Lipid nanoparticle conjugate, and preparation method therefor and use thereof

Abstract

Provided in the present invention are a lipid nanoparticle conjugate, and a preparation method therefor and the use thereof. In particular, an antibody is conjugated to the surface of an LNP via the N-glycosylation site of an Fc heavy chain by using a copper-free click chemistry reaction, thereby obtaining an LNP with specific cell or tissue targeting properties. Preferably, the N-glycosylation site is located on the asparagine residue at position 297 of the Fc heavy chain.

Description

Lipid nanoparticle conjugates, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of targeted delivery. Specifically, this invention relates to a lipid nanoparticle conjugate for targeted delivery of cargo molecules to target cells or tissues. This invention also relates to a method for preparing the lipid nanoparticle conjugate and its applications. Background Technology

[0002] Lipid nanoparticles (LNPs) are among the most efficient carriers for nucleic acid delivery, exhibiting high safety and low toxicity. However, LNP development has primarily been limited to intramuscular injection of mRNA vaccines and intravenous liver delivery. This is because, in the blood, untargeted LNPs are adsorbed by apolipoprotein E (ApoE) and accumulate in the liver by binding to low-density lipoprotein receptors on liver cells. Therefore, delivering nucleic acid drugs to specific sites requires the design of LNPs with targeting functions. Targeting LNPs can be achieved by conjugating specific antibodies to their surface. However, most current antibody functionalization modifications involve modifying the amino or carboxyl groups on the antibody and then conjugating them to functional groups on LNPs. Since amino or carboxyl sites on antibodies are not unique, a single antibody may have multiple LNP conjugation sites, leading to multiple LNPs on one antibody, which can easily aggregate and result in heterogeneous particles. Some modification sites may be located in the variable region of the antibody, affecting the antibody's recognition function after modification and / or conjugation.

[0003] PCT publication WO2023248125A1 discloses a targeted lipid nanoparticle. This involves transfecting 293F cells with a vector encoding an anti-CD117 VHH antibody and a sorting enzyme-recognized peptide sequence added to its C-terminus. After the cells express the corresponding antibody, a specific peptide sequence modified with azidation is added under the catalysis of the sorting enzyme, and finally, it is conjugated to an LNP modified with DBCO. CD117 is a surface marker of various types of hematopoietic cells. Conjugating an antibody that specifically binds to CD117 to lipid nanoparticles enables the targeted delivery of multiple components required for gene editing to hematopoietic cells.

[0004] PCT Publication No. WO2024102770A1 discloses a specifically oriented connector comprising a first click handle containing a tetrazine (Tz) ring and a second click handle containing trans-cyclooctene (TCO). Homogeneous coupling and higher coupling efficiency are achieved through the specifically oriented connector (Tz ring-TCO).

[0005] There is still a need in this field for improved methods to achieve highly efficient tissue and cell targeting after extensive conjugation of antibodies to lipid nanoparticles. Summary of the Invention

[0006] The inventors modified the N-glycosylation site of the Fc heavy chain of the antibody, especially the N-glycosylation site on the asparagine residue (N297), and then used click chemistry to selectively couple the antibody to the modified LNP, thereby obtaining an LNP with specific cell or tissue targeting.

[0007] The advantages of this invention are that the antibody modification sites in the LNP conjugate are fixed, ranging from 1 to 4. Due to steric hindrance, one antibody can only be linked to a maximum of two LNPs, reducing LNP aggregation and improving particle homogeneity. The LNP conjugate of this invention also maintains the antibody's highly efficient tissue targeting.

[0008] Therefore, in one aspect, an LNP conjugate is provided, the LNP conjugate comprising (a) lipid nanoparticles (LNP) and (b) an antibody or antigen-binding fragment thereof having cell or tissue targeting, wherein the antibody or antigen-binding fragment thereof is coupled to the surface of the LNP via an N-glycosylation site of the Fc heavy chain.

[0009] In one embodiment, the antibody or its antigen-binding fragment is IgG. In another embodiment, the antibody or its antigen-binding fragment has an IgG1, IgG2, IgG3, or IgG4 isotype. In a preferred embodiment, the antibody or its antigen-binding fragment has an IgG1 isotype. In another preferred embodiment, the antibody or its antigen-binding fragment has an IgG2 isotype. In one embodiment, the N-glycosylation site is located at the 297th asparagine residue of the Fc heavy chain, according to the EU index amino acid residue number.

[0010] In one embodiment, the antibody or its antigen-binding fragment is coupled to the surface of the LNP via click chemistry. In one embodiment, the click chemistry is a copper-free click chemistry. In one embodiment, the click chemistry includes, but is not limited to, azide / alkyne chemistry. In a preferred embodiment, the click chemistry is azide / alkyne chemistry.

[0011] In one embodiment, the Fc heavy chain has an azide functional group at its N-glycosylation site. In one embodiment, the azide functional group is galactosyl azide.

[0012] In one embodiment, the LNP has an alkyne functional group on its surface. In one embodiment, the alkyne functional group is dibenzocyclooctyne (DBCO). In one embodiment, the alkyne functional group is PEG-DBCO.

[0013] In one embodiment, each antibody or its antigen-binding fragment in the LNP conjugate is conjugated to one or two LNPs.

[0014] In one embodiment, the antibody or its antigen-binding fragment specifically binds to cell surface antigens, including but not limited to: CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR 1. CTLA-4, DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinogen C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3. Mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 integrins), tendinin C, TRAIL-R2, or vimentin.

[0015] In one embodiment, the LNP comprises, or is composed of, polymer-coupled lipids, cationic lipids, structural lipids, and sterols. In one embodiment, the polymer-coupled lipid comprises PEG-coupled lipids. In one embodiment, the sterol comprises cholesterol. In one embodiment, the cationic lipid comprises aminolipids. In one embodiment, the structural lipid comprises phospholipids.

[0016] In one embodiment, the LNP further comprises a therapeutic or diagnostic agent, wherein the therapeutic or diagnostic agent is encapsulated in or attached to the LNP. In one embodiment, the therapeutic agent is a nucleic acid. In another embodiment, the therapeutic agent is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

[0017] In another aspect, a pharmaceutical composition is provided comprising an LNP conjugate as described herein and a pharmaceutically acceptable excipient.

[0018] In another aspect, a method for preparing lipid nanoparticle (LNP) conjugates as described herein is provided, the method comprising conjugating an antibody or an antigen-binding fragment thereof having an N-glycosylation site in the Fc heavy chain to the surface of the LNP by click chemistry.

[0019] In one embodiment, the antibody or its antigen-binding fragment is IgG. In another embodiment, the antibody or its antigen-binding fragment has an IgG1, IgG2, IgG3, or IgG4 isotype. In a preferred embodiment, the antibody or its antigen-binding fragment has an IgG1 isotype. In another preferred embodiment, the antibody or its antigen-binding fragment has an IgG2 isotype. In one embodiment, the N-glycosylation site is located at the 297th asparagine residue of the Fc heavy chain, according to the EU index amino acid residue number.

[0020] In one embodiment, the antibody or its antigen-binding fragment is coupled to the surface of the LNP via click chemistry. In one embodiment, the click chemistry is a copper-free click chemistry. In one embodiment, the click chemistry includes, but is not limited to, azide / alkyne chemistry. In a preferred embodiment, the click chemistry is azide / alkyne chemistry.

[0021] In one embodiment, the Fc heavy chain has an azide functional group at its N-glycosylation site. In one embodiment, the azide functional group is galactosyl azide.

[0022] In one embodiment, the LNP has an alkyne functional group on its surface. In one embodiment, the alkyne functional group is dibenzocyclooctyne (DBCO). In one embodiment, the alkyne functional group is PEG-DBCO.

[0023] In one embodiment, each antibody in the LNP conjugate is conjugated to one or two LNPs.

[0024] In one embodiment, the antibody or its antigen-binding fragment specifically binds to cell surface antigens, including but not limited to: CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR 1. CTLA-4, DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinogen C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3. Mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 integrins), tendinin C, TRAIL-R2, or vimentin.

[0025] In one embodiment, the LNP comprises, or is composed of, polymer-coupled lipids, cationic lipids, structural lipids, and sterols. In one embodiment, the polymer-coupled lipid comprises PEG-coupled lipids. In one embodiment, the sterol comprises cholesterol. In one embodiment, the cationic lipid comprises aminolipids. In one embodiment, the structural lipid comprises phospholipids.

[0026] In one embodiment, the LNP further comprises a therapeutic or diagnostic agent, wherein the therapeutic or diagnostic agent is encapsulated in or attached to the LNP. In one embodiment, the therapeutic agent is a nucleic acid. In another embodiment, the therapeutic agent is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

[0027] In one embodiment, a method for preparing lipid nanoparticle (LNP) conjugates as described herein is provided, the method comprising the following steps:

[0028] (a) Provide an antibody or antigen-binding fragment thereof with an N-glycosylation site in the Fc heavy chain;

[0029] (b) Contact the antibody or its antigen-binding fragment with galactosidase to remove galactose residues at the N-glycosylation site of the Fc heavy chain of the antibody or its antigen-binding fragment, exposing N-acetylglucosamine residues;

[0030] (c) Linking UDP-galactosyl azide to exposed N-acetylglucosamine residues by contacting the antibody or its antigen-binding fragment obtained in step (b) with β-1,4-galactosyltransferase; and

[0031] (d) The antibody or its antigen-binding fragment obtained in step (c) is mixed with an LNP modified with dibenzocyclooctylene (DBCO) on its surface, and the antibody or its antigen-binding fragment is site-coupled to the LNP surface by click chemistry.

[0032] In another aspect, a method for delivering cargo molecules into cells is provided, the method comprising contacting an LNP conjugate, as described herein, with a cell expressing a cell surface antigen under conditions conducive to the entry of the cargo molecule into the cell, wherein the LNP comprises a cargo molecule, and wherein the antibody or an antigen-binding fragment thereof is specifically capable of binding to the cell surface antigen.

[0033] In one embodiment, the cargo molecule is a therapeutic or diagnostic agent, wherein the therapeutic or diagnostic agent is encapsulated in or attached to the LNP. In one embodiment, the therapeutic agent is a nucleic acid. In another embodiment, the therapeutic agent is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

[0034] In one embodiment, the antibody or its antigen-binding fragment specifically binds to cell surface antigens, including but not limited to: CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR 1. CTLA-4, DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinogen C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3. Mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 integrins), tendinin C, TRAIL-R2, or vimentin.

[0035] In one embodiment, the cell is a mammalian cell.

[0036] In one embodiment, the method is an in vivo method. In another embodiment, the method is an in vitro method.

[0037] In another aspect, lipid nanoparticle (LNP) conjugates, as described herein, are provided for delivering cargo molecules to cells.

[0038] In another aspect, the use of lipid nanoparticle (LNP) conjugates, as described herein, in the preparation of pharmaceuticals for delivering cargo molecules into cells is provided.

[0039] Other features and advantages of this disclosure will be apparent from the accompanying drawings, detailed embodiments and claims. Attached Figure Description

[0040] The invention will be better understood in conjunction with the accompanying drawings.

[0041] Figure 1A shows the RPS results of the uncoupled antibody LNP-luciferase mRNA.

[0042] Figure 1B shows the RPS results of site-specific conjugation of CD31 antibody to LNP-luciferase mRNA.

[0043] Figure 2 shows the in vitro imaging results of major organs in the in vivo experiments of unconjugated LNP and CD31 antibody-conjugated LNP.

[0044] Figure 3A shows the RPS results of the unconjugated antibody LNP-Cre mRNA.

[0045] Figure 3B shows the RPS results of site-specific conjugation of CD31 antibody to LNP-Cre mRNA.

[0046] Figure 4 shows the tdTomato fluorescence results of lung sections in in vivo experiments with unconjugated LNP and CD31 antibody-conjugated LNP. L and H represent the low-dose and high-dose groups, respectively.

[0047] Figure 5 shows the SDS-PAGE electrophoresis images of non-site-coupled antibodies and site-coupled antibodies. Detailed Implementation Plan

[0048] This disclosure provides a novel lipid nanoparticle (LNP) conjugate, a method for preparing the lipid nanoparticle (LNP) conjugate, pharmaceutical compositions containing the conjugate, and various uses thereof.

[0049] General definition

[0050] When used in conjunction with numerical values, the terms "about," "approximately," or "approximately" refer to a set or range of values. In some embodiments, "about X" includes a range of values ​​of ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numerical value. In some embodiments, the term "about" refers to a range of values ​​that are 5% more or less than a specified value. In some embodiments, the term "about" refers to a range of values ​​that are 2% more or less than a specified value. In some embodiments, the term "about" refers to a range of values ​​that are 1% more or less than a specified value.

[0051] Unless otherwise specified herein, the enumeration of value ranges is intended only as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually enumerated herein. Unless otherwise specified, the ranges used herein include both ends of the range. In some embodiments, the expressions "x is an integer between 1 and 6" and "x is an integer between 1 and 6" both mean "x is 1, 2, 3, 4, 5, or 6," that is, the terms "between X and Y" and "within the range of X to Y" include X and Y, as well as integers between them.

[0052] It should be understood that throughout the specification, when a composition is described as having, including, or comprising specific components, it is contemplated that the composition also substantially consists of or is composed of the listed components. Similarly, when a method or procedure is described as having, including, or comprising specific procedure steps, it is contemplated that the method or procedure also substantially consists of or is composed of the listed procedure steps. Furthermore, it should be understood that the order of steps or the sequence of certain actions is irrelevant as long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0053] Unless otherwise specified, all percentages and ratios used in this document are by weight.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the specification, the singular form also includes the plural unless the context clearly requires otherwise. Although those methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not acknowledged as prior art to the claimed invention. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be restrictive.

[0055] The following provides various definitions and a detailed explanation of the invention. It should be understood that this content is not restrictive.

[0056] Antibody

[0057] The LNP conjugates disclosed herein consist of LNPs covalently linked to an antibody. The antibody represents a targeting mechanism against a specific site of action. Upon reaching the site, the LNP conjugate can perform its designed function in a targeted manner, rather than spreading systemically throughout the subject's body. This targeting approach enables treatment with drugs that would otherwise require very high doses, making systemic administration toxic.

[0058] As used herein, the term "antibody" is used in the broadest sense and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, provided they exhibit the desired antigen-binding activity. Antibodies are also glycoproteins, containing sugar chains in certain amino acid residues. Antibody amino acids are numbered according to the Kabat EU index (see Kabat, EA et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

[0059] The term "antigen-binding fragment" refers to a molecule, other than a complete antibody, that contains a portion of a complete antibody and binds to an antigen bound by the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies, linear antibodies, single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0060] In one specific embodiment, the antibody or its antigen-binding fragment comprises an N-glycosylation site on the Fc heavy chain, specifically an asparagine residue at position 297 (N297). It should be understood that N297 described in this disclosure corresponds to the IgG amino acid number according to the EU index; other antibody subclasses, such as IgA, IgM, IgE, and IgD, may also contain glycosylation sites corresponding to IgG N297, but with slightly different amino acid positions. In one embodiment, the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment, the antibody has an IgG1 isotype, and the N-glycosylation site is located on an asparagine residue at position 297. In another embodiment, the antibody has an IgG2 isotype, and the N-glycosylation site is located on an asparagine residue at position 297.

[0061] Antibodies that "specifically bind" or "target" antigens or cell surface antigens, as used herein, preferentially associate with said antigens or cell surface antigens via intermolecular interactions. In some embodiments, the antibody may preferentially associate with the antigen or cell surface antigen at a Kd of less than about 50 nM, less than about 5 nM, or less than 500 pM. Techniques for measuring the binding affinity of antibodies to antigens are well known, such as surface plasmon resonance (SPR).

[0062] In one embodiment, the antibody specifically binds to cell surface antigens, including but not limited to: CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR1, CT LA-4, DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinogen C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3. Mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 integrins), tendinin C, TRAIL-R2, or vimentin.

[0063] In some implementations, the antibody targets the following cell surface markers: 5T4, CA-125, CEA, CDH6, CD3, CD11b, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD51, CD-103, CTLA-4, CEACAM5, Clec9A, CSFR1, DEC205, EpCAM, HER2, EGFR (HER1), FAP, fibronectin-EDB, folate receptor, GCC (GUCY2C), HGF, integrin αvβ3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, NaPi2b, PTK7, phosphatidylserine, prostate-specific antigen, PDGFRα, TAG-72, tendinin C, TRAIL-R2, VEGF-A, and VEGFR2. In this implementation scheme, the antibodies include, but are not limited to, abagovomab, adecatumumab, alacizumab, attumomab, anatumomab, arcitumomab, bavituximab, and bevacizumab. Bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, citatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab ab), farletuzumab, figitumumab, gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab, lexatumumab, lintuzumab, lucarumumab, matuzumab, mitumomab, naaptumomab estafenatox, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab Rituximab, Robatumumab, Satumomab, Siboruzumab, Taplitumomab, Tenatumomab, Ticilimumab, Tigatuzumab, Trastuzumab Tositumomab, tremelimumab, tucotuzumabcelmoleukin, volociximab, and zalutumumab.

[0064] In some implementations, the antibody is a bispecific antibody.

[0065] It should be understood that the antibodies in this disclosure are not limited to any specific antibody, as long as the antibody contains an N-single-chain glycosylation site of the Fc heavy chain, particularly the glycosylation site corresponding to the asparagine residue (N297) at position 297 of IgG.

[0066] lipid nanoparticles

[0067] As used herein, the term “lipid” refers to a group of organic compounds, including but not limited to esters of fatty acids, which are typically poorly soluble in water but soluble in many nonpolar organic solvents. While lipids are generally poorly soluble in water, some classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and may be soluble in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be classified into at least three categories: (1) “simple lipids,” including fats and oils as well as waxes; (2) “complex lipids,” including phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) “derived lipids,” such as steroids. In addition, as used herein, lipids also include lipid-like compounds. The term “lipid-like compound,” also simply “lipid,” refers to lipid-like compounds (e.g., amphiphilic compounds with lipid-like physical properties).

[0068] The term "lipid nanoparticle" or "LNP" refers to particles with a maximum dimension in the nanometer (nm) range (e.g., 1-1000 nm) containing one or more types of lipid molecules. Lipid nanoparticles are capable of carrying aqueous solutions, compounds, drugs, or other substances, such as one or more nucleic acid molecules, collectively referred to as cargo molecules, within compartments (i.e., internal cavities or spaces) surrounded by at least one lipid bilayer. In some embodiments, the lipid nanoparticles provided herein have a maximum dimension of 1 μm or less (e.g., ≤1 μm, ≤900 nm, ≤800 nm, ≤700 nm, ≤600 nm, ≤500 nm, ≤400 nm, ≤300 nm, ≤200 nm, ≤175 nm, ≤150 nm, ≤125 nm, ≤100 nm, ≤75 nm, ≤50 nm or less), for example, when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or other methods. In one embodiment, the lipid nanoparticles provided herein have at least one dimension ranging from about 40 to about 200 nm. In one implementation, at least one dimension is in the range of about 40 to about 100 nm.

[0069] In some embodiments, the LNP comprises a non-lipid cargo molecule partially or completely encapsulated within a lipid shell. Specifically, in some embodiments, the cargo molecule is a negatively charged molecule (e.g., mRNA encoding a viral protein), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, cationic lipids can interact with the negatively charged cargo molecule and facilitate the incorporation and / or encapsulation of the cargo molecule into the LNP during its formation. Other lipids that can constitute part of an LNP, as described herein, include, but are not limited to, neutral lipids (also known as structural lipids) and charged lipids, such as steroids, polymer-coupled lipids, and various zwitterionic lipids.

[0070] The term "cationic lipid" refers to a lipid that carries a positive charge at any pH or hydrogen ion activity in its environment, or a lipid that is capable of carrying a positive charge in response to the pH or hydrogen ion activity of its environment (e.g., its intended use environment). Therefore, the term "cationic" includes both "permanently cationic" and "cationizable." In some embodiments, the positive charge in the cationic lipid is caused by the presence of quaternary nitrogen atoms. In some embodiments, the cationic lipid comprises zwitterionic lipids that carry a positive charge in the intended use environment (e.g., at physiological pH).

[0071] In one embodiment, the cationic lipid can be any lipid carrying a net positive charge at a selected pH value (e.g., physiological pH). Exemplary cationic lipids are described below. In one embodiment, the cationic lipid has a pKa greater than 6.25. In one embodiment, the cationic lipid has a pKa greater than 6.5. In one embodiment, the cationic lipid has a pKa greater than 6.1, 6.2, 6.3, 6.35, 6.4, 6.45, 6.55, 6.6, 6.65, or 6.7.

[0072] Examples of cationic lipids include, but are not limited to, N,N-diolenoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), N-(2,3-diolenoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0073] In a preferred embodiment, the cationic lipid is an amino lipid. Suitable amino lipids that can be used in this invention include those described in WO2009 / 096558, which is incorporated herein by reference in its entirety. Representative amino lipids include 1,2-dilinolenicooxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinolenicooxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-linolenicothio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linolenicooxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-linolenicooxy-3-trimethylaminopropane chloride (DLin-TMA·Cl), 1,2-dilinolenicooxy-3-trimethylaminopropane chloride (DLin-TAP·Cl), 1,2-linolenicooxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N The following are listed: 1,2-linolenicooxy-3-(2-N,N-dimethylamino)-1,2-propanediol (DLinAP), 3-(N,N-diolenicooxy)-1,2-propanediol (DOAP), 1,2-linolenicooxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-linolenicooxy-4-dimethylaminomethyl-[1,3]-dioxopropane (DLin-K-DMA), 1,2-linolenicooxy-N,N-dimethyl-3-aminopropane (DLin-DMA), methyl 4-(N,N-dimethylamino)butyrate (dilinolenico)ester (DLin-MC3-DMA), 2,2-linolenicooxy-4-(2-dimethylaminoethyl)-[1,3]-dioxopropane (DLin-KC2-DMA), and 1,2-diolenicooxy-3-dimethylaminopropane (DODMA).

[0074] In other embodiments, cationic lipids mentioned in US20180000953 were used, such as 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazineethylamine (KL10), 14,25-bistridecyl-15,18,21,24-tetraaza-octacosane (KL25), and 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,2Z)-octadecyl-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLin) DMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,2Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,2Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)).

[0075] In some embodiments, based on the total lipid content of the LNP, the LNP contains cationic lipids between about 20 mol% and about 75 mol%. In some embodiments, based on the total lipid content of the LNP, the LNP contains cationic lipids between about 30 mol% and about 55 mol%. In some embodiments, based on the total lipid content of the LNP, the LNP contains more than about 30 mol%, such as more than about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or more cationic lipids. In some embodiments, based on the total lipid content of the LNP, the LNP contains less than about 55 mol%, such as less than about 50 mol%, about 45 mol%, about 40 mol%, about 35 mol%, about 30 mol%, or less cationic lipids.

[0076] The term "structural lipid," also known as neutral lipid, refers to any lipid molecule present in an uncharged form or a neutral zwitterionic form within a selected pH value or range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions of the environment in which the lipid is intended for use, such as physiological pH. Suitable structural lipids support particle formation during manufacturing. Structural lipids can be phospholipids. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0077] Exemplary structural lipids include, but are not limited to, zwitterionic lipids, such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (transDOPE). In a preferred embodiment, the structural lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The structural lipids provided in this article can be synthetic or derived from natural sources or compounds.

[0078] In another embodiment, the structural lipid is any lipid that carries a negative charge at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups attached to neutral lipids.

[0079] Other suitable structural lipids include glycolipids (e.g., monosalivary ganglioside GM1).

[0080] In some embodiments, based on the total lipid content of the LNP, the LNP contains between about 5 mol% and about 40 mol% of structured lipids. In some embodiments, based on the total lipid content of the LNP, the LNP contains more than about 5 mol% of structured lipids, such as more than about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, or more. In some embodiments, based on the total lipid content of the LNP, the LNP contains less than about 40 mol% of structured lipids, such as less than about 35 mol%, about 30 mol%, about 25 mol%, about 20 mol%, about 15 mol%, about 10 mol%, about 5 mol%, or less.

[0081] In one embodiment, the molar ratio of cationic lipid to structural lipid ranges from about 2:1 to about 8:1. In one embodiment, the lipid nanoparticles comprise 5 to 10 mol% of structural lipid.

[0082] The term "polymer-coupled lipid" refers to a molecule comprising both a lipid moiety and a polymer moiety. Without being bound by theory, it is conceivable that polymer-coupled lipids in lipid nanoparticles can improve colloidal stability and / or reduce protein uptake by the nanoparticles. An example of a polymer-coupled lipid is a polyethylene glycol-modified lipid (PEG-lipid), wherein the polymer moiety comprises polyethylene glycol. Exemplary polymer-coupled lipids that can be used in this disclosure include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG lipids can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0083] Any lipid described herein, including components of the lipid nanoparticles described herein, may be coupled with polyethylene glycol (PEG). In some embodiments, the PEGylated form of any lipid described herein may be coupled with an antibody or its antigen-binding fragment. In a preferred embodiment, the polymer-coupled lipid is 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE) coupled with PEG. In some embodiments, the PEG may be from PEG500 (molecular weight 500) to PEG20000 (molecular weight 20000).

[0084] In one embodiment, based on the total lipid content of the LNP, the LNP contains about 0.5 to 5.0 mol% of polymer-coupled lipids. In one embodiment, based on the total lipid content of the LNP, the LNP contains about 1.0 to 2.5 mol% of polymer-coupled lipids. In one embodiment, based on the total lipid content of the LNP, the LNP contains about 1.7 mol% of polymer-coupled lipids. In one embodiment, based on the total lipid content of the LNP, the LNP contains about 1.5 mol% of polymer-coupled lipids.

[0085] In one embodiment, the molar ratio of the cationic lipid to the polymer-coupled lipid ranges from about 35:1 to about 25:1. In another embodiment, the molar ratio of the cationic lipid to the polymer-coupled lipid ranges from about 100:1 to about 20:1.

[0086] Lipid nanoparticles may also contain sterols, such as cholesterol or phytosterols and their derivatives. Without being bound by theory, it is considered that sterols can stabilize the amphiphilic structure of nanoparticles, such as, but not limited to, the lipid bilayer structure of nanoparticles. Exemplary sterols and their derivatives that can be used in this disclosure include, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, tomatine glycoside, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisolone, and hydrocortisone) or combinations thereof.

[0087] In some embodiments, based on total lipids, the LNP contains between about 20 mol% and about 50 mol% of sterols. In some embodiments, based on total lipids, the LNP contains more than about 20 mol%, for example, more than about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol% or more of sterols. In some embodiments, based on total lipids, the LNP contains less than about 50 mol%, for example, less than about 45 mol%, about 40 mol%, about 35 mol%, about 30 mol%, about 25 mol%, about 20 mol% or less of sterols.

[0088] In one embodiment, the molar ratio of cationic lipids to sterols ranges from about 10:1 to 1:1. In another embodiment, the molar ratio of cationic lipids to sterols ranges from about 5:1 to 1:1. In one embodiment, sterols are present at a concentration of 32 to 40 mol% based on total lipids of LNP.

[0089] In some embodiments, the LNP comprises cationic lipids, structural lipids, sterols, and polymer-coupled lipids, such as any one of the cationic lipids, structural lipids, sterols, and polymer-coupled lipids described herein. In some embodiments, the LNP comprises i) cationic lipids between about 30 mol% and about 55 mol%, and ii) structural lipids between about 5 mol% and about 40 mol%.

[0090] In one embodiment, the lipid nanoparticles comprise:

[0091] i) about 20 to 65 mol% cationic lipids; ii) about 5 to 40 mol% structural lipids; iii) about 20 to 50 mol% sterols; and iv) polymer-coupled lipids.

[0092] In one embodiment, the lipid nanoparticles comprise:

[0093] i) about 40 to 55 mol% cationic lipids; ii) about 5 to about 15 mol% structural lipids; iii) about 35 to 50 mol% sterols; and iv) about 2 to 10 mol% polymer-coupled lipids.

[0094] In one embodiment, the lipid nanoparticles comprise:

[0095] i) about 45 to 55 mol% cationic lipids; ii) about 6 to 10 mol% structural lipids; iii) about 40 to 48 mol% sterols; and iv) about 1 to 2.5 mol% polymer-coupled lipids.

[0096] In one embodiment, the lipid nanoparticles comprise cationic lipids, DSPC, cholesterol, and PEG-lipids, as well as mRNA. In one embodiment, the molar ratio of cationic lipids, DSPC, cholesterol, and PEG-lipids is approximately 50:10:38.5:1.5. In another embodiment, the molar ratio of cationic lipids, DSPC, cholesterol, and PEG-lipids is approximately 50:10:38:2.

[0097] As used in this article, “Mol%” refers to the Mole of a component relative to the total moles of all lipid components in the LNP (i.e., cationic lipids, structural lipids, sterols, and polymer-coupled lipids).

[0098] The physical properties of lipid nanoparticles can vary depending on their composition. For example, nanoparticles containing lipids with different structures may have different properties. Similarly, the properties of lipid nanoparticles can depend on the absolute or relative amounts of their components. For instance, nanoparticles containing a higher molar fraction of phospholipids may have different properties than those containing a lower molar fraction. Properties can also vary depending on the methods and conditions used to prepare the nanoparticles.

[0099] Lipid nanoparticles can be characterized using a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticles. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure various properties of nanoparticles, such as particle size, polydispersity index, and zeta potential.

[0100] The lipid nanoparticles according to the present invention have an average particle size of about 15 nm to about 300 nm. In some embodiments, the average particle size is greater than 300 nm. In some embodiments, the diameter of the lipid nanoparticles is about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. In one embodiment, the diameter of the lipid nanoparticles is about 50 nm to about 150 nm. Compared with larger particles, smaller particles generally exhibit increased cycle life in vivo. In one embodiment, the diameter of the lipid particles is about 15 nm to about 50 nm.

[0101] Lipid nanoparticles can be relatively homogeneous. The polydispersity index (PDI) can be used to indicate the homogeneity of the nanoparticle composition, such as the particle size distribution of the nanoparticles. A smaller PDI (e.g., less than 0.3) typically indicates a narrower particle size distribution. The PDI of a nanoparticle composition can range from about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the PDI of the nanoparticles can be from about 0.10 to about 0.20.

[0102] The zeta potential describes the surface charge of nanoparticle components. Nanoparticles with relatively low charges (positive or negative) are typically preferred because substances with higher charges can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticles can be about -10mV to about +20mV, about -10mV to about +15mV, about -10mV to about +10mV, about -10mV to about +5mV, about -10mV to about 0mV, about -10mV to about -5mV, about -5mV to about +20mV, about -5mV to about +15mV, about -5mV to about +10mV, about -5mV to about +5mV, about -5mV to about 0mV, about 0mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about +5mV to about +20mV, about +5mV to about +15mV, or about +5mV to about +10mV.

[0103] The encapsulation efficiency of therapeutic and / or preventative agents describes the amount of therapeutic and / or preventative agents that, after preparation, are encapsulated by or otherwise associated with the nanoparticles relative to the initial amount provided. Ideally, the encapsulation efficiency is very high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or preventative agents in a solution containing the nanoparticles before and after decomposition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or preventative agents (e.g., RNA) in solution. For the nanoparticles described herein, the encapsulation efficiency of the therapeutic and / or preventative agents can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some implementations, the packaging efficiency can be at least 90%.

[0104] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated in a coated capsule, film, or tablet. Capsules, films, or tablets containing the nanoparticle compositions described herein may have any useful size, tensile strength, hardness, or density.

[0105] Lipid nanoparticles according to embodiments of the present invention can be prepared by standard T-tube mixing techniques, turbulent mixing, grinding mixing, accelerated stirring-sequential self-assembly, or passive mixing in which all components self-assemble into nanoparticles. Various methods have been developed to formulate lipid nanoparticles (LNPs) containing gene therapies. Suitable methods are disclosed, for example, in U.S. Patent Nos. 5,753,613 and 6,734,171. These methods involve mixing pre-formed lipid particles with a nucleic acid therapeutic agent (NAT) in the presence of ethanol, or mixing lipids dissolved in ethanol with an aqueous medium containing NAT, to obtain lipid particles with a NAT encapsulation efficiency of 65-95%. Both methods rely on the presence of cationic lipids to achieve NAT encapsulation and stabilizers to inhibit the aggregation and formation of large structures. The performance of the resulting lipid particle system (including size and NAT encapsulation efficiency) is sensitive to various formulation parameters, such as ionic strength, lipid and ethanol concentrations, pH, NAT concentration, and mixing rate. See J Drug Target. 2016 Nov; 24(9):821-835.

[0106] Microfluidic two-phase droplet technology has been applied to produce monodisperse polymeric microparticles for drug delivery or to produce large vesicles for encapsulating cells, proteins, or other biomolecules. Fluid dynamics flow focusing (a common microfluidic technique that provides rapid mixing of reagents) has been demonstrated to produce monodisperse liposomes with controlled sizes.

[0107] Typically, current formulation procedures struggle to control parameters such as relative lipid and NAT concentrations, as well as mixing rates, leading to variations in NAT properties both within and between formulations. Automated micromixing instruments like NanoAssemblr address these challenges. TM The instrument (Precision NanoSystems Inc., Vancouver, Canada) enables the rapid and controlled fabrication of nanomedicines (liposomes, lipid nanoparticles, and polymer nanoparticles).

[0108] NanoAssemblr TM The instrument enables controlled molecular self-assembly of nanoparticles via a microfluidic mixing cylinder, which allows for the mixing of nanoparticle components at nanoliter, microliter, or even larger millisecond scales, in customized or parallel settings. This rapid, small-scale mixing allows for reproducible control over particle synthesis and quality, which is impossible in larger instruments.

[0109] Preferred methods include incorporating NanoAssemblr, etc. TM Spark TM Ingnite TM Benchtop TM and Blaze TM A microfluidic mixing device is used to encapsulate nearly 100% of the nucleic acids used in the formation process into particles in a single step. In one embodiment, lipid nanoparticles are prepared by encapsulating approximately 90% to approximately 95% of the nucleic acids used in the formation process into the particles.

[0110] In embodiments of the invention, an apparatus for biomicrofluidic mixing is used to prepare the lipid particles and therapeutic formulations of the invention. The apparatus includes a first reagent stream and a second reagent stream supplied to a microfluidic mixer, and the lipid particles are collected from an outlet, or in other embodiments, into a sterile environment.

[0111] The first stream includes the therapeutic agent in a first solvent. A suitable first solvent includes a solvent in which the therapeutic agent is soluble and miscible with a second solvent. A suitable first solvent includes an aqueous buffer. Representative first solvents include citrate and acetate buffers.

[0112] The second stream comprises a lipid mixture in a second solvent. Suitable second solvents include those in which cationic lipids are soluble and miscible with the first solvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and alcohols. Representative second solvents include 90% aqueous ethanol or anhydrous ethanol.

[0113] In one embodiment of the invention, a suitable device includes one or more microchannels (i.e., channels with a maximum size of less than 1 mm). In one embodiment, the diameter of the microchannel is from about 20 μm to about 300 μm. In one embodiment, at least one region of the microchannel has a main flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation angular to the main direction as described in U.S. Patent Publication No. 20040262223 (e.g., an interlaced herringbone mixer), or a bifurcated annular flow as described in U.S. Patent Publication No. 2018093232. To achieve maximum mixing rate, it is advantageous to avoid undue fluid resistance before the mixing region. Therefore, one example of the device has non-microfluidic channels with a size greater than 1000 micrometers to deliver fluid to a single mixing channel.

[0114] Less automated micromixing methods and instruments, such as those disclosed in Zhang, S. et al., Chem. Eng. J. 144, 2008, 324-328 and Strook A. et al., Science 295, 2002, 647-651, can also be used to produce the formulations of this invention. A more primitive system involving T-tube mixing is disclosed in Jeffs LB et al., A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. PharmRes. 2005; 22(3):362-72.

[0115] Coupling methods

[0116] The lipid nanoparticle conjugates described in this article consist of an antibody, a linker, and lipid nanoparticles (payload). Each component of the conjugate plays a crucial role in ensuring its stability and minimizing potential side effects. An ideal conjugate should effectively reach the target tissue or cells without prematurely releasing any off-target payload, and exert its payload efficacy within the target tissue or cells without affecting normal healthy cells. To develop an effective lipid nanoparticle conjugate, factors such as the selection of antigen, antibody, payload, and linker must be considered.

[0117] antigen selection

[0118] The target antigen should help distinguish the target tissue or cell from normal tissue or cells, thereby reducing off-target toxicity. Therefore, selecting a suitable target antigen is the first step in developing ideal lipid nanoparticle conjugates. An ideal antigen must possess certain characteristics:

[0119] 1) It should be overexpressed on the surface of the target cells compared to normal cells;

[0120] 2) The binding site of the target antigen should face the outer surface of the target cell, rather than the interior. This allows lipid nanoparticle conjugates diffusing from blood vessels to bind to the target antigen before internalization;

[0121] 3) The target antigen should not enter the systemic circulation to avoid unnecessary binding of lipid nanoparticle conjugates outside the target tissue; and

[0122] 4) The target antigen should have the ability to internalize and bind lipid nanoparticle conjugates.

[0123] In one embodiment, the target antigen selected in this disclosure is a cell surface antigen expressed on the target cell. In one embodiment, the target antigen selected in this disclosure may include, but is not limited to, CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR1, CTLA- 4. DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinogen C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3. Mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 integrins), tendinin C, TRAIL-R2, or vimentin.

[0124] Antibody selection

[0125] In ideal lipid nanoparticle conjugates, antibodies are crucial carriers for the specific binding of target antigens. Antibodies must possess high binding affinity for the target antigen and low immunogenicity. Furthermore, antibodies should exhibit a long plasma half-life and rapid internalization. Five types of antibodies exist in human serum, with immunoglobulin G (IgG) being the most abundant, accounting for approximately 70-85% of total antibodies, with a half-life of about 21 days. Due to its richest antibody diversity and ability to initiate immune responses, IgG is the most commonly used antibody in lipid nanoparticle conjugates. In addition, IgG antibodies include isotypes IgG1, IgG2, IgG3, and IgG4, with IgG1 being the most frequently used antibody in lipid nanoparticle conjugates and proven to be an effective immune effector.

[0126] Antibody glycosylation is a type of post-translational modification that can occur via oligosaccharides added to antibodies through two types of covalent bonds—bonds on asparagine residues (N-oligosaccharides) or bonds on serine / threonine residues (O-oligosaccharides) (Alter, G. et al., Semin Immunol., 2018, Vol. 39: 102-110); and significantly affects the therapeutic function of antibodies (Walsh, G. and Jefferis, R., Nat. Biotechnol., 2006, Vol. 24: 1241-1252; Jefferis, R., Nat. Rev. Drug Discov., 2009, Vol. 8, No. 3: 226-234; Dalziel, M. et al., Science, 2014, Vol. 343: No. 6166: 1235681). It is noteworthy that all IgG antibodies are glycosylated at the conserved residue 297 in their Fc region (Alter G. et al., ibid.).

[0127] When referring to residues in the constant region (Fc) of the immunoglobulin heavy chain, the term "EU number" or "EU index" is commonly used. It refers to the residue number of a human IgG1 EU antibody. It is calculated by aligning the antibody sequence to the Eu antibody sequence (Edelman, GM et al., Proc Natl Acad Sci USA, 1969, Vol. 63, No. 1: pp. 78-85; Kabat et al., ibid.), such that each residue homologous to a residue in the Eu antibody will have the same residue number as that Eu residue.

[0128] The vast majority of monoclonal antibodies are expressed by CHO, SP2 / 0, and NS0 cells, and N-glycosylation is the most common form of glycosylation. Core glycosylation typically occurs in the endoplasmic reticulum, catalyzed by oligosaccharide transferases (OSTs), which transfer a pre-synthesized glycan (core glycan) to the Asn in the Asn-X-(Ser / Thr) sequence of the target protein, where X represents any amino acid except Pro. In IgG antibodies, this motif is primarily located in the CH2 domain of the heavy chain, and the glycosylation site in most antibodies is only at approximately N297 of the Fc region of the heavy chain.

[0129] The N-oligosaccharide linked to residue 297 is composed of a conservative dual-antenna core structure (Liu, L., J Pharm Sci., 2015, Vol. 104, No. 6: pp. 1866-1884). This conservative dual-antenna core structure consists of two covalently linked N-acetylglucosamine (GlcNAc) residues further linked to mannose and connected to two other mannose residues in a 1,3- and 1,6-branched manner (Alter, G. et al., ibid.). Additional monosaccharides, including two galactoses, one fucose, two GlcNAcs, and / or two sialic acids (Alter, G. et al., ibid.), can extend the core structure, resulting in considerable structural and functional heterogeneity (Jefferis, R., Biochem J., 1990, Vol. 268, No. 3: pp. 529-537; Rudd, PM., Science, 2001, Vol. 291, No. 5512: pp. 2370-2376; Liu, L., ibid.). At least 30 structures (glycoforms) of N-oligosaccharides for IgG N297 conjugation have been reported (Alter, G. et al., ibid.). Because both heavy chains of an antibody can be glycosylated, significant oligosaccharide heterogeneity may also exist within a single antibody molecule. Oligosaccharides can directly affect the binding of Fc to FcγR. The steric hindrance at N297 on Fc hinders the binding of Fc and FcγRⅢa, thus reducing antibody-mediated ADCC activity. NMR spectroscopy revealed differences in the conformation of the hinge region when different oligosaccharides are present in Fc N297. Because the antibody hinge region interacts with FcγR, glycosylation at N297 indirectly affects the interaction between Fc and FcγR.

[0130] In one embodiment, the antibody selected in this disclosure is an IgG antibody. In one embodiment, the antibody selected in this disclosure has an IgG1, IgG2, IgG3, or IgG4 isotype, comprising an N-glycosylation site located on an asparagine residue at position 297. In a preferred embodiment, the antibody has an IgG1 isotype, and the N-glycosylation site is located on an asparagine residue at position 297. In another preferred embodiment, the antibody has an IgG2 isotype, and the N-glycosylation site is located on an asparagine residue at position 297.

[0131] In one embodiment, the antibody specifically binds to cell surface antigens, including but not limited to: CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR1, CTLA-4, DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folic acid receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER 2. VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 integrins), tendinin C, TRAIL-R2, or vimentin.

[0132] Selection of connector

[0133] The linker acts as a bridge between the antibody and the lipid nanoparticles (payload), playing a crucial role in the stability and effectiveness of the conjugate. Furthermore, payload release also largely depends on the type and properties of the linker. An ideal linker should possess high water solubility to prevent payload aggregation and premature release during systemic circulation. Generally, stable and effective conjugates can be obtained by modifying these three components: the antibody, the linker, and the payload. The three main factors affecting linker stability and payload release are the coupling site, steric hindrance, and linker length.

[0134] The coupling site is a major factor affecting the stability of conjugates and a key factor in their therapeutic efficacy. Coupling sites can resist payload loss mediated by the reverse Michael reaction in systemic circulation and can also affect the uncoupling of nonspecific valine-citrulline in vivo. Furthermore, coupling sites can improve the efficacy of conjugates by reducing metabolic load, demonstrating their crucial role in the stability of conjugates.

[0135] Steric hindrance during coupling creates a steric barrier, thus altering the stability of the conjugate. Another factor is that the linker length affects the steric barrier through the distance between the antibody and the payload, leading to variations in conjugate stability. Shorter linkers generally result in better stability compared to longer linkers. However, shorter linkers bring the payload closer to the steric barrier provided by the antibody. The linker should be stable in systemic circulation and bind to the target cell under chemical or enzymatic action. Effective unbinding of the linker to release a sufficient amount of payload is a key factor in conjugate effectiveness.

[0136] By selecting specific coupling sites and linkers, this disclosure provides a stable conjugate in which the number of antibody modification sites is fixed and ranges from 1 to 4; due to steric hindrance, an antibody can only be linked to a maximum of two LNPs, reducing LNP aggregation and improving particle homogeneity; and also maintaining the antibody's efficient tissue targeting.

[0137] In one embodiment, the coupling site is an N-glycosylation site in the Fc region of the antibody's heavy chain. In a preferred embodiment, the N-glycosylation site is located on an asparagine residue at position 297.

[0138] In one embodiment, lipid nanoparticles can be chemically coupled to antibodies via streptavidin / biotin bonds, thiols / maleimides, azides / alkynes, tetrazines / cyclooctene, and other chemistries. As used herein, the term "click chemistry" refers to a biocompatible reaction primarily designed to bind a selected substrate to a specific biomolecule. Click chemistry reactions are undisturbed by water, produce very few and non-toxic byproducts, and are characterized by a high thermodynamic driving force that rapidly and irreversibly drives them to achieve high yields of a single reaction product under conditions of high reaction specificity.

[0139] In one embodiment, the antibody is conjugated to the surface of the LNP via click chemistry, preferably copper-free click chemistry.

[0140] The term "copper-free click chemistry" as used herein refers to a chemical synthesis method for the rapid and efficient synthesis of useful new molecules based on carbon-heteroatom (CXC) linkages without cytotoxic transition metal catalysts. Copper-free click chemistry can utilize the reaction between cyclooctyne and azides via strain-promoted azide-acetylene click chemistry. This reaction is copper-free and proceeds due to the high activation energy of the strained molecules. Commonly used reagents in copper-free click chemistry include dibenzocyclooctyne (DBCO), transcyclooctene (TCO), and cyclopropanecyclooctyne (BCN). Azide-containing compounds can react with acetylene-containing compounds or small biomolecules via azide-acetylene click chemistry to form stable 1,2,3-triazole linkages.

[0141] In one embodiment, the antibody is coupled to the surface of the LNP via azide / acetylene chemistry.

[0142] In one embodiment, the coupling site corresponds to the N-glycosylation site of the asparagine residue at position 297 of the Fc region of the antibody heavy chain.

[0143] In one implementation, each antibody is conjugated to up to two LNPs.

[0144] In one embodiment, a method for preparing lipid nanoparticle (LNP) conjugates as described herein is provided, the method comprising the following steps:

[0145] (a) Provide an antibody or antigen-binding fragment thereof with an N-glycosylation site in the Fc heavy chain;

[0146] (b) Contact the antibody or its antigen-binding fragment with galactosidase to remove galactose residues at the N-glycosylation site of the Fc heavy chain of the antibody or its antigen-binding fragment, exposing N-acetylglucosamine residues;

[0147] (c) Linking UDP-galactosyl azide to exposed N-acetylglucosamine residues by contacting the antibody or its antigen-binding fragment obtained in step (b) with β-1,4-galactosyltransferase; and

[0148] (d) The antibody or its antigen-binding fragment obtained in step (c) is mixed with an LNP modified with dibenzocyclooctylene (DBCO) on its surface, and the antibody or its antigen-binding fragment is site-coupled to the LNP surface by click chemistry.

[0149] In one embodiment, the LNP with a surface modified with dibenzocyclooctyne (DBCO) contains DBCO covalently linked to PEG.

[0150] Galactosidases are a class of enzymes that hydrolyze substances containing galactosidic bonds (such as lactose). In one embodiment, the galactosidase is a β-galactosidase. In one embodiment, the N-oligosaccharide linked to residue 297 of the Fc region of the heavy chain of the antibody according to the invention comprises one or two galactose residues, which can be cleaved by a galactosidase to expose one or two N-acetylglucosamine residues. β-1,4-galactosyltransferase (B4GALT1) catalyzes a reaction involving UDP-galactose and N-acetylglucosamine for the production of galactoseβ-1,4-N-acetylglucosamine. These enzymes are known to those skilled in the art and are readily available commercially.

[0151] Other methods known in the art can also be used to produce the LNP conjugates disclosed herein. For example, conjugation can be mediated by ligand-receptor pairs, such as biotin-streptavidin pairs. In this case, one member of the ligand-receptor pair can be linked to an antibody, and the other member can be linked to an LNP.

[0152] use

[0153] The LNP conjugates of this invention can be used for systemic or local delivery of peptides. As used herein, the term "therapeutic peptide" is intended to include any amino acid chain delivered into cells that elicits a desired effect. Peptides are short chains of amino acids, ranging from 2 to 50 amino acids in length, as opposed to proteins, which have longer chains (50 amino acids or more) and typically possess tertiary and / or quaternary structures. The amino acids in a peptide are linked together in sequence by bonds called peptide bonds.

[0154] The LNP conjugates of this invention can be used for systemic or local delivery of nucleic acids. As used herein, "nucleic acid" is a polymer or polymer segment having a nucleobase sequence in the form of a backbone formed of nucleotides or analogs. The term "nucleic acid" also refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single-stranded, double-stranded, or contain portions of both double-stranded and single-stranded sequences, as appropriate.

[0155] As used herein, the term "nucleic acid therapeutic agent (NAT)" is intended to include any oligonucleotide or polynucleotide delivered to cells to elicit a desired effect. Fragments containing up to 50 nucleotides are generally referred to as oligonucleotides, while longer fragments are referred to as polynucleotides. In some embodiments, oligonucleotides are 20-50 nucleotides in length. In some embodiments, polynucleotides are 996-4500 nucleotides in length, as is the case with messenger RNA.

[0156] The delivery method and LNP conjugate can be readily adapted to deliver any suitable therapeutic agent for treating any disease or condition from which such treatment would benefit.

[0157] In some contexts, the terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to single- and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by phosphodiester bonds, such as 3'-5' and 2'-5' anti-linking bonds, or 3'-3' and 5'-5' branching structures or nucleotide-linking analogs. Polynucleotides may have associated counterions such as H+, NH4+, trialkylammonium, Mg2+, Na+, etc. Polynucleotides may consist entirely of deoxyribonucleotides, entirely of ribonucleotides, or a chimeric mixture thereof. Polynucleotides may contain nucleotide-linking bonds, nucleobases, and / or sugar analogs.

[0158] Currently, NATs are being actively sought in an increasing number of preclinical and clinical studies. These NATs include deoxyribonucleic acid (DNA), complementary DNA, complete genes, ribonucleic acid (RNA), oligonucleotides, and ribozymes, for gene therapy targeting a variety of diseases, such as cancer, infectious diseases, genetic disorders, and neurodegenerative diseases. In some embodiments, the nucleic acid therapeutic agent (NAT) is incorporated into lipid nanoparticles during their formation. In other embodiments, the nucleic acid therapeutic agent (NAT) is incorporated into lipid nanoparticles after their formation.

[0159] The nucleic acids present in the lipid nanoparticles according to the present invention include any known form of nucleic acid. The nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference agents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNAs, mRNAs, and triplet-forming oligonucleotides.

[0160] In one embodiment, the nucleic acid is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

[0161] The term "mRNA" stands for messenger RNA, a single-stranded ribonucleic acid responsible for protein synthesis in biological cells. mRNA serves as a template for ribosomes, directing tRNA (transfer RNA) to carry the corresponding amino acids to the ribosomes, where they assemble into proteins according to the codon sequence on the mRNA.

[0162] The term "saRNA" stands for self-replicating RNA, such as RNA derived from viral replicons, which can be used for protein expression. It results in the production of numerous copies of the original RNA, leading to high levels and sustained expression of the target protein.

[0163] The term "circRNA" stands for circular RNA, referring to polynucleotides that form a circular structure through covalent bonds. circRNA is a single-stranded RNA that forms a 3'-5' covalently closed circular structure. circRNAs can be produced through various mechanisms. A primary method is backsplicing, a non-canonical splicing process mediated by the spliceosome. In this process, a downstream splice donor site joins an upstream splice acceptor site, forming a covalently closed circular structure. In addition, circRNAs can also be generated through chemical ligation, enzymatic ligation, and ribozyme methods. These alternative methods enable the formation of circular RNA structures, expanding the variety of circRNAs available for research and therapeutic applications. Unlike linear mRNA, circRNAs do not require a 5'-cap or a 3'-poly(A) tail to maintain stability. The closed circular structure of circRNAs protects them from exonuclease-mediated degradation, makes them resistant to various mechanisms of RNA turnover, and results in a longer half-life compared to their linear mRNA counterparts.

[0164] The term "siRNA" stands for small interfering RNA, also known as short interfering RNA or silencing RNA. It is a type of double-stranded RNA molecule, approximately 20-25 base pairs in length. siRNA plays a role in the RNA interference (RNAi) pathway by degrading mRNA molecules complementary to its sequence, thereby preventing the translation of specific genes and achieving gene silencing.

[0165] The term "pDNA," short for plasmid DNA, refers to DNA molecules that are separable from chromosomal DNA in cells and can replicate independently. Plasmids range in size from less than 1,000 nucleotides to tens of thousands of nucleotides. The most common form is small, circular, double-stranded DNA. Plasmids can be synthesized and delivered to mammalian cells for therapeutic purposes. Synthetic plasmids are used as vectors in molecular cloning to drive the replication of recombinant DNA sequences within the host organism. Plasmids can be introduced into cells via transformation using physical methods, such as electroporation, or chemical means, such as transfection enhanced via lipid particles as described in this invention. Compared to physical techniques, these lipid-based vector systems offer several advantages, including: i) high biocompatibility and low toxicity in cellular and tissue systems; ii) relative ease of fabrication; iii) the lipophilic matrix is ​​less susceptible to corrosion phenomena observed in polymer systems; and iv) an increased circulating half-life in vivo because they are not observable in the immune system.

[0166] The term "ssDNA" stands for single-stranded DNA, which is DNA composed of a single nucleotide chain. Unlike typical double-stranded DNA (dsDNA), it lacks a complementary strand. ssDNA differs from dsDNA in its molecular hydrodynamic properties, absorption spectrum, and base reactivity. In living organisms, ssDNA is present during DNA replication and transcription, serving as a template to guide the synthesis of new strands. Some viruses, such as single-stranded DNA viruses, use ssDNA as their genetic material.

[0167] As used herein, “N / P” is the ratio of the molar number of amino groups in a cationic lipid to the molar number of phosphate groups in mRNA / DNA. In one embodiment, the N / P ranges from 2:1 to 30:1, for example, from 3:1 to 22:1. In one embodiment, the N / P ranges from 6:1 to 20:1, or from 2:1 to 12:1. Exemplary N / P ranges include about 3:1, about 6:1, about 12:1, and about 22:1, with the most preferred ratio being from 4:1 to 6:1.

[0168] In some embodiments, the LNP contains a total lipid to mRNA weight ratio of about 10:1 to about 30:1, for example, about 10:1 to about 20:1, about 15:1 to about 25:1, or about 20:1 to about 30:1. In some embodiments, the LNP contains a total lipid to circular RNA or circularized precursor RNA weight ratio greater than about 10:1, for example, greater than about 15:1, 20:1, 25:1, 30:1, or greater. In some embodiments, the LNP contains a total lipid to circular RNA or circularized precursor RNA weight ratio less than about 30:1, for example, less than about 25:1, 20:1, 15:1, 10:1, or smaller. In some embodiments, the total lipid to circular RNA or circularized precursor RNA weight ratio can be adjusted based on other components of the pharmaceutical composition, the individual to be administered, and / or the route of administration. For example, the amount of circular RNA or circularized precursor RNA in the LNP is measured using absorption spectroscopy (e.g., UV-Vis spectroscopy).

[0169] The LNP conjugate according to the present invention can be an aqueous formulation or a frozen formulation thereof (e.g., an aqueous formulation stored at about -20°C or lower, such as about -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C or -80°C).

[0170] The following describes pharmaceutical compositions comprising LNP conjugates and methods for delivering nucleic acid therapeutic agents via LNP conjugates of the present invention.

[0171] In some embodiments, the present invention provides a method for delivering cargo molecules into cells. In one embodiment, the cargo molecule is a nucleic acid, and therefore the delivery method is also referred to as transfection. Transfection is a technique commonly used in molecular biology for introducing nucleic acid therapeutic agents (or NATs) from outside the cell into the intracellular space for the purpose of transcription, translation, and expression of the delivered gene. Transfection efficiency is generally defined as: i) the percentage of cells in the total treated population showing positive expression of the delivered gene, as measured by protein quantification methods, such as live-cell imaging (for detecting fluorescent proteins), flow cytometry, or ELISA; or ii) the intensity or amount of protein expressed by the treated cells. These methods can be implemented by contacting the LNP conjugate of the present invention with cells for a period of time sufficient for intracellular delivery to occur.

[0172] Typical applications also include the use of well-known procedures to deliver siRNA intracellularly to knock out or silence specific cellular targets. Optionally, applications also include the delivery of DNA or mRNA sequences encoding therapeutically useful peptides. In this way, treatment of genetic diseases is provided by delivering defective or deleted gene products. The methods of the present invention can be performed in vitro, ex vivo, or in vivo. For example, the LNP conjugates of the present invention can also be used in vivo to deliver nucleic acids to cells using methods known to those skilled in the art. In another embodiment, the LNP conjugates of the present invention can be used to deliver nucleic acids to ex vivo patient cell samples and then return them to the patient.

[0173] In one embodiment, the present invention provides a method for regulating the expression of a target polynucleotide or polypeptide. These methods typically involve contacting cells with an LNP conjugate of the present invention, said LNP conjugate binding to a nucleic acid capable of regulating the expression of the target polynucleotide or polypeptide. As used herein, the term "regulation" refers to altering the expression of a target polynucleotide or polypeptide. Regulation can mean increasing or enhancing, or it can mean decreasing or reducing.

[0174] In related embodiments, the present invention provides a method for treating a disease or condition characterized by peptide overexpression in a subject, comprising providing the subject with a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from siRNA, microRNA, antisense oligonucleotide and plasmid capable of expressing siRNA, microRNA or antisense oligonucleotide, and wherein said siRNA, microRNA or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding a peptide or a complement thereof.

[0175] In related embodiments, the present invention provides a method for treating a disease or condition in a subject characterized by insufficient peptide expression, comprising providing the subject with a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from mRNA, self-amplified RNA (SAM), self-replicating DNA, or plasmids, comprising a nucleic acid therapeutic agent that specifically encodes or expresses an insufficiently expressed peptide or its complement.

[0176] As used herein, “pharmaceutically acceptable” refers to compounds, conjugates, materials, compositions, and dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with tissues in humans and animals without excessive toxicity, irritation, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0177] As used herein, the term "treatment" describes the management and care of a patient for the purpose of combating a disease, condition, or symptom, and includes administering the conjugates or pharmaceutical compositions of this disclosure to alleviate or eliminate symptoms or complications of the disease, condition, or symptom. The term "treatment" may also include treatment in in vitro cell or animal models.

[0178] As used in this article, the term "prevention" describes reducing or eliminating the onset of symptoms or complications of this disease, condition, or ailment.

[0179] The term "subject" refers to an animal, preferably a mammal, and most preferably a human, used as a subject of treatment, observation, or experimentation.

[0180] The term “therapeutic effective amount” refers to the amount of an active compound or agent that elicits a biological or medical response in an tissue system, animal, or human being explored by researchers, veterinarians, physicians, or other clinicians, including the reduction or partial reduction of symptoms of the disease, syndrome, condition, or symptom being treated.

[0181] "Therapeutic effective amount" refers to the amount of conjugate as defined herein that is sufficient to effectively treat or prevent disease when given to a patient requiring such treatment. The amount of a given conjugate corresponding to this amount will depend on factors such as the specific conjugate (e.g., the potency (pIC) of the conjugate). 50 ), efficacy (EC) 50 The duration of treatment and the time of administration of the conjugate (time between doses and time of dose, e.g., before / during / after a meal) should vary depending on the mammalian being treated (e.g., weight), the specific conjugate and its characteristics (e.g., pharmacokinetic properties), the disease or condition and its severity, and the specific composition and method used, but can still be determined by those skilled in the art.

[0182] The term "composition" or "pharmaceutical composition" means a product containing a therapeutically effective amount of a specified ingredient, or any product produced directly or indirectly from a combination of specified amounts of the specified ingredients.

[0183] For internal administration, parenteral administration (e.g., intra-articular, intravenous, intraperitoneal, subcutaneous, intrathecal, intradermal, intratracheal, intraosseous, or intramuscular) of the drug composition is preferred. In certain embodiments, the drug composition is administered intravenously, intrathecally, or intraperitoneally. Other routes of administration include topical (skin, eyes, mucous membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.

[0184] For ex vivo applications, it is preferable to apply the pharmaceutical composition to a biological sample that has been removed from an organism, then wash the cells and return them to the organism. The organism can be a mammal, particularly a human. This method is used for applications such as cell reprogramming, gene repair, or immunotherapy.

[0185] As used herein, the term "pharmaceuticalally acceptable excipient" means an excipient that is generally safe and non-toxic and is not biologically or otherwise undesirable for the preparation of a pharmaceutical composition, and includes excipients acceptable for veterinary and human pharmaceutical use. As used in the specification and claims, "pharmaceuticalally acceptable excipient" includes one or more excipients.

[0186] Pharmaceutically acceptable excipients used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, surfactants and / or emulsifiers, preservatives, buffers, lubricants and / or oils. Such excipients may optionally be included in the pharmaceutical compositions of the present invention. Various excipients used to formulate pharmaceutical compositions and techniques used to prepare such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, ARGennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient media is contemplated herein, except that any conventional excipient medium may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or interacting with any other component of the pharmaceutical composition in a harmful manner in other respects.

[0187] In one embodiment, pharmaceutically acceptable excipients that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, potassium sorbate, mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0188] To facilitate the delivery of the pharmaceutical compositions disclosed herein, any mild fixed oil, including synthetic monoglycerides or diglycerides, may be used. Fatty acids, such as oleic acid and its glyceride derivatives, may be used to prepare injectable formulations, such as natural, pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants, which are commonly used to formulate pharmaceutically acceptable dosage forms, including emulsions and suspensions.

[0189] Other commonly used surfactants, such as polysorbate ( Compounds), sorbitol esters ( Compounds and other emulsifiers or bioavailability enhancers commonly used to manufacture pharmaceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes.

[0190] Alternatively, the pharmaceutical compositions disclosed herein can be administered rectally as suppositories. These can be prepared by mixing the conjugate with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thus melting in the rectum to release the drug. These materials include cocoa butter, beeswax, and polyethylene glycol.

[0191] The pharmaceutical compositions disclosed herein can also be administered via nasal aerosol or inhalation. These compositions are prepared according to techniques recognized in the field of pharmaceutical formulation and can be prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption enhancers, fluorocarbons, and / or other conventional solubilizers or dispersants.

[0192] In the embodiments described herein, the formulations of the pharmaceutical compositions may be prepared by any method known or subsequently developed in the field of pharmacology. Typically, such preparation methods involve the steps of combining the active ingredient with excipients and / or one or more other auxiliary ingredients.

[0193] The pharmaceutical compositions according to this disclosure may be prepared, packaged, and / or marketed in batches as a single unit dose and / or as multiple single unit doses. As used herein, a “unit dose” means a pharmaceutical composition comprising a discrete amount of a predetermined amount of the active ingredient. The amount of the active ingredient may generally be equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of such dose, including but not limited to half or one-third of such dose.

[0194] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any other components in the pharmaceutical composition according to this disclosure may vary depending on the age, sex, weight, and / or condition of the treated subject, and further may vary depending on the route of administration of the composition. For example, the composition may contain 0.1% to 99% (w / w) of the active ingredient.

[0195] In some implementations, the particle size of lipid particles can be increased and / or decreased. Changes in particle size may help counteract biological responses such as, but not limited to, inflammation, or may increase the biological effects of NAT delivered to mammals by altering their biodistribution.

[0196] This disclosure also provides kits for delivering cargo molecules using LNP conjugates for intended therapeutic or diagnostic purposes. Such kits may include one or more containers containing one or more pharmaceutical compositions comprising one or more LNP conjugates disclosed herein and one or more pharmaceutically acceptable excipients.

[0197] In some embodiments, the kit may include instructions for use for any of the methods described herein. The instructions may include a description of administering the pharmaceutical composition to a subject to achieve the intended activity in a human patient. The kit may further include a description of selecting suitable human patients for treatment based on determining whether the human patient requires treatment. In some embodiments, the instructions include a description of administering the pharmaceutical composition to a human patient requiring treatment.

[0198] The following embodiments are provided for illustrative purposes and not for limiting the scope of the invention.

[0199] Example 1: Antibody Modification

[0200] Using SiteClick TM An antibody azidomodification kit (from Invitrogen, catalog number S20026) was used to modify the antibody azidomodification according to the manufacturer's instructions. In short, the antibody was ultrafiltered to an azide-free buffer, and β-galactosidase was used to remove the galactose residues on the glycan chain (N297), exposing the N-acetylglucosamine residues. Then, β-1,4-galactosyltransferase was used to ligate UDP-galactose azide to the exposed N-acetylglucosamine residues. Finally, ultrafiltration purification was performed to obtain an antibody modified with an azide at a specific asparagine residue (N297) on the Fc heavy chain. The antibody used in this example was a CD31 antibody (InVivoMAb anti-mouse CD31 (PECAM-1), from Bio X Cell, catalog number BE0377), which has lung-targeting properties.

[0201] Example 2: LNP Preparation

[0202] The lipid used in this embodiment is:

[0203] SM-102 (a cationic lipid), from MCE, catalog number HY-134541;

[0204] DSPC, from Aladdin, catalog number D130429-1g;

[0205] Cholesterol, from Sigma, catalog number C8667-5G;

[0206] DMG-PEG2000, from MCE, catalog number HY-112764; and

[0207] DSPE-PEG2000-DBCO, from Huateng Pharmaceutical, catalog number LP096048-2K.

[0208] The mRNA used in this embodiment is:

[0209] HiExpress TM Firefly luciferase IVT mRNA (m1Psi modified), from VectorBuilder, catalog number NR1021-1000; and

[0210] HiExpress TM Cre IVT mRNA (m1Psi modified), from VectorBuilder, catalog number NR1121-1000.

[0211] The preparation steps are as follows:

[0212] 4.55 mg of lipid SM-102, 1.01 mg of DSPC, 1.88 mg of cholesterol, 0.48 mg of DMG-PEG2000, and 0.20 mg of DSPE-PEG200-DBCO were placed in five 1.5 ml EP tubes. Anhydrous ethanol was added to dissolve the lipids, bringing the concentration of each lipid solution to 40 mM. The solutions were sonicated for 2 min, and then all lipids were thoroughly mixed into one EP tube, with a total volume of 320 μl. The mixture (ethanol phase) was drawn up using a 1 ml syringe.

[0213] In another EP tube, mix sodium acetate buffer (pH 4) and 346 μg mRNA to bring the final concentration of sodium acetate buffer (pH 4) to 50 mM, resulting in a total solution volume of 960 μl. Draw this solution (aqueous phase) into a 1 ml syringe.

[0214] The two phases were mixed using microfluidic mixing technology with the NanoAssemblr Ignite™ instrument (from Precision NanoSystems). The flow rate ratio (FRR) was set to 3:1, and the total flow rate was set to 12 ml / min. The mixed sample was diluted with 25 volumes of 15 mM Tris-HCl, concentrated using an Amicon Ultra-15 mL 10 kDa ultrafiltration tube, with three ultrafiltration buffer changes. The sample was centrifuged at 2800 × g, 12 °C, for 15 min, and filtered sterilized to obtain LNP.

[0215] Example 3: Preparation and characterization of antibody-conjugated LNPs

[0216] The azide-modified antibody prepared in Example 1 was mixed with the LNP prepared in Example 2 at a mass ratio of 1:1, and the coupling reaction was carried out overnight. After filtration and sterilization, the antibody-conjugated LNP was obtained.

[0217] LNP particle size and potential were measured using resistive pulse sensing (RPS) or dynamic light scattering (DLS) methods. The polydispersity index (PDI) of LNPs was measured using DLS, and the encapsulation efficiency was measured using the RiboGreen method. Details are as follows:

[0218] After diluting the sample 1000 times, RPS was measured using a Nanocoulter G nanoparticle size analyzer (manufactured by Rockchip).

[0219] After diluting the sample 50 times, DLS was detected using the Zetasizer Ultra instrument (manufacturer Malvern Panalytical).

[0220] LNP encapsulation efficiency was determined using the Quant-iT RiboGreen RNA Kit (from Invitrogen, catalog number R11490).

[0221] All tests were conducted in accordance with the manufacturer's instructions.

[0222] Example 4: In vivo experiment

[0223] LNP was injected via the tail vein into 6-8 week old female ICR mice (from Guangzhou Bojin Biotechnology Co., Ltd.) at a dose of 0.5 mg / kg body weight. Six hours post-injection, major organs (lung, liver, spleen, heart, and kidney) were dissected for in vitro imaging. Specifically, 45 mg of Pierce was weighed in a clean bench in the dark. TM D-fluorescein monopotassium salt (from Thermo Fisher, catalog number 88292) was dissolved in 3 ml of 1×PBS, sterilized by filtration through a 0.22 μm filter, and prepared as a 15 mg / ml substrate solution. The substrate was injected intraperitoneally at a rate of 10 μl / g. After 10 min, the mice were dissected, and lungs, liver, spleen, heart, and kidneys were harvested and imaged using an AniView100 multimodal animal in vivo imaging system (from Boluteng). The results are shown in Figure 2.

[0224] LNP was injected via the tail vein into 6-week-old male Ai9 mice (from Guangzhou Bojin Biotechnology Co., Ltd.). The injection doses were low (L, 0.1 mg / kg body weight) and high (H, 0.4 mg / kg body weight). 72 hours post-injection, cardiac perfusion was performed, and lungs were dissected, fixed, dehydrated, and frozen sectioned for observation of tdTomato red fluorescence expression. The negative control group was the PBS-injected group. Results are shown in Figure 4.

[0225] result:

[0226] (1) The mRNA encapsulated by LNP is luciferase mRNA. RPS results of unconjugated antibody LNP showed an average particle size of 76 nm (Figure 1A). DLS results showed a PDI of 0.061 and an average zeta potential of -7.78 mV.

[0227] RiboGreen measured the encapsulation efficiency at 90.1%. RPS results for site-directed conjugation of CD31 antibody to LNP showed an average particle size of 93 nm (Figure 1B). DLS results showed a PDI of 0.015 and an average zeta potential of -8.17 mV. RiboGreen measured the encapsulation efficiency at 90.1%.

[0228] (2) As shown in Figure 2, in the in vivo experiment, the CD31 antibody was site-conjugated to LNP and expressed in the lungs, followed by the liver and spleen; the unconjugated LNP was expressed in the liver and spleen, but not in the lungs. This demonstrates that the CD31 antibody is site-conjugated to LNP and achieves targeted expression in the lungs in vivo.

[0229] (3) The mRNA encapsulated by LNP was Cre mRNA. RPS results for unconjugated LNP showed an average particle size of 82 nm (Figure 3A). DLS results showed a PDI of 0.150 and an average zeta potential of -7.34 mV. RiboGreen measured the encapsulation efficiency as 96.9%. RPS results for site-conjugated CD31 antibody to LNP showed an average particle size of 83 nm (Figure 3B). DLS results showed a PDI of 0.077 and an average zeta potential of -4.16 mV. RiboGreen measured the encapsulation efficiency as 100%.

[0230] (4) As shown in Figure 4, in the in vivo experiment, the CD31 antibody was site-conjugated to LNP and targeted to the lungs for expression, while the unconjugated LNP was not expressed in the lungs, which proved that the CD31 antibody was site-conjugated to LNP and achieved targeting of the lungs in vivo.

[0231] Example 5: Detection of antibody modification level

[0232] First, use SiteClick. TMAn antibody azidomodification kit was used to modify the CD31 antibody according to the manufacturer's instructions. The antibody was ultrafiltered to an azide-free buffer, and galactose residues on the glycan chain (N297) were removed using β-galactosidase (reaction conditions shown in Table 1) to expose N-acetylglucosamine residues. Then, UDP-galactose azide was ligated to the exposed N-acetylglucosamine residues using β-1,4-galactosyltransferase. The antibody was purified by ultrafiltration to obtain an antibody modified with an azide at a specific asparagine residue (N297) on the Fc heavy chain. SiteClick was used as the method of modification. TM Alexa Fluor TM 488sDIBO Alkyne (Thermo, catalog number: S10904) dye was used for fluorescent labeling of the modification sites according to the manufacturer's instructions. 5 μl of SiteClick dye was added to the azide-modified antibody described above. TM Alexa Fluor TM 488s DIBO Alkyne was used for overnight coupling reaction. Finally, ultrafiltration purification was performed to obtain the dye-labeled antibody. 2 μl of the dye-labeled antibody was taken, and the absorbance at 495 nm and 280 nm was measured using a NanoDrop Eight spectrophotometer (Thermo). The absorbance was then calculated using the following formula: (Moles / L) dye =A 495 / 73,000 (Moles / L) IgG =[A 280 –(0.134×A 495 Modification level = (Moles) / 203,000 dye / (Moles) IgG

[0233] Using the modification method of this application, it is possible to modify approximately 1.16 mol of azide in 1 mol of antibody (see Table 1).

[0234] Table 1 Antibody modification conditions and degree of modification

[0235] Example 6: SDS-PAGE detection of antibody site-conjugated / non-site-conjugated products

[0236] Preparation steps of non-site-directed conjugation products: Antibody modification was performed according to the manufacturer's instructions (Thermo) for SATA molecules (N-succinic acid, S-acetyl mercaptoethylene glycol ester). A certain amount of CD31 antibody was taken, and the buffer was changed to PB buffer (0.1M phosphate, 0.15M sodium chloride, dissolved in ultrapure water, pH 7.3). The antibody was then reacted with SATA at room temperature for 30 min, with a SATA to antibody molar ratio of 9:1. After ultrafiltration to remove excess reactants, deprotection buffer (0.5M hydroxylamine hydrochloride, 25mM EDTA, dissolved in PB buffer, pH 7.3) was added, and the mixture was incubated at room temperature for 2 h, with an antibody volume to deprotection buffer volume ratio of 10:1. Ultrafiltration was then performed, and the buffer was changed to PB buffer containing 10mM EDTA to obtain the thiol-modified antibody. Weigh a certain amount of DSPE-PEG2000-maleimide (from Avanti, catalog number: 880126P-50MG), dissolve it in PB buffer containing 10mM EDTA, and add a portion to the thiol-modified antibody. The molar ratio between the maleimide group and the antibody is 28:1. After coupling reaction at 25℃ for 2 hours, incubate overnight at 4℃.

[0237] Site-directed coupling product preparation steps: First, use SiteClick... TM An antibody azidomodification kit was used to modify CD31 antibody with azido groups according to the manufacturer's instructions. The antibody was ultrafiltered to a buffer without azido groups. β-galactosidase was used to remove the galactose residues on the glycan chain (N297), exposing the N-acetylglucosamine residues. Then, β-1,4-galactosyltransferase was used to ligate UDP-galactose azide to the exposed N-acetylglucosamine residues. The mixture was purified by ultrafiltration to obtain an antibody modified with azido groups at specific asparagine residues (N297) on the Fc heavy chain. A certain amount of DSPE-PEG2000-DBCO was weighed, dissolved in 1×Tris buffer, and a portion was added to the azidomodified antibody. The molar ratio of DBCO groups to antibody was 28:1. The coupling reaction was carried out overnight at 25°C.

[0238] SDS-PAGE assay procedure: CD31 antibody, non-site-directed conjugated product, and site-directed conjugated product are added to SDS-PAGE protein loading buffer (5X) (from Beyotime, catalog number: P0286-15ml), and heated at 95℃ for 5 min. SDS-PAGE electrophoresis is performed using a 4-20% precast gel (from GenScript, catalog number: M00928) under the following conditions: 150V, 50 min. After electrophoresis, the sample is stained with Coomassie Brilliant Blue staining solution (from Beyotime, catalog number: P0003M) for 1 h, followed by destaining.

[0239] As shown in Figure 5, the band at 50kD represents the antibody heavy chain, and the band at 25kD represents the antibody light chain. The bands above the heavy and light chains are the conjugation products. In non-site-directed conjugation, multiple bands appear above the heavy chain and also above the light chain, indicating that conjugation sites are generated on both the heavy and light chains, and the number of sites is large, resulting in heterogeneous conjugation products. In contrast, in site-directed conjugation, the products are mainly concentrated in a single band of the heavy chain, indicating that the conjugation products are more homogeneous.

Claims

1. A lipid nanoparticle (LNP) conjugate, characterized in that... The LNP conjugate comprises (a) lipid nanoparticles (LNPs) and (b) an antibody or antigen-binding fragment thereof with cell or tissue targeting, wherein the antibody or antigen-binding fragment thereof is coupled to the surface of the LNP via an N-glycosylation site of the Fc heavy chain. According to the amino acid residue numbering in the EU index, the N-glycosylation site is located on the 297th asparagine residue in the Fc heavy chain. The Fc heavy chain has an azide functional group at its N-glycosylation site, the LNP has an alkyne functional group on its surface, and the antibody or its antigen-binding fragment is coupled to the surface of the LNP by click chemistry.

2. The LNP conjugate according to claim 1, characterized in that... The antibody or its antigen-binding fragment is IgG, and optionally the antibody or its antigen-binding fragment has an IgG1, IgG2, IgG3 or IgG4 isotype.

3. The LNP conjugate according to claim 1 or 2, characterized in that... The antibody or its antigen-binding fragment is coupled to the surface of the LNP by copper-free click chemistry; Optionally, the Fc heavy chain has galactosylation sites at the N-glycosylation sites, and the LNP has dibenzocyclooctylene (DBCO) on its surface, more preferably PEG-DBCO.

4. The LNP conjugate according to any one of claims 1-3, characterized in that... Each antibody or its antigen-binding fragment is conjugated to one or two LNPs.

5. The LNP conjugate according to any one of claims 1-4, characterized in that... The antibody or its antigen-binding fragment specifically binds to antigens selected from the following: CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR1, CTLA-4, D EC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin, tendinin C, TRAIL-R2, and vimentin.

6. The LNP conjugate according to any one of claims 1-5, characterized in that... The LNP comprises polymer-coupled lipids, cationic lipids, structural lipids, and sterols. Optionally, the LNP further comprises a therapeutic agent or a diagnostic agent, wherein the therapeutic agent or diagnostic agent is encapsulated in or attached to the LNP; Optionally, the therapeutic agent is a nucleic acid; Optionally, the therapeutic agent is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

7. A pharmaceutical composition comprising the LNP conjugate of any one of claims 1-6 and a pharmaceutically acceptable excipient.

8. A method for preparing lipid nanoparticle (LNP) conjugates, characterized in that... The method involves coupling an antibody or its antigen-binding fragment having an N-glycosylation site in the Fc heavy chain to the surface of the LNP via click chemistry. According to the amino acid residue numbering in the EU index, the N-glycosylation site is located on the 297th asparagine residue in the Fc heavy chain. The Fc heavy chain has an azide functional group at its N-glycosylation site, and the LNP has an alkyne functional group on its surface.

9. The method according to claim 8, characterized in that... The antibody or its antigen-binding fragment is IgG, and optionally the antibody or its antigen-binding fragment has an IgG1, IgG2, IgG3 or IgG4 isotype.

10. The method according to claim 8 or 9, characterized in that... The antibody or its antigen-binding fragment is coupled to the surface of the LNP via copper-free click chemistry; and / or The Fc heavy chain has galactose azide at its N-glycosylation site, and the LNP has dibenzocyclooctylene (DBCO) on its surface, more preferably PEG-DBCO.

11. The method according to any one of claims 8-10, characterized in that Each antibody or its antigen-binding fragment is conjugated to one or two LNPs.

12. The LNP conjugate according to any one of claims 8-11, characterized in that... The antibody or its antigen-binding fragment specifically binds to antigens selected from the following: CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR1, CTLA-4, D EC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin, tendinin C, TRAIL-R2, and vimentin.

13. The LNP conjugate according to any one of claims 8-12, characterized in that... The LNP comprises polymer-coupled lipids, cationic lipids, structural lipids, and sterols. Optionally, the LNP further comprises a therapeutic agent or a diagnostic agent, wherein the therapeutic agent or diagnostic agent is encapsulated in or attached to the LNP; Optionally, the therapeutic agent is a nucleic acid; Optionally, the therapeutic agent is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

14. A method for preparing lipid nanoparticle (LNP) conjugates, characterized in that the method comprises the following steps: (a) Provide an antibody or an antigen-binding fragment thereof having an N-glycosylation site in the Fc heavy chain, wherein the N-glycosylation site is located at the 297th asparagine residue in the Fc heavy chain according to the amino acid residue number in the EU index; (b) Contact the antibody or its antigen-binding fragment with galactosidase to remove galactose residues at the N-glycosylation site of the Fc heavy chain of the antibody or its antigen-binding fragment, exposing N-acetylglucosamine residues; (c) Linking UDP-galactosyl azide to exposed N-acetylglucosamine residues by contacting the antibody or its antigen-binding fragment obtained in step (b) with β-1,4-galactosyltransferase; and (d) The antibody or its antigen-binding fragment obtained in step (c) is mixed with an LNP modified with dibenzocyclooctylene (DBCO) on its surface, and the antibody or its antigen-binding fragment is site-coupled to the LNP surface by click chemistry.

15. A method for delivering cargo molecules into cells, characterized in that... The method comprises contacting the LNP conjugate of any one of claims 1-6 with a cell expressing a cell surface antigen under conditions that facilitate the entry of cargo molecules into the cell, wherein the LNP comprises cargo molecules, and wherein the antibody or its antigen-binding fragment specifically binds to the cell surface antigen.

16. The method according to claim 15, characterized in that... The cargo molecule is a therapeutic or diagnostic agent, wherein the therapeutic or diagnostic agent is encapsulated in or attached to the LNP; Optionally, the therapeutic agent is a nucleic acid; Optionally, the therapeutic agent is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

17. The method according to claim 15 or 16, characterized in that... The cell surface antigens are selected from CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD117, CD138, CD141, CD326, CD5, ASPGR, CLEC9A, PD-1, CEA, Clec9A, CSFR1, CTLA-4, DEC205, E GFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tendinin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate-specific antigen, adrenergic receptor-β2, Claudine 3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, coilin-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin, tendinin C, TRAIL-R2, and vimentin.

18. The method according to any one of claims 15-17, characterized in that... The cells are mammalian cells, and / or the method is an in vitro method.

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