem cell surface markers that can act as tLNP-targeting antigens can also effectively serve as targets for immune cell reprogramming agents. These antibodies, together with peptides containing their antigen-binding domains, constitute means for binding cell surface markers or for binding immune cells and stem cells.

[0514] In some embodiments, CD2 is a cell surface antigen that targets, and the binding moiety comprises an antigen-binding domain of an anti-CD2 antibody. CD2 contains three well-characterized epitopes (T11.1, T11.2, and T11.3 / CD2R). T11.3 / CD2R is located proximal to the membrane and its exposure increases with T cell activation and CD2 aggregation. Therefore, in some such embodiments, the anti-CD2 antigen-binding domain is derived from RPA-2.10; OKT11, UMCD2, 0.1, and 3T4-8B5 (T11.1 epitope); 9.6 and 1OLD2-4C1 (T11.2 epitope); 1Mono2A6 (T11.3 epitope), ciprolizumab (T11.2 / T11.3 epitope), HuMCD2, TS2 / 18, TS1 / 8, AB75, LT-2, T6.3, MEM-65, OTI4E4, or their antigen-binding moieties. Additionally, CD2 ligand CD58 (LFA-3) can be used as a CD2 binding moiety, as can afasette (CD58-Fc fusion). Each of these constitutes a means for binding CD2 (Li et al., 1996, J Mol Biol. 263:209-26; Binder et al., 2020, Front Immunol. 9:11:1090).

[0515] In some embodiments, CD3 targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-CD3 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from moromuzumab-CD3 (OKT3), terprelimin, olelizumab, vexizumab, cisvirizumab, teratozumab, enastatumab, pavurustatumab, vexutuzumab, ozontuzumab, or their antigen-binding moieties. Each of these constitutes a means for binding CD3.

[0516] In some embodiments, CD4 is a cell surface antigen that targets, and the binding moiety comprises an antigen-binding domain of an anti-CD4 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from ipalzumab, inelolizumab, semzuvolimab, zamumab, trelizumab, UB-421, priximab, MTRX1011A, cililumab, crixaximab, keliximab, M-T413, TRX1, hB-F5, MAX.16H5, IT208, or their antigen-binding moieties. Each of these constitutes a means for binding CD4.

[0517] In some embodiments, CD5 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD5 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in 5D7, UCHT2, L17F12, H65, HE3, OKT1, MAT304, and WO1989006968, WO2008121160, US8,679,500, WO2010022737, WO2019108863, WO2022040608, or WO2022127844 (all contents of each of these teachings concerning anti-CD5 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD5.

[0518] In some implementations, CD7 is a cell surface antigen that targets and binds to an antigen-binding domain containing an anti-CD7 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in TH-69, 3A1E, 3A1F, Huly-m2, WT1, YTH3.2.6, T3-3A1, grisnilimab, and those disclosed in US10,106,609, WO2017213979, WO2018098306, US11447548, WO2022136888, WO2020212710, WO2021160267, WO2022095802, WO2022095803, WO2022151851, or WO2022257835 (all contents of each of these teachings concerning anti-CD7 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD7.

[0519] In some embodiments, CD8 targets a cell surface antigen and binds to an antigen-binding domain containing an anti-CD8 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from crefmirlimab (IAB22M), 3B5, SP-16, LT8, 17D8, MEM-31, MEM-87, RIV11, UCHT4, YTC182.20, RPA-T8, OKT8, SK1, 51.1, TRX2, MT807-R1, HIT8α, C8 / 144B, RAVB3, SIDI8BEE, BU88, EPR26538-16, 2ST8.5H7, and US10. Those disclosed in 414,820, WO2015184203, WO2017134306, WO2019032661, WO2020060924, US10,730,944, WO2019033043, WO2021046159, WO2021127088, WO2022081516, US11,535,869 or WO2023004304 (all contents of each of these teachings concerning anti-CD8 antibodies and their properties are incorporated herein by reference) or their antigen-binding portions. Additionally, the humanized anti-CD8 antibodies described in U.S. Provisional Patent Application No. 63 / 610,917, filed December 15, 2023, and U.S. Provisional Patent Application No. 23-1742-US-PRO2, filed May 31, 2024, all teachings of which relate to these humanized anti-CD8 antibodies and their properties or antigen-binding portions thereof are incorporated herein by reference. Each of the aforementioned anti-CD8 antibodies constitutes a means for binding CD8.

[0520] In some embodiments, tLNP targets CD10, a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-CD10 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from hybridoma-generated antibodies, FR4D11, or REA877, as indicated in NITE BP-02489 (disclosed in WO2018235247, all of which teaches about anti-CD10 antibodies and their properties and is incorporated herein by reference), or their antigen-binding moieties. Each of these constitutes a means for binding CD10.

[0521] In some embodiments, CD11b is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-CD11b antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from ASD141 or MAB107, and those disclosed in US20150337039, US10,738,121, WO2016197974, US10,919,967, or WO2022147338 (all the teachings of each of which are incorporated herein by reference regarding anti-CD11b antibodies and their properties), or their antigen-binding portions. Each of these constitutes a means for binding CD11b.

[0522] In some embodiments, CD13 is a target cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-CD13 antibody. CD13 is also known as aminopeptidase N (APN). Therefore, in some such embodiments, the antigen-binding domain is derived from MT95-4 or Nbl57 (disclosed in WO2021072312, all of which teaches about anti-CD13 antibodies and their properties and is incorporated herein by reference), and those disclosed in WO2023037015 (all of which teaches about anti-CD13 antibodies and their properties and is incorporated herein by reference), or their antigen-binding moieties. Each of these constitutes a means for binding CD13.

[0523] In some embodiments, CD14 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD14 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from atemblizumab or r18D11, and those disclosed in WO2018191786 or WO2015140591 (all teachings of each of which concerning anti-CD14 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD14.

[0524] In some embodiments, CD16a is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-CD16a antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in AFM13, sdA1, sdA2, or hu3G8-5.1-N297Q, and US11535672, WO2018158349, WO2007009065, US10385137, WO2017064221, US10,758,625, WO2018039626, WO2018152516, WO2021076564, WO2022161314, or WO2023274183 (all contents of each of these teachings concerning anti-CD16A antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD16a.

[0525] In some embodiments, CD25 is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-CD25 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from daclizumab, basiliximab, camidanlumab, tesirine, inomomab, RO7296682, HuMax-TAC, CYT-91000, STI-003, RTX-003, or their antigen-binding moieties. Each of these constitutes a means for binding CD25.

[0526] In some embodiments, CD28 targets a cell surface antigen and binds to an antigen-binding domain containing an anti-CD28 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from GN1412, acacicolcept, lulizumab, prerulbumab, cilalizumab, FR104CD, and davoceticept, as well as US8,454,959, US8,785,604, US11,548,947, US11,530,268, US11,453,721, and US11, Those disclosed in WO2002030459, WO2002047721, US20170335016, US20200181260, US11608376, WO2020127618, WO2021155071, or WO2022056199 (all contents of each of these teachings concerning anti-CD28 antibodies and their properties are incorporated herein by reference) or their antigen-binding portions. Each of these constitutes a means for binding CD28.

[0527] In some embodiments, CD29 is a target cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-CD29 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from OS2966, 6D276, 12G10, REA1060, or their antigen-binding moieties. Each of these constitutes a means for binding CD29.

[0528] In some embodiments, CD32A is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-CD32A antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in VIB9600, humanized IV.3, humanized AT-10, or MDE-8, and US9,688,755, US9,284,375, US9,382,321, US11306145, or WO2022067394 (all contents of each of these teachings concerning anti-CD32A antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD32A.

[0529] In some embodiments, CD34 is a target cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-CD34 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from h4C8, 9C5, 2E10, 5B12, REA1164, C5B12, C2e10, or their antigen-binding moieties. Each of these constitutes a means for binding CD34.

[0530] In some embodiments, CD40 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD40 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in cifurtilimab, sotilimab, iscalimab, dacetuzumab, selicrelumab, bleselumab, lucarumumab, or mitazalimab, as well as those disclosed in US10633444 (all contents of each of which teachings concerning anti-CD40 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD40.

[0531] In some implementations, CD44 is a target cell surface antigen, and the binding portion contains an antigen-binding domain of an anti-CD44 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in RO5429083, VB6-008, PF-03475952 or RG7356, and WO2008144890, US8,383,117, WO2008079246, US20100040540, WO2015076425, US9,220,772, US20140308301, WO2020159754, WO2021160269, WO2021178896, WO2022022749, WO2022022720 or WO2022243838 (all the teachings of each of which are incorporated herein by reference regarding anti-CD44 antibodies and their properties are included herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD44.

[0532] In some embodiments, CD45 is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-CD45 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in Entomologumab, BC8-B10, and WO2023183927, WO2023235772, US7,825,222, WO2017009473, WO2021186056, US9,701,756, US9,701,756, WO2020092654, WO2022040088, WO2022040577, WO2022064191, WO2022063853, or WO2024064771 (all contents of each of these teachings concerning anti-CD45 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for combining CD45.

[0533] In some embodiments, CD56 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD56 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from lovotozumab, adcitmer, or propimidosum, and those disclosed in WO2012138537, US10,548,987, US10,730,941, or US20230144142 (all the teachings of each of which pertain to anti-CD56 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD56.

[0534] In some embodiments, CD64 is a target cell surface antigen, and the binding portion comprises an antigen-binding domain of an anti-CD64 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from HuMAb 611 or H22, and those disclosed in US7,378,504, WO2014083379, US20170166638, or WO2022155608 (all teachings of each of which are incorporated herein by reference regarding anti-CD64 antibodies and their properties), or their antigen-binding portions. Each of these constitutes a means for binding CD64.

[0535] In some embodiments, CD68 is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-CD68 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from Ki-M7, PG-M1, 514H12, ABM53F5, 3F7C6, 3F7D3, Y1 / 82A, EPR20545, CDLA68-1, LAMP4-824, or their antigen-binding moieties. Each of these constitutes a means for binding CD68.

[0536] In some embodiments, CD70 targets a cell surface antigen and binds to an antigen-binding domain comprising an anti-CD70 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from cutuzumab, vortexuzumab, MDX-1203, MDX-1411, AMG-172, SGN-CD70A, ARX305, PRO1160, and US9,765,148, US8,124,738, IS10,266,604, WO2021138264, US9,701,752, US10,108,123, WO2014158821, US10,689,456, WO2017062271, and US11,046,7 75. Those disclosed in US11,377,500, WO2021055437, WO2021245603, WO2022002019, WO2022078344, WO2022105914, WO2022143951, WO2023278520, WO2022226317, WO2022262101, US11,613,584, or WO2023072307 (all contents of each of these teachings concerning anti-CD70 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD70.

[0537] In some embodiments, CD73 targets a cell surface antigen and binds to an antigen-binding domain containing an anti-CD73 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from oleclumab, uliedlimab, mupadolimab, AK119, IBI325, BMS-986179, NZV930, JAB-BX102, Sym024, TB19, TB38, HBM1007, 3F7, mAb19, Hu001-MMAE, IPH5301, or INCA00186, and... US9,938,356, US10,584,169, WO2022083723, WO2022037531, WO2021213466, WO2022083049, US10,822,426, WO2 021259199, US10,100,129, US11,312,783, US11,174,319, US11,634,500, WO2021138467, WO2017118613, US9,3 88,249, WO2020216697, US11180554, US11,530,273, WO2019173692, WO2019170131, US11,312,785, WO20200985 99. WO2020143836, WO2020143710, US11,034,771, US11,299,550, WO2020253568, WO2021017892, WO2021032173 Those disclosed in WO2021032173, WO2021097223, WO2021205383, WO2021227307, WO2021241729, WO2022096020, WO2022105881, WO2022179039, WO2022214677, or WO2022242758 (all contents of each of these teachings concerning anti-CD73 antibodies and their properties are incorporated herein by reference) or their antigen-binding portions. Each of these constitutes a means for binding CD73.

[0538] In some embodiments, CD90 is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-CD90 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from REA897, OX7, 5E10, K117, L127, or their antigen-binding portions. Each of these constitutes a means for binding CD90.

[0539] In some embodiments, CD105 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD105 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from capuciximab, TRC205, or huRH105, and those disclosed in US8,221,753, US9,926,375, WO2010039873, WO2010032059, WO2012149412, WO2015118031, WO2021118955, US20220233591, or US20230075244 (all contents of each of these teachings concerning anti-CD105 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD105.

[0540] In some embodiments, CD117 is a cell surface antigen that targets and binds to an antigen-binding domain containing an anti-CD117 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from briquilimab, barzolvolimab, CDX-0158, LOP628, MGTA-117, NN2101, CK6, JSP191, Ab85, 104D2, or SR1, and US7,915,391, WO2022159737, US9540443, WO2015050959, US9,789,203, US8,552,157, US10,406,179, US... Those disclosed in 9,932,410, WO2019084067, WO2020219770, US10,611,838, WO2020076105, WO2021107566, US11,208,482, WO2021044008, WO2021099418, WO2022187050 or WO2023026791, WO2021188590 (all the teachings of each of these documents regarding anti-CD117 antibodies and their properties are incorporated herein by reference) or their antigen-binding portions. Each of these constitutes a means for binding CD117.

[0541] In some embodiments, CD133 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD133 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from AC133, 293C3, CMab-43, or RW03, and those disclosed in WO2018045880, US8,722,858, US9,249,225, WO2014128185, US10,711,068, US10,106,623, WO2018072025, or WO2022022718 (all teachings of each of these documents regarding anti-CD133 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD133.

[0542] In some embodiments, CD137 targets a cell surface antigen and the binding portion contains an antigen-binding domain comprising an anti-CD137 antibody. CD137 is also known as 4-1BB. Therefore, in some such embodiments, the antigen-binding domain is derived from YH004, urelumab (BMS-663513), utomilumab (PF-05082566), ADG106, LVGN6051, PRS-343, and WO2005035584, WO2012032433, WO2017123650, US11,203,643, US11,242,395, and US11,555. Those disclosed in WO20230067770, US11,535,678, US11,440,966, WO2019092451, US10,174,122, US11,242,385, US10,716,851, WO2020011966, WO2020011964, or US11,447,558 (all contents of each of these teachings concerning CD137 antibodies and their properties are incorporated herein by reference) or their antigen-binding portions. Each of these constitutes a means for binding CD137.

[0543] In some embodiments, CD146 is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-CD146 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in iplemab, ABX-MA1, huAA98, M2H, or IM1-24-3, and US10,407,506, US10,414,825, US6,924,360, US9,447,190, WO2014000338, US9,782,500, WO2018220467, US11,427,648, WO2019133639, WO2019137309, WO2020132190, or WO2022082073 (all contents of each of these teachings concerning CD146 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD146.

[0544] In some embodiments, CD166 is a cell surface antigen that targets, and the binding moiety comprises an antigen-binding domain of an anti-CD166 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in propultumab, AZN-L50, REA442, or AT002, and US10,745,481, US11,220,544, or WO2008117049 (all contents of each of these teachings concerning CD166 antibodies and their properties are incorporated herein by reference), or their antigen-binding moieties. Each of these constitutes a means for binding CD166.

[0545] In some embodiments, CD200 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD200 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in samalizumab, OX-104, REA1067, B7V3V2, HPAB-0260-YJ, or TTI-CD200, and WO2007084321 or WO2019126536 (all teachings of each of which are incorporated herein by reference regarding CD200 antibodies and their properties), or their antigen-binding portions. Each of these constitutes a means for binding CD200.

[0546] In some embodiments, CD205 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD205 antibody. CD205 is also referred to as DEC205. Thus, in some such embodiments, the antibody comprises 3G9-2D2 (a component of CDX-1401) or LY75_A1 (a component of MEN1309), and those disclosed in US8,236,318, US10,081,682, or US11,365,258 (all the teachings of each of which are incorporated herein by reference regarding anti-CD205 antibodies and their properties), or their antigen-binding portions. Each of these constitutes a means for binding CD205.

[0547] In some embodiments, CD271 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-CD271 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in REA844 or REAL709, and WO2022166802 (all the teachings of that document regarding anti-CD271 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD271.

[0548] In some embodiments, BMPR2 is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-BMPR2 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in TAB-071CL (Creative Biolabs catalog number), and US11,292,846 or WO2021174198 (all teachings of each of which, as to their contents, pertain to anti-BMPR2 antibodies and their properties, are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding BMPR2.

[0549] In some embodiments, tight junction protein 18.2 (CLDN 18.2) targets cell surface antigens and binds to an antigen-binding domain containing an antibody against tight junction protein 18.2. Therefore, in some such embodiments, the antigen-binding domain is derived from zolbetuximab, osemitamab, RC118, IBI-343, AZD0901, M108, SYSA1801, TORL-2-307-ADC, LM-302, ASKB589, gresonitamab, SPX-101, SKB315, Q-1802, GIVASTOMIG, LCAR-C18S, SOT102, CT041, and WO2013167259, WO2021032157, WO2021254481, and WO2022007808. The following are disclosed in WO2021008463, WO2022111616, WO2018006882, WO2020147321, WO2019219089, US20200040101, WO2020025792, WO2020139956, WO2020135201, US20240228610, WO2021218874, WO2021027850, WO2021129765, WO2022068854, WO2021111003 (all contents of each of these teachings concerning antibodies against tight junction protein 18.2 and their properties are incorporated herein by reference) or their antigen-binding portions. Each of these constitutes a means for binding tight junction protein 18.2.

[0550] In some implementations, CTLA-4 targets a cell surface antigen and the binding portion contains an antigen-binding domain of an anti-CTLA-4 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from botensilimab, ipilimumab, nurulimab, quavonlimab, tremelimumab, zalifrelimab, ADG116, ADG126, ADU-1604, AGEN1181, BCD-145, BMS-986218, BMS-986249, BT-007, CS1002, GIGA-564, HBM4003, IBI310, JK08, JMW-3B3, JS007, KD6001, KN044, ONC-392, REGN4659, TG6050, XTX101, YH001, or their antigen-binding portions. Each of these constitutes a means for binding CTLA-4.

[0551] In some embodiments, GD2 targets a cell surface antigen and binds to an antigen-binding domain containing an anti-GD2 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from denutoxicumab, glyliximab, nacituzumab, nivatuzumab, EMD 273063, hu14.18k322A, MORAb-028, 3F8BiAb, BCD-245, KM666, ATL301, icotumab, and US9,777,068, US9,315,585, WO2004055056, US9,617,349, US9,493,740, US20210002384, and US8507. Those disclosed in WO2001023573, WO2012071216, WO2018010846, US8,951,524, WO2023280880, US9,856,324, WO2015132604, WO2017055385, WO2019059771, and WO2020020194, or their antigen-binding portions. Each of these constitutes a means for binding GD2.

[0552] In some embodiments, GITR targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-GITR antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from ragifilimab, TRX518, MK-4166, AMG 228, MEDI1873, BMS-986156, REGN6569, ASP1951, MK-1248, FRA154, GWN323, JNJ-64164711, ATOR-1144, or their antigen-binding moieties. Each of these constitutes a means for binding GITR.

[0553] In some embodiments, the low-affinity IL-2 receptor is a target cell surface antigen (CD122 and / or CD132), and the binding moiety comprises an antigen-binding domain of an anti-IL-2 receptor antibody. Therefore, in some such embodiments, anti-CD122 antibodies include ANV419, FB102, MiK-β-1, and the anti-CD122 antibodies disclosed in WO2011127324, WO2017021540, WO2022212848, WO2022221409, WO2023078113, US20230272090, and WO2024073723, or their antigen-binding moieties. Therefore, in some such embodiments, anti-CD132 antibodies include REGN7257, as well as the anti-CD132 antibodies disclosed in WO2020160242, WO2017021540, WO2022212848, WO2023078113, and US20230272089, or their antigen-binding portions. Each of these constitutes a means for binding a low-affinity IL-2 receptor (CD122 or CD132, as appropriate).

[0554] In some embodiments, the high-affinity IL-2 receptor targets a cell surface antigen (CD25), and the binding moiety comprises an antigen-binding domain of an anti-IL-2 receptor antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from daklizumab, balithimab, calimumab, vopitug, enomozumab, HuMAx-TAC, Xenopax, STI-003, RA8, RTX-003, and anti-CD25 antibodies disclosed in WO2023031403, WO2006108670, WO2019175223, WO2019175215, WO2019175226, WO2004045512, WO2022104009, and WO2020102591, or their antigen-binding moieties. Each of these constitutes a means for binding the high-affinity IL-2 receptor (CD25).

[0555] In some embodiments, the IL-7 receptor (CD127) is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-IL-7 receptor antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from anti-CD127 antibodies disclosed in PF-06342674, GSK2618960, OSE-127, lusvertikimab, beempikibart, and WO2011104687, WO2011094259, WO2013056984, WO2015189302, WO2017062748, WO2020154293, WO2020254827, WO2021222227, and WO2023201316, or their antigen-binding moieties. Each of these constitutes a means for binding CD127.

[0556] In some embodiments, the IL-12 receptor targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-IL-12 receptor antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from CBYY-I0413, REA333, or their antigen-binding moieties. Each of these constitutes a means for binding the IL-12 receptor.

[0557] In some embodiments, the IL-15 receptor α targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-IL-15 receptor α antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from MAB1472-100, MAB5511, JM7A4, 5E3E1, JM7A4, 2639B, or their antigen-binding moieties. Each of these constitutes a means for binding the IL-15 receptor α.

[0558] In some embodiments, the IL-18 receptor α targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-IL-18 receptor α antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from H44 or its antigen-binding moiety. Each of these constitutes a means for binding the IL-18 receptor α.

[0559] In some embodiments, the IL-21 receptor targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-IL-21 receptor antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from 1D1C2, 19F5, 18A5, REA233, or their antigen-binding moieties. Each of these constitutes a means for binding the IL-21 receptor α.

[0560] In some embodiments, LAG-3 is a cell surface antigen that targets the cell surface, and the binding portion contains an antigen-binding domain of an anti-LAG-3 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from relatlimab, tebotelimab, favezelimab, fianlimab, miptenalimab, HLX26, ieramilimab, GSK2831781, INCAGN2385, RO7247669, encelimab, FS118, SHR-1802, Sym022, IBI110, IBI323, bavunalimab, EMB-02, ABL501, INCA32459, AK129, or their antigen-binding portions. Each of these constitutes a means for binding LAG-3.

[0561] In some embodiments, MSCA-1 is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-MSCA-1 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from REAL219, W8B2, X9C3, or their antigen-binding moieties. Each of these constitutes a means for binding MSCA-1.

[0562] In some embodiments, OX40 is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-OX40 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from MEDI6469, ivuxolimab, rocatinlimab, GSK3174998, BMS-986178, vonlerizumab, INCAGN1949, tavolimab, BGB-A445, INBRX-106, BAT6026, telazorlimab, ATOR-1015, MEDI6383, cudarolimab, FS120, HFB301001, EMB-09, HLX51, Hu222, ABM193, or their antigen-binding moieties. Each of these constitutes a means for binding OX40.

[0563] In some embodiments, PD-1 targets a cell surface antigen and binds to an antigen-binding domain containing an anti-PD-1 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from nivolumab, pembrolizumab, camrelizumab, toripalimab, sintilimab, tislelizumab, cemiplimab, spartalizumab, serplulimab, cadonilimab, penpulimab, dostarlimab, zimberelimab, retifanlimab, and putolimab. Enlimab, Pidilizumab, Balstilimab, Ezabenlimab, AK112, Geptanolimab, Cetrelimab, Prolgolimab, Tebotelimab, Sasanlimab, SG001, Vudalimab, MEDI5752, Rulonilimab, Peresolimab, IBI318, Bulgarimab, MEDI0680, Pimivalimab, QL1706, AMG 404, RO7121661, lorigerlimab, nofazinlimab, sindelizumab, or their antigen-binding moieties. Each of these constitutes a means for binding PD-1.

[0564] In some embodiments, PODXL targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-PODXL antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from MAI1738, HPAB-3334LY, HPAB-MO612-YC, REA246, REA157, or their antigen-binding moieties. Each of these constitutes a means for binding PODXL.

[0565] In some embodiments, Sca-1 is a cell surface antigen that targets the cell surface, and the binding portion contains an antigen-binding domain of an anti-Sca-1 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from CPP32 4-1-18, 2D4-C9-F1, AMM22070N, or their antigen-binding portions. Each of these constitutes a means for binding Sca-1.

[0566] In some embodiments, SSEA-3 targets a cell surface antigen, and the binding portion comprises an antigen-binding domain of an anti-SSEA-3 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in MC631, 2A9, 8A7, ND-742, 3H420, and US11,643,456 or WO2021138378 (all contents of each of which teach about anti-SSEA-3 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding SSEA-3.

[0567] In some embodiments, SSEA-4 is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-SSEA-4 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in ch28 / 11, REA101, MC-813-70, ND-942-80, and US11,446,379, US10,273,295, US11,643,456, WO2019190952, or WO2021044039 (all contents of each of these teachings concerning anti-SSEA-4 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding SSEA-4.

[0568] In some embodiments, Stro-1 targets a cell surface antigen, and the binding moiety comprises an antigen-binding domain of an anti-Stro-1 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from those disclosed in STRO-1, TUSP-2, and US20130122022 (all the teachings of that document regarding anti-Stro-1 antibodies and their properties are incorporated herein by reference), or their antigen-binding moieties. Each of these constitutes a means for binding Stro-1.

[0569] In some embodiments, Stro-4 is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-Stro-4 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from STRO-4, ivengumab, 4C5, and those disclosed in US7,722,869, US20110280881, US9,115,192, US10,273,294, US10,457,726, WO2023091148 (all contents of each of these teachings concerning anti-Stro-4 antibodies and their properties are incorporated herein by reference), or their antigen-binding moieties. Each of these constitutes a means for binding Stro-4 (also known as heat shock protein-90).

[0570] In some embodiments, SUSD2 is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-SUSD2 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from REA795, CBXS-3571, CBXS-1650, CBXS-1989, CBXS-1671, CBXS1990, CBXS-3676, 1279B, EPR8913(2), W5C5, or their antigen-binding moieties. Each of these constitutes a means for binding SUSD2.

[0571] In some embodiments, TIM-3 is a cell surface antigen that targets the cell surface, and the binding moiety comprises an antigen-binding domain of an anti-TIM-3 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from TQB2618, sabatolimab, cobolimab, RO7121661, INCAGN02390, AZD7789, surzebiclimab, LY3321367, Sym023, BMS-986258, SHR-1702, LY3415244, LB1410, or their antigen-binding moieties. Each of these constitutes a means for binding TIM-3.

[0572] In some implementations, TREM2 targets cell surface antigens and the binding portion contains an antigen-binding domain of an anti-TREM2 antibody. Therefore, in some such embodiments, the antigen-binding domain is derived from PI37012, and those disclosed in US10,508,148, US10,676,525, WO2017058866, US11,186,636, US11,124,567, WO2020055975, US11,492,402, WO2020121195, WO2023012802, WO2021101823, WO2023047100, WO2022032293, WO2022241082, WO2023039450, or WO2023039612 (all the teachings of each of which are incorporated herein by reference regarding anti-TREM2 antibodies and their properties are included herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding TREM2.

[0573] In some embodiments, the G protein-coupled receptor class C group 5 member D (GPRC5D) targets cell surface antigens and binds to an antigen-binding domain containing an anti-GPRC5D antibody. Therefore, in some such embodiments, the anti-GPRC5D antigen-binding domain is derived from talquetamab, forimtamig, BMS-986393, IBI-3003, QLS32015, SIM0500, or EPR28376-41, or disclosed in WO2018017786, WO2016090329, WO2022174813, WO2023236889, WO2018147245, WO2024079015, WO2019154890, WO20210188. 59, WO2021018925, WO2020092854, WO2024031091, WO2020148677, WO2022175255, WO2022222910, WO2022247804, WO2022247756, WO2023078382, WO2023125728, WO2023143537, WO2024046239 or WO2024131962 (all contents of each of these teachings concerning anti-GPRC5D antibodies and their properties are incorporated herein by reference) or their antigen-binding portions. Each of these constitutes a means for binding GPRC5D.

[0574] In some embodiments, FCRL5 (CD307E) is a cell surface antigen that targets, and the binding portion comprises an antigen-binding domain of an anti-FCRL5 antibody. Therefore, in some such embodiments, the antigen-binding domain of the anti-FCRL5 antibody is derived from civastastatin, 2A10H7, 307307, 2A10D6, EPR27365-87, EPR26948-19, or EPR26948-67, or disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558 ​​(all contents of each of these teachings concerning anti-FCRL5 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding FCRL5.

[0575] In some embodiments, LRRC15 is a cell surface antigen that targets the cell surface, and the binding portion comprises an antigen-binding domain of an anti-LRRC15 antibody. Therefore, in some such embodiments, the antigen-binding domain of the anti-LRRC15 antibody is derived from samrotamab or DUNP19, or disclosed in WO2005037999, WO2021022304, WO2024081729, WO2021102332, WO2021202642, WO2022157094, or WO2024158047 (all teachings of each of which, concerning anti-LRRC15 antibodies and their properties, are incorporated herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding LRRC15.

[0576] In a further embodiment, tLNP targets tumor cells. In some embodiments, the tumor cells express one of the aforementioned antigens, and tLNP is targeted to the tumor expressing the antigen using the same methods described above. In other embodiments, tLNP targets some other tumor antigens, such as those listed in U.S. Provisional Application No. 63 / 371,742, filed August 17, 2022 (titled "CONDITIONING FOR IN VIVO IMMUNE CELL ENGINEERING"), all of which teaches and is not contradictory to this disclosure regarding the use of tLNP for the delivery of nucleic acids to tumor cells, and all such teachings are incorporated herein by reference.

[0577] Nucleic acid

[0578] In some embodiments, the disclosed LNP and tLNP include a payload that comprises or is composed of one or more nucleic acid substances. In some embodiments, the LNP or tLNP payload contains only one nucleic acid substance, while in other embodiments, the LNP or tLNP payload contains multiple nucleic acid substances, such as two, three, or four nucleic acid substances. For example, in embodiments in which the payload comprises a nucleic acid encoding a CAR or immune cell connective (ICE), the payload may comprise or consist of the following: 1) a single nucleic acid material encoding a single type of CAR or ICE; 2) a single nucleic acid material encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), such as bicistronic or polycistronic mRNA, wherein each CAR and / or ICE is specific to the same target antigen; 3) a single nucleic acid material encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), such as bicistronic or polycistronic mRNA, wherein at least one CAR and / or ICE is specific to a target antigen different from the other target antigens; 4) two or more nucleic acid materials encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), wherein each CAR and / or ICE is specific to the same target antigen; 5) two or more nucleic acid materials encoding two or more types of CAR or ICE (or a mixture of CAR and ICE), wherein at least one CAR and / or ICE is specific to a target antigen different from the other target antigens. When two or more CARs and / or ICEs are specific for the same target antigen, they can be specific for the same or different epitopes of the same target antigen. Further variations will be apparent to those skilled in the art (e.g., multiple bicistronic or polycistronic nucleic acids, nucleic acids encoding TCRs, etc.). The nucleic acid can be RNA or DNA. The nucleic acid can be polycistronic, such as bicistronic.

[0579] In some embodiments, the LNP or tLNP of this disclosure further comprises a nucleic acid payload. In various embodiments, the nucleic acid is mRNA, self-replicating RNA, circular RNA, siRNA, miRNA, DNA, gene editing components (e.g., guide RNA, tracr RNA, sgRNA), gene writing components, mRNA encoding gene or base editing proteins, zinc finger nucleases, TALENs, CRISPR nucleases (such as Cas9), DNA molecules to be inserted or used as repair templates, or combinations thereof. In some embodiments, the nucleic acid comprises small interfering RNA (siRNA), microRNA (miRNA), or antisense oligonucleotides (ASO). In some embodiments, the nucleic acid comprises self-replicating RNA or circular RNA. In some embodiments, the mRNA encodes a reprogramming agent or contains or encodes an opsonizing agent. In some embodiments, the mRNA (linear, circular, or self-replicating) contains a miRNA binding site. In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR). In other embodiments, the mRNA encodes a gene editing or base editing or gene writing protein. In some embodiments, the nucleic acid is guide RNA. In some implementations, the LNP or tLNP contains mRNA encoding a gene-editing, base-editing, or gene-writing protein, and one or more guide RNAs. CRISPR nucleases may have altered activities; for example, they may be modified to be nicking enzymes instead of producing double-stranded nicks, or they may bind to a sequence specified by the guide RNA but lack enzymatic activity. Base-editing proteins are typically fusion proteins containing a deaminase domain and a sequence-specific DNA-binding domain (such as an inactive CRISPR nuclease).

[0580] In some implementations, the reprogramming agent comprises an immune receptor (e.g., a chimeric antigen receptor or a T-cell receptor) or an immune cell connector (e.g., a bispecific T-cell connector (BiTE), a bispecific killer cell connector (BiKE), a trispecific killer cell connector (TriKE), a dual affinity retargeting antibody (DART), a TRIDENT (connecting two DART units or a DART unit and a Fab domain), a macrophage connector (e.g., BiME), an innate cell connector, etc.).

[0581] In some embodiments, the nucleic acid is RNA, such as mRNA, and the RNA contains at least one modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine, N... 1 -Methylpseudouridine, 5-methylcytosine, 5-methyluridine, N 6-Methyladenosine, 2'-O-methyluridine, or 2-thiouridine. In some embodiments, all uridines are substituted with modified nucleosides. Further disclosure of modified nucleosides and their uses can be found in U.S. Patent No. 8,278,036, the teachings of which are incorporated herein by reference.

[0582] In some embodiments, the reprogramming agent encodes a gene / genome editing component or a gene / genome editing component. In some embodiments, the gene / genome editing component is the guide RNA of an RNA-guided nuclease or other nuclease editing enzyme, a clustered regularly spaced short palindromic repeat RNA (crisprRNA), or a trans-activated clustered regularly spaced short palindromic repeat RNA (tracrRNA). In some embodiments, the gene / genome editing component is a nucleic acid-encoded enzyme, such as an RNA-guided nuclease, a gene or base editing protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a large-scale nuclease, a transposase, or a CRISPR nuclease (e.g., Cas9 or Cas12). In some embodiments, the gene / genome editing component is the DNA to be inserted or DNA that serves as a template in gene or genome editing (e.g., a template for repairing double-strand breaks).

[0583] In some embodiments comprising multiple reagents, the nucleic acid may be polycistronic. In other embodiments comprising multiple reagents or components, each reagent or component is encoded or contained as a separate nucleic acid substance. In some embodiments involving multiple payload nucleic acid substances, two or more nucleic acid substances are encapsulated together in a single LNP substance. In other embodiments, a subset of the payload nucleic acid substances to be delivered (e.g., a single nucleic acid substance) is encapsulated in one LNP or tLNP substance, while another subset of the nucleic acid substances is encapsulated in another LNP or tLNP substance. Different (t)LNP substances may differ only in the payload they contain. Different (t)LNP substances may be combined in a single formulation or pharmaceutical composition for administration.

[0584] Methods for preparing LNP or tLNP

[0585] In some aspects, this disclosure provides a method for preparing LNPs or tLNPs, the method comprising mixing an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of a lipid in proportions disclosed herein. In certain embodiments, the mixing is rapid.

[0586] The aqueous solution can be buffered to a pH of about 3 to about 5, for example, but not limited to, citrate or acetate. In various embodiments, the alcohol can be ethanol, isopropanol, tert-butanol, or a combination thereof. In some embodiments, rapid mixing is achieved by pumping the two solutions through a T-joint or using an impingement jet mixer. Microfluidic mixing via a staggered herringbone mixer (SHM) or hydrodynamic mixer (microfluidic hydrodynamic focusing), microfluidic bifurcation mixer, and microfluidic baffle mixer can also be used. After LNP formation, they are diluted with a buffer (e.g., phosphate, HEPES, or Tris) in a pH range of 6 to 8.5 to reduce the alcohol (ethanol) concentration. The diluted LNP is purified by tangential flow filtration (TFF) against a buffer (e.g., phosphate, HEPES, or Tris) in a pH range of 6 to 8.5 using dialysis, ultrafiltration, or percolation to remove the alcohol. Alternatively, size exclusion chromatography can be used. Once the alcohol has been completely removed, the buffer solution is replaced with a similar buffer containing a cryoprotectant (e.g., glycerol or a sugar such as sucrose, trehalose, or mannose). The LNP is concentrated to the desired concentration, then filtered through a 0.2 μm filter, such as a polyethersulfone (PES) or modified PES filter, and filled into glass vials, stoppered, capped, and frozen for storage. In an alternative embodiment, a lyophilization protectant is used, and the LNP is lyophilized for storage rather than as a cryo-liquid. Other methods for preparing LNPs can be found, for example, in U.S. Patent Application Publications US2020 / 0297634, US2013 / 0115274, and International Patent Application Publication WO2017 / 048770, all of which teach the production of LNPs and are incorporated herein by reference.

[0587] One aspect is a method for preparing tLNPs, which includes rapidly mixing an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of a lipid as disclosed for LNPs. In some embodiments, the lipid mixture includes a functionalized PEG-lipid for subsequent conjugation to a target moiety. As used herein, a functionalized PEG-lipid refers to a PEG-lipid whose PEG moiety has been derivatized with a chemically reactive group (such as maleimide, N-hydroxysuccinimide (NHS) ester, Cys, azides, alkynes, etc.) that can be used to conjugate the target moiety to the PEG-lipid, and thus to an LNP containing the PEG-lipid. In other embodiments, the functionalized PEG-lipid is inserted into the LNP after the initial formation of the LNP from other components. In either type of embodiment, the target moiety is conjugated to the functionalized PEG-lipid after the formation of the functionalized PEG-lipid containing the LNP. Conjugation schemes can be found, for example, Parhiz et al., 2018, J. Controlled Release 291:106-115 and Tombacz et al., 2021, Molecular Therapy 29(11):3293-3304, all of which teach the following about the conjugation of PEG-lipids with the binding moiety and are incorporated herein by reference. Alternatively, the targeting moiety may be conjugated with the PEG-lipid prior to the insertion of the pre-formed LNP.

[0588] In some embodiments of the tLNP preparation method, the method includes:

[0589] i) The initial LNP is formed by mixing all components of the tLNP (excluding one or more functionalized PEG-lipids and one or more targeted moieties) in proportions disclosed herein;

[0590] ii) Preconjugated tLNPs are formed by mixing initial LNPs with one or more functionalized PEG-lipids; and

[0591] iii) A tLNP is formed by concatenating a pre-concatenated tLNP with one or more target portions.

[0592] In some embodiments of the tLNP preparation method, the method includes:

[0593] i) Preconjugated tLNPs are formed by mixing all components of tLNP (including one or more functionalized PEG-lipids, in addition to one or more targeting moieties) in proportions disclosed herein; and

[0594] ii) Forming a tLNP by concatenating a pre-concatenated tLNP with one or more target portions.

[0595] In some embodiments of the tLNP preparation method, the method includes:

[0596] i) Forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids to one or more targeting moieties; and

[0597] ii) tLNP is formed by mixing all components of tLNP (including one or more conjugated functionalized PEG-lipids) in proportions disclosed herein.

[0598] In some embodiments of the tLNP preparation method, the method includes:

[0599] i) Forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids with one or more target moieties;

[0600] ii) To form LNP by mixing all components of tLNP (excluding one or more conjugated functionalized PEG-lipids); and

[0601] iii) tLNP is formed by mixing the initial LNP with one or more conjugated functionalized PEG-lipids.

[0602] After conjugation, as disclosed above for LNP, tLNP is purified and stored by dialysis, tangential flow filtration or size exclusion chromatography.

[0603] The encapsulation efficiency of LNP or tLNP for nucleic acids is typically determined by adding a nucleic acid-binding fluorescent dye to both intact and lysed aliquots of the final LNP or tLNP formulation to measure the amounts of unencapsulated nucleic acids and total nucleic acids, respectively. Encapsulation efficiency is usually expressed as a percentage and calculated as 100 × (TU) / T, where T is the total amount of nucleic acids and U is the amount of unencapsulated nucleic acids. In various embodiments, encapsulation efficiencies are ≥80%, ≥85%, ≥90%, or ≥95%.

[0604] Methods for delivering payloads into cells

[0605] In other respects, this document discloses methods for delivering nucleic acids (or other negatively charged payloads) into cells, comprising contacting the cells with the LNPs or tLNPs disclosed herein. Therefore, each of the genera, subgenera, and / or species of the LNPs or tLNPs disclosed herein (including those based on the inclusion or exclusion of specific lipids, specific lipid compositions, specific payloads, and / or specific target moieties) can be used to define the scope of methods for delivering payloads to cells. In some embodiments, contact occurs ex vivo. In some embodiments, contact occurs in vitro. In some embodiments, contact occurs in vivo. In some embodiments, LNPs or tLNPs are contacted with target cells in vivo via systemic or local administration. In some embodiments, in vivo contact includes intravenous, intramuscular, subcutaneous, intralesional, intranodal, or intralymphatic administration. In some embodiments, administration is via intravenous or subcutaneous infusion or injection. In some embodiments, administration is via intraperitoneal or intralesional infusion or injection. In a further embodiment, hepatocyte transfection is reduced compared to tLNPs containing conventional ionizable cationic lipids (such as ALC-0315). In some embodiments, LNP or tLNP is administered 1 to 3 times per week for 1, 2, 3, or 4 weeks. In some embodiments, as disclosed above, toxicity is limited to (or largely limited to) grade 0, 1, or 2.

[0606] Compared to widely used existing LNP compositions, such as those containing ALC-0315, the LNP and tLNP compositions and formulations disclosed herein exhibit reduced toxicity. In various embodiments, toxicity can be described as observable toxicity, substantial toxicity, serious toxicity, or acceptable toxicity or dose-limiting toxicity (such as, but not limited to, maximum tolerated dose (MTD)). Observable toxicity refers to an effect that is negligible or minor, although a change is observed. Substantial toxicity refers to a negative impact on the patient's overall health or quality of life. In some cases, substantial toxicity can be mitigated or resolved through other ongoing medical interventions. Serious toxicity refers to an effect that requires acute medical intervention and / or dose reduction or treatment interruption. The acceptability of toxicity will be affected by the specific disease being treated and its severity, as well as the availability of mitigating medical interventions. In some embodiments, toxicity is limited to (or largely limited to) observable toxicity. In some embodiments, toxicity is limited to (or largely limited to) grade 0, 1, or 2.

[0607] In some embodiments, the payload is a nucleic acid, and the delivery method is a transfection method. In some embodiments, the nucleic acid payload comprises mRNA, circular RNA, self-amplifying RNA, or guide RNA. Nucleic acid structures, particularly mRNA structures, and single RNA molecules encoding specific polypeptides well suited for delivery via LNP or tLNP are disclosed in U.S. Provisional Patent Application No. 63 / 595,753, filed November 2, 2023; U.S. Provisional Patent Application No. 63 / 611,092, filed December 15, 2023; and U.S. Provisional Patent Application No. 23-1871-US-PRO3, filed May 31, 2024, all contents of which teach about nucleic acid payloads for in vivo transfection and their design are incorporated herein by reference.

[0608] In some embodiments, the payload comprises nucleic acid encoding an immune receptor or immune cell connector, and the delivery method is also a method of reprogramming immune cells. In some embodiments, the payload comprises nucleic acid encoding a BRM or being a BRM, and the delivery method is also a method of providing an opsonizing agent. In various embodiments, the BRM or opsonizing agent is a γ-chain receptor cytokine, such as IL-2, IL-7, IL-15, IL-15 / 15Rα, IL-21; an immunomodulatory cytokine, such as IL-12, IL-18; a chemokine, such as RANTES, IP10, MIG; or another BRM, such as Flt3, GM-CSF, and G-CSF.

[0609] In some implementations, the payload comprises nucleic acid encoding a gene / genome editing enzyme and / or guide RNA or other components of the gene / genome editing system, and the delivery method is also a method of reprogramming cells. In some cases, the cells are immune cells. In some cases, the cells are HSCs. In some cases, the cells are MSCs.

[0610] In some embodiments, including delivery of a payload to immune cells, binding is performed on lymphocyte surface molecules or monocyte surface molecules. Lymphocyte surface molecules include CD2, CD3, CD4, CD5, CD7, CD8, CD28, 4-1BB (CD137), CD166, CTLA-4, OX40, PD-1, GITR, LAG-3, TIM-3, CD25, low-affinity IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, and IL-21 receptor. Monocyte surface molecules include CD5, CD14, CD16a, CD32, CD40, CD11b (Mac-1), CD64, DEC205, CD68, and TREM2. Exemplary antibodies that can provide antigen-binding domains to bind to these surface molecules are disclosed herein. As noted, such antibodies, whether individually or collectively, constitute a means of binding immune cells (or leukocytes) or lymphocytes or monocytes.

[0611] In some embodiments, including delivery of a payload to stem cells, binding is performed on HSC surface molecules or MSC surface molecules. HSC surface molecules include CD117, CD34, CD44, CD90 (Thy1), CD105, CD133, BMPR2, and Sca-1. MSC surface molecules include CD70, CD105, CD73, Stro-1, SSEA-4, CD271, CD146, GD2, SSEA-3, SUSD2, Stro-4, MSCA-1, CD56, CD200, PODXL, CD13, CD29, CD44, and CD10. Exemplary antibodies that can provide antigen-binding domains to bind these surface molecules are disclosed above. As noted, such antibodies, individually and collectively, constitute a means for binding stem cells or HSCs or MSCs.

[0612] Treatment

[0613] In some respects, this disclosure provides methods for treating diseases or conditions, including administering the disclosed tLNP to a subject in need. Each of the genera, subgenera, and / or species of the LNP or tLNP disclosed herein, including those based on the inclusion or exclusion of specific lipids, specific lipid compositions, specific payloads, and / or specific target moieties, can be used to define the scope of treatment methods.

[0614] In some embodiments, the subject is a human. In some embodiments, tLNP is administered systemically. In some embodiments, tLNP is administered via intravenous or subcutaneous infusion or injection. In some embodiments, tLNP is administered locally. In some embodiments, tLNP is administered via intraperitoneal or intralesional infusion or injection.

[0615] In further embodiments, tLNP can be administered in combination with standards of care for specific indications, such as corticosteroids (e.g., prednisone) used to treat myositis or lupus nephritis. In some cases, myositis is also treated with methotrexate, which can be combined with immunosuppressants (e.g., azathioprine, mycophenolate mofetil, tacrolimus), which are often required in addition to corticosteroids. For membranous nephropathy, cyclic steroids and cyclophosphamide can be used in combination with the tLNP of this disclosure. In other cases, anti-IL-6 (such as tocilizumab) can also be used as pretreatment or in combination with the tLNP of this disclosure. These combinations can be administered simultaneously or sequentially.

[0616] In some implementations, the disease or condition is an autoimmune disease. Examples of autoimmune diseases include, but are not limited to, myocarditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sidnam's chorea, myasthenia gravis, systemic lupus erythematosus, fibrotic alveolitis, multiple sclerosis, rheumatic fever, polyglandular syndrome, agranulocytosis, autoimmune hemolytic anemia, bullous pemphigoid, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia rheumatica, pernicious anemia, rapidly progressive glomerulonephritis, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, allergic reactions, insulin-resistant diabetes mellitus, psoriasis, diabetes mellitus, Addison's disease, and Graves' disease. Diabetes mellitus, endometriosis, celiac disease, Crohn's disease, allergic purpura, ulcerative colitis, Goodpassu syndrome, thromboangiitis obliterans, Sjögren's syndrome, aplastic anemia, rheumatoid arthritis, sarcoidosis, scleritis, T-cell-mediated or B-cell-mediated autoimmune diseases, B-cell-mediated (antibody-mediated) autoimmune diseases, necrotizing myopathy, chronic inflammatory demyelinating polyneuropathy (CIDP), neuromyelitis optica (NMO)-associated myositis, neuromyelitis optica spectrum disorders, pemphigus vulgaris, systemic sclerosis, antisynthetic enzyme syndrome (idiopathic inflammatory myopathy), lupus nephritis, membranous nephropathy, Fanconi anemia, and vasculitis.

[0617] In some implementations, autoimmune diseases are either T-cell-mediated or B-cell-mediated autoimmune diseases. In some cases, B-cell-mediated autoimmune diseases include myositis (such as antisynthetic enzyme antibody-associated myositis), lupus nephritis, membranous nephropathy, systemic lupus erythematosus, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, pemphigus vulgaris, rheumatoid arthritis, dermatomyositis, immune-mediated necrotizing myopathy (IMNM), antisynthetic enzyme syndrome, polymyositis, systemic sclerosis, and diffuse cutaneous myositis. Systemic sclerosis, localized cutaneous systemic sclerosis, anti-synthesizing enzyme syndrome (idiopathic inflammatory myopathy), stiff-person syndrome, myeloid oligodendrocyte glycoprotein autoantibody-associated disease (MOGAD), amyloid light chain amyloidosis, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, inactive secondary progressive multiple sclerosis, Sjögren's syndrome, IgA nephropathy, IgG4-related disease, or Fanconi anemia. In some embodiments, B-cell-mediated autoimmune diseases are myositis, lupus nephritis, membranous nephropathy, scleroderma, systemic lupus erythematosus, myasthenia gravis, ANCA-associated vasculitis, multiple sclerosis, or pemphigus vulgaris. In some embodiments, B-cell-mediated autoimmune diseases are myositis, lupus nephritis, membranous nephropathy, or scleroderma. In some embodiments, B-cell-mediated autoimmune diseases are myositis. In some cases, myositis is antisynthetic enzyme myositis. In certain implementations, B-cell-mediated autoimmune diseases are systemic lupus erythematosus, myasthenia gravis, ANCA-associated vasculitis, multiple sclerosis, or pemphigus vulgaris.

[0618] In some implementations, the disease or condition is rejection of allogeneic organ or tissue transplants. Pre-existing antibodies and / or B cells, in their role as antigen-presenting cells, can promote rapid immune rejection through known mechanisms; therefore, depleting large numbers of B cells can help prevent allogeneic graft rejection.

[0619] In some implementations, the disease or condition is cancer. Examples of cancer include, but are not limited to, carcinoma, sarcoma, and blood cancers. In some implementations, blood cancers are lymphoma, leukemia, or myeloma. In some cases, blood cancers are B-lineage or T-lineage cancers. In some cases, B-lineage cancers are multiple myeloma, diffuse large B-cell lymphoma, acute myeloid leukemia, mantle cell lymphoma, follicular lymphoma, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, or myelodysplastic syndrome. In some implementations, the cancer is a sarcoma. In some implementations, the cancer is carcinoma, such as breast cancer, colon cancer, ovarian cancer, lung cancer, testicular cancer, or pancreatic cancer. In some implementations, the cancer is melanoma.

[0620] In some implementations, the disease or condition is a genetic disease or condition, such as a single-gene inherited disease. In other cases, the genetic disease or condition is a hemoglobinopathic disorder, such as sickle cell disease or beta-thalassemia.

[0621] In some implementations, the disease or condition is a fibrotic disease or condition. In some cases, the fibrotic disease is cardiac fibrosis, arthritis, idiopathic pulmonary fibrosis, and non-alcoholic steatohepatitis (also known as metabolic dysfunction-related steatohepatitis). In other cases, the condition involves tumor-associated fibroblasts.

[0622] In some embodiments, the tLNP of this disclosure comprises a nucleic acid encoding a chimeric antigen receptor (CAR). Receptors are chimeric because they combine antigen binding and T cell activation functions into a single receptor. In some embodiments, the nucleic acid encoding the CAR refers to one or more nucleic acid substances encoding one or more CARs; for example, a single or multiple nucleic acid substances encoding a single CAR species, or multiple nucleic acid substances encoding multiple CAR species. In some cases, these multiple CAR species have the same specificity, while in others they have multiple specificities. In some embodiments, the CAR of this disclosure is multispecific, such as bispecific, comprising multiple antigen-binding moieties, each antigen-binding moiety specific to a single antigen. For example, the CAR in LCAR-AIO targets three antigens—CD19, CD20, and CD22 (see Blood (2021) 138 (Supplement 1): 1700). In some embodiments, the CAR may comprise an extracellular binding domain, a transmembrane domain, and one or more intracellular signal transduction domains that specifically bind to the target antigen. In some embodiments, the CAR may further include one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, or one or more intracellular co-stimulatory domains. Domains may be directly adjacent to each other, or there may be one or more amino acids connecting the domains. The signal peptide may be derived from an antibody, TCR, CD8, or other type 1 membrane protein, preferably a protein expressed in T or other immune cells. The transmembrane domain may be associated with any potential intracellular domain or a domain derived from another type 1 membrane protein, such as TCRα, β or ζ chain, CD3ε, CD4, CD8, or CD28, and other possibilities known in the art. The transmembrane domain may also include a hinge domain located between the extracellular binding domain and a hydrophobic transmembrane region of the transmembrane domain. In some, but not all, embodiments, the hinge domain and the transmembrane domain are consecutive sequences in a protein of the same origin. In some cases, the hinge and transmembrane domain are derived from CD28. In other cases, the hinge and transmembrane domain are derived from CD8α. Intracellular signal transduction domains may be derived from the CD3ζ chain, DAP10, DAP12, FcγRIII, FcsRI, or an immune receptor tyrosine-based activation motif (ITAM) carrying a cytoplasmic domain, as well as other possibilities known in the art. Intracellular co-stimulatory domains may be derived from CD27, CD28, 4-1BB, OX40, or ICOS, as well as other possibilities known in the art.

[0623] In some embodiments, the CAR is used to treat diseases or conditions associated with target cells expressing an antigen targeted by the CAR. For example, in some embodiments, anti-CD19 or anti-CD20 CARs can be used to target and treat B-cell malignancies or B-cell-mediated autoimmune conditions or diseases (e.g., having an immune cell-targeting portion, such as an anti-CD8 antibody). In other embodiments, anti-FAP CARs can be used to target and treat solid tumors or fibrosis (e.g., cardiac fibrosis, cancer-associated fibroblasts), which may also have an immune cell-targeting portion, such as an anti-CD8 antibody.Examples of CARs that may be used according to the embodiments described herein include US 7,446,190, US 9,328,156, US 11,248,058, US20190321404, WO2019119822, WO2019159193, WO2020011706, WO2022125837 and WO2024086190 (anti-CD19), US10,287,35 (anti-CD19), US 10,442,867 and US2021 / 0363245 (anti-CD19 and anti-CD20), US 10,543,263 (anti-CD22), WO2016149578 (anti-CD19 and anti-CD22), US 10,316,101, US 11,623,961, WO2015052538, WO2016166630, WO2017130223, WO2017173256, WO2019085102, WO2019241426, WO2020065330, WO2020038146, WO2020190737, WO2021091945 (anti-BCMA), WO2016130598 (anti-BCMA and multiligand glycan-1), US 10,426,797 (anti-CD33), US 10,844,128 (anti-CD123), US 10,428,141, US 10,752,684, US The CARs disclosed in WO2016187216, WO2017156479, WO2018197675, WO2020014366 and WO2020198531 (anti-ROR1), WO2022247756, WO2020148677, WO2020092854 and US20230331872 (anti-GPRC5D), WO2016090337, WO2022263855 and WO2024047558 ​​(anti-FCRL5) and US2021 / 0087295 (anti-FAP), each of which is incorporated by reference because it generally teaches all that is concerned with the structure and function of CARs, and all that is concerned with the antigen specificity and target indications of CARs is inconsistent with this disclosure. Each CAR constitutes a means of targeting a specific antigen with immune cells (such as T cells).

[0624] Exemplary target antigens that may be specific to CAR, TCR, or ICE include, but are not limited to, B cell maturation agents (BCMA). †‡ CA9 †‡ CD4 †‡ CD5 †‡ CD19*†‡ CD20 (MS4A1)* †‡ CD22* †‡ FCRL5 †‡ GPRC5D †‡ CD23* †‡ CD30 (TNFRSF8)* †‡ CD33* †‡ CD38* †‡ CD44* ‡ CD70* †‡ CD133 ‡ CD174, CD274 (PD-L1)* †‡ CD276 (B7-H3) †‡ CEACAM5* †‡ CLL1 ‡ CSPG4* ‡ EGFR* †‡ EGFRvIII*, EPCAM* †‡ EPHA2* ‡ ERBB2* ‡ FAP* †‡ FOLH1, FORR1* †‡ GD2* †‡ GPC3* †‡ ,GPNMB* ‡ IL1RAP †‡ IL3RA* ‡ IL13RA2* ‡ Kappa*, KDR (VEGFR2)* ‡ CD171 (L1CAM)* ‡ Lambda*, MET* ‡ MSLN (mesothelin)* †‡ MUC1* †‡ NCAM1 (CD56)* ‡ PD-1 (CD279) †‡ PSCA ‡ ROR1 †‡ CD138 (SDC1)* ‡ CD319 (SLAMF7)* †‡ CD248 (TEM1) ‡ ULBP1, ULBP2, and G protein-coupled receptor family C5 member D (GPRC5D) †‡ (Related to leukemia); CD319 (SLAMF7)* †‡ CD38*†‡ CD138 †‡ GPRC5D †‡ CD267 (TACI) ‡ and BCMA †‡ (Associated with multiple myeloma); and GD2* †‡ GPC3* †‡ HER2* †‡ EGFR* †‡ , EGFRvIII*, CD276 (B7H3) †‡ PSMA* †‡ PSCA ‡ CAIX (CA9) †‡ CD171 (L1-CAM)* ‡ CEA* ‡ CSPG4* ‡ EPHA2* ‡ FAP* †‡ LRRC15 †‡ ,FOLR1* †‡ IL-13Rα* †‡ mesothelin* †‡ MUC1* †‡ MUC16* †‡ TROP2* †‡ Tight junction protein 18.2 †‡ and ROR1 †‡ (Related to solid tumors). (* indicates an exemplary antibody with the specificity shown, from which the binding moiety can be derived, which can be found in Table 9 or Table 10 of U.S. Patent No. 11,326,182B2.) † Exemplary antibodies with the indicated specificity are shown, from which the binding moiety can be derived; these can be found in Wilkinson & Hale, 2022. Both references cited above are incorporated herein by reference. ‡Exemplary antibodies having the specificity shown are described, from which the binding moiety can be derived and are available in the Therapeutic Antibody Database (TABS) at tabs.craic.com. Other suitable antibodies can be found in Appendix A, all of which contains teachings on individual antibodies and the antigens they bind, incorporated herein by reference. Many of these target antigens are themselves receptors and, if expressed on immune cells, can bind to their ligands. Thus, in some embodiments, the extracellular binding domain of the CAR contains a ligand of the receptor expressed on the target cell. In a further embodiment, the extracellular binding domain of the CAR contains a ligand-binding domain of the receptor against the ligand expressed on the target cell. The advantages of the aspects and embodiments disclosed herein are independent of the specificity of the binding moiety. Therefore, the binding specificity is generally unknown in the disclosed aspects and embodiments. In some embodiments, a specific binding specificity may be required.

[0625] In some embodiments, the tLNP contains a nucleic acid encoding an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the nucleic acid contains mRNA. Examples of anti-CD19 CARs include those containing a CD19-binding moiety derived from human antibody 47G4 or mouse antibody FMC63. FMC63 and its derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997) and PCT applications WO 2018 / 213337 and WO 2015 / 187528, the entire contents of each of these documents (all teachings concerning anti-CD19 CARs and their uses) are incorporated herein by reference. A 47G4-based CAR is disclosed in U.S. Patent No. 10,287,350, all teachings of which concerning anti-CD19 CARs and their uses are incorporated herein by reference. In some cases, the anti-CD19 CAR is a CAR found in tesalonyl, lisocabtagene maraleucel, axicabtagene ciloleucel, or brexucabtagene autoleucel. Each of these CARs constitutes a means for targeting CD19 with immune cells, such as T cells. The entire contents of each of the foregoing references in this paragraph (all teachings concerning the design, structure, and activity of anti-CD19 CARs) are incorporated herein by reference. In any of the above-described tLNP embodiments, certain embodiments include tLNPs encapsulating an RNA-encoded CD19 CAR payload and having a T-cell targeting portion, such as an anti-CD8 antibody.

[0626] In some implementations, the tLNP contains a nucleic acid encoding an anti-CD20 chimeric antigen receptor (CAR). CD20 is an antigen found on the surface of B cells as early as the pre-B phase, and its levels gradually increase until the B cells mature; it is also an antigen found on cells in most B-cell tumors. CD20-positive cells are sometimes also found in cases of Hodgkin's disease, myeloma, and thymoma. In some implementations, the nucleic acid contains mRNA. Examples of anti-CD20 CARs include those containing a CD20-binding moiety derived from an antibody specific for CD20, including, for example, Leu16, IF5, 1.5.3, rituximab, ostuzumab, teimozumab, oflamumab, tositumumab, vetouzumab, utuzumab, and ozretrimumab. In some embodiments, the anti-CD20 CAR is derived from a CD20-specific CAR, including, for example, MB-106 (Fred Hutchinson Cancer Research Center, see Shadman et al., Blood 134(Supplement 1):3235 (2019)), UCART20 (Cellectis, www.cellbiomedgroup.com), or C-CAR066 (Cellular Biomedicine Group, see Liang et al., J. Clin. Oncol. 39(15) Supplement:2508 (2021)). In some embodiments, the extracellular binding domain of the anti-CD20 CAR comprises an scFv derived from a Leu16 monoclonal antibody, which includes a heavy chain variable region (V) of Leu16 linked via a linker. H ) and light chain variable region (V L See Wu et al., Protein Engineering. 14(12):1025-1033 (2001). Each of these CARs constitutes a means for targeting CD20 with immune cells, such as T cells. The entire contents of each of the foregoing references in this paragraph (all teachings on the design, structure, and activity of anti-CD20 CARs) are incorporated herein by reference. In any of the above-described tLNP embodiments, some embodiments include tLNPs encapsulating a CD20 CAR payload encoded by RNA and having a T-cell targeting portion, such as an anti-CD8 antibody.

[0627] In some embodiments, the tLNP contains a nucleic acid encoding an anti-BCMA chimeric antigen receptor (CAR). BCMA is a member of the tumor necrosis family receptors (TNFR) expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. BCMA expression has recently been associated with many cancers, such as multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the nucleic acid contains mRNA. Examples of anti-BCMA CARs include those containing a BCMA-binding moiety derived from C11D5.3, a mouse monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO 2010 / 104949. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another mouse monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT application publication WO2010104949. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from a mouse monoclonal antibody with high specificity for human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018). See also PCT application publication WO2012163805. In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable segments (VHHs) of both heavy chains that can bind to two epitopes of BCMA, as described in Zhao et al., J. Hematol. Oncol. 11(1):141 (2018), also known as LCAR-B38M. See also PCT Application Publication WO 2018 / 028647. In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy chain variable domain (FHVH), as described in Lam et al., Nat. Commun. 11(1):283 (2020), also known as FHVH33. See also PCT Application Publication WO 2019 / 006072. In some embodiments, the extracellular binding domain of the BCMA CAR comprises scFv derived from CT103A (or CAR0085), as described in U.S. Patent No. 11,026,975 B2. Other anti-BCMA CARs are disclosed in U.S. Patent Application Publication Nos. 2020 / 0246381 and 2020 / 0339699.Other anti-BCMA CARs include Allo-605 (described in U.S. Patent Publication No. 20200261503), CT053 (described in U.S. Patent No. 11,525,006), Descartes-08 (described in U.S. Patent No. 10,934,337), LCAR-B38M (described in U.S. Patent No. 10,934,363), PersonGen anti-BCMA CAR (described in CN114763383), Pregene Bio anti-BCMA CAR (described in U.S. Patent Publication No. 20220218746), the CAR in ciltacabtagene autoleucel (described in the binding portion of U.S. Patent No. 20170051068), and the CAR in idecabtagene vicleucel (described in U.S. Patent No. 10,383,929). Each of these CARs constitutes a means for targeting BCMA with immune cells, such as T cells. Other antibodies containing an anti-BCMA antigen-binding domain that can be used to construct CARs include AMG224 (described in U.S. Patent No. 9,243,058, along with other anti-BCMA antibodies), EMB-06 (described in U.S. Patent Publication No. US20230002489, along with other anti-BCMA antibodies), HPN217 (described in U.S. Patent No. 11,136,403), MEDI2228 (described in U.S. Patent No. 10,988,546), REGN5459 (described in U.S. Patent No. 11,384,153), SAR445514 (described in U.S. Patent Publication No. 20240034816), SEA-BCMA (described in U.S. Patent No. 11,078,291), and TNB-383B (described in U.S. Patent No. 11,078,291). The following are described in U.S. Patent Publication No. 11,505,606: TQB2934 (described in U.S. Patent Publication No. 20230193292), WV078 (described in U.S. Patent Publication No. 11,492,409), anucatumab (described in U.S. Patent Publication No. 10,683,369), belantumab (described in U.S. Patent Publication No. 9,273,141), enatumab (described in U.S. Patent Publication No. 11,814,435), ispectamab (described in U.S. Patent Publication No. 20210130483), rivosaitumab (described in U.S. Patent Publication No. 11,919,965), pavurutumab (described in U.S. Patent Publication No. 11,419,933), and teritumab (described in U.S. Patent Publication No. 10,072,088). Bispecific anti-BCMA and anti-CD19 CARs are described in WO2022007650.The entire contents of each of the foregoing references in this paragraph (all teachings on the design, structure, and activity of anti-BCMA CARs and anti-BCMA antibodies that may provide an antigen-binding domain for a CAR or immune cell connector) are incorporated herein by reference. In any of the above-described tLNP embodiments, some embodiments include tLNPs encapsulating an RNA-encoded BCMA CAR payload and having a T-cell targeting portion (such as an anti-CD8 antibody).

[0628] In some implementations, the tLNP contains a nucleic acid encoding an anti-GPRC5D chimeric antigen receptor (CAR). GPRC5D is a G protein-coupled receptor with no known ligand, and its function in human tissues is unclear. However, this receptor is expressed in myeloma cell lines and bone marrow plasma cells from patients with multiple myeloma. GPRC5D has been identified as an immunotherapeutic target in multiple myeloma and Hodgkin's lymphoma. Examples of anti-GPRC5D CARs include CARs containing a GPRC5D binding moiety, such as MCARH109 (Mailankody et al., N Engl J Med. 387(13): 1196-1206 (2022)), BMS-986393, or OriCAR-017 (Rodriguez-Otero et al., Blood Cancer J. 14(1): 24 (2024)). Examples of anti-GPRC5D CARs include CARs containing a GPRC5D binding moiety derived from an antibody specific for GPRC5D, such as taquituzumab (Pillarisetti et al., Blood 135:1232-43 (2020)) or voritumumab. In some embodiments, the extracellular binding domain of the anti-GPRC5D CAR comprises an scFv derived from a 6D9 mouse antibody specific for human GPRC5D (see creative-biolabs.com / car-t / anti-gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm). In some implementations, the extracellular binding domain of the GPRC5D CAR comprises an scFv of an anti-GPRC5D antibody linked to a 4-1BB or CD28 co-stimulatory domain and a CD3ζ signaling domain, as described in Mailankody et al., N Engl J Med. 387(13): 1196-1206 (2022); creative-biolabs.com / car-t / anti-gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm; and Rodriguez-Otero et al., Blood Cancer J. 14(1): 24 (2024). The entire contents of each of the foregoing references in this paragraph (all contents teaching the design, structure, and activity of anti-GPRC5D CARs and anti-GPRC5D antibodies that may provide an antigen-binding domain for a CAR or immune cell adapter) are incorporated herein by reference, and each example constitutes a means for binding GPRC5D.In any of the above-described tLNP implementations, some implementations include tLNPs encapsulating an RNA-encoded anti-GPRC5DCAR payload and having a T-cell targeting portion (such as an anti-CD8 antibody).

[0629] In some implementations, the tLNP contains a nucleic acid encoding an anti-FCRL5 chimeric antigen receptor (CAR). FCRL5 (Fc receptor-like 5), also known as FCRH5, BXMAS1, CD307, CD307E, and IRTA2, is a protein marker expressed on the surface of plasma cells in patients with multiple myeloma. Furthermore, contact with FCRL5 stimulates B cell proliferation; therefore, FCRL5 has been identified as an immunotherapeutic target for this disease. Examples of anti-FCRL5 CARs include CARs containing an FCRL5 binding moiety, such as those described in WO2016090337, WO2017096120, WO2022263855, and WO2024047558. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises an scFv specific to FCRL5, such as ET200-31, ET200-39, ET200-69, ET200-104, ET200-105, ET200-109, or ET200-117. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises an scFv derived from a mouse antibody specific to human FCRL5. Such antibodies include 7D11, F25, F56, and F119, as cited in Polson et al., Int. Immunol., 18(9): 1363-1373 (2006); Franco et al., J. Immunol.190(11): 5739-5746 (2013); Ise et al., Clin. CancerRes.11(1): 87-96 (2005); and Ise et al., Clin. Chem. Lab. Med.44(5): 594-602 (2006), all of which are incorporated herein by reference. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR includes a binding moiety derived from an antigen-binding domain of an anti-FCRL5 antibody or nanobody, including civastimab, 2A10H7, 307307, 2A10D6, 13G9, 10A8, 509f6, EPR27365-87, EPR26948-19, or EPR26948-67, or as disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR includes a binding moiety derived from an antibody-drug conjugate targeting FCRL5, such as the binding moiety described in Elkins et al., Mol. Cancer Ther. 11(10): 2222-2232 (2012).In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR is coupled with a co-stimulatory domain (such as a 4-1BB or CD28 co-stimulatory domain) and a signal transduction domain (such as a CD3ζ signal transduction domain). The entire contents of each of the foregoing references in this paragraph (all teachings on the design, structure, properties, and activity of anti-FCRL5 CARs and anti-FCRL5 antibodies that may provide an antigen-binding domain for a CAR or immune cell connector) are incorporated herein by reference. Each example constitutes a means for binding FCRL5. In any of the above-described tLNP embodiments, some embodiments include tLNPs encapsulating an RNA-encoded FCRL5 CAR payload and having a T-cell targeting portion (such as an anti-CD8 antibody).

[0630] In some embodiments, the tLNP contains a nucleic acid encoding one or more CARs targeting multiple antigens. In some embodiments, the tLNP contains different mRNAs encapsulated together in a single tLNP, wherein each mRNA encodes a single-specific CAR. For example, the tLNP may contain mRNA encoding anti-CD19 CAR and mRNA encoding anti-CD20 CAR, mRNA encoding anti-CD19 CAR and mRNA encoding anti-BCMA CAR, mRNA encoding anti-GPRC5D CAR and mRNA encoding anti-BCMA CAR, or mRNA encoding anti-FCRL5 CAR and mRNA encoding anti-BCMA CAR. In some embodiments, the tLNP contains a single mRNA encoding a bicistronic mRNA that encodes two single-specific CARs. For example, the bicistronic mRNA may encode anti-CD19 CAR and anti-CD20 CAR, anti-CD19 CAR and anti-BCMA CAR, anti-GPRC5D CAR and anti-BCMA CAR, or anti-FCRL5 CAR and anti-BCMA CAR. In some embodiments, the tLNP contains a single mRNA encoding a multispecific CAR. In some embodiments, the tLNP contains a single mRNA encoding a bispecific CAR. For example, the mRNA may encode a bispecific CAR against CD19 and CD20, a bispecific CAR against CD19 and BCMA, a bispecific CAR against GPRC5D and BCMA, or a bispecific CAR against FCRL5 and BCMA. In some embodiments, multiple tLNPs may be co-formulated with each tLNP containing one mRNA. In some cases, one mRNA encodes one monospecific CAR. For example, two tLNPs can be co-formulated with one tLNP containing mRNA encoding anti-CD19 CAR and another containing mRNA encoding anti-CD20 CAR, one tLNP containing mRNA encoding anti-C19 CAR and another containing mRNA encoding anti-BCMA CAR, one tLNP containing mRNA encoding anti-GPRC5D CAR and another containing mRNA encoding anti-BCMA CAR, or one tLNP containing mRNA encoding anti-FCRL5 CAR and another containing mRNA encoding anti-BCMA CAR. In some embodiments, multiple tLNPs can be combined and administered simultaneously or sequentially, wherein each tLNP contains one mRNA. In some cases, one mRNA encodes a single-specific CAR.For example, two tLNPs can be administered simultaneously or sequentially in combination, wherein one tLNP contains mRNA encoding anti-CD19 CAR and another tLNP contains mRNA encoding anti-CD20 CAR, one tLNP contains mRNA encoding anti-C19 CAR and another tLNP contains mRNA encoding anti-BCMA CAR, one tLNP contains mRNA encoding anti-GPRC5D CAR and another tLNP contains mRNA encoding anti-BCMA CAR, or one tLNP contains mRNA encoding anti-FCRL5 CAR and another tLNP contains mRNA encoding anti-BCMA CAR. Targeting can be mediated by any of the CARs described herein. In addition to combinations of two specific CARs, higher-order combinations are also possible, particularly using bispecific and trispecific CARs. Following these patterns, further implementations consist of other tLNPs or combinations of tLNPs with necessary modifications, containing nucleic acids encoding one or more CARs that target a variety of antigens specific to these and other CARs disclosed herein.

[0631] Cell therapies involving the administration of genetically engineered cells to patients often require depleting or ablation conditioning to facilitate the implantation of engineered cells (e.g., T cells or HSCs). In the context of in vivo engineering and reprogramming, such conditioning can be counterproductive, as it eliminates the cells to be engineered. Instead, activation and / or adjuvant conditioning can be used to increase the number of cells suitable for engineering, mobilize them to pathological sites, make pathological sites (e.g., the tumor microenvironment) more treatable, enhance therapeutic efficacy, etc., as appropriate for a specific disease and primary treatment. Conditioners include biological response modifiers (BRMs) that can be delivered directly to the subject or encoded in nucleic acid molecules, including mRNA, and delivered to the subject using the LNP and tLNP compositions and formulations disclosed herein.

[0632] Therefore, some aspects are methods for conditioning a subject receiving an engineered agent, which include providing the subject with a tLNP containing a nucleic acid molecule encoding the conditioning agent before, during, or after administration of the engineered agent. In various embodiments, the encoded conditioning agent comprises a γ-chain receptor agonist, an inflammatory chemokine, a pan-activating cytokine, an antigen-presenting cell activity enhancer, an immune checkpoint inhibitor, or an anti-CCR4 antibody. In some embodiments, the γ-chain receptor cytokine comprises IL-15, IL-2, IL-7, or IL-21. In some embodiments, the immune checkpoint inhibitor comprises an anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-Tim-3, or anti-LAG-3 antibody. In some embodiments, the inflammatory chemokine comprises CCL2, CCL3, CCL4, CCL5, CCL11, CXCL1, CXCL2, CXCL-8, CXCL9, CXCL10, or CXCL11. In some embodiments, the antigen-presenting cell activity enhancer comprises Flt-3 ligand, gm-CSF, or IL-18. In some embodiments, the pan-activating cytokine comprises IL-12 of IL-18. In some embodiments, the opsonizer comprises transcription factors, such as those selected from the group consisting of: activating T cell nuclear factor (NFAT), NF-κB, T-bet, signal transducer and activator of transcription 4 (STAT4), Blimp-1, c-Jun, and ameserioles, and the tLNP targets T cells. In some embodiments, the tLNP encapsulating the nucleic acid-encoded opsonizer is administered systemically, e.g., via intravenous or subcutaneous infusion or injection. In other embodiments, the tLNP is administered locally, e.g., via intralesional or intraperitoneal injection or infusion. In some embodiments, the nucleic acid molecules encoding the opsonizer and the engineered agent are encapsulated in the same tLNP, while in other embodiments, they are encapsulated in separate tLNPs. These two modes of opsonide delivery are described in more detail in PCT application PCT / US 2023 / 072426, all of which teaches, without contradiction with this disclosure, all matters relating to the opsonide and the delivery of its LNP or tLNP are incorporated herein by reference. In some embodiments, the nucleic acid comprises mRNA.

[0633] The term "treating" or "treatment" broadly encompasses any kind of therapeutic activity, including any activity that alleviates, cures, or prevents a disease or aspect thereof in a person or other animal, or otherwise affects the structure or any function of the body of a person or other animal. Therapeutic activities include administering the drugs, dosage forms, and drug compositions described herein to a patient, particularly the various treatment methods disclosed herein, whether performed by a healthcare professional, the patient himself / herself, or any other person. Therapeutic activities include orders, instructions, and recommendations from healthcare professionals (such as physicians, physician assistants, nurse practitioners, etc.) and then actions taken against them by any other person, including other healthcare professionals or the patient himself / herself. In some embodiments, the order, instruction, and recommendation aspect of therapeutic activities may also include encouraging, inducing, or compelling the selection of a particular drug or combination thereof for the treatment of a condition—and actually using that drug—through insurance coverage for approved drugs, denial of coverage for alternative drugs, including drugs on a drug formulary, exclusion of alternative drugs from a drug formulary, or provision of financial incentives for the use of the drug (as may be done by an insurance company or pharmacy benefit management company). In some implementation schemes, treatment activities may also include encouraging, inducing, or compelling the selection of a specific medication for the treatment of a condition—and actually using that medication—through policies or standards of practice that may be established by hospitals, clinics, health maintenance organizations, medical practices, or physician groups. All such orders, instructions, and recommendations should be considered as conditional upon receiving the benefits of treatment. In some cases, patients also receive financial benefits from adhering to such orders, instructions, and recommendations. In some cases, healthcare professionals also receive financial benefits from adhering to such orders, instructions, and recommendations.

[0634] Some implementations of these treatments involve administering an effective amount of the compound or composition disclosed herein. Some cases involve a therapeutic (or preventative) effective amount. A therapeutically effective amount is not necessarily a clinically effective amount; that is, while there may be a therapeutic benefit compared to no treatment, the treatment may not be equivalent to or superior to standard treatment available at a given point in time. Other cases involve a pharmacologically effective amount, which is the amount or dose that produces an effect relevant to or reasonably predictable in relation to therapeutic (or preventative) efficacy. As used herein, the term “therapeuticly effective amount” is synonymous with “therapeuticly effective dose” and means the minimum dose of the compound or composition disclosed herein required to achieve the desired therapeutic or preventative effect. Similarly, a pharmacologically effective dose means the minimum dose of the compound or composition disclosed herein required to achieve the desired pharmacological effect. Some implementations refer to an amount sufficient to prevent or disrupt the disease process or reduce the degree or duration of pathology. Some implementations refer to a dose sufficient to alleviate symptoms associated with the disease or condition being treated.

[0635] The following examples are intended to illustrate various embodiments. Therefore, the specific embodiments discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that such equivalent embodiments are included herein. Furthermore, all references cited in this disclosure are incorporated herein by reference in their entirety as if fully set forth herein.

[0636] Example

[0637] Example 1: Synthesis of 2-(2-(tert-butoxy)-2-oxoethyl)propane-1,3-dimethyldinonanoate (1)

[0638]

[0639] Under nitrogen atmosphere, nonanoic acid (76.86 g, 0.486 mol) was added to a solution of tert-butyl 4-hydroxy-3-(hydroxymethyl)butyrate (Org. Proc. Res. Dev. 2011, 15, 515; 44.0 g, 0.231 mol) in acetonitrile (900 mL), which was cooled in an ice-water bath. Then, DMAP (28.22 g, 0.231 mol) and EDC-HCl (97.8 g, 0.513 mol) were added. The mixture was stirred for 1 hour, then warmed to room temperature and stirred for 12 hours. The solution was poured into n-heptane (1.40 L) and water (0.9 L), and the organic phase was separated. The organic phase was washed twice with a MeOH:10% citric acid aqueous solution (0.90 L), and then twice with a mixture of MeOH:H₂O:triethylamine (0.90 L, 3:1:0.1). The organic phase was then washed with 10% NaCl aqueous solution, dried over Na2SO4, filtered, and concentrated under vacuum to obtain 2-(2-(tert-butoxy)-2-oxoethyl)propane-1,3-dimethyldinonanoate 1 (90.20 g, HPLC purity 96.3%, 0.185 mol, 80% yield), which was a pale yellow viscous liquid.

[0640] 1 H-NMR (300MHz, CDCl3): δ = 4.12 (m, 4H), 2.53 (m, 1H), 2.29-2.34(6H), 1.52-1.64 (4H), 1.46 (s, 9H), 1.16-1.37 (24H), 0.89 (t, J = 7.0Hz, 6H); LCMS: RT = 1.748 for C 27 H 50 O6 minus tert-butyl + H + Calculated value: 415.31. Measured value: 415.20.

[0641] Example 2: Synthesis of 4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butyric acid (2)

[0642]

[0643] Over a 30-minute period, TFA (208.46 g, 1.83 mol, 140 mL) was added to a 0.41 L solution of toluene (90.0 g, 96.3% purity, 0.184 mol) cooled in an ice-water bath under nitrogen. After the addition was complete, the mixture was warmed to 15 °C and stirred for 18 hours. The cooled solution was poured into n-heptane (1.50 L), and the resulting solution was extracted with 5% potassium phosphate aqueous solution (2.0 L), and the aqueous phase was collected. The organic phase was extracted with MeOH:H₂O:triethylamine (2.0 L, 5:1:0.1), and the combined aqueous phases were poured into n-heptane (1.80 L) and 1.2 M HCl aqueous solution (1.0 L). The organic layer was separated, washed with MeOH:water (1.0L, 1:1), dried with Na2SO4, filtered and concentrated under vacuum to obtain acid 2 (69.0g, HPLC purity 96.1%, 0.177mol, 96% yield), which was a pale yellow viscous oily substance.

[0644] 1 H-NMR (300MHz, CDCl3): δ = 4.13 (m, 4H), 2.58 (m, 1H), 2.48 (m, 2H), 2.32 (m, 4H), 1.63 (m, 4H), 1.20-1.37 (24H), 0.89 (t, J = 7.0Hz, 6H); LCMS:RT = 1.723 for C 23 H 42 O6+H + The calculated value is 415.31. The measured value is 415.30.

[0645] Example 3: Synthesis of rel((2S,4R)-1-benzylazine-2,4-diyl)diethanol (3)

[0646]

[0647] Over a 15-minute period, LiAlH4 (2.74 g, 74.08 mmol) was added in portions to a commercially available diethyl ether solution (10.0 g, 34.32 mmol) of rel-(2S,4R)-1-benzylazetane-2,4-dicarboxylic acid diethyl ester (200 mL) cooled under nitrogen in an ice-water bath. The cooling bath was removed, and the mixture was warmed to room temperature and stirred for 5 hours. The mixture was cooled in an ice-water bath, and the reaction was carefully quenched with ice water (200 mL). The organic phase was separated, the aqueous phase was extracted with EtOAc (3 × 300 mL), the combined organic phases were washed with brine (2 × 300 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give crude product 3 (4.00 g, 83.3% HPLC purity, 16.1 mmol, 47% yield), a yellow viscous liquid.

[0648] 1 H-NMR (300MHz, CDCl3): δ = 7.26–7.33 (5H), 4.03 (m, 1H), 3.62–3.77 (3H), 3.49–3.63 (4H), 2.26 (brs, 2H), 2.13 (m, 2H); LCMS: RT = 0.91, for C 12 H 17 NO2+H + The calculated value is 208.13. The measured value is 208.10.

[0649] Example 4: Synthesis of rel-(2S,4R)-2,4-bis(hydroxymethyl)azonylbutane-1-carboxylic acid tert-butyl ester (4)

[0650]

[0651] Pd(OH)₂ (0.80 g, 20% w / w) and BOC₂O (5.27 g, 2.15 mmol) were added to a solution of 3 (4.00 g, 16.1 mmol) in 200 mL of EtOH, and the mixture was placed under 30 psi of hydrogen. The mixture was shaken under 30 psi of hydrogen for 20 hours, and then filtered through a diatomaceous earth pad to remove the catalyst. The filter cake was washed with EtOH (50 mL), and the combined filtrates were concentrated under vacuum to give crude product 4, a yellow oil. Crude product 4 was purified by chromatography on a silica gel column (250 g, 100-200 mesh), packed with petroleum ether-EtOA (4:1) and eluted. The fractions containing 4 were combined and concentrated under vacuum to provide 4 (2.10 g, HPLC purity 97.9%, 9.20 mmol, 57% yield), a clear pale yellow oil.

[0652] 1H-NMR (300MHz, CDCl3): δ = 4.31 (m, 2H), 3.80 (dd, J = 11.7, 2.7Hz,2H), 3.67 (dd, J = 11.7, 5.7Hz, 2H), 2.93 (brs, 2H), 2.22 (m, 1H), 1.94 (m,1H), 1.49 (s, 9H).

[0653] Example 5: ((((rel-(2S,4R)-1-(tert-butoxycarbonyl)azacyclobutane-2,4-diyl)bis(methylene) Synthesis of bis(2-oxoethane-2,1-diyl)bis(propane-2,1,3-triyl)tetranonanoate (5)

[0654]

[0655] Under nitrogen atmosphere, at room temperature, 2 (8.01 g, 19.331 mmol), DMAP (1.12 g, 9.205 mmol), and EDC-HCl (4.41 g, 23.012 mmol) were added sequentially to a solution of 4 (2.00 g, 9.205 mmol) in 100 mL of dichloromethane. The reaction mixture was stirred at room temperature for 18 hours, then concentrated under vacuum, and the residue was dissolved in n-heptane (500 mL). The resulting solution was washed with MeOH / water (10:1, 2 × 200 mL), and the organic phase was concentrated under vacuum to give crude product 5. Crude product 5 was purified by chromatography on a silica gel column (500 g, 100-200 mesh), packed with n-heptane, and eluted with a gradient of n-heptane:EtOAc from 100:0 to 90:10. The qualified fractions were combined and concentrated under vacuum to obtain 5 (7.00 g, HPLC purity 97.33%, 6.93 mmol, 75.3% yield), which was a light yellow oily substance.

[0656] 1 H-NMR (400MHz, CDCl3): δ = 4.25-4.37 (6H), 4.18 (m, 8H), 2.62 (m,2H), 2.50 (m, 4H), 2.42 (m, 1H), 2.33 (t, J = 7.0Hz, 8H), 1.83 (m, 1H), 1.52-1.60 (8H), 1.45 (s, 9H), 1.18-1.37 (40H), 0.89 (t, J = 7.9Hz, 12H); LCMS: RT =2.24 for C 56 H 99 NO 14 The calculated value of Na+ is 1032.70, and the measured value is also 1032.70.

[0657] Example 6: Rel-(2S,4R)-2,4-bis(((4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butyryl)oxy Synthesis of (6)-methyl)azonium butane-1-onium trifluoroacetate

[0658]

[0659] Trifluoroacetic acid (14 mL, 20.85 g, 0.183 mol) was added to a solution of 5 (7.00 g, 6.928 mmol) in dichloromethane (35 mL) over a 10-minute period under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours and then concentrated under vacuum to give crude product 6. Ammonium salt 6 was dissolved in n-heptane (500 mL), and the solution was washed with brine (2 × 200 mL) and water (200 mL), and then concentrated under vacuum to give 6 (6.80 g, HPLC purity 91.82%) as a pale yellow oil.

[0660] 1 H-NMR (400MHz, CDCl3): δ = 4.77 (brs, 2H), 4.44 (m, 4H), 4.02-4.25 (9H), 2.72 (brm, 1H), 2.38-2.5 (8H), 2.33 (m, 8H), 1.51-1.62 (8H), 1.20-1.37 (40H), 0.88 (m, 12H); LCMS: RT = 1.72, for C 51 H 92 NO 12 The calculated value is 910.55. The measured value is 910.70.

[0661] Example 7: ((((rel-(2S,4R)-1-(1H-imidazol-1-carbonyl)azacyclobutane-2,4-diyl)bis(methylene) Synthesis of bis(2-oxoethane-2,1-diyl)bis(propane-2,1,3-triyl)tetranonanoate (7)

[0662]

[0663] CDI (10.44 g, 64.44 mmol) and Et3N (3.26 g, 32.27 mmol) were added to a solution of 6 (6.50 g, 91.82% purity by HPLC) in 180 mL of dichloromethane cooled under nitrogen in an ice-water bath. The mixture was stirred for 1 hour, then warmed to room temperature and stirred for 18 hours. The reaction was quenched with 0.5 M HCl aqueous solution (150 mL), and the organic phase was separated. The aqueous layer was extracted with dichloromethane (2 × 300 mL), the combined organic phases were concentrated under vacuum, and the residual oil was dissolved in n-heptane (300 mL). The n-heptane solution was washed with MeOH / water (5:1, 2 × 200 mL) and the solvent was removed under vacuum to give 7 (6.00 g, 97.24% purity by HPLC, 5.97 mmol, 92% yield, in 2 steps), a pale yellow oil.

[0664] 1H-NMR (400MHz, CDCl3): δ = 8.09 (s, 1H), 7.39 (s, 1H), 7.11 (s, 1H), 4.52 (m, 2H), 4.44 (m, 4H), 4.12 (m, 8H), 2.75 (m, 1H), 2.57 (m, 2H), 2.43(m, 4H), 2.28 (m, 8H), 2.07 (m, 1H), 1.52-1.64 (8H), 1.18-1.32 (40H), 0.88(t, J = 7.20Hz, 12H); LCMS: RT = 1.74 for C 55 H 93 N3O 13 + H + The calculated value is 1004.68, and the measured value is 1004.70.

[0665] Example 8: ((((rel-(2S,4R)-1-((2-(dimethylamino)ethoxy)carbonyl)azacyclobutane-2,4- Di(methylene)di(oxy)di(2-oxoethane-2,1-di)di(propane-2,1,3-triyl)tetranonanoate Synthesis of (CICL-221)

[0666]

[0667] Methyl trifluoromethanesulfonate (MeOTf, 1.14 g, 6.947 mmol) was added to a CH3CN (120 mL) solution of 7 (5.80 g, 5.775 mmol) cooled in an ice-water bath under nitrogen over a 5-minute period. The mixture was stirred at 0 °C for 2 hours, and then a CH3CN solution of trimethylamine (2.4 M, 11.55 mL, 27.7 mmol) was added over a 10-minute period, followed by a one-time addition of 2-dimethylaminoethanol (0.77 g, 8.638 mmol). The mixture was stirred at 0 °C for 1 hour, then heated to 60 °C and stirred for 120 hours. The reaction mixture was concentrated under vacuum, and the residue was dissolved in n-heptane (400 mL). The solution was washed with MeOH / water (5:1, 2 × 150 mL), and the organic phase was dried over Na2SO4. The crude CICL-221 solution was obtained by filtration in heptane, and silica gel (15 g, type: ZCX-2, 100-200 mesh) was added. The solvent was removed under vacuum, and the impregnated silica gel was placed on top of a combined flash column of silica gel (150 g, type: ZCX-2, 100-200 mesh). The column was eluted with a gradient of n-heptane:ethyl acetate from 100:0 to 35:65. The qualified fractions (eluted at 50:50) were combined and concentrated under vacuum to give CICL-221 (1.876 g, 1.83 mmol, 32%) as a pale yellow oil.

[0668] 1¹H-NMR (400MHz, CDCl₃): δ = 4.27 (m, 8H), 4.13 (m, 8H), 2.58 (m, 4H), 2.40–2.52 (5H), 2.23–2.34 (14H), 1.89 (m, 1H), 1.60 (m, 8H), 1.18–1.38 (40H), 0.88 (t, J = 6.90Hz, 12H); HPLC (RT = 20.11) 96.57% purity; LCMS: RT = 1.215, for C 56 H 100 N2O 14 + H + The calculated value is 1025.73. The measured value is 1025.70.

[0669] Example 9: Synthesis of rel-(2R,4R)-2,4-bis(hydroxymethyl)azonylbutane-1-carboxylic acid tert-butyl ester (9)

[0670]

[0671] 10% Pd / C (1.60 g, 20% w / w) and BOC2O (12.48 g, 57.16 mmol) were added to a solution of 8 (7.90 g, 38.11 mmol, Tetrahedron Asymmetry 2001, 12, 605) in 240 mL of EtOH, and the mixture was placed under 30 psi of hydrogen. The mixture was shaken under 30 psi of hydrogen for 30 h, and then filtered through a diatomaceous earth pad to remove the catalyst. The filter cake was washed with EtOH (100 mL), and the combined filtrates were concentrated under vacuum to give crude product 9, a yellow oily substance. Crude product 9 was purified by chromatography on a silica gel column (300 g, 100-200 mesh), packed with petroleum ether-EtOA (4:1) and eluted. The fractions containing 9 were combined and concentrated under vacuum to provide 9 (6.90 g, HPLC purity 98.05%, 31.63 mmol, 83% yield) as a clear, pale yellow oil.

[0672] 1 H-NMR (300MHz, CDCl3): δ = 4.58–4.23 (3H), 3.93–3.62 (4H), 2.32 (brs, 1H), 2.00 (brm, 2H), 1.47 (s, 9H); LCMS: RT = 0.897, for C 10 H 19 NO4+H + The calculated value is 218.14. The measured value is 218.10.

[0673] Example 10: ((((rel-(2R,4R)-1-(tert-butoxycarbonyl)azacyclobutane-2,4-diyl)bis(methylene) Synthesis of bis(2-oxoethane-2,1-diyl)bis(propane-2,1,3-triyl)tetranonanoate (10)

[0674]

[0675] Under nitrogen atmosphere, 2 (8.01 g, 19.331 mmol), DMAP (1.12 g, 9.205 mmol), and EDC-HCl (4.41 g, 23.012 mmol) were added sequentially to a solution of 9 (2.00 g, 9.205 mmol) in 100 mL of dichloromethane. The reaction mixture was stirred at room temperature for 18 hours, then concentrated under vacuum, and the residue was dissolved in n-heptane (500 mL). The resulting solution was washed with MeOH / water (10:1, 2 × 200 mL), and the organic phase was concentrated under vacuum to give crude product 10. Crude product 10 was purified by chromatography on a silica gel column (500 g, 100-200 mesh), packed with n-heptane, and eluted with a gradient of n-heptane:EtOAc from 100:0 to 90:10. The qualified fractions were combined and concentrated under vacuum to obtain 10 (7.00 g, HPLC purity 95.94%, 6.93 mmol, 75.3% yield), which was a light yellow oily substance.

[0676] 1 H-NMR (300MHz, CDCl3): δ = 4.20–4.52 (5H), 4.13 (m, 8H), 2.60 (m, 2H), 2.46 (m, 4H), 2.30 (t, J = 7.5Hz, 8H), 2.19 (m, 2H), 1.52–1.65 (9H), 1.40 (s, 9H), 1.18–1.30 (40H), 0.88 (m, 12H); LCMS: RT = 2.235, for C 56 H 99 NO 14 The calculated value of Na+ is 1032.70, and the measured value is 1032.80.

[0677] Example 11: Rel-(2R,4R)-2,4-bis(((4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butyryl) Synthesis of (11)-(oxy)-(methyl)-(azacyclobutane-1-onium trifluoroacetate)

[0678]

[0679] Trifluoroacetic acid (14 mL, 20.85 g, 0.183 mol) was added to a solution of 10 (7.00 g, 6.928 mmol) in dichloromethane (35 mL) over a 10-minute period under nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours and then concentrated under vacuum to give crude product 11. Trifluoroacetic acid ammonium salt 11 was dissolved in n-heptane (500 mL), and the solution was washed with brine (2 × 200 mL) and water (200 mL), and then concentrated under vacuum to give 6 (6.80 g, HPLC purity 91.63%), a pale yellow oil.

[0680] 1 H-NMR (300MHz, CDCl3): δ = 4.66 (brm, 2H), 4.46 (m, 4H), 4.16 (m, 8H), 2.42–2.65 (9H), 2.30 (t, J = 7.4Hz, 8H), 1.55–1.65 (9H), 1.20–1.40 (40H), 0.88 (m, 12H); LCMS: RT = 1.724, for C 51 H 92 NO 12 The calculated value is 910.66, and the measured value is 910.70.

[0681] Example 12: ((((rel-(2R,4R)-1-(1H-imidazol-1-carbonyl)azacyclobutane-2,4-diyl)bis(imidazolium) Synthesis of methyl(2-oxoethane-2,1-diyl)bis(propane-2,1,3-triyl)tetranonanoate (12)

[0682]

[0683] CDI (10.44 g, 64.44 mmol) and Et3N (3.26 g, 32.27 mmol) were added to a solution of 11 (6.50 g, 91.63% purity by HPLC) in dichloromethane (180 mL) cooled under nitrogen in an ice-water bath. The mixture was stirred for 1 hour, then warmed to room temperature and stirred for 18 hours. The reaction was quenched with 0.5 M HCl aqueous solution (150 mL), and the organic phase was separated. The aqueous layer was extracted with dichloromethane (2 × 300 mL), the combined organic phases were concentrated under vacuum, and the residual oil was dissolved in n-heptane (300 mL). The n-heptane solution was washed with MeOH / water (5:1, 2 × 200 mL) and the solvent was removed under vacuum to give 12 (6.00 g, 96.67% purity by HPLC, 5.97 mmol, 93% yield, 2 steps), a pale yellow oil.

[0684] 1H-NMR (300MHz, CDCl3): δ = 0.87 (s, 1H), 7.32 (s, 1H), 7.12 (s, 1H), 4.85 (brm, 2H), 4.08–4.30 (12H), 2.57 (m, 2H), 2.26–2.51 (14H), 1.62 (m, 8H), 1.23–1.40 (40H), 0.89 (t, J = 6.9Hz, 12H); LCMS: RT 1.743, for C 55 H 93 N3O 13 + H + The calculated value is 1004.68, and the measured value is 1004.70.

[0685] Example 13: ((((rel-(2S,4R)-1-((2-(dimethylamino)ethoxy)carbonyl)azacyclobutane-2, 4-Diyl)bis(methylene))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(propane-2,1,3-triyl)tetranonanoic acid Synthesis of ester (CICL-222)

[0686]

[0687] Under nitrogen atmosphere, a solution of 12 (5.80 g, 5.78 mmol) of acetonitrile (120 mL) was cooled in an ice-water bath, and then MeOTf (1.14 g, 6.95 mmol) was added over a 5-minute period. The mixture was stirred at 0 °C for 2 hours, and then trimethylamine (11.55 mL, 2 M THF solution, 23.10 mmol) was added over a 5-minute period, followed by a one-time addition of 2-dimethylaminoethanol (0.77 g, 8.64 mmol). The resulting solution was stirred at 0 °C for 1 hour, then warmed to room temperature, and subsequently heated in a 75 °C oil bath for 8 days. After cooling to room temperature, the solvent was removed under vacuum, and the residue was dissolved in n-heptane (400 mL). The n-heptane solution was washed with MeOH / water (5:1, 2 × 150 mL), and the organic phase was dried over Na₂SO₄. The crude CICL-222 was obtained by filtration and vacuum concentration, dissolved in CH3CN (10 mL), and purified by preparative reversed-phase HPLC (XB-phenyl column 19 × 250 mm, 5 μM; mobile phase A: water / 0.1% TFA; mobile phase B: CH3CN; flow rate 20 mL / min; gradient 50% B to 90% B over 18 min; wavelength 200 nM). The qualified fraction was concentrated under vacuum to remove CH3CN, and the pH of the aqueous residue was adjusted to 8.0 with 2% Na2CO3 aqueous solution. The aqueous phase was extracted with n-heptane (3 × 100 mL), and the combined organic phases were washed with MeOH / water (5:1, 2 × 100 mL), water (200 mL), and dried (Na2SO4). The sample was filtered and concentrated under vacuum to obtain CICL-222 (1.26 g, 1.23 mmol, HPLC purity 97%, 21% yield), which was a clear, colorless, viscous oil.

[0688] 1 H-NMR (300MHz, CDCl3): δ = 4.31-4.57 (5H), 4.08-4.22 (11H), 2.61 (brm, 4H), 2.49 (m, 4H), 2.22-2.35 (16H), 1.63 (m, 8H), 1.20-1.40 (40H), 0.90 (t, J = 6.6Hz, 12H); LCMS: RT 1.588, for C 56 H 100 N2O 14 + H + The calculated value is 1025.73. The measured value is 1025.70.

[0689] Example 14: 2-(2-(((2S,4R)-1-(tert-butoxycarbonyl)-4-((4-(nonanoyloxy)-3-((nonanoyloxy) (14) methyl)butyryl)oxy)pyrrolidone-2-yl)methoxy)-2-oxoethyl)propane-1,3-dimethyldinonanoate Synthesis

[0690]

[0691] Under nitrogen atmosphere, 2 (12.0 g, 28.94 mmol), DMAP (1.80 g, 14.73 mmol), and EDC-HCl (7.00 g, 36.70 mmol) were added sequentially to a CH3CN (60 mL) solution of 13 (3.10 g, 14.27 mmol, TCI America #B3662). The mixture was stirred at room temperature for 18 hours and then concentrated under vacuum to give crude product 14. Crude product 14 was dissolved in n-heptane (60 mL) and washed with MeOH / 10% citric acid aqueous solution (5:1, 2 × 60 mL) and water (60 mL). The organic phase was concentrated under vacuum, and the residue was dissolved in CH2Cl2 (20 mL). Silica gel (25 g, type ZCX-2, 200-300 mesh) was added to the solution of crude product 14, and the solvent was removed under vacuum to provide silica gel impregnated with crude product 14. The silica gel was placed on top of a combined flash column of silica gel (210 g, type ZCX-2, 200-300 mesh), and the column was eluted with a CH2Cl2 / MeOH gradient (100:0 to 90:10). The qualified fractions were combined and concentrated under vacuum to give 14 (5.80 g, 5.82 mmol, 41%), a viscous, pale yellow oil.

[0692] 1 H-NMR (300MHz, CDCl3): δ = 5.28 (brm, 1H), 4.04-4.37 (8H), 3.61 (brm,1H), 2.55 (m, 2H), 2.42 (m, 4H), 2.32 (t, J = 7.5Hz, 8H), 2.18 (brm, 2H),1.60-1.75 (10H), 1.5 (9H), 1.22-1.38 (40H), 0.88 (m, 12H); LCMS: RT 2.017 for C 55 H 97 NO 14 The calculated value of Na+ is 1032.70. The measured value is 1032.60.

[0693] Example 15: (2S,4R)-4-((4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butyryl)oxy)-2- (((4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butyryl)oxy)methyl)pyrrolidine-1-onium trifluoroacetate (15) Synthesis

[0694]

[0695] TFA (12 mL, 17.87 g, 157 mmol) was added to a 36 mL solution of 14 (5.80 g, 5.74 mmol) cooled to 20 °C under nitrogen over a 15-minute period. After the addition was complete, the mixture was stirred at 20 °C for 2 hours and then concentrated under vacuum to give crude product 15. Crude trifluoroacetate ammonium salt 15 was dissolved in n-heptane (60 mL), and the resulting solution was washed with 10% K₂HPO₄ aqueous solution (30 mL) and water (2 × 60 mL). The organic layer was concentrated under vacuum to give 15 (5.50 g, HPLC purity 85%, 5.37 mmol, 94% yield), as a viscous yellow oil.

[0696] 1 H-NMR (300MHz, CDCl3): δ = 5.50 (m, 1H), 4.62 (m, 1H), 4.26-4.36 (3H), 4.08-4.24 (8H), 3.89 (m, 1H), 3.72 (m, 1H), 2.61 (m, 2H), 2.25-2.49 (15H), 1.63 (m, 8H), 1.20-1.43 (40H), 0.90 (t, J = 6.6Hz, 12H); LCMS: RT 1.679, for C 51 H 92 NO 12 The calculated value is 910.66, and the measured value is 910.60.

[0697] Example 16: 2-(2-(((2S,4R)-1-(1H-imidazol-1-carbonyl)-4-((4-(nonanoyloxy)-3-((nonanoyl) oxy)methyl)butyryl)oxy)pyrrolidone-2-yl)methoxy)-2-oxoethyl)propane-1,3-dimethyldinonanoate Synthesis of (16)

[0698]

[0699] CDI (3.48 g, 21.48 mmol) and Et3N (1.09 g, 10.74 mmol) were added sequentially to a CH2Cl2 (60 mL) solution of 15 (5.50 g, 5.37 mmol) cooled to 20 °C under nitrogen. After the addition was complete, the solution was warmed to room temperature and the mixture was stirred for 20 hours. The solution was poured into a 1 M HCl aqueous solution (70 mL) to separate the organic phase. The aqueous layer was extracted with CH2Cl2 (3 × 70 mL), and the combined organic phases were concentrated under vacuum to give crude product 16. Crude product 16 was dissolved in n-heptane (150 mL), and the solution was washed with MeOH / water (2 × 150 mL) and brine (150 mL) and dried over Na2SO4. The solution was filtered and concentrated under vacuum to give 16 (4.80 g, 4.78 mmol, 89% yield) as a pale yellow oil.

[0700] 1 H-NMR (300MHz, CDCl3): δ = 8.75 (s, 1H), 7.56 (s, 1H), 7.32 (s, 1H), 5.37 (m, 1H), 4.80 (m, 1H), 4.68 (m, 2H), 3.99-4.28 (10H), 3.75 (d, J = 12Hz,1H), 2.38-2.60 (4H), 2.25-2.37 (11H), 1.62 (m, 8H), 1.24-1.40 (40H), 0.90 (t,J = 6.6Hz, 12H); LCMS: RT 1.709 for C 55 H 93 N3O 13 + H + The calculated value is 1004.68. The measured value is 1004.60.

[0701] Example 17: 2-(2-(((2S,4R)-1-((2-(dimethylamino)ethoxy)carbonyl)-4-((4-(nonanoyloxy) 3-((nonanoyloxy)methyl)butyryl)oxy)pyrrolidine-2-yl)methoxy)-2-oxoethyl)propane-1,3-di Synthesis of dinonanoate (CICL-207)

[0702]

[0703] MeOTf (0.862 g, 5.25 mmol) was added to a 50 mL solution of CH3CN (4.80 g, 4.78 mmol) cooled under nitrogen in an ice-water bath over a 5-minute period. The mixture was stirred at 0 °C for 2 hours, and then a THF solution of trimethylamine (2.0 M, 7.20 mL, 14.40 mmol) was added over a 10-minute period, followed by a one-time addition of 2-dimethylaminoethanol (0.638 g, 7.16 mmol). The mixture was stirred at 0 °C for 1 hour, then heated to 60 °C and stirred for 120 hours. The reaction mixture was concentrated under vacuum, and the residue was dissolved in n-heptane (100 mL). The solution was washed with MeOH / water (5:1, 2 × 100 mL) and MeOH / 5.6% citric acid aqueous solution (10:1). The organic phase was separated, and the citric acid / MeOH aqueous phase was extracted with n-heptane (5 × 50 mL). Add 200 mL of n-heptane and 100 mL of 4.6% Na₂CO₃ aqueous solution to the MeOH / citric acid aqueous phase, and separate the organic phase. Wash with MeOH / water (5:1, 100 mL). The separated organic phase was dried over Na₂SO₄ and filtered. The filtrate was concentrated under vacuum to obtain CICL-207 (4.00 g, HPLC purity 84.1%), which was further purified by reversed-phase preparative HPLC. CICL-207 was dissolved in CH₃CN (10 mL) and purified by chromatography (Xselect column CSH phenyl-hexyl 19 × 250 mm, 5 μM; mobile phase A: water / 0.1% TFA; mobile phase B: CH₃CN; flow rate 20 mL / min; gradient 65% B to 85% B over 10 min; wavelength 200 nM; retention time (min): 9.5). The qualified fraction was concentrated under vacuum to remove CH3CN, and the pH of the aqueous residue was adjusted to 7.0 with saturated Na2CO3 aqueous solution. The aqueous phase was extracted with n-heptane (3 × 100 mL), and the combined organic phases were dried over Na2SO4. The mixture was filtered and concentrated under vacuum to give CICL-207 (2.11 g, 2.06 mmol, HPLC purity 99%, 43% yield), as a clear, colorless, viscous oil.

[0704] 1 H-NMR (300MHz, CDCl3): δ = 5.29 (m, 1H), 4.18–4.40 (5H), 4.11 (m, 8H), 3.60 (m, 2H), 2.48–2.70 (4H), 2.02–2.45 (20H), 1.61 (m, 8H), 1.19–1.39 (40H), 0.88 (m, 12H); LCMS: RT 1.127, for C 56 H1...

Claims

1. An ionizable cationic lipid having the structure of formula M2, Where X is , , , , , , , , , , , , , , , , , , , , , or ; Y is O, S, NH, or NCH3; Z is O, NH, or NCH3; Each R 1 Independently selected from C7-C 11 Alkyl or C7-C 11 alkenyl; and A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 (CH2) 1-4 Or CH2-CH=CH-CH2; or A 1 For (CH2)0, A 2 For (CH2)1, A 3 For (CH2)1, A 4 It is (CH2)0, and A 5 For (CH2)1; or A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)0; or A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 For (CH2)1; or A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)2 or CH=CH, and The wavy bond indicates that any relative or absolute stereoconfiguration or mixture of stereoconfigurations of the corresponding ring atom can be assumed.

2. The ionizable cationic lipid according to claim 1, wherein A 1 For (CH2)0, A 2 For (CH2)0, A 3 For (CH2)1, A 4 It is (CH2)1, and A 5 (CH2) 1-4 Or CH2-CH=CH-CH2.

3. The ionizable cationic lipid according to claim 1, wherein A 1 For (CH2)0, A 2 For (CH2)1, A 3 For (CH2)1, A 4 It is (CH2)0, and A 5 It is (CH2)1.

4. The ionizable cationic lipid according to claim 1, wherein A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)0.

5. The ionizable cationic lipid according to claim 1, wherein A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)1.

6. The ionizable cationic lipid according to claim 1, wherein A 1 For (CH2)1, A 2 For (CH2)1, A 3 For (CH2)0, A 4 It is (CH2)0, and A 5 It is (CH2)2 or CH=CH.

7. The ionizable cationic lipid according to any one of claims 1 to 6, wherein R 1 It is (CH2)7CH3.

8. The ionizable cationic lipid according to claim 1, wherein the ionizable cationic lipid has the following structure: The wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

9. The ionizable cationic lipid according to claim 1, wherein the ionizable cationic lipid has the following structure: The wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

10. The ionizable cationic lipid according to claim 1, wherein the ionizable cationic lipid has the following structure: The wavy bond indicates that any relative or absolute stereo configuration of the corresponding ring atom can be assumed.

11. The ionizable cationic lipid according to any one of claims 1 to 10, wherein X is... .

12. The ionizable cationic lipid according to claim 11, wherein X is... .

13. The ionizable cationic lipid according to any one of claims 1 to 12, wherein Y is O.

14. The ionizable cationic lipid according to any one of claims 1 to 11, wherein Z is O.

15. A lipid nanoparticle (LNP) comprising at least one ionizable cationic lipid according to any one of claims 1 to 14.

16. A targeted lipid nanoparticle (tLNP) comprising at least one ionizable cationic lipid and a functionalized PEG-lipid according to any one of claims 1 to 14, wherein the functionalized PEG-lipid has been conjugated with a binding moiety.

17. The LNP of claim 15 or the tLNP of claim 16, wherein the LNP or the tLNP further comprises one or more of phospholipids, sterols, auxiliary lipids, nonfunctionalized PEG-lipids, or combinations thereof.

18. The LNP or tLNP according to claim 17, wherein the LNP or tLNP comprises at least one phospholipid, wherein the phospholipid comprises dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearate phosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), or 1,2-disarachidonico-sn-glycerol-3-phosphocholine (DAPC) or combinations thereof.

19. The LNP or tLNP according to claim 17, wherein the LNP or tLNP comprises at least one sterol, wherein the sterol comprises cholesterol, campesterol, sitosterol, stigmasterol, or a combination thereof.

20. The LNP or tLNP according to any one of claims 17 to 19, wherein the PEG-lipid comprises a PEG portion with a molecular weight (MW) of 1000 Da to 5000 Da.

21. The LNP or tLNP according to any one of claims 17 to 20, wherein the PEG-lipid comprises having C 14 -C 18 Fatty acids with varying fatty acid chain lengths.

22. The LNP or tLNP according to any one of claims 17 to 21, wherein the at least one ionizable cationic lipid is present in an amount ranging from about 40 mol% to about 65 mol%.

23. The LNP or tLNP according to any one of claims 17 to 22, wherein the LNP or the tLNP comprises an amount of phospholipid ranging from about 7 mol% to about 30 mol%, a amount of sterol ranging from about 20 mol% to about 45 mol%, at least one auxiliary lipid ranging from about 1 mol% to about 30 mol%, at least one nonfunctionalized PEG-lipid ranging from about 0.1 mol% to about 5 mol%, or at least one functionalized PEG-lipid ranging from about 0.1 mol% to about 5 mol%, or any combination thereof.

24. The LNP according to any one of claims 17 to 23, wherein the LNP comprises a functionalized PEG-lipid.

25. The tLNP according to any one of claims 17 to 23, wherein the binding portion comprises an antigen, a ligand-binding domain of a receptor, an antigen-binding domain of a receptor ligand or an antibody, or an antigen-binding fragment thereof.

26. The LNP according to any one of claims 15 to 24 or the tLNP according to any one of claims 17 to 23 or 25, wherein the LNP or the tLNP further comprises a biologically active payload nucleic acid.

27. A method for delivering nucleic acids into cells, the method comprising contacting the cells with an LNP or tLNP according to claim 26.

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