Lipid nanoparticle conjugate as well as preparation method and application thereof

Abstract

The invention provides a lipid nanoparticle conjugate as well as a preparation method and application thereof. In particular, an antibody is coupled to the surface of the LNP through an N-glycosylation site of an Fc heavy chain by using a copper-free click chemical reaction, thereby obtaining the LNP having a specific cell or tissue targeting property. Preferably, the N-glycosylation site is located on the asparagine residue at the 297th position of the Fc heavy chain.

Description

Technical Field

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

[0002] Lipid nanoparticles (LNPs) are one of the most effective carriers for nucleic acid delivery, with high safety and low toxicity. However, the development of LNPs has mainly been limited to mRNA vaccines administered by intramuscular injection and liver delivery by intravenous injection. This is because in the blood, non-targeted LNPs are adsorbed by apolipoprotein E (ApoE) and enriched in the liver by binding to the low-density lipoprotein receptor of liver cells. Therefore, designing LNPs with targeted functions is required to deliver nucleic acid drugs to specific sites. Among them, conjugating a specific antibody to the surface of LNPs can achieve the targeted function of LNPs. However, most current antibody functionalization modifications are carried out on the amino or carboxyl groups of the antibody and then conjugated to the functional groups on the LNPs. The amino or carboxyl sites on the antibody are not unique, and there may be many sites for conjugating with LNPs on one antibody after modification, resulting in multiple LNPs conjugated to one antibody, prone to aggregation, and the particles are heterogeneous. Some modification sites may be located in the variable region of the antibody, and the recognition function of the antibody will be affected after modification and / or conjugation.

[0003] PCT Publication No. WO2023248125A1 discloses a targeted lipid nanoparticle. By transfecting a vector encoding an anti-CD117 VHH antibody with a sortase recognition peptide sequence added to the C-terminus into 293F cells, after the cells express the corresponding antibody, a specific peptide sequence modified with azide is added under the catalysis of sortase, and finally conjugated to the LNP with DBCO modification. CD117 is a surface marker of various types of hematopoietic cells. Conjugating an antibody that specifically binds to it to the lipid nanoparticle enables the targeted delivery of multiple components required for gene editing to hematopoietic cells.

[0004] There is still a need in the art for improved methods to achieve efficient tissue and cell targeting after conjugating a wide range of antibodies with lipid nanoparticles. Summary of the Invention

[0005] The inventors modified the N-glycosylation site on the Fc heavy chain of the antibody, especially the N-glycosylation site at the asparagine residue (N297), and conjugated the antibody to the LNP with surface modification by click chemistry, thereby obtaining LNPs with specific cell or tissue targeting.

[0006] The advantages of the present invention are that the antibody modification sites in the LNP conjugate are fixed, and the number is between 1 and 4. Due to steric hindrance, one antibody can at most be connected to two LNPs, reducing LNP aggregation and improving particle homogeneity. The LNP conjugate of the present invention also maintains the high-efficiency tissue targeting of the antibody.

[0007] Accordingly, in one aspect, there is provided an LNP conjugate comprising (a) a lipid nanoparticle (LNP) and (b) an antibody or an antigen-binding fragment thereof having cell or tissue targeting properties, wherein the antibody or the antigen-binding fragment thereof is conjugated to the surface of the LNP through an N-glycosylation site of the Fc heavy chain.

[0008] In one embodiment, the antibody or the antigen-binding fragment thereof is IgG. In another embodiment, the antibody or the antigen-binding fragment thereof has an IgG1, IgG2, IgG3 or IgG4 isotype. In a preferred embodiment, the antibody or the antigen-binding fragment thereof has an IgG1 isotype. In another preferred embodiment, the antibody or the antigen-binding fragment thereof has an IgG2 isotype. In one embodiment, according to the amino acid residue numbering of the EU index, the N-glycosylation site is located at the asparagine residue at position 297 of the Fc heavy chain.

[0009] In one embodiment, the antibody or the antigen-binding fragment thereof is conjugated to the surface of the LNP by click chemistry. In one embodiment, the click chemistry is 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.

[0010] In one embodiment, the N-glycosylation site of the Fc heavy chain has an azide functional group. In one embodiment, the azide functional group is galactose azide.

[0011] In one embodiment, the surface of the LNP has an alkyne functional group. In one embodiment, the alkyne functional group is dibenzocyclooctyne (DBCO). In one embodiment, the alkyne functional group is PEG-DBCO.

[0012] In one embodiment, in the LNP conjugate, each antibody or the antigen-binding fragment thereof is conjugated to 1 or 2 LNPs.

[0013] In one embodiment, the antibody or its antigen-binding fragment specifically binds to a cell surface antigen, which includes 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, tenascin 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, frizzled protein-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin (including α v β3, α v β5, α v β6, α1β4, α4β1, α5β1, α6β4 integrin), tenascin C, TRAIL-R2 or vimentin.

[0014] In one embodiment, the LNP comprises or consists of a polymer-conjugated lipid, a cationic lipid, a structural lipid, and a sterol. In one embodiment, the polymer-conjugated lipid comprises a PEG-conjugated lipid. In one embodiment, the sterol comprises cholesterol. In one embodiment, the cationic lipid comprises an amino lipid. In one embodiment, the structural lipid comprises a phospholipid.

[0015] In one embodiment, the LNP further comprises a therapeutic agent or a diagnostic agent, wherein the therapeutic agent or the diagnostic agent is encapsulated in the LNP 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.

[0016] In another aspect, a pharmaceutical composition is provided, which comprises the LNP conjugate as described herein and a pharmaceutically acceptable excipient.

[0017] In another aspect, a method for preparing a lipid nanoparticle (LNP) conjugate 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.

[0018] In one embodiment, the antibody or the antigen-binding fragment thereof is IgG. In another embodiment, the antibody or the antigen-binding fragment thereof has an IgG1, IgG2, IgG3 or IgG4 isotype. In a preferred embodiment, the antibody or the antigen-binding fragment thereof has an IgG1 isotype. In another preferred embodiment, the antibody or the antigen-binding fragment thereof has an IgG2 isotype. In one embodiment, according to the amino acid residue numbering of the EU index, the N-glycosylation site is located at the asparagine residue at position 297 of the Fc heavy chain.

[0019] In one embodiment, the antibody or the antigen-binding fragment thereof is conjugated to the surface of the LNP by click chemistry. In one embodiment, the click chemistry is 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.

[0020] In one embodiment, the N-glycosylation site of the Fc heavy chain has an azide functional group. In one embodiment, the azide functional group is galactose azide.

[0021] In one embodiment, the surface of the LNP has an alkyne functional group. In one embodiment, the alkyne functional group is dibenzocyclooctyne (DBCO). In one embodiment, the alkyne functional group is PEG-DBCO.

[0022] In one embodiment, in the LNP conjugate, each antibody is conjugated to 1 or 2 LNPs.

[0023] In one embodiment, the antibody or its antigen-binding fragment specifically binds to a cell surface antigen, which includes 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, tenascin 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, frizzled protein-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin (including α v β3, α v β5, α v β6, α1β4, α4β1, α5β1, α6β4 integrin), tenascin C, TRAIL-R2 or vimentin.

[0024] In one embodiment, the LNP comprises or consists of a polymer-conjugated lipid, a cationic lipid, a structural lipid, and a sterol. In one embodiment, the polymer-conjugated lipid comprises a PEG-conjugated lipid. In one embodiment, the sterol comprises cholesterol. In one embodiment, the cationic lipid comprises an amino lipid. In one embodiment, the structural lipid comprises a phospholipid.

[0025] In one embodiment, the LNP further comprises a therapeutic agent or a diagnostic agent, wherein the therapeutic agent or the diagnostic agent is encapsulated in the LNP 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.

[0026] In one embodiment, a method for preparing a lipid nanoparticle (LNP) conjugate as described herein is provided, the method comprising the following steps:

[0027] (a) providing an antibody or an antigen-binding fragment thereof having an N-glycosylation site in the Fc heavy chain;

[0028] (b) contacting the antibody or an antigen-binding fragment thereof with galactosidase to remove galactose residues at the N-glycosylation site of the Fc heavy chain of the antibody or an antigen-binding fragment thereof, exposing N-acetylglucosamine residues;

[0029] (c) contacting the antibody or an antigen-binding fragment thereof obtained in step (b) with β-1,4-galactosyltransferase to ligate UDP-galactose azide to the exposed N-acetylglucosamine residues; and

[0030] (d) mixing the antibody or an antigen-binding fragment thereof obtained in step (c) with LNPs bearing dibenzocyclooctyne (DBCO) modification on the surface, and site-specifically conjugating the antibody or an antigen-binding fragment thereof to the surface of the LNPs through a click chemical reaction.

[0031] In another aspect, a method for delivering a cargo molecule into a cell 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 contains the cargo molecule, and wherein the antibody or an antigen-binding fragment thereof can specifically bind to the cell surface antigen.

[0032] In one embodiment, the cargo molecule is a therapeutic agent or a diagnostic agent, wherein the therapeutic agent 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.

[0033] In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to a cell surface antigen, which includes 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, tenascin 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, frizzled protein-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin (including α v β3, α v β5, α v β6, α1β4, α4β1, α5β1, α6β4 integrin), tenascin C, TRAIL-R2 or vimentin.

[0034] In one embodiment, the cell is a mammalian cell.

[0035] In one embodiment, the method is an in vivo method. In another embodiment, the method is an in vitro method.

[0036] In another aspect, there is provided a lipid nanoparticle (LNP) conjugate as described herein for delivering a cargo molecule to a cell.

[0037] In another aspect, there is provided the use of a lipid nanoparticle (LNP) conjugate as described herein in the preparation of a medicament for delivering a cargo molecule to a cell.

[0038] Other features and advantages of the present disclosure will be apparent from the accompanying drawings, specific embodiments, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present invention will be better understood in conjunction with the accompanying drawings.

[0040] Figure 1A The RPS results of uncoupled antibody LNP-luciferase mRNA are shown.

[0041] Figure 1B The RPS results of CD31 antibody site-specifically conjugated LNP-luciferase mRNA are shown.

[0042] Figure 2 The ex vivo imaging results of major organs in the in vivo experiments of uncoupled antibody LNP and CD31 antibody site-specifically conjugated LNP are shown.

[0043] Figure 3A The RPS results of uncoupled antibody LNP-Cre mRNA are shown.

[0044] Figure 3B The RPS results of CD31 antibody site-specifically conjugated LNP-Cre mRNA are shown.

[0045] Figure 4 The tdTomato fluorescence results of lung sections in the in vivo experiments of uncoupled antibody LNP and CD31 antibody site-specifically conjugated LNP are shown. L and H represent the low-dose group and the high-dose group, respectively. Specific embodiments

[0046] The present disclosure provides a new lipid nanoparticle (LNP) conjugate, a method for preparing a lipid nanoparticle (LNP) conjugate, a pharmaceutical composition containing them, and various uses thereof.

[0047] General definition

[0048] When used in conjunction with a numerical value, the terms "about", "approximately" or "approximate" mean to include 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" means a range of values that are 5% more or less than the specified value. In some embodiments, the term "about" means a range of values that are 2% more or less than the specified value. In some embodiments, the term "about" means a range of values that are 1% more or less than the specified value.

[0049] Unless otherwise indicated herein, the recitation of a range of values is merely intended to be a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise specified, the ranges used herein include the two end values of the range. In some embodiments, the statements “x is an integer between 1 and 6” and “x is an integer of 1-6” both mean “x is 1, 2, 3, 4, 5, or 6”, i.e., the terms “between X and Y” and “in the range of X to Y” include X and Y and the integers therebetween.

[0050] It should be understood that throughout the specification, when a composition is described as having, including, or comprising a particular component, it is contemplated that the composition also consists essentially of or consists of the recited components. Similarly, when a method or procedure is described as having, including, or comprising a particular procedural step, it is contemplated that the method or procedure also consists essentially of or consists of the recited procedural steps. Further, it should be understood that as long as the present invention remains operable, the order of steps or the order of performing certain actions is immaterial. In addition, two or more steps or actions can be performed simultaneously.

[0051] Unless otherwise indicated, all percentages and ratios used herein are by weight.

[0052] 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 forms also include the plural unless the context clearly dictates otherwise. Although those similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present disclosure, the following describes suitable methods and materials. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art of the claimed invention. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0053] Various definitions and a detailed explanation of the present invention are provided below. It should be understood that these are not restrictive.

[0054] Antibody

[0055] The LNP conjugates of the present disclosure consist of LNPs covalently linked to antibodies. The antibody represents a targeting mechanism for a specific site of action. After reaching the site, the LNP conjugate is able to perform its designed function in a targeted manner rather than diffusing throughout the entire body of the subject systemically. This targeted approach enables treatment with drugs that would otherwise require very high doses that are toxic when administered systemically.

[0056] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity. An antibody is also a glycoprotein that contains sugar chains in some amino acid residues. The numbering of antibody amino acids follows the Kabat EU index (see Kabat, E.A. et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

[0057] The term "antigen-binding fragment" refers to a molecule other than a full antibody that comprises a portion of a full antibody and binds to an antigen to which the full antibody binds. Examples of antigen-binding fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0058] In a particular embodiment, the antibody or its antigen-binding fragment comprises an N-glycosylation site of the Fc heavy chain, particularly the asparagine residue at position 297 (N297). It should be understood that the N297 described in the present disclosure corresponds to the IgG amino acid numbering according to the EU index, and other antibody isotypes, such as IgA, IgM, IgE, and IgD, may also contain glycosylation sites corresponding to IgGN297, 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 at the asparagine residue at position 297. In another embodiment, the antibody has an IgG2 isotype, and the N-glycosylation site is located at the asparagine residue at position 297.

[0059] As used herein, an antibody that "specifically binds" or "targets" an antigen or cell surface antigen associates preferentially with the antigen or cell surface antigen via intermolecular forces. In some embodiments, the antibody can have a K d that preferentially associates with the antigen or cell surface antigen. Techniques for measuring the binding affinity of an antibody for an antigen are well known, such as surface plasmon resonance (SPR).

[0060] In one embodiment, the antibody specifically binds to a cell surface antigen, which includes 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, tenascin 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, frizzled protein-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin (including α v β3, α v β5, α v β6, α1β4, α4β1, α5β1, α6β4 integrin), tenascin C, TRAIL-R2 or vimentin.

[0061] In some embodiments, the antibody is directed against 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 α vβ3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, NaPi2b, PTK7, phosphatidylserine, prostate specific antigen, PDGFRα, TAG-72, tenascin C, TRAIL-R2, VEGF-A and VEGFR2. In such embodiments, the antibodies include but are not limited to abagovomab, adecatumumab, alacizumab, altumomab, anatumomab, arcitumomab, bavituximab, bevacizumab Bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, citatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab, matuzumab, mitumomab, naptumomab estafenatox, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab Rilotumumab, robatumumab, satumomab, sibrotuzumab, taplitumomab, tenatumomab, ticilimumab, tigatuzumab, trastuzumab Tositumomab, tremelimumab, tucotuzumab celmoleukin, volociximab, and zalutumumab.

[0062] In some embodiments, the antibody is a bispecific antibody.

[0063] It should be understood that the antibodies of the present disclosure are not limited to any specific antibody, provided that the antibody contains an N - single chain glycosylation site of the Fc heavy chain, particularly a glycosylation site corresponding to the asparagine residue (N297) at position 297 of IgG.

[0064] Lipid nanoparticle

[0065] 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 characterized by poor solubility in water but solubility in many non - polar organic solvents. Although lipids generally have poor solubility in water, there are some classes of lipids (e.g., lipids modified by polar groups such as DMG - PEG2000) that have limited water solubility and can dissolve 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, oils, and waxes; (2) "compound lipids", including phospholipids and glycolipids (e.g., DMPE - PEG2000); (3) "derived lipids", such as steroids. In addition, as used herein, lipids also include lipid - like compounds. The term "lipid - like compound", also simply referred to as "lipidoid", refers to lipid - like compounds (e.g., amphiphilic compounds having lipid - like physical properties).

[0066] The term "lipid nanoparticle" or "LNP" refers to particles with a maximum dimension in the nanometer (nm) range (e.g., 1 - 1000 nm), which contain one or more types of lipid molecules. Lipid nanoparticles are capable of carrying an aqueous solution, compound, drug, or other substance, such as one or more nucleic acid molecules, collectively referred to herein as cargo molecules, within a compartment (i.e., an internal cavity or space) 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 in the range of about 40 to about 200 nm. In one embodiment, at least one dimension is in the range of about 40 to about 100 nm.

[0067] In some embodiments, the LNP contains non-lipid cargo molecules that are partially or fully encapsulated within a lipid shell. In particular, in some embodiments, the cargo molecules are negatively charged molecules (e.g., mRNA encoding viral proteins), and the lipid component of the LNP contains at least one cationic lipid. Without being bound by theory, the cationic lipid can interact with the negatively charged cargo molecules and facilitate the incorporation and / or encapsulation of the cargo molecules into the LNP during LNP formation. Other lipids that can form part of the LNP as described herein include, but are not limited to, neutral lipids (also referred to as structural lipids) and charged lipids, such as steroids, polymer-conjugated lipids, and various zwitterionic lipids.

[0068] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or that is capable of becoming positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., its intended use environment). Thus, the term "cationic" encompasses both "permanent cation" and "cationizable" cases. In certain embodiments, the positive charge in the cationic lipid is caused by the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid contains a zwitterionic lipid that is positively charged in the environment in which it is intended to be used (e.g., at physiological pH).

[0069] In one embodiment, the cationic lipid can be any lipid that bears a net positive charge at a selected pH value (e.g., physiological pH). Exemplary cationic lipids are described below. In one embodiment, the pKa of the cationic lipid is greater than 6.25. In one embodiment, the pKa of the cationic lipid is greater than 6.5. In one embodiment, the cationic lipid has a pKa greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65, or greater than 6.7.

[0070] Examples of cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearoyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and N-(1,2-dimyristyloxypropyl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0071] In a preferred embodiment, the cationic lipid is an amino lipid. Suitable amino lipids for use in the present invention include those described in WO2009 / 096558, which is incorporated herein by reference in its entirety. Representative amino lipids include 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-linoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-linoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA·Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP·Cl), 1,2-linoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-linoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-linoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-linoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-linoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoeyl) methyl ester (DLin-MC3-DMA), 2,2-linoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and 1,2-dioleyloxy-3-dimethylaminopropane (DODMA).

[0072] In other embodiments, the cationic lipids mentioned in US20180000953 are used, such as 3-(dilauryldiamino)-N1,N1,4-tris(dodecyl)-1-piperazineethanamine (KL10), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-octatriacontane (KL25), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)).

[0073] In some embodiments, based on the total lipids of the LNP, the LNP contains between about 20 Mol% and about 75 Mol% of cationic lipids. In some embodiments, based on the total lipids of the LNP, the LNP contains between about 30 Mol% and about 55 Mol% of cationic lipids. In some embodiments, based on the total lipids of the LNP, the LNP contains more than about 30 Mol% of cationic lipids, such as more than about 35 Mol%, about 40 Mol%, about 45 Mol%, about 50 Mol%, about 55 Mol% or more of cationic lipids. In some embodiments, based on the total lipids of the LNP, the LNP contains less than about 55 Mol% of cationic lipids, such as less than about 50 Mol%, about 45 Mol%, about 40 Mol%, about 35 Mol%, about 30 Mol% or less of cationic lipids.

[0074] The term "structural lipid", also known as neutral lipid, refers to any lipid molecule that exists in an uncharged form or a neutral zwitterionic form at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of intended use of the lipid, such as physiological pH. Suitable structural lipids support the formation of particles during the manufacturing process. Structural lipids can be phospholipids. Representative structural lipids include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, diacyl phosphatidylglycerol, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside.

[0075] Exemplary structural lipids include, but are not limited to, zwitterionic lipids such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In a preferred embodiment, the structural lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The structural lipids provided herein can be synthetic or derived from natural sources or compounds.

[0076] In another embodiment, the structural lipid is any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol such as dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacyl phosphatidylserine, diacyl phosphatidic acid, and other anionic modifying groups attached to neutral lipids.

[0077] Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0078] In some embodiments, based on the total lipids of the LNP, the LNP contains from about 5 Mol% to about 40 Mol% of structural lipids. In some embodiments, based on the total lipids of the LNP, the LNP contains greater than about 5 Mol% of structural lipids, such as greater than about 10 Mol%, about 15 Mol%, about 20 Mol%, about 25 Mol%, about 30 Mol%, about 35 Mol%, about 40 Mol% or more of structural lipids. In some embodiments, based on the total lipids of the LNP, the LNP contains less than about 40 Mol% of structural 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 of structural lipids.

[0079] 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 nanoparticle contains 5 to 10 Mol% of structural lipids.

[0080] The term "polymer-conjugated lipid" refers to a molecule that contains a lipid moiety and a polymer moiety. Without being bound by theory, it is contemplated that polymer-conjugated lipids in lipid nanoparticles can enhance colloidal stability and / or reduce protein uptake by the nanoparticles. An example of a polymer-conjugated lipid is a polyethylene glycolylated lipid (PEG-lipid), wherein the polymer moiety comprises polyethylene glycol. Exemplary polymer-conjugated lipids useful in the present 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, the PEG-lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0081] Any lipid described herein, including components of the lipid nanoparticles described herein, can be conjugated to polyethylene glycol. In some embodiments, a polyethylene glycolylated form of any lipid described herein can be conjugated to an antibody or an antigen-binding fragment thereof. In a preferred embodiment, the polymer-conjugated lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated to polyethylene glycol. In some embodiments, the PEG can be PEG500 (molecular weight 500) to PEG20000 (molecular weight 20000).

[0082] In one embodiment, based on the total lipids of the LNP, the LNP contains about 0.5 to 5.0 Mol% of the polymer-conjugated lipid. In one embodiment, based on the total lipids of the LNP, the LNP contains about 1.0 to 2.5 Mol% of the polymer-conjugated lipid. In one embodiment, based on the total lipids of the LNP, the LNP contains about 1.7 Mol% of the polymer-conjugated lipid. In one embodiment, based on the total lipids of the LNP, the LNP contains about 1.5 Mol% of the polymer-conjugated lipid.

[0083] In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid ranges from about 35:1 to about 25:1. In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid ranges from about 100:1 to about 20:1.

[0084] The lipid nanoparticles may also comprise a sterol, such as cholesterol or phytosterol and derivatives thereof. Without being bound by theory, it is believed that sterols can stabilize the amphiphilic structure of the nanoparticles, such as but not limited to the lipid bilayer structure of the nanoparticles. Exemplary sterols and derivatives thereof that can be used in the present disclosure include but are not limited to cholesterol, Fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatoside, ursolic acid, α-tocopherol, and mixtures thereof. In certain embodiments, the sterol is cholesterol. In some embodiments, the sterol comprises cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0085] In some embodiments, based on the total lipids of the LNP, the LNP comprises between about 20 Mol% and about 50 Mol% of sterol. In some embodiments, based on the total lipids of the LNP, the LNP comprises greater than about 20 Mol%, such as greater than about 25 Mol%, about 30 Mol%, about 35 Mol%, about 40 Mol%, about 45 Mol%, about 50 Mol% or more of sterol. In some embodiments, based on the total lipids of the LNP, the LNP comprises less than about 50 Mol%, such as less than about 45 Mol%, about 40 Mol%, about 35 Mol%, about 30 Mol%, about 25 Mol%, about 20 Mol% or less of sterol.

[0086] In one embodiment, the molar ratio of cationic lipid to sterol ranges from about 10:1 to 1:1. In one embodiment, the molar ratio of cationic lipid to sterol ranges from about 5:1 to 1:1. In one embodiment, based on the total lipids of the LNP, the sterol is present at a concentration of 32 to 40 Mol%.

[0087] In some embodiments, the LNP comprises a cationic lipid, a structural lipid, a sterol, and a polymer-conjugated lipid, such as any of the cationic lipids, structural lipids, sterols, and polymer-conjugated lipids described herein. In some embodiments, the LNP comprises i) between about 30 Mol% and about 55 Mol% of cationic lipid, and ii) between about 5 Mol% and about 40 Mol% of structural lipid.

[0088] In one embodiment, the lipid nanoparticles comprise:

[0089] i) from about 20 to 65 Mol% of cationic lipid; ii) from about 5 to 40 Mol% of structural lipid; iii) from about 20 to 50 Mol% of sterol; and iv) polymer-conjugated lipid.

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

[0091] i) about 40 to 55 Mol% of cationic lipid; ii) about 5 to about 15 Mol% of structural lipid; iii) about 35% to 50 Mol% of sterol; and iv) about 2 to 10 Mol% of polymer-conjugated lipid.

[0092] In one embodiment, the lipid nanoparticle comprises:

[0093] i) about 45 to 55 Mol% of cationic lipid; ii) about 6 to 10 Mol% of structural lipid; iii) about 40 to 48 Mol% of sterol; and iv) about 1 to 2.5 Mol% of polymer-conjugated lipid.

[0094] In one embodiment, the lipid nanoparticle comprises a cationic lipid, DSPC, cholesterol, and PEG-lipid as well as mRNA. In one embodiment, the molar ratio of the cationic lipid, DSPC, cholesterol, and PEG-lipid is about 50:10:38.5:1.5. In one embodiment, the molar ratio of the cationic lipid, DSPC, cholesterol, and PEG-lipid is about 50:10:38:2.

[0095] As used herein, "Mol%" refers to the Mol% of a component relative to the total moles of all lipid components in the LNP (i.e., the total moles of cationic lipid, structural lipid, sterol, and polymer-conjugated lipid).

[0096] The physical properties of the lipid nanoparticles may depend on their composition. For example, nanoparticles containing different structural lipids may have different properties. Similarly, the properties of the lipid nanoparticles may depend on the absolute or relative amounts of their components. For example, nanoparticles containing a higher mole fraction of phospholipids may have different properties from nanoparticles containing a lower mole fraction of phospholipids. The properties may also vary according to the method and conditions for preparing the nanoparticles.

[0097] Lipid nanoparticles can be characterized by 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 the nanoparticles. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine the particle size. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure various properties of the nanoparticles, such as particle size, polydispersity index, and zeta potential.

[0098] 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. Smaller particles generally exhibit increased circulation lifetimes in vivo compared to larger particles. In one embodiment, the diameter of the lipid particles is about 15 nm to about 50 nm.

[0099] The lipid nanoparticles can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticles. A smaller (e.g., less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of the nanoparticle composition can range from about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticles can be about 0.10 to about 0.20.

[0100] The zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticles with a relatively low charge (positive or negative) are generally desired because substances with higher charges can have adverse interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticles can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0101] The encapsulation efficiency of a therapeutic and / or prophylactic agent illustrates the amount of the therapeutic and / or prophylactic agent encapsulated by or otherwise associated with the nanoparticles after preparation relative to the initial amount provided. Ideally, the encapsulation efficiency is very high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic and / or prophylactic agent in a solution containing the nanoparticles before and after decomposing the nanoparticles with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For the nanoparticles described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent 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 certain embodiments, the encapsulation efficiency can be at least 90%.

[0102] The nanoparticle composition can optionally comprise one or more coatings. For example, the nanoparticle composition can be formulated in a capsule, film or tablet having a coating. The capsule, film or tablet containing the nanoparticle composition described herein can have any useful size, tensile strength, hardness or density.

[0103] Lipid nanoparticles according to embodiments of the present invention can be prepared by standard T-tube mixing techniques, turbulent mixing, milling mixing, facilitated stirring sequential self-assembly, or passive mixing of all components into nanoparticles. A variety of methods have been developed to formulate lipid nanoparticles (LNPs) containing gene drugs. Suitable methods are disclosed, for example, in U.S. Patent No. 5,753,613 and U.S. Patent No. 6,734,171. These methods include 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 an NAT encapsulation efficiency of 65-95%. Both methods rely on the presence of cationic lipids to achieve encapsulation of NAT and stabilizers to inhibit aggregation and formation of large structures. The performance of the resulting lipid particle system (including size and NAT encapsulation efficiency) is sensitive to a variety of formulation parameters, such as ionic strength, lipid and ethanol concentration, pH, NAT concentration and mixing rate. See J Drug Target. 2016 Nov;24(9):821-835.

[0104] Microfluidic two-phase droplet technology has been applied to produce monodisperse polymer microparticles for drug delivery or large vesicles for encapsulating cells, proteins, or other biomolecules. It has been demonstrated that monodisperse liposomes with controlled size can be produced using hydrodynamic flow focusing, a common microfluidic technique that provides rapid mixing of reagents.

[0105] Typically, it is difficult to control parameters such as relative lipid and NAT concentrations, as well as mixing rates, during mixing using current formulation procedures, resulting in variations in NAT properties within and between formulations. Automated micro-mixing instruments such as NanoAssemblr TM instrument (Precision NanoSystems Inc, Vancouver, Canada) enable the rapid and controlled manufacture of nanodrugs (liposomes, lipid nanoparticles, and polymer nanoparticles). NanoAssemblr TM instrument enables the controlled molecular self-assembly of nanoparticles via a microfluidic mixing cartridge, which allows for millisecond mixing of nanoparticle components in nanoliter, microliter, or larger scales in a customized or parallelized manner. Rapid mixing at small scales allows for reproducible control of particle synthesis and quality, which is not possible in larger instruments.

[0106] Preferred methods incorporate microfluidic mixing devices such as NanoAssemblr TM , Spark TM , Ignite TM , Benchtop TM and Blaze TM to encapsulate nearly 100% of the nucleic acid used in the formation process in the particles in one step. In one embodiment, lipid nanoparticles are prepared by encapsulating approximately 90% to approximately 95% of the nucleic acid used in the formation process in the particles.

[0107] In an embodiment of the present invention, a device for bio-microfluidic mixing is used to prepare the lipid particles and therapeutic formulations of the present invention. The device includes a first reagent stream and a second reagent stream, which are supplied to a microfluidic mixer, and the lipid particles are collected from the outlet or, in other embodiments, enter a sterile environment.

[0108] The first stream includes a therapeutic agent in a first solvent. Suitable first solvents include solvents in which the therapeutic agent is soluble and miscible with the second solvent. Suitable first solvents include aqueous buffers. Representative first solvents include citrate and acetate buffers.

[0109] The second stream includes a lipid hybrid material in a second solvent. Suitable second solvents include solvents in which the cationic lipid is soluble and which are 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 absolute ethanol.

[0110] In one embodiment of the invention, suitable devices include one or more microchannels (i.e., channels with a maximum dimension less than 1 mm). In one embodiment, the diameter of the microchannels 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 forming an angle with the main direction as described in U.S. Patent Publication No. 20040262223 (e.g., a staggered herringbone mixer), or a bifurcated annular flow as described in U.S. Patent Publication No. 2018093232. To achieve maximum mixing rates, it is advantageous to avoid undue fluid resistance prior to the mixing region. Thus, an example of a device has a non-microfluidic channel greater than 1000 microns in size to deliver fluid to a single mixing channel.

[0111] Less automated micro-mixing 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 the present invention. A more primitive system involving T-tube mixing is disclosed in Jeffs L B et al., A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. PharmRes. 2005;22(3):362 - 72.

[0112] Conjugation method

[0113] The lipid nanoparticle conjugates described herein are composed of an antibody, a linker, and a lipid nanoparticle (payload). Each component of the conjugate plays a crucial role in stabilizing the conjugate and minimizing potential side effects. An ideal conjugate should be able to effectively reach the target tissue or cell without prematurely releasing any off-target payload and exert the efficacy of the payload in the target tissue or cell without affecting normal healthy cells. To develop an effective lipid nanoparticle conjugate, factors such as the selection of the antigen, antibody, payload, and linker must be considered.

[0114] Selection of antigen

[0115] The target antigen should help distinguish the target tissue or cell from the normal tissue or cell, thereby reducing off-target toxicity. Therefore, selecting a suitable target antigen is the first step in developing an ideal lipid nanoparticle conjugate. An ideal antigen must have certain characteristics:

[0116] 1) It should be overexpressed on the surface of the target cell compared to normal cells;

[0117] 2) The binding site of the target antigen should face the outer surface of the target cell, rather than the interior of the target cell. This enables the lipid nanoparticle conjugate diffusing from the blood vessel to bind to the target antigen before internalization;

[0118] 3) The target antigen should not enter the systemic circulation to avoid unnecessary binding of the lipid nanoparticle conjugate outside the target tissue; and

[0119] 4) The target antigen should have the potency to internalize and bind the lipid nanoparticle conjugate.

[0120] In one embodiment, the target antigen selected in the present disclosure is a cell surface antigen expressed on the target cell. In one embodiment, the target antigen selected in the present 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, tenascin 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, frizzled protein-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin (including α v β3, α v β5, αv β6, α1β4, α4β1, α5β1, α6β4 integrins), tenascin C, TRAIL-R2, or vimentin.

[0121] Selection of antibody

[0122] In an ideal lipid nanoparticle conjugate, the antibody is an important carrier that specifically binds to the target antigen. The antibody must have a high binding affinity for the target antigen and low immunogenicity. In addition, the antibody should have the ability to maintain a long plasma half-life and rapid internalization. Five types of antibodies are present in human serum, and among them, immunoglobulin G (IgG) is the most abundant serum antibody, accounting for approximately 70 - 85% of the total antibodies, with a half-life of approximately 21 days. Since IgG has the most abundant antibody repertoire and the ability to initiate immune effector functions, IgG is the most commonly used antibody in lipid nanoparticle conjugates. In addition, IgG antibodies include IgG1, IgG2, IgG3, and IgG4 isotypes, and among them, IgG1 is the most used antibody in lipid nanoparticle conjugates and has been proven to be an effective immune effector.

[0123] Antibody glycosylation is a type of post-translational modification that can occur via the addition of oligosaccharides to the antibody through two types of covalent bonds - bonds on asparagine residues (N-glycans) or bonds on serine / threonine residues (O-glycans) (Alter, G. et al., Semin Immunol., 2018, Vol. 39: pp. 102 - 110); and significantly affects the therapeutic functions of antibodies (Walsh, G. and Jefferis, R., Nat. Biotechnol., 2006, Vol. 24: pp. 1241 - 1252; Jefferis, R., Nat. Rev. Drug Discov., 2009, Vol. 8 No. 3: pp. 226 - 234; Dalziel, M. et al., Science, 2014, Vol. 343: No. 6166: p. 1235681). Notably, all IgG antibodies are glycosylated at the conserved residue 297 in their Fc region (Alter G. et al., ibid.).

[0124] When referring to residues in the constant region of the immunoglobulin heavy chain (Fc), the "EU numbering" or "EU index" is usually used. It refers to the residue numbering of the human IgG1 EU antibody. It is calculated by aligning the antibody sequence with the Eu antibody sequence (Edelman, G.M. 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.

[0125] Most monoclonal antibodies are expressed by CHO, SP2 / 0, and NS0 cells, and N-glycosylation is the most common form of glycosylation. Core glycosylation usually occurs in the endoplasmic reticulum, catalyzed by oligosaccharyltransferase (OST), which transfers a pre-synthesized sugar chain (core sugar chain) to the Asn in the Asn-X-(Ser / Thr) sequence of the target protein, where X represents any amino acid except Pro. In IgG-type antibodies, this motif is mainly located in the CH2 domain of the heavy chain, and the glycosylation site of most antibodies is only at about N297 of the heavy chain Fc.

[0126] The N-glycan linked to the 297th residue consists of a conserved biantennary core structure (Liu, L., J Pharm Sci., 2015, Vol. 104, No. 6: pp. 1866-1884), which is composed of two covalently linked N-acetylglucosamine (GlcNAc) residues further linked to mannose and linked 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, P.M., Science, 2001, Vol. 291, No. 5512: pp. 2370-2376; Liu, L., ibid.). At least 30 structures (glycoforms) have been reported for the IgGN297-linked N-glycans (Alter, G. et al., ibid.). Since both heavy chains of the antibody can be glycosylated, there may also be significant oligosaccharide heterogeneity within a single antibody molecule. Oligosaccharides can directly affect the binding of Fc to FcγR. The oligosaccharide at N297 on Fc sterically hinders the binding of Fc to FcγRⅢa, resulting in a decrease in antibody-mediated ADCC. Nuclear magnetic resonance shows that when different oligosaccharides are present at Fc N297, there are differences in the conformation of its hinge region. Since the hinge region of the antibody interacts with FcγR, glycosylation at position N297 indirectly affects the interaction between Fc and FcγR.

[0127] In one embodiment, the selected antibody in the present disclosure is an IgG antibody. In one embodiment, the selected antibody in the present disclosure has an IgG1, IgG2, IgG3, or IgG4 isotype, which contains an N-glycosylation site at the asparagine residue at position 297. In a preferred embodiment, the antibody has an IgG1 isotype, and the N-glycosylation site is at the asparagine residue at position 297. In another preferred embodiment, the antibody has an IgG2 isotype, and the N-glycosylation site is at the asparagine residue at position 297.

[0128] In one embodiment, the antibody specifically binds to a cell surface antigen, which includes 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, tenascin 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, frizzled protein-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including α v β3, α v β5, α v β6, α1β4, α4β1, α5β1, α6β4 integrins), tenascin C, TRAIL-R2, or vimentin.

[0129] Selection of linker

[0130] The linker is the bridge between the antibody and the lipid nanoparticle (payload), playing an important role in the stability and effectiveness of the conjugate. In addition, the release of the payload also mainly depends on the type and properties of the linker. An ideal linker should have high water solubility to prevent the formation of payload aggregates and premature release of the payload in the systemic circulation. Generally, the three components of the antibody, linker, and payload can be modified to obtain a stable and effective conjugate. The three main factors affecting linker stability and payload release are the conjugation site, steric hindrance, and linker length.

[0131] The conjugation site is a major factor affecting the stability of the conjugate and is also a key factor affecting its efficacy. The conjugation site can resist payload loss mediated by the retro-Michael reaction in the systemic circulation and can also affect the deconjugation of non-specific valine-citrulline in the body. The conjugation site can also improve the efficacy of the conjugate by reducing the metabolic load, demonstrating that the conjugation site plays a crucial role in the stability of the conjugate.

[0132] Conjugation steric hindrance creates a steric barrier, thus changing 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, resulting in changes in the stability of the conjugate. Compared with a linker with a longer length, a shorter linker can make the conjugate have better stability. However, a shorter linker brings the payload closer to the steric barrier provided by the antibody. The linker should be stable in the systemic circulation and bind at the target cell under chemical or enzymatic action. The effective de-binding of the linker to release a sufficient amount of the payload is a key factor in the effectiveness of the conjugate.

[0133] By selecting specific conjugation sites and linkers, the present disclosure provides a stable conjugate in which the antibody modification sites are fixed and the number is between 1 and 4; due to steric hindrance, an antibody can be linked to at most two LNPs, reducing LNP aggregation and improving particle homogeneity; and the high-efficiency tissue targeting of the antibody is also maintained.

[0134] In one embodiment, the conjugation site is the N-glycosylation site of the heavy chain Fc region of the antibody. In a preferred embodiment, the N-glycosylation site is located at the asparagine residue at position 297.

[0135] In one embodiment, the lipid nanoparticles can be conjugated to the antibody via streptavidin / biotin bonds, thiol / maleimide chemistry, azide / alkyne chemistry, tetrazine / norbornene chemistry, and other chemistries. As used herein, the term "click chemistry" refers to biocompatible reactions primarily aimed at binding selected substrates to specific biomolecules. Click chemical reactions are not interfered with by water, produce few and non-toxic by-products, and are characterized by a high thermodynamic driving force that can rapidly and irreversibly drive it to obtain a high yield of a single reaction product with high reaction specificity.

[0136] In one embodiment, the antibody is conjugated to the surface of the LNP by click chemistry, preferably copper-free click chemistry.

[0137] The term "copper-free click chemistry" as used herein refers to a chemical synthesis method for rapidly and efficiently synthesizing useful new molecules based on the formation of carbon-heteroatom bonds (C-X-C) in the absence of cytotoxic transition metal catalysts. Copper-free click chemistry can be achieved through strain-promoted azide-alkyne click chemistry, utilizing the reaction between cyclooctyne and azide. This reaction does not contain copper and proceeds due to the high activation energy of the strained molecule. In copper-free click chemistry, commonly used reagents include dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO), and bicyclo[6.1.0]nonyne (BCN). Azide-containing compounds can react with alkyne-containing compounds or small biomolecules through azide-alkyne click chemical reactions to form stable 1,2,3-triazole linkages.

[0138] In one embodiment, the antibody is conjugated to the surface of the LNP via azide / alkyne chemistry.

[0139] In one embodiment, the conjugation site corresponds to the N-glycosylation site of the asparagine residue at position 297 of the Fc region of the antibody heavy chain.

[0140] In one embodiment, each antibody is conjugated to at most 2 LNPs.

[0141] In one embodiment, a method for preparing a lipid nanoparticle (LNP) conjugate as described herein is provided, the method comprising the following steps:

[0142] (a) Providing an antibody or an antigen-binding fragment thereof having an N-glycosylation site in the Fc heavy chain;

[0143] (b) Contacting the antibody or an antigen-binding fragment thereof with galactosidase to remove the galactose residue at the N-glycosylation site of the Fc heavy chain of the antibody or an antigen-binding fragment thereof, exposing the N-acetylglucosamine residue;

[0144] (c) contacting the antibody or antigen-binding fragment thereof obtained in step (b) with β-1,4-galactosyltransferase to attach UDP-galactose azide to the exposed N-acetylglucosamine residue; and

[0145] (d) mixing the antibody or antigen-binding fragment thereof obtained in step (c) with an LNP modified with dibenzocyclooctyne (DBCO) on its surface, and site-specifically conjugating the antibody or antigen-binding fragment thereof to the surface of the LNP through click chemistry.

[0146] In one embodiment, the LNP modified with dibenzocyclooctyne (DBCO) on its surface comprises DBCO covalently linked to PEG.

[0147] Galactosidase refers to a class of enzymes that hydrolyze substances containing galactoside bonds (such as lactose). In one embodiment, the galactosidase is β-galactosidase. In one embodiment, the N-glycan linked to the 297th residue of the heavy chain Fc region of the antibody according to the present invention comprises 1 or 2 galactoses, which can be removed by galactosidase, thereby exposing 1 or 2 N-acetylglucosamine residues. β-1,4-galactosyltransferase (B4GALT1) catalyzes the reaction involving UDP-galactose and N-acetylglucosamine for the production of galactose β-1,4-N-acetylglucosamine. These enzymes are all known to those skilled in the art and are readily commercially available.

[0148] Other methods known in the art can also be used to produce the LNP conjugates disclosed herein. For example, the conjugation can be mediated by a ligand-receptor pair, such as the biotin-streptavidin pair. In this case, one member of the ligand-receptor pair can be linked to the antibody, and the other member can be linked to the LNP.

[0149] Use

[0150] The LNP conjugates of the present 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 to cause a desired effect. A peptide is a short chain of amino acids, with a length of 2 - 50 amino acids, as opposed to a protein with a longer chain (50 amino acids or more), which typically has a tertiary and / or quaternary structure. The amino acids in a peptide are linked to each other in sequence by a bond called a peptide bond.

[0151] The LNP conjugates of the present 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 with a backbone formed by nucleotides or their analogs. The term "nucleic acid" also refers to ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phospho-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single-stranded, double-stranded, or contain portions of double-stranded and single-stranded sequences, as appropriate.

[0152] As used herein, the term "nucleic acid therapeutic (NAT)" is intended to include any oligonucleotide or polynucleotide that is delivered into cells to cause 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, the oligonucleotide has a length of 20 - 50 nucleotides. In some embodiments, the polynucleotide has a length of 996 - 4500 nucleotides, as in the case of messenger RNA.

[0153] The delivery methods and LNP conjugates can be readily adapted to deliver any suitable therapeutic agent for treating any disease or disorder that would benefit from such treatment.

[0154] In some cases, the terms "polynucleotide" and "oligonucleotide" may be used interchangeably and refer to single-stranded and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bonds, e.g., 3'-5' and 2'-5' inverted linkages, e.g., 3'-3' and 5'-5' branched structures or internucleotide linkage analogs. Polynucleotides can have associated counterions such as H+, NH4+, trialkylammonium, Mg2+, Na+, etc. Polynucleotides can consist entirely of deoxyribonucleotides, entirely of ribonucleotides, or a chimeric mixture thereof. Polynucleotides can contain internucleotide linkages, nucleobases, and / or sugar analogs.

[0155] Currently, NATs are being actively pursued in an increasing number of preclinical and clinical studies. These NATs include deoxyribonucleic acid, complementary deoxyribonucleic acid, full genes, ribonucleic acid, 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 (NAT) is incorporated into lipid nanoparticles during its formation. In other embodiments, the nucleic acid therapeutic (NAT) is incorporated into lipid nanoparticles after its formation.

[0156] 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 regions and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNAs, mRNAs, and oligonucleotides forming triplexes.

[0157] In one embodiment, the nucleic acid is selected from mRNA, saRNA, circRNA, siRNA, pDNA, and ssDNA.

[0158] The term "mRNA" stands for messenger ribonucleic acid, which is a single-stranded ribonucleic acid responsible for protein synthesis in biological cells. As a template for ribosomes, mRNA guides tRNA (transfer RNA) to bring the corresponding amino acids to the ribosome and assemble them into proteins according to the codon sequence on the mRNA.

[0159] 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 large copies of the original RNA, causing high-level and sustained expression of the target protein.

[0160] The term "circRNA" stands for circular RNA, which refers to polyribonucleotides that form a circular structure through covalent bonds. circRNA is a single-stranded RNA that can form a 3'-5' covalent closed loop. circRNA can be generated by various mechanisms. A major method is back-splicing, which is a non-classical splicing process mediated by the spliceosome. During this process, the downstream splice donor site is connected to the upstream splice acceptor site to form a covalent closed loop. In addition, circRNA can also be generated by chemical ligation, enzymatic ligation, and ribozyme methods. These alternative methods can form circular RNA structures, expanding the variety of circRNAs available for research and therapeutic applications. Different from linear mRNA, circRNA does not require a 5'-cap or 3'-poly(A) tail to maintain stability. The closed-loop structure of circRNA can protect them from exonuclease-mediated degradation, making them resistant to various mechanisms of RNA turnover and having a longer half-life compared to their linear mRNA counterparts.

[0161] The term "siRNA" stands for small interfering RNA, also known as short interfering RNA or silencing RNA, which is a class of double-stranded RNA molecules with a length of approximately 20 - 25 base pairs. siRNA functions in the RNA interference (RNAi) pathway, preventing the translation of specific genes by degrading mRNA molecules complementary to the siRNA sequence, thereby achieving gene silencing effects.

[0162] The term "pDNA" stands for plasmid DNA, which is a DNA molecule that is separated from chromosomal DNA in cells and can replicate independently. The size of plasmids ranges from less than 1000 nucleotides to tens of thousands of nucleotides. The most common form is a small circular double-stranded DNA. Plasmids can be synthesized and delivered into mammalian cells for therapeutic purposes. Synthetic plasmids are used as vectors in molecular cloning to drive the replication of recombinant DNA sequences in host organisms. Plasmids can be introduced into cells via transformation using physical methods such as electroporation or chemical means such as transfection enhanced by lipid particles in the present invention. Compared with physical techniques, these lipid-based vector systems have several advantages, including: i) high biocompatibility and low toxicity in cell and tissue systems; ii) relatively easy to manufacture; iii) the lipophilic matrix is not easily affected by the corrosion phenomena observed in polymer systems; iv) increased in vivo circulation half-life due to their invisibility to the immune system.

[0163] The term "ssDNA" stands for single-stranded DNA, which is composed of a single nucleotide chain and lacks the other complementary strand compared to the usual double-stranded DNA (dsDNA). ssDNA differs from dsDNA in terms of molecular hydrodynamic properties, absorption spectra, base reaction properties, etc. In living organisms, ssDNA appears 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.

[0164] As used herein, "N / P" is the ratio of the number of moles of the amine group of the cationic lipid to the number of moles of the phosphate group of mRNA / DNA. In one embodiment, the range of N / P is from 2:1 to 30:1, such as from 3:1 to 22:1. In one embodiment, the range of N / P is from 6:1 to 20:1, or from 2:1 to 12:1. Exemplary N / P ranges include approximately 3:1, approximately 6:1, approximately 12:1, and approximately 22:1, and the most preferred ratio is from 4:1 to 6:1.

[0165] In some embodiments, the LNP comprises a total lipid to mRNA weight ratio of from about 10:1 to about 30:1, such as from about 10:1 to about 20:1, from about 15:1 to about 25:1, or from about 20:1 to about 30:1. In some embodiments, the LNP comprises a total lipid to circular RNA or circularized precursor RNA weight ratio greater than about 10:1, such as greater than about 15:1, 20:1, 25:1, 30:1 or greater. In some embodiments, the LNP comprises a total lipid to circular RNA or circularized precursor RNA weight ratio less than about 30:1, such as less than about 25:1, 20:1, 15:1, 10:1 or less. In some embodiments, the weight ratio of total lipid to circular RNA or circularized precursor RNA can be adjusted according to 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 (such as ultraviolet-visible spectroscopy).

[0166] The LNP conjugate according to the 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).

[0167] The following describes pharmaceutical compositions comprising LNP conjugates and methods of delivering nucleic acid therapeutics by the LNP conjugates of the invention.

[0168] In certain embodiments, the invention provides methods for delivering cargo molecules into cells. In one embodiment, the cargo molecule is a nucleic acid, and thus the delivery method is also referred to as transfection. Transfection is a commonly used technique in molecular biology for introducing nucleic acid therapeutics (or NATs) from outside the cell into the intracellular space for the purpose of transcribing, translating, and expressing the delivered gene. Transfection efficiency is typically defined as: i) the percentage of cells in the total treated population that show 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 invention with the cells for a period of time sufficient to effect intracellular delivery.

[0169] Typical applications also include the use of well-known procedures to provide intracellular delivery of siRNA to knock out or silence specific cellular targets. Optionally, the applications also include the delivery of DNA or mRNA sequences encoding therapeutically useful polypeptides. In this way, the treatment of genetic diseases is provided by supplying a defective or missing gene product. The methods of the present invention can be practiced in vitro, ex vivo, or in vivo. For example, using methods known to those skilled in the art, the LNP conjugates of the present invention can also be used to deliver nucleic acids to cells in vivo. In another embodiment, the LNP conjugates of the present invention can be used to deliver nucleic acids to a patient's cell sample ex vivo and then returned to the patient.

[0170] In one embodiment, the present invention provides methods of modulating the expression of a target polynucleotide or polypeptide. These methods generally comprise contacting a cell with an LNP conjugate of the present invention that is bound to a nucleic acid capable of modulating the expression of the target polynucleotide or polypeptide. As used herein, the term "modulating" refers to altering the expression of a target polynucleotide or polypeptide. Modulating can mean increasing or enhancing, or it can mean decreasing or reducing.

[0171] In a related embodiment, the present invention provides methods of treating a disease or disorder in a subject characterized by overexpression of a polypeptide, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from siRNA, microRNA, antisense oligonucleotides, and plasmids capable of expressing siRNA, microRNA, or antisense oligonucleotides, and wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide or its complement.

[0172] In a related embodiment, the present invention provides methods of treating a disease or disorder in a subject characterized by underexpression of a polypeptide, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from mRNA, self-amplifying RNA (SAM), self-replicating DNA, or plasmids that comprise a nucleic acid therapeutic agent that specifically encodes or expresses the underexpressed polypeptide or its complement.

[0173] As used herein, the term "pharmaceutically acceptable" refers to those compounds, conjugates, materials, compositions, and dosage forms that, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0174] As used herein, the term "treatment" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes administering a conjugate or pharmaceutical composition of the present disclosure to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treatment" can also include the treatment of in vitro cell or animal models.

[0175] As used herein, the term "prevent" describes reducing or eliminating the onset of symptoms or complications of such disease, condition or disorder.

[0176] The term "subject" refers to an animal, preferably a mammal, and most preferably a human, that is the object of treatment, observation or experiment.

[0177] The term "therapeutically effective amount" refers to the amount of an active compound or agent that elicits a biological or medical response in a tissue system, animal or human being sought by a researcher, veterinarian, physician or other clinician, including alleviating or partially alleviating the symptoms of the disease, syndrome, condition or disorder being treated.

[0178] "Therapeutically effective amount" is intended to mean an amount of the conjugate as defined herein that is sufficient to effectively treat or prevent when administered to a patient in need of such treatment. The amount of a given conjugate corresponding to such amount will depend on factors such as the specific conjugate (e.g., the potency (pIC 50 ), efficacy (EC 50 ), and biological half-life), the disease state and its severity, the identity of the patient in need of treatment (e.g., age, size and weight), but can still be routinely determined by those skilled in the art. Similarly, the duration of treatment and the dosing period (the period between doses and the timing of the dose, e.g., before / during / after a meal) of the conjugate should vary depending on the identity of the mammal in need of treatment (e.g., weight), the specific conjugate and its properties (e.g., pharmacokinetic properties), the disease or disorder and its severity, and the specific composition and method used, but can still be determined by those skilled in the art.

[0179] The term "composition" or "pharmaceutical composition" refers to a product containing a therapeutically effective amount of a designated ingredient and any product directly or indirectly resulting from the combination of a designated amount of the designated ingredients.

[0180] For in vivo administration, parenteral (e.g., intra-articular, intravenous, intraperitoneal, subcutaneous, intrathecal, intradermal, intratracheal, intraosseous or intramuscular) administration of the pharmaceutical composition is preferred. In certain embodiments, the pharmaceutical composition is administered intravenously, intrathecally or intraperitoneally. Other routes of administration include topical (skin, eye, mucosa), oral, pulmonary, intranasal, sublingual, rectal and vaginal.

[0181] For ex vivo applications, it is preferred to administer the pharmaceutical composition to a biological sample that has been removed from an organism, and then the cells are washed and returned to the organism. The organism can be a mammal, particularly a human. This method is used for, for example, cell reprogramming, gene repair or immunotherapy, etc.

[0182] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that is generally safe and non-toxic and is not otherwise undesirable for use in preparing a pharmaceutical composition, and includes excipients acceptable for veterinary as well as human pharmaceutical use. "Pharmaceutically acceptable excipient" as used in the specification and claims includes one and more excipients.

[0183] Pharmaceutically acceptable excipients used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, surfactants and / or emulsifiers, preservatives, buffering agents, lubricants and / or oils. Such excipients may optionally be included in the pharmaceutical compositions of the present invention. The various excipients used to formulate pharmaceutical compositions and the techniques for preparing the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient media is contemplated herein, except where any conventional excipient medium may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition in some other way.

[0184] In one embodiment, pharmaceutically acceptable excipients useful in the compositions of the present invention include, but are not limited to, ion exchange agents, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, 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 carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene block polymers, polyethylene glycol, and lanolin.

[0185] To aid in the delivery of the pharmaceutical compositions disclosed herein, any mild fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, may be used in the preparation of injectables, such as natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions.

[0186] Other commonly used surfactants, such as polysorbates ( compounds), sorbitan esters ( Emulsifiers or bioavailability enhancers, such as (compounds), and other agents commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

[0187] Alternatively, the pharmaceutical compositions disclosed herein may be administered in the form of suppositories for rectal administration. These may be prepared by mixing the conjugate with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and will thus melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0188] The pharmaceutical compositions disclosed herein may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well recognized in the pharmaceutical formulation art and may be formulated as a saline solution, using benzyl alcohol or other suitable preservatives, absorption promoters, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0189] In embodiments, the formulations of the pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology or developed hereafter. Generally, such preparation methods include the step of combining the active ingredient with an excipient and / or one or more other accessory ingredients.

[0190] The pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold as a single unit dose and / or as multiple single unit dose batches. As used herein, "unit dose" refers to a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient will 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 one-half or one-third of such dose.

[0191] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in the pharmaceutical compositions according to the present disclosure may vary depending on the age, sex, weight, and / or medical condition of the subject being treated and further depending on the route of administration of the composition. For example, the composition may contain from 0.1% to 99% (w / w) of the active ingredient.

[0192] In some embodiments, the particle size of the lipid particles may be increased and / or decreased. Variation in the particle size may be capable of helping to counter biological responses such as, but not limited to, inflammation, or may increase the biological effect delivered to the NAT of a mammal by altering the biodistribution.

[0193] The present disclosure also provides a kit for using an LNP conjugate to deliver a cargo molecule for a desired therapeutic or diagnostic purpose. Such a kit may include one or more containers that contain one or more pharmaceutical compositions, which comprise one or more LNP conjugates disclosed herein and one or more pharmaceutically acceptable excipients.

[0194] 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 desired activity in a human patient. The kit may further include a description of selecting a suitable human patient 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 in need of treatment.

[0195] The following examples are provided for illustrative purposes and not for limiting the invention.

[0196] Example 1: Antibody modification

[0197] Using SiteClick TM Antibody azide modification kit (from Invitrogen, catalog number S20026), according to the manufacturer's instructions, the antibody was modified with azide. Briefly, the antibody was ultrafiltered and exchanged into a buffer without azide groups, the galactose residue on the sugar chain (N297) was removed using β-galactosidase to expose the N-acetylglucosamine residue, and then UDP-galactose azide was linked to the exposed N-acetylglucosamine residue using β-1,4-galactosyltransferase. Finally, it was ultrafiltered and purified to obtain an antibody with azide modification at the N-monosaccharide glycosylation site on the specific asparagine residue (N297) of 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.

[0198] Example 2: LNP preparation

[0199] The lipids used in this example were:

[0200] SM-102 (a cationic lipid), from MCE, catalog number HY-134541;

[0201] DSPC, from Aladdin, catalog number D130429-1g;

[0202] Cholesterol, from Sigma, catalog number C8667-5G;

[0203] DMG-PEG2000, from MCE, Catalog No. HY-112764; and

[0204] DSPE-PEG2000-DBCO, from Huateng Pharmaceutical, Catalog No. LP096048-2K.

[0205] The mRNA used in this example is:

[0206] HiExpress TM Firefly luciferase IVT mRNA (m1Psi modified), from VectorBuilder, Catalog No. NR1021-1000; and

[0207] HiExpress TM Cre IVT mRNA (m1Psi modified), from VectorBuilder, Catalog No. NR1121-1000.

[0208] The preparation steps are as follows:

[0209] Weigh 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, and place them in five 1.5 ml EP tubes. Add anhydrous ethanol to dissolve the lipids, so that the concentration of each lipid solution is 40 mM. Use ultrasound to promote dissolution for 2 min, and then mix all the lipids evenly into one EP tube, with a total volume of 320 μl. Aspirate this mixed solution (ethanol phase) with a 1 ml syringe.

[0210] In another EP tube, mix sodium acetate buffer (pH 4) and 346 μg of mRNA, so that the final concentration of sodium acetate buffer (pH 4) is 50 mM, and the total volume of the resulting solution is 960 μl. Aspirate this solution (aqueous phase) with a 1 ml syringe.

[0211] Using the instrument NanoAssemblr IgniteTM (from Precision NanoSystems), mix the two phases using microfluidic mixing technology. Set the FRR (flow rate ratio) to 3:1 and the total flow rate to 12 ml / min. Dilute the mixed sample with 25 volumes of 15 mM Tris-HCl, concentrate it using an Amicon Ultra-15 mL 10 kDa ultrafiltration tube, perform ultrafiltration and buffer exchange three times, with centrifugation conditions of 2800×g, 12 °C, 15 min, and filter and sterilize to obtain LNP.

[0212] Example 3: Preparation and characterization of site-specific conjugated LNP with antibody

[0213] Mix the azide-modified antibody prepared in Example 1 and the LNP prepared in Example 2 at a mass ratio of 1:1, conduct the coupling reaction overnight, and obtain the antibody-site-specifically conjugated LNP after filtration and sterilization.

[0214] Measure the particle size and potential of LNP by resistive pulse sensing (RPS) or dynamic light scattering (DLS) methods, measure the polydispersity index PDI of LNP by DLS method, and measure the encapsulation efficiency of LNP by RiboGreen method. Details are as follows:

[0215] After diluting the sample 1000 times, conduct RPS detection with the instrument Nanocoulter G nanoparticle counter (manufacturer: Ruixin Zhizao).

[0216] After diluting the sample 50 times, conduct DLS detection with the instrument Zetasizer Ultra (manufacturer: Malvern Panalytical).

[0217] Use the Quant-iT RiboGreen RNA kit (from Invitrogen, catalog number R11490) to detect the encapsulation efficiency of LNP.

[0218] All detections are carried out according to the manufacturer's instructions.

[0219] Example 4: In vivo experiment

[0220] Inject LNP into 6-8-week-old female ICR mice (from Guangzhou Bojin Biotechnology Co., Ltd.) via the tail vein at an injection dose of 0.5 mg / kg of mouse body weight. Dissect and take the main organs (lung, liver, spleen, heart, kidney) for ex vivo imaging 6 h after injection. Specifically, weigh 45 mg of Pierce TM D-luciferin monopotassium salt (from Thermo Fisher, catalog number 88292) in the ultra-clean bench in the dark, dissolve it with 3 ml of 1×PBS, filter and sterilize it with a 0.22 μm filter to prepare a 15 mg / ml substrate solution. Inject the substrate intraperitoneally at 10 μl / g, wait for 10 min, dissect the mice, take the lung, liver, spleen, heart, and kidney organs, and place them in the AniView100 multimode in vivo imaging system (from Boluteng) for imaging. The results are shown in Figure 2 .

[0221] LNP was injected into 6-week-old male Ai9 mice (from Guangzhou Bojin Biotechnology Co., Ltd.) via the tail vein at low dose (group denoted as L, 0.1 mg / kg body weight of mice) and high dose (group denoted as H, 0.4 mg / kg body weight of mice). Cardiac perfusion was performed 72 h after injection, and the lungs were dissected for fixation, dehydration, and cryosectioning to observe the expression of tdTomato red fluorescence. The negative control group was the PBS injection group. The results are shown in Figure 4 .

[0222] Results:

[0223] (1) The mRNA encapsulated by LNP was luciferase mRNA. The RPS results of uncoupled antibody LNP showed that the average particle size was 76 nm ( Figure 1A ). The DLS results showed that the PDI was 0.061 and the average zeta potential was -7.78 mV. RiboGreen measurement showed an encapsulation efficiency of 90.1%. The RPS results of CD31 antibody site-specifically conjugated LNP showed that the average particle size was 93 nm ( Figure 1B ). The DLS results showed that the PDI was 0.015 and the average zeta potential was -8.17 mV. RiboGreen measurement showed an encapsulation efficiency of 90.1%.

[0224] (2) As Figure 2 shown, in the in vivo experiment, CD31 antibody site-specifically conjugated LNP was targeted to the lungs for expression, followed by the liver and spleen; uncoupled antibody LNP was expressed in the liver and spleen and not in the lungs. It was demonstrated that CD31 antibody was site-specifically conjugated to LNP and achieved in vivo lung targeting.

[0225] (3) The mRNA encapsulated by LNP was Cre mRNA. The RPS results of uncoupled antibody LNP showed that the average particle size was 82 nm ( Figure 3A ). The DLS results showed that the PDI was 0.150 and the average zeta potential was -7.34 mV. RiboGreen measurement showed an encapsulation efficiency of 96.9%. The RPS results of CD31 antibody site-specifically conjugated LNP showed that the average particle size was 83 nm ( Figure 3B ). The DLS results showed that the PDI was 0.077 and the average zeta potential was -4.16 mV. RiboGreen measurement showed an encapsulation efficiency of 100%.

[0226] (4) As Figure 4 shown, in the in vivo experiment, CD31 antibody site-specifically conjugated LNP was targeted to the lungs for expression, and uncoupled antibody LNP was not expressed in the lungs, demonstrating that CD31 antibody was site-specifically conjugated to LNP and achieved in vivo lung targeting.

Claims

1. A lipid nanoparticle (LNP) conjugate, characterized in that The LNP conjugate comprises (a) a lipid nanoparticle (LNP) and (b) an antibody or an antigen-binding fragment thereof with cell or tissue targeting, wherein the antibody or the antigen-binding fragment thereof is coupled to the surface of the LNP through the N-glycosylation site of the Fc heavy chain.

2. The LNP conjugate according to claim 1, characterized in that The antibody or antigen-binding fragment thereof is an IgG, optionally wherein the antibody or antigen-binding fragment thereof is of an IgG1, IgG2, IgG3 or IgG4 isotype, preferably an IgG1 or IgG2 isotype, and optionally wherein the N-glycosylation site is located at asparagine residue 297 of the Fc heavy chain according to the EU index for amino acid residue numbering.

3. The LNP conjugate according to claim 1 or 2, characterized in that coupling the antibody or antigen-binding fragment thereof to the surface of the LNP by click chemistry, preferably copper-free click chemistry; Optionally, the click chemistry includes but is not limited to azide / alkyne chemistry; Optionally, the Fc heavy chain has an azide functional group on its N-glycosylation site, preferably galactose azide, and the LNP has an alkyne functional group on its surface, preferably dibenzocyclooctyne (DBCO), more preferably PEG-DBCO.

4. The LNP conjugate according to any one of claims 1-3, characterised in that Each antibody or antigen-binding fragment thereof is coupled to 1 or 2 LNPs.

5. The LNP conjugate according to any one of claims 1-4, characterised in that The antibody or antigen-binding fragment thereof specifically binds to an antigen selected from the group consisting of 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, tenascin 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, Frizzled-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin, tenascin C, TRAIL-R2, and vimentin.

6. The LNP conjugate according to any one of claims 1-5, characterised 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 the diagnostic agent is encapsulated in the LNP 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 according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. A method for preparing a lipid nanoparticle (LNP) conjugate, characterized in that The method comprises coupling an antibody or an antigen-binding fragment thereof having an N-glycosylation site in the Fc heavy chain to the surface of LNP by click chemistry.

9. The method according to claim 8, characterized in that The antibody or antigen-binding fragment thereof is an IgG, optionally wherein the antibody or antigen-binding fragment thereof is of an IgG1, IgG2, IgG3 or IgG4 isotype, preferably an IgG1 or IgG2 isotype, and optionally wherein the N-glycosylation site is located at asparagine residue 297 of the Fc heavy chain according to the EU index for amino acid residue numbering.

10. The method according to claim 8 or 9, characterized in that coupling the antibody or antigen-binding fragment thereof to the surface of LNP by copper-free click chemistry; and / or The click chemistry includes but is not limited to azide / alkyne chemistry; and / or The Fc heavy chain has an azide functional group on its N-glycosylation site, preferably galactose azide, and the LNP has an alkyne functional group on its surface, preferably dibenzocyclooctyne (DBCO), more preferably PEG-DBCO.

11. The method according to any one of claims 8 to 10, characterized in that Each antibody or antigen-binding fragment thereof is coupled to 1 or 2 LNPs.

12. The LNP conjugate according to any one of claims 8 to 11, characterized in that The antibody or antigen-binding fragment thereof specifically binds to an antigen selected from the group consisting of 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, tenascin 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, Frizzled-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin, tenascin C, TRAIL-R2, and vimentin.

13. The LNP conjugate according to any one of claims 8 to 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 the diagnostic agent is encapsulated in the LNP 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 a lipid nanoparticle (LNP) conjugate, characterized in that the method comprises the following steps: (a) providing an antibody or an antigen-binding fragment thereof having an N-glycosylation site in the Fc heavy chain; (b) contacting the antibody or antigen-binding fragment thereof with β-galactosidase to remove the galactose residue on the N-glycosylation site of the Fc heavy chain of the antibody or antigen-binding fragment thereof to expose the N-acetylglucosamine residue; (c) linking UDP-galactose azide to the exposed N-acetylglucosamine residue by contacting the antibody or antigen-binding fragment thereof obtained in step (b) with β-1,4-galactosyltransferase; and (d) mixing the antibody or antigen-binding fragment thereof obtained in step (c) with LNPs having a surface modified with dibenzocyclooctyne (DBCO), and site-specifically coupling the antibody or antigen-binding fragment thereof to the surface of the LNPs by a click chemistry reaction.

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

16. The method according to claim 15, characterized in that The cargo molecule is a therapeutic agent or a diagnostic agent, wherein the therapeutic agent or the diagnostic agent is encapsulated in the LNP 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 antigen is 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, tenascin 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, Frizzled-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin, tenascin C, TRAIL-R2, and vimentin.

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

Images