Lipid nanoparticle formulations and compositions

Abstract

Disclosed are compositions of lipid nanoparticles (LNPs) comprising an ionizable cationic lipid, a phospholipid, a sterol, and a PEG-lipid (non-functionalized and optionally functionalized). The functionalized PEG-lipid may be conjugated to a binding moiety to form a targeted LNP (tLNP). The disclosed tLNP preferentially delivers a nucleic acid molecule or other negatively charged payload to a cell expressing a cell surface antigen recognized by the binding moiety of the tLNP and has better tolerance compared to LNP and tLNP comprising ionizable cationic lipids found in commercially available LNP-containing medicaments.

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Application No. 63 / 595,201, filed November 1, 2023; U.S. Provisional Application No. 63 / 520,303, filed August 17, 2023; U.S. Provisional Application No. 63 / 510,061, filed June 23, 2023; and U.S. Provisional Application No. 63 / 505,424, filed May 31, 2023; the disclosures of these U.S. Provisional Applications are expressly incorporated herein by reference.

[0002] Reference to electronic sequence listing 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 May 31, 2024, named “24-0226-WO_SequenceListing.xml”, and has a size of 8,266 bytes. Background Technology

[0003] Lipid compositions have been used in the laboratory to deliver nucleic acids into cells. Early compositions 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 ONPATTRO. ® The extent to which two mRNA-based SARS-CoV-2 vaccines have been approved by regulatory authorities and entered the market.

[0004] Following administration, the ability to control which tissues or cells take up LNPs is limited. Intravenously administered LNPs are primarily absorbed in the liver, lungs, or spleen, largely depending on net charge and particle size. Combinations 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. LNPs can be redirected to other tissues or cell types by conjugating binding moieties specific to target tissues or cell types, such as conjugating peptides containing antigen-binding domains from antibodies to LNPs. 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 enter the cytoplasm. Current compositions release only 2–5% of the administered RNA into the cytoplasm (see, e.g., Gilleron et al.). Nat. Biotechnol . 31:638-646, 2013, and Munson et al., Commun. Biol (4:211-224, 2021). Therefore, issues remain regarding off-target delivery, inefficient release of nucleic acids into the cytoplasm, and toxicity associated with the accumulation of component lipids.

[0005] Therefore, this disclosure provides lipid nanoparticles and lipid nanoparticle formulations and compositions to meet the urgent needs in the art. Attached Figure Description

[0006] Figures 1A to 1B This demonstrates improved LNP and tLNP function using lipids and compositions as disclosed herein. Figure 1A The transfection rate (percentage of cells expressing mRNA) and expression level (in equivalent soluble fluorescent dye molecules (MESF)) of spleen T cells from mice administered various LNPs or tLNPs were depicted. Figure 1B The transfection rate (percentage of cells expressing mRNA) and expression level (in mean fluorescence intensity (MFI)) of hepatocytes from mice administered various LNPs or tLNPs were depicted.

[0007] Figure 1C The transfection rate (percentage of T cells expressing mRNA) and expression level (in mean fluorescence intensity (MFI)) of spleen T cells from C57BL / 6 mice administered with targeted LNP (tLNP), which comprises a lipid composition that varies only in the structure of ionizable cationic lipids, were depicted. The binding moiety of tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry.

[0008] Figures 2A to 2F The transfection rate or transfection rate and expression level (in MFI) of tLNP with different input antibody densities were depicted, and the antibody density was expressed as the ratio of conjugated antibody to mRNA (w / w). Figure 2A The graph shows the transfection rate and expression level in mouse spleen T cells cultured in vitro. Figures 2C to 2E The results of in vivo transfection with tLNP are shown, demonstrating spleen T cells ( Figure 2C CD45 − Hepatocytes (hepatocytes; Figure 2D ) and CD45 + / CD11 + Hepatocytes (Kupffer cells; Figure 2E The graph shows the transfection rate versus expression level. Figure 2B Presented with Figure 2C Same transfection rate data, but a bar chart of the ratio of binding agent. Figure 2F The graph shows the transfection rate and expression level in blood T cells of cynomolgus monkeys (non-human primates) cultured in vitro.

[0009] Figures 3A to 3C Transfection rate (percentage of T cells expressing mRNA) and expression level (MFI) were depicted in cells from C57BL / 6 mice administered tLNP, which comprises a tLNP composition containing the ionizable cationic lipid CICL1 and differs only in the N / P ratio. These were further compared to a baseline composition BF1, in which the ionizable cationic lipid is ALC-0315. BF1 and F1 are compared with... Figure 1C The composition containing ALC-0315 and CICL1 is identical. The binding moiety of tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry. Spinal T cells ( Figure 3A CD45 − hepatocytes ( Figure 3B ) and CD45 + CD11b + hepatocytes ( Figure 3C (Data).

[0010] Figures 4A to 4C Transfection rate (percentage of T cells expressing mRNA) and expression level (in mean fluorescence intensity (MFI)) of cells from C57BL / 6 mice administered tLNP, which comprises a tLNP composition containing the ionizable cationic lipid CICL1 but altering only the amounts of CICL1 and cholesterol. The mol% of CICL1 were 42 (F4), 50 (F1), and 58 (F5), with compensatory adjustments made for the mol% of cholesterol. These were further compared to the baseline composition BF1. The binding moiety of tLNP is an anti-CD5 antibody, and the payload is mRNA encoding mCherry. Splenic T cells (…) were presented. Figure 4A ), CD45−hepatocytes ( Figure 4B ) and CD45 + CD11b + hepatocytes ( Figure 4C (Data).

[0011] Figures 5A to 5F The transfection rate (percentage of T cells expressing mRNA) and expression level (in MFI) of tLNPs from C57BL / 6 mice administered tLNPs containing different nonfunctionalized PEG-lipids with varying lipid anchoring lengths targeting spleen T cells were depicted. Figure 5A and 5D CD45 − hepatocytes ( Figure 5B and 5E ) and CD45 + CD11b +hepatocytes ( Figure 5C and 5F ).

[0012] Figures 6A to 6C Transfection rate (percentage of T cells expressing mRNA) and expression level (in terms of equivalent soluble fluorescent dye molecules (MESF)) were depicted in cells from C57BL / 6 mice administered tLNP containing varying amounts of nonfunctionalized PEG-lipids but the same amount of total PEG-lipids, targeting spleen T cells. Figure 6A CD45 − hepatocytes ( Figure 6B ) and CD45 + CD11b + hepatocytes ( Figure 6C ).

[0013] Figures 7A to 7C The transfection rate (percentage of T cells expressing mRNA) and expression level (in MESF) of tLNP from C57BL / 6 mice administered tLNP, which contains various combinations of functionalized and nonfunctionalized PEG-lipids targeting spleen T cells, were depicted. Figure 7A CD45 − hepatocytes ( Figure 7B ) and CD45 + CD11b + hepatocytes ( Figure 7C ).

[0014] Figures 8A to 8C The transfection rate (percentage of T cells expressing mRNA) and expression level (in MFI) were depicted in cells from C57BL / 6 mice administered tLNP, which contains two different amounts of phospholipids and two different nonfunctionalized PEG-lipids targeting spleen T cells. Figure 8A CD45 − hepatocytes ( Figure 8B ) and CD45 + CD11b + hepatocytes ( Figure 8C ).

[0015] Figures 9A to 9B The transfection rate (percentage of T cells expressing mRNA) and expression level (mRNA) were depicted in cells from C57BL / 6 mice. Figure 9A and Figure 9B (Calculated as MESF or MFI respectively), the mice were administered tLNP (modified with a targeting antibody), LNP modified with an irrelevant antibody, and LNP without an antibody, targeting spleen T cells ( Figure 9A CD45-hepatocytes ( Figure 9B ).

[0016] Figures 9C to 9D The images show bioluminescence of the whole animal and individual organs of the C57BL / 6 mouse. Figure 9C ) and quantitative bioluminescence of individual organs ( Figure 9D The mice were administered BF1 LNP, CD5-targeting BF1 tLNP, F9 LNP, or CD5-targeting F9 tLNP.

[0017] Figures 10A to 10F The study described the results in NSG mice implanted with human peripheral blood lymphocytes and administered up to five doses of the tLNP composition F9 twice a week, which targets CD5 or CD8 and encapsulates mRNA encoding mCherry or anti-CD19 CAR. Figures 10A to 10E Describes the application of targeted CD5 + or CD8 + Following tLNP in cells, CD4 levels in the blood and spleen of NSG mice transplanted with human peripheral blood lymphocytes were increased. + CD8 + A bar chart showing the mCherry transfection rate or expression level (in MFI) in total T cells. Transfection rates in blood and spleen T cells are shown separately. Figure 10A and Figure 10C The results of expression levels in blood and spleen T cells were shown in... Figure 10B and Figure 10D middle. Figure 10E The study depicted the levels of CD4 in the blood of mice 24 hours after receiving the third dose. + CD8 + A bar chart showing the CAR transfection rate of total T cells. Figure 10F Tumor burden and / or clearance are depicted in mouse groups treated with tLNPs targeting CD5 carrying mCherry or anti-CD19 CAR mRNA, or tLNPs targeting CD8 carrying anti-CD19 CAR mRNA. X represents mice that died due to graft-versus-host disease and / or tumor burden, as is evident from the figures. Spots seen on the entire animal in some images are artifacts.

[0018] Figures 11A to 11L The levels of various liver enzymes and acute-phase proteins in rats administered baseline BF1 LNP or BF1 tLNP and tLNP with F5 were depicted. Figures 11A to 11C The levels of three acute-phase proteins, particularly α1-acid glycoprotein, were depicted at 6 and 24 hours post-administration in rats administered a specified dose of BF1 LNP. Figure 11A ), α2-macroglobulin ( Figure 11B ) and haptoglobin ( Figure 11C ). Figures 11D to 11EThe aspartate aminotransferases (ASTs) from comparative compositions BF1 to F5 in rats were plotted. Figure 11D ) and alanine aminotransferase (ALT) Figure 11E The second study compared the median 24-hour liver enzyme levels with the dosage. Figures 11F to 11J The responses of the same series of markers to BF1 and F5 tLNPs are shown. Figures 11K to 11L The levels of ALT and AGP were shown in rats carrying anti-human CD8 F9 tLNP or encapsulated mRNA encoding mCherry targeting CD5 tLNP, respectively, after injection of a specified dose of mRNA encapsulated with anti-CD19 CAR. Figure 11K The horizontal gray band in the figure represents the range of ALT observed in 21 primordial rats in a previous study.

[0019] Figures 12A to 12L It describes various data from two experiments involving non-human primates (cynomolgus monkeys). Figures 12A to 12D Involving the first study, and Figures 12E to 12J This involves a second study. Figure 12A The diagram shows the time-varying responses of liver enzymes ALT and AST in individual cynomolgus monkeys after administration of BF1 (top panel) and F5 (bottom panel), respectively. "Lower limit of normal" and "Upper limit of normal" indicate the normal ranges for these liver enzymes. Figure 12B Cytokine secretion of IL-6, MCP-1, IOL-2, IFNγ, TNFα, TGFα, IL-8, and gm-CSF was shown in the same animals. Figure 12C Following the administration of the F5 composition encapsulating mCherry mRNA and targeting CD5 tLNP, individual animals showed elevated blood T cells (in mCherry mRNA) at 4 and 24 hours. + A graph showing the transfection efficiency (based on a percentage of cells). Figure 12D This is a bar graph depicting the T cell transfection efficiency in spleen, lymph nodes, and bone marrow tissues 24 hours after administration of an F5-targeted CD5 tLNP protocol encapsulated with mCherry mRNA. Figures 12E to 12F An embodiment of the composition F9, which encapsulates anti-CD19 CAR mRNA, targeting tLNPs of CD5 and CD8 in a single administration is described. Figure 12E Or administer F9-targeted CD5-based tLNPs at lower doses multiple times. Figure 12F Following this, the liver enzymes ALT and AST in individual cynomolgus monkeys responded over time. Figure 12G Following the administration of an infusion of F9-targeted tLNPs encapsulated with anti-CD19 CAR mRNA targeting CD5 and CD8, individual animals showed elevated blood T cell counts (in CAR mRNA) at 8 and 24 hours. +A graph showing the transfection efficiency (based on a percentage of cells). Figure 12H This is a bar chart depicting the T cell transfection efficiency in spleen, lymph nodes, bone marrow, and liver tissues 24 hours after administration of an F9-encapsulated anti-CD19 CAR mRNA targeting CD5 and CD8 tLNP. An asterisk indicates data from bone marrow and lymph node biopsy samples collected from two animals 24 hours post-infusion, observed 96 hours post-infusion, while other animals were euthanized 24 hours post-infusion, and necropsy samples from all four tissues were evaluated. Figure 12I This is a graph showing the transfection efficiency at each time point after each infusion of an F9-encapsulated anti-CD19 CAR mRNA targeting CD5 tLNP in a multi-dose infusion regimen. Figure 12J Box plots show CAR expression levels in individual animals 24 hours after a single administration of an F9-based regimen encapsulated with anti-CD19 CAR mRNA targeting CD5 and CD8 tLNPs. The caption above each plot indicates the target and payload dose. Figure 12K This is a compilation of liver enzyme data from three studies following a single administration of various tLNPs. Figure 12L The levels of liver enzymes during three administrations of CD8-targeting tLNP were depicted.

[0020] Figures 13A to 13H It involves the biodegradability of ionizable cationic lipids. Figure 13A A conceptual biodegradation scheme for CICL1 is depicted (above the line), along with the starting compounds and end products of the biodegradation (below the line). The disclosed ionizable cationic lipids with the CICL structure can be biodegraded according to this conceptual scheme without being bound by any particular theory. However, according to this scheme, esterase cleavage or other hydrolysis of CICL1 is expected to produce tetraol B and 4 equivalents of nonanoic acid. Cyclization should then result in 2 equivalents of butyrolactone C and 1 equivalent of diol D. Esterase hydrolysis of C will result in 2 equivalents of diol-acid E. Figure Figure 13B The amount of ionizable cationic lipids detected in plasma, liver, and spleen was depicted 24 hours after administration of CD5-targeting LNPs containing ALC-0315 or CICL1 to cynomolgus monkeys (n=2). Figures 13C to 13E The time-dependent disappearance of ionizable cationic lipids CICL1 and ALC-0315 in plasma, spleen, and liver after administration of F9 and BF1 tLNP to mice was depicted and plotted as lipid concentration measurements based on LC-MS; mean ± standard deviation (SD) is shown. tLNP was targeted to mouse CD8 and encapsulated with mRNA encoding anti-human CD19 CAR. Figures 13F to 13H The study described the effects of digital PCR detection and normalization to spiked controls (plasma) or GAPDH (spleen and liver) derived from 13C to... Figure 13EThe time course of disappearance of encapsulated mRNA in plasma, spleen, and liver of the same mice.

[0021] Figure 14 Transfection efficiency (left panel) and expression levels (gMFI; right panel) of human T cells from two donors (top panel (donor 1) and bottom panel (donor 2)) are depicted as the percentage of mCherry-positive cells. tLNP contains an F5 lipid composition and encapsulates mCherry mRNA, targeting CD2, CD5, CD4, or CD8. CD3 is plotted. + CD4 + and CD8 + Cellular data.

[0022] Figure 15 This study depicts the percentage of CD3 knockout in primary human T cells after disruption of the TRAC gene at different time points following transfection of SpCas9 mRNA and a single guide RNA (sgRNA) targeting the TRAC locus with tLNP modified with an anti-CD5 monoclonal antibody (chimeric 5D7, also known as ch5D7) using different doses of the payload (μg of SpCas9 mRNA + sgRNA). Experiments were performed using T cells obtained from two donors (top panel (donor #1) and bottom panel (donor #2)). CD8 + T cells and CD4 + The results for T cells are presented separately in the left and right images, respectively.

[0023] Figure 16 The payload (μg of SpCas9 mRNA + sgRNA) for various doses of both the first and second doses was depicted, representing the percentage of CD3 knockout in primary human T cells following TRAC gene disruption after transfection with SpCas9 mRNA and sgRNA targeting the TRAC locus using tLNP modified with an anti-CD5 monoclonal antibody. CD8 + T cells and CD4 + The T cell results are presented separately in the left and right graphs. The bars represent the average of the results from the two donors (circles and triangles).

[0024] Figure 17This plot depicts the payload (μg of SpCas9 mRNA + sgRNA) at various doses for both the first and second doses, representing the percentage of CD3 knockout following TRAC gene disruption in primary human T cells after transfection with tLNPs modified with anti-CD8 monoclonal antibody (chRPA-T8-tLNP) or a combination of tLNPs modified with anti-CD8 monoclonal antibody (chRPA-T8-tLNP) and tLNPs modified with anti-CD5 monoclonal antibody (ch5D7-tLNP), monoclonal antibody transfection of SpCas9 mRNA and sgRNA targeting the TRAC locus. In each of the four bar subplots, the results for a single dose are grouped on the left, and the results for a second dose are grouped on the right. Zero and first- and second-dose data are plotted in both the single-dose and second-dose groups. CD8 + T cells and CD4 + The T cell results are presented separately in the upper and lower bar charts. The bars represent the average results from three donors (circles, squares, and triangles). CD8 from donor 1... + A representative flow cytometry histogram of fluorescence versus event (cell) count for T cells (round) at each 3 μg dose condition is shown on the far right.

[0025] Figure 18 This plot depicts the percentage of CD3 knockout in human T cells transplanted into NSG mice following administration / transfection of ch5D7-tLNP only, chRPA-T8-tLNP only, or ch5D7-tLNP + chRPA-T8-tLNP (administered as a single or double dose of tLNP). Five mice were given the initial dose, three of which were re-administered (squares), and two received a single dose only (circles). Three mice did not receive a dose but were replotted in each group for comparison. Columns represent the mean of two or three mice. Total T cells, CD4+ + T cells and CD8 + The T-cell results are shown in the left, middle, and right graphs, respectively. In each graph, single-dose data are grouped on the left, and re-dose data are grouped on the right.

[0026] Figures 19A to 19B A depiction of CD34 from human peripheral blood (i.e., hematopoietic stem cells) + Cell transfection rate over time ( Figure 19A ) and level of expression ( Figure 19BThe cells were contacted in vitro with CD117-targeting LNPs at three different doses, as shown in the illustration. The tLNPs were targeted with one of four anti-CD117 antibodies: 104D2 (#1), JSP191 (#2), CK6 (#3), and Ab85 (#4). #1 was non-antagonistic, while the other three were antagonistic. tLNPs carrying the anti-HIV gp120 antibody telopavir were used as a negative control.

[0027] Figures 20A to 20D . Figures 20A to 20B The mCherry transfection rate in human T cells was depicted 24 hours after transfection with one of four formulations of CD5-targeting tLNPs encapsulated in lipid composition F5 containing mCherry mRNA. Figure 20A ) and level of expression ( Figure 20B Untransfected cells were used as a negative control. Each of the four tLNP formulations uses a different antibody conjugation chemistry. Figures 20C to 20D The study depicted the mCherry transfection rate in human T cells 24 hours after administration of one of four formulations of a lipid composition F5 encapsulating mCherry mRNA targeting CD5 tLNPs to NSG mice implanted with human PBMCs. Figure 20C ) and level of expression ( Figure 20D Untransfected cells (PBS) were used as a negative control. Each of the four tLNP formulations used a different antibody conjugation chemistry. Detailed Implementation

[0028] This disclosure provides lipid nanoparticles (LNPs) and LNP compositions, as well as pharmaceutical compositions (or formulations), for delivering payloads (such as one or more nucleic acid substances) to target cell types or tissues. The LNPs may comprise functionalized polyethylene glycol (PEG)-lipids that may be conjugated to or conjugated to a binding moiety that binds to surface markers on the target cell type, thereby constituting a targeted LNP (tLNP). The binding moiety guides the tLNP to the target tissue or target cell type, such that the tLNP is delivered to the target cell type along with an encapsulated nucleic acid molecule or other negatively charged payload. Transfection can be performed in vivo (also known as in situ), in vitro, or ex vivo. This disclosure further provides methods for reprogramming or opsonizing cells by transfecting or contacting cells with tLNPs. Methods for preparing LNPs containing functionalized PEG-lipids and methods for preparing tLNPs are also provided.

[0029] While this disclosure can be implemented in various forms, the following description of several embodiments is made with the understanding that this disclosure is to be regarded as an example of the invention and is not intended to limit the invention to the specific embodiments shown.

[0030] While this disclosure can be implemented in various forms, the following description of several embodiments is made with the understanding that this disclosure is to be regarded as an example of the invention and is not intended to limit the invention to the specific embodiments shown.

[0031] The headings are provided for convenience only and should not be construed as limiting the invention in any way. Embodiments shown under any heading may be combined with embodiments shown under any other heading.

[0032] In the event of any conflict between any material incorporated herein by reference and this disclosure, this disclosure shall prevail.

[0033] Before elaborating on this disclosure in more detail, it may be helpful to provide abbreviations and definitions for certain terms used herein. Additional definitions are set forth in this disclosure.

[0034] definition 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.

[0035] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives.

[0036] 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.

[0037] 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.

[0038] Unless the context otherwise requires, throughout this specification and claims, the word “comprising” and its variations such as “including” shall be construed as having an open, inclusive meaning, that is, as meaning “including but not limited to”. As used herein, the terms “comprising” and “including” are used synonymously.

[0039] 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.

[0040] 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. Typically, a “derivative” differs from an “analogue” in that the parent compound can be the starting material for producing the “derivative,” while the parent compound may not necessarily be used as the starting material for producing the “analogue.” 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) modifications of the parent compound, but they 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.

[0041] 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.

[0042] 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 that 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.

[0043] 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.

[0044] As used herein, “transfection” 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 between transduction (the transfer of genetic material from cells to cells or from viruses to cells) and transformation (the uptake of extracellular genetic material through natural cellular processes). As used herein, phrases such as “delivering nucleic acids into cells” are synonymous with transfection.

[0045] 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.

[0046] 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.

[0047] 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 were developed by Anzalone et al. Nature Biotechnology 38:824-844, 2020, Sakuma, Gene and Genome EditingA review has been made in 3-4:100017, 2022 and Zhou et al., MedComm 3(3):e155, 2022, and all the contents of each of these documents concerning the components and uses of this technique are incorporated herein by reference to the extent that they do not conflict with this disclosure.

[0048] 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.

[0049] 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.

[0050] The LNP disclosed herein is a multi-component composition comprising a payload and multiple lipid components, including ionizable cationic lipids, unfunctionalized and / or functionalized PEG-lipids, phospholipids, and sterols. In some aspects, the payload comprises one or more nucleic acid molecules or other negatively charged molecules. That is, in some embodiments, the payload comprises only a single nucleic acid substance or other negatively charged molecule (or consists of such substance or molecule), while in other embodiments, the payload comprises multiple nucleic acid substances or other negatively charged molecules, such as 2, 3, or 4 such substances or molecules. In some embodiments in which the payload comprises multiple nucleic acid substances or other negatively charged molecules, up to and including more than one substance of all substances reacts with the same target or encodes a polypeptide that reacts with the same target. As used herein, “LNP composition” refers to the lipid components present in the LNP, their molar ratios relative to each other (e.g., mol%), and the ratio of the payload to total lipids. The ratio of payload to total lipids can be expressed as weight to weight (w / w), or, for nucleic acid molecules, as an N / P ratio, where "N / P ratio" refers to the ratio of positively charged lipid amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. The tLNP disclosed herein is a multi-component composition comprising an LNP and a binding moiety. As used herein, "tLNP composition" refers to an LNP composition having the same characteristics as an LNP composition with a binding moiety added as a targeting moiety, and the density of the binding moiety on the tLNP can be expressed as a w / w to payload ratio. As used herein, "LNP formulation" or "tLNP formulation" refers to the complete corresponding composition plus buffer, carrier, solvent, and / or other excipients, which may also be referred to as a pharmaceutical composition.

[0051] LNP Composition 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 used to guide or deflect tLNPs to binding sites (or multiple sites) for specific cell types.

[0052] 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, dosage always refers to the amount of payload provided. In some embodiments, the payload comprises one or more nucleic acid molecules. In some cases, the N / P ratio is about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 6, about 5 to about 6, or about 6. In some cases, 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, the LNP or tLNP comprises about 40 mol% to about 62 mol% of ionizable cationic lipids. In some embodiments, the LNP or tLNP comprises about 1 mol% to about 2 mol% of total PEG-lipids. In some embodiments, the LNP or tLNP comprises about 0.1 mol% to about 0.3 mol%, such as about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% of functionalized PEG-lipids. In some embodiments, the binding moiety is conjugated to the functionalized PEG-lipids. In some cases, the tLNP is an LNP that also contains an antibody (e.g., intact IgG) as a binding moiety, present at an antibody:mRNA ratio (w / w) of about 0.3 to about 1.0.

[0053] Overview of the Improved LNP The LNP (and its tLNP) disclosed in this paper represents a significant improvement over existing LNP technologies in delivery to target cell types (such as T cells or hematopoietic stem cells (HSCs)) and detargeting undesirable cells or tissues (such as hepatocytes and liver). Figure 1B It is evident that, compared to ionizable lipids from existing technologies (Comirnaty) ® Compared to ALC-0315 (BF1, hollow circle) used in the present invention, the use of the cationic lipids of the present invention in the LNP composition (F5, gray circle) significantly improved hepatocyte detargeting. Further addition of the binding moiety to the LNP of the present invention also improved detargeting in mice (e.g., Figure 1A and Figure 1B ) and non-human primates (e.g., Example 12, Figures 12A to 12J In vivo, the desired level of transfection for the cell type was achieved. For example, Figure 1AThe tLNPs targeting CD5 (triangles) showed that they could transfect spleen T cells, but LNPs without a binding portion (circles) or with irrelevant antibodies (squares) were indistinguishable from the background. Furthermore, tLNPs containing the ionizable cationic lipids of this disclosure (e.g., F5, gray triangle; and F9, black triangle) showed greater targeting to spleen T cells than those containing the prior art lipid ALC-0315 (BF1, hollow triangle). (It must be noted that F9, the CD5-targeting tLNP (black triangle), is also different from other tLNPs because the mCherry mRNA contains N...) 1 (Using methylpseuuridine instead of 5-methoxyuridine used in other tLNPs). Overall, liver detargeting is due to two effects: the lipid composition of the LNP and the addition of the targeting moiety. Therefore, the LNPs and tLNPs disclosed herein represent improvements in the targeted delivery of commercial LNPs or tLNPs.

[0054] Ionizable cationic lipids Ionizable cationic lipids are useful components for compounding with negatively charged payloads and for facilitating the delivery of payloads to the cytoplasm of cells following endocytosis. Therefore, each of the classes and types of ionizable cationic lipids disclosed below can be used to define the scope of embodiments of the LNP and tLNP compositions and pharmaceutical compositions disclosed herein, as well as methods of using them. In some embodiments, ionizable cationic lipids of LNPs having a measured pKa of 6 to 7 can remain substantially neutral in the bloodstream and interstitial space, but ionize upon uptake into cells with endosomal acidification. Upon acidification in the endosomal space, the lipids become protonated and more tightly associated with the phosphate backbone of nucleic acids, which destabilizes the LNP structure and facilitates the release of nucleic acids from the LNP into the cytoplasm (also known as endosome escape). Therefore, the ionizable cationic lipids disclosed herein constitute means for destabilizing the LNP structure (when ionized) or for facilitating nucleic acid release or endosome escape.

[0055] In some implementations, the ionizable cationic lipid has a CICL structure: Where R is or .

[0056] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL1.

[0057] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL2.

[0058] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL3.

[0059] In some implementations, when R is (Right now When CICL is ionized cationic lipid, it is called CICL4.

[0060] In some implementations, the ionizable cationic lipid has the structure CICL-IE: Where R is or .

[0061] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.

[0062] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.2.

[0063] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.3.

[0064] In some implementations, when R is (Right now When CICL-IE is used, the ionizable cationic lipid is called CICL250.4.

[0065] The synthesis of lipids having CICL or CICL-IE structures is described in U.S. Patent Application Publication No. 2023 / 0320995 and U.S. Patent Application No. 63 / 632,940, respectively, and all contents taught in each of them concerning the synthesis of such lipids are incorporated herein by reference in their entirety.

[0066] The ionizable cationic lipids disclosed herein have a branched structure to impart a conical rather than cylindrical shape to the lipids, and such a structure facilitates endosome cleavage activity. Greater endosome cleavage activity leads to more efficient release of bioactive payloads (e.g., one or more nucleic acid molecules).

[0067] 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 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 8.4 to about 8.7 or 8.4 to 8.7. The ionizable cationic lipids described herein may have a cLogD in the range of about 9 to about 18, for example, about 10 to about 18, or about 10 to about 16, about 10 to about 14, or about 11 to about 18, or about 11 to about 15, or about 11 to about 14. The ionizable cationic lipids described herein may have a cLogD ranging from 9 to 18, for example, from 10 to 18, or 10 to 16, or 10 to 14, or 11 to 18, or 11 to 15, or 11 to 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 lipids have 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 pKa ranges of about 6 to about 7 or 6 to 7 measured in LNPs, which facilitates ionization in endosomes after delivery to cells.

[0068] In some embodiments, a slightly higher alkalinity may be desired and can be obtained from ionizable cationic lipids having c-pKa and cLogD within the ranges disclosed herein. In some embodiments, the cLogD of the ionizable cationic lipids of this disclosure is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or within the range defined by any pair of these values. Lipid design also considers the potential biodegradable pathways of the target lipids, such as via esterases in plasma, liver, and other tissues. Another consideration in lipid design is the fate of the ionizable lipid fragments resulting from degradation (such as after esterase cleavage). Preferably, the resulting fragments are rapidly cleared from the body without requiring hepatic oxidative metabolism.

[0069] In some embodiments, the LNP or tLNP comprises about 35 mol% to about 65 mol%, about 40 mol% to about 62 mol%, or about 54 mol% to about 60 mol% of ionizable cationic lipids. In some embodiments, the lipid composition is at least 40 mol% and / or no more than 62 mol% of ionizable cationic lipids. In some embodiments, the LNP of tLNP comprises about 54 mol%, about 58 mol%, or about 62 mol% of ionizable cationic lipids. In a further embodiment, the LNP comprises 35 mol% to 65 mol%, 40 mol% to 62 mol%, or 54 mol% to 60 mol% of ionizable cationic lipids. In an even further embodiment, the LNP has at least 40 mol% or no more than 62 mol% of ionizable cationic lipids. In some embodiments, the LNP comprises 54 mol%, 58 mol%, or 62 mol% of ionizable cationic lipids.

[0070] Sterols The disclosed LNPs and tLNPs contain sterols. Sterols refer to a subgroup of steroids containing at least one hydroxyl (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, 20(S)-hydroxycholesterol, 22-hydroxycholesterol, or phytosterols, or combinations thereof. In further embodiments, the phytosterols include campesterol, sitosterol, or stigmasterol, or combinations thereof. In some embodiments, 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 obtaining high transfection efficiency. While β-sitosterol and stigmasterol perform well, the use of vitamin D2, D3, and calcipotriol (analogs lacking complete cholesterol bodies) and betulin, lupeol, ursolic acid, and oleanolic acid (containing ring 5) should be avoided. Sterols fill the spaces between other lipids in LNPs or tLNPs and affect the shape of LNPs or tLNPs. Sterols also control the flowability of lipid compositions and reduce temperature dependence. Therefore, sterols (such as cholesterol, ergosterol, 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. Some embodiments specifically include one or more of the above-mentioned sterols, while other embodiments specifically exclude one or more of the above-mentioned sterols.

[0071] 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 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 is about 25 mol% of 20-hydroxycholesterol and about 75 mol% of cholesterol. In some cases, the sterol is about 25 mol% of β-sitosterol and about 75 mol% of cholesterol. In some cases, the sterol is about 50 mol% of β-sitosterol and about 50 mol% of cholesterol. 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% 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 is 25 mol% 20-hydroxycholesterol and 75 mol% cholesterol. In a further case, the sterol is 25 mol% β-sitosterol and 75 mol% cholesterol. In an even further case, the sterol is 50 mol% β-sitosterol and 50 mol% cholesterol.

[0072] Phospholipids The disclosed LNPs and tLNPs contain phospholipids. In various embodiments of the LNPs or tLNPs disclosed herein, the phospholipids comprise dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), distearate-glycerol-phosphate (18:0 PA, DSGP), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyl-glycerol-phosphate (18:1 PA, DOGP), or arachidonicylphosphatidylcholine (DAPC) or combinations thereof. Phospholipids can contribute to the formation of a membrane surrounding the core of the LNP or tLNP, whether monolayer, bilayer, or multilayer. Furthermore, phospholipids (such as DSPC, DMPC, DPPC, DAPC) impart structural stability and rigidity to the membrane. Phospholipids (such as DOPE) impart cohesion. Additional phospholipids (such as DMPG, which acquires a negative charge at physiological pH) promote charge regulation. Therefore, phospholipids constitute means for promoting membrane formation, for imparting membrane stability and rigidity, for imparting fusion, and for charge regulation. Some embodiments specifically include one or more of the above-mentioned phospholipids, while other embodiments specifically exclude one or more of the above-mentioned phospholipids.

[0073] 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.

[0074] PEG-lipids 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, dibenzocyclooctylene (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.

[0075] The disclosed LNP and tLNP contain PEG-lipids, i.e., lipids conjugated with polyethylene glycol. Typically, the lipids are C14-C20 lipids. In some embodiments, the PEG-lipids contain C18 and / or C20 lipids. Common PEG-lipids fall 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-dispalmitoyl-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). The glycerol in these lipids is chiral. Therefore, in some embodiments, the PEG-lipid is racemic. Alternatively, an optically pure enantiomer 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 embodiments, the enantiomer excess is ≥98%. In some embodiments, the enantiomer excess is ≥99%. Other uses of PEG-lipids, as well as PEG-lipids and functionalized PEG-lipids and LNPs containing them, are disclosed in International Application No. PCT / US2023 / 017648 (Publication No. WO2023196445A1), all of which teaches about PEG-lipids and is incorporated herein by reference.

[0076] The above example is presented as methoxy polyethylene glycol, but the terminus is not necessarily methoxy. For any non-functionalized 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 (non-functionalized) 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.

[0077] In some embodiments, the LNP or tLNP comprises about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol% of a PEG-lipid, which comprises 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 a non-functionalized PEG lipid. 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.

[0078] 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.

[0079] 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 polyethylene formulations of these sizes 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 would have a molecular weight of 986, n=45 would have a molecular weight of 1998, and n=113 would have a molecular weight of 4990. n≈22 to 113 is used to represent PEG-lipids containing a 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 boundaries will have n values ​​outside that range. For a single formulation, n≈22 is used to indicate a PEG-lipid containing the PEG portion from PEG-1000, n≈45 is used to indicate a PEG-lipid containing the PEG portion from PEG-2000, n≈67 is used to indicate a PEG-lipid containing the PEG portion from PEG-3000, n≈90 is used to indicate a PEG-lipid containing the PEG portion from PEG-4000, and n≈113 is used to indicate a PEG-lipid containing the PEG portion from PEG-5000. Some embodiments include a PEG portion within a range defined by any pair of the foregoing n or average molecular weight values. Some embodiments include a PEG portion of PEG-1000, PEG-2000, or PEG-5000. 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.

[0080] The PEG moiety provides a hydrophilic surface on LNPs, inhibiting their aggregation or coalescence, thus contributing to their stability and reducing polydispersity. 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 inhibition of binding can lead to an increased proportion of LNPs reaching other tissues and cell types. These plasma proteins also include opsonins, which reduce recognition by the reticuloendothelial system due to inhibition of binding.

[0081] The PEG moiety can also be functionalized to act as an attachment site for the binding moiety, which in turn acts as the targeting moiety. Conjugating the cell- or tissue-specific binding moiety to the PEG moiety 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 act as a means of inhibiting LNP binding, and PEG-lipids conjugated to the binding moiety can act as a means of LNP targeting. As used herein, the term "functionalized PEG-lipid" and similar constructs generally refer to both unreacted and reacted entities. Even after conjugation (formation of tLNPs) has occurred, the lipid composition of the LNP can be described with reference to the reactive substance. For example, a lipid composition can be described as containing DSPE-PEG-maleimide and, moreover, the binding moiety, without explicitly stating that the maleimide will have been converted to succinimidide (or hydrolyzed succinimidide) during the reaction to form the conjugate. Similarly, if the reactive group is bromomaleimide, it will be maleimide after conjugation. Even if not explicitly stated, these differences in the chemical nomenclature of unreacted and reacted substances should be understood implicitly. 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, the functionalized PEG-lipid is partially functionalized with maleimide, such as DSPE-PEG-2000-MAL.

[0082] In some embodiments, the PEG-lipid and / or functionalized PEG-lipid comprises a scaffold selected from formula S1, S2, S3, or S4: 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 C14 -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 C 20 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.

[0083] In some embodiments, the PEG portion is PEG-500 to PEG-5000, such as PEG-500, PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000. In some cases, the PEG portion is PEG-2000. In some embodiments, the PEG unit has a MW of 2000 Da. In some cases, MW2000... PEG-lipids include 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), and DMPE-PEG200 (1,2- Dimyristico-glycerol-3-phosphate ethanolamine-3-methoxy polyethylene glycol-2000, DPPE-PEG2000 (1,2-dipalmitoyl-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 glycerol moiety is racemic. Alternatively, an optically pure enantiomer of the glycerol moiety, i.e., a purely chiral glycerol moiety, can be used.

[0084] Adhesion Several suitable chemical approaches for binding PEG partially conjugated to PEG-lipids include maleimide (see Parhiz et al., Journal of Controlled Release 291:106-115, 2018) and click chemistry (see Kolb et al., Angewandte Chemie International Edition 40(11):2004-2021, 2001; and Evans, Australian Journal of Chemistry 60(6):384-395, 2007). 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, lipid-PEG-alkynylimide, and lipid-PEG-alkynylene reactions, as disclosed in PCT / US23 / 17648 entitled PEG-Lipids and Lipid Nanoparticles, all of which teaches conjugation chemistry and alternative PEG-lipids and 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 (collectively, “maleimide”), alkanoamide, or alkynylimide. Alternatively, an alkyne can be added to the thiol group or ε-amino group of lysine to participate in click chemistry reactions.

[0085] To modify the ε-amino group of the lysine-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 Quick Spin 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. Purification can be performed using a Sepharose CL-4B gel filtration column (Sigma-Aldrich). The tLNP (LNP conjugated with the targeting antibody) can be cryopreserved at -80°C until needed. Others conjugate antibodies with free functionalized PEG-lipids and then incorporate the conjugated lipids into pre-formed LNPs. However, we have found that the procedure of this invention is more controllable and produces more consistent results.

[0086] 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: 1), 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.

[0087] For intact antibodies and other forms containing the Fc region, site-specific conjugation to either (or both) of the two specific lysine residues (Lys248 and Lys288) can be achieved using AJICAP. ® One of the reagents can be used to achieve this without any alteration or extension of the natural antibody sequence (see, for example, Matsuda et al., Molecular Pharmaceutics 18:4058-4066, 2021; Fujii et al., Bioconjugate Chemistry 34(4):728-738, 2023; and WO2019 / 240,287; all of these documents teach everything about the conjugation of antibodies with AJICAP reagents, which is incorporated herein by reference. AJICAP reagents are modified affinity peptides that bind to a specific locus on the Fc and react with an adjacent lysine residue. The peptide is then cleaved with a base to leave a thiol-functionalized lysine residue, which can then be conjugated by, for example, a maleimide or haloamide reaction). Functionalization with azides or dibenzocyclooctylene (DBCO) for conjugation via click chemistry is also possible.

[0088] 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, the conjugation is via a cysteine ​​residue in a natural or added antibody sequence. In other embodiments, the conjugation is via a sorting enzyme A substrate sequence. In still other embodiments, the conjugation is via a specific lysine residue (Lys248 or Lys288) in the Fc region.

[0089] Nucleic acid 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 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. In some embodiments, the mRNA encodes a reprogramming agent or contains or encodes a regulator. In some embodiments, the nucleic acid contains small interfering RNA (siRNA), microRNA (miRNA), or antisense oligonucleotide (ASO). In some embodiments, the nucleic acid contains self-replicating RNA or circular RNA.

[0090] 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 (ICE), etc.).

[0091] 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 the 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.

[0092] 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).

[0093] 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.

[0094] Combined part 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 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.

[0095] 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 to 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 for 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 the teachings of this document regarding single antibodies and the various antibody forms that can be constructed are incorporated herein by reference. U.S. Patent No. 11,326,182 (especially its Table 9, Antibodies for Cancer, Inflammation, and the Immune System) is a source of sequence and other information for a wide range of antibodies, including many antibodies that do not have INNs, and all of the teachings of this document regarding single antibodies are incorporated herein by reference. For antibodies mentioned in the art, sequence information is not always readily available, even when it is 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 part of tLNPs without any sequence information. Even where sequence information is required, those skilled in the art are fully capable of sequencing (or having it sequenced by a contract laboratory) antibody proteins so that the variable regions of the antibody can be incorporated into scFvs, bivalent antibodies, microantibodies, or some other antibody form, or humanized. When selecting from available antibodies in the art for developing 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 manufacture, antibody affinity, and cross-reactivity with homologous antigens in the model species to be used for product development.

[0096] 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.

[0097] 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 a complete or whole antibody, as well as antibody fragments and constructs containing the antigen-binding portion of the whole antibody. While typical natural antibodies have a pair of heavy and light chains, camelids (from 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), bivalent antibodies, 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 species of antibody, regardless of its origin or production. Antibodies can be obtained through immunization, selection from natural or immune libraries (e.g., via 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 Medicine Names (INNs) have been proposed or recommended is Wilkinson & Hale, 2022. MAbs 14(1):2123299, including its supplementary tables, all that this document 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 Antibodies for Cancer, Inflammation and the Immune System) is a source of sequences and other information on a wide range of antibodies, including many antibodies that do not have INNs, and all that this document teaches about single antibodies is incorporated herein by reference.

[0098] If the antibody or other binding moiety (or its fusion protein) is equal to or greater than 10 5 M −1 When an antibody or other binding moiety (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 / 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 −1At 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).

[0099] Bivalent antibodies are dimers of scFv fragments, consisting of V molecules non-covalently or covalently linked together via small peptide linkers. H and V L 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, the antibody and its antigen-binding moiety constitute a means of binding to cell surface molecules. In various embodiments, depending on the antibody's specificity, the cell can be an immune cell, leukocyte, lymphocyte, monocyte, stem cell, HSC, or MSC.

[0100] 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.

[0101] 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).

[0102] The binding density on the tLNP can be defined based on the amount of antibody input based on the conjugation reaction or the ratio (w / w) of antibody (binding agent) to mRNA as measured in the tLNP. For intact antibodies (e.g., whole IgG), in some embodiments, the preferred ratios for the input or final measured binding 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 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 ratio. In some embodiments, if the binding agent differs in size from the intact antibody (e.g., scFv, bivalent antibody, or microantibody, etc.), the w / w ratio is adjusted for the different sizes of the binding agent.

[0103] In some implementations, the LNP or tLNP contains a binding moiety derived from an antibody containing 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 (41 BB) †‡ 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* ‡ CD45 †‡ 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 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 shown specificity are shown, from which the binding moiety can be derived; these can be found in the Therapeutic Antibody Database (TABS) at tabs.craic.com. Other suitable antibodies can be found in Appendix A. The following paragraphs provide a non-exhaustive list of known antibodies that bind to cell surface markers 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. 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.

[0104] In some implementations, tLNP targets CD2 +The cell, and the binding portion contains the antigen-binding domain of the 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 antibody includes: 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, OT14E4, or their antigen-binding portions. Additionally, the CD2 ligand CD58 (LFA-3) can be used as a CD2 binding moiety, as can afasicept (a CD58-Fc fusion compound). Each of these constitutes a means of binding CD2 (Li et al., 1996, J Mol Biol. 263:209-26; Binder et al., 2020). Front Immunol . 9:11:1090).

[0105] In some implementations, tLNP targets CD3 + The cell, and the binding portion contains an antigen-binding domain of an anti-CD3 antibody. Therefore, in some such embodiments, the antibody includes moromuzumab-CD3 (OKT3), telizumab, oxizumab, vexizumab, cevostatin, teritolumab, enastatin, pavurustatin, vexolumab, onitolumab, or their antigen-binding portions. Each of these constitutes a means for binding CD3.

[0106] In some implementations, tLNP targets CD4 + Cells, and the binding portion contains an antigen-binding domain of an anti-CD4 antibody. Therefore, in some such embodiments, the antibody includes ipalzumab, inelolizumab, semzuvolimab, zamumab, trelizumab, UB-421, priximab, MTRX1011A, cililizumab, crixaximab, keliximab, M-T413, TRX1, hB-F5, MAX.16H5, IT208, or their antigen-binding portions. Each of these constitutes a means for binding CD4.

[0107] In some implementations, tLNP targets CD5. +Cells, and the binding portion comprises an antigen-binding domain of an anti-CD5 antibody. Therefore, in some such embodiments, the antibody includes 5D7, UCHT2, L17F12, H65, HE3, OKT1, MAT304, and those disclosed in WO1989006968, WO2008121160, U.S. Patent No. 8,679,500, WO2010022737, WO2019108863, WO2022040608, or WO2022127844 (all teachings of each of which are incorporated herein by reference regarding anti-CD5 antibodies and their properties are included herein by reference), or their antigen-binding portions. Each of these constitutes a means for binding CD5.

[0108] In some implementations, tLNP targets CD7. + Cells, and the binding portion comprises an antigen-binding domain of an anti-CD7 antibody. Therefore, in some such embodiments, the antibody includes TH-69, 3A1E, 3A1F, Huly-m2, WT1, YTH3.2.6, T3-3A1, grisnilimab, and those disclosed in U.S. Patent Nos. 10,106,609, WO2017213979, WO2018098306, U.S. Serial No. 11 / 447,548, WO2022136888, WO2020212710, WO2021160267, WO2022095802, WO2022095803, WO2022151851, or WO2022257835 (all teachings of each of which regarding 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.

[0109] In some implementations, tLNP targets CD8. +Cells, and binding to an antigen-binding domain containing an anti-CD8 antibody. Therefore, in some such embodiments, the antibody includes 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 U.S. Patent No. 10,414,8 20. Those disclosed in WO2015184203, WO2017134306, WO2019032661, WO2020060924, U.S. Patent No. 10,730,944, WO2019033043, WO2021046159, WO2021127088, WO2022081516, U.S. Patent No. 11,535,869, or WO2023004304 (all the teachings of each of these concerning anti-CD8 antibodies and their properties are incorporated herein by reference), or their antigen-binding portions. Additionally, humanized anti-CD8 antibodies are described in U.S. Application Serial No. 18 / 983,294, filed December 16, 2024, and all the teachings of that document concerning these humanized anti-CD8 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of the aforementioned anti-CD8 antibodies constitutes a means for binding CD8.

[0110] In some implementations, tLNP targets CD10. + Cells, and the binding portion contains the antigen-binding domain of an anti-CD10 antibody. Therefore, in some such embodiments, the antibody includes a hybridoma-generated antibody, FR4D11, or REA877, as indicated by accession number 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 portions. Each of these constitutes a means for binding CD10.

[0111] In some implementations, tLNP targets CD11b +Cells, and the binding portion comprises an antigen-binding domain of an anti-CD11b antibody. Therefore, in some such embodiments, the antibody includes ASD141 or MAB107, and those disclosed in US20150337039, US Patent No. 10,738,121, WO2016197974, US Patent No. 10,919,967, or WO2022147338 (all 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.

[0112] In some implementations, tLNP targets CD13. + The cell, and the binding portion contains the antigen-binding domain of the anti-CD13 antibody. CD13 is also known as aminopeptidase N (APN). Therefore, in some such embodiments, the antibody includes 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 portions. Each of these constitutes a means for binding CD13.

[0113] In some implementations, tLNP targets CD14. + The cell, and the binding portion contains the antigen-binding domain of the anti-CD14 antibody. Therefore, in some such embodiments, the antibody includes atemblimab or r18D11, and those disclosed in WO2018191786 or WO2015140591 (all teachings of each of which are incorporated herein by reference regarding anti-CD14 antibodies and their properties), or their antigen-binding portions. Each of these constitutes a means for binding CD14.

[0114] In some implementations, tLNP targets CD16a +Cells, and the binding portion comprises an antigen-binding domain of an anti-CD16a antibody. Therefore, in some such embodiments, the antibody includes AFM13, sdA1, sdA2, or hu3G8-5.1-N297Q, and those disclosed in U.S. Serial Nos. 11 / 535,672, WO2018158349, WO2007009065, U.S. Serial No. 10 / 385,137, WO2017064221, U.S. Patent Nos. 10,758,625, WO2018039626, WO2018152516, WO2021076564, WO2022161314, or WO2023274183, all of which teach in each of these documents regarding anti-CD16A antibodies and their properties or antigen-binding portions thereof, and are incorporated herein by reference. Each of these constitutes a means for binding CD16a.

[0115] In some implementations, tLNP targets CD25. + The cell, and the binding portion contains an antigen-binding domain of an anti-CD25 antibody. Therefore, in some such embodiments, the antibody includes daclizumab, basiliximab, camidanlumab, tesirine, inomomab, R07296682, HuMax-TAC, CYT-91000, STI-003, RTX-003, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD25.

[0116] In some implementations, tLNP targets CD28. +Cells, and binding to an antigen-binding domain containing an anti-CD28 antibody. Therefore, in some such embodiments, the antibody includes GN1412, abatacept, lulizumab, prezalumab, theralizumab, FR104CD, and davoceticept, as well as U.S. Patent Nos. 8,454,959, 8,785,604, 11,548,947, 11,530,268, and 11,4 The contents of those disclosed in WO2002030459, WO2002047721, US20170335016, US20200181260, US Serial No. 11 / 608,376, WO2020127618, WO2021155071, or WO2022056199, all teachings of each of these documents concerning anti-CD28 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding CD28.

[0117] In some implementations, tLNP targets CD29. + The cell, and the binding portion contains an antigen-binding domain of an anti-CD29 antibody. Therefore, in some such embodiments, the antibody includes OS2966, 6D276, 12G10, REA1060, or their antigen-binding portions. Each of these constitutes a means for binding CD29.

[0118] In some implementations, tLNP targets CD32A. + Cells, and the binding portion comprises an antigen-binding domain of an anti-CD32A antibody. Therefore, in some such embodiments, the antibody includes VIB9600, humanized IV.3, humanized AT-10, or MDE-8, and those disclosed in U.S. Patent Nos. 9,688,755, 9,284,375, 9,382,321, U.S. Serial No. 11 / 306,145, or WO2022067394, all of which teach in each of these documents regarding anti-CD32A antibodies and their properties or antigen-binding portions thereof, and all such teachings are incorporated herein by reference. Each of these constitutes a means for binding CD32A.

[0119] In some implementations, tLNP targets CD34. +The cell, and the binding portion contains an antigen-binding domain of an anti-CD34 antibody. Therefore, in some such embodiments, the antibody includes h4C8, 9C5, 2E10, 5B12, REA1164, C5B12, C2e10, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD34.

[0120] In some implementations, tLNP targets CD40. + Cells, and the binding portion comprises an antigen-binding domain of an anti-CD40 antibody. Therefore, in some such embodiments, the antibody includes cifurtilimab, sotilimab, iscalimab, dacetuzumab, selicrelumab, bleselumab, lucarumumab, or mitazalimab, as disclosed in U.S. Serial No. 10 / 633,444, all of which teach in each of these documents regarding anti-CD40 antibodies and their properties or their antigen-binding portion are incorporated herein by reference. Each of these constitutes a means for binding CD40.

[0121] In some implementations, tLNP targets CD44. + The cell, and the binding portion contains the antigen-binding domain of the anti-CD44 antibody. Therefore, in some such embodiments, the antibody includes RO5429083, VB6-008, PF-03475952, or RG7356, as well as those disclosed in WO2008144890, U.S. Patent No. 8,383,117, WO2008079246, US20100040540, WO2015076425, U.S. Patent No. 9,220,772, US20140308301, WO2020159754, WO2021160269, WO2021178896, WO2022022749, WO2022022720, or WO2022243838, all of which teach in each of these documents regarding anti-CD44 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding CD44.

[0122] In some implementations, tLNP targets CD45. +Cells, and the binding portion comprises an antigen-binding domain of an anti-CD45 antibody. Therefore, in some such embodiments, the antibody includes apamistamab, BC8-B10, and those disclosed in WO2023183927, WO2023235772, U.S. Patent Nos. 7,825,222, WO2017009473, WO2021186056, U.S. Patent Nos. 9,701,756, 9,701,756, WO2020092654, WO2022040088, WO2022040577, WO2022064191, WO2022063853, or WO2024064771, all of which teach in each of these documents regarding anti-CD45 antibodies and their properties or their antigen-binding portion are incorporated herein by reference. Each of these constitutes a means for binding CD45.

[0123] In some implementations, tLNP targets CD56. + Cells, and the binding portion comprises an antigen-binding domain of an anti-CD56 antibody. Therefore, in some such embodiments, the antibody includes lorvotuzumab, adcitmer, or promiximab, as well as those disclosed in WO2012138537, U.S. Patent Nos. 10,548,987, 10,730,941, or US20230144142, all of which teach in each of these documents regarding anti-CD56 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding CD56.

[0124] In some implementations, tLNP targets CD64. + The cell, and the binding portion comprises an antigen-binding domain of an anti-CD64 antibody. Therefore, in some such embodiments, the antibody includes HuMAb 611 or H22, and those disclosed in U.S. Patent Nos. 7,378,504, WO2014083379, US20170166638, or WO2022155608, all of which teach in each of these documents regarding anti-CD64 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding CD64.

[0125] In some implementations, tLNP targets CD68. +The cell, and the binding portion contains an antigen-binding domain of an anti-CD68 antibody. Therefore, in some such embodiments, the antibody includes Ki-M7, PG-M1, 514H12, ABM53F5, 3F7C6, 3F7D3, Y1 / 82A, EPR20545, CDLA68-1, LAMP4-824, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD68.

[0126] In some implementations, tLNP targets CD70. + Cells, and the binding portion contains an antigen-binding domain comprising an anti-CD70 antibody. Therefore, in some such embodiments, the antibody includes cusatuzumab, vorsetuzumab, MDX-1203, MDX-1411, AMG-172, SGN-CD70A, ARX305, PRO1160, and U.S. Patent Nos. 9,765,148, 8,124,738, 110,266,604, WO2021138264, 9,701,752, 10,108,123, WO2014158821, 10,689,456, WO2017062271, and 1... The following documents, disclosed in U.S. Patent Nos. 1,046,775, 11,377,500, WO2021055437, WO2021245603, WO2022002019, WO2022078344, WO2022105914, WO2022143951, WO2023278520, WO2022226317, WO2022262101, U.S. Patent No. 11,613,584, or WO2023072307, all teachings of each of these documents concerning anti-CD70 antibodies and their properties or antigen-binding portions thereof are incorporated herein by reference. Each of these constitutes a means for binding CD70.

[0127] In some implementations, tLNP targets CD73. +Cells, and binding portions containing antigen-binding domains of anti-CD73 antibodies. Therefore, in some such embodiments, the antibodies include oleclumab, ultraedlimab, mupadolimab, AK119, IB1325, BMS-986179, NZV930, JAB-BX102, Sym024, TB19, TB38, HBM1007, 3F7, mAb19, Hu001-MMAE, IPH5301, or INCA00186, and U.S. Patent No. 9,990. 38,356, 10,584,169, WO2022083723, WO2022037531, WO2021213466, WO2022083049, US Patent No. 10,822,426, WO2021259199, US Patent No. 10,100,129, 11,312,783, 11,174,319, 11,634,500, WO2021138467, WO2017118613, US Patent No. 9,388,249 WO2020216697, US Serial No. 11 / 180,554, US Patent No. 11,530,273, WO2019173692, WO2019170131, US Patent No. 11,312,785, WO2020098599, WO2020143836, WO2020143710, US Patent No. 11,034,771, 11,299,550, WO2020253568, WO2021017892, WO202103 The contents of WO2021032173, WO2021097223, WO2021205383, WO2021227307, WO2021241729, WO2022096020, WO2022105881, WO2022179039, WO2022214677, or WO2022242758, all of which teach in each of these documents regarding anti-CD73 antibodies and their properties or their antigen-binding portions, are incorporated herein by reference. Each of these constitutes a means for binding CD73.

[0128] In some implementations, tLNP targets CD90. + The cell, and the binding portion contains an antigen-binding domain of an anti-CD90 antibody. Therefore, in some such embodiments, the antibody includes REA897, OX7, 5E10, K117, L127, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD90.

[0129] In some implementations, tLNP targets CD105. + Cells, and the binding portion comprises an antigen-binding domain of an anti-CD105 antibody. Therefore, in some such embodiments, the antibody includes carotuximab, TRC205, or huRH105, as well as those disclosed in U.S. Patent Nos. 8,221,753, 9,926,375, WO2010039873, WO2010032059, WO2012149412, WO2015118031, WO2021118955, US20220233591, or US20230075244, all of which teach in each of these documents regarding anti-CD105 antibodies and their properties or their antigen-binding portion are incorporated herein by reference. Each of these constitutes a means for binding CD105.

[0130] In some implementations, tLNP targets CD117. + Cells, and binding to an antigen-binding domain containing an anti-CD117 antibody. Therefore, in some such embodiments, the antibody includes briquilimab, barzolvolimab, CDX-0158, LOP628, MGTA-117, NN2101, CK6, JSP191, Ab85, 104D2, or SR1, and U.S. Patent Nos. 7,915,391, WO2022159737, 9,540,443, WO2015050959, 9,789,203, 8,552,157, 10,406,179, 9 The contents of those disclosed in U.S. Patent Nos. 10,611,838, 10,2020076105, 10,2021107566, 11,208,482, 10,21044008, 10,21099418, 10,22187050, or 20,23026791, 10,21188590, all of which teach in each of these documents concerning anti-CD117 antibodies and their properties or antigen-binding portions thereof, are incorporated herein by reference. Each of these constitutes a means for binding CD117.

[0131] In some implementations, tLNP targets CD133. +Cells, and the binding portion comprises an antigen-binding domain of an anti-CD133 antibody. Therefore, in some such embodiments, the antibody includes AC133, 293C3, CMab-43, or RWO3, as well as those disclosed in WO2018045880, U.S. Patent Nos. 8,722,858, 9,249,225, WO2014128185, U.S. Patent Nos. 10,711,068, 10,106,623, WO2018072025, or WO2022022718, all of which teach in each of these documents regarding anti-CD133 antibodies and their properties or their antigen-binding portion are incorporated herein by reference. Each of these constitutes a means for binding CD133.

[0132] In some implementations, tLNP targets CD137. + Cells, and binding to a portion containing an antigen-binding domain of an anti-CD137 antibody. CD137 is also known as 4-11BB. Therefore, in some such embodiments, the antibody includes YH004, urelumab (BMS-663513), utomilumab (PF-05082566), ADG106, LVGN6051, PRS-343, and WO2005035584, WO2012032433, WO2017123650, US Patent Nos. 11,203,643, 11,242,395, 11,555,077, U... Those disclosed in US Patent Nos. 120230067770, 11,535,678, 11,440,966, WO2019092451, 10,174,122, 11,242,385, 10,716,851, WO2020011966, WO2020011964, or 11,447,558, all teachings of each of these documents concerning anti-CD137 antibodies and their properties or antigen-binding portions thereof are incorporated herein by reference. Each of these constitutes a means for binding CD137.

[0133] In some implementations, tLNP targets CD146. +Cells, and the binding portion comprises an antigen-binding domain of an anti-CD146 antibody. Therefore, in some such embodiments, the antibody includes imaprelimab, ABX-MA1, huAA98, M2H, or IM1-24-3, and those disclosed in U.S. Patent Nos. 10,407,506, 10,414,825, 6,924,360, 9,447,190, WO2014000338, U.S. Patent Nos. 9,782,500, WO2018220467, U.S. Patent Nos. 11,427,648, WO2019133639, WO2019137309, WO2020132190, or WO2022082073, all of which teach in each of these documents regarding CD146 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding CD146.

[0134] In some implementations, tLNP targets CD166. + Cells, and the binding portion comprises an antigen-binding domain of an anti-CD166 antibody. Therefore, in some such embodiments, the antibody includes praluzatamab, AZN-L50, REA442, or AT002, as well as those disclosed in U.S. Patent Nos. 10,745,481, 11,220,544, or WO2008117049, all of which teach, by reference, all matters relating to CD166 antibodies and their properties or their antigen-binding portions herein. Each of these constitutes a means for binding CD166.

[0135] In some implementations, tLNP targets CD200. + Cells, and the binding portion contains an antigen-binding domain of an anti-CD200 antibody. Therefore, in some such embodiments, the antibody includes samalizumab, OX-104, REA1067, B7V3V2, HPAB-0260-YJ, or TTI-CD200, as well as those disclosed in WO2007084321 or WO2019126536, all of which teach in each of these documents regarding CD200 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding CD200.

[0136] In some implementations, tLNP targets CD205. +The cell, and the binding portion contains an antigen-binding domain of an anti-CD205 antibody. CD205 is also known as DEC205. Thus, in some such embodiments, the antibody includes 3G9-2D2 (a component of CDX-1401) or LY75_A1 (a component of MEN1309) and those disclosed in U.S. Patent Nos. 8,236,318, 10,081,682 or 11,365,258, all of which teach in each of these documents regarding anti-CD205 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding CD205.

[0137] In some implementations, tLNP targets CD271. + The cell, and the binding portion contains the antigen-binding domain of the anti-CD271 antibody. Therefore, in some such embodiments, the antibody includes REA844 or REAL709, as well as those disclosed in WO2022166802, all of which teaches about anti-CD271 antibodies and their properties or their antigen-binding portions and is incorporated herein by reference. Each of these constitutes a means for binding CD271.

[0138] In some implementations, tLNP targets BMPR2. + Cells, and the binding portion contains an antigen-binding domain of an anti-BMPR2 antibody. Therefore, in some such embodiments, the antibody includes those disclosed in TAB-071 CL (Creative Biolabs catalog number) and U.S. Patent Nos. 11,292,846 or WO2021174198, all of which teach, by reference, all contents relating to anti-BMPR2 antibodies and their properties or their antigen-binding portions herein. Each of these constitutes a means for binding BMPR2.

[0139] In some implementations, tLNP targets CTLA-4. +Cells, and the binding portion contains the antigen-binding domain of the anti-CTLA-4 antibody. Therefore, in some such embodiments, the antibody includes 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.

[0140] In some implementations, tLNP targets GD2. + Cells, and binding to an antigen-binding domain comprising an anti-GD2 antibody. Therefore, in some such embodiments, the antibody includes dinutuximab, ganglidiximab, naxitamab, nivatrotamab, EMD 273063, hu14.18k322A, MORAb-028, 3F8BiAb, BCD-245, KM666, ATL301, Ektomab, and U.S. Patent Nos. 9,777,068, 9,315,585, WO2004055056, 9,617,349, 9,493,740, US20210002384, and U.S. Patent Nos. 8,5 Those disclosed in WO2001023573, WO2012071216, WO2018010846, U.S. Patent No. 8,951,524, WO2023280880, U.S. Patent No. 9,856,324, WO2015132604, WO2017055385, WO2019059771, and WO2020020194, or their antigen-binding portions. Each of these constitutes a means for binding GD2.

[0141] In some implementations, tLNP targets GITR +Cells, and the binding portion contains an antigen-binding domain of an anti-GITR antibody. Therefore, in some such embodiments, the antibody includes ragifilimab, TRX518, MK-4166, AMG 228, MED11873, BMS-986156, REGN6569, ASP1951, MK-1248, FRA154, GWN323, JNJ-64164711, ATOR-1144, or an antigen-binding portion thereof. Each of these constitutes a means for binding GITR.

[0142] In some implementations, tLNP targets the low-affinity IL-2 receptor. + Cells (CD122) + and / or CD132 + The antibody comprises an antigen-binding domain of an anti-IL-2 receptor antibody. Therefore, in some such embodiments, the anti-CD122 antibody includes the anti-CD122 antibody or its antigen-binding portion disclosed in ANV419, FB102, MiK-β-1, and WO2011127324, WO2017021540, WO2022212848, WO2022221409, WO2023078113, US20230272090, and WO2024073723. Therefore, in some such embodiments, the anti-CD132 antibody includes the anti-CD132 antibody or its antigen-binding portion disclosed in REGN7257 and WO2020160242, WO2017021540, WO2022212848, WO2023078113, and US20230272089. Each of these constitutes a means for binding to low-affinity IL-2 receptors (CD122 or CD132, depending on the case).

[0143] In some implementations, tLNP targets the high-affinity IL-2 receptor. + Cells (CD25) +The antibody comprises an antigen-binding domain of an anti-IL-2 receptor antibody. Therefore, in some such embodiments, the antibody includes daklizumab, baliximab, calimumab, vopitug, enomozumab, HuMAx-TAC, Xenopax, STI-003, RA8, RTX-003, and the anti-CD25 antibody or its antigen-binding portion disclosed in WO2023031403, WO2006108670, WO2019175223, WO2019175215, WO2019175226, WO2004045512, WO2022104009, and WO2020102591. Each of these constitutes a means for binding to a high-affinity IL-2 receptor (CD25).

[0144] In some implementations, tLNP targets the IL-7 receptor. + (CD127) + The antibody binds to cells and the binding portion comprises an antigen-binding domain of an anti-IL-7 receptor antibody. Therefore, in some such embodiments, the antibody includes anti-CD127 antibodies or their antigen-binding portions disclosed in WO2011104687, WO2011094259, WO2013056984, WO2015189302, WO2017062748, WO2020154293, WO2020254827, WO2021222227, and WO2023201316. Each of these constitutes a means for binding CD127.

[0145] In some implementations, tLNP targets the IL-12 receptor. + The cell, and the binding portion contains an antigen-binding domain of an anti-IL-12 receptor antibody. Therefore, in some such embodiments, the antibody includes CBYY-10413, REA333, or their antigen-binding portions. Each of these constitutes a means for binding to the IL-12 receptor.

[0146] In some implementations, tLNP targets IL-15 receptor α + Cells, and the binding portion comprises an antigen-binding domain of an anti-IL-15 receptor α antibody. Therefore, in some such embodiments, the antibody includes MAB1472-100, MAB5511, JM7A4, 5E3E1, JM7A4, 2639B, or its antigen-binding portion. Each of these constitutes a means for binding IL-15 receptor α.

[0147] In some implementations, tLNP targets IL-18 receptor α + The cell, and the binding portion contains an antigen-binding domain of an anti-IL-18 receptor α antibody. Therefore, in some such embodiments, the antibody includes H44 or its antigen-binding portion. Each of these constitutes a means for binding IL-18 receptor α.

[0148] In some implementations, tLNP targets the IL-21 receptor. + The cell, and the binding portion comprises an antigen-binding domain of an anti-IL-21 receptor antibody. Therefore, in some such embodiments, the antibody includes 1D1C2, 19F5, 18A5, REA233, or an antigen-binding portion thereof. Each of these constitutes a means for binding to the IL-21 receptor α.

[0149] In some implementations, tLNP targets LAG-3. + Cells, and the binding portion contains an antigen-binding domain of an anti-LAG-3 antibody. Therefore, in some such embodiments, the antibody includes relatlimab, tebotelimab, favezelimab, fianlimab, miptenalimab, HLX26, ieramilimab, GSK2831781, INCAGN2385, R07247669, encelimab, FS118, SHR-1802, Sym022, IB1110, 1B1323, bavunalimab, EMB-02, ABL501, INCA32459, AK129, or an antigen-binding portion thereof. Each of these constitutes a means for binding to LAG-3.

[0150] In some implementations, tLNP targets MSCA-1 + The cell, and the binding portion contains an antigen-binding domain of an anti-MSCA-1 antibody. Therefore, in some such embodiments, the antibody includes REAL219, W8B2, X9C3, or its antigen-binding portion. Each of these constitutes a means for binding to MSCA-1.

[0151] In some implementations, tLNP targets OX40. +Cells, and the binding portion contains an antigen-binding domain of an anti-OX40 antibody. Therefore, in some such embodiments, the antibody includes MED16469, ivuxolimab, rocatinlimab, GSK3174998, BMS-986178, vonlerizumab, INCAGN1949, tavolimab, BGB-A445, INBRX-106, BAT6026, telazorlimab, ATOR-1015, MED16383, cudarolimab, FS120, HFB301001, EMB-09, HLX51, Hu222, ABM193, or an antigen-binding portion thereof. Each of these constitutes a means for binding OX40.

[0152] In some implementations, tLNP targets PD-1 +Cells, and binding to an antigen-binding domain containing an anti-PD-1 antibody. Therefore, in some such embodiments, the antibody includes nivolumab, pembrolizumab, camrelizumab, toripalimab, sintilimab, tislelizumab, cemiplimab, spartalizumab, serplulimab, cadonilimab, penpulimab, dostarlimab, zimberelimab, retifanlimab, and puctolimab. nlimab), pidilizumab, balstilimab, ezabenlimab, AK112, geptanolimab, cetrelimab, prolgolimab, tebotelimab, sasanlimab, SG001, vudalimab, MED15752, rulonilimab, peresolimab, IB1318, budigalimab, MED10680, pimivalimab, QL1706, AMG 404, RO7121661, lorigerlimab, nofazinlimab, sindelizumab, or their antigen-binding moiety. Each of these constitutes a means for binding PD-1.

[0153] In some implementations, tLNP targets PODXL + The cell, and the binding portion contains an antigen-binding domain of an anti-PODXL antibody. Therefore, in some such embodiments, the antibody includes MA11738, HPAB-3334LY, HPAB-M0612-YC, REA246, REA157, or their antigen-binding portions. Each of these constitutes a means for binding PODXL.

[0154] In some implementations, tLNP targets Sca-1. +The cell, and the binding portion contains an antigen-binding domain of an anti-Sca-1 antibody. Therefore, in some such embodiments, the antibody includes CPP32 4-1-18, 2D4-C9-F1, AMM22070N, or its antigen-binding portion. Each of these constitutes a means for binding SCA-1.

[0155] In some implementations, tLNP targets SSEA-3. + Cells, and the binding portion comprises an antigen-binding domain of an anti-SSEA-3 antibody. Therefore, in some such embodiments, the antibody includes MC631, 2A9, 8A7, ND-742, 3H420, and those disclosed in U.S. Patent Nos. 11,643,456 or WO2021138378, all of which teach in each of these documents regarding anti-SSEA-3 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding SSEA-3.

[0156] In some implementations, tLNP targets SSEA-4. + Cells, and the binding portion comprises an antigen-binding domain of an anti-SSEA-4 antibody. Therefore, in some such embodiments, the antibody includes ch28 / 11, REA101, MC-813-70, ND-942-80, and those disclosed in U.S. Patent Nos. 11,446,379, 10,273,295, 11,643,456, WO2019190952, or WO2021044039, all of which teach in each of these documents regarding anti-SSEA-4 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding SSEA-4.

[0157] In some implementations, tLNP targets Stro-1. + The cells, and the binding portion contains an antigen-binding domain of an anti-Stro-1 antibody. Therefore, in some such embodiments, the antibody includes STRO-1, TUSP-2, and those disclosed in US20130122022, all of which teaches about anti-Stro-1 antibodies and their properties or their antigen-binding portions and is incorporated herein by reference. Each of these constitutes a means for binding Stro-1.

[0158] In some implementations, tLNP targets Stro-4. +Cells, and the binding portion contains an antigen-binding domain of an anti-Stro-4 antibody. Therefore, in some such embodiments, the antibody includes STRO-4, ivengumab, 4C5, and those disclosed in U.S. Patent Nos. 7,722,869, US20110280881, 9,115,192, 10,273,294, 10,457,726, and WO2023091148, all of which teach in each of these documents regarding anti-Stro-4 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding Stro-4 (also known as heat shock protein-90).

[0159] In some implementations, tLNP targets SUSD2. + Cells, and the binding portion contains an antigen-binding domain of an anti-SUSD2 antibody. Therefore, in some such embodiments, the antibody includes REA795, CBXS-3571, CBXS-1650, CBXS-1989, CBXS-1671, CBXS1990, CBXS-3676, 1279B, EPR8913(2), W5C5, or an antigen-binding portion thereof. Each of these constitutes a means for binding SUSD2.

[0160] In some implementations, tLNP targets TIM-3. + Cells, and the binding portion contains an antigen-binding domain of an anti-TIM-3 antibody. Therefore, in some such embodiments, the antibody includes TQB2618, sabatolimab, cobolimab, R07121661, INCAGN02390, AZD7789, surzebiclimab, LY3321367, Sym023, BMS-986258, SHR-1702, LY3415244, LB1410, or an antigen-binding portion thereof. Each of these constitutes a means for binding TIM-3.

[0161] In some implementations, tLNP targets TREM2. +Cells, and the binding portion contains the antigen-binding domain of the anti-TREM2 antibody. Therefore, in some such embodiments, the antibody includes P137012 and those disclosed in U.S. Patent Nos. 10,508,148, 10,676,525, WO2017058866, 11,186,636, 11,124,567, WO2020055975, 11,492,402, WO2020121195, WO2023012802, WO2021101823, WO2023047100, WO2022032293, WO2022241082, WO2023039450, or WO2023039612, all of which teach in each of these documents regarding anti-TREM2 antibodies and their properties or their antigen-binding portions are incorporated herein by reference. Each of these constitutes a means for binding TREM2.

[0162] 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 as described above. In other embodiments, tLNP targets some other tumor antigens, such as those listed in International Application Publication No. WO2024040195A1, all of which teaches the use of tLNP consistent with this disclosure for the delivery of nucleic acids to tumor cells and is incorporated herein by reference.

[0163] Methods for preparing LNP or tLNP 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. The aqueous solution may be buffered at a pH of about 3 to about 5, for example, but not limited to, citrate or acetate. In various embodiments, the alcohol may be ethanol, isopropanol, tert-butanol, or combinations thereof. In some embodiments, rapid mixing may be achieved by pumping the two solutions through a T-connector or using an impingement jet mixer. Microfluidic mixing via a staggered herringbone mixer (SHM) or a hydrodynamic mixer (microfluidic hydrodynamic focusing), a microfluidic bifurcation mixer, and a microfluidic baffle mixer may also be used. After the LNPs are formed, they may be diluted with a buffer (e.g., phosphate, HEPES, or Tris) in a pH range of about 6 to about 8.5 to reduce the alcohol (ethanol) concentration. Diluted LNP can be purified by tangential flow filtration (TFF) using a buffer (e.g., phosphate, HEPES, or Tris) with a pH range of about 6 to about 8.5 to remove alcohol, via dialysis, ultrafiltration, or percolation. Alternatively, size exclusion chromatography can be used. Once the alcohol has been completely removed, the buffer can be replaced with a similar buffer containing a cryoprotectant (e.g., glycerol or a sugar such as sucrose, trehalose, or mannose). The LNP can be concentrated to the desired concentration, then filtered through a 0.2 μm filter, such as polyethersulfone (PES) or a modified PES filter, filled into glass vials, stoppered, capped, and frozen for storage. In an alternative embodiment, a lyophilization protectant can be used, and the LNP can be lyophilized for storage rather than as a cryoprotectant. Further methods for preparing LNPs can be found in, for example, US20200297634, US20130115274 and WO2017 / 048770, all of which teach about the generation of LNPs and are incorporated herein by reference.

[0164] 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 in which the PEG moiety has been derivatized with a chemically reactive group (such as maleimide, NHO ester, Cys, azide, alkyne, 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, for example, in Example 1 and Parhiz et al., J. Controlled Release 291:106-115, 2018 and Tombacz et al., Molecular Therapy The full content of the literature teaching on the conjugation of PEG-lipids with binding moieties, found in 29(11):3293-3304, 2021, is incorporated herein by reference. Alternatively, the targeting moieties may be conjugated with PEG-lipids prior to the insertion of the pre-formed LNP.

[0165] In some embodiments of the tLNP preparation method, the method includes: 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 fractions) in proportions disclosed herein; ii) Preconjugated tLNPs are formed by mixing initial LNPs with one or more functionalized PEG-lipids; and iii) A tLNP is formed by concatenating a pre-concatenated tLNP with one or more target portions.

[0166] In some embodiments of the tLNP preparation method, the method includes: 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 ii) Forming a tLNP by concatenating a pre-concatenated tLNP with one or more target portions.

[0167] In some embodiments of the tLNP preparation method, the method includes: i) Forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids with one or more targeted moieties; and ii) tLNP is formed by mixing all components of tLNP (including one or more conjugated functionalized PEG-lipids) in proportions disclosed herein.

[0168] In some embodiments of the tLNP preparation method, the method includes: i) Forming one or more conjugated functionalized PEG-lipids by conjugating one or more functionalized PEG-lipids with one or more targeting moieties; ii) To form LNP by mixing all components of tLNP (excluding one or more conjugated functionalized PEG-lipids) in the proportions disclosed herein; and iii) tLNP is formed by mixing the initial LNP with one or more conjugated functionalized PEG-lipids.

[0169] After conjugation, as disclosed above for LNP, tLNP is purified and stored by dialysis, tangential flow filtration or size exclusion chromatography.

[0170] 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%.

[0171] Methods for delivering payloads into cells In some aspects, this document discloses methods for delivering nucleotides (or other negatively charged payloads) into cells, comprising contacting cells with tLNPs of any of the foregoing aspects. Therefore, each of the genera, subgenera, and / or species of LNPs or tLNPs disclosed herein (including those based on the inclusion or exclusion of specific lipids, specific lipid compositions, specific payloads, and / or specific targeting moieties) can be used to define the scope of methods for delivering payloads into cells. In some embodiments, contact occurs ex vivo. In some embodiments, contact occurs in vivo. In some cases, in vivo contact includes intravenous, intramuscular, subcutaneous, intranodal, or intralymphatic administration. In further cases, hepatocyte transfection is reduced compared to tLNPs containing conventional ionizable cationic lipids such as ALC-0315.

[0172] 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 cessation. 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.

[0173] 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, well suited for delivery via LNP or tLNP, and single RNA molecules encoding specific polypeptides are disclosed in U.S. Application Serial No. 18 / 934,237, filed November 1, 2024, all of which teaches about nucleic acid payloads for in vivo transfection and their design, and is incorporated herein by reference.

[0174] 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 FIT3, GM-CSF, and G-CSF.

[0175] 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.

[0176] 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 above. As noted, such antibodies, individually and collectively, constitute a means of binding to immune cells (or leukocytes) or lymphocytes or monocytes.

[0177] 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.

[0178] Treatment 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.

[0179] 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.

[0180] 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.

[0181] 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, Graves' disease. Stokes disease, 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 autoimmune diseases, 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.

[0182] In some implementations, autoimmune diseases are T-cell-mediated or B-cell-mediated autoimmune diseases. In some cases, B-cell-mediated autoimmune diseases include myositis (such as antisynthetic 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, antisynthetic syndrome, polymyositis, systemic sclerosis, diffuse cutaneous systemic sclerosis, localized cutaneous systemic sclerosis, antisynthetic syndrome (idiopathic inflammatory myopathy), multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, inactive secondary progressive multiple sclerosis, Sjögren's syndrome, IgA nephropathy, or Fanconi anemia. In some embodiments, the B-cell-mediated autoimmune disease is myositis, lupus nephritis, membranous nephropathy, scleroderma, systemic lupus erythematosus, myasthenia gravis, ANCA-associated vasculitis, multiple sclerosis, or pemphigus vulgaris. In some embodiments, the B-cell-mediated autoimmune disease is myositis. In some cases, the myositis is antisynthetic enzyme myositis. In some embodiments, the B-cell-mediated autoimmune disease is systemic lupus erythematosus, myasthenia gravis, ANCA-associated vasculitis, multiple sclerosis, or pemphigus vulgaris.

[0183] 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, consuming large numbers of B cells can help prevent allogeneic graft rejection.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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, one or more 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. In some embodiments, the CAR may comprise an extracellular binding domain specifically binding to a target antigen, a transmembrane domain, and one or more intracellular signal transduction domains. In some embodiments, the CAR may also comprise 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 antibodies, TCR, CD8, or other type 1 membrane proteins, preferably proteins 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 chains, 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. The intracellular signal transduction domain may be derived from the CD3ζ chain, DAP10, DAP12, FcγRIII, FcsRI, or an immune receptor tyrosine-based activation motif (ITAM) carrying a cytoplasmic domain, and other possibilities known in the art. Intracellular costimulatory domains may be derived from CD27, CD28, 4-1 BB, OX40, or ICOS, as well as other possibilities known in the art.

[0188] 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 those disclosed in U.S. Patent Nos. 7,446,190 (anti-CD19), 10,287,35 (anti-CD19), US2021 / 0363245 (anti-CD19 and anti-CD20), 10,543,263 (anti-CD22), 10,426,797 (anti-CD33), 10,844,128 (anti-CD123), 10,428,141 (anti-ROR1), and US2021 / 0087295 (anti-FAP). To the extent that they do not contradict this disclosure, all teachings in each of these documents regarding CAR structure and function, as well as regarding CAR antigen specificity and target indications, are incorporated herein by reference.

[0189] Exemplary target antigens that may be specific to CAR, TCR, or ICE include, but are not limited to, B cell maturation agents (BCMA). †‡ CA9 †‡ CD5 †‡ CD19* †‡ CD20 (MS4A1)* †‡ CD22* †‡ CD23* †‡ CD30 (TNFRSF8)* †‡ CD33* †‡ CD38* †‡ CD44* ‡ CD70* †‡ CD133 ‡ CD174, CD274 (PD-L1)* †‡ CD276 (B7-H3) †‡ CEACAM5* †‡ CLL1V, CSPG4* ‡ EGFR* †‡ EGFRvIII*, EPCAM* †‡ EPHA2* ‡ ERBB2*‡ FAP* †‡ FOLH1, FORR1* †‡ GD2* †‡ GPC3* †‡ ,GPNMB* ‡ IL1RAP †‡ IL3RA* ‡ IL13RA2* ‡ κ*, KDR (VEGFR2)* ‡ CD171 (L1CAM)* ‡ ,λ*,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* †‡ HER2* †‡ EGFR* †‡ , EGFRvIII*, CD276 (B7H3) †‡ PSMA* †‡ PSCA ‡ CAIX (CA9) †‡ CD171 (L1-CAM)* ‡ CEA* ‡ CSPG4* ‡ EPHA2* ‡ FAP* †‡ LRRC15 †‡ ,FOLR1* †‡ IL-13Rα* †‡ mesothelin* †‡ MUC1*†‡ MUC16* †‡ 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 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 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 are available in Appendix A. 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.

[0190] 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 tesalonide, lekimeronide, akimeronide, or brenucide. 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).

[0191] 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, oxotuzumab, teimozumab, oflamumab, tositumumab, onituzumab, vetouzumab, ututuzumab, 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). The entire contents of each of the foregoing references in this paragraph (all teachings concerning 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 an RNA-encoded CD20 CAR payload and having a T-cell targeting portion (such as an anti-CD8 antibody).

[0192] 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 number 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 number 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 number 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 BCMACAR comprises scFv derived from CT103A (or CAR0085), as described in U.S. Patent No. 11,026,975B2.Other anti-BCMA CARs are disclosed in U.S. Patent Application Publications 2020 / 0246381 and 2020 / 0339699. Other anti-BCMA CARs include Allo-605 (described in U.S. Patent Publication 20200261503), CT053 (described in U.S. Patent Publication 11,525,006), Descartes-08 (described in U.S. Patent Publication 10,934,337), LCAR-B38M (described in U.S. Patent Publication 10,934,363), PersonGen anti-BCMA CAR (described in CN114763383), PregeneBio anti-BCMA CAR (described in U.S. Patent Publication US20220218746), the CAR in Cedar-Loncin (described in the binding portion of US20170051068), and the CAR in Acilancin (described in U.S. Patent Publication 10,383,929). Other antibodies containing an anti-BCMA antigen-binding domain that can be used to construct CARs include AMG224 (described in US Patent No. 9,243,058 along with other anti-BCMA antibodies), EMB-06 (described in US Patent Publication No. US20230002489 along with other anti-BCMA antibodies), HPN217 (described in US Patent No. 11,136,403), MED12228 (described in US Patent No. 10,988,546), REGN5459 (described in US Patent No. 11,384,153), SAR445514 (described in US Patent Publication No. 20240034816), SEA-BCMA (described in US Patent No. 11,078,291), and TNB-383B (described in US Patent No. 11,078,291). The following are patents: patent number 11,505,606, TQB2934 (described in U.S. Patent Publication No. 20230193292), WV078 (described in U.S. Patent No. 11,492,409), anucatumab (described in U.S. Patent No. 10,683,369), belantumab (described in U.S. Patent No. 9,273,141), enatumab (described in U.S. Patent No. 11,814,435), ispectamab (described in U.S. Patent Publication No. 20210130483), rivosaitumab (described in U.S. Patent No. 11,919,965), pavurutumab (described in U.S. Patent No. 11,419,933), and teritumab (described in U.S. Patent No. 10,072,088). The entire contents of each of the preceding references in this paragraph (all teachings on the design, structure, and activity of anti-BCMA CARs and anti-BCMA antibodies that provide antigen-binding domains for CARs or immune cell connectors) are incorporated herein by reference.In any of the above-described tLNP implementations, some implementations include tLNPs encapsulated with an RNA-encoded BCMA CAR payload and having a T-cell targeting portion (such as an anti-CD8 antibody).

[0193] 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 nucleotide molecules, including mRNA, and delivered to the subject using the LNP and tLNP compositions and formulations disclosed herein.

[0194] 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 nucleotide 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.

[0195] The term "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 implementations, 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 prescription set, or exclusion of alternative drugs from a drug prescription set, or providing financial incentives for the use of the drug (as may be done by an insurance company or pharmacy benefit management company), etc. 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.

[0196] 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. The effective dose or amount of the compounds or compositions disclosed herein can be readily determined by a person skilled in the art by considering all criteria (e.g., the rate of excretion of the compound or composition used, the pharmacodynamics of the compound or composition used, the nature of other compounds contained in the composition, the particular route of administration, the specific characteristics of the individual, medical history and risk factors (e.g., age, weight, general health status, etc.), the individual's response to treatment, or any combination thereof) and utilizing their best judgment representing the individual. Exemplary doses are also disclosed in the examples below.

[0197] Tolerance 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 LNP containing the ionizable cationic lipid ALC-0315 used in the study caused an increase in liver enzyme and acute-phase protein levels in rats at a single dose of 1 mg / kg. Only antibody attachment to the baseline LNP partially reversed this increase, and the reversal was greater if the antibody directed the LNP to some other tissue (i.e., tLNP). However, for LNP, antibody-conjugated LNP, and tLNP, the use of the ionizable cationic lipids disclosed herein reduced delivery to the liver and associated liver enzyme and acute-phase protein levels to a greater extent. The tLNP composition containing CICL1 is generally well tolerated in rats and non-human primates (NHP) at single doses up to at least 3 mg / kg. Example

[0198] The following examples are intended to illustrate various embodiments of the invention. Therefore, the specific embodiments discussed should not be construed as limiting the scope of the invention. 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 the invention, 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, provided that they do not contradict or are inconsistent with this disclosure. Some embodiments utilize donor materials, and the donors are individually numbered in each embodiment, such that donor 1 in one embodiment is not necessarily the same as or different from donor 1 in another embodiment or donor 2 in another embodiment, unless otherwise expressly stated.

[0199] Example 1: Establishing a benchmark Relatively little experience exists in the tLNP field, and much of it relies on compositions and formulations developed for non-targeted LNPs. The first step to achieving superior performance is establishing a baseline from which improvements in one or more parameters can be assessed. One of the main determinants of LNP encapsulation and transfection efficiency is the cationic lipid used. Therefore, tLNPs incorporating CICL1 plus functionalized PEG-lipids into a base lipid composition will be compared with tLNPs containing one of the seven known ionizable cationic lipids in the art within the same lipid composition. The payload is a CleanCap encoding the fluorescent protein mCherry (Trilink). ® mCherry 5-methoxyuridine (5 moU) mRNA.

[0200] Ionizable cationic lipids used for benchmarking include [(4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid, 1-octylnonyl ester (SM-102), (6Z,9Z,28Z,31Z)-heptadecano-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC-3) (each a component of an FDA-approved product), and bis(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonamido)nonadecanedioate (lipid A9). Ionizable cationic lipids 10a, 10f, and 10p are also used. They are described in J. Med. Chem. 63 All the contents of this literature teaching about the structure and properties of these lipids in :12992-13012, 2020 are incorporated herein by reference.

[0201] The lipid composition commonly used in this experiment is an ionizable cationic lipid:DSPC:cholesterol:DMG-PEG-2000:DSPE-PEG-2000-maleimide in a ratio of 50:10:38.5:1.4:0.1. Unless otherwise stated, the terminal group of the unfunctionalized PEG is methoxy throughout the examples. The N / P ratio (the ratio of positively charged lipid amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups) is 6. After initial LNP formation, the SATA-modified anti-CD5 antibody is partially reacted with maleimide to provide the final tLNP.

[0202] mRNA was encapsulated in LNPs containing DSPE-PEG-2000-maleimide using a self-assembly method, in which an aqueous solution of mRNA at pH 3.5 was rapidly mixed with a lipid solution dissolved in ethanol, followed by stepwise phosphate and Tris buffer dilution and tangential flow filtration (TFF) purification. The LNPs were stored at 4°C until conjugation. Next, anti-CD5 mAb was conjugated to the LNPs to generate tLNPs. Purified rat anti-mouse CD5 antibody clone 53-7.3 (BioLegend) was conjugated to the LNPs via N-succinimide S-acetylsioacetate (SATA)-maleimide conjugation chemistry. The antibody was modified with SATA (Sigma-Aldrich) to introduce thiol groups at accessible lysine residues, thereby allowing conjugation with maleimide. SATA was deprotected using 0.5 M hydroxylamine, followed by removal of unreacted components via a G-25 Sephadex Quick Spin Protein column (Roche Applied Science, Indianapolis, IN). The reactive thiol groups on the antibody were then conjugated to the maleimide moiety on the LNP using thioether conjugation chemistry. Purification was performed using a Sepharose CL-4B gel filtration column (Sigma-Aldrich). The tLNP (LNP conjugated with the targeting antibody) was frozen at -80°C. Others have conjugated antibodies with free functionalized PEG-lipids and then incorporated the conjugated lipids into pre-formed LNPs. However, the procedure disclosed herein is more controlled and produces more consistent results.

[0203] The particle size (hydrodynamic diameter) and polydispersity index of targeted lipid nanoparticles were determined using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Size measurements were performed in a disposable capillary cell at 25°C in Tris buffer at pH 7.4. Size measurements were performed using a non-invasive backscattering system (NIBS) with a scattering angle of 173°. Quant-iT was used. ™ The RiboGreen RNA Assay Kit (Invitrogen) measures mRNA content. Encapsulation efficiency is calculated by measuring the amount of unencapsulated mRNA. This assay measures the amount of unencapsulated mRNA in RiboGreen. ® The fluorescence intensity (Fi) when the reagent is added to the LNP is measured and compared with the total fluorescence intensity (Ft) of the RNA content obtained when the LNP is cleaved by 1% Triton X-100, where encapsulation% = (Ft - Fi) / Ft × 100).

[0204] As shown in Table 1, all these LNP compositions have an acceptable hydrodynamic diameter and polydispersity index in the range of 50-150 nm and a PDI ≤ 0.2. Encapsulation efficiency of ≥80% is acceptable, but ≥85% and ≥90% are preferred.

[0205] To evaluate the performance of tLNPs, they were used to transfect mouse T cells by injection into live mice, and their ability to generate mouse T cells expressing the mCherry reporter gene in vivo was assessed. All tLNP test samples were thawed at room temperature for 30 minutes and then diluted 1:2 with sterile water for injection to achieve a final dose concentration of 100 μg mRNA / mL. 100 μL (10 μg mRNA) of each test sample was then injected via tail vein into 8-week-old female C57BI / 6 mice. All treated mice were then sacrificed 24 hours post-treatment, and their spleens were collected. Each spleen was then dissociated into a single-cell suspension and stained with antibodies to identify T cells, B cells, monocytes, and non-hematopoietic cells. The expression of mCherry in immune cell subsets and non-hematopoietic cells in the stained samples was then analyzed by flow cytometry. Data analysis was performed using FlowJo (version 10.8.1) and GraphPad Prism (9.4.1).

[0206] like Figure 1CAs observed, all tLNP compositions successfully transfected spleen T cells, although with varying transfection rates (the proportion of cells expressing mCherry) and expression levels (as determined by the mean fluorescence intensity (MFI) of the mCherry signal). The tLNP composition containing CICL1 (F1) achieved comparable or better performance than the tLNP composition containing the benchmark ionizable cationic lipid in both transfection rate and expression level. The tLNP composition containing ALC-0315 was selected as the benchmark composition (BF1) for further experiments.

[0207] Several CICL1-based lipid compositions have been prepared (Table 1B below) and found to successfully form LNPs. The properties of many of these LNPs and the tLNPs made from them are shown in the following examples.

[0208] Table 1B. LNP Compositions Composition Code Lipid composition [ratio] N / P BF1 ALC-0315:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:38.5:1.4:0.1] 6 F1 CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:38.5:1.4:0.1] 6 F2 CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:38.5:1.3:0.2] 3 F3 CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:38.5:1.425:0.075] 9 F4 CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[42:10:46.5:1.4:0.1] 6 F5 CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F6 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[35:10:53.5:1.4:0.1] 6 F7 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[42:10:46.5:1.4:0.1] 6 F8 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:38.5:1.4:0.1] 6 F9 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F10 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[62:10:26.5:1.4:0.1] 6 F11 CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[58:7:33.5:1.4:0.1] 6 F12 CICL1:DSPC:CHOL:DPG-PEG(2k):DSPE-PEG(2k)-MAL[58:7:33.5:1.4:0.1] 6 F13 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:7:33.5:1.4:0.1] 6 F14 CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-MAL[58:7:34:0.9:0.1] 6 F15 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30:1.9:0.1] 6 F16 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:39.5:0.4:0.1] 6 F17 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:39:0.9:0.1] 6 F18 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:38.5:1.4:0.1] 6 F19 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:38:1.9:0.1] 6 F20 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:37.5:2.4:0.1] 6 F21 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[50:10:37:2.9:0.1] 6 F22 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:31:0.9:0.1] 6 F23 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30:1.9:0.1] 6 F24 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:29.5:2.4:0.1] 6 F25 CICL1:DSPC:CHOL:DSPE-PEG(0.75k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F26 CICL1:DSPC:CHOL:DSPE-PEG(1k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F27 CICL1:DSPC:CHOL:DMPE-PEG(1k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F29 CICL1:DSPC:CHOL:DSG-PEG(5k):DSPE-PEG(5k)-MA[58:10:31.4:0.5:0.1] 6 F30 CICL1:DSPC:CHOL:DMG-PEG(2k):DSG-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F31 CICL1:DSPC:CHOL:DSG-PEG(2k):DSG-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F32 CICL1:DSPC:CHOL:DSPE-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F33 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(5k)-MAL[58:10:30.5:1.4:0.1] 6 F34 CICL1:DSPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:13:27.5:1.4:0.1] 6 F35 CICL1:DMPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F36 CICL1:DMPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F37 CICL1:DPPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F38 CICL1:DAPC:CHOL:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:30.5:1.4:0.1] 6 F40 CICL1:DSPC:CHOL:20(S)-hydroxycholesterol:DSG-PEG(2k):DSPE-PEG(2k)-MAL [58:10:22.9:7.6:1.4:0.1] 6 F41 CICL1:DSPC:CHOL:β-sitosterol:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:22.9:7.6:1.4:0.1] 6 F42 CICL1:DSPC:CHOL:β-sitosterol:DSG-PEG(2k):DSPE-PEG(2k)-MAL[58:10:15.25:15.25:1.4:0.1] 6

[0209] Example 2: Evaluation of the effect of binder presence and density To assess the effect of the presence of the binding moiety on LNPs and the amount of the conjugated binding moiety (binding agent density), various amounts of anti-CD5 antibody were conjugated to LNPs containing BF1 lipid and mRNA compositions (including compositions without antibody). Antibodies were added to the conjugation reaction at an antibody:mRNA ratio in the range of 0 to 2 (w / w) (essentially as described in Example 1). Maleimide in zero-antibody samples did not react. After conjugation, total protein assays using BOA (dicaprinic acid) and Ribogreen assays for mRNA content were performed. ® The final tLNP antibody to mRNA weight ratio (binding density) was determined by assay. tLNP was also characterized by DLS. As shown in Table 2 (below), BF1-based LNPs and tLNPs have similar physicochemical properties and encapsulation efficiencies.

[0210] BF1-based LNPs and tLNPs were evaluated in vitro on mouse spleen T cells. Without antibody conjugation, BF1-based LNPs did not show mCherry expression in mouse spleen T cells. For tLNPs with antibody:mRNA ratios (w / w) ranging from 0.2 to 0.5, transfection rates were substantially the same, but decreased as the conjugate density increased above this range. Figures 2A to 2B Expression levels (as determined by mean fluorescence intensity (MFI)) decrease with increasing antibody density. These data suggest that, for tLNP assay implementations, the expected range for both the input antibody ratio and the measured tLNP binding ratio is 0.2 to 0.5.

[0211] The same LNPs and tLNPs were also evaluated in vivo in wild-type C57BL / 6 mice. Without antibody conjugation, BF1-based LNPs did not express mCherry in mouse spleen T cells in vivo. Figure 2C However, compared to tLNP targeting CD5, it showed better performance in hepatic CD45- cells (hepatocyte staining) and hepatic CD45- cells. + CD11b + Higher transfection rates and at least similar expression levels were observed in cells (Kupffer cell staining). Figures 2D to 2E Conversely, tLNP showed significant transfection rates and expression levels in splenic T cells. In liver CD45... − Cells and liver CD45 + CD11b + In cells, all BF1-based tLNPs showed comparable mCherry expression, although both transfection rate and expression level were slightly lower in hepatocytes compared to LNPs, as was the case in Kupffer cells. Figures 2D to 2E Therefore, antibody conjugation to LNPs, in addition to increasing transfection rates in cells expressing the target antigen (CD5-expressing T cells in this example), also inhibited transfection rates in the liver. Transfection rates showed a bell-shaped curve as antibody density increased in mouse spleen T cells, with the peak of the infused antibody ratio occurring in the range of 0.5 to 1, and the peak of the ratio measured in tLNPs occurring in the range of approximately 0.5 to approximately 0.7. Figure 2B (Table 2A). However, values ​​of 0.2 to 2.0 for the ratio of input antibody to payload provide useful tLNP ( Figures 2B to 2C ).

[0212] To confirm the applicability of the preferred binder density across species, additional tLNPs were prepared with an input antibody:mRNA ratio (w / w) ranging from 0.1 to 1.0, but the antibody was chimeric 5D7 (ch5D7). 5D7 is a mouse antibody that recognizes CD5 in human and non-human primates (NHP; at least rhesus monkeys and cynomolgus monkeys). In the chimeric antibody, the mouse constant domain was replaced with a human sequence. The tLNPs were evaluated as previously described, including the final measured binder ratio, and are shown in Table 2B (below).

[0213] The ability of these tLNPs to transfect NHP hematologic T cells in vitro was then tested. Transfection rates were substantially similar for ingress-binding ratios from 0.3 to 1.0, with the highest expression levels observed for tLNPs at ingress-binding ratios of 0.3 and 0.5. Figure 2F This effectively demonstrates that for the input binding ratio of full-length IgG antibodies, the preferred binding density is the same in both species.

[0214] Based on all this data, for all tLNPs in subsequent embodiments, an input binder ratio of 0.5 to 0.7 is used.

[0215] Example 3: Assessment of the impact of the N / P ratio Generally, it is desirable to maximize the amount of payload (mRNA) in the dose, but sufficient lipids, especially ionizable cationic lipids, are also required for tLNPs to function optimally as delivery carriers for releasing the payload into the cytoplasm. To determine the sensitivity of tLNP performance to changes in mRNA content, the N / P ratio was varied while keeping other parameters constant.

[0216] To facilitate comparability within and between experiments, a fixed dose of mRNA was used. Therefore, if the lipid ratio remained constant, variations in the N / P ratio would result in a greater or lesser amount of each lipid (including functionalized PEG-lipids) in the dose. This would result in doses with more or less conjugated binding moieties without compensatory adjustments. Therefore, while the total amount of PEG-lipids was kept constant in this experiment, the amount of functionalized PEG-lipids (i.e., DSPE-PEG-2000-maleimide) was adjusted to at least partially mitigate any such effect. Thus, for the general lipid composition of CICL 1:DSPC:cholesterol:DMG-PEG-2000:DSPE-PEG-2000, the lipid ratio was 50:10:38.5:(1.5-X):X. When N / P was 3, X was 0.2; when N / P was 6, X was 0.1; and when N / P was 9, X was 0.075. The formation of tLNP and the conjugation of antiCD5 antibody are described above.

[0217] As shown in Table 3, all tLNPs exhibited similar physicochemical properties and encapsulation efficiency. It was found that altering the N / P ratio within this range had almost no effect on T cell expression. Figure 3A Although the differences between these compositions and the reference composition (BF1) are greater than those in previous experiments.

[0218] In addition to maximizing expression in T cells, it is generally desirable to minimize expression in the liver. Therefore, hepatic CD45-cells (hepatocytes) and hepatic CD45 expression were evaluated. + CD11b + mCherry expression in cells (Kupffer cells). Here, the N / P ratio plays a surprisingly significant role, with tLNPs at N / P ratios of 3 and 6 producing significantly lower expression levels in hepatocytes and Kupffer cells than tLNPs at an N / P ratio of 9 or the baseline tLNP containing ALC-0315. Figures 3B to 3C Therefore, considering expression in the liver and T cells, an N / P ratio of 3 to 6 was considered superior to an N / P ratio of 9. Compositions with an N / P ratio of 6 produced slightly better encapsulation efficiency and were selected for evaluation of other parameters.

[0219] Example 4: Evaluation of the effect of ionizable cationic lipid content As mentioned above, one of the main determinants of (t)LNP performance is the ionizable cationic lipid. Here, we varied the proportion of ionizable cationic lipids in the composition for preparing tLNP, which contained CICL1:DSPC:cholesterol:DMG-PEG-2000:DSPE-PEG-2000-maleimide in a ratio of Y:10:(38.5-Y):1.4:0.1, where Y was 42 (F4), 50 (F1), or 58 (F5), and N / P was 6. The formation of tLNP and the conjugation of the anti-CD5 antibody were described above.

[0220] The apparent (i.e., measured) pKa of ionizable lipids in lipid nanoparticles was determined using sodium 6-(p-toluidine)-2-naphthalenesulfonate (TNS salt, Toronto Research Chemicals, Toronto, ON, Canada). Lipid nanoparticles were diluted to a total lipid concentration of 1 mM in 1× Dulbecco's PBS. A 1 mg / mL stock solution of TNS salt in DMSO was prepared and then further diluted with distilled water to a working solution of 60 μg / mL (179 mM). The diluted lipid nanoparticle sample was further diluted to 90 μM total lipids in 165 μL of a buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, resulting in a final TNS concentration of 1.33 μg / mL (4 μM) at a pH range of 3.5 to 12.2. After mixing in pipettes and incubating in the dark at room temperature for 15 minutes, fluorescence intensity was measured at room temperature using excitation and emission wavelengths of 321 and 445 nm, respectively, in a BioTek Synergy H1 plate reader. The fluorescence signal was subtracted from the blank and plotted against pH, then analyzed using nonlinear (Boltzmann) regression analysis, where the apparent pKa was determined as the pH at which half-maximum fluorescence intensity was produced, as calculated using the Henderson-Hasselbalch equation.

[0221] The physicochemical properties of these tLNPs are comparable, including the measured pKa. The measured pKa of the ionizable cationic lipids in the tLNPs is in the range of 6 to 7, which allows for ionization upon endosome acidification and promotes the release of endosome contents from the (t)LNPs into the cytoplasm. The proportion of ionizable cationic lipids in the (t)LNPs may affect the observed pKa, although, as seen in Table 4, the effect is small in this case. There is a tendency for particle size to increase with increasing ionizable cationic lipids, but it remains within a preferred range.

[0222] tLNP was administered to C57BL / 6 mice via tail vein injection. Spleen and liver were harvested and decomposed, and mCherry expression in splenic T cells, hepatocytes, and Kupffer cells was assessed by flow cytometry. A clear trend of increasing transfection rate and expression level was observed with increasing CICL1 content, with the 58% CICL1 composition (F5) showing superior performance compared to the previous 50% CICL1 composition (F1) and the baseline composition. Figure 4A The same trend was observed in hepatocytes and Kupffer cells, but the differences between the compositions were significantly reduced, and both transfection rate and expression level were much lower than the baseline BF1 (). Figures 4B to 4C This demonstrates that the content of ionizable cationic lipids is an important factor in T cell-targeted delivery via tLNPs, with higher levels being advantageous within the test range. Therefore, 58% CICL1 (F5) was used as a baseline for evaluating other parameters.

[0223] Example 5: Evaluation of PEG-lipid anchoring length To evaluate the effects of PEG-lipids with different anchored carbon chain lengths, tLNPs with the general composition CICL1:DSPC:CHOL:PEG-lipid:DSPE-PEG-2000-MAL [58:7:33.5:1.4:0.1] were prepared, essentially as described above, wherein the nonfunctionalized PEG-lipid was DMG-PEG-2000 (F11), DPG-PEG-2000 (F12), or DSG-PEG-2000 (F13). As previously stated, the N / P ratio was 6, and the mRNA was CleanCap encoding the fluorescent protein mCherry (Trilink). ® mCherry 5-methoxyuridine (5 moU) mRNA was detected and recognized by the entire IgG of mouse CD5 conjugated to DSPE-PEG(2k)-MAL. Physicochemical characterization is reported in Table 5A.

[0224] The ability of these tLNPs to transfect cells in vivo was tested by injection into C57BL / 6 mice. For spleen T cells, both transfection rate and expression level (equivalent soluble fluorescent dye molecule (MESF)) significantly increased with increasing anchor chain length, with the transfection rate and expression level of tLNPs containing DSG-PEG-2000 (F13) being almost three times that of tLNPs containing DMG-PEG-2000 (F11). Figure 5A Meanwhile, the expression of compositions F12 and F13 was lowest in hepatocytes (CD45-hepatocytes) (transfection rate <2%). Figure 5B Kupffer cells (CD45) + / CD11 + Transfection of hepatocytes with F12 and F13 was similarly reduced, and only about half that with F11, although the expression level in transfected Kupffer cells appeared to be significantly higher than that in hepatocytes. Figures 5B to 5C The increased transfection rate and expression levels achieved in T cells (increased) and hepatocytes (decreased) with the increased anchoring chain length of nonfunctionalized PEG-lipids reflect the increased tLNP cycle time due to reduced PEG-lipid shedding and the resulting inhibition of apoE-mediated delivery to the liver.

[0225] Although the tLNPs in the previous examples contained 10% DSPC, the compositions of F11-F13 contained 7% DSPC, resulting in a compensatory reduction in cholesterol content. In the tLNPs, at a 10% DSPC content, the effect of PEG-lipids on the carbon chain length was evaluated using the F5 lipid composition compared to a lipid composition in which DMG-PEG-2000 in F5 was replaced by DSG-PEG-2000. That is, the lipid composition of the tLNPs was CICL1:DSPC:CHOL:PEG-lipid:DSPE-PEG-2000-MAL [58:10:30.5:1.4:0.1], where the PEG-lipid was either DMG-PEG-2000 (F5) or DSG-PEG-2000 (F9). These tLNPs differ from those described above in that they encapsulate N1-methylpseudouridine (m1ψ)-substituted mRNA encoding the fluorescent protein mCherry. The mRNA replaced by M1ψ was generally expressed at higher levels than the mRNA replaced by 5 molU, but there was no clear reason to suggest that this change would have a substantial impact on transfection rate. As previously stated, the N / P ratio was 6, and the entire IgG recognizing mouse CD5 was conjugated to DSPE-PEG-2000-MAL. Physicochemical characterization is reported in Table 5B.

[0226] After injection into C57BL / 6 mice, improved performance of tLNP with longer-chain PEG-lipids was again observed in spleen and hepatocytes. Figures 5D to 5F ).

[0227] Example 6: Evaluation of PEG-lipid content The effects of increasing nonfunctionalized lipid content and total PEG-lipid content were evaluated by comparing tLNP with lipid compositions having nonfunctionalized lipid content reduced from 1.4% to 0.9% (with a compensatory reduction in cholesterol and subsequently a reduction in total PEG-lipids). Specifically, the lipid contents were CICL1:DSPC:CHOL:PEG-DMG2000:DSPE-PEG-2000-MAL [58:7:33.5:1.4:0.1] (F11) and [58:7:34:0.9:0.1] (F14). As previously mentioned, the N / P ratio was 6, and the mRNA was CleanCap encoding the fluorescent protein mCherry (Trilink). ® mCherry 5-methoxyuridine (5 moU) mRNA was detected and recognized by conjugation of the entire IgG of mouse CD5 with DSPE-PEG-2000-MAL. Physicochemical characterization is reported in Table 6.

[0228] When injected into C57BL / 6 mice, both compositions showed similar transfection rates and expression levels in mouse spleen T cells and liver Kupffer cells, but F14 tLNP provided lower transfection rates and expression levels. Figures 6A to 6C However, taking into account other factors, including favorable physicochemical properties, potential toxicity and stability, and our other strategies to avoid hepatocyte uptake, we continue to use 1.5% as our total PEG-lipid ratio.

[0229] Example 7. Evaluation of various functionalized / unfunctionalized PEG-lipid combinations Several potential options exist for functionalized and non-functionalized PEG-lipids used in tLNPs. PEG-lipids can be longer or shorter, and the lipids can be diacylglycerols or diacylphospholipids. To gain some insight into the impact of these variables, various combinations were tested. For functionalized PEG-lipids anchored to the binding moiety (after conjugation), it is important that the lipid does not detach from the tLNP. Therefore, only longer carbon chain lipids were selected, particularly PEG-lipids based on DSPE (diacylphospholipids) and DSG (diacylglycerols). For non-functionalized lipids, DMG-PEG-2000 (a shorter carbon chain lipid) was used in addition to DSG-PEG-2000 and DSPE-PEG2000.

[0230] tLNPs were prepared by encapsulating m1ψ-substituted mCherry mRNA with an N / P ratio of 6 and a typical lipid composition of CICL1:DSPC:CHOL:PEG-lipid:PEG-MAL lipid [58:7:33.5:1.4:0.1]. Essentially as described above, intact mouse CD5 IgG conjugated with PEG-MAL lipids was recognized. Specific PEG-lipid and PEG-MAL lipid combinations and the physicochemical characterization of tLNPs are reported in Table 7.

[0231] As seen in the table, all these particles exhibit similar physicochemical properties. However, aggregation was observed during the purification of F32 tLNP, suggesting that the lipid composition may not be the optimal combination for forming stable nanoparticles.

[0232] After injection into C57BL / 6 mice, tLNPs with the F9 and F31 combination showed significantly better transfection rates and expression levels in spleen T cells than the other three combinations. Figure 7A Both F9 and F31 contain long-chain diacylglycerol DSG-PEG2000 as their non-functionalized PEG-lipids, indicating that not only lipid tail length but also the glycerol-based scaffold, as opposed to phosphatidyl scaffolds, is important for performance. Comparisons of F5 and F9 tLNPs with F30 and F31 tLNPs, respectively, showed that, in the test implementation, the use of DSPE-based and DSG-based functionalized PEG-lipids had no major impact on T cell performance.

[0233] However, in hepatocytes, tLNPs with the F31 composition and tLNPs with the F30 composition provided fairly high transfection rates and expression levels, i.e., both containing functionalized diacylglycerol, while tLNPs with F9 or F32 (both containing diacylphospholipids DSPE) provided very low transfection and expression levels. Therefore, the use of phospholipids in functionalized PEG-lipids appears to be crucial for avoiding liver transfection. tLNPs with the F5 composition provided moderate performance; this composition also contains DSPE-based functionalized lipids, but also shorter-chain, DMG-based non-functionalized lipids. Figure 7B This reflects the importance of lipid carbon chain length seen in Example 5, but further indicates that the lipid scaffold (glycerol or phospholipid) is of little importance in preventing hepatocyte uptake.

[0234] In Kupffer cells, tLNPs with the F9, F31, and F32 compositions provided low transfection and expression levels, again reflecting the importance of longer carbon chain lengths in nonfunctionalized PEG-lipids for avoiding hepatic uptake of tLNPs. The slightly lower expression of F9 tLNP compared to F32 tLNP supports the aforementioned preference for glycerol-based lipid scaffolds in nonfunctionalized PEG-lipids to avoid hepatic uptake. tLNPs with the F5 composition showed moderate levels, while tLNPs with the F30 composition showed considerably higher transfection and expression levels in Kupffer cells. Figure 7C A performance comparison of F5 and F30 tLNPs containing different nonfunctionalized PEG-lipids revealed the important role of their common component DSPE-PEG-MAL (phosphatidyl-functionalized PEG-lipid) in preventing hepatic uptake of tLNPs.

[0235] Overall, F9 tLNP containing DSG-PEG2000 and DSPE-PEG2000-MAL showed the best performance in transfecting T cells, but avoided uptake in the liver.

[0236] Example 8. Assessment of the effect of phospholipid content Phospholipid content can affect the formation, structure, and stability of LNPs, as well as other characteristics. Here, the proportion of DSPC was varied, and cholesterol content was compensatorily adjusted in the context of DMG-PEG2000 or DSG-PEG2000 as non-functionalized PEG-lipids. Specifically, tLNPs contained CICL1:DSPC:CHOL:DMG-PEG2000:DSPE-PEG2000-MAL in a ratio of [58:M:(40.5-M):1.4:0.1], where M was 10 (F5) or 7 (F11), or CICL1:DSPC:CHOL:DSG-PEG2000:DSPE-PEG2000-MAL in a ratio of [58:N:(40.5-N):1.4:0.1], where N was 10 (F9) or 7 (F13). tLNPs were prepared by encapsulating m1ψ-substituted mCherry mRNA with an N / P ratio of 6. The formation of tLNPs and the conjugation of anti-CD5 antibodies are described above. Table 8 reports the specific PEG-lipid and DSPC combinations and the physicochemical characterization of tLNPs.

[0237] As shown in Table 8, tLNPs (F11 and F13) with only 7% DSPC had lower mRNA encapsulation efficiency and slightly larger particle size, indicating that 7% DSPC may not be sufficient to provide a stable structure for LNPs.

[0238] After injection into C57BL / 6 mice, with the corresponding 7% DSPC forms (F11 and F13, respectively); Figure 8A Compared to 10% DSPC (F5 and F9) and either DMG or DSG-based nonfunctionalized PEG-lipids, tLNPs provided significantly higher mCherry expression in mouse spleen T cells. This superior performance is likely at least partly due to the greater stability of 10% DSPC-containing tLNPs compared to those with 7% DSPC. Comparison of two pairs of DMG- and DSG-based tLNPs with the same DSPC content further demonstrates the superiority of longer carbon chain anchoring on PEG-lipids. F11 tLNPs exhibited significantly higher mCherry transfection and expression levels in both hepatocytes and Kupffer cells than F5 tLNPs, suggesting that 10% DSPC is more conducive to avoiding hepatic uptake. Figures 8B to 8C In hepatocytes, F13 tLNP exhibits performance comparable to or slightly weaker than F9 tLNP. However, given the already low transfection and expression levels resulting from the use of DSG-PEG2000, and considering the superior performance of the F9 composition in T cells compared to the F13 composition, F9 tLNP remains the better option for delivery to T cells overall.

[0239] Example 9. Effect of antibody conjugation on delivery loci Conventional LNPs primarily deliver their payload to the liver after intravenous administration. Here, we investigated the effect of antibody modification on payload delivery of LNPs. Unmodified LNPs, LNPs modified with unrelated IgG, and tLNPs modified with anti-mouse CD5 antibody were prepared using a BF1 and F5 combination and administered to C57BL / 6 mice via tail vein injection. Only the anti-CD5 tLNP provided a significant transfection rate in splenic T cells ( Figure 9A For CD45 + Hepatocytes (liver cells) showed a transfection rate of nearly 25% with BF1 LNP. Modifying BF1 LNP with unrelated IgG reduced the hepatocyte transfection rate by approximately half, while modifying BF1 LNP with anti-CD5 antibody (true tLNP) reduced the transfection rate to less than 10%. Figure 9B F5 LNP already had a low transfection rate on hepatocytes, less than 5%, and adding antibodies in this experiment did not further reduce it. Therefore, adding antibodies or altering ionizable lipids both reduced delivery to the liver.

[0240] In further studies, nine-week-old female C57BI / 6 mice were intravenously injected via the tail vein with BF1 or F9 LNPs encapsulating luciferase mRNA or tLNPs targeting CD5. Bioluminescence images were collected from all mice in prone and supine positions 6 hours post-injection. Figure 9C After whole-animal imaging, mice were euthanized, and the following tissues were perfused and collected: liver, spleen, lung, kidney (both), heart, and brain. The tissues were placed in fluorescein-pre-filled black polystyrene plates with gaps between them, and bioluminescent images were collected. Figure 9C Quantitative results () Figure 9D Data from the right kidney (not shown) is similar to that from the left kidney, showing that the use of CICL1 reduced bioluminescence in the liver by about 2 logs compared to the BF1 composition. However, for tLNP containing CICL1, bioluminescence in the spleen was similar to that of the BF1 composition and about 1 log higher than that caused by its application in the liver.

[0241] These compositions exhibit a general pattern of bioluminescence in the spleen, lungs, kidneys, heart, and brain, with the tLNP compositions containing CICL1 typically producing minimal bioluminescence in each non-target organ.

[0242] Example 10. Comparison of tLNP targeting CD5 and CD8 in a mouse tumor model LNPs containing the F9 composition were prepared and conjugated with humanized 5D7 (anti-CD5) antibody or chimeric RPA-T8 (anti-CD8) antibody to generate tLNPs. The tLNPs encapsulated N-terminated polymorphic antibodies encoding anti-CD19 CARs. 1 5-methylpseuuridine-substituted mRNA (tLNPs targeting CD5 and CD8) or 5-methoxyuridine-substituted mRNA encoding mCherry (tLNPs targeting CD5 only).

[0243] NSG mice (approximately 10 weeks old) were purchased from The Jackson Laboratory and acclimatized for at least 5 days. Ten million human T cells were injected intravenously via the tail vein. Ten days after T cell engraftment, 5 × 10⁵ cells were injected intravenously. 5Nalm6 cells (Nalm6-Luc) constitutively expressing firefly luciferase were used. Five days after tumor cell engraftment, T cell engraftment (frequency of circulating human CD45+ cells) and tumor burden (assessed by luciferase signal) were evaluated in mice, and mice were grouped based on similar mean values ​​of the two readings. Starting from day 18 post-T cell engraftment, mice in each group were intravenously injected twice weekly with one of the tLNPs containing 20 μg mRNA, for a total of 5 doses. Tumor cell burden was assessed twice weekly by bioluminescence imaging (BLI) of luciferase signal. Additionally, 24 hours after the third dose, mCherry expression and CAR expression on T cells in peripheral blood samples were analyzed by flow cytometry (using an antibody conjugated to the scFv linker).

[0244] tLNPs modified with anti-CD5 and anti-CD8 antibodies in CD8 + Similar transfection rates were achieved in blood T cells. The overall transfection rate on T cells with CD8-targeting tLNPs (F9 CD8, solid triangles) was lower than that with CD5-targeting tLNPs (F9 CD5, solid circles), because they could not transfect CD4. + T cells ( Figure 10A PBS (solid squares) was applied as a negative control. tLNPs targeting CD8 (F9 CD8, solid triangles) were observed on CD8. + The expression level of tLNP (F9 CD5, solid circle) was higher in total T cells than in CD5-targeting cells, which may reflect the higher density of CD8 on the cell surface compared to CD5. Figure 10B A similar pattern was observed in spleen T cells. Figures 10C to 10D CD4 cells treated with tLNP targeting CD8 were observed in blood. + Aberrant expression in T cells ( Figure 10B ) is an artifact of a small number of events in flow cytometry and was not observed in results from spleen T cells that collected a larger number of events. Figure 10D These data support the correlation between data obtained using tLNPs targeting CD5 and tLNPs targeting other cell surface antigens.

[0245] For CAR, in total T cells and CD8 + In both types of T cells, the transfection efficiency of tLNP targeting CD8 (F9 CD8) was higher than that of tLNP targeting CD5 (F9 CD5). Figure 10E Although the transfection rate was significantly lower than that observed with mCherry mRNA, this may reflect that CAR mRNA has not yet been optimized for in vivo transfection using tLNP.

[0246] Assessment of tumor burden via luciferase signaling showed that, in general, tumors grew in the mCherry-treated group, while the tumor burden was significantly reduced in the group receiving the encoded anti-CD19 CAR, regardless of whether they were treated with composition F9 targeting tLNP with CD5 (CD5) or CD8 (CD8). Figure 10F In fact, in most treated mice, significant complete clearance was achieved on day 2 after the first administration of tLNP.

[0247] Example 11. Tolerance of BF1 LNP, BF1 tLNP, F5 LNP and tLNP Following administration to rats, unmodified BF1 LNP showed a significant dose-response increase in liver enzymes (data not shown) and acute-phase proteins (>1000 IU / L at 3 mg / kg), with a significant increase at doses ≥1 mg / kg. Figures 11A to 11C A second head-to-head comparison of compositions BF1 and F5 in rats confirmed the liver enzyme elevation observed with BF1, however, no similar liver enzyme elevation was observed with F5 LNP. Figures 11D to 11E For BF1 tLNP, the increase was significantly reduced, although some increase in acute-phase proteins remained, especially at 3 mg / kg. In contrast, F5 tLNP showed no significant increase in liver enzymes or acute-phase proteins at up to 3 mg / kg (the highest dose tested). Figures 11F to 11J The binding site in this experiment was the anti-CD5 antibody mentioned in Example 2, which does not recognize rat CD5, therefore there was no targeting effect here, and the mere presence of the conjugated antibody is the main reason for the greater tolerability of tLNP, consistent with the effect seen with IgG in Example 9. However, the antibody is a neutral factor for the difference in tolerability between BF1 tLNP and F5 tLNP. Therefore, the different ionizable cationic lipids may be the reason why F5 tLNP is better tolerable than BF1 tLNP.

[0248] In further studies, rats were administered different doses of F9 tLNP targeting rat CD5 encapsulated with mCherry mRNA (up to 5 mg / kg) and F9 tLNP targeting human CD8 encapsulated with anti-CD19 CAR mRNA (up to 6 mg / kg). Liver enzyme and acute-phase protein levels were reassessed. The CD8 binding moiety was not cross-linked to rat CD8. ALT ( Figure 11K ) and AGP ( Figure 11L The results showed that even at these higher doses, tLNP containing CICL1 was well tolerated.

[0249] Example 12. Tolerance to T cell transfection and administration of tLNP to non-human primates The first study was conducted to evaluate the tolerability and engineering efficiency of a single administration of CD5-targeting tLNPs with an RNA payload encoding mCherry. The CD5-targeting compositions BF1 and F5 tLNPs were administered as a single dose ranging from 0.5 mg mRNA / kg to 3.0 mg mRNA / kg. Clinical observations, clinicopathological markers, and toxicological biomarkers were evaluated to assess safety and tolerability. Pharmacological evaluation was performed by flow cytometry detection of mCherry in immune cell subsets in peripheral blood and tissues (spleen, bone marrow, lymph nodes, and liver).

[0250] For each CD5-targeting tLNP of composition BF1 or F5, paired cynomolgus monkeys were administered a dose containing 0.5, 1.0, 2.0, or 3.0 mg mRNA / kg via intravenous infusion at a rate of 6.25 mL / kg / h over 1 hour, with additional control animals receiving PBS. Whole blood, serum, and plasma were collected at 4, 8, and 24 hours post-administration, both at baseline and after test administration. Under sedation, biopsy samples of lymph nodes, liver, and bone marrow tissue were collected 24 hours post-administration. Animals were euthanized 24 hours post-administration for full necropsy. Since the modified tLNP anti-CD5 antibody mentioned in Example 2 recognizes cynomolgus monkey CD5, the tLNP provides true targeting in this model.

[0251] Liver function tests showed that for BF1 compositions containing 2.0 and 3.0 mg / kg of ionizable cationic lipid ALC-0315, ALT increased at 24 hours post-infusion, and AST increased at 4 and 24 hours post-infusion. Figure 12A (See above figure). In contrast, the F5 composition containing the ionizable cationic lipid CICL1 was well tolerated even at the highest tested dose of 3 mg / kg, with no or only slight elevation observed ( Figure 12A (See the image below). (The abnormally elevated AST level in an animal receiving a 1 mg / kg dose is likely related to the procedure rather than the test sample).

[0252] Cytokine secretion of IL-6, MCP-1, IL-2, IFNγ, TNFα, TGFα, IL-8, and gm-CSF was also assessed after tLNP administration. Following tLNP administration, IL-6 and MCP-1 showed minimal to slight transient increases, but remained at low levels. Figure 12B ).

[0253] For animals receiving the F5 composition containing CD5-targeting tLNPs, transfection efficiency was assessed by flow cytometry at 4 and 24 hours post-infusion, to determine the levels of mCherry in the blood. + Percentage of T cells. At 4 hours, up to approximately 30% of total T cells were mCherry. +All animals treated with tLNP had approximately 4-6% mCherry levels within 24 hours. + T cells demonstrated successful transfection with a certain degree of persistent expression. Figure 12C The transfection efficiency of spleen, lymph nodes, and bone marrow T cells in tissues collected during autopsy 24 hours after infusion was also evaluated. Expression frequency showed a significant trend towards higher frequencies with increasing dose, at the two highest doses ( Figure 12D ), 9% to 15% of mCherry in the spleen + T cells demonstrated dose-proportional transfection. Either tLNPs successfully reached and transfected T cells in these tissues, or the transfected T cells migrated into the tissues after transfection.

[0254] A second study was conducted using F9 tLNPs to evaluate the tolerability and engineering efficiency of the following: (1) a single administration of a CD5-targeting tLNP (humanized 5D7-targeting antibody) with an mRNA payload encoding an anti-CD19 CAR, (2) a single administration of a CD8-targeting tLNP (chimeric RPA-T8-targeting antibody) with an mRNA payload also encoding an anti-CD19 CAR, and (3) repeated administrations (3 doses) of a CD5-targeting tLNP. The CD5-targeting tLNP was administered as a single dose of 3.0 mg / kg (Group 2, n=2) or as repeated doses of 1.0 mg / kg at 72-hour intervals (Q72h) three times (Group 5, n=2). The CD8-targeting tLNP was administered as a single dose of 3.0 mg / kg, with terminal necropsy at 24 hours post-administration (Group 4, n=2) or 96 hours post-administration (Group 3, n=2). As a control, PBS was administered (Group 1, n=1). All tLNPs tested in the second study were administered using composition F9. The tLNP formulation (tLNP composition plus buffer and excipients) was administered via IV infusion at 6.25 mL / kg / h, with a total administration duration of 1 hour. Clinical observations, clinicopathological markers, and toxicological biomarkers were evaluated to assess safety and tolerability. Pharmacological evaluation was performed by flow cytometry of anti-CD19 CARs in immune cell subsets in peripheral blood and tissues (spleen, bone marrow, lymph nodes, and liver). It should be noted that anti-human CD19 CARs do not exhibit significant pharmacological activity in NHPs; therefore, the tolerability demonstrated in this study is solely related to the biochemical composition of the tLNPs (including the encapsulated mRNA) and does not reflect any effect on the immunomodulatory activity of the CAR.

[0255] Liver function tests showed transient increases in AST and / or ALT in a few animals that received a single dose of 3 mg mRNA / kg tLNP, but the highest increases were observed in the PBS control animals, indicating that the increases were procedure-related rather than test-sample-related. Figure 12ENo increase in ALT or AST was observed in animals that received three doses of CD5-targeting tLNP at a dose of 1 mg mRNA / kg. Figure 12F These data again demonstrate that tLNPs (especially the ionizable cationic lipid CICL1) are well tolerated.

[0256] For animals receiving a single administration, transfection efficiency was assessed by flow cytometry at 8 and 24 hours post-infusion, as well as at 24 hours, to determine the level of anti-CD19 CAR in the blood. + Percentage of T cells. This replicates the general pattern observed with mCherry mRNA payload expression, with a higher percentage of CARs observed at earlier time points. + T cells, but a large number of CARs were still observed 24 hours after infusion. + T cells ( Figure 12G CAR was also observed in the spleen, bone marrow, lymph nodes, and liver. + T cells suggest that the liver detargeting effect of the tLNP composition disclosed in this paper may be limited and does not extend to liver-resident T cells. Figure 12H Repeat administration 24 hours after the third infusion was able to maintain or increase CAR during the experiment. + The proportion of T cells ( Figure 12I At a dose of 3 mg / kg, transfection with tLNPs targeting CD5 and CD8 resulted in similar expression of CAR molecules in multiple immune cell subtypes in the spleen, including helper T cells, cytotoxic T cells, B cells, NK cells, and lineage-negative cells. Figure 12J For the mRNA encoding anti-CD19 CAR used in this experiment, the number of CAR molecules per cell was approximately 100–1000 equivalent soluble fluorescent dye molecules (MESF). However, it should be noted that the mRNA encoding anti-CD19 CAR has not been optimized for tLNP-mediated in vivo transfection, and several times higher levels of expression can be achieved with optimized mRNA.

[0257] A third study was also conducted in NHP, in which F9 tLNP targets CD8 and encapsulates mRNA encoding anti-CD19 CAR. Single-dose liver enzyme results from all three studies (dose range 3.5 to 3.0 mg mRNA / kg) were compiled into a single graph of AST and ALT levels for a more comprehensive assessment. Figure 12K Regarding AST, many monkeys showed a slight elevation (below 1000 U / L) above the nominal normal range (grey shaded area), and in fact, several monkeys had elevated AST levels before tLNP administration, which typically decreased after administration. Before tLNP administration, most monkeys had ALT levels within the normal range or slightly elevated. Figure 12K These results did not indicate any substantial toxicity associated with the administration of the F5 or F9 tLNP compositions.

[0258] Two additional studies further evaluating repeated dosing utilized three administrations at 72-hour intervals of tLNP containing composition F9 and encapsulated mRNA encoding an anti-CD19 CAR. The dose range was 0.1 mg mRNA / kg–2.0 mg mRNA / kg. Mild to moderate transient increases in AST or ALT were observed in some monkeys. Figure 12L These increases do not impair the generally good biochemical tolerance of tLNP and its components.

[0259] Example 13. Biodegradability of CICL1 in non-human primates and mice The determining factor for tLNP tolerability is ionizable cationic lipids. While other lipid components in tLNP are either already present in the body or diet, or very similar, ionizable cationic lipids are not. This study was conducted to assess and compare the clearance of ionizable cationic lipids in the blood, liver, and spleen following administration of tLNP containing ionizable cationic lipids.

[0260] CICL structures are designed to promote biodegradation, rather than necessarily involving oxidative degradation in the liver, such as... Figure 13A The conceptual biodegradation scheme is shown. Although this study does not prove it to be correct, the following data are consistent with the conceptual biodegradation scheme.

[0261] Samples were obtained from cynomolgus monkeys that were intravenously administered BF1 or F5 tLNP (two of each) at a dose of 3 mg mRNA / kg in the aforementioned examples. The tLNP was conjugated to the anti-CD5 as the targeting component mentioned in Example 2. Twenty-four hours after administration, the animals were sacrificed, and plasma, liver, and spleen tissues were collected from two animals in each group. The presence of these ionizable cationic lipids was assessed by mass spectrometry to evaluate their biodegradability and biodistribution.

[0262] In short, plasma was stabilized by adding 10 μL of formic acid per mL of K2EDTA plasma for mass spectrometry analysis. A Geno / Grinder filter set to 1500 rpm for two minutes was used. ® (SPEX) ® SamplePrep homogenized spleen and liver tissues to 1 tissue volume (v / w) in 19 homogenization buffers (50:40:10 0.1% formic acid in 1:1 methanol:acetone / 10mM ammonium formate (containing 0.2% formic acid) / dimethylformamide (DMF)).

[0263] Calibration curves and quality control samples were prepared using a control matrix (plasma or homogenate) spiked with CICL1 or ALC-0315 in DMF. Extraction was performed on wet ice by adding 300 μL of lysis solvent (0.1% formic acid in 1:1 methanol:acetonitrile) to 25 μL of sample containing 50 μL of internal standard (1:1 isopropanol:water). The extract was mixed at 1500 rpm for four minutes and then centrifuged at 3000 × g for ten minutes. 200 μL of the supernatant was transferred to a clean 96-well plate for LC / MS / MS analysis.

[0264] Chromatographic analysis was performed on a Waters XBridge BEH C8 column (130 Å, 2.5 μm, 2.1 × 30 mm) using the following gradient. Mobile phase A (10 mM ammonium formate aqueous solution containing 0.2% formic acid) was held at 20% for 0.3 min, then reduced to 5% over 1.2 min. This condition was maintained for 1 min, then restored to 80% B (0.1% formic acid in 1:1 methanol:acetonitrile) over 0.1 min. The column was then held at 80% B for 1.4 min. The LC flow rate was maintained at 0.5 mL / min, and the autosampler was maintained at 2 °C–8 °C. Mass spectrometry was obtained using atmospheric pressure ionization (Sciex) with a triple quadrupole apparatus.

[0265] Quantification was performed using 1 / X2 quadratic regression, with the peak area of ​​transition 1013.6→968.4 (CICL1) or 766.6→748.4 (ALC-0315) relative to the peak area of ​​the internal standard transition 1040.6→995.4.

[0266] Within 24 hours of administration, CICL1 was present in very small amounts in the blood, liver, and spleen. At this time, residual ALC-0315 (Comirnaty) remained in the blood. ® The concentration of the ionizable cationic lipids used was significantly higher, approximately 6 times higher. The concentrations of ALC-0315 in the liver and spleen were approximately 14 times and 11 times higher, respectively. In contrast, the concentrations of CICL1 in blood, liver, and spleen differed by less than 2 times, and the concentrations in the liver and spleen were 1 / 127 and 1 / 35 of ALC-0315, respectively. Figure 13B ).

[0267] These clearance data indicate that, at least in the initial steps, CICL1 is more biodegradable than ALC-0315. Furthermore, these data are consistent with the greater tolerance of CICL1 relative to ALC-0315 seen in Examples 11 and 12.

[0268] To obtain more detailed data, similar experiments were performed in mice, where the time course of ionizable cationic lipid and encapsulated mRNA disappearance could also be obtained. Mice were intravenously administered 2 mg / kg tLNP targeting mouse CD8 and encapsulated with anti-human CD19 CAR compositions BF1 (containing ALC-0315) or F9 (containing CICL1), and monitored for 7 days. At each time point, three mice were sacrificed, and liver, spleen, and plasma were processed for lipid concentration assays based on LC-MS and digital PCR detection of mRNA. In plasma, mRNA levels were normalized relative to known amounts of mRNA spiked in controls. In spleen and liver, mRNA levels were normalized relative to the mRNA of the housekeeping gene GAPDH.

[0269] In plasma, both lipids initially disappeared at similar rates, but by 24 hours, the curves began to diverge, with CICL1 disappearing significantly faster. At the end of the 7-day study, plasma CICL1 levels had returned to baseline, while significant amounts of ALC-0315 were still detected. Figure 13C In the spleen and liver, ALC-0315 levels remained almost unchanged over 7 days, while CICL1 levels decreased in the spleen and liver at 1 hour and 2 hours after administration, respectively. Figures 13D to 13E The mRNA levels in these three tissues were not correlated with ionizable cationic lipids. In plasma, the mRNA levels of F9 tLNP (containing CICL1) remained relatively stable for about 6 hours before rapidly decaying, while the mRNA levels of BF1 tLNP (containing ALC-0315) were significantly lower and decayed more rapidly from the start. Figure 13F The mRNA levels in the spleen were largely similar between the two lipid compositions. Figure 13G Although both tLNPs target CD8, BF1 tLNP delivers significantly more mRNA to the liver than F9 tLNP. The reduced hepatic delivery characteristics of CICL1 are related to its biodegradation properties and the effect of tLNPs on CD8. + Targeted combination with T cells offers the prospect of minimizing toxicity associated with the accumulation of ionizable lipids in the liver.

[0270] Example 14. mRNA delivery to T cells can be mediated by tLNPs targeting multiple T cell surface antigens. To evaluate whether the LNP compositions disclosed herein perform similarly well to different T cell targeting modalities, F5 tLNPs encapsulating mCherry mRNA, conjugated with anti-CD2, anti-CD5, anti-CD4, and anti-CD8 antibodies, were prepared. Human T cells from two donors (donor 1 and donor 2, not necessarily related to donors similarly identified in other examples) were transfected using tLNPs, each well containing 0.6 μg mRNA and 2 × 10⁵ cells. Twenty-four hours post-transfection, cells were stained with either anti-CD3 / anti-CD4 or anti-CD3 / anti-CD8 antibodies, and mCherry expression was analyzed by flow cytometry.

[0271] All four tLNPs targeting four different T cell markers CD2, CD5, CD4, and CD8 are located on CD3. + CD4 + and CD8 + Transfection efficiency on T cells (mCherry) + Both cellular percentage (%) and mCherry expression level (gMFI) showed similar good performance. As expected, tLNPs targeting CD4 and CD8 only provided CD8 expression. + and CD4 + Minimal transfection of cells ( Figure 14 While this does not mean that all antibodies targeting CD2, CD5, CD4, CD8, or other T cell markers will transfect T cells similarly well, as antibody affinity and recognized epitopes can affect transfection efficiency (data not shown), it does indicate that the tLNP composition is permissible for targeting specific moieties.

[0272] Example 15. In vitro delivery of CRISPR gene editing components Delivering gene-editing components into cells typically requires the delivery of at least two nucleic acids, such as the mRNA encoding an enzyme and the guide RNA. These two components differ in size; for example, Cas9 from Staphylococcus aureus Cas9 (SpCas9) requires mRNA close to 5 kb (kilobases), while the guide RNA can be on the order of 100 bases. Both differ in size from the mCherry and CAR mRNAs used in the examples above. Furthermore, enzymes used for base editing, lead editing, and similar techniques are often fusions of two or more enzymes. Therefore, we tested the ability of the disclosed tLNP composition to encapsulate and deliver two RNA materials of different sizes, larger and smaller than the mRNAs already used. Specifically, the F5 lipid composition was used to encapsulate SpCas9 mRNA and a single-stranded guide RNA (sgRNA) specific to the T-cell receptor α constant (TRAC) locus. Transfection and knockout efficiencies were evaluated.

[0273] According to the manufacturer's manual, use EasySep ™ The Human T Cell Isolation Kit (#100-0695, STEMCELL Technologies) isolates T cells from fresh leukocyte isolates. In short, the leukocyte isolate is treated with ammonium chloride solution (#07800, STEMCELL Technologies) to lyse residual red blood cells and platelets, and then treated with EasySep. ™ Wash with buffer (#20144, STEMCELL Technologies) and centrifuge gently. EasySep ™ The procedure involves magnetic beads and antibody complexes that recognize non-T cell antigens, enabling them to interact with a magnet (Easy 250 EasySep). ™ T cells were isolated after removing non-T cells from a magnet (#100-0821, STEMCELL Technologies). The isolated T cells were frozen in CryoStor. ® T cells were stored in CS10 (#210502, STEMCELL Technologies) and in LN2 for future use. T cell purity and phenotype were analyzed and confirmed by flow cytometry (Table 9).

[0274] Lipid composition F5 was used to encapsulate the mRNA encoding SpCas9 and the single-stranded guide RNA (sgRNA) with an N / P ratio of 6. Essentially as described above, the sgRNA has the target sequence GUCUCUCAGCUGGUACA (SEQ ID NO.:2), a 2'-O-methyl modification at the first three and last three bases, and a 3' phosphate thioester bond between the first three and last two bases (ordered from Synthego and modified using the CRISPRevolution sgRNA EZ kit). The SpCas9 mRNA is approximately 4.7 kb in length, and the sgRNA is 100 nucleotides in length, significantly longer and shorter than the CAR mRNA (approximately 1.9 kb) and mCherry mRNA (approximately 1 and 1.1 kb) used in Examples 1-11 above, respectively. An anti-CD5 antibody (chimeric 5D7) was conjugated to the LNP to provide tLNP (ch5D7-tLNP), which was frozen until use, essentially as described above. The N / P ratio was 6, and the mRNA:sgRNA ratio was 3:1 (w / w), or approximately 3:47 (mol / mol) considering the size difference. Previous work has shown that mRNA:sgRNA ratios of 9:1 and 1:1 (w / w) resulted in transfection rates that were approximately one-third lower than those of 3:1 (w / w) (data not shown).

[0275] Three days before tLNP transfection, isolated T cells from two donors were thawed and placed in a container containing OpTmizer. ™ T cell expansion basal culture medium (A10485, Gibco), OpTimizer ™ T-cell expansion supplement (A10484, Gibco), heat-inactivated human serum (HP1022HI, Valley Biomed), GlutaMax ™ T cells were cultured in complete T cell culture medium supplemented with 35050061 (Gibco), Pen / Strep (1514032, Gibco), and human IL-2 (202-IL, R&D). Immediately after thawing, cells were cultured in DynaBeads for T cell expansion and activation. ™ Human T cell activator CD3 / CD28 (#11161D, ThermoFisher) activated T cells at a 1:1 (bead:cell) ratio for 3 days. On the day of tLNP transfection, Easy 250 EasySep was first used. ™ Magnets (#100-0821, STEMCELL Technologies) debead activated T cells and deliver them at 1×10 6 Cells were resuspended at 200 μL / well in complete T cell culture medium. Prior to tLNP transfection, cells were loaded at 200 μL / well (2 × 10⁻⁶ cells / mL). 5 (1 cell / well) was further seeded into 96-well plates. To maintain cell culture, cells were inoculated with complete T-cell medium at 2 × 10⁻⁶ every 2–3 days. 5 Up to 1×10 6 / mL passaged T cells.

[0276] On the day of the experiment, tLNP was thawed and equilibrated to room temperature on the lab bench. Once completely thawed, tLNP was diluted in sterile water for injection. tLNP in doses providing 0 μg (i.e., no tLNP), 0.1 μg, 0.3 μg, 0.9 μg, 2.7 μg, or 8.1 μg effective loadings was diluted at 1 × 10⁻⁶. 6 Introduce cells at a rate of 1 cell / mL into 96-well plates. After incubation for 1 hour, 2 hours, 4 hours, or 24 hours, wash the cells three times with PBS to remove free tLNPs (no 8.1 μg dose was available for the 4-hour and 24-hour time points). Passage the transfected T cells and incubate at 2 × 10⁻⁶ cells / mL. 5 Up to 1×10 6 / mL was maintained in complete T cell culture medium for 4 days, and then collected for knockout analysis.

[0277] Four days after tLNP transfection, T cells were collected and stained with Aqua Live / Dead (#L34965, Invitrogen), anti-CD3-APC / Cy7 (BDB55775, BD), anti-CD4-BV650 (317436, BioLegend), and anti-CD8-FITC (300906, BioLegend) (see Table 9), and analyzed by multicolor flow cytometry. In the absence of the TCRα chain, CD3 is not expressed on the cell surface. Therefore, TRAC knockout can be assessed by CD3 staining.

[0278] like Figure 15 As observed, at 1 hour, a 0.9 μg dose was effective against CD8. + Approximately 9% knockout was achieved in T cells. Higher doses did increase knockout, but not proportionally to the increase in dose, possibly reflecting CD5 binding saturation at higher doses. Long-term exposure led to CD8... + T-cell knockout increased to approximately 15%, and the dose response was more pronounced.

[0279] Example 16. In vitro re-administration of CRISPR gene editing components Assess whether the second exposure to tLNP as described in the preceding examples would increase the knockout frequency. Thaw the tLNP and equilibrate it to room temperature on the lab bench. Once fully thawed, dilute the tLNP in sterile water for injection. Dilute the tLNP in amounts providing 0 μg (i.e., no tLNP), 0.1 μg, 0.3 μg, 0.9 μg, 2.7 μg, or 8.1 μg payload at 1 × 10⁻⁶. 6 Cells / mL were introduced into cells seeded in 96-well plates. After 1 hour, cells were washed three times with PBS to remove free tLNPs. For wells that were re-doped, cells were transfected with a second tLNP, providing a payload of 0.3 μg or 2.7 μg, 24 hours after the first transfection, and incubated again for 1 hour, followed by washing with PBS as before. Transfected T cells showed a growth rate of 2 × 10⁶ cells / mL after the re-doping. 5 Up to 1×10 6 The cells were passaged in 1 mL of complete T cell culture medium and maintained for 4 days, and then collected as described above for knockout analysis by flow cytometry.

[0280] like Figure 16 As observed, at each dose tested, the second dose increased the knockout percentage, with larger second doses exhibiting greater efficacy. These results suggest that any limitations on transfection due to target cell surface antigen saturation, and consequently any limitations on the knockout percentage, can be overcome by re-administration.

[0281] Example 17. In vitro re-administration and dual targeting of CRISPR gene editing components This experiment evaluated the knockout efficiency using tLNPs targeting CD8 and combinations of tLNPs targeting both CD8 and CD5, as well as re-dose administration. Human T cell isolation and culture were as described above, as were the tLNPs, except that some LNPs encapsulating SpCas9 mRNA and sgRNA payloads were conjugated with an anti-CD8 antibody (chimeric RPA-T8, also known as chRPA-T8) instead of an anti-CD5 antibody (e.g., ch5D7).

[0282] On day 1 after transfection, tLNP was thawed and equilibrated to room temperature on the lab bench. Once completely thawed, tLNP was diluted in sterile water for injection. Anti-CD8-tLNP (chRPA-T8-tLNP) was provided with effective loads of 0 μg (i.e., no tLNP), 0.03 μg, 0.3 μg, or 3 μg. Figure 17 ) with 1×10 6 / mL was introduced into cells seeded in 96-well plates. For co-delivery of anti-CD5-tLNP and anti-CD8-tLNP, tLNP was premixed at a 3:1 wt / wt payload ratio and administered with a total payload of 0 μg, 0.3 μg, 0.9 μg, or 3 μg. After 1 hour of tLNP incubation, cells were washed three times with PBS to remove free tLNP. 24 hours after the first transfection, cells were transfected with a second dose of tLNP containing 3 μg payload. This second tLNP administration was also incubated with the cells for 1 hour, followed by PBS washing. Transfected T cells were passaged and, after the second transfection, were seeded at 2 × 10⁶ cells / mL. 5 Up to 1×10 6 / mL was maintained in complete T cell culture medium for 5 days, and then collected for knockout analysis.

[0283] Six days after the first tLNP transfection, T cells were collected and stained with Aqua Live / Dead (#L34965, ThermoFisher), anti-CD3-APC / Cy7 (BDB55775, BD), anti-CD4-BV650 (317436, BioLegend), and anti-CD8-FITC (300906, BioLegend), and analyzed by multicolor flow cytometry.

[0284] like Figure 16 As seen in Figure B (top left), a single 0.3 μg dose of the CD8-targeting tLNP (chRPA-T8-tLNP) can effectively target CD8. + A 30% to 40% CD3 knockout was achieved in cells, but increasing the single dose to 3.0 μg did not further increase CD3 knockout. However, for both the initial doses of 0.3 μg and 3.0 μg, the second dose of 3.0 μg resulted in CD8 knockout. +The percentage of CD3 knockout in cells increased to approximately 50%. Figure 16 A, top left figure). For both 0.3 μg and 3.0 μg doses, tLNPs targeting CD8 and CD5 (ch5D7-tLNP) were observed. + Co-delivery of chRPA-T8-tLNP in CD8 + Approximately 35% to 50% of CD3 knockout was achieved in cells, depending on the donor ( Figure 17 (See upper middle figure). For both initial doses of 0.3 μg and 3.0 μg, a repeat administration of 3.0 μg tLNP increased CD3 knockout to approximately 50-70%, depending on the donor ( Figure 16 B, upper middle image). In Figure 17 The right-hand panel shows representative flow cytometry histograms from each of the four conditions. Compared to the single-dose histogram, the histogram for cells that were re-dosed showed a wider peak and reached higher brightness, reflecting the slower CD3 turnover and a one-day reduction in the number of days elapsed since the last exposure to tLNP at the time of assessment. As expected, there was no significant CD3 knockout in CD4 cells transfected with tLNP targeting CD8 alone. Figure 17 (See bottom left image).

[0285] Example 18. In vivo delivery of CRISPR gene editing components To evaluate the ability of the disclosed tLNP to deliver gene-editing components to human T cells in vivo, tLNP targeting CD5 and CD8 was administered, alone or in combination, to NSG mice with transplanted human T cells in one or two doses. As in other gene-editing embodiments, tLNP was used with an F5 lipid composition having an overall N / p ratio of 6 and a 3:1 (w / w) SpCas9:sgRNA payload, as described above.

[0286] On day -20, 1×10⁻⁶ cells were transplanted into NSG mice. 6 Personal T cells (mice were also infused with 500,000 Nalm6, which was irrelevant to this experiment). Five mice in each group were transfected with tLNPs targeting CD5 or CD8, or a 3:1 mixture of tLNPs targeting CD5:CD8 (w / w payload), and three untreated mice were also included. On day 0, tLNPs containing 30 μg payload in storage buffer were diluted to 100 μl with sterile water for injection and then infused into the tail vein of the mice. For repeated administration, tLNPs containing 20 μg payload were infused into three of the five mice in each group 24 hours after the first infusion. Five days after the first infusion, blood samples were collected and stained with anti-human CD45, CD3, CD4, and CD8 antibodies. Total human T cells were gated to hCD45. + Cells. Using CD4 + and CD8+ T cell gating was used to study CD4 and CD8 T cell subsets. TRAC knockout T cells were gated to CD3- and CD4- T cells from the entire human T cell population. + / CD3- or CD8 + / CD3- T cells. From CD3- / CD45 respectively + CD3-CD4 + / CD45 + CD3-CD8 + / CD45 + Cellular calculation of total T cells, CD4 + and CD8 + The percentage of T cells with TRAC (CD3) knockout.

[0287] The results are presented in Figure 18 This study demonstrated that SpCas9 and sgRNA are effectively delivered to T cells in vivo. In this experiment, CD4... + T cell editing is less than 10%, and re-administration has minimal impact. A single dose of tLNP targeting CD5 (ch5D7-tLNP) or CD8 (chRPA-T8-tLNP), or a mixture of both tLNPs, is used, along with total T or CD8... + Cell editing was approximately 15% or 20%, respectively. In all cases, re-dose increased the percentage of CD3 knockout, but for treatments including tLNP targeting CD8, the percentage of CD8 knockout was lower. + This is most evident in the group, where the editing rate is approximately 40%. Figure 18 ).

[0288] Example 19.CD117 + In vitro transfection of cells To evaluate the ability of the disclosed tLNP composition to deliver mRNA to human HSCs, tLNPs encapsulating mCherry mRNA and containing various anti-CD117 antibodies as binding sites were transfected in vitro with CD34 cells. + Cells. Essentially as described above, tLNPs of composition F5 were prepared using four different anti-CD117 antibodies: 104D2 (#1), JSP191 (#2), CK6 (#3), and Ab85 (#4). 104D2 does not block the action of SCF-1 (the ligand of CD117), while the other three anti-CD117 antibodies are blocking antibodies. tLNPs of composition F5 conjugated with telopavir (an anti-HIV-1 gp120 antibody) were prepared as a negative control.

[0289] Peripheral blood CD34 of Plexus mobilized +Hematopoietic stem cells were obtained from STEMCELL Technologies (#70075.1). One day before tLNP transfection, cells were thawed in X-VIVO 10 medium (#BP04-743Q, Lonza) containing 3% human AB serum (#HP1022, Valley Biomed). The cells were then gently centrifuged and resuspended in CD34 pre-stimulated medium, which consisted of X-VIVO 10 medium (#BP04-743Q, Lonza), 3% human AB serum (#HP1022, ValleyBiomed), 100 ng / mL FIt3-L (#308-FK / CF, R&D Systems), 10 ng / mL TPO (#130-096-479, Miltenyi), 60 ng / mL IL-3 (#130-095-069, Miltenyi), 1 μM UM729 (#72332, STEMCELL Tech), and 1 μM StemRegenin1 (#72344, STEMCELL Tech).

[0290] On the day of tLNP transfection, cells were loaded with 2×10⁻⁶ cells per cell line. 5 Reseed tLNPs at 100 cells / mL in fresh CD34 pre-stimulated medium. Thaw tLNPs and equilibrate to room temperature on the lab bench. Once completely thawed, dilute tLNPs in sterile water for injection. Dilute tLNPs in amounts providing 0 μg (i.e., no tLNP), 2.5 μg, 5 μg, or 9 μg effective loadings at 1 × 10⁻⁶ cells / mL. 5 Cells were introduced per well into 48-well plates. After 1 hour of tLNP incubation, the cells were washed three times with PBS to remove free tLNPs. The transfected cells were then passaged into new 48-well tissue culture plates and maintained in CD34 amplification medium containing 1× StemSpan CD34. + StemSpan SFEM II (#09655, STEMCELL Tech) is an amplification supplement (#02691, STEMCELL Tech).

[0291] The 48-well plates were then placed in an IncuCyte Live-Cell Analysis System (Sartorius) to acquire images at 10× magnification in both the phase and red channels for 72 hours. Image analysis was performed using IncuCyte software to calculate mCherry. + % is ((total red area (per well) / total phase area (per well)) (%)), and MFI is (integral intensity of red (per well) / total red area (per well)).

[0292] Targeting CD117 with 104D2 + Cellular tLNPs showed productive transfection of HSCs with a clear dose-response, where for three increasing doses, mCherry + Cell percentage peaked at approximately 30% to 55% 24 to 36 hours after tLNP introduction. Figure 19A The expression level also peaked within the same time frame. Figure 19B The transfection rates of the three anti-CD117 blocking antibodies were less than approximately 4%. Therefore, the disclosed tLNP composition can be used to transfect HSCs in addition to T cells.

[0293] Example 20. Comparison of combined partial conjugation chemistry Several chemicals can be used to conjugate the binding moiety to LNPs. Three chemicals for conjugating antibodies to LNPs containing maleimide-functionalized PEG-lipids are compared here. One chemical, AJICAP, is site-selective, while the other two chemicals, SATA and TCEP, are not.

[0294] To activate the antibody via SATA chemistry and tLNP conjugation, an anti-CD5 (ch5D7) antibody was conjugated to an LNP encapsulating mCherry mRNA via N-succinimide-S-acetylthioacetate (SATA)-maleimide conjugation chemistry. The anti-CD5 antibody in phosphate-buffered saline (PBS) was modified with SATA (Sigma-Aldrich) (SATA:Ab molar ratio 5) to introduce a thiol group at an accessible lysine residue, allowing conjugation with maleimide. The SATA was deprotected with sufficient 0.5M hydroxylamine, and unreacted fractions were removed via a Zeba spin desalting column (Thermo Scientific, Rockford, IL). The reactive thiol group on the antibody was then conjugated to the maleimide portion of the LNP using thioether conjugation chemistry. The conjugated tLNP (LNP conjugated with the targeting antibody) was purified using a Sepharose CL-4B gel filtration column (Sigma-Aldrich). The tLNP was frozen at -80°C until use.

[0295] For antibody activation via TCEP chemistry and tLNP conjugation, anti-CD5 antibody in PBS was conjugated by first partially reducing the cysteine ​​bonds in the antibody with tris(2-carboxy)phosphine (TCEP, TCEP:Ab molar ratio 0.25) to generate thiol groups, followed by removal of unreacted components via a Zeba spin desalting column (Thermo Scientific, Rockford, IL). Then, thioether conjugation chemistry was used to partially conjugate the reactive thiol groups on the antibody to the maleimide on the LNP. The conjugated tLNP (LNP conjugated with the targeting antibody) was purified using TFF (tangential flow filtration). The tLNP was frozen at -80°C until use.

[0296] For AJICAP tLNP conjugation, thiolated antibodies are typically prepared by Ajinomoto Bio-Pharma Services as described in WO2019 / 240,287. Anti-CD5 AjiCap monothiolated or dithiolated antibodies are prepared in 20 mM acetate at pH 5.5 for conjugation. The antibody is thiolated at Lys248 of the heavy chain (EU number, or see Fujii T et al.). Bioconjugate Chem (2023, 34, 4, 728-738). To obtain an antibody in which only one heavy chain is thiolated (monothiol), the desired substance was purified from the incomplete reaction. Two antibodies were conjugated via the maleimide portion of the LNP using thioether conjugation chemistry. The conjugated tLNP (LNP conjugated with the targeting antibody) was purified using a Sepharose CL-4B gel filtration column (Sigma-Aldrich). The tLNP was frozen at -80°C until use.

[0297] The physicochemical properties of tLNP are within acceptable limits and are presented in Table 10.

[0298] The ability of these tLNPs to deliver mCherry mRNA into human T cells was tested in in vitro and in vivo models.

[0299] To evaluate in vitro transfection, human T cells from a healthy donor were infected with an anti-CD3 / CD28 antibody (Dynabeads). ® Human T-activators (CD3 / CD28) were activated, and tLNPs targeting CD5 were transfected with an mCherry mRNA payload to provide 0.6 μg mRNA per 200,000 cells in vitro. tLNPs were incubated with T cells in a 37°C CO2 incubator for 1 hour, and then transfected with medium (RPM 11640). +The cells were washed away with 10% FBS (supplemented with 100 IU / mL hIL-2). T cells were then incubated with culture medium in a 37°C CO2 incubator for 24 hours, followed by CD3 assay by flow cytometry. + mCherry expression in T cells. Comparable transfection efficiencies (mCherry) were observed with all four tLNP formulations. + CD3 + T cell percentage) and expression level (mean fluorescence intensity) Figures 20A to 20B ).

[0300] To evaluate in vivo transfection, NSG mice (approximately 10 weeks old) were purchased from The Jackson Laboratory and acclimatized for at least 5 days. Ten million human PBMCs were injected intravenously via the tail vein. Nineteen days after PBMC transfer, circulating human CD45 was assessed by flow cytometry. + Cell frequency, and based on the human CD45 in the blood of animals. + The average value of the cells was grouped similarly.

[0301] On day 20, mice were intravenously injected with tLNP containing 10 μg mRNA (0.5 mg / kg). 24 hours post-injection, spleen samples were obtained, processed, and the presence of mCherry expression on human T cells was analyzed by flow cytometry. Comparable transfection efficiencies (mCherry expression) were observed with all four tLNP formulations. + CD3 + T cell percentage) and expression level (mean fluorescence intensity) Figures 20C to 20D ).

[0302] Example 21. Dual conjugation of antibody and peptide to LNP This capability was demonstrated by conjugating LNPs modified with two different peptide moieties using complementary chemistry with two distinct functionalized PEG-lipid LNP components. Specifically, peptide fragments containing an anti-mouse CD5 binding moiety and mouse CD47 were conjugated to an LNP comprising CICL1:DSPC:CHOL:DMG-PEG(2k):DSPE-PEG(2k)-Mal:DSPE-PEG(2k)-DBCO at a ratio of 58:10:30.5:1.3:0.1:0.1. However, this strategy can also be used to conjugate LNPs with two antibodies, each with different specificities.

[0303] Anti-CD5 antibodies can act as a targeting component of tLNP. Targeting T cells with antibodies is also expected to reduce uptake by hepatocytes, consistent with the above findings.

[0304] CD47 is a protein that promotes the "don't eat me" signal, binding to signal regulatory protein α (SIRPα) on macrophages to prevent phagocytosis [Tsai et al., 2008, ]. J Cell Biol. 180(5):989-1003. doi:10.1083 / jcb.200708043. PMID:18332220; PMCID:PMC2265407]. The interaction between CD47 and SIRPα is highly conserved across various mammalian species, enabling the use of CD47 modifications on drug delivery carriers to enhance delivery efficiency. It has been reported that short peptide sequences in CD47 have a binding affinity for SIRPα that is 10 times greater than that of the full-length CD47 protein [Rodriguez et al., 2013, Science 339(6122):971-5. doi: 10.1126 / science.1229568.PMID:23430657; PMCID:PMC3966479]. Therefore, conjugating this CD47 peptide NYTCEVTELSREGKTVIELK (SEQ ID NO: 3) with tLNP can prolong in vivo circulation by inhibiting macrophage uptake.

[0305] The CD47 peptide was synthesized using an azide moiety, which is a functional group that can be conjugated to a dibenzocyclooctyne (DBCO) moiety via a copper-strain-promoted SPAAC click reaction. The azide was attached to the N-terminus of the peptide separated by a polyethylene glycol (PEG4) linker, resulting in the structure N3-PEG4-NYTCEVTELSREGKTVIELK-NH2 (SEQ ID NO: 4). Therefore, the azide moiety on the CD47 peptide allows for covalent conjugation to LNPs containing DBCO-functionalized lipids. The advantages of using these functional groups (e.g., azides and DBCO) in click chemistry for tLNP formulations include: 1) rapid reaction kinetics, requiring no additional catalyst to initiate the reaction; 2) biocompatibility across a wide pH range; and 3) providing additional or alternative conjugation strategies for the thiol-maleimide chemistry described above for antibody-LNP conjugation.

[0306] The LNP intermediate was formed essentially as described in Example 1. The LNP contained 0.1 mol% DSPE-PEG (2000)-DBCO and 0.1 mol% DSPE-PEG (2000)-maleimide and had good physicochemical properties (Table 11).

[0307] Essentially, as described above, antibodies were modified with SATA and conjugated with LNPs. The system was designed for the reaction of SATA-modified antibodies with maleimide-functionalized PEG-lipids. Antibody addition was successful, and the physicochemical properties of the LNPs did not change significantly (Table 12). The protein (antibody):mRNA ratio was determined by BCA assay.

[0308] A second conjugation step was used to link the CD47 peptide. The system was designed to react azide-modified peptides with DBCO-functionalized PEG-lipids in a click chemistry reaction. Conjugation was performed with peptide-to-DBCO molar ratios of 1:5, 1:10, and 1:20. Successful conjugation was reflected by an increase in particle size and protein:mRNA ratio (Table 13), with the protein portion of the ratio indicating the sum of the antibody and CD47 peptide.

[0309] Successful double conjugation was only observed when mCD5 was conjugated first, followed by CD47 peptide (the opposite data were not shown).

[0310] Implementation Plan Implementation Scheme 1: A lipid nanoparticle (LNP) comprising a lipid composition, said lipid composition comprising: Approximately 35 mol% to approximately 65 mol% of ionizable cationic lipids having the following structure Where R is or PEG-lipids comprising approximately 0.5 mol% to approximately 3 mol% of functionalized and non-functionalized PEG-lipids. Phospholipids of approximately 7 mol% to approximately 13 mol%, and Sterols, approximately 27 mol% to approximately 50 mol%.

[0311] Implementation Scheme 2: The LNP according to Implementation Scheme 1 further includes a payload having a net negative charge, said payload being selected from peptides, polypeptides, proteins, small molecules, nucleic acids, and combinations thereof.

[0312] Implementation Scheme 3: The LNP according to Implementation Scheme 2, wherein the payload comprises nucleic acid molecules.

[0313] Implementation Scheme 4: The LNP according to Implementation Scheme 3, wherein the N / P ratio is about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 7, or about 6.

[0314] Implementation Scheme 5: The LNP according to Implementation Scheme 3 or 4, wherein the nucleic acid includes mRNA.

[0315] Implementation Scheme 6: The LNP according to Implementation Scheme 3 or 5, wherein the nucleic acid molecule comprises circular RNA, self-replicating RNA, microRNA (miRNA), siRNA, antisense RNA, tracking RNA (trRNA), single-stranded guide RNA (sgRNA), DNA, or a genetic engineering system.

[0316] Implementation Scheme 7: The LNP according to Implementation Scheme 5 or 6, wherein the mRNA encodes a T cell receptor (TCR), a chimeric antigen receptor (CAR), or an immune cell connector.

[0317] Implementation Scheme 8: The LNP according to Implementation Scheme 5 or 6, wherein the mRNA encodes a polypeptide containing a gene-editing nuclease.

[0318] Implementation Scheme 9: The LNP according to any one of Implementation Schemes 1 to 8, wherein the PEG-lipid comprises about 0.1 mol% to about 0.3 mol% of functionalized PEG-lipid.

[0319] Implementation Scheme 10: The LNP according to Implementation Scheme 9, wherein the nonfunctionalized PEG-lipid and the functionalized PEG-PEG-lipid are not the same PEG-lipid.

[0320] Implementation Scheme 11: The LNP according to Implementation Scheme 10, wherein the functionalized PEG-lipid comprises diacylphosphatidylethanolamine and the nonfunctionalized PEG-lipid comprises diacylglycerol.

[0321] Implementation Scheme 12: The LNP according to Implementation Scheme 11, wherein the functionalized PEG-lipid comprises distearate phosphatidylethanolamine (DSPE).

[0322] Implementation Scheme 13: The LNP according to Implementation Scheme 11 or 12, wherein the nonfunctionalized PEG-lipid comprises distearate (DSG).

[0323] Implementation Scheme 14: The LNP according to any one of Implementation Schemes 1 to 13, wherein the PEG portion of the PEG lipid has a molecular weight of about 1,000 to about 5,000.

[0324] Implementation Scheme 15: The LNP according to Implementation Scheme 14, wherein for both the functionalized and nonfunctionalized PEG-lipids, the PEG portion is PEG2000.

[0325] Implementation Scheme 16: The LNP according to Implementation Scheme 15, wherein the PEG portion of the functionalized PEG-lipid is larger than the PEG portion of the nonfunctionalized PEG-lipid.

[0326] Implementation Scheme 17: The LNP according to any one of Implementation Schemes 9 to 16, wherein the PEG portion of the functionalized PEG-lipid comprises a terminal maleimide portion.

[0327] Implementation Scheme 18: The LNP according to any one of Implementation Schemes 9 to 17 further comprises a binding portion conjugated to the functionalized PEG-lipid.

[0328] Implementation Scheme 19: The LNP according to Implementation Scheme 18, wherein the functionalized PEG-lipid is conjugated to the binding portion via a succinimide moiety, a hydrolyzed succinimide moiety, or a thiomaleimide moiety.

[0329] Implementation Scheme 20: The LNP according to Implementation Scheme 18 or 19, wherein the binding portion comprises an antibody or its antigen-binding domain.

[0330] Implementation Scheme 21: The LNP according to Implementation Scheme 20, wherein the binding portion is an intact antibody, microantibody, F(ab)2, F(ab), bivalent antibody, single-chain Fv (scFv) or nanobody.

[0331] Implementation Scheme 22: The LNP according to any one of Implementation Schemes 18 to 21, wherein the binding portion specifically binds to: a) Surface proteins of immune cells, selected from the following groups: CD2, CD3, CD4, CD5, CD7, CD8, CD28, 4-1BB, CD166, CTLA-4, GITR, LAG-3, OX40, PD-1, TIM-3, CD25, low-affinity IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor, CD14, CD16a, CD32, CD40, CD11b (Mac-1), CD64, DEC205, and TREM2; b) Hematopoietic stem cell (HSC) surface proteins selected from the group consisting of: CD117, CD34, CD44, CD45, CD90, CD105, CD133, BMPR2, and Sca-1, or wherein the binding portion specifically binds to mesenchymal stem cell (MSC) surface proteins selected from the group consisting of: 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; or c) Mesenchymal stem cell (MSC) surface proteins selected from the group consisting of: 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.

[0332] Implementation Scheme 23: The LNP according to any one of Implementation Schemes 18 to 21, wherein the binding portion specifically binds to hematopoietic stem cell (HSC) surface proteins, the hematopoietic stem cell surface proteins being selected from the group consisting of: CD117, CD34, CD44, CD45, CD90, CD105, CD133, BMPR2 and Sca-1.

[0333] Implementation Scheme 24: The LNP according to any one of Implementation Schemes 18 to 21, wherein the binding portion specifically binds to mesenchymal stem cell (MSC) surface proteins selected from the group consisting of: 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.

[0334] Implementation Scheme 25: The LNP according to any one of Implementation Schemes 1 to 24, wherein the lipid composition has about 1 mol% to about 2 mol% of total PEG-lipids.

[0335] Implementation Scheme 26: The LNP according to any one of Implementation Schemes 1 to 25, wherein the lipid composition has about 0.1 mol% of functionalized PEG-lipid.

[0336] Implementation Scheme 27: The LNP according to any one of Implementation Schemes 1 to 26, wherein the lipid composition has about 40 mol% to about 60 mol% of ionizable cationic lipids.

[0337] Implementation Scheme 28: The LNP according to Implementation Scheme 27, wherein the lipid composition has about 58 mol% ionizable cationic lipids.

[0338] Implementation Scheme 29: The LNP according to any one of Implementation Schemes 1 to 28, wherein the lipid composition has about 10 mol% phospholipids.

[0339] Implementation Scheme 30: The LNP according to Implementation Scheme 29, wherein the phospholipid is distearate phosphatidylcholine.

[0340] Implementation Scheme 31: The LNP according to Implementation Scheme 29, wherein the phospholipid is arachidophosphatidylcholine.

[0341] Implementation Scheme 32: The LNP according to any one of Implementation Schemes 1 to 31, wherein the lipid composition comprises about 33 mol% to about 38 mol% of sterols.

[0342] Implementation Scheme 33: The LNP according to any one of Implementation Schemes 1 to 32, wherein the sterols include cholesterol.

[0343] Implementation Scheme 34: The LNP according to any one of claims 1 to 33, wherein the sterols include phytosterols.

[0344] Implementation Scheme 35: The LNP according to Implementation Scheme 34, wherein the phytosterols include campesterol, sitosterol or stigmasterol or combinations thereof.

[0345] Implementation Scheme 36: The LNP according to Implementation Scheme 33, wherein the sterol is cholesterol.

[0346] Implementation Scheme 37: The LNP according to Implementation Scheme 4 contains CICL1:DSPC:cholesterol:DSG-PEG2000:DSPE-PEG2000-maleimide in a ratio of 58:7:33.5:1.4:0.1 or 58:10:30.5:1.4:0.1.

[0347] Implementation Scheme 38: The LNP according to Implementation Scheme 4 contains CICL1:DSPC:cholesterol:DPG-PEG2000:DSPE-PEG2000-maleimide in a ratio of 58:7:33.5:1.4:0.1 or 58:10:30.5:1.4:0.1.

[0348] Implementation Scheme 39: The LNP according to any one of Implementation Schemes 1 to 38 comprises: (a) a payload comprising mRNA encoding a chimeric antigen receptor specific to human CD19, human CD20, human CD19 and human CD20; human BCMA; human FAP; or a payload comprising mRNA encoding an RNA-directed nuclease and / or a guide RNA; and (b) a binding portion comprising an antibody or an antigen-binding fragment thereof specific to human CD5, human CD8 or human CD2, wherein the antibody or antigen-binding fragment thereof is covalently linked to the functionalized PEG-lipid via lysine or cysteine ​​residues of the antibody or binding fragment thereof.

[0349] Implementation Scheme 40: The LNP according to any one of Implementation Schemes 1 to 38 comprises: (a) an mRNA encoding an RNA-guided nuclease and a payload of a guide RNA; and (b) a binding portion of an antibody or an antigen-binding fragment thereof that is specific to human CD5, human CD8 or human CD2, wherein the antibody or the antigen-binding fragment thereof is covalently linked to the functionalized PEG-lipid via a lysine or cysteine ​​residue of the antibody or the binding fragment thereof.

[0350] Embodiment 41: A composition comprising LNP according to any one of Embodiments 1 to 40, and further comprising one or more pharmaceutically acceptable carriers or excipients.

[0351] Implementation Scheme 42: A method for delivering a payload to immune cells or stem cells, comprising contacting the LNP according to any one of Implementation Schemes 1 to 40 with the immune cells or stem cells of a subject.

[0352] Implementation Scheme 43: The method according to Implementation Scheme 42, wherein delivery of the payload includes transfecting the immune cells or stem cells.

[0353] Implementation Scheme 44: The method according to Implementation Scheme 42 or 43, wherein the contact is performed in vivo, in vitro or ex vivo.

[0354] Implementation Scheme 45: A method for reprogramming immune cells or stem cells, comprising administering to a subject an LNP according to any one of Implementation Schemes 1 to 40 or a composition according to Implementation Scheme 41.

[0355] Implementation Scheme 46: The method according to any one of Implementation Schemes 42 to 45, wherein the stem cells are hematopoietic stem cells (HSCs) or MSCs.

[0356] Implementation Scheme 47: The method according to any one of Implementation Schemes 42 to 45, wherein the immune cell is a T cell.

[0357] Implementation Scheme 48: A method of treating a disease, comprising administering to a subject in need the LNP according to any one of Implementation Schemes 1 to 40 or the composition according to Implementation Scheme 41.

[0358] Implementation Scheme 49: A method for conditioning a subject receiving an engineered agent, comprising providing the subject with an LNP according to any one of Implementation Schemes 1 to 38 before, simultaneously with or after administration of the engineered agent, the LNP comprising a nucleic acid encoding the conditioning agent.

[0359] Implementation Scheme 50: The method according to any one of Implementation Schemes 42 to 49, comprising intravenous administration to the subject.

[0360] Implementation Scheme 51: A method for reprogramming immune cells or hematopoietic stem cells (HSCs), comprising contacting an LNP according to any one of Implementation Schemes 1 to 40 with the immune cells or HSCs, wherein the reprogrammed immune cells or HSCs do not contain DNA encoding a reprogramming agent.

[0361] Implementation Scheme 52: The method according to Implementation Scheme 51, wherein the reprogrammed immune cell is a CAR-T cell.

[0362] Implementation Scheme 53: A reprogrammed immune cell prepared by means of the method according to Implementation Scheme 51 or 52.

[0363] Implementation Scheme 54: A method for reprogramming an HSC, comprising contacting the HSC with an LNP according to any one of Implementation Schemes 1 to 40, wherein the reprogrammed HSC does not contain DNA encoding components of a gene editing system.

[0364] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, as each individual publication, patent and patent application is specifically and individually indicated to the extent of its inclusion herein by reference.

[0365] While some embodiments have been illustrated and described in detail in the accompanying drawings and the foregoing description, such illustrations and descriptions are intended to be illustrative rather than restrictive. Other variations of the disclosed embodiments can be understood and implemented in the practical claims by studying the drawings, the disclosure, and the appended claims. The mere fact that certain measures or features are recited in mutually different dependent claims does not indicate that combinations of those measures or features cannot be used. Any reference numerals in the claims should not be construed as limiting the scope.

Claims

1. A lipid nanoparticle (LNP) comprising a lipid composition, said lipid composition comprising: Approximately 35 mol% to approximately 65 mol% of ionizable cationic lipids having the following structure Where R is or PEG-lipids comprising approximately 0.5 mol% to approximately 3 mol% of functionalized and non-functionalized PEG-lipids. Phospholipids of approximately 7 mol% to approximately 13 mol%, and Sterols, approximately 27 mol% to approximately 50 mol%.

2. The LNP of claim 1, further comprising a payload having a net negative charge, said payload being selected from peptides, polypeptides, proteins, small molecules, nucleic acids, and combinations thereof.

3. The LNP according to claim 2, wherein the payload comprises a nucleic acid molecule.

4. The LNP according to claim 3, wherein the N / P ratio is about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 7, or about 6.

5. The LNP according to claim 3 or 4, wherein the nucleic acid comprises mRNA.

6. The LNP according to claim 3 or 5, wherein the nucleic acid molecule comprises circular RNA, self-replicating RNA, microRNA (miRNA), siRNA, antisense RNA, guide RNA (gRNA), crirRNA (crRNA), tracking RNA (trRNA), single-stranded guide RNA (sgRNA), DNA, or a genetic engineering system.

7. The LNP according to claim 5 or 6, wherein the mRNA encodes a T-cell receptor (TOR), a chimeric antigen receptor (CAR), or an immune cell connector.

8. The LNP according to claim 5 or 6, wherein the mRNA encodes a polypeptide comprising a gene-editing nuclease.

9. The LNP according to any one of claims 1 to 8, wherein the PEG-lipid comprises about 0.1 mol% to about 0.3 mol% of functionalized PEG-lipid.

10. The LNP of claim 9, wherein the nonfunctionalized PEG-lipid and the functionalized PEG-PEG-lipid are not the same PEG-lipid.

11. The LNP of claim 10, wherein the functionalized PEG-lipid comprises diacylphosphatidylethanolamine, and the nonfunctionalized PEG-lipid comprises diacylglycerol.

12. The LNP of claim 11, wherein the functionalized PEG-lipid comprises distearate (DSPE).

13. The LNP according to claim 11 or 12, wherein the nonfunctionalized PEG-lipid comprises distearate (DSG).

14. The LNP according to any one of claims 1 to 13, wherein the PEG portion of the PEG lipid has a molecular weight of about 1,000 to about 5,000.

15. The LNP of claim 14, wherein the PEG portion is PEG2000 for both the functionalized PEG-lipid and the nonfunctionalized PEG-lipid.

16. The LNP of claim 15, wherein the PEG portion of the functionalized PEG-lipid is larger than the PEG portion of the nonfunctionalized PEG-lipid.

17. The LNP according to any one of claims 9 to 16, wherein the PEG portion of the functionalized PEG-lipid comprises a terminal maleimide portion.

18. The LNP according to any one of claims 9 to 17, further comprising a binding portion conjugated to the functionalized PEG-lipid.

19. The LNP of claim 18, wherein the functionalized PEG-lipid is conjugated to the binding portion via a succinimide moiety, a hydrolyzed succinimide moiety, or a thiomaleimide moiety.

20. The LNP of claim 18 or 19, wherein the binding portion comprises an antibody or its antigen-binding domain.

21. The LNP of claim 20, wherein the binding portion is an intact antibody, a microantibody, F(ab)2, F(ab), a bivalent antibody, a single-chain Fv (scFv), or a nanobody.

22. The LNP according to any one of claims 18 to 21, wherein the binding portion specifically binds to: a) Surface proteins of immune cells, selected from the following groups: CD2, CD3, CD4, CD5, CD7, CD8, CD28, 4-1BB, CD166, CTLA-4, GITR, LAG-3, OX40, PD-1, TIM-3, CD25, low-affinity IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor, CD14, CD16a, CD32, CD40, CD11b (Mac-1), CD64, DEC205, and TREM2; b) Hematopoietic stem cell (HSC) surface proteins selected from the group consisting of: CD117, CD34, CD44, CD45, CD90, CD105, CD133, BMPR2, and Sca-1, or wherein the binding portion specifically binds to mesenchymal stem cell (MSC) surface proteins selected from the group consisting of: 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; or c) Mesenchymal stem cell (MSC) surface proteins selected from the group consisting of: 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.

23. The LNP according to any one of claims 18 to 21, wherein the binding portion specifically binds to hematopoietic stem cell (HSC) surface proteins, the hematopoietic stem cell surface proteins being selected from the group consisting of: CD117, CD34, CD44, CD45, CD90, CD105, CD133, BMPR2, and Sca-1.

24. The LNP according to any one of claims 18 to 21, wherein the binding portion specifically binds to mesenchymal stem cell (MSC) surface proteins, said MSC surface proteins being selected from the group consisting of: 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.

25. The LNP according to any one of claims 1 to 24, wherein the lipid composition has about 1 mol% to about 2 mol% of total PEG-lipids.

26. The LNP according to any one of claims 1 to 25, wherein the lipid composition has about 0.1 mol% of functionalized PEG-lipid.

27. The LNP according to any one of claims 1 to 26, wherein the lipid composition has about 40 mol% to about 60 mol% of ionizable cationic lipids.

28. The LNP of claim 27, wherein the lipid composition has about 58 mol% of ionizable cationic lipids.

29. The LNP according to any one of claims 1 to 28, wherein the lipid composition has about 10 mol% phospholipids.

30. The LNP according to claim 29, wherein the phospholipid is distearate phosphatidylcholine.

31. The LNP according to claim 29, wherein the phospholipid is arachidophosphatidylcholine.

32. The LNP according to any one of claims 1 to 31, wherein the lipid composition comprises about 33 mol% to about 38 mol% of sterols.

33. The LNP according to any one of claims 1 to 32, wherein the sterol comprises cholesterol.

34. The LNP according to any one of claims 1 to 33, wherein the sterols include phytosterols.

35. The LNP according to claim 34, wherein the phytosterol comprises campesterol, sitosterol, or stigmasterol or a combination thereof.

36. The LNP according to claim 33, wherein the sterol is cholesterol.

37. The LNP according to claim 4, comprising CICL1:DSPC:cholesterol:DSG-PEG2000:DSPE-PEG2000-maleimide in a ratio of 58:7:33.5:1.4:0.1 or 58:10:30.5:1.4:0.

1.

38. The LNP according to claim 4, comprising CICL1:DSPC:cholesterol:DPG-PEG2000:DSPE-PEG2000-maleimide in a ratio of 58:7:33.5:1.4:0.1 or 58:10:30.5:1.4:0.

1.

39. The LNP according to any one of claims 1 to 38, comprising: a. A payload containing mRNA encoding a chimeric antigen receptor specific to human CD19, human CD20, human BCMA, or combinations thereof; human FAP; or a payload containing mRNA encoding an RNA-directed nuclease and / or a guide RNA; and b. Containing a binding portion of an antibody or antigen-binding fragment thereof that is specific to human CD5, human CD8, or human CD2, wherein the antibody or antigen-binding fragment thereof is covalently linked to the functionalized PEG-lipid via a lysine or cysteine ​​residue of the antibody or binding fragment thereof.

40. The LNP according to any one of claims 1 to 38, comprising: a. The mRNA containing the encoding RNA-guided nuclease and the payload of the guide RNA; and b. Containing a binding portion of an antibody or antigen-binding fragment thereof that is specific to human CD5, human CD8, or human CD2, wherein the antibody or antigen-binding fragment thereof is covalently linked to the functionalized PEG-lipid via a lysine or cysteine ​​residue of the antibody or binding fragment thereof.

41. A composition comprising LNP according to any one of claims 1 to 40, and further comprising one or more pharmaceutically acceptable carriers or excipients.

42. A method of delivering a payload to immune cells or stem cells, comprising contacting the LNP according to any one of claims 1 to 40 with the immune cells or stem cells of a subject.

43. The method of claim 42, wherein delivery of the payload comprises transfecting the immune cells or stem cells.

44. The method of claim 42 or 43, wherein the contact is performed in vivo, in vitro, or ex vivo.

45. A method for reprogramming immune cells or stem cells, comprising administering to a subject the LNP according to any one of claims 1 to 40 or the composition according to claim 41.

46. ​​The method according to any one of claims 42 to 45, wherein the stem cell is a hematopoietic stem cell (HSC) or MSC.

47. The method according to any one of claims 42 to 45, wherein the immune cell is a T cell.

48. A method of treating a disease, comprising administering to a subject in need the LNP according to any one of claims 1 to 40 or the composition according to claim 41.

49. A method of conditioning a subject receiving an engineered agent, comprising providing the subject with an LNP according to any one of claims 1 to 38 before, simultaneously with or after administration of the engineered agent, the LNP comprising a nucleic acid encoding the conditioning agent.

50. The method according to any one of claims 42 to 49, comprising intravenous administration to the subject.

51. A method of reprogramming immune cells or hematopoietic stem cells (HSCs), comprising contacting an LNP according to any one of claims 1 to 40 with the immune cells or HSCs, wherein the reprogrammed immune cells or HSCs do not contain DNA encoding a reprogramming agent.

52. The method of claim 51, wherein the reprogrammed immune cell is a CAR-T cell.

53. A reprogrammed immune cell prepared by the method according to claim 51 or 52.

54. A method of reprogramming an HSC, comprising contacting the HSC with an LNP according to any one of claims 1 to 40, wherein the reprogrammed HSC does not contain DNA encoding components of a gene editing system.

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